Isac Almeida de Medeiros2 and Darizy Flaacutevia Silva1
1 Laboratorio de Fisiologia e Farmacologia Endocrina e Cardiovascular Departamento de BiorregulacaoInstituto de Ciencias da Saude Universidade Federal da Bahia Avenida Reitor Miguel Calmon Vale do Canela40110-902 Salvador BA Brazil
2 Laboratorio de Farmacologia Cardiovascular Centro de Biotecnologia Universidade Federal da Paraıba Cidade Universitaria58051-900 Joao Pessoa PB Brazil
3 Laboratorio de Neurociencias Instituto de Ciencias da Saude Universidade Federal da Bahia Avenida Reitor Miguel CalmonVale do Canela 40110-902 Salvador BA Brazil
4Nucleo de Investigacoes Quımico-Farmaceuticas Centro de Ciencias da Saude Universidade do Vale do ItajaıRua Uruguai 458 Centro 88302-202 Itajaı SC Brazil
5 Facultad de Ciencias Quımicas y Farmacia Universidad de San Carlos de Guatemala (USAC)01012 Ciudad de Guatemala Guatemala
Correspondence should be addressed to Darizy Flavia Silva darizygmailcom
Received 23 March 2013 Revised 7 July 2013 Accepted 8 July 2013
Academic Editor Jae Youl Cho
Copyright copy 2013 Milena Ramos Reis et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited
Assays in vitro and in vivo were performed on extract from roots and leaves from the Valeriana prionophylla Standl (VPR andVPF resp) In phenylephrine (1 120583M) precontracted rings VPR (001ndash300 120583gmL) induced a concentration-dependent relaxation(maximum response (MR) = 754 plusmn 40 EC
50= 597 (38ndash93) 120583gmL 119899 = 6]) this effect was significantly modified after
removal of the endothelium (EC50= 396 (272ndash576) 120583gmL 119875 lt 005) However VPF-induced vasorelaxation was less effective
compared to VPR When rings were preincubated with L-NAME (100120583M) or indomethacin (10 120583M) the endothelium-dependentrelaxation induced by VPR was significantly attenuated (MR = 209 plusmn 23 342 plusmn 29 resp 119875 lt 0001) In rings denudedendothelium precontracted with KCl (80mM) or in preparations pretreated with KCl (20mM) or tetraethylammonium (1 or3mM) the vasorelaxant activity of VPR was significantly attenuated (MR = 400 plusmn 82 119899 = 5 505 plusmn 60 493 plusmn 64 468 plusmn62 resp 119875 lt 001) In contrast neither glibenclamide (10120583M) barium chloride (30120583M) nor 4-aminopyridine (1mM) affectedVPR-induced relaxation Taken together these results demonstrate that hypotension induced by VPR seems to involve at least inpart a vascular component Furthermore endothelium-independent relaxation induced by VPR involves K+ channels activationmost likely due to BKCa channels in the rat superior mesenteric artery
1 Introduction
The Valerianaceae family is well known for an abundanceof different species of the genus Valeriana L used in the
folk medicine for the treatment of psychosomatic disorderssuch as anxiety and insomnia [1 2] Valeriana prionophyllaStandl is a species distributed throughout Latin Americanmainly Costa Rica Guatemala and Mexico and is known
2 Evidence-Based Complementary and Alternative Medicine
as ldquoValeriana del monterdquo Studies performed in animals havedemonstrated that rhizomes of this species affect centralnervous system activity demonstrating sedative hypnoticanxiolytic and antidepressive effects [3 4] However despiteits widespread use by the population for psychosomaticdisorders possible peripheral effect of this species is not wellcharacterized it is important to investigate a possible periph-eral cardiovascular action and thus differentiate betweencentral and peripheral effects in the cardiovascular system
Furthermore phytochemical analysis of rhizomes of Vprionophylla identified the presence of valepotriates [5] andnew lignans isolated from these roots have demonstratedvasorelaxant activity in isolated rat aorta artery rings [6]demonstrating a potential effect on peripheral systems Lig-nans are secondary plant metabolites that exist in the phenyl-propanoid pathway and have been identified and isolatedfrom approximately 70 different families of all plant originsthe majority of which are those used in popular medicine [7]Lignans in the cardiovascular system have demonstrated atherapeutic potential as a cardiotonic agent by the inhibitionof phosphodiesterase III which is responsible for metab-olizing the second messenger cAMP (35-cyclic adenosinemonophosphate) Moreover some lignans also demonstrateantagonistic activity of the platelet-activating factor (PAF)receptor and blocking of the L-type Ca2+ channel [8]
Many cardiovascular disorders such as hypertensionangina and heart failure are often treated with vasodilatordrugs that act directly on the vascular smooth musclecausing vasodilation indirectly by stimulating the release ofendogenous vasorelaxant factors or by inhibiting the releaseof vasoconstrictive factors [9]
Thus the aim of this study was to evaluate the peripheralactions of Valeriana prionophylla Standl in the cardiovas-cular system and the mechanisms underlying the vascularresponse induced by this species in isolated rat mesentericartery
2 Materials and Methods
21 Drugs and Solutions The drugs used in this studywere Cremophor EL dimethyl sulphoxide (DMSO) L-phenylephrine chloride (Phe) acetylcholine chloride (Ach)glibenclamide tetraethylammonium 4-aminopyridine andbarium chloride (SIGMA) All compounds were dissolvedin distilled water except glibenclamide that was dissolvedin DMSO The composition of Tyrodersquos solution used wasas follows (mM) NaCl 1583 KCl 40 CaCl
2 20 MgCl
2
105 NaH2PO4 042 NaHCO
3 100 and glucose 56 K+-
depolarizing solutions (KCl 20 and 80mM) were preparedby replacing 20 or 80mM KCl in Tyrodersquos solution withequimolar NaCl respectively
22 Plant Material Valeriana prionophylla Standl Valerian-aceae were collected from cultivations in Tierra Blanca Con-cepcion Tutuapa San Marcos (15∘ 148081015840N 91∘ 554301015840W)Guatemala a vegetative zone that resides in a very humid andlow mountainous forest Three-year-old rhizomes and rootswere dug up washed and shade-dried Botanical samples
were determined by Mario Veliz at Herbarium BIGU Schoolof Biology USAC and a voucher sample deposited (no49183)
23 Preparation of Ethanol Extract Drymaterial was groundwetted with 50 ethanol and placed in a stainless steelpercolator 50 ethanol was added to obtain a tincture whichwas concentrated in a rotavapor Fresh ethanol was addedfor five consecutive days and the extract was concentratedThe final drying was performed in a vacuum dryer with silicagel as described by Holzmann et al [3] The average yield ofthe extractable solids was 2852 For in vitro experimentsextracts of the roots and leaves from the Valeriana priono-phylla Standl species (VPR and VPF resp) were dissolvedin a mixture of distilled waterCremophor and diluted to thedesired concentrationswith distilledwater just before use thefinal concentration of Cremophor in the bath never exceeded001
24 Animals Male Wistar rats (250ndash300 g) were used forall experiments Animals were housed under controlledtemperature (21plusmn1∘C) exposed to a 12 h light-dark cycle withfree access to food (Purina Brazil) and tap water The studywas carried out in accordance with the Guide for the CareandUse of LaboratoryAnimals as adopted by theUSNationalInstitutes of Health
25 Tissue Preparation Rats were euthanized and superiormesenteric artery was removed cleaned from connectivetissue and fat as described by Silva and colleagues [10]Whenever appropriate the endothelium was removed bygently rubbing the intimal surface of the vessels Rings (1-2mm) were suspended in organ baths containing 10mLof Tyrodersquos solution gassed with a mixture of 95 O
2
and 5 CO2 maintained at 37∘C and at pH 74 Isometric
tension was recorded under a resting tension of 075 g Thesolution was changed every 15min during a stabilizationperiod of 1 hr to prevent the accumulation of metabolites[11] The isometric contractile force was recorded by a forcetransducer (MLT020 ADInstruments Australia) coupledto an amplifier-recorder (ML870P com LabChart versao70 ADInstruments Australia) and to a computer equippedwith a data acquisition software The presence of functionalendothelium was assessed by the ability of Ach (10120583M) toinduce more than 90 relaxation of pre-contracted vesselswith Phe (10 120583M) and the absence less than 10 of relaxationinduced by Ach
26 Effects of VPF and VPR in Phenylephrine-Induced Con-tractions In this experiment sustained Phe-induced con-tractions were obtained in isolated rat superior mesentericartery rings with or without endothelium In the tonic phaseof the second contraction induced by Phe (1 120583M) increasingcumulative concentrations of VPF and VPR (001 003 0103 1 3 10 30 100 and 300 120583gmL) were separately andcontinually added to the bath until a maximum response forthe added extract was observed as indicated by a plateauresponse (approximately 4ndash6min)
Evidence-Based Complementary and Alternative Medicine 3
27 Investigation of Nitric Oxide and COX-Derived Prod-ucts Participation in the Vasorelaxation Mediated by VPRTo investigate the involvement of NO and prostanoidsendothelium-intact rings were incubated for 30min withL-NAME (10minus4M) or indomethacin (10minus5M) (endothelialnitric oxide synthase (eNOS) or cyclooxygenase inhibitorsresp) before the second application of Phe (1 120583M) and thevasorelaxation induced byVPR (300 120583gmL)was investigatedbefore and after the addition of the inhibitors
28 Effect of VPR on Contraction Induced by Depolarizationwith High K+ Concentration After the stabilization periodrings without endothelium were precontracted with highpotassium concentration solution (KCl 80mM) and inthe tonic phase different concentrations of VPR (001ndash300 120583gmL) were added cumulatively to the organ bath andrelaxations were measured as previously described
29 Investigation of K+ Channel Participation in the Vasore-laxation Elicited by VPR To investigate the involvementof K+ channels in the first set of experiments vasorelax-ation with VPR was performed in vessels with denudedendothelium that were precontracted with Phe in Tyrodersquossolution with elevated K+ (20mM equimolar replacementof NaCl with KCl) to attenuate K+ efflux [10] Additionallyin the second group of experiments endothelium-denudedrings were incubated for 30min with putative K+ channelblockers before the second application of Phe (1 120583M) and theinhibition was calculated by comparing the response elicitedby VPR before and after the addition of the inhibitorsPreparations were exposed to tetraethylammonium (TEA3mM) a nonselective K+ channel blocker TEA (1mM)a large-conductance calcium-activated K+ channel (BKCa)selective blocker in this concentration 4-aminopyridine (4-AP) (1mM) a voltage-dependent K+ (KV) channel blockerglibenclamide (Glib) (10 120583M) an ATP-sensitive K+ (KATP)channel blocker and barium chloride (BaCl
2) (30 120583M) an
inward rectifier K+ (Kir) channel blocker
210 Investigation of Effect of VPR on CaCl2-Induced Con-
tractions To investigate the hypothesis that VPR act throughthe blockade of extracellular calcium influx in endothelium-denuded rings cumulative concentrations of CaCl
2(10minus6ndash
10minus2M) were added in medium containing Ca2+-free depo-larizing solution (KCl 60mM) in absence (control) or inpresence of VPR (300120583gmL)
211 Measurement of Mean Arterial Pressure and Heart Ratein Nonanesthetized Normotensive Rats The day before theexperimental session a catheter (PE
50) filled with hep-
arinized saline solution (1000UmL) was inserted into theleft carotid artery under ketaminexylazine anesthesia andexteriorized at the nape of the animalrsquos neck to permit bloodpressure recording An additional catheter was placed inthe right femoral vein to allow intravenous drug adminis-tration After 24 hours arterial pressure was continuouslymonitored through the carotid catheter connected to a bloodpressure transducer (World Precision Instruments) whose
signal was amplified and digitally recorded by an analog-to-digital interface (AqDados application for data acquisitionLynx Tecnologia Eletronica Ltda version 70 Sao PauloBrazil) and recorded (1 kHz) on a microcomputer for lateranalysis Mean arterial pressure (MAP) was calculated fromsystolic and diastolic pressure data while heart rate (HR) wasdetermined by the pulsation of arterial pressure using theAcqKnowledge software program version 357 developed byBiopac Systems Inc California USA
212 Statistical Analysis Data are presented as the mean plusmnSEM and n represents a number of rings prepared fromdifferent rats Concentration-response curves to VPR andVPF were based on the percent relaxation of the agonist-induced contraction A 100 relaxation was assigned whenthe precontracted rings returned to the baseline values Thecurves were fitted using a variable slope sigmoid fittingroutine in GraphPad Prism50 (Graph Pad Software Inc LaJolla CA USA) EC
50(concentration required to relax the
induced tone by half and maximum response (MR) valueswere calculated from the fitted sigmoidal curves Statisticalanalyses were performed by comparing 119864max and EC50 valuesbetween groups from independent observations UnpairedStudentrsquos t-test or one-way ANOVAs were used to compare2 3 or more groups respectively with the addition ofBonferronirsquos multiple comparisons post-test Two-sided 119875 lt005 was considered statistically significant
3 Results
31 Effects of VPR andVPF in Phenylephrine-InducedContrac-tions In isolated rat superior mesenteric artery rings VPR(001ndash300 120583gmL) decreased in a concentration-dependentmanner following Phe-induced contraction (1 120583M) (EC
50
values = 596 (38minus92) 120583gmL) (Figure 1(a)) In the absenceof the vascular endothelium the potency of VPR was signif-icantly shifted (EC
50values = 3963 (272minus576)120583gmL 119875 lt
005) (Figure 1(a) Table 1) whereas VPF (001ndash300120583gmL)induced a vasorelaxant effect in the presence and absence ofvascular endothelium (maximum response (MR) = 2939 plusmn94MR = 2705plusmn62 resp 119899 = 4) (Figure 1(b)) howeverthis response was reduced in comparison to that obtained byVPR (MR = 7543 plusmn 408 MR = 7033 plusmn 458 119875 lt 005resp)
32 Effect of VPR on Endothelium-Intact Rings in the Presenceof L-NAME or Indomethacin To investigate the involve-ment of NO and prostanoids endothelium-intact ringswere preincubated for 30min with L-NAME (100120583M) orindomethacin (10120583M) before the second contraction withPhe (1 120583M) Figure 2 shows that the vasorelaxant activity ofVPR (300 120583gmL) was significantly reduced in the presenceof these inhibitors
33 Effects of VPR on Contraction Induced by High K+ Con-centration As illustrated in Figure 3 following contractionwith KCl 80mM VPR-mediated relaxation of endothelium-removed rings was significantly reduced when compared
4 Evidence-Based Complementary and Alternative Medicine
0
20
40
60
80
minus2 minus1 0 1 2 3
Intact endotheliumDenuded endothelium
Rela
xatio
n (
)
log[VPR] (120583gmL)
(a)
0
20
40
60
80
minus2 minus1 0 1 2 3
Intact endotheliumDenuded endothelium
Rela
xatio
n (
)
log[VPF] (120583gmL)
(b)
Figure 1 Vasorelaxant effect induced by VPR Concentration-response curves to VPR or VPF (001ndash300120583gmL cumulatively) precontractedwith 1 120583Mphenylephrine (Phe) in mesenteric artery rings (with endothelium) and after removal of endothelium (a) concentration-responsecurves showing the relaxant effect of VPR 119899 = 6 (b) responses induced by VPF 119899 = 4 The response is expressed as a percentage relaxationof the Phe-induced contraction Values are mean plusmn SEM
to the contractile response induced by Phe (Table 1) asdemonstrated by the displacement of the concentration-response curve to the right
34 Effect of VPR on Arteries Treated with KCl 20mMTo evaluate the involvement of K+ channels in the VPR-mediated vasorelaxant response experiments were per-formed in endothelium-denuded rings incubated with KCl20mM and precontracted with Phe As shown in Figure 4(a)the VPR-mediated vasorelaxation was reduced with signifi-cant alterations in pharmacological efficacy when comparedto the control (Table 1)
35 Effect of VPR on Endothelium-Denuded Rings in the Pres-ence of Different K+ Channel Inhibitors Figure 4(a) showsthat the vasorelaxant activity of VPR at the highest concen-tration was significantly rightward shifted in the presenceof TEA (3mM) (Table 1) However in the presence of Glib(10 120583M) 4-AP (1mM) or BaCl
2(30 120583M) VPR-mediated
relaxation in rings lacking the vascular endothelium andprecontracted with Phe was not significantly reduced Inthe presence of TEA (1mM) the vasorelaxant response wassignificantly attenuated (Figures 4(b) and 4(c) Table 1)
36 Effect of VPR on CaCl2-Induced Contractions To investi-
gate the hypothesis of the residual vasorelaxant effect inducedby VPR being the same as in the presence of the K+ channelsinhibitors concentration-response curve was induced with
37 VPR Effect on MAP and HR in Nonanesthetized RatsAs Figure 6 shows in nonanesthetized normotensive ratsintravenous bolus injections of VPR (1 5 10 and 20mgkg)induced hypotension (MAP = minus1318 plusmn 16 minus1854 plusmn 64minus1636 plusmn 56 and minus2832 plusmn 34mmHg resp 119899 = 5) (Figures6(a) and 6(b)) associated with tachycardia (HR = 1164 plusmn 555024 plusmn 140 6145 plusmn 80 and 6988 plusmn 84 bpm resp 119899 = 5)(Figure 6(c))
4 Discussion
The biological effects of Valeriana prionophylla Standl in thecardiovascular system have yet to be fully elucidated Thisstudy aimed to increase our understanding of this naturalmedicine and highlight its potential pharmacological contri-bution Specifically this work demonstrated cardiovascularpharmacological effects of VPR in rats using in vitro and invivo protocols
Initially we evaluated the peripheral vascular effects ofVPR and VPF by performing in vitro experiments using ratsuperior mesenteric artery rings VPR significantly reducePhe-induced contractions a selective 120572
1-adrenergic receptor
agonist in a concentration-dependent manner [12] VPF also
Evidence-Based Complementary and Alternative Medicine 5
50
40
30
20
10
0
lowast
lowastlowastlowast
VPRVPR + indomethacinVPR + L-NAME
Max
imum
resp
onse
()
Figure 2 Participation of NO and COX-derived products in thevasorelaxation induced by VPR in mesenteric artery rings Effect ofVPR (300 120583gmL) in rings intact endothelium precontracted withPhe (1120583M) in the absence or in the presence of L-NAME (100120583M)or indomethacin (10 120583M) Values are mean plusmn SEM 119899 = 5 and 119899 = 5respectively
Phe (1120583M)KCl (80mM)
0
20
40
60
80
minus2 minus1 0 1 2 3
Rela
xatio
n (
)
log[VPR] (120583gmL)
Figure 3 High K+ extracellular decreased relaxant effect of VPRConcentration-response curves showing the relaxant effect of VPR(001ndash300 120583gmL cumulatively) in rings denuded endothelium con-tracted with Phe or KCl 80mM Values are expressed as mean plusmnSEM 119899 = 5
Table 1 Participation of K+ channels in the vasorelaxation inducedby VPR in mesenteric artery rings precontracted with phenyle-phrine
Values are expressed as mean plusmn SEM unpaired Studentrsquos 119905-tests wereused to examine the difference between denuded endothelium and intactendothelium one-way ANOVAs with the addition of Bonferronirsquos multiplecomparisons post hoc test were used to compare denuded endothelium(control) with different inhibitors groups No significance versus intactendothelium lowast119875 lt 005 versus intact endothelium lowastlowast119875 lt 001 versusdenuded endothelium
119875 lt 001 versus denuded endothelium nsnosignificance versus denuded endothelium
induced a vasorelaxant effect in mesenteric artery rings butthis response was reduced in comparison to that obtained byVPR
It is well known that the endothelium is an importantregulator of the vascular tone releasing endothelium-derivedrelaxing factors including NO COX-derived products andendothelium-derived hyperpolarization factors (EDHFs) [1314] To investigate the participation of these factors in thevasorelaxant effect induced by VPR we performed exper-iments in the absence of functional endothelium Underthese conditions VPR vasorelaxant maximum response wasnot significantly altered however the potency of VPR wasreduced in absence of vascular endothelium Therefore toinvestigate the role of endothelium-derived relaxing fac-tors such as NO and COX-derived products assays wereperformed with L-NAME (eNOS blocker) or indomethacin(COX inhibitor) In these conditions vasorelaxant responsemediated by VPR was significantly reduced suggesting thatVPR inhibit the releasing of endothelial NO and prostanoids
This suggests that the presence of a functional endothe-lium is important but not essential to the relaxation responsemediated by VPR and that an endothelium-independentpathway is also involved in this effect which led to the subse-quent experiments exploring VPR-mediated vasorelaxationin the absence of the endothelium
Vascular tone the contractile activity of vascular smoothmuscle cells (VSMCs) is the major determinant of bloodflow resistance through the circulation Therefore vasculartone plays an important role in the regulation of bloodpressure and the blood distribution [15] Due to the fact thatmany drugs exert their antihypertensive effects by decreasingperipheral vascular resistance by direct action on vascularsmooth muscle we investigated the mechanisms underlying
6 Evidence-Based Complementary and Alternative Medicine
20
40
60
80
minus2 minus1 0 1 2 3
Rela
xatio
n (
)
0
PhePhe + KCl (20mM)Phe + TEA (3mM)
log[VPR] (120583gmL)
(a)
20
40
60
80
minus2 minus1 0 1 2 3Re
laxa
tion
()
0
PhePhe + GlibPhe + 4-AP
log[VPR] (120583gmL)
(b)
PheTEA (1mM)BaCl2
20
40
60
80
minus2 minus1 0 1 2 3
Rela
xatio
n (
)
0
log[VPR] (120583gmL)
(c)
Figure 4 Participation of K+ channels in the vasorelaxation induced by VPR in mesenteric artery rings Concentration-response curvesshowing the relaxant effect of VPR (001ndash300 120583gmL cumulatively) in rings denuded endothelium precontracted with Phe (1120583M) in theabsence or in the presence of (a) KCl 20mM or TEA (3mM) (b) 4-AP (1mM) or Glib (10120583M) (c) TEA (1mM) or BaCl
2(30120583M) Values are
expressed as mean plusmn SEM
Evidence-Based Complementary and Alternative Medicine 7
ControlVPR (300120583gmL)
minus6 minus5 minus4 minus3 minus2 minus1
120
100
80
60
40
20
0
Con
trac
tion
()
log[CaCl2] (M)
Figure 5 Effect of VPR on CaCl2-induced contraction in
endothelium-denuded mesenteric artery rings Concentration-response curves for CaCl
2were determined in Ca2+-free solution
containing KCl (60mM) The curves were determined in theabsence (control) and after incubation with VPR (300120583gmL)Values are expressed as mean plusmn SEM 119899 = 4
endothelium-independent relaxation induced by VPR inmesenteric artery rings
The literature reports that VSMC contraction inducedby high extracellular K+ concentration is mediated by anincrease in membrane depolarization and consequently anincrease in Ca2+ influx through voltage-dependent calciumchannel (CaV) [16] Furthermore the sustained contractiongenerated by a high external K+ concentration may also bemediated by Ca2+ release from the sarcoplasmic reticulum[17] resulting in Ca2+ entry through store-operated channels(SOC) andor transient receptor potential (TRP) channels[18]
Endothelium removed mesenteric rings were precon-tractedwith a depolarizing K+ solution (KCl 80mM) in orderto observe VPR responses before nonspecific generated con-tractions and averting activation of membrane receptors Inthe presence of this depolarizing solution the concentration-response curve induced by VPR was significantly shifted tothe right when compared to the response of VPR in thepresence of Phe changing both the efficacy and potency ofVPR
Based on these preliminary results we speculate thatthis VPR-mediated vasorelaxant effect requires activation ofpotassium channels (K+ channels) in VSMCsThe opening ofthese channels in VSMCs results in an increase in K+ perme-ability leading to K+ efflux The exit of K+ from the VSMCinduces membrane hyperpolarization and consequently the
closing of voltage-operated Ca2+ channels leading to vasore-laxation [19 20] Membrane potential regulation in VSMCsis an important factor in the maintenance of vascular toneAdditionally K+ channels are effector proteins that contributeto membrane potential regulation in electrically excitablecells such as VSMCs Therefore drugs that activate thesechannels could be beneficial in hypertension treatment [21]
Thus to evaluate the involvement of K+ channels inVPR-mediated vasorelaxant response experiments were per-formed in endothelium-denuded arterial rings incubatedwith KCl 20mM This procedure partially decreases mem-brane efflux of K+ due to the increase in extracellular K+concentration from 4mM to 20mM resulting in a reductionin the electrochemical gradient [22] Under these conditionsthe maximum response to VPR was significantly attenuatedwhich was also observed when rings were exposed to KCl80mM suggesting the participation ofK+ channels in vasore-laxation induced by VPR
To confirm the involvement of K+ channels the prepa-rations were pretreated with TEA (3mM) a nonselective K+channel blocker [23] In the presence of this pharmacolog-ical tool the vasorelaxant response induced by increasingconcentrations of VPR was significantly attenuated corrob-orating our previous observation that VPR appears to havea significant effect on the open probability of K+ channels inmesenteric artery rings
Several types of K+ channels are expressed in membraneof VSMCs and each of these channels plays an importantrole in the control and maintenance of the contractile toneof the arterial muscle [24] In VSMCs the variety of K+ chan-nels subtypes identified includes voltage-dependent K+ (KV)channels whose activity is increased upon membrane depo-larization and are important regulators of vascular smoothmuscle membrane potential in response to a depolarizingstimulus [25] large-conductance Ca2+-activated K+ (BKCa)channels which respond to changes in the concentration ofintracellular Ca2+ regulating membrane potential and havean important role in control of myogenic tone in smallerresistance arteries [26] ATP-sensitive K+ (KATP) channelswhich respond actively to changes in cellular metabolism andare targets of a variety of relaxing stimuli [27] and inwardrectifier K+ (KIR) channels regulatingmembrane potential ofvarious types of resistance vessels of small diameter [28]
Thus to investigate the possible participation of thesedifferent K+ channels subtypes experimental preparationswere separately preincubated with different selective blockersof these channels In the presence of a KATP channels blockerGlib (10 120583M) or a KV channels blocker 4-AP (1mM) themaximum effect of VPRwas not changedThe same responseoccurred in rings preincubated with BaCl
2(30 120583M) Kir
channels blocker suggesting that KATP KV and Kir channelsdo not participate in VPR-mediated vasorelaxation
The KCa channels are divided into a subfamily of small(SKCa) intermediate (IKCa) and large (BKCa) conductanceThe BKCa channels are expressed in vascular tissues with ahigh density they are a major component that mediates thedegree of depolarization and contraction in vascular smoothmuscle they are preferentially expressed in vascular smooth
8 Evidence-Based Complementary and Alternative Medicine
150
100
50
Art
eria
l pre
ssur
e (m
mH
g)
2000 3000 4000 5000 6000 7000 8000
Time (s)
VPR(20mgkg)
(a)
0
minus10
minus20
minus30
minus40
ΔM
AP
(mm
Hg)
(b)
100
80
60
40
20
0
1 5 10 20
VPR (mgkg)
ΔH
R (b
pm)
(c)
Figure 6 Effect of VPR on MAP and HR in nonanesthetized rats (a) Representative tracing showed the hypotension induced by additionof VPR (20mgkg) (b) Changes in mean arterial pressure (MAP mmHg) (c) Changes in heart rate (HR bpm) induced by the acuteadministration of increasing doses of VPR (mgkg iv) Values are expressed by mean plusmn SEM 119899 = 5
muscle while the SKCa and IKCa are expressed in a varietyof cell types such as secretory epithelial cells fibroblastsT lymphocytes melanoma cells granulocytes macrophageserythrocytes cultured cell lines and endothelial cells [29 30]BKCa channels act by inhibiting the increase of intracellu-lar Ca2+ Furthermore several recent findings support theimportance of BKCa channels in the regulation of vascularsmooth muscle tone and regulation of blood pressure [31]
The data shows that VPR-mediated vasorelaxation atthe highest concentration was significantly attenuated inrings preincubated with TEA (1mM) which preferentiallyblocks BKCa channels at concentrations lower than 1mM[32] These results suggest that BKCa channels mediate VPR-induced vasorelaxation demonstrating that in addition to theneuronal actions VPR exhibits important peripheral vasculareffects as well
Therefore in vivo experiments were performed to eval-uate VPR effects on cardiovascular parameters in nonanes-thetized normotensive rats in order to avoid the influencesof anesthesia and postsurgical stress In these animals VPRinduced a hypotensive response associated with tachycardia
The hypotensive effect caused by VPR may be due todecreased peripheral vascular resistance and the tachycardiamay be a reflection of the decrease in blood pressure
Taken together these results demonstrate that VPRinduce vasodilatation probably involving K+ channel acti-vation most likely through BKCa channels in smoothmuscle inner from rat superior mesenteric artery Fur-thermore endothelium-derived relaxing factors (NO andprostanoids) are involved in endothelium relaxation depen-dent on VPR
K+ channelKATP ATP-sensitive K+ channelKir Inward rectifier K+ channelKV Voltage-dependent K+ channelMAP Mean arterial pressureMR Maximum responseNO Nitric oxidePAF Platelet-activating factorPhe PhenylephrineK+ channel Potassium channelSEM Standard error of meanSKCa Small-conductance calcium-activated K+
channelSOC Store operated channelsTEA TetraethylammoniumTRP Transient receptor potentialVPR Roots from the Valeriana prionophyllaVPF Leaves from the Valeriana prionophyllaVSMC Vascular smooth muscle cells
Conflict of Interests
The authors declare that there is no conflict of interests
Acknowledgments
This work was supported by grants from Conselho Nacionalde Desenvolvimento Cientıfico e Tecnologico (CNPq)Coordenacao de Aperfeicoamento de Pessoal de Nıvel Supe-rior (CAPES) Consejo Nacional de Ciencia y Tecnologıa deGuatemala (FODECYT 102-2006) and Fundacao de Amparoa Pesquisa do Estado da Bahia (FAPESB)
References
[1] E A Carlini ldquoPlants and the central nervous systemrdquo Phar-macology Biochemistry and Behavior vol 75 no 3 pp 501ndash5122003
[2] V Schulz H Rudolf and V E Tyler Rational Phytotherapy APhysicisnrsquos Guide to Herbal Medicine 2004
[3] I Holzmann V Cechinel-Filho T C Mora et al ldquoEvaluationof behavioral and pharmacological effects of hydroalcoholicextract of Valeriana prionophylla Standl from GuatemalardquoEvidence-Based Complementary and Alternative Medicine vol2011 Article ID 312320 9 pages 2011
[4] D L Nash and J V A Dieterle ldquoFlora of guatemalardquo FieldianaBotany vol 24 no 11 p 297 1976
[5] S Chavadej H Becker and F Weberling ldquoFurther investi-gations of valepotriates in the Valerianaceaerdquo PharmaceutischWeekblad vol 7 no 4 pp 167ndash168 1985
[6] A L Piccinelli S Arana A Caceres R D Di Villa BiancaR Sorrentino and L Rastrelli ldquoNew lignans from the rootsof Valeriana prionophylla with antioxidative and vasorelaxant
activitiesrdquo Journal of Natural Products vol 67 no 7 pp 1135ndash1140 2004
[7] G M Massanet E Pando F Rodriguez-Luis and E ZubialdquoLignans a reviewrdquo Fitoterapia vol 60 no 1 pp 3ndash35 1989
[8] E LGhisalberti ldquoCardiovascular activity of naturally occurringlignansrdquo Phytomedicine vol 4 no 2 pp 151ndash166 1997
[9] A M Gurney ldquoMechanisms of drug-induced vasodilationrdquoJournal of Pharmacy and Pharmacology vol 46 no 4 pp 242ndash251 1994
[10] D F Silva I G A Araujo J G F Albuquerque et alldquoRotundifolone-induced relaxation is mediated by BKCa chan-nel activation and Ca
119907channel inactivationrdquo Basic and Clinical
Pharmacology and Toxicology vol 109 no 6 pp 465ndash475 2011[11] B M Altura and B T Altura ldquoDifferential effects of substrate
depletion on drug-induced contractions of rabbit aortardquo TheAmerican Journal of Physiology vol 219 no 6 pp 1698ndash17051970
[12] W-J Zang C W Balke and W G Wier ldquoGraded 1205721-adrenoceptor activation of arteries involves recruitment ofsmooth muscle cells to produce ldquoall or nonerdquo Ca2+ signalsrdquo CellCalcium vol 29 no 5 pp 327ndash334 2001
[13] R F Furchgott and J V Zawadzki ldquoThe obligatory role ofendothelial cells in the relaxation of arterial smooth muscle byacetylcholinerdquo Nature vol 288 no 5789 pp 373ndash376 1980
[14] M Feletou R Kohler and P M Vanhoutte ldquoNitric oxideorchestrator of endothelium-dependent responsesrdquo Annals ofMedicine vol 44 no 7 pp 694ndash716 2012
[15] Y Tanaka Y Mochizuki H Tanaka and K Shigenobu ldquoSig-nificant role of neuronal non-N-type calcium channels in thesympathetic neurogenic contraction of rat mesenteric arteryrdquoBritish Journal of Pharmacology vol 128 no 7 pp 1602ndash16081999
[16] T Godfraind and A Kaba ldquoBlockade or reversal of the contrac-tion induced by calcium and adrenaline in depolarized arterialsmooth musclerdquo British Journal of Pharmacology vol 36 no 3pp 549ndash560 1969
[17] S Kobayashi H Kanaide and M Nakamura ldquoK+-depolarization induces a direct release of Ca2+ fromintracellular storage sites in cultured vascular smooth musclecells from rat aortardquo Biochemical and Biophysical ResearchCommunications vol 129 no 3 pp 877ndash884 1985
[18] B Ay Y S Prakash C M Pabelick and G C Sieck ldquoStore-operated Ca2+ entry in porcine airway smooth musclerdquo Ameri-can Journal of Physiology vol 286 no 5 pp L909ndashL917 2004
[19] G M Dick and J D Tune ldquoRole of potassium channels incoronary vasodilationrdquo Experimental Biology andMedicine vol235 no 1 pp 10ndash22 2010
[20] EAKo JHan I D Jung andW S Park ldquoPhysiological roles ofK+ channels in vascular smoothmuscle cellsrdquo Journal of SmoothMuscle Research vol 44 no 2 pp 65ndash81 2008
[21] K Lawson ldquoPotassiumchannel openers as potential therapeuticweapons in ion channel diseaserdquo Kidney International vol 57no 3 pp 838ndash845 2000
[22] D X P Brochet and P D Langton ldquoDual effect of initial [K]on vascular tone in rat mesenteric arteriesrdquo Pflugers ArchivEuropean Journal of Physiology vol 453 no 1 pp 33ndash41 2006
[23] F Jiang C G Li and M J Rand ldquoRole of potassium channelsin the nitrergic nerve stimulation-induced vasodilatation in theguinea-pig isolated basilar arteryrdquo British Journal of Pharmacol-ogy vol 123 no 1 pp 106ndash112 1998
10 Evidence-Based Complementary and Alternative Medicine
[24] N B Standen and J M Quayle ldquoK+ channel modulation inarterial smooth musclerdquo Acta Physiologica Scandinavica vol164 no 4 pp 549ndash557 1998
[25] E A Ko W S Park A L Firth N Kim J X-J Yuan and JHan ldquoPathophysiology of voltage-gatedK+ channels in vascularsmooth muscle cells modulation by protein kinasesrdquo Progressin Biophysics and Molecular Biology vol 103 no 1 pp 95ndash1012010
[26] F Sun E Hayama Y Katsube R Matsuoka and T NakanishildquoThe role of the large-conductance voltage-dependent andcalcium-activated potassium (BKCa) channels in the regulationof rat ductus arteriosus tonerdquo Heart and Vessels vol 25 no 6pp 556ndash564 2010
[27] M T Nelson and J M Quayle ldquoPhysiological roles andproperties of potassium channels in arterial smooth musclerdquoAmerican Journal of Physiology vol 268 no 4 pp C799ndashC8221995
[28] T Karkanis S Li J G Pickering and S M Sims ldquoPlasticityof KIR channels in human smooth muscle cells from internalthoracic arteryrdquo American Journal of Physiology vol 284 no 6pp H2325ndashH2334 2003
[29] A Schwab ldquoFunction and spatial distribution of ion channelsand transporters in cell migrationrdquo American Journal of Physi-ology vol 280 no 5 pp F739ndashF747 2001
[30] H Ouadid-Ahidouch M Roudbaraki P Delcourt A Ahi-douchN Joury andN Prevarskaya ldquoFunctional andmolecularidentification of intermediate-conductance Ca2+-activated K+channels in breast cancer cells association with cell cycleprogressionrdquo American Journal of Physiology vol 287 no 1 ppC125ndashC134 2004
[31] J Ledoux M E Werner J E Brayden and M T NelsonldquoCalcium-activated potassium channels and the regulation ofvascular tonerdquo Physiology vol 21 no 1 pp 69ndash78 2006
[32] PD LangtonMTNelson YHuang andN B Standen ldquoBlockof calcium-activated potassium channels inmammalian arterialmyocytes by tetraethylammonium ionsrdquo American Journal ofPhysiology vol 260 no 3 pp H927ndashH934 1991
2 Evidence-Based Complementary and Alternative Medicine
as ldquoValeriana del monterdquo Studies performed in animals havedemonstrated that rhizomes of this species affect centralnervous system activity demonstrating sedative hypnoticanxiolytic and antidepressive effects [3 4] However despiteits widespread use by the population for psychosomaticdisorders possible peripheral effect of this species is not wellcharacterized it is important to investigate a possible periph-eral cardiovascular action and thus differentiate betweencentral and peripheral effects in the cardiovascular system
Furthermore phytochemical analysis of rhizomes of Vprionophylla identified the presence of valepotriates [5] andnew lignans isolated from these roots have demonstratedvasorelaxant activity in isolated rat aorta artery rings [6]demonstrating a potential effect on peripheral systems Lig-nans are secondary plant metabolites that exist in the phenyl-propanoid pathway and have been identified and isolatedfrom approximately 70 different families of all plant originsthe majority of which are those used in popular medicine [7]Lignans in the cardiovascular system have demonstrated atherapeutic potential as a cardiotonic agent by the inhibitionof phosphodiesterase III which is responsible for metab-olizing the second messenger cAMP (35-cyclic adenosinemonophosphate) Moreover some lignans also demonstrateantagonistic activity of the platelet-activating factor (PAF)receptor and blocking of the L-type Ca2+ channel [8]
Many cardiovascular disorders such as hypertensionangina and heart failure are often treated with vasodilatordrugs that act directly on the vascular smooth musclecausing vasodilation indirectly by stimulating the release ofendogenous vasorelaxant factors or by inhibiting the releaseof vasoconstrictive factors [9]
Thus the aim of this study was to evaluate the peripheralactions of Valeriana prionophylla Standl in the cardiovas-cular system and the mechanisms underlying the vascularresponse induced by this species in isolated rat mesentericartery
2 Materials and Methods
21 Drugs and Solutions The drugs used in this studywere Cremophor EL dimethyl sulphoxide (DMSO) L-phenylephrine chloride (Phe) acetylcholine chloride (Ach)glibenclamide tetraethylammonium 4-aminopyridine andbarium chloride (SIGMA) All compounds were dissolvedin distilled water except glibenclamide that was dissolvedin DMSO The composition of Tyrodersquos solution used wasas follows (mM) NaCl 1583 KCl 40 CaCl
2 20 MgCl
2
105 NaH2PO4 042 NaHCO
3 100 and glucose 56 K+-
depolarizing solutions (KCl 20 and 80mM) were preparedby replacing 20 or 80mM KCl in Tyrodersquos solution withequimolar NaCl respectively
22 Plant Material Valeriana prionophylla Standl Valerian-aceae were collected from cultivations in Tierra Blanca Con-cepcion Tutuapa San Marcos (15∘ 148081015840N 91∘ 554301015840W)Guatemala a vegetative zone that resides in a very humid andlow mountainous forest Three-year-old rhizomes and rootswere dug up washed and shade-dried Botanical samples
were determined by Mario Veliz at Herbarium BIGU Schoolof Biology USAC and a voucher sample deposited (no49183)
23 Preparation of Ethanol Extract Drymaterial was groundwetted with 50 ethanol and placed in a stainless steelpercolator 50 ethanol was added to obtain a tincture whichwas concentrated in a rotavapor Fresh ethanol was addedfor five consecutive days and the extract was concentratedThe final drying was performed in a vacuum dryer with silicagel as described by Holzmann et al [3] The average yield ofthe extractable solids was 2852 For in vitro experimentsextracts of the roots and leaves from the Valeriana priono-phylla Standl species (VPR and VPF resp) were dissolvedin a mixture of distilled waterCremophor and diluted to thedesired concentrationswith distilledwater just before use thefinal concentration of Cremophor in the bath never exceeded001
24 Animals Male Wistar rats (250ndash300 g) were used forall experiments Animals were housed under controlledtemperature (21plusmn1∘C) exposed to a 12 h light-dark cycle withfree access to food (Purina Brazil) and tap water The studywas carried out in accordance with the Guide for the CareandUse of LaboratoryAnimals as adopted by theUSNationalInstitutes of Health
25 Tissue Preparation Rats were euthanized and superiormesenteric artery was removed cleaned from connectivetissue and fat as described by Silva and colleagues [10]Whenever appropriate the endothelium was removed bygently rubbing the intimal surface of the vessels Rings (1-2mm) were suspended in organ baths containing 10mLof Tyrodersquos solution gassed with a mixture of 95 O
2
and 5 CO2 maintained at 37∘C and at pH 74 Isometric
tension was recorded under a resting tension of 075 g Thesolution was changed every 15min during a stabilizationperiod of 1 hr to prevent the accumulation of metabolites[11] The isometric contractile force was recorded by a forcetransducer (MLT020 ADInstruments Australia) coupledto an amplifier-recorder (ML870P com LabChart versao70 ADInstruments Australia) and to a computer equippedwith a data acquisition software The presence of functionalendothelium was assessed by the ability of Ach (10120583M) toinduce more than 90 relaxation of pre-contracted vesselswith Phe (10 120583M) and the absence less than 10 of relaxationinduced by Ach
26 Effects of VPF and VPR in Phenylephrine-Induced Con-tractions In this experiment sustained Phe-induced con-tractions were obtained in isolated rat superior mesentericartery rings with or without endothelium In the tonic phaseof the second contraction induced by Phe (1 120583M) increasingcumulative concentrations of VPF and VPR (001 003 0103 1 3 10 30 100 and 300 120583gmL) were separately andcontinually added to the bath until a maximum response forthe added extract was observed as indicated by a plateauresponse (approximately 4ndash6min)
Evidence-Based Complementary and Alternative Medicine 3
27 Investigation of Nitric Oxide and COX-Derived Prod-ucts Participation in the Vasorelaxation Mediated by VPRTo investigate the involvement of NO and prostanoidsendothelium-intact rings were incubated for 30min withL-NAME (10minus4M) or indomethacin (10minus5M) (endothelialnitric oxide synthase (eNOS) or cyclooxygenase inhibitorsresp) before the second application of Phe (1 120583M) and thevasorelaxation induced byVPR (300 120583gmL)was investigatedbefore and after the addition of the inhibitors
28 Effect of VPR on Contraction Induced by Depolarizationwith High K+ Concentration After the stabilization periodrings without endothelium were precontracted with highpotassium concentration solution (KCl 80mM) and inthe tonic phase different concentrations of VPR (001ndash300 120583gmL) were added cumulatively to the organ bath andrelaxations were measured as previously described
29 Investigation of K+ Channel Participation in the Vasore-laxation Elicited by VPR To investigate the involvementof K+ channels in the first set of experiments vasorelax-ation with VPR was performed in vessels with denudedendothelium that were precontracted with Phe in Tyrodersquossolution with elevated K+ (20mM equimolar replacementof NaCl with KCl) to attenuate K+ efflux [10] Additionallyin the second group of experiments endothelium-denudedrings were incubated for 30min with putative K+ channelblockers before the second application of Phe (1 120583M) and theinhibition was calculated by comparing the response elicitedby VPR before and after the addition of the inhibitorsPreparations were exposed to tetraethylammonium (TEA3mM) a nonselective K+ channel blocker TEA (1mM)a large-conductance calcium-activated K+ channel (BKCa)selective blocker in this concentration 4-aminopyridine (4-AP) (1mM) a voltage-dependent K+ (KV) channel blockerglibenclamide (Glib) (10 120583M) an ATP-sensitive K+ (KATP)channel blocker and barium chloride (BaCl
2) (30 120583M) an
inward rectifier K+ (Kir) channel blocker
210 Investigation of Effect of VPR on CaCl2-Induced Con-
tractions To investigate the hypothesis that VPR act throughthe blockade of extracellular calcium influx in endothelium-denuded rings cumulative concentrations of CaCl
2(10minus6ndash
10minus2M) were added in medium containing Ca2+-free depo-larizing solution (KCl 60mM) in absence (control) or inpresence of VPR (300120583gmL)
211 Measurement of Mean Arterial Pressure and Heart Ratein Nonanesthetized Normotensive Rats The day before theexperimental session a catheter (PE
50) filled with hep-
arinized saline solution (1000UmL) was inserted into theleft carotid artery under ketaminexylazine anesthesia andexteriorized at the nape of the animalrsquos neck to permit bloodpressure recording An additional catheter was placed inthe right femoral vein to allow intravenous drug adminis-tration After 24 hours arterial pressure was continuouslymonitored through the carotid catheter connected to a bloodpressure transducer (World Precision Instruments) whose
signal was amplified and digitally recorded by an analog-to-digital interface (AqDados application for data acquisitionLynx Tecnologia Eletronica Ltda version 70 Sao PauloBrazil) and recorded (1 kHz) on a microcomputer for lateranalysis Mean arterial pressure (MAP) was calculated fromsystolic and diastolic pressure data while heart rate (HR) wasdetermined by the pulsation of arterial pressure using theAcqKnowledge software program version 357 developed byBiopac Systems Inc California USA
212 Statistical Analysis Data are presented as the mean plusmnSEM and n represents a number of rings prepared fromdifferent rats Concentration-response curves to VPR andVPF were based on the percent relaxation of the agonist-induced contraction A 100 relaxation was assigned whenthe precontracted rings returned to the baseline values Thecurves were fitted using a variable slope sigmoid fittingroutine in GraphPad Prism50 (Graph Pad Software Inc LaJolla CA USA) EC
50(concentration required to relax the
induced tone by half and maximum response (MR) valueswere calculated from the fitted sigmoidal curves Statisticalanalyses were performed by comparing 119864max and EC50 valuesbetween groups from independent observations UnpairedStudentrsquos t-test or one-way ANOVAs were used to compare2 3 or more groups respectively with the addition ofBonferronirsquos multiple comparisons post-test Two-sided 119875 lt005 was considered statistically significant
3 Results
31 Effects of VPR andVPF in Phenylephrine-InducedContrac-tions In isolated rat superior mesenteric artery rings VPR(001ndash300 120583gmL) decreased in a concentration-dependentmanner following Phe-induced contraction (1 120583M) (EC
50
values = 596 (38minus92) 120583gmL) (Figure 1(a)) In the absenceof the vascular endothelium the potency of VPR was signif-icantly shifted (EC
50values = 3963 (272minus576)120583gmL 119875 lt
005) (Figure 1(a) Table 1) whereas VPF (001ndash300120583gmL)induced a vasorelaxant effect in the presence and absence ofvascular endothelium (maximum response (MR) = 2939 plusmn94MR = 2705plusmn62 resp 119899 = 4) (Figure 1(b)) howeverthis response was reduced in comparison to that obtained byVPR (MR = 7543 plusmn 408 MR = 7033 plusmn 458 119875 lt 005resp)
32 Effect of VPR on Endothelium-Intact Rings in the Presenceof L-NAME or Indomethacin To investigate the involve-ment of NO and prostanoids endothelium-intact ringswere preincubated for 30min with L-NAME (100120583M) orindomethacin (10120583M) before the second contraction withPhe (1 120583M) Figure 2 shows that the vasorelaxant activity ofVPR (300 120583gmL) was significantly reduced in the presenceof these inhibitors
33 Effects of VPR on Contraction Induced by High K+ Con-centration As illustrated in Figure 3 following contractionwith KCl 80mM VPR-mediated relaxation of endothelium-removed rings was significantly reduced when compared
4 Evidence-Based Complementary and Alternative Medicine
0
20
40
60
80
minus2 minus1 0 1 2 3
Intact endotheliumDenuded endothelium
Rela
xatio
n (
)
log[VPR] (120583gmL)
(a)
0
20
40
60
80
minus2 minus1 0 1 2 3
Intact endotheliumDenuded endothelium
Rela
xatio
n (
)
log[VPF] (120583gmL)
(b)
Figure 1 Vasorelaxant effect induced by VPR Concentration-response curves to VPR or VPF (001ndash300120583gmL cumulatively) precontractedwith 1 120583Mphenylephrine (Phe) in mesenteric artery rings (with endothelium) and after removal of endothelium (a) concentration-responsecurves showing the relaxant effect of VPR 119899 = 6 (b) responses induced by VPF 119899 = 4 The response is expressed as a percentage relaxationof the Phe-induced contraction Values are mean plusmn SEM
to the contractile response induced by Phe (Table 1) asdemonstrated by the displacement of the concentration-response curve to the right
34 Effect of VPR on Arteries Treated with KCl 20mMTo evaluate the involvement of K+ channels in the VPR-mediated vasorelaxant response experiments were per-formed in endothelium-denuded rings incubated with KCl20mM and precontracted with Phe As shown in Figure 4(a)the VPR-mediated vasorelaxation was reduced with signifi-cant alterations in pharmacological efficacy when comparedto the control (Table 1)
35 Effect of VPR on Endothelium-Denuded Rings in the Pres-ence of Different K+ Channel Inhibitors Figure 4(a) showsthat the vasorelaxant activity of VPR at the highest concen-tration was significantly rightward shifted in the presenceof TEA (3mM) (Table 1) However in the presence of Glib(10 120583M) 4-AP (1mM) or BaCl
2(30 120583M) VPR-mediated
relaxation in rings lacking the vascular endothelium andprecontracted with Phe was not significantly reduced Inthe presence of TEA (1mM) the vasorelaxant response wassignificantly attenuated (Figures 4(b) and 4(c) Table 1)
36 Effect of VPR on CaCl2-Induced Contractions To investi-
gate the hypothesis of the residual vasorelaxant effect inducedby VPR being the same as in the presence of the K+ channelsinhibitors concentration-response curve was induced with
37 VPR Effect on MAP and HR in Nonanesthetized RatsAs Figure 6 shows in nonanesthetized normotensive ratsintravenous bolus injections of VPR (1 5 10 and 20mgkg)induced hypotension (MAP = minus1318 plusmn 16 minus1854 plusmn 64minus1636 plusmn 56 and minus2832 plusmn 34mmHg resp 119899 = 5) (Figures6(a) and 6(b)) associated with tachycardia (HR = 1164 plusmn 555024 plusmn 140 6145 plusmn 80 and 6988 plusmn 84 bpm resp 119899 = 5)(Figure 6(c))
4 Discussion
The biological effects of Valeriana prionophylla Standl in thecardiovascular system have yet to be fully elucidated Thisstudy aimed to increase our understanding of this naturalmedicine and highlight its potential pharmacological contri-bution Specifically this work demonstrated cardiovascularpharmacological effects of VPR in rats using in vitro and invivo protocols
Initially we evaluated the peripheral vascular effects ofVPR and VPF by performing in vitro experiments using ratsuperior mesenteric artery rings VPR significantly reducePhe-induced contractions a selective 120572
1-adrenergic receptor
agonist in a concentration-dependent manner [12] VPF also
Evidence-Based Complementary and Alternative Medicine 5
50
40
30
20
10
0
lowast
lowastlowastlowast
VPRVPR + indomethacinVPR + L-NAME
Max
imum
resp
onse
()
Figure 2 Participation of NO and COX-derived products in thevasorelaxation induced by VPR in mesenteric artery rings Effect ofVPR (300 120583gmL) in rings intact endothelium precontracted withPhe (1120583M) in the absence or in the presence of L-NAME (100120583M)or indomethacin (10 120583M) Values are mean plusmn SEM 119899 = 5 and 119899 = 5respectively
Phe (1120583M)KCl (80mM)
0
20
40
60
80
minus2 minus1 0 1 2 3
Rela
xatio
n (
)
log[VPR] (120583gmL)
Figure 3 High K+ extracellular decreased relaxant effect of VPRConcentration-response curves showing the relaxant effect of VPR(001ndash300 120583gmL cumulatively) in rings denuded endothelium con-tracted with Phe or KCl 80mM Values are expressed as mean plusmnSEM 119899 = 5
Table 1 Participation of K+ channels in the vasorelaxation inducedby VPR in mesenteric artery rings precontracted with phenyle-phrine
Values are expressed as mean plusmn SEM unpaired Studentrsquos 119905-tests wereused to examine the difference between denuded endothelium and intactendothelium one-way ANOVAs with the addition of Bonferronirsquos multiplecomparisons post hoc test were used to compare denuded endothelium(control) with different inhibitors groups No significance versus intactendothelium lowast119875 lt 005 versus intact endothelium lowastlowast119875 lt 001 versusdenuded endothelium
119875 lt 001 versus denuded endothelium nsnosignificance versus denuded endothelium
induced a vasorelaxant effect in mesenteric artery rings butthis response was reduced in comparison to that obtained byVPR
It is well known that the endothelium is an importantregulator of the vascular tone releasing endothelium-derivedrelaxing factors including NO COX-derived products andendothelium-derived hyperpolarization factors (EDHFs) [1314] To investigate the participation of these factors in thevasorelaxant effect induced by VPR we performed exper-iments in the absence of functional endothelium Underthese conditions VPR vasorelaxant maximum response wasnot significantly altered however the potency of VPR wasreduced in absence of vascular endothelium Therefore toinvestigate the role of endothelium-derived relaxing fac-tors such as NO and COX-derived products assays wereperformed with L-NAME (eNOS blocker) or indomethacin(COX inhibitor) In these conditions vasorelaxant responsemediated by VPR was significantly reduced suggesting thatVPR inhibit the releasing of endothelial NO and prostanoids
This suggests that the presence of a functional endothe-lium is important but not essential to the relaxation responsemediated by VPR and that an endothelium-independentpathway is also involved in this effect which led to the subse-quent experiments exploring VPR-mediated vasorelaxationin the absence of the endothelium
Vascular tone the contractile activity of vascular smoothmuscle cells (VSMCs) is the major determinant of bloodflow resistance through the circulation Therefore vasculartone plays an important role in the regulation of bloodpressure and the blood distribution [15] Due to the fact thatmany drugs exert their antihypertensive effects by decreasingperipheral vascular resistance by direct action on vascularsmooth muscle we investigated the mechanisms underlying
6 Evidence-Based Complementary and Alternative Medicine
20
40
60
80
minus2 minus1 0 1 2 3
Rela
xatio
n (
)
0
PhePhe + KCl (20mM)Phe + TEA (3mM)
log[VPR] (120583gmL)
(a)
20
40
60
80
minus2 minus1 0 1 2 3Re
laxa
tion
()
0
PhePhe + GlibPhe + 4-AP
log[VPR] (120583gmL)
(b)
PheTEA (1mM)BaCl2
20
40
60
80
minus2 minus1 0 1 2 3
Rela
xatio
n (
)
0
log[VPR] (120583gmL)
(c)
Figure 4 Participation of K+ channels in the vasorelaxation induced by VPR in mesenteric artery rings Concentration-response curvesshowing the relaxant effect of VPR (001ndash300 120583gmL cumulatively) in rings denuded endothelium precontracted with Phe (1120583M) in theabsence or in the presence of (a) KCl 20mM or TEA (3mM) (b) 4-AP (1mM) or Glib (10120583M) (c) TEA (1mM) or BaCl
2(30120583M) Values are
expressed as mean plusmn SEM
Evidence-Based Complementary and Alternative Medicine 7
ControlVPR (300120583gmL)
minus6 minus5 minus4 minus3 minus2 minus1
120
100
80
60
40
20
0
Con
trac
tion
()
log[CaCl2] (M)
Figure 5 Effect of VPR on CaCl2-induced contraction in
endothelium-denuded mesenteric artery rings Concentration-response curves for CaCl
2were determined in Ca2+-free solution
containing KCl (60mM) The curves were determined in theabsence (control) and after incubation with VPR (300120583gmL)Values are expressed as mean plusmn SEM 119899 = 4
endothelium-independent relaxation induced by VPR inmesenteric artery rings
The literature reports that VSMC contraction inducedby high extracellular K+ concentration is mediated by anincrease in membrane depolarization and consequently anincrease in Ca2+ influx through voltage-dependent calciumchannel (CaV) [16] Furthermore the sustained contractiongenerated by a high external K+ concentration may also bemediated by Ca2+ release from the sarcoplasmic reticulum[17] resulting in Ca2+ entry through store-operated channels(SOC) andor transient receptor potential (TRP) channels[18]
Endothelium removed mesenteric rings were precon-tractedwith a depolarizing K+ solution (KCl 80mM) in orderto observe VPR responses before nonspecific generated con-tractions and averting activation of membrane receptors Inthe presence of this depolarizing solution the concentration-response curve induced by VPR was significantly shifted tothe right when compared to the response of VPR in thepresence of Phe changing both the efficacy and potency ofVPR
Based on these preliminary results we speculate thatthis VPR-mediated vasorelaxant effect requires activation ofpotassium channels (K+ channels) in VSMCsThe opening ofthese channels in VSMCs results in an increase in K+ perme-ability leading to K+ efflux The exit of K+ from the VSMCinduces membrane hyperpolarization and consequently the
closing of voltage-operated Ca2+ channels leading to vasore-laxation [19 20] Membrane potential regulation in VSMCsis an important factor in the maintenance of vascular toneAdditionally K+ channels are effector proteins that contributeto membrane potential regulation in electrically excitablecells such as VSMCs Therefore drugs that activate thesechannels could be beneficial in hypertension treatment [21]
Thus to evaluate the involvement of K+ channels inVPR-mediated vasorelaxant response experiments were per-formed in endothelium-denuded arterial rings incubatedwith KCl 20mM This procedure partially decreases mem-brane efflux of K+ due to the increase in extracellular K+concentration from 4mM to 20mM resulting in a reductionin the electrochemical gradient [22] Under these conditionsthe maximum response to VPR was significantly attenuatedwhich was also observed when rings were exposed to KCl80mM suggesting the participation ofK+ channels in vasore-laxation induced by VPR
To confirm the involvement of K+ channels the prepa-rations were pretreated with TEA (3mM) a nonselective K+channel blocker [23] In the presence of this pharmacolog-ical tool the vasorelaxant response induced by increasingconcentrations of VPR was significantly attenuated corrob-orating our previous observation that VPR appears to havea significant effect on the open probability of K+ channels inmesenteric artery rings
Several types of K+ channels are expressed in membraneof VSMCs and each of these channels plays an importantrole in the control and maintenance of the contractile toneof the arterial muscle [24] In VSMCs the variety of K+ chan-nels subtypes identified includes voltage-dependent K+ (KV)channels whose activity is increased upon membrane depo-larization and are important regulators of vascular smoothmuscle membrane potential in response to a depolarizingstimulus [25] large-conductance Ca2+-activated K+ (BKCa)channels which respond to changes in the concentration ofintracellular Ca2+ regulating membrane potential and havean important role in control of myogenic tone in smallerresistance arteries [26] ATP-sensitive K+ (KATP) channelswhich respond actively to changes in cellular metabolism andare targets of a variety of relaxing stimuli [27] and inwardrectifier K+ (KIR) channels regulatingmembrane potential ofvarious types of resistance vessels of small diameter [28]
Thus to investigate the possible participation of thesedifferent K+ channels subtypes experimental preparationswere separately preincubated with different selective blockersof these channels In the presence of a KATP channels blockerGlib (10 120583M) or a KV channels blocker 4-AP (1mM) themaximum effect of VPRwas not changedThe same responseoccurred in rings preincubated with BaCl
2(30 120583M) Kir
channels blocker suggesting that KATP KV and Kir channelsdo not participate in VPR-mediated vasorelaxation
The KCa channels are divided into a subfamily of small(SKCa) intermediate (IKCa) and large (BKCa) conductanceThe BKCa channels are expressed in vascular tissues with ahigh density they are a major component that mediates thedegree of depolarization and contraction in vascular smoothmuscle they are preferentially expressed in vascular smooth
8 Evidence-Based Complementary and Alternative Medicine
150
100
50
Art
eria
l pre
ssur
e (m
mH
g)
2000 3000 4000 5000 6000 7000 8000
Time (s)
VPR(20mgkg)
(a)
0
minus10
minus20
minus30
minus40
ΔM
AP
(mm
Hg)
(b)
100
80
60
40
20
0
1 5 10 20
VPR (mgkg)
ΔH
R (b
pm)
(c)
Figure 6 Effect of VPR on MAP and HR in nonanesthetized rats (a) Representative tracing showed the hypotension induced by additionof VPR (20mgkg) (b) Changes in mean arterial pressure (MAP mmHg) (c) Changes in heart rate (HR bpm) induced by the acuteadministration of increasing doses of VPR (mgkg iv) Values are expressed by mean plusmn SEM 119899 = 5
muscle while the SKCa and IKCa are expressed in a varietyof cell types such as secretory epithelial cells fibroblastsT lymphocytes melanoma cells granulocytes macrophageserythrocytes cultured cell lines and endothelial cells [29 30]BKCa channels act by inhibiting the increase of intracellu-lar Ca2+ Furthermore several recent findings support theimportance of BKCa channels in the regulation of vascularsmooth muscle tone and regulation of blood pressure [31]
The data shows that VPR-mediated vasorelaxation atthe highest concentration was significantly attenuated inrings preincubated with TEA (1mM) which preferentiallyblocks BKCa channels at concentrations lower than 1mM[32] These results suggest that BKCa channels mediate VPR-induced vasorelaxation demonstrating that in addition to theneuronal actions VPR exhibits important peripheral vasculareffects as well
Therefore in vivo experiments were performed to eval-uate VPR effects on cardiovascular parameters in nonanes-thetized normotensive rats in order to avoid the influencesof anesthesia and postsurgical stress In these animals VPRinduced a hypotensive response associated with tachycardia
The hypotensive effect caused by VPR may be due todecreased peripheral vascular resistance and the tachycardiamay be a reflection of the decrease in blood pressure
Taken together these results demonstrate that VPRinduce vasodilatation probably involving K+ channel acti-vation most likely through BKCa channels in smoothmuscle inner from rat superior mesenteric artery Fur-thermore endothelium-derived relaxing factors (NO andprostanoids) are involved in endothelium relaxation depen-dent on VPR
K+ channelKATP ATP-sensitive K+ channelKir Inward rectifier K+ channelKV Voltage-dependent K+ channelMAP Mean arterial pressureMR Maximum responseNO Nitric oxidePAF Platelet-activating factorPhe PhenylephrineK+ channel Potassium channelSEM Standard error of meanSKCa Small-conductance calcium-activated K+
channelSOC Store operated channelsTEA TetraethylammoniumTRP Transient receptor potentialVPR Roots from the Valeriana prionophyllaVPF Leaves from the Valeriana prionophyllaVSMC Vascular smooth muscle cells
Conflict of Interests
The authors declare that there is no conflict of interests
Acknowledgments
This work was supported by grants from Conselho Nacionalde Desenvolvimento Cientıfico e Tecnologico (CNPq)Coordenacao de Aperfeicoamento de Pessoal de Nıvel Supe-rior (CAPES) Consejo Nacional de Ciencia y Tecnologıa deGuatemala (FODECYT 102-2006) and Fundacao de Amparoa Pesquisa do Estado da Bahia (FAPESB)
References
[1] E A Carlini ldquoPlants and the central nervous systemrdquo Phar-macology Biochemistry and Behavior vol 75 no 3 pp 501ndash5122003
[2] V Schulz H Rudolf and V E Tyler Rational Phytotherapy APhysicisnrsquos Guide to Herbal Medicine 2004
[3] I Holzmann V Cechinel-Filho T C Mora et al ldquoEvaluationof behavioral and pharmacological effects of hydroalcoholicextract of Valeriana prionophylla Standl from GuatemalardquoEvidence-Based Complementary and Alternative Medicine vol2011 Article ID 312320 9 pages 2011
[4] D L Nash and J V A Dieterle ldquoFlora of guatemalardquo FieldianaBotany vol 24 no 11 p 297 1976
[5] S Chavadej H Becker and F Weberling ldquoFurther investi-gations of valepotriates in the Valerianaceaerdquo PharmaceutischWeekblad vol 7 no 4 pp 167ndash168 1985
[6] A L Piccinelli S Arana A Caceres R D Di Villa BiancaR Sorrentino and L Rastrelli ldquoNew lignans from the rootsof Valeriana prionophylla with antioxidative and vasorelaxant
activitiesrdquo Journal of Natural Products vol 67 no 7 pp 1135ndash1140 2004
[7] G M Massanet E Pando F Rodriguez-Luis and E ZubialdquoLignans a reviewrdquo Fitoterapia vol 60 no 1 pp 3ndash35 1989
[8] E LGhisalberti ldquoCardiovascular activity of naturally occurringlignansrdquo Phytomedicine vol 4 no 2 pp 151ndash166 1997
[9] A M Gurney ldquoMechanisms of drug-induced vasodilationrdquoJournal of Pharmacy and Pharmacology vol 46 no 4 pp 242ndash251 1994
[10] D F Silva I G A Araujo J G F Albuquerque et alldquoRotundifolone-induced relaxation is mediated by BKCa chan-nel activation and Ca
119907channel inactivationrdquo Basic and Clinical
Pharmacology and Toxicology vol 109 no 6 pp 465ndash475 2011[11] B M Altura and B T Altura ldquoDifferential effects of substrate
depletion on drug-induced contractions of rabbit aortardquo TheAmerican Journal of Physiology vol 219 no 6 pp 1698ndash17051970
[12] W-J Zang C W Balke and W G Wier ldquoGraded 1205721-adrenoceptor activation of arteries involves recruitment ofsmooth muscle cells to produce ldquoall or nonerdquo Ca2+ signalsrdquo CellCalcium vol 29 no 5 pp 327ndash334 2001
[13] R F Furchgott and J V Zawadzki ldquoThe obligatory role ofendothelial cells in the relaxation of arterial smooth muscle byacetylcholinerdquo Nature vol 288 no 5789 pp 373ndash376 1980
[14] M Feletou R Kohler and P M Vanhoutte ldquoNitric oxideorchestrator of endothelium-dependent responsesrdquo Annals ofMedicine vol 44 no 7 pp 694ndash716 2012
[15] Y Tanaka Y Mochizuki H Tanaka and K Shigenobu ldquoSig-nificant role of neuronal non-N-type calcium channels in thesympathetic neurogenic contraction of rat mesenteric arteryrdquoBritish Journal of Pharmacology vol 128 no 7 pp 1602ndash16081999
[16] T Godfraind and A Kaba ldquoBlockade or reversal of the contrac-tion induced by calcium and adrenaline in depolarized arterialsmooth musclerdquo British Journal of Pharmacology vol 36 no 3pp 549ndash560 1969
[17] S Kobayashi H Kanaide and M Nakamura ldquoK+-depolarization induces a direct release of Ca2+ fromintracellular storage sites in cultured vascular smooth musclecells from rat aortardquo Biochemical and Biophysical ResearchCommunications vol 129 no 3 pp 877ndash884 1985
[18] B Ay Y S Prakash C M Pabelick and G C Sieck ldquoStore-operated Ca2+ entry in porcine airway smooth musclerdquo Ameri-can Journal of Physiology vol 286 no 5 pp L909ndashL917 2004
[19] G M Dick and J D Tune ldquoRole of potassium channels incoronary vasodilationrdquo Experimental Biology andMedicine vol235 no 1 pp 10ndash22 2010
[20] EAKo JHan I D Jung andW S Park ldquoPhysiological roles ofK+ channels in vascular smoothmuscle cellsrdquo Journal of SmoothMuscle Research vol 44 no 2 pp 65ndash81 2008
[21] K Lawson ldquoPotassiumchannel openers as potential therapeuticweapons in ion channel diseaserdquo Kidney International vol 57no 3 pp 838ndash845 2000
[22] D X P Brochet and P D Langton ldquoDual effect of initial [K]on vascular tone in rat mesenteric arteriesrdquo Pflugers ArchivEuropean Journal of Physiology vol 453 no 1 pp 33ndash41 2006
[23] F Jiang C G Li and M J Rand ldquoRole of potassium channelsin the nitrergic nerve stimulation-induced vasodilatation in theguinea-pig isolated basilar arteryrdquo British Journal of Pharmacol-ogy vol 123 no 1 pp 106ndash112 1998
10 Evidence-Based Complementary and Alternative Medicine
[24] N B Standen and J M Quayle ldquoK+ channel modulation inarterial smooth musclerdquo Acta Physiologica Scandinavica vol164 no 4 pp 549ndash557 1998
[25] E A Ko W S Park A L Firth N Kim J X-J Yuan and JHan ldquoPathophysiology of voltage-gatedK+ channels in vascularsmooth muscle cells modulation by protein kinasesrdquo Progressin Biophysics and Molecular Biology vol 103 no 1 pp 95ndash1012010
[26] F Sun E Hayama Y Katsube R Matsuoka and T NakanishildquoThe role of the large-conductance voltage-dependent andcalcium-activated potassium (BKCa) channels in the regulationof rat ductus arteriosus tonerdquo Heart and Vessels vol 25 no 6pp 556ndash564 2010
[27] M T Nelson and J M Quayle ldquoPhysiological roles andproperties of potassium channels in arterial smooth musclerdquoAmerican Journal of Physiology vol 268 no 4 pp C799ndashC8221995
[28] T Karkanis S Li J G Pickering and S M Sims ldquoPlasticityof KIR channels in human smooth muscle cells from internalthoracic arteryrdquo American Journal of Physiology vol 284 no 6pp H2325ndashH2334 2003
[29] A Schwab ldquoFunction and spatial distribution of ion channelsand transporters in cell migrationrdquo American Journal of Physi-ology vol 280 no 5 pp F739ndashF747 2001
[30] H Ouadid-Ahidouch M Roudbaraki P Delcourt A Ahi-douchN Joury andN Prevarskaya ldquoFunctional andmolecularidentification of intermediate-conductance Ca2+-activated K+channels in breast cancer cells association with cell cycleprogressionrdquo American Journal of Physiology vol 287 no 1 ppC125ndashC134 2004
[31] J Ledoux M E Werner J E Brayden and M T NelsonldquoCalcium-activated potassium channels and the regulation ofvascular tonerdquo Physiology vol 21 no 1 pp 69ndash78 2006
[32] PD LangtonMTNelson YHuang andN B Standen ldquoBlockof calcium-activated potassium channels inmammalian arterialmyocytes by tetraethylammonium ionsrdquo American Journal ofPhysiology vol 260 no 3 pp H927ndashH934 1991
Evidence-Based Complementary and Alternative Medicine 3
27 Investigation of Nitric Oxide and COX-Derived Prod-ucts Participation in the Vasorelaxation Mediated by VPRTo investigate the involvement of NO and prostanoidsendothelium-intact rings were incubated for 30min withL-NAME (10minus4M) or indomethacin (10minus5M) (endothelialnitric oxide synthase (eNOS) or cyclooxygenase inhibitorsresp) before the second application of Phe (1 120583M) and thevasorelaxation induced byVPR (300 120583gmL)was investigatedbefore and after the addition of the inhibitors
28 Effect of VPR on Contraction Induced by Depolarizationwith High K+ Concentration After the stabilization periodrings without endothelium were precontracted with highpotassium concentration solution (KCl 80mM) and inthe tonic phase different concentrations of VPR (001ndash300 120583gmL) were added cumulatively to the organ bath andrelaxations were measured as previously described
29 Investigation of K+ Channel Participation in the Vasore-laxation Elicited by VPR To investigate the involvementof K+ channels in the first set of experiments vasorelax-ation with VPR was performed in vessels with denudedendothelium that were precontracted with Phe in Tyrodersquossolution with elevated K+ (20mM equimolar replacementof NaCl with KCl) to attenuate K+ efflux [10] Additionallyin the second group of experiments endothelium-denudedrings were incubated for 30min with putative K+ channelblockers before the second application of Phe (1 120583M) and theinhibition was calculated by comparing the response elicitedby VPR before and after the addition of the inhibitorsPreparations were exposed to tetraethylammonium (TEA3mM) a nonselective K+ channel blocker TEA (1mM)a large-conductance calcium-activated K+ channel (BKCa)selective blocker in this concentration 4-aminopyridine (4-AP) (1mM) a voltage-dependent K+ (KV) channel blockerglibenclamide (Glib) (10 120583M) an ATP-sensitive K+ (KATP)channel blocker and barium chloride (BaCl
2) (30 120583M) an
inward rectifier K+ (Kir) channel blocker
210 Investigation of Effect of VPR on CaCl2-Induced Con-
tractions To investigate the hypothesis that VPR act throughthe blockade of extracellular calcium influx in endothelium-denuded rings cumulative concentrations of CaCl
2(10minus6ndash
10minus2M) were added in medium containing Ca2+-free depo-larizing solution (KCl 60mM) in absence (control) or inpresence of VPR (300120583gmL)
211 Measurement of Mean Arterial Pressure and Heart Ratein Nonanesthetized Normotensive Rats The day before theexperimental session a catheter (PE
50) filled with hep-
arinized saline solution (1000UmL) was inserted into theleft carotid artery under ketaminexylazine anesthesia andexteriorized at the nape of the animalrsquos neck to permit bloodpressure recording An additional catheter was placed inthe right femoral vein to allow intravenous drug adminis-tration After 24 hours arterial pressure was continuouslymonitored through the carotid catheter connected to a bloodpressure transducer (World Precision Instruments) whose
signal was amplified and digitally recorded by an analog-to-digital interface (AqDados application for data acquisitionLynx Tecnologia Eletronica Ltda version 70 Sao PauloBrazil) and recorded (1 kHz) on a microcomputer for lateranalysis Mean arterial pressure (MAP) was calculated fromsystolic and diastolic pressure data while heart rate (HR) wasdetermined by the pulsation of arterial pressure using theAcqKnowledge software program version 357 developed byBiopac Systems Inc California USA
212 Statistical Analysis Data are presented as the mean plusmnSEM and n represents a number of rings prepared fromdifferent rats Concentration-response curves to VPR andVPF were based on the percent relaxation of the agonist-induced contraction A 100 relaxation was assigned whenthe precontracted rings returned to the baseline values Thecurves were fitted using a variable slope sigmoid fittingroutine in GraphPad Prism50 (Graph Pad Software Inc LaJolla CA USA) EC
50(concentration required to relax the
induced tone by half and maximum response (MR) valueswere calculated from the fitted sigmoidal curves Statisticalanalyses were performed by comparing 119864max and EC50 valuesbetween groups from independent observations UnpairedStudentrsquos t-test or one-way ANOVAs were used to compare2 3 or more groups respectively with the addition ofBonferronirsquos multiple comparisons post-test Two-sided 119875 lt005 was considered statistically significant
3 Results
31 Effects of VPR andVPF in Phenylephrine-InducedContrac-tions In isolated rat superior mesenteric artery rings VPR(001ndash300 120583gmL) decreased in a concentration-dependentmanner following Phe-induced contraction (1 120583M) (EC
50
values = 596 (38minus92) 120583gmL) (Figure 1(a)) In the absenceof the vascular endothelium the potency of VPR was signif-icantly shifted (EC
50values = 3963 (272minus576)120583gmL 119875 lt
005) (Figure 1(a) Table 1) whereas VPF (001ndash300120583gmL)induced a vasorelaxant effect in the presence and absence ofvascular endothelium (maximum response (MR) = 2939 plusmn94MR = 2705plusmn62 resp 119899 = 4) (Figure 1(b)) howeverthis response was reduced in comparison to that obtained byVPR (MR = 7543 plusmn 408 MR = 7033 plusmn 458 119875 lt 005resp)
32 Effect of VPR on Endothelium-Intact Rings in the Presenceof L-NAME or Indomethacin To investigate the involve-ment of NO and prostanoids endothelium-intact ringswere preincubated for 30min with L-NAME (100120583M) orindomethacin (10120583M) before the second contraction withPhe (1 120583M) Figure 2 shows that the vasorelaxant activity ofVPR (300 120583gmL) was significantly reduced in the presenceof these inhibitors
33 Effects of VPR on Contraction Induced by High K+ Con-centration As illustrated in Figure 3 following contractionwith KCl 80mM VPR-mediated relaxation of endothelium-removed rings was significantly reduced when compared
4 Evidence-Based Complementary and Alternative Medicine
0
20
40
60
80
minus2 minus1 0 1 2 3
Intact endotheliumDenuded endothelium
Rela
xatio
n (
)
log[VPR] (120583gmL)
(a)
0
20
40
60
80
minus2 minus1 0 1 2 3
Intact endotheliumDenuded endothelium
Rela
xatio
n (
)
log[VPF] (120583gmL)
(b)
Figure 1 Vasorelaxant effect induced by VPR Concentration-response curves to VPR or VPF (001ndash300120583gmL cumulatively) precontractedwith 1 120583Mphenylephrine (Phe) in mesenteric artery rings (with endothelium) and after removal of endothelium (a) concentration-responsecurves showing the relaxant effect of VPR 119899 = 6 (b) responses induced by VPF 119899 = 4 The response is expressed as a percentage relaxationof the Phe-induced contraction Values are mean plusmn SEM
to the contractile response induced by Phe (Table 1) asdemonstrated by the displacement of the concentration-response curve to the right
34 Effect of VPR on Arteries Treated with KCl 20mMTo evaluate the involvement of K+ channels in the VPR-mediated vasorelaxant response experiments were per-formed in endothelium-denuded rings incubated with KCl20mM and precontracted with Phe As shown in Figure 4(a)the VPR-mediated vasorelaxation was reduced with signifi-cant alterations in pharmacological efficacy when comparedto the control (Table 1)
35 Effect of VPR on Endothelium-Denuded Rings in the Pres-ence of Different K+ Channel Inhibitors Figure 4(a) showsthat the vasorelaxant activity of VPR at the highest concen-tration was significantly rightward shifted in the presenceof TEA (3mM) (Table 1) However in the presence of Glib(10 120583M) 4-AP (1mM) or BaCl
2(30 120583M) VPR-mediated
relaxation in rings lacking the vascular endothelium andprecontracted with Phe was not significantly reduced Inthe presence of TEA (1mM) the vasorelaxant response wassignificantly attenuated (Figures 4(b) and 4(c) Table 1)
36 Effect of VPR on CaCl2-Induced Contractions To investi-
gate the hypothesis of the residual vasorelaxant effect inducedby VPR being the same as in the presence of the K+ channelsinhibitors concentration-response curve was induced with
37 VPR Effect on MAP and HR in Nonanesthetized RatsAs Figure 6 shows in nonanesthetized normotensive ratsintravenous bolus injections of VPR (1 5 10 and 20mgkg)induced hypotension (MAP = minus1318 plusmn 16 minus1854 plusmn 64minus1636 plusmn 56 and minus2832 plusmn 34mmHg resp 119899 = 5) (Figures6(a) and 6(b)) associated with tachycardia (HR = 1164 plusmn 555024 plusmn 140 6145 plusmn 80 and 6988 plusmn 84 bpm resp 119899 = 5)(Figure 6(c))
4 Discussion
The biological effects of Valeriana prionophylla Standl in thecardiovascular system have yet to be fully elucidated Thisstudy aimed to increase our understanding of this naturalmedicine and highlight its potential pharmacological contri-bution Specifically this work demonstrated cardiovascularpharmacological effects of VPR in rats using in vitro and invivo protocols
Initially we evaluated the peripheral vascular effects ofVPR and VPF by performing in vitro experiments using ratsuperior mesenteric artery rings VPR significantly reducePhe-induced contractions a selective 120572
1-adrenergic receptor
agonist in a concentration-dependent manner [12] VPF also
Evidence-Based Complementary and Alternative Medicine 5
50
40
30
20
10
0
lowast
lowastlowastlowast
VPRVPR + indomethacinVPR + L-NAME
Max
imum
resp
onse
()
Figure 2 Participation of NO and COX-derived products in thevasorelaxation induced by VPR in mesenteric artery rings Effect ofVPR (300 120583gmL) in rings intact endothelium precontracted withPhe (1120583M) in the absence or in the presence of L-NAME (100120583M)or indomethacin (10 120583M) Values are mean plusmn SEM 119899 = 5 and 119899 = 5respectively
Phe (1120583M)KCl (80mM)
0
20
40
60
80
minus2 minus1 0 1 2 3
Rela
xatio
n (
)
log[VPR] (120583gmL)
Figure 3 High K+ extracellular decreased relaxant effect of VPRConcentration-response curves showing the relaxant effect of VPR(001ndash300 120583gmL cumulatively) in rings denuded endothelium con-tracted with Phe or KCl 80mM Values are expressed as mean plusmnSEM 119899 = 5
Table 1 Participation of K+ channels in the vasorelaxation inducedby VPR in mesenteric artery rings precontracted with phenyle-phrine
Values are expressed as mean plusmn SEM unpaired Studentrsquos 119905-tests wereused to examine the difference between denuded endothelium and intactendothelium one-way ANOVAs with the addition of Bonferronirsquos multiplecomparisons post hoc test were used to compare denuded endothelium(control) with different inhibitors groups No significance versus intactendothelium lowast119875 lt 005 versus intact endothelium lowastlowast119875 lt 001 versusdenuded endothelium
119875 lt 001 versus denuded endothelium nsnosignificance versus denuded endothelium
induced a vasorelaxant effect in mesenteric artery rings butthis response was reduced in comparison to that obtained byVPR
It is well known that the endothelium is an importantregulator of the vascular tone releasing endothelium-derivedrelaxing factors including NO COX-derived products andendothelium-derived hyperpolarization factors (EDHFs) [1314] To investigate the participation of these factors in thevasorelaxant effect induced by VPR we performed exper-iments in the absence of functional endothelium Underthese conditions VPR vasorelaxant maximum response wasnot significantly altered however the potency of VPR wasreduced in absence of vascular endothelium Therefore toinvestigate the role of endothelium-derived relaxing fac-tors such as NO and COX-derived products assays wereperformed with L-NAME (eNOS blocker) or indomethacin(COX inhibitor) In these conditions vasorelaxant responsemediated by VPR was significantly reduced suggesting thatVPR inhibit the releasing of endothelial NO and prostanoids
This suggests that the presence of a functional endothe-lium is important but not essential to the relaxation responsemediated by VPR and that an endothelium-independentpathway is also involved in this effect which led to the subse-quent experiments exploring VPR-mediated vasorelaxationin the absence of the endothelium
Vascular tone the contractile activity of vascular smoothmuscle cells (VSMCs) is the major determinant of bloodflow resistance through the circulation Therefore vasculartone plays an important role in the regulation of bloodpressure and the blood distribution [15] Due to the fact thatmany drugs exert their antihypertensive effects by decreasingperipheral vascular resistance by direct action on vascularsmooth muscle we investigated the mechanisms underlying
6 Evidence-Based Complementary and Alternative Medicine
20
40
60
80
minus2 minus1 0 1 2 3
Rela
xatio
n (
)
0
PhePhe + KCl (20mM)Phe + TEA (3mM)
log[VPR] (120583gmL)
(a)
20
40
60
80
minus2 minus1 0 1 2 3Re
laxa
tion
()
0
PhePhe + GlibPhe + 4-AP
log[VPR] (120583gmL)
(b)
PheTEA (1mM)BaCl2
20
40
60
80
minus2 minus1 0 1 2 3
Rela
xatio
n (
)
0
log[VPR] (120583gmL)
(c)
Figure 4 Participation of K+ channels in the vasorelaxation induced by VPR in mesenteric artery rings Concentration-response curvesshowing the relaxant effect of VPR (001ndash300 120583gmL cumulatively) in rings denuded endothelium precontracted with Phe (1120583M) in theabsence or in the presence of (a) KCl 20mM or TEA (3mM) (b) 4-AP (1mM) or Glib (10120583M) (c) TEA (1mM) or BaCl
2(30120583M) Values are
expressed as mean plusmn SEM
Evidence-Based Complementary and Alternative Medicine 7
ControlVPR (300120583gmL)
minus6 minus5 minus4 minus3 minus2 minus1
120
100
80
60
40
20
0
Con
trac
tion
()
log[CaCl2] (M)
Figure 5 Effect of VPR on CaCl2-induced contraction in
endothelium-denuded mesenteric artery rings Concentration-response curves for CaCl
2were determined in Ca2+-free solution
containing KCl (60mM) The curves were determined in theabsence (control) and after incubation with VPR (300120583gmL)Values are expressed as mean plusmn SEM 119899 = 4
endothelium-independent relaxation induced by VPR inmesenteric artery rings
The literature reports that VSMC contraction inducedby high extracellular K+ concentration is mediated by anincrease in membrane depolarization and consequently anincrease in Ca2+ influx through voltage-dependent calciumchannel (CaV) [16] Furthermore the sustained contractiongenerated by a high external K+ concentration may also bemediated by Ca2+ release from the sarcoplasmic reticulum[17] resulting in Ca2+ entry through store-operated channels(SOC) andor transient receptor potential (TRP) channels[18]
Endothelium removed mesenteric rings were precon-tractedwith a depolarizing K+ solution (KCl 80mM) in orderto observe VPR responses before nonspecific generated con-tractions and averting activation of membrane receptors Inthe presence of this depolarizing solution the concentration-response curve induced by VPR was significantly shifted tothe right when compared to the response of VPR in thepresence of Phe changing both the efficacy and potency ofVPR
Based on these preliminary results we speculate thatthis VPR-mediated vasorelaxant effect requires activation ofpotassium channels (K+ channels) in VSMCsThe opening ofthese channels in VSMCs results in an increase in K+ perme-ability leading to K+ efflux The exit of K+ from the VSMCinduces membrane hyperpolarization and consequently the
closing of voltage-operated Ca2+ channels leading to vasore-laxation [19 20] Membrane potential regulation in VSMCsis an important factor in the maintenance of vascular toneAdditionally K+ channels are effector proteins that contributeto membrane potential regulation in electrically excitablecells such as VSMCs Therefore drugs that activate thesechannels could be beneficial in hypertension treatment [21]
Thus to evaluate the involvement of K+ channels inVPR-mediated vasorelaxant response experiments were per-formed in endothelium-denuded arterial rings incubatedwith KCl 20mM This procedure partially decreases mem-brane efflux of K+ due to the increase in extracellular K+concentration from 4mM to 20mM resulting in a reductionin the electrochemical gradient [22] Under these conditionsthe maximum response to VPR was significantly attenuatedwhich was also observed when rings were exposed to KCl80mM suggesting the participation ofK+ channels in vasore-laxation induced by VPR
To confirm the involvement of K+ channels the prepa-rations were pretreated with TEA (3mM) a nonselective K+channel blocker [23] In the presence of this pharmacolog-ical tool the vasorelaxant response induced by increasingconcentrations of VPR was significantly attenuated corrob-orating our previous observation that VPR appears to havea significant effect on the open probability of K+ channels inmesenteric artery rings
Several types of K+ channels are expressed in membraneof VSMCs and each of these channels plays an importantrole in the control and maintenance of the contractile toneof the arterial muscle [24] In VSMCs the variety of K+ chan-nels subtypes identified includes voltage-dependent K+ (KV)channels whose activity is increased upon membrane depo-larization and are important regulators of vascular smoothmuscle membrane potential in response to a depolarizingstimulus [25] large-conductance Ca2+-activated K+ (BKCa)channels which respond to changes in the concentration ofintracellular Ca2+ regulating membrane potential and havean important role in control of myogenic tone in smallerresistance arteries [26] ATP-sensitive K+ (KATP) channelswhich respond actively to changes in cellular metabolism andare targets of a variety of relaxing stimuli [27] and inwardrectifier K+ (KIR) channels regulatingmembrane potential ofvarious types of resistance vessels of small diameter [28]
Thus to investigate the possible participation of thesedifferent K+ channels subtypes experimental preparationswere separately preincubated with different selective blockersof these channels In the presence of a KATP channels blockerGlib (10 120583M) or a KV channels blocker 4-AP (1mM) themaximum effect of VPRwas not changedThe same responseoccurred in rings preincubated with BaCl
2(30 120583M) Kir
channels blocker suggesting that KATP KV and Kir channelsdo not participate in VPR-mediated vasorelaxation
The KCa channels are divided into a subfamily of small(SKCa) intermediate (IKCa) and large (BKCa) conductanceThe BKCa channels are expressed in vascular tissues with ahigh density they are a major component that mediates thedegree of depolarization and contraction in vascular smoothmuscle they are preferentially expressed in vascular smooth
8 Evidence-Based Complementary and Alternative Medicine
150
100
50
Art
eria
l pre
ssur
e (m
mH
g)
2000 3000 4000 5000 6000 7000 8000
Time (s)
VPR(20mgkg)
(a)
0
minus10
minus20
minus30
minus40
ΔM
AP
(mm
Hg)
(b)
100
80
60
40
20
0
1 5 10 20
VPR (mgkg)
ΔH
R (b
pm)
(c)
Figure 6 Effect of VPR on MAP and HR in nonanesthetized rats (a) Representative tracing showed the hypotension induced by additionof VPR (20mgkg) (b) Changes in mean arterial pressure (MAP mmHg) (c) Changes in heart rate (HR bpm) induced by the acuteadministration of increasing doses of VPR (mgkg iv) Values are expressed by mean plusmn SEM 119899 = 5
muscle while the SKCa and IKCa are expressed in a varietyof cell types such as secretory epithelial cells fibroblastsT lymphocytes melanoma cells granulocytes macrophageserythrocytes cultured cell lines and endothelial cells [29 30]BKCa channels act by inhibiting the increase of intracellu-lar Ca2+ Furthermore several recent findings support theimportance of BKCa channels in the regulation of vascularsmooth muscle tone and regulation of blood pressure [31]
The data shows that VPR-mediated vasorelaxation atthe highest concentration was significantly attenuated inrings preincubated with TEA (1mM) which preferentiallyblocks BKCa channels at concentrations lower than 1mM[32] These results suggest that BKCa channels mediate VPR-induced vasorelaxation demonstrating that in addition to theneuronal actions VPR exhibits important peripheral vasculareffects as well
Therefore in vivo experiments were performed to eval-uate VPR effects on cardiovascular parameters in nonanes-thetized normotensive rats in order to avoid the influencesof anesthesia and postsurgical stress In these animals VPRinduced a hypotensive response associated with tachycardia
The hypotensive effect caused by VPR may be due todecreased peripheral vascular resistance and the tachycardiamay be a reflection of the decrease in blood pressure
Taken together these results demonstrate that VPRinduce vasodilatation probably involving K+ channel acti-vation most likely through BKCa channels in smoothmuscle inner from rat superior mesenteric artery Fur-thermore endothelium-derived relaxing factors (NO andprostanoids) are involved in endothelium relaxation depen-dent on VPR
K+ channelKATP ATP-sensitive K+ channelKir Inward rectifier K+ channelKV Voltage-dependent K+ channelMAP Mean arterial pressureMR Maximum responseNO Nitric oxidePAF Platelet-activating factorPhe PhenylephrineK+ channel Potassium channelSEM Standard error of meanSKCa Small-conductance calcium-activated K+
channelSOC Store operated channelsTEA TetraethylammoniumTRP Transient receptor potentialVPR Roots from the Valeriana prionophyllaVPF Leaves from the Valeriana prionophyllaVSMC Vascular smooth muscle cells
Conflict of Interests
The authors declare that there is no conflict of interests
Acknowledgments
This work was supported by grants from Conselho Nacionalde Desenvolvimento Cientıfico e Tecnologico (CNPq)Coordenacao de Aperfeicoamento de Pessoal de Nıvel Supe-rior (CAPES) Consejo Nacional de Ciencia y Tecnologıa deGuatemala (FODECYT 102-2006) and Fundacao de Amparoa Pesquisa do Estado da Bahia (FAPESB)
References
[1] E A Carlini ldquoPlants and the central nervous systemrdquo Phar-macology Biochemistry and Behavior vol 75 no 3 pp 501ndash5122003
[2] V Schulz H Rudolf and V E Tyler Rational Phytotherapy APhysicisnrsquos Guide to Herbal Medicine 2004
[3] I Holzmann V Cechinel-Filho T C Mora et al ldquoEvaluationof behavioral and pharmacological effects of hydroalcoholicextract of Valeriana prionophylla Standl from GuatemalardquoEvidence-Based Complementary and Alternative Medicine vol2011 Article ID 312320 9 pages 2011
[4] D L Nash and J V A Dieterle ldquoFlora of guatemalardquo FieldianaBotany vol 24 no 11 p 297 1976
[5] S Chavadej H Becker and F Weberling ldquoFurther investi-gations of valepotriates in the Valerianaceaerdquo PharmaceutischWeekblad vol 7 no 4 pp 167ndash168 1985
[6] A L Piccinelli S Arana A Caceres R D Di Villa BiancaR Sorrentino and L Rastrelli ldquoNew lignans from the rootsof Valeriana prionophylla with antioxidative and vasorelaxant
activitiesrdquo Journal of Natural Products vol 67 no 7 pp 1135ndash1140 2004
[7] G M Massanet E Pando F Rodriguez-Luis and E ZubialdquoLignans a reviewrdquo Fitoterapia vol 60 no 1 pp 3ndash35 1989
[8] E LGhisalberti ldquoCardiovascular activity of naturally occurringlignansrdquo Phytomedicine vol 4 no 2 pp 151ndash166 1997
[9] A M Gurney ldquoMechanisms of drug-induced vasodilationrdquoJournal of Pharmacy and Pharmacology vol 46 no 4 pp 242ndash251 1994
[10] D F Silva I G A Araujo J G F Albuquerque et alldquoRotundifolone-induced relaxation is mediated by BKCa chan-nel activation and Ca
119907channel inactivationrdquo Basic and Clinical
Pharmacology and Toxicology vol 109 no 6 pp 465ndash475 2011[11] B M Altura and B T Altura ldquoDifferential effects of substrate
depletion on drug-induced contractions of rabbit aortardquo TheAmerican Journal of Physiology vol 219 no 6 pp 1698ndash17051970
[12] W-J Zang C W Balke and W G Wier ldquoGraded 1205721-adrenoceptor activation of arteries involves recruitment ofsmooth muscle cells to produce ldquoall or nonerdquo Ca2+ signalsrdquo CellCalcium vol 29 no 5 pp 327ndash334 2001
[13] R F Furchgott and J V Zawadzki ldquoThe obligatory role ofendothelial cells in the relaxation of arterial smooth muscle byacetylcholinerdquo Nature vol 288 no 5789 pp 373ndash376 1980
[14] M Feletou R Kohler and P M Vanhoutte ldquoNitric oxideorchestrator of endothelium-dependent responsesrdquo Annals ofMedicine vol 44 no 7 pp 694ndash716 2012
[15] Y Tanaka Y Mochizuki H Tanaka and K Shigenobu ldquoSig-nificant role of neuronal non-N-type calcium channels in thesympathetic neurogenic contraction of rat mesenteric arteryrdquoBritish Journal of Pharmacology vol 128 no 7 pp 1602ndash16081999
[16] T Godfraind and A Kaba ldquoBlockade or reversal of the contrac-tion induced by calcium and adrenaline in depolarized arterialsmooth musclerdquo British Journal of Pharmacology vol 36 no 3pp 549ndash560 1969
[17] S Kobayashi H Kanaide and M Nakamura ldquoK+-depolarization induces a direct release of Ca2+ fromintracellular storage sites in cultured vascular smooth musclecells from rat aortardquo Biochemical and Biophysical ResearchCommunications vol 129 no 3 pp 877ndash884 1985
[18] B Ay Y S Prakash C M Pabelick and G C Sieck ldquoStore-operated Ca2+ entry in porcine airway smooth musclerdquo Ameri-can Journal of Physiology vol 286 no 5 pp L909ndashL917 2004
[19] G M Dick and J D Tune ldquoRole of potassium channels incoronary vasodilationrdquo Experimental Biology andMedicine vol235 no 1 pp 10ndash22 2010
[20] EAKo JHan I D Jung andW S Park ldquoPhysiological roles ofK+ channels in vascular smoothmuscle cellsrdquo Journal of SmoothMuscle Research vol 44 no 2 pp 65ndash81 2008
[21] K Lawson ldquoPotassiumchannel openers as potential therapeuticweapons in ion channel diseaserdquo Kidney International vol 57no 3 pp 838ndash845 2000
[22] D X P Brochet and P D Langton ldquoDual effect of initial [K]on vascular tone in rat mesenteric arteriesrdquo Pflugers ArchivEuropean Journal of Physiology vol 453 no 1 pp 33ndash41 2006
[23] F Jiang C G Li and M J Rand ldquoRole of potassium channelsin the nitrergic nerve stimulation-induced vasodilatation in theguinea-pig isolated basilar arteryrdquo British Journal of Pharmacol-ogy vol 123 no 1 pp 106ndash112 1998
10 Evidence-Based Complementary and Alternative Medicine
[24] N B Standen and J M Quayle ldquoK+ channel modulation inarterial smooth musclerdquo Acta Physiologica Scandinavica vol164 no 4 pp 549ndash557 1998
[25] E A Ko W S Park A L Firth N Kim J X-J Yuan and JHan ldquoPathophysiology of voltage-gatedK+ channels in vascularsmooth muscle cells modulation by protein kinasesrdquo Progressin Biophysics and Molecular Biology vol 103 no 1 pp 95ndash1012010
[26] F Sun E Hayama Y Katsube R Matsuoka and T NakanishildquoThe role of the large-conductance voltage-dependent andcalcium-activated potassium (BKCa) channels in the regulationof rat ductus arteriosus tonerdquo Heart and Vessels vol 25 no 6pp 556ndash564 2010
[27] M T Nelson and J M Quayle ldquoPhysiological roles andproperties of potassium channels in arterial smooth musclerdquoAmerican Journal of Physiology vol 268 no 4 pp C799ndashC8221995
[28] T Karkanis S Li J G Pickering and S M Sims ldquoPlasticityof KIR channels in human smooth muscle cells from internalthoracic arteryrdquo American Journal of Physiology vol 284 no 6pp H2325ndashH2334 2003
[29] A Schwab ldquoFunction and spatial distribution of ion channelsand transporters in cell migrationrdquo American Journal of Physi-ology vol 280 no 5 pp F739ndashF747 2001
[30] H Ouadid-Ahidouch M Roudbaraki P Delcourt A Ahi-douchN Joury andN Prevarskaya ldquoFunctional andmolecularidentification of intermediate-conductance Ca2+-activated K+channels in breast cancer cells association with cell cycleprogressionrdquo American Journal of Physiology vol 287 no 1 ppC125ndashC134 2004
[31] J Ledoux M E Werner J E Brayden and M T NelsonldquoCalcium-activated potassium channels and the regulation ofvascular tonerdquo Physiology vol 21 no 1 pp 69ndash78 2006
[32] PD LangtonMTNelson YHuang andN B Standen ldquoBlockof calcium-activated potassium channels inmammalian arterialmyocytes by tetraethylammonium ionsrdquo American Journal ofPhysiology vol 260 no 3 pp H927ndashH934 1991
4 Evidence-Based Complementary and Alternative Medicine
0
20
40
60
80
minus2 minus1 0 1 2 3
Intact endotheliumDenuded endothelium
Rela
xatio
n (
)
log[VPR] (120583gmL)
(a)
0
20
40
60
80
minus2 minus1 0 1 2 3
Intact endotheliumDenuded endothelium
Rela
xatio
n (
)
log[VPF] (120583gmL)
(b)
Figure 1 Vasorelaxant effect induced by VPR Concentration-response curves to VPR or VPF (001ndash300120583gmL cumulatively) precontractedwith 1 120583Mphenylephrine (Phe) in mesenteric artery rings (with endothelium) and after removal of endothelium (a) concentration-responsecurves showing the relaxant effect of VPR 119899 = 6 (b) responses induced by VPF 119899 = 4 The response is expressed as a percentage relaxationof the Phe-induced contraction Values are mean plusmn SEM
to the contractile response induced by Phe (Table 1) asdemonstrated by the displacement of the concentration-response curve to the right
34 Effect of VPR on Arteries Treated with KCl 20mMTo evaluate the involvement of K+ channels in the VPR-mediated vasorelaxant response experiments were per-formed in endothelium-denuded rings incubated with KCl20mM and precontracted with Phe As shown in Figure 4(a)the VPR-mediated vasorelaxation was reduced with signifi-cant alterations in pharmacological efficacy when comparedto the control (Table 1)
35 Effect of VPR on Endothelium-Denuded Rings in the Pres-ence of Different K+ Channel Inhibitors Figure 4(a) showsthat the vasorelaxant activity of VPR at the highest concen-tration was significantly rightward shifted in the presenceof TEA (3mM) (Table 1) However in the presence of Glib(10 120583M) 4-AP (1mM) or BaCl
2(30 120583M) VPR-mediated
relaxation in rings lacking the vascular endothelium andprecontracted with Phe was not significantly reduced Inthe presence of TEA (1mM) the vasorelaxant response wassignificantly attenuated (Figures 4(b) and 4(c) Table 1)
36 Effect of VPR on CaCl2-Induced Contractions To investi-
gate the hypothesis of the residual vasorelaxant effect inducedby VPR being the same as in the presence of the K+ channelsinhibitors concentration-response curve was induced with
37 VPR Effect on MAP and HR in Nonanesthetized RatsAs Figure 6 shows in nonanesthetized normotensive ratsintravenous bolus injections of VPR (1 5 10 and 20mgkg)induced hypotension (MAP = minus1318 plusmn 16 minus1854 plusmn 64minus1636 plusmn 56 and minus2832 plusmn 34mmHg resp 119899 = 5) (Figures6(a) and 6(b)) associated with tachycardia (HR = 1164 plusmn 555024 plusmn 140 6145 plusmn 80 and 6988 plusmn 84 bpm resp 119899 = 5)(Figure 6(c))
4 Discussion
The biological effects of Valeriana prionophylla Standl in thecardiovascular system have yet to be fully elucidated Thisstudy aimed to increase our understanding of this naturalmedicine and highlight its potential pharmacological contri-bution Specifically this work demonstrated cardiovascularpharmacological effects of VPR in rats using in vitro and invivo protocols
Initially we evaluated the peripheral vascular effects ofVPR and VPF by performing in vitro experiments using ratsuperior mesenteric artery rings VPR significantly reducePhe-induced contractions a selective 120572
1-adrenergic receptor
agonist in a concentration-dependent manner [12] VPF also
Evidence-Based Complementary and Alternative Medicine 5
50
40
30
20
10
0
lowast
lowastlowastlowast
VPRVPR + indomethacinVPR + L-NAME
Max
imum
resp
onse
()
Figure 2 Participation of NO and COX-derived products in thevasorelaxation induced by VPR in mesenteric artery rings Effect ofVPR (300 120583gmL) in rings intact endothelium precontracted withPhe (1120583M) in the absence or in the presence of L-NAME (100120583M)or indomethacin (10 120583M) Values are mean plusmn SEM 119899 = 5 and 119899 = 5respectively
Phe (1120583M)KCl (80mM)
0
20
40
60
80
minus2 minus1 0 1 2 3
Rela
xatio
n (
)
log[VPR] (120583gmL)
Figure 3 High K+ extracellular decreased relaxant effect of VPRConcentration-response curves showing the relaxant effect of VPR(001ndash300 120583gmL cumulatively) in rings denuded endothelium con-tracted with Phe or KCl 80mM Values are expressed as mean plusmnSEM 119899 = 5
Table 1 Participation of K+ channels in the vasorelaxation inducedby VPR in mesenteric artery rings precontracted with phenyle-phrine
Values are expressed as mean plusmn SEM unpaired Studentrsquos 119905-tests wereused to examine the difference between denuded endothelium and intactendothelium one-way ANOVAs with the addition of Bonferronirsquos multiplecomparisons post hoc test were used to compare denuded endothelium(control) with different inhibitors groups No significance versus intactendothelium lowast119875 lt 005 versus intact endothelium lowastlowast119875 lt 001 versusdenuded endothelium
119875 lt 001 versus denuded endothelium nsnosignificance versus denuded endothelium
induced a vasorelaxant effect in mesenteric artery rings butthis response was reduced in comparison to that obtained byVPR
It is well known that the endothelium is an importantregulator of the vascular tone releasing endothelium-derivedrelaxing factors including NO COX-derived products andendothelium-derived hyperpolarization factors (EDHFs) [1314] To investigate the participation of these factors in thevasorelaxant effect induced by VPR we performed exper-iments in the absence of functional endothelium Underthese conditions VPR vasorelaxant maximum response wasnot significantly altered however the potency of VPR wasreduced in absence of vascular endothelium Therefore toinvestigate the role of endothelium-derived relaxing fac-tors such as NO and COX-derived products assays wereperformed with L-NAME (eNOS blocker) or indomethacin(COX inhibitor) In these conditions vasorelaxant responsemediated by VPR was significantly reduced suggesting thatVPR inhibit the releasing of endothelial NO and prostanoids
This suggests that the presence of a functional endothe-lium is important but not essential to the relaxation responsemediated by VPR and that an endothelium-independentpathway is also involved in this effect which led to the subse-quent experiments exploring VPR-mediated vasorelaxationin the absence of the endothelium
Vascular tone the contractile activity of vascular smoothmuscle cells (VSMCs) is the major determinant of bloodflow resistance through the circulation Therefore vasculartone plays an important role in the regulation of bloodpressure and the blood distribution [15] Due to the fact thatmany drugs exert their antihypertensive effects by decreasingperipheral vascular resistance by direct action on vascularsmooth muscle we investigated the mechanisms underlying
6 Evidence-Based Complementary and Alternative Medicine
20
40
60
80
minus2 minus1 0 1 2 3
Rela
xatio
n (
)
0
PhePhe + KCl (20mM)Phe + TEA (3mM)
log[VPR] (120583gmL)
(a)
20
40
60
80
minus2 minus1 0 1 2 3Re
laxa
tion
()
0
PhePhe + GlibPhe + 4-AP
log[VPR] (120583gmL)
(b)
PheTEA (1mM)BaCl2
20
40
60
80
minus2 minus1 0 1 2 3
Rela
xatio
n (
)
0
log[VPR] (120583gmL)
(c)
Figure 4 Participation of K+ channels in the vasorelaxation induced by VPR in mesenteric artery rings Concentration-response curvesshowing the relaxant effect of VPR (001ndash300 120583gmL cumulatively) in rings denuded endothelium precontracted with Phe (1120583M) in theabsence or in the presence of (a) KCl 20mM or TEA (3mM) (b) 4-AP (1mM) or Glib (10120583M) (c) TEA (1mM) or BaCl
2(30120583M) Values are
expressed as mean plusmn SEM
Evidence-Based Complementary and Alternative Medicine 7
ControlVPR (300120583gmL)
minus6 minus5 minus4 minus3 minus2 minus1
120
100
80
60
40
20
0
Con
trac
tion
()
log[CaCl2] (M)
Figure 5 Effect of VPR on CaCl2-induced contraction in
endothelium-denuded mesenteric artery rings Concentration-response curves for CaCl
2were determined in Ca2+-free solution
containing KCl (60mM) The curves were determined in theabsence (control) and after incubation with VPR (300120583gmL)Values are expressed as mean plusmn SEM 119899 = 4
endothelium-independent relaxation induced by VPR inmesenteric artery rings
The literature reports that VSMC contraction inducedby high extracellular K+ concentration is mediated by anincrease in membrane depolarization and consequently anincrease in Ca2+ influx through voltage-dependent calciumchannel (CaV) [16] Furthermore the sustained contractiongenerated by a high external K+ concentration may also bemediated by Ca2+ release from the sarcoplasmic reticulum[17] resulting in Ca2+ entry through store-operated channels(SOC) andor transient receptor potential (TRP) channels[18]
Endothelium removed mesenteric rings were precon-tractedwith a depolarizing K+ solution (KCl 80mM) in orderto observe VPR responses before nonspecific generated con-tractions and averting activation of membrane receptors Inthe presence of this depolarizing solution the concentration-response curve induced by VPR was significantly shifted tothe right when compared to the response of VPR in thepresence of Phe changing both the efficacy and potency ofVPR
Based on these preliminary results we speculate thatthis VPR-mediated vasorelaxant effect requires activation ofpotassium channels (K+ channels) in VSMCsThe opening ofthese channels in VSMCs results in an increase in K+ perme-ability leading to K+ efflux The exit of K+ from the VSMCinduces membrane hyperpolarization and consequently the
closing of voltage-operated Ca2+ channels leading to vasore-laxation [19 20] Membrane potential regulation in VSMCsis an important factor in the maintenance of vascular toneAdditionally K+ channels are effector proteins that contributeto membrane potential regulation in electrically excitablecells such as VSMCs Therefore drugs that activate thesechannels could be beneficial in hypertension treatment [21]
Thus to evaluate the involvement of K+ channels inVPR-mediated vasorelaxant response experiments were per-formed in endothelium-denuded arterial rings incubatedwith KCl 20mM This procedure partially decreases mem-brane efflux of K+ due to the increase in extracellular K+concentration from 4mM to 20mM resulting in a reductionin the electrochemical gradient [22] Under these conditionsthe maximum response to VPR was significantly attenuatedwhich was also observed when rings were exposed to KCl80mM suggesting the participation ofK+ channels in vasore-laxation induced by VPR
To confirm the involvement of K+ channels the prepa-rations were pretreated with TEA (3mM) a nonselective K+channel blocker [23] In the presence of this pharmacolog-ical tool the vasorelaxant response induced by increasingconcentrations of VPR was significantly attenuated corrob-orating our previous observation that VPR appears to havea significant effect on the open probability of K+ channels inmesenteric artery rings
Several types of K+ channels are expressed in membraneof VSMCs and each of these channels plays an importantrole in the control and maintenance of the contractile toneof the arterial muscle [24] In VSMCs the variety of K+ chan-nels subtypes identified includes voltage-dependent K+ (KV)channels whose activity is increased upon membrane depo-larization and are important regulators of vascular smoothmuscle membrane potential in response to a depolarizingstimulus [25] large-conductance Ca2+-activated K+ (BKCa)channels which respond to changes in the concentration ofintracellular Ca2+ regulating membrane potential and havean important role in control of myogenic tone in smallerresistance arteries [26] ATP-sensitive K+ (KATP) channelswhich respond actively to changes in cellular metabolism andare targets of a variety of relaxing stimuli [27] and inwardrectifier K+ (KIR) channels regulatingmembrane potential ofvarious types of resistance vessels of small diameter [28]
Thus to investigate the possible participation of thesedifferent K+ channels subtypes experimental preparationswere separately preincubated with different selective blockersof these channels In the presence of a KATP channels blockerGlib (10 120583M) or a KV channels blocker 4-AP (1mM) themaximum effect of VPRwas not changedThe same responseoccurred in rings preincubated with BaCl
2(30 120583M) Kir
channels blocker suggesting that KATP KV and Kir channelsdo not participate in VPR-mediated vasorelaxation
The KCa channels are divided into a subfamily of small(SKCa) intermediate (IKCa) and large (BKCa) conductanceThe BKCa channels are expressed in vascular tissues with ahigh density they are a major component that mediates thedegree of depolarization and contraction in vascular smoothmuscle they are preferentially expressed in vascular smooth
8 Evidence-Based Complementary and Alternative Medicine
150
100
50
Art
eria
l pre
ssur
e (m
mH
g)
2000 3000 4000 5000 6000 7000 8000
Time (s)
VPR(20mgkg)
(a)
0
minus10
minus20
minus30
minus40
ΔM
AP
(mm
Hg)
(b)
100
80
60
40
20
0
1 5 10 20
VPR (mgkg)
ΔH
R (b
pm)
(c)
Figure 6 Effect of VPR on MAP and HR in nonanesthetized rats (a) Representative tracing showed the hypotension induced by additionof VPR (20mgkg) (b) Changes in mean arterial pressure (MAP mmHg) (c) Changes in heart rate (HR bpm) induced by the acuteadministration of increasing doses of VPR (mgkg iv) Values are expressed by mean plusmn SEM 119899 = 5
muscle while the SKCa and IKCa are expressed in a varietyof cell types such as secretory epithelial cells fibroblastsT lymphocytes melanoma cells granulocytes macrophageserythrocytes cultured cell lines and endothelial cells [29 30]BKCa channels act by inhibiting the increase of intracellu-lar Ca2+ Furthermore several recent findings support theimportance of BKCa channels in the regulation of vascularsmooth muscle tone and regulation of blood pressure [31]
The data shows that VPR-mediated vasorelaxation atthe highest concentration was significantly attenuated inrings preincubated with TEA (1mM) which preferentiallyblocks BKCa channels at concentrations lower than 1mM[32] These results suggest that BKCa channels mediate VPR-induced vasorelaxation demonstrating that in addition to theneuronal actions VPR exhibits important peripheral vasculareffects as well
Therefore in vivo experiments were performed to eval-uate VPR effects on cardiovascular parameters in nonanes-thetized normotensive rats in order to avoid the influencesof anesthesia and postsurgical stress In these animals VPRinduced a hypotensive response associated with tachycardia
The hypotensive effect caused by VPR may be due todecreased peripheral vascular resistance and the tachycardiamay be a reflection of the decrease in blood pressure
Taken together these results demonstrate that VPRinduce vasodilatation probably involving K+ channel acti-vation most likely through BKCa channels in smoothmuscle inner from rat superior mesenteric artery Fur-thermore endothelium-derived relaxing factors (NO andprostanoids) are involved in endothelium relaxation depen-dent on VPR
K+ channelKATP ATP-sensitive K+ channelKir Inward rectifier K+ channelKV Voltage-dependent K+ channelMAP Mean arterial pressureMR Maximum responseNO Nitric oxidePAF Platelet-activating factorPhe PhenylephrineK+ channel Potassium channelSEM Standard error of meanSKCa Small-conductance calcium-activated K+
channelSOC Store operated channelsTEA TetraethylammoniumTRP Transient receptor potentialVPR Roots from the Valeriana prionophyllaVPF Leaves from the Valeriana prionophyllaVSMC Vascular smooth muscle cells
Conflict of Interests
The authors declare that there is no conflict of interests
Acknowledgments
This work was supported by grants from Conselho Nacionalde Desenvolvimento Cientıfico e Tecnologico (CNPq)Coordenacao de Aperfeicoamento de Pessoal de Nıvel Supe-rior (CAPES) Consejo Nacional de Ciencia y Tecnologıa deGuatemala (FODECYT 102-2006) and Fundacao de Amparoa Pesquisa do Estado da Bahia (FAPESB)
References
[1] E A Carlini ldquoPlants and the central nervous systemrdquo Phar-macology Biochemistry and Behavior vol 75 no 3 pp 501ndash5122003
[2] V Schulz H Rudolf and V E Tyler Rational Phytotherapy APhysicisnrsquos Guide to Herbal Medicine 2004
[3] I Holzmann V Cechinel-Filho T C Mora et al ldquoEvaluationof behavioral and pharmacological effects of hydroalcoholicextract of Valeriana prionophylla Standl from GuatemalardquoEvidence-Based Complementary and Alternative Medicine vol2011 Article ID 312320 9 pages 2011
[4] D L Nash and J V A Dieterle ldquoFlora of guatemalardquo FieldianaBotany vol 24 no 11 p 297 1976
[5] S Chavadej H Becker and F Weberling ldquoFurther investi-gations of valepotriates in the Valerianaceaerdquo PharmaceutischWeekblad vol 7 no 4 pp 167ndash168 1985
[6] A L Piccinelli S Arana A Caceres R D Di Villa BiancaR Sorrentino and L Rastrelli ldquoNew lignans from the rootsof Valeriana prionophylla with antioxidative and vasorelaxant
activitiesrdquo Journal of Natural Products vol 67 no 7 pp 1135ndash1140 2004
[7] G M Massanet E Pando F Rodriguez-Luis and E ZubialdquoLignans a reviewrdquo Fitoterapia vol 60 no 1 pp 3ndash35 1989
[8] E LGhisalberti ldquoCardiovascular activity of naturally occurringlignansrdquo Phytomedicine vol 4 no 2 pp 151ndash166 1997
[9] A M Gurney ldquoMechanisms of drug-induced vasodilationrdquoJournal of Pharmacy and Pharmacology vol 46 no 4 pp 242ndash251 1994
[10] D F Silva I G A Araujo J G F Albuquerque et alldquoRotundifolone-induced relaxation is mediated by BKCa chan-nel activation and Ca
119907channel inactivationrdquo Basic and Clinical
Pharmacology and Toxicology vol 109 no 6 pp 465ndash475 2011[11] B M Altura and B T Altura ldquoDifferential effects of substrate
depletion on drug-induced contractions of rabbit aortardquo TheAmerican Journal of Physiology vol 219 no 6 pp 1698ndash17051970
[12] W-J Zang C W Balke and W G Wier ldquoGraded 1205721-adrenoceptor activation of arteries involves recruitment ofsmooth muscle cells to produce ldquoall or nonerdquo Ca2+ signalsrdquo CellCalcium vol 29 no 5 pp 327ndash334 2001
[13] R F Furchgott and J V Zawadzki ldquoThe obligatory role ofendothelial cells in the relaxation of arterial smooth muscle byacetylcholinerdquo Nature vol 288 no 5789 pp 373ndash376 1980
[14] M Feletou R Kohler and P M Vanhoutte ldquoNitric oxideorchestrator of endothelium-dependent responsesrdquo Annals ofMedicine vol 44 no 7 pp 694ndash716 2012
[15] Y Tanaka Y Mochizuki H Tanaka and K Shigenobu ldquoSig-nificant role of neuronal non-N-type calcium channels in thesympathetic neurogenic contraction of rat mesenteric arteryrdquoBritish Journal of Pharmacology vol 128 no 7 pp 1602ndash16081999
[16] T Godfraind and A Kaba ldquoBlockade or reversal of the contrac-tion induced by calcium and adrenaline in depolarized arterialsmooth musclerdquo British Journal of Pharmacology vol 36 no 3pp 549ndash560 1969
[17] S Kobayashi H Kanaide and M Nakamura ldquoK+-depolarization induces a direct release of Ca2+ fromintracellular storage sites in cultured vascular smooth musclecells from rat aortardquo Biochemical and Biophysical ResearchCommunications vol 129 no 3 pp 877ndash884 1985
[18] B Ay Y S Prakash C M Pabelick and G C Sieck ldquoStore-operated Ca2+ entry in porcine airway smooth musclerdquo Ameri-can Journal of Physiology vol 286 no 5 pp L909ndashL917 2004
[19] G M Dick and J D Tune ldquoRole of potassium channels incoronary vasodilationrdquo Experimental Biology andMedicine vol235 no 1 pp 10ndash22 2010
[20] EAKo JHan I D Jung andW S Park ldquoPhysiological roles ofK+ channels in vascular smoothmuscle cellsrdquo Journal of SmoothMuscle Research vol 44 no 2 pp 65ndash81 2008
[21] K Lawson ldquoPotassiumchannel openers as potential therapeuticweapons in ion channel diseaserdquo Kidney International vol 57no 3 pp 838ndash845 2000
[22] D X P Brochet and P D Langton ldquoDual effect of initial [K]on vascular tone in rat mesenteric arteriesrdquo Pflugers ArchivEuropean Journal of Physiology vol 453 no 1 pp 33ndash41 2006
[23] F Jiang C G Li and M J Rand ldquoRole of potassium channelsin the nitrergic nerve stimulation-induced vasodilatation in theguinea-pig isolated basilar arteryrdquo British Journal of Pharmacol-ogy vol 123 no 1 pp 106ndash112 1998
10 Evidence-Based Complementary and Alternative Medicine
[24] N B Standen and J M Quayle ldquoK+ channel modulation inarterial smooth musclerdquo Acta Physiologica Scandinavica vol164 no 4 pp 549ndash557 1998
[25] E A Ko W S Park A L Firth N Kim J X-J Yuan and JHan ldquoPathophysiology of voltage-gatedK+ channels in vascularsmooth muscle cells modulation by protein kinasesrdquo Progressin Biophysics and Molecular Biology vol 103 no 1 pp 95ndash1012010
[26] F Sun E Hayama Y Katsube R Matsuoka and T NakanishildquoThe role of the large-conductance voltage-dependent andcalcium-activated potassium (BKCa) channels in the regulationof rat ductus arteriosus tonerdquo Heart and Vessels vol 25 no 6pp 556ndash564 2010
[27] M T Nelson and J M Quayle ldquoPhysiological roles andproperties of potassium channels in arterial smooth musclerdquoAmerican Journal of Physiology vol 268 no 4 pp C799ndashC8221995
[28] T Karkanis S Li J G Pickering and S M Sims ldquoPlasticityof KIR channels in human smooth muscle cells from internalthoracic arteryrdquo American Journal of Physiology vol 284 no 6pp H2325ndashH2334 2003
[29] A Schwab ldquoFunction and spatial distribution of ion channelsand transporters in cell migrationrdquo American Journal of Physi-ology vol 280 no 5 pp F739ndashF747 2001
[30] H Ouadid-Ahidouch M Roudbaraki P Delcourt A Ahi-douchN Joury andN Prevarskaya ldquoFunctional andmolecularidentification of intermediate-conductance Ca2+-activated K+channels in breast cancer cells association with cell cycleprogressionrdquo American Journal of Physiology vol 287 no 1 ppC125ndashC134 2004
[31] J Ledoux M E Werner J E Brayden and M T NelsonldquoCalcium-activated potassium channels and the regulation ofvascular tonerdquo Physiology vol 21 no 1 pp 69ndash78 2006
[32] PD LangtonMTNelson YHuang andN B Standen ldquoBlockof calcium-activated potassium channels inmammalian arterialmyocytes by tetraethylammonium ionsrdquo American Journal ofPhysiology vol 260 no 3 pp H927ndashH934 1991
Evidence-Based Complementary and Alternative Medicine 5
50
40
30
20
10
0
lowast
lowastlowastlowast
VPRVPR + indomethacinVPR + L-NAME
Max
imum
resp
onse
()
Figure 2 Participation of NO and COX-derived products in thevasorelaxation induced by VPR in mesenteric artery rings Effect ofVPR (300 120583gmL) in rings intact endothelium precontracted withPhe (1120583M) in the absence or in the presence of L-NAME (100120583M)or indomethacin (10 120583M) Values are mean plusmn SEM 119899 = 5 and 119899 = 5respectively
Phe (1120583M)KCl (80mM)
0
20
40
60
80
minus2 minus1 0 1 2 3
Rela
xatio
n (
)
log[VPR] (120583gmL)
Figure 3 High K+ extracellular decreased relaxant effect of VPRConcentration-response curves showing the relaxant effect of VPR(001ndash300 120583gmL cumulatively) in rings denuded endothelium con-tracted with Phe or KCl 80mM Values are expressed as mean plusmnSEM 119899 = 5
Table 1 Participation of K+ channels in the vasorelaxation inducedby VPR in mesenteric artery rings precontracted with phenyle-phrine
Values are expressed as mean plusmn SEM unpaired Studentrsquos 119905-tests wereused to examine the difference between denuded endothelium and intactendothelium one-way ANOVAs with the addition of Bonferronirsquos multiplecomparisons post hoc test were used to compare denuded endothelium(control) with different inhibitors groups No significance versus intactendothelium lowast119875 lt 005 versus intact endothelium lowastlowast119875 lt 001 versusdenuded endothelium
119875 lt 001 versus denuded endothelium nsnosignificance versus denuded endothelium
induced a vasorelaxant effect in mesenteric artery rings butthis response was reduced in comparison to that obtained byVPR
It is well known that the endothelium is an importantregulator of the vascular tone releasing endothelium-derivedrelaxing factors including NO COX-derived products andendothelium-derived hyperpolarization factors (EDHFs) [1314] To investigate the participation of these factors in thevasorelaxant effect induced by VPR we performed exper-iments in the absence of functional endothelium Underthese conditions VPR vasorelaxant maximum response wasnot significantly altered however the potency of VPR wasreduced in absence of vascular endothelium Therefore toinvestigate the role of endothelium-derived relaxing fac-tors such as NO and COX-derived products assays wereperformed with L-NAME (eNOS blocker) or indomethacin(COX inhibitor) In these conditions vasorelaxant responsemediated by VPR was significantly reduced suggesting thatVPR inhibit the releasing of endothelial NO and prostanoids
This suggests that the presence of a functional endothe-lium is important but not essential to the relaxation responsemediated by VPR and that an endothelium-independentpathway is also involved in this effect which led to the subse-quent experiments exploring VPR-mediated vasorelaxationin the absence of the endothelium
Vascular tone the contractile activity of vascular smoothmuscle cells (VSMCs) is the major determinant of bloodflow resistance through the circulation Therefore vasculartone plays an important role in the regulation of bloodpressure and the blood distribution [15] Due to the fact thatmany drugs exert their antihypertensive effects by decreasingperipheral vascular resistance by direct action on vascularsmooth muscle we investigated the mechanisms underlying
6 Evidence-Based Complementary and Alternative Medicine
20
40
60
80
minus2 minus1 0 1 2 3
Rela
xatio
n (
)
0
PhePhe + KCl (20mM)Phe + TEA (3mM)
log[VPR] (120583gmL)
(a)
20
40
60
80
minus2 minus1 0 1 2 3Re
laxa
tion
()
0
PhePhe + GlibPhe + 4-AP
log[VPR] (120583gmL)
(b)
PheTEA (1mM)BaCl2
20
40
60
80
minus2 minus1 0 1 2 3
Rela
xatio
n (
)
0
log[VPR] (120583gmL)
(c)
Figure 4 Participation of K+ channels in the vasorelaxation induced by VPR in mesenteric artery rings Concentration-response curvesshowing the relaxant effect of VPR (001ndash300 120583gmL cumulatively) in rings denuded endothelium precontracted with Phe (1120583M) in theabsence or in the presence of (a) KCl 20mM or TEA (3mM) (b) 4-AP (1mM) or Glib (10120583M) (c) TEA (1mM) or BaCl
2(30120583M) Values are
expressed as mean plusmn SEM
Evidence-Based Complementary and Alternative Medicine 7
ControlVPR (300120583gmL)
minus6 minus5 minus4 minus3 minus2 minus1
120
100
80
60
40
20
0
Con
trac
tion
()
log[CaCl2] (M)
Figure 5 Effect of VPR on CaCl2-induced contraction in
endothelium-denuded mesenteric artery rings Concentration-response curves for CaCl
2were determined in Ca2+-free solution
containing KCl (60mM) The curves were determined in theabsence (control) and after incubation with VPR (300120583gmL)Values are expressed as mean plusmn SEM 119899 = 4
endothelium-independent relaxation induced by VPR inmesenteric artery rings
The literature reports that VSMC contraction inducedby high extracellular K+ concentration is mediated by anincrease in membrane depolarization and consequently anincrease in Ca2+ influx through voltage-dependent calciumchannel (CaV) [16] Furthermore the sustained contractiongenerated by a high external K+ concentration may also bemediated by Ca2+ release from the sarcoplasmic reticulum[17] resulting in Ca2+ entry through store-operated channels(SOC) andor transient receptor potential (TRP) channels[18]
Endothelium removed mesenteric rings were precon-tractedwith a depolarizing K+ solution (KCl 80mM) in orderto observe VPR responses before nonspecific generated con-tractions and averting activation of membrane receptors Inthe presence of this depolarizing solution the concentration-response curve induced by VPR was significantly shifted tothe right when compared to the response of VPR in thepresence of Phe changing both the efficacy and potency ofVPR
Based on these preliminary results we speculate thatthis VPR-mediated vasorelaxant effect requires activation ofpotassium channels (K+ channels) in VSMCsThe opening ofthese channels in VSMCs results in an increase in K+ perme-ability leading to K+ efflux The exit of K+ from the VSMCinduces membrane hyperpolarization and consequently the
closing of voltage-operated Ca2+ channels leading to vasore-laxation [19 20] Membrane potential regulation in VSMCsis an important factor in the maintenance of vascular toneAdditionally K+ channels are effector proteins that contributeto membrane potential regulation in electrically excitablecells such as VSMCs Therefore drugs that activate thesechannels could be beneficial in hypertension treatment [21]
Thus to evaluate the involvement of K+ channels inVPR-mediated vasorelaxant response experiments were per-formed in endothelium-denuded arterial rings incubatedwith KCl 20mM This procedure partially decreases mem-brane efflux of K+ due to the increase in extracellular K+concentration from 4mM to 20mM resulting in a reductionin the electrochemical gradient [22] Under these conditionsthe maximum response to VPR was significantly attenuatedwhich was also observed when rings were exposed to KCl80mM suggesting the participation ofK+ channels in vasore-laxation induced by VPR
To confirm the involvement of K+ channels the prepa-rations were pretreated with TEA (3mM) a nonselective K+channel blocker [23] In the presence of this pharmacolog-ical tool the vasorelaxant response induced by increasingconcentrations of VPR was significantly attenuated corrob-orating our previous observation that VPR appears to havea significant effect on the open probability of K+ channels inmesenteric artery rings
Several types of K+ channels are expressed in membraneof VSMCs and each of these channels plays an importantrole in the control and maintenance of the contractile toneof the arterial muscle [24] In VSMCs the variety of K+ chan-nels subtypes identified includes voltage-dependent K+ (KV)channels whose activity is increased upon membrane depo-larization and are important regulators of vascular smoothmuscle membrane potential in response to a depolarizingstimulus [25] large-conductance Ca2+-activated K+ (BKCa)channels which respond to changes in the concentration ofintracellular Ca2+ regulating membrane potential and havean important role in control of myogenic tone in smallerresistance arteries [26] ATP-sensitive K+ (KATP) channelswhich respond actively to changes in cellular metabolism andare targets of a variety of relaxing stimuli [27] and inwardrectifier K+ (KIR) channels regulatingmembrane potential ofvarious types of resistance vessels of small diameter [28]
Thus to investigate the possible participation of thesedifferent K+ channels subtypes experimental preparationswere separately preincubated with different selective blockersof these channels In the presence of a KATP channels blockerGlib (10 120583M) or a KV channels blocker 4-AP (1mM) themaximum effect of VPRwas not changedThe same responseoccurred in rings preincubated with BaCl
2(30 120583M) Kir
channels blocker suggesting that KATP KV and Kir channelsdo not participate in VPR-mediated vasorelaxation
The KCa channels are divided into a subfamily of small(SKCa) intermediate (IKCa) and large (BKCa) conductanceThe BKCa channels are expressed in vascular tissues with ahigh density they are a major component that mediates thedegree of depolarization and contraction in vascular smoothmuscle they are preferentially expressed in vascular smooth
8 Evidence-Based Complementary and Alternative Medicine
150
100
50
Art
eria
l pre
ssur
e (m
mH
g)
2000 3000 4000 5000 6000 7000 8000
Time (s)
VPR(20mgkg)
(a)
0
minus10
minus20
minus30
minus40
ΔM
AP
(mm
Hg)
(b)
100
80
60
40
20
0
1 5 10 20
VPR (mgkg)
ΔH
R (b
pm)
(c)
Figure 6 Effect of VPR on MAP and HR in nonanesthetized rats (a) Representative tracing showed the hypotension induced by additionof VPR (20mgkg) (b) Changes in mean arterial pressure (MAP mmHg) (c) Changes in heart rate (HR bpm) induced by the acuteadministration of increasing doses of VPR (mgkg iv) Values are expressed by mean plusmn SEM 119899 = 5
muscle while the SKCa and IKCa are expressed in a varietyof cell types such as secretory epithelial cells fibroblastsT lymphocytes melanoma cells granulocytes macrophageserythrocytes cultured cell lines and endothelial cells [29 30]BKCa channels act by inhibiting the increase of intracellu-lar Ca2+ Furthermore several recent findings support theimportance of BKCa channels in the regulation of vascularsmooth muscle tone and regulation of blood pressure [31]
The data shows that VPR-mediated vasorelaxation atthe highest concentration was significantly attenuated inrings preincubated with TEA (1mM) which preferentiallyblocks BKCa channels at concentrations lower than 1mM[32] These results suggest that BKCa channels mediate VPR-induced vasorelaxation demonstrating that in addition to theneuronal actions VPR exhibits important peripheral vasculareffects as well
Therefore in vivo experiments were performed to eval-uate VPR effects on cardiovascular parameters in nonanes-thetized normotensive rats in order to avoid the influencesof anesthesia and postsurgical stress In these animals VPRinduced a hypotensive response associated with tachycardia
The hypotensive effect caused by VPR may be due todecreased peripheral vascular resistance and the tachycardiamay be a reflection of the decrease in blood pressure
Taken together these results demonstrate that VPRinduce vasodilatation probably involving K+ channel acti-vation most likely through BKCa channels in smoothmuscle inner from rat superior mesenteric artery Fur-thermore endothelium-derived relaxing factors (NO andprostanoids) are involved in endothelium relaxation depen-dent on VPR
K+ channelKATP ATP-sensitive K+ channelKir Inward rectifier K+ channelKV Voltage-dependent K+ channelMAP Mean arterial pressureMR Maximum responseNO Nitric oxidePAF Platelet-activating factorPhe PhenylephrineK+ channel Potassium channelSEM Standard error of meanSKCa Small-conductance calcium-activated K+
channelSOC Store operated channelsTEA TetraethylammoniumTRP Transient receptor potentialVPR Roots from the Valeriana prionophyllaVPF Leaves from the Valeriana prionophyllaVSMC Vascular smooth muscle cells
Conflict of Interests
The authors declare that there is no conflict of interests
Acknowledgments
This work was supported by grants from Conselho Nacionalde Desenvolvimento Cientıfico e Tecnologico (CNPq)Coordenacao de Aperfeicoamento de Pessoal de Nıvel Supe-rior (CAPES) Consejo Nacional de Ciencia y Tecnologıa deGuatemala (FODECYT 102-2006) and Fundacao de Amparoa Pesquisa do Estado da Bahia (FAPESB)
References
[1] E A Carlini ldquoPlants and the central nervous systemrdquo Phar-macology Biochemistry and Behavior vol 75 no 3 pp 501ndash5122003
[2] V Schulz H Rudolf and V E Tyler Rational Phytotherapy APhysicisnrsquos Guide to Herbal Medicine 2004
[3] I Holzmann V Cechinel-Filho T C Mora et al ldquoEvaluationof behavioral and pharmacological effects of hydroalcoholicextract of Valeriana prionophylla Standl from GuatemalardquoEvidence-Based Complementary and Alternative Medicine vol2011 Article ID 312320 9 pages 2011
[4] D L Nash and J V A Dieterle ldquoFlora of guatemalardquo FieldianaBotany vol 24 no 11 p 297 1976
[5] S Chavadej H Becker and F Weberling ldquoFurther investi-gations of valepotriates in the Valerianaceaerdquo PharmaceutischWeekblad vol 7 no 4 pp 167ndash168 1985
[6] A L Piccinelli S Arana A Caceres R D Di Villa BiancaR Sorrentino and L Rastrelli ldquoNew lignans from the rootsof Valeriana prionophylla with antioxidative and vasorelaxant
activitiesrdquo Journal of Natural Products vol 67 no 7 pp 1135ndash1140 2004
[7] G M Massanet E Pando F Rodriguez-Luis and E ZubialdquoLignans a reviewrdquo Fitoterapia vol 60 no 1 pp 3ndash35 1989
[8] E LGhisalberti ldquoCardiovascular activity of naturally occurringlignansrdquo Phytomedicine vol 4 no 2 pp 151ndash166 1997
[9] A M Gurney ldquoMechanisms of drug-induced vasodilationrdquoJournal of Pharmacy and Pharmacology vol 46 no 4 pp 242ndash251 1994
[10] D F Silva I G A Araujo J G F Albuquerque et alldquoRotundifolone-induced relaxation is mediated by BKCa chan-nel activation and Ca
119907channel inactivationrdquo Basic and Clinical
Pharmacology and Toxicology vol 109 no 6 pp 465ndash475 2011[11] B M Altura and B T Altura ldquoDifferential effects of substrate
depletion on drug-induced contractions of rabbit aortardquo TheAmerican Journal of Physiology vol 219 no 6 pp 1698ndash17051970
[12] W-J Zang C W Balke and W G Wier ldquoGraded 1205721-adrenoceptor activation of arteries involves recruitment ofsmooth muscle cells to produce ldquoall or nonerdquo Ca2+ signalsrdquo CellCalcium vol 29 no 5 pp 327ndash334 2001
[13] R F Furchgott and J V Zawadzki ldquoThe obligatory role ofendothelial cells in the relaxation of arterial smooth muscle byacetylcholinerdquo Nature vol 288 no 5789 pp 373ndash376 1980
[14] M Feletou R Kohler and P M Vanhoutte ldquoNitric oxideorchestrator of endothelium-dependent responsesrdquo Annals ofMedicine vol 44 no 7 pp 694ndash716 2012
[15] Y Tanaka Y Mochizuki H Tanaka and K Shigenobu ldquoSig-nificant role of neuronal non-N-type calcium channels in thesympathetic neurogenic contraction of rat mesenteric arteryrdquoBritish Journal of Pharmacology vol 128 no 7 pp 1602ndash16081999
[16] T Godfraind and A Kaba ldquoBlockade or reversal of the contrac-tion induced by calcium and adrenaline in depolarized arterialsmooth musclerdquo British Journal of Pharmacology vol 36 no 3pp 549ndash560 1969
[17] S Kobayashi H Kanaide and M Nakamura ldquoK+-depolarization induces a direct release of Ca2+ fromintracellular storage sites in cultured vascular smooth musclecells from rat aortardquo Biochemical and Biophysical ResearchCommunications vol 129 no 3 pp 877ndash884 1985
[18] B Ay Y S Prakash C M Pabelick and G C Sieck ldquoStore-operated Ca2+ entry in porcine airway smooth musclerdquo Ameri-can Journal of Physiology vol 286 no 5 pp L909ndashL917 2004
[19] G M Dick and J D Tune ldquoRole of potassium channels incoronary vasodilationrdquo Experimental Biology andMedicine vol235 no 1 pp 10ndash22 2010
[20] EAKo JHan I D Jung andW S Park ldquoPhysiological roles ofK+ channels in vascular smoothmuscle cellsrdquo Journal of SmoothMuscle Research vol 44 no 2 pp 65ndash81 2008
[21] K Lawson ldquoPotassiumchannel openers as potential therapeuticweapons in ion channel diseaserdquo Kidney International vol 57no 3 pp 838ndash845 2000
[22] D X P Brochet and P D Langton ldquoDual effect of initial [K]on vascular tone in rat mesenteric arteriesrdquo Pflugers ArchivEuropean Journal of Physiology vol 453 no 1 pp 33ndash41 2006
[23] F Jiang C G Li and M J Rand ldquoRole of potassium channelsin the nitrergic nerve stimulation-induced vasodilatation in theguinea-pig isolated basilar arteryrdquo British Journal of Pharmacol-ogy vol 123 no 1 pp 106ndash112 1998
10 Evidence-Based Complementary and Alternative Medicine
[24] N B Standen and J M Quayle ldquoK+ channel modulation inarterial smooth musclerdquo Acta Physiologica Scandinavica vol164 no 4 pp 549ndash557 1998
[25] E A Ko W S Park A L Firth N Kim J X-J Yuan and JHan ldquoPathophysiology of voltage-gatedK+ channels in vascularsmooth muscle cells modulation by protein kinasesrdquo Progressin Biophysics and Molecular Biology vol 103 no 1 pp 95ndash1012010
[26] F Sun E Hayama Y Katsube R Matsuoka and T NakanishildquoThe role of the large-conductance voltage-dependent andcalcium-activated potassium (BKCa) channels in the regulationof rat ductus arteriosus tonerdquo Heart and Vessels vol 25 no 6pp 556ndash564 2010
[27] M T Nelson and J M Quayle ldquoPhysiological roles andproperties of potassium channels in arterial smooth musclerdquoAmerican Journal of Physiology vol 268 no 4 pp C799ndashC8221995
[28] T Karkanis S Li J G Pickering and S M Sims ldquoPlasticityof KIR channels in human smooth muscle cells from internalthoracic arteryrdquo American Journal of Physiology vol 284 no 6pp H2325ndashH2334 2003
[29] A Schwab ldquoFunction and spatial distribution of ion channelsand transporters in cell migrationrdquo American Journal of Physi-ology vol 280 no 5 pp F739ndashF747 2001
[30] H Ouadid-Ahidouch M Roudbaraki P Delcourt A Ahi-douchN Joury andN Prevarskaya ldquoFunctional andmolecularidentification of intermediate-conductance Ca2+-activated K+channels in breast cancer cells association with cell cycleprogressionrdquo American Journal of Physiology vol 287 no 1 ppC125ndashC134 2004
[31] J Ledoux M E Werner J E Brayden and M T NelsonldquoCalcium-activated potassium channels and the regulation ofvascular tonerdquo Physiology vol 21 no 1 pp 69ndash78 2006
[32] PD LangtonMTNelson YHuang andN B Standen ldquoBlockof calcium-activated potassium channels inmammalian arterialmyocytes by tetraethylammonium ionsrdquo American Journal ofPhysiology vol 260 no 3 pp H927ndashH934 1991
6 Evidence-Based Complementary and Alternative Medicine
20
40
60
80
minus2 minus1 0 1 2 3
Rela
xatio
n (
)
0
PhePhe + KCl (20mM)Phe + TEA (3mM)
log[VPR] (120583gmL)
(a)
20
40
60
80
minus2 minus1 0 1 2 3Re
laxa
tion
()
0
PhePhe + GlibPhe + 4-AP
log[VPR] (120583gmL)
(b)
PheTEA (1mM)BaCl2
20
40
60
80
minus2 minus1 0 1 2 3
Rela
xatio
n (
)
0
log[VPR] (120583gmL)
(c)
Figure 4 Participation of K+ channels in the vasorelaxation induced by VPR in mesenteric artery rings Concentration-response curvesshowing the relaxant effect of VPR (001ndash300 120583gmL cumulatively) in rings denuded endothelium precontracted with Phe (1120583M) in theabsence or in the presence of (a) KCl 20mM or TEA (3mM) (b) 4-AP (1mM) or Glib (10120583M) (c) TEA (1mM) or BaCl
2(30120583M) Values are
expressed as mean plusmn SEM
Evidence-Based Complementary and Alternative Medicine 7
ControlVPR (300120583gmL)
minus6 minus5 minus4 minus3 minus2 minus1
120
100
80
60
40
20
0
Con
trac
tion
()
log[CaCl2] (M)
Figure 5 Effect of VPR on CaCl2-induced contraction in
endothelium-denuded mesenteric artery rings Concentration-response curves for CaCl
2were determined in Ca2+-free solution
containing KCl (60mM) The curves were determined in theabsence (control) and after incubation with VPR (300120583gmL)Values are expressed as mean plusmn SEM 119899 = 4
endothelium-independent relaxation induced by VPR inmesenteric artery rings
The literature reports that VSMC contraction inducedby high extracellular K+ concentration is mediated by anincrease in membrane depolarization and consequently anincrease in Ca2+ influx through voltage-dependent calciumchannel (CaV) [16] Furthermore the sustained contractiongenerated by a high external K+ concentration may also bemediated by Ca2+ release from the sarcoplasmic reticulum[17] resulting in Ca2+ entry through store-operated channels(SOC) andor transient receptor potential (TRP) channels[18]
Endothelium removed mesenteric rings were precon-tractedwith a depolarizing K+ solution (KCl 80mM) in orderto observe VPR responses before nonspecific generated con-tractions and averting activation of membrane receptors Inthe presence of this depolarizing solution the concentration-response curve induced by VPR was significantly shifted tothe right when compared to the response of VPR in thepresence of Phe changing both the efficacy and potency ofVPR
Based on these preliminary results we speculate thatthis VPR-mediated vasorelaxant effect requires activation ofpotassium channels (K+ channels) in VSMCsThe opening ofthese channels in VSMCs results in an increase in K+ perme-ability leading to K+ efflux The exit of K+ from the VSMCinduces membrane hyperpolarization and consequently the
closing of voltage-operated Ca2+ channels leading to vasore-laxation [19 20] Membrane potential regulation in VSMCsis an important factor in the maintenance of vascular toneAdditionally K+ channels are effector proteins that contributeto membrane potential regulation in electrically excitablecells such as VSMCs Therefore drugs that activate thesechannels could be beneficial in hypertension treatment [21]
Thus to evaluate the involvement of K+ channels inVPR-mediated vasorelaxant response experiments were per-formed in endothelium-denuded arterial rings incubatedwith KCl 20mM This procedure partially decreases mem-brane efflux of K+ due to the increase in extracellular K+concentration from 4mM to 20mM resulting in a reductionin the electrochemical gradient [22] Under these conditionsthe maximum response to VPR was significantly attenuatedwhich was also observed when rings were exposed to KCl80mM suggesting the participation ofK+ channels in vasore-laxation induced by VPR
To confirm the involvement of K+ channels the prepa-rations were pretreated with TEA (3mM) a nonselective K+channel blocker [23] In the presence of this pharmacolog-ical tool the vasorelaxant response induced by increasingconcentrations of VPR was significantly attenuated corrob-orating our previous observation that VPR appears to havea significant effect on the open probability of K+ channels inmesenteric artery rings
Several types of K+ channels are expressed in membraneof VSMCs and each of these channels plays an importantrole in the control and maintenance of the contractile toneof the arterial muscle [24] In VSMCs the variety of K+ chan-nels subtypes identified includes voltage-dependent K+ (KV)channels whose activity is increased upon membrane depo-larization and are important regulators of vascular smoothmuscle membrane potential in response to a depolarizingstimulus [25] large-conductance Ca2+-activated K+ (BKCa)channels which respond to changes in the concentration ofintracellular Ca2+ regulating membrane potential and havean important role in control of myogenic tone in smallerresistance arteries [26] ATP-sensitive K+ (KATP) channelswhich respond actively to changes in cellular metabolism andare targets of a variety of relaxing stimuli [27] and inwardrectifier K+ (KIR) channels regulatingmembrane potential ofvarious types of resistance vessels of small diameter [28]
Thus to investigate the possible participation of thesedifferent K+ channels subtypes experimental preparationswere separately preincubated with different selective blockersof these channels In the presence of a KATP channels blockerGlib (10 120583M) or a KV channels blocker 4-AP (1mM) themaximum effect of VPRwas not changedThe same responseoccurred in rings preincubated with BaCl
2(30 120583M) Kir
channels blocker suggesting that KATP KV and Kir channelsdo not participate in VPR-mediated vasorelaxation
The KCa channels are divided into a subfamily of small(SKCa) intermediate (IKCa) and large (BKCa) conductanceThe BKCa channels are expressed in vascular tissues with ahigh density they are a major component that mediates thedegree of depolarization and contraction in vascular smoothmuscle they are preferentially expressed in vascular smooth
8 Evidence-Based Complementary and Alternative Medicine
150
100
50
Art
eria
l pre
ssur
e (m
mH
g)
2000 3000 4000 5000 6000 7000 8000
Time (s)
VPR(20mgkg)
(a)
0
minus10
minus20
minus30
minus40
ΔM
AP
(mm
Hg)
(b)
100
80
60
40
20
0
1 5 10 20
VPR (mgkg)
ΔH
R (b
pm)
(c)
Figure 6 Effect of VPR on MAP and HR in nonanesthetized rats (a) Representative tracing showed the hypotension induced by additionof VPR (20mgkg) (b) Changes in mean arterial pressure (MAP mmHg) (c) Changes in heart rate (HR bpm) induced by the acuteadministration of increasing doses of VPR (mgkg iv) Values are expressed by mean plusmn SEM 119899 = 5
muscle while the SKCa and IKCa are expressed in a varietyof cell types such as secretory epithelial cells fibroblastsT lymphocytes melanoma cells granulocytes macrophageserythrocytes cultured cell lines and endothelial cells [29 30]BKCa channels act by inhibiting the increase of intracellu-lar Ca2+ Furthermore several recent findings support theimportance of BKCa channels in the regulation of vascularsmooth muscle tone and regulation of blood pressure [31]
The data shows that VPR-mediated vasorelaxation atthe highest concentration was significantly attenuated inrings preincubated with TEA (1mM) which preferentiallyblocks BKCa channels at concentrations lower than 1mM[32] These results suggest that BKCa channels mediate VPR-induced vasorelaxation demonstrating that in addition to theneuronal actions VPR exhibits important peripheral vasculareffects as well
Therefore in vivo experiments were performed to eval-uate VPR effects on cardiovascular parameters in nonanes-thetized normotensive rats in order to avoid the influencesof anesthesia and postsurgical stress In these animals VPRinduced a hypotensive response associated with tachycardia
The hypotensive effect caused by VPR may be due todecreased peripheral vascular resistance and the tachycardiamay be a reflection of the decrease in blood pressure
Taken together these results demonstrate that VPRinduce vasodilatation probably involving K+ channel acti-vation most likely through BKCa channels in smoothmuscle inner from rat superior mesenteric artery Fur-thermore endothelium-derived relaxing factors (NO andprostanoids) are involved in endothelium relaxation depen-dent on VPR
K+ channelKATP ATP-sensitive K+ channelKir Inward rectifier K+ channelKV Voltage-dependent K+ channelMAP Mean arterial pressureMR Maximum responseNO Nitric oxidePAF Platelet-activating factorPhe PhenylephrineK+ channel Potassium channelSEM Standard error of meanSKCa Small-conductance calcium-activated K+
channelSOC Store operated channelsTEA TetraethylammoniumTRP Transient receptor potentialVPR Roots from the Valeriana prionophyllaVPF Leaves from the Valeriana prionophyllaVSMC Vascular smooth muscle cells
Conflict of Interests
The authors declare that there is no conflict of interests
Acknowledgments
This work was supported by grants from Conselho Nacionalde Desenvolvimento Cientıfico e Tecnologico (CNPq)Coordenacao de Aperfeicoamento de Pessoal de Nıvel Supe-rior (CAPES) Consejo Nacional de Ciencia y Tecnologıa deGuatemala (FODECYT 102-2006) and Fundacao de Amparoa Pesquisa do Estado da Bahia (FAPESB)
References
[1] E A Carlini ldquoPlants and the central nervous systemrdquo Phar-macology Biochemistry and Behavior vol 75 no 3 pp 501ndash5122003
[2] V Schulz H Rudolf and V E Tyler Rational Phytotherapy APhysicisnrsquos Guide to Herbal Medicine 2004
[3] I Holzmann V Cechinel-Filho T C Mora et al ldquoEvaluationof behavioral and pharmacological effects of hydroalcoholicextract of Valeriana prionophylla Standl from GuatemalardquoEvidence-Based Complementary and Alternative Medicine vol2011 Article ID 312320 9 pages 2011
[4] D L Nash and J V A Dieterle ldquoFlora of guatemalardquo FieldianaBotany vol 24 no 11 p 297 1976
[5] S Chavadej H Becker and F Weberling ldquoFurther investi-gations of valepotriates in the Valerianaceaerdquo PharmaceutischWeekblad vol 7 no 4 pp 167ndash168 1985
[6] A L Piccinelli S Arana A Caceres R D Di Villa BiancaR Sorrentino and L Rastrelli ldquoNew lignans from the rootsof Valeriana prionophylla with antioxidative and vasorelaxant
activitiesrdquo Journal of Natural Products vol 67 no 7 pp 1135ndash1140 2004
[7] G M Massanet E Pando F Rodriguez-Luis and E ZubialdquoLignans a reviewrdquo Fitoterapia vol 60 no 1 pp 3ndash35 1989
[8] E LGhisalberti ldquoCardiovascular activity of naturally occurringlignansrdquo Phytomedicine vol 4 no 2 pp 151ndash166 1997
[9] A M Gurney ldquoMechanisms of drug-induced vasodilationrdquoJournal of Pharmacy and Pharmacology vol 46 no 4 pp 242ndash251 1994
[10] D F Silva I G A Araujo J G F Albuquerque et alldquoRotundifolone-induced relaxation is mediated by BKCa chan-nel activation and Ca
119907channel inactivationrdquo Basic and Clinical
Pharmacology and Toxicology vol 109 no 6 pp 465ndash475 2011[11] B M Altura and B T Altura ldquoDifferential effects of substrate
depletion on drug-induced contractions of rabbit aortardquo TheAmerican Journal of Physiology vol 219 no 6 pp 1698ndash17051970
[12] W-J Zang C W Balke and W G Wier ldquoGraded 1205721-adrenoceptor activation of arteries involves recruitment ofsmooth muscle cells to produce ldquoall or nonerdquo Ca2+ signalsrdquo CellCalcium vol 29 no 5 pp 327ndash334 2001
[13] R F Furchgott and J V Zawadzki ldquoThe obligatory role ofendothelial cells in the relaxation of arterial smooth muscle byacetylcholinerdquo Nature vol 288 no 5789 pp 373ndash376 1980
[14] M Feletou R Kohler and P M Vanhoutte ldquoNitric oxideorchestrator of endothelium-dependent responsesrdquo Annals ofMedicine vol 44 no 7 pp 694ndash716 2012
[15] Y Tanaka Y Mochizuki H Tanaka and K Shigenobu ldquoSig-nificant role of neuronal non-N-type calcium channels in thesympathetic neurogenic contraction of rat mesenteric arteryrdquoBritish Journal of Pharmacology vol 128 no 7 pp 1602ndash16081999
[16] T Godfraind and A Kaba ldquoBlockade or reversal of the contrac-tion induced by calcium and adrenaline in depolarized arterialsmooth musclerdquo British Journal of Pharmacology vol 36 no 3pp 549ndash560 1969
[17] S Kobayashi H Kanaide and M Nakamura ldquoK+-depolarization induces a direct release of Ca2+ fromintracellular storage sites in cultured vascular smooth musclecells from rat aortardquo Biochemical and Biophysical ResearchCommunications vol 129 no 3 pp 877ndash884 1985
[18] B Ay Y S Prakash C M Pabelick and G C Sieck ldquoStore-operated Ca2+ entry in porcine airway smooth musclerdquo Ameri-can Journal of Physiology vol 286 no 5 pp L909ndashL917 2004
[19] G M Dick and J D Tune ldquoRole of potassium channels incoronary vasodilationrdquo Experimental Biology andMedicine vol235 no 1 pp 10ndash22 2010
[20] EAKo JHan I D Jung andW S Park ldquoPhysiological roles ofK+ channels in vascular smoothmuscle cellsrdquo Journal of SmoothMuscle Research vol 44 no 2 pp 65ndash81 2008
[21] K Lawson ldquoPotassiumchannel openers as potential therapeuticweapons in ion channel diseaserdquo Kidney International vol 57no 3 pp 838ndash845 2000
[22] D X P Brochet and P D Langton ldquoDual effect of initial [K]on vascular tone in rat mesenteric arteriesrdquo Pflugers ArchivEuropean Journal of Physiology vol 453 no 1 pp 33ndash41 2006
[23] F Jiang C G Li and M J Rand ldquoRole of potassium channelsin the nitrergic nerve stimulation-induced vasodilatation in theguinea-pig isolated basilar arteryrdquo British Journal of Pharmacol-ogy vol 123 no 1 pp 106ndash112 1998
10 Evidence-Based Complementary and Alternative Medicine
[24] N B Standen and J M Quayle ldquoK+ channel modulation inarterial smooth musclerdquo Acta Physiologica Scandinavica vol164 no 4 pp 549ndash557 1998
[25] E A Ko W S Park A L Firth N Kim J X-J Yuan and JHan ldquoPathophysiology of voltage-gatedK+ channels in vascularsmooth muscle cells modulation by protein kinasesrdquo Progressin Biophysics and Molecular Biology vol 103 no 1 pp 95ndash1012010
[26] F Sun E Hayama Y Katsube R Matsuoka and T NakanishildquoThe role of the large-conductance voltage-dependent andcalcium-activated potassium (BKCa) channels in the regulationof rat ductus arteriosus tonerdquo Heart and Vessels vol 25 no 6pp 556ndash564 2010
[27] M T Nelson and J M Quayle ldquoPhysiological roles andproperties of potassium channels in arterial smooth musclerdquoAmerican Journal of Physiology vol 268 no 4 pp C799ndashC8221995
[28] T Karkanis S Li J G Pickering and S M Sims ldquoPlasticityof KIR channels in human smooth muscle cells from internalthoracic arteryrdquo American Journal of Physiology vol 284 no 6pp H2325ndashH2334 2003
[29] A Schwab ldquoFunction and spatial distribution of ion channelsand transporters in cell migrationrdquo American Journal of Physi-ology vol 280 no 5 pp F739ndashF747 2001
[30] H Ouadid-Ahidouch M Roudbaraki P Delcourt A Ahi-douchN Joury andN Prevarskaya ldquoFunctional andmolecularidentification of intermediate-conductance Ca2+-activated K+channels in breast cancer cells association with cell cycleprogressionrdquo American Journal of Physiology vol 287 no 1 ppC125ndashC134 2004
[31] J Ledoux M E Werner J E Brayden and M T NelsonldquoCalcium-activated potassium channels and the regulation ofvascular tonerdquo Physiology vol 21 no 1 pp 69ndash78 2006
[32] PD LangtonMTNelson YHuang andN B Standen ldquoBlockof calcium-activated potassium channels inmammalian arterialmyocytes by tetraethylammonium ionsrdquo American Journal ofPhysiology vol 260 no 3 pp H927ndashH934 1991
Evidence-Based Complementary and Alternative Medicine 7
ControlVPR (300120583gmL)
minus6 minus5 minus4 minus3 minus2 minus1
120
100
80
60
40
20
0
Con
trac
tion
()
log[CaCl2] (M)
Figure 5 Effect of VPR on CaCl2-induced contraction in
endothelium-denuded mesenteric artery rings Concentration-response curves for CaCl
2were determined in Ca2+-free solution
containing KCl (60mM) The curves were determined in theabsence (control) and after incubation with VPR (300120583gmL)Values are expressed as mean plusmn SEM 119899 = 4
endothelium-independent relaxation induced by VPR inmesenteric artery rings
The literature reports that VSMC contraction inducedby high extracellular K+ concentration is mediated by anincrease in membrane depolarization and consequently anincrease in Ca2+ influx through voltage-dependent calciumchannel (CaV) [16] Furthermore the sustained contractiongenerated by a high external K+ concentration may also bemediated by Ca2+ release from the sarcoplasmic reticulum[17] resulting in Ca2+ entry through store-operated channels(SOC) andor transient receptor potential (TRP) channels[18]
Endothelium removed mesenteric rings were precon-tractedwith a depolarizing K+ solution (KCl 80mM) in orderto observe VPR responses before nonspecific generated con-tractions and averting activation of membrane receptors Inthe presence of this depolarizing solution the concentration-response curve induced by VPR was significantly shifted tothe right when compared to the response of VPR in thepresence of Phe changing both the efficacy and potency ofVPR
Based on these preliminary results we speculate thatthis VPR-mediated vasorelaxant effect requires activation ofpotassium channels (K+ channels) in VSMCsThe opening ofthese channels in VSMCs results in an increase in K+ perme-ability leading to K+ efflux The exit of K+ from the VSMCinduces membrane hyperpolarization and consequently the
closing of voltage-operated Ca2+ channels leading to vasore-laxation [19 20] Membrane potential regulation in VSMCsis an important factor in the maintenance of vascular toneAdditionally K+ channels are effector proteins that contributeto membrane potential regulation in electrically excitablecells such as VSMCs Therefore drugs that activate thesechannels could be beneficial in hypertension treatment [21]
Thus to evaluate the involvement of K+ channels inVPR-mediated vasorelaxant response experiments were per-formed in endothelium-denuded arterial rings incubatedwith KCl 20mM This procedure partially decreases mem-brane efflux of K+ due to the increase in extracellular K+concentration from 4mM to 20mM resulting in a reductionin the electrochemical gradient [22] Under these conditionsthe maximum response to VPR was significantly attenuatedwhich was also observed when rings were exposed to KCl80mM suggesting the participation ofK+ channels in vasore-laxation induced by VPR
To confirm the involvement of K+ channels the prepa-rations were pretreated with TEA (3mM) a nonselective K+channel blocker [23] In the presence of this pharmacolog-ical tool the vasorelaxant response induced by increasingconcentrations of VPR was significantly attenuated corrob-orating our previous observation that VPR appears to havea significant effect on the open probability of K+ channels inmesenteric artery rings
Several types of K+ channels are expressed in membraneof VSMCs and each of these channels plays an importantrole in the control and maintenance of the contractile toneof the arterial muscle [24] In VSMCs the variety of K+ chan-nels subtypes identified includes voltage-dependent K+ (KV)channels whose activity is increased upon membrane depo-larization and are important regulators of vascular smoothmuscle membrane potential in response to a depolarizingstimulus [25] large-conductance Ca2+-activated K+ (BKCa)channels which respond to changes in the concentration ofintracellular Ca2+ regulating membrane potential and havean important role in control of myogenic tone in smallerresistance arteries [26] ATP-sensitive K+ (KATP) channelswhich respond actively to changes in cellular metabolism andare targets of a variety of relaxing stimuli [27] and inwardrectifier K+ (KIR) channels regulatingmembrane potential ofvarious types of resistance vessels of small diameter [28]
Thus to investigate the possible participation of thesedifferent K+ channels subtypes experimental preparationswere separately preincubated with different selective blockersof these channels In the presence of a KATP channels blockerGlib (10 120583M) or a KV channels blocker 4-AP (1mM) themaximum effect of VPRwas not changedThe same responseoccurred in rings preincubated with BaCl
2(30 120583M) Kir
channels blocker suggesting that KATP KV and Kir channelsdo not participate in VPR-mediated vasorelaxation
The KCa channels are divided into a subfamily of small(SKCa) intermediate (IKCa) and large (BKCa) conductanceThe BKCa channels are expressed in vascular tissues with ahigh density they are a major component that mediates thedegree of depolarization and contraction in vascular smoothmuscle they are preferentially expressed in vascular smooth
8 Evidence-Based Complementary and Alternative Medicine
150
100
50
Art
eria
l pre
ssur
e (m
mH
g)
2000 3000 4000 5000 6000 7000 8000
Time (s)
VPR(20mgkg)
(a)
0
minus10
minus20
minus30
minus40
ΔM
AP
(mm
Hg)
(b)
100
80
60
40
20
0
1 5 10 20
VPR (mgkg)
ΔH
R (b
pm)
(c)
Figure 6 Effect of VPR on MAP and HR in nonanesthetized rats (a) Representative tracing showed the hypotension induced by additionof VPR (20mgkg) (b) Changes in mean arterial pressure (MAP mmHg) (c) Changes in heart rate (HR bpm) induced by the acuteadministration of increasing doses of VPR (mgkg iv) Values are expressed by mean plusmn SEM 119899 = 5
muscle while the SKCa and IKCa are expressed in a varietyof cell types such as secretory epithelial cells fibroblastsT lymphocytes melanoma cells granulocytes macrophageserythrocytes cultured cell lines and endothelial cells [29 30]BKCa channels act by inhibiting the increase of intracellu-lar Ca2+ Furthermore several recent findings support theimportance of BKCa channels in the regulation of vascularsmooth muscle tone and regulation of blood pressure [31]
The data shows that VPR-mediated vasorelaxation atthe highest concentration was significantly attenuated inrings preincubated with TEA (1mM) which preferentiallyblocks BKCa channels at concentrations lower than 1mM[32] These results suggest that BKCa channels mediate VPR-induced vasorelaxation demonstrating that in addition to theneuronal actions VPR exhibits important peripheral vasculareffects as well
Therefore in vivo experiments were performed to eval-uate VPR effects on cardiovascular parameters in nonanes-thetized normotensive rats in order to avoid the influencesof anesthesia and postsurgical stress In these animals VPRinduced a hypotensive response associated with tachycardia
The hypotensive effect caused by VPR may be due todecreased peripheral vascular resistance and the tachycardiamay be a reflection of the decrease in blood pressure
Taken together these results demonstrate that VPRinduce vasodilatation probably involving K+ channel acti-vation most likely through BKCa channels in smoothmuscle inner from rat superior mesenteric artery Fur-thermore endothelium-derived relaxing factors (NO andprostanoids) are involved in endothelium relaxation depen-dent on VPR
K+ channelKATP ATP-sensitive K+ channelKir Inward rectifier K+ channelKV Voltage-dependent K+ channelMAP Mean arterial pressureMR Maximum responseNO Nitric oxidePAF Platelet-activating factorPhe PhenylephrineK+ channel Potassium channelSEM Standard error of meanSKCa Small-conductance calcium-activated K+
channelSOC Store operated channelsTEA TetraethylammoniumTRP Transient receptor potentialVPR Roots from the Valeriana prionophyllaVPF Leaves from the Valeriana prionophyllaVSMC Vascular smooth muscle cells
Conflict of Interests
The authors declare that there is no conflict of interests
Acknowledgments
This work was supported by grants from Conselho Nacionalde Desenvolvimento Cientıfico e Tecnologico (CNPq)Coordenacao de Aperfeicoamento de Pessoal de Nıvel Supe-rior (CAPES) Consejo Nacional de Ciencia y Tecnologıa deGuatemala (FODECYT 102-2006) and Fundacao de Amparoa Pesquisa do Estado da Bahia (FAPESB)
References
[1] E A Carlini ldquoPlants and the central nervous systemrdquo Phar-macology Biochemistry and Behavior vol 75 no 3 pp 501ndash5122003
[2] V Schulz H Rudolf and V E Tyler Rational Phytotherapy APhysicisnrsquos Guide to Herbal Medicine 2004
[3] I Holzmann V Cechinel-Filho T C Mora et al ldquoEvaluationof behavioral and pharmacological effects of hydroalcoholicextract of Valeriana prionophylla Standl from GuatemalardquoEvidence-Based Complementary and Alternative Medicine vol2011 Article ID 312320 9 pages 2011
[4] D L Nash and J V A Dieterle ldquoFlora of guatemalardquo FieldianaBotany vol 24 no 11 p 297 1976
[5] S Chavadej H Becker and F Weberling ldquoFurther investi-gations of valepotriates in the Valerianaceaerdquo PharmaceutischWeekblad vol 7 no 4 pp 167ndash168 1985
[6] A L Piccinelli S Arana A Caceres R D Di Villa BiancaR Sorrentino and L Rastrelli ldquoNew lignans from the rootsof Valeriana prionophylla with antioxidative and vasorelaxant
activitiesrdquo Journal of Natural Products vol 67 no 7 pp 1135ndash1140 2004
[7] G M Massanet E Pando F Rodriguez-Luis and E ZubialdquoLignans a reviewrdquo Fitoterapia vol 60 no 1 pp 3ndash35 1989
[8] E LGhisalberti ldquoCardiovascular activity of naturally occurringlignansrdquo Phytomedicine vol 4 no 2 pp 151ndash166 1997
[9] A M Gurney ldquoMechanisms of drug-induced vasodilationrdquoJournal of Pharmacy and Pharmacology vol 46 no 4 pp 242ndash251 1994
[10] D F Silva I G A Araujo J G F Albuquerque et alldquoRotundifolone-induced relaxation is mediated by BKCa chan-nel activation and Ca
119907channel inactivationrdquo Basic and Clinical
Pharmacology and Toxicology vol 109 no 6 pp 465ndash475 2011[11] B M Altura and B T Altura ldquoDifferential effects of substrate
depletion on drug-induced contractions of rabbit aortardquo TheAmerican Journal of Physiology vol 219 no 6 pp 1698ndash17051970
[12] W-J Zang C W Balke and W G Wier ldquoGraded 1205721-adrenoceptor activation of arteries involves recruitment ofsmooth muscle cells to produce ldquoall or nonerdquo Ca2+ signalsrdquo CellCalcium vol 29 no 5 pp 327ndash334 2001
[13] R F Furchgott and J V Zawadzki ldquoThe obligatory role ofendothelial cells in the relaxation of arterial smooth muscle byacetylcholinerdquo Nature vol 288 no 5789 pp 373ndash376 1980
[14] M Feletou R Kohler and P M Vanhoutte ldquoNitric oxideorchestrator of endothelium-dependent responsesrdquo Annals ofMedicine vol 44 no 7 pp 694ndash716 2012
[15] Y Tanaka Y Mochizuki H Tanaka and K Shigenobu ldquoSig-nificant role of neuronal non-N-type calcium channels in thesympathetic neurogenic contraction of rat mesenteric arteryrdquoBritish Journal of Pharmacology vol 128 no 7 pp 1602ndash16081999
[16] T Godfraind and A Kaba ldquoBlockade or reversal of the contrac-tion induced by calcium and adrenaline in depolarized arterialsmooth musclerdquo British Journal of Pharmacology vol 36 no 3pp 549ndash560 1969
[17] S Kobayashi H Kanaide and M Nakamura ldquoK+-depolarization induces a direct release of Ca2+ fromintracellular storage sites in cultured vascular smooth musclecells from rat aortardquo Biochemical and Biophysical ResearchCommunications vol 129 no 3 pp 877ndash884 1985
[18] B Ay Y S Prakash C M Pabelick and G C Sieck ldquoStore-operated Ca2+ entry in porcine airway smooth musclerdquo Ameri-can Journal of Physiology vol 286 no 5 pp L909ndashL917 2004
[19] G M Dick and J D Tune ldquoRole of potassium channels incoronary vasodilationrdquo Experimental Biology andMedicine vol235 no 1 pp 10ndash22 2010
[20] EAKo JHan I D Jung andW S Park ldquoPhysiological roles ofK+ channels in vascular smoothmuscle cellsrdquo Journal of SmoothMuscle Research vol 44 no 2 pp 65ndash81 2008
[21] K Lawson ldquoPotassiumchannel openers as potential therapeuticweapons in ion channel diseaserdquo Kidney International vol 57no 3 pp 838ndash845 2000
[22] D X P Brochet and P D Langton ldquoDual effect of initial [K]on vascular tone in rat mesenteric arteriesrdquo Pflugers ArchivEuropean Journal of Physiology vol 453 no 1 pp 33ndash41 2006
[23] F Jiang C G Li and M J Rand ldquoRole of potassium channelsin the nitrergic nerve stimulation-induced vasodilatation in theguinea-pig isolated basilar arteryrdquo British Journal of Pharmacol-ogy vol 123 no 1 pp 106ndash112 1998
10 Evidence-Based Complementary and Alternative Medicine
[24] N B Standen and J M Quayle ldquoK+ channel modulation inarterial smooth musclerdquo Acta Physiologica Scandinavica vol164 no 4 pp 549ndash557 1998
[25] E A Ko W S Park A L Firth N Kim J X-J Yuan and JHan ldquoPathophysiology of voltage-gatedK+ channels in vascularsmooth muscle cells modulation by protein kinasesrdquo Progressin Biophysics and Molecular Biology vol 103 no 1 pp 95ndash1012010
[26] F Sun E Hayama Y Katsube R Matsuoka and T NakanishildquoThe role of the large-conductance voltage-dependent andcalcium-activated potassium (BKCa) channels in the regulationof rat ductus arteriosus tonerdquo Heart and Vessels vol 25 no 6pp 556ndash564 2010
[27] M T Nelson and J M Quayle ldquoPhysiological roles andproperties of potassium channels in arterial smooth musclerdquoAmerican Journal of Physiology vol 268 no 4 pp C799ndashC8221995
[28] T Karkanis S Li J G Pickering and S M Sims ldquoPlasticityof KIR channels in human smooth muscle cells from internalthoracic arteryrdquo American Journal of Physiology vol 284 no 6pp H2325ndashH2334 2003
[29] A Schwab ldquoFunction and spatial distribution of ion channelsand transporters in cell migrationrdquo American Journal of Physi-ology vol 280 no 5 pp F739ndashF747 2001
[30] H Ouadid-Ahidouch M Roudbaraki P Delcourt A Ahi-douchN Joury andN Prevarskaya ldquoFunctional andmolecularidentification of intermediate-conductance Ca2+-activated K+channels in breast cancer cells association with cell cycleprogressionrdquo American Journal of Physiology vol 287 no 1 ppC125ndashC134 2004
[31] J Ledoux M E Werner J E Brayden and M T NelsonldquoCalcium-activated potassium channels and the regulation ofvascular tonerdquo Physiology vol 21 no 1 pp 69ndash78 2006
[32] PD LangtonMTNelson YHuang andN B Standen ldquoBlockof calcium-activated potassium channels inmammalian arterialmyocytes by tetraethylammonium ionsrdquo American Journal ofPhysiology vol 260 no 3 pp H927ndashH934 1991
8 Evidence-Based Complementary and Alternative Medicine
150
100
50
Art
eria
l pre
ssur
e (m
mH
g)
2000 3000 4000 5000 6000 7000 8000
Time (s)
VPR(20mgkg)
(a)
0
minus10
minus20
minus30
minus40
ΔM
AP
(mm
Hg)
(b)
100
80
60
40
20
0
1 5 10 20
VPR (mgkg)
ΔH
R (b
pm)
(c)
Figure 6 Effect of VPR on MAP and HR in nonanesthetized rats (a) Representative tracing showed the hypotension induced by additionof VPR (20mgkg) (b) Changes in mean arterial pressure (MAP mmHg) (c) Changes in heart rate (HR bpm) induced by the acuteadministration of increasing doses of VPR (mgkg iv) Values are expressed by mean plusmn SEM 119899 = 5
muscle while the SKCa and IKCa are expressed in a varietyof cell types such as secretory epithelial cells fibroblastsT lymphocytes melanoma cells granulocytes macrophageserythrocytes cultured cell lines and endothelial cells [29 30]BKCa channels act by inhibiting the increase of intracellu-lar Ca2+ Furthermore several recent findings support theimportance of BKCa channels in the regulation of vascularsmooth muscle tone and regulation of blood pressure [31]
The data shows that VPR-mediated vasorelaxation atthe highest concentration was significantly attenuated inrings preincubated with TEA (1mM) which preferentiallyblocks BKCa channels at concentrations lower than 1mM[32] These results suggest that BKCa channels mediate VPR-induced vasorelaxation demonstrating that in addition to theneuronal actions VPR exhibits important peripheral vasculareffects as well
Therefore in vivo experiments were performed to eval-uate VPR effects on cardiovascular parameters in nonanes-thetized normotensive rats in order to avoid the influencesof anesthesia and postsurgical stress In these animals VPRinduced a hypotensive response associated with tachycardia
The hypotensive effect caused by VPR may be due todecreased peripheral vascular resistance and the tachycardiamay be a reflection of the decrease in blood pressure
Taken together these results demonstrate that VPRinduce vasodilatation probably involving K+ channel acti-vation most likely through BKCa channels in smoothmuscle inner from rat superior mesenteric artery Fur-thermore endothelium-derived relaxing factors (NO andprostanoids) are involved in endothelium relaxation depen-dent on VPR
K+ channelKATP ATP-sensitive K+ channelKir Inward rectifier K+ channelKV Voltage-dependent K+ channelMAP Mean arterial pressureMR Maximum responseNO Nitric oxidePAF Platelet-activating factorPhe PhenylephrineK+ channel Potassium channelSEM Standard error of meanSKCa Small-conductance calcium-activated K+
channelSOC Store operated channelsTEA TetraethylammoniumTRP Transient receptor potentialVPR Roots from the Valeriana prionophyllaVPF Leaves from the Valeriana prionophyllaVSMC Vascular smooth muscle cells
Conflict of Interests
The authors declare that there is no conflict of interests
Acknowledgments
This work was supported by grants from Conselho Nacionalde Desenvolvimento Cientıfico e Tecnologico (CNPq)Coordenacao de Aperfeicoamento de Pessoal de Nıvel Supe-rior (CAPES) Consejo Nacional de Ciencia y Tecnologıa deGuatemala (FODECYT 102-2006) and Fundacao de Amparoa Pesquisa do Estado da Bahia (FAPESB)
References
[1] E A Carlini ldquoPlants and the central nervous systemrdquo Phar-macology Biochemistry and Behavior vol 75 no 3 pp 501ndash5122003
[2] V Schulz H Rudolf and V E Tyler Rational Phytotherapy APhysicisnrsquos Guide to Herbal Medicine 2004
[3] I Holzmann V Cechinel-Filho T C Mora et al ldquoEvaluationof behavioral and pharmacological effects of hydroalcoholicextract of Valeriana prionophylla Standl from GuatemalardquoEvidence-Based Complementary and Alternative Medicine vol2011 Article ID 312320 9 pages 2011
[4] D L Nash and J V A Dieterle ldquoFlora of guatemalardquo FieldianaBotany vol 24 no 11 p 297 1976
[5] S Chavadej H Becker and F Weberling ldquoFurther investi-gations of valepotriates in the Valerianaceaerdquo PharmaceutischWeekblad vol 7 no 4 pp 167ndash168 1985
[6] A L Piccinelli S Arana A Caceres R D Di Villa BiancaR Sorrentino and L Rastrelli ldquoNew lignans from the rootsof Valeriana prionophylla with antioxidative and vasorelaxant
activitiesrdquo Journal of Natural Products vol 67 no 7 pp 1135ndash1140 2004
[7] G M Massanet E Pando F Rodriguez-Luis and E ZubialdquoLignans a reviewrdquo Fitoterapia vol 60 no 1 pp 3ndash35 1989
[8] E LGhisalberti ldquoCardiovascular activity of naturally occurringlignansrdquo Phytomedicine vol 4 no 2 pp 151ndash166 1997
[9] A M Gurney ldquoMechanisms of drug-induced vasodilationrdquoJournal of Pharmacy and Pharmacology vol 46 no 4 pp 242ndash251 1994
[10] D F Silva I G A Araujo J G F Albuquerque et alldquoRotundifolone-induced relaxation is mediated by BKCa chan-nel activation and Ca
119907channel inactivationrdquo Basic and Clinical
Pharmacology and Toxicology vol 109 no 6 pp 465ndash475 2011[11] B M Altura and B T Altura ldquoDifferential effects of substrate
depletion on drug-induced contractions of rabbit aortardquo TheAmerican Journal of Physiology vol 219 no 6 pp 1698ndash17051970
[12] W-J Zang C W Balke and W G Wier ldquoGraded 1205721-adrenoceptor activation of arteries involves recruitment ofsmooth muscle cells to produce ldquoall or nonerdquo Ca2+ signalsrdquo CellCalcium vol 29 no 5 pp 327ndash334 2001
[13] R F Furchgott and J V Zawadzki ldquoThe obligatory role ofendothelial cells in the relaxation of arterial smooth muscle byacetylcholinerdquo Nature vol 288 no 5789 pp 373ndash376 1980
[14] M Feletou R Kohler and P M Vanhoutte ldquoNitric oxideorchestrator of endothelium-dependent responsesrdquo Annals ofMedicine vol 44 no 7 pp 694ndash716 2012
[15] Y Tanaka Y Mochizuki H Tanaka and K Shigenobu ldquoSig-nificant role of neuronal non-N-type calcium channels in thesympathetic neurogenic contraction of rat mesenteric arteryrdquoBritish Journal of Pharmacology vol 128 no 7 pp 1602ndash16081999
[16] T Godfraind and A Kaba ldquoBlockade or reversal of the contrac-tion induced by calcium and adrenaline in depolarized arterialsmooth musclerdquo British Journal of Pharmacology vol 36 no 3pp 549ndash560 1969
[17] S Kobayashi H Kanaide and M Nakamura ldquoK+-depolarization induces a direct release of Ca2+ fromintracellular storage sites in cultured vascular smooth musclecells from rat aortardquo Biochemical and Biophysical ResearchCommunications vol 129 no 3 pp 877ndash884 1985
[18] B Ay Y S Prakash C M Pabelick and G C Sieck ldquoStore-operated Ca2+ entry in porcine airway smooth musclerdquo Ameri-can Journal of Physiology vol 286 no 5 pp L909ndashL917 2004
[19] G M Dick and J D Tune ldquoRole of potassium channels incoronary vasodilationrdquo Experimental Biology andMedicine vol235 no 1 pp 10ndash22 2010
[20] EAKo JHan I D Jung andW S Park ldquoPhysiological roles ofK+ channels in vascular smoothmuscle cellsrdquo Journal of SmoothMuscle Research vol 44 no 2 pp 65ndash81 2008
[21] K Lawson ldquoPotassiumchannel openers as potential therapeuticweapons in ion channel diseaserdquo Kidney International vol 57no 3 pp 838ndash845 2000
[22] D X P Brochet and P D Langton ldquoDual effect of initial [K]on vascular tone in rat mesenteric arteriesrdquo Pflugers ArchivEuropean Journal of Physiology vol 453 no 1 pp 33ndash41 2006
[23] F Jiang C G Li and M J Rand ldquoRole of potassium channelsin the nitrergic nerve stimulation-induced vasodilatation in theguinea-pig isolated basilar arteryrdquo British Journal of Pharmacol-ogy vol 123 no 1 pp 106ndash112 1998
10 Evidence-Based Complementary and Alternative Medicine
[24] N B Standen and J M Quayle ldquoK+ channel modulation inarterial smooth musclerdquo Acta Physiologica Scandinavica vol164 no 4 pp 549ndash557 1998
[25] E A Ko W S Park A L Firth N Kim J X-J Yuan and JHan ldquoPathophysiology of voltage-gatedK+ channels in vascularsmooth muscle cells modulation by protein kinasesrdquo Progressin Biophysics and Molecular Biology vol 103 no 1 pp 95ndash1012010
[26] F Sun E Hayama Y Katsube R Matsuoka and T NakanishildquoThe role of the large-conductance voltage-dependent andcalcium-activated potassium (BKCa) channels in the regulationof rat ductus arteriosus tonerdquo Heart and Vessels vol 25 no 6pp 556ndash564 2010
[27] M T Nelson and J M Quayle ldquoPhysiological roles andproperties of potassium channels in arterial smooth musclerdquoAmerican Journal of Physiology vol 268 no 4 pp C799ndashC8221995
[28] T Karkanis S Li J G Pickering and S M Sims ldquoPlasticityof KIR channels in human smooth muscle cells from internalthoracic arteryrdquo American Journal of Physiology vol 284 no 6pp H2325ndashH2334 2003
[29] A Schwab ldquoFunction and spatial distribution of ion channelsand transporters in cell migrationrdquo American Journal of Physi-ology vol 280 no 5 pp F739ndashF747 2001
[30] H Ouadid-Ahidouch M Roudbaraki P Delcourt A Ahi-douchN Joury andN Prevarskaya ldquoFunctional andmolecularidentification of intermediate-conductance Ca2+-activated K+channels in breast cancer cells association with cell cycleprogressionrdquo American Journal of Physiology vol 287 no 1 ppC125ndashC134 2004
[31] J Ledoux M E Werner J E Brayden and M T NelsonldquoCalcium-activated potassium channels and the regulation ofvascular tonerdquo Physiology vol 21 no 1 pp 69ndash78 2006
[32] PD LangtonMTNelson YHuang andN B Standen ldquoBlockof calcium-activated potassium channels inmammalian arterialmyocytes by tetraethylammonium ionsrdquo American Journal ofPhysiology vol 260 no 3 pp H927ndashH934 1991
K+ channelKATP ATP-sensitive K+ channelKir Inward rectifier K+ channelKV Voltage-dependent K+ channelMAP Mean arterial pressureMR Maximum responseNO Nitric oxidePAF Platelet-activating factorPhe PhenylephrineK+ channel Potassium channelSEM Standard error of meanSKCa Small-conductance calcium-activated K+
channelSOC Store operated channelsTEA TetraethylammoniumTRP Transient receptor potentialVPR Roots from the Valeriana prionophyllaVPF Leaves from the Valeriana prionophyllaVSMC Vascular smooth muscle cells
Conflict of Interests
The authors declare that there is no conflict of interests
Acknowledgments
This work was supported by grants from Conselho Nacionalde Desenvolvimento Cientıfico e Tecnologico (CNPq)Coordenacao de Aperfeicoamento de Pessoal de Nıvel Supe-rior (CAPES) Consejo Nacional de Ciencia y Tecnologıa deGuatemala (FODECYT 102-2006) and Fundacao de Amparoa Pesquisa do Estado da Bahia (FAPESB)
References
[1] E A Carlini ldquoPlants and the central nervous systemrdquo Phar-macology Biochemistry and Behavior vol 75 no 3 pp 501ndash5122003
[2] V Schulz H Rudolf and V E Tyler Rational Phytotherapy APhysicisnrsquos Guide to Herbal Medicine 2004
[3] I Holzmann V Cechinel-Filho T C Mora et al ldquoEvaluationof behavioral and pharmacological effects of hydroalcoholicextract of Valeriana prionophylla Standl from GuatemalardquoEvidence-Based Complementary and Alternative Medicine vol2011 Article ID 312320 9 pages 2011
[4] D L Nash and J V A Dieterle ldquoFlora of guatemalardquo FieldianaBotany vol 24 no 11 p 297 1976
[5] S Chavadej H Becker and F Weberling ldquoFurther investi-gations of valepotriates in the Valerianaceaerdquo PharmaceutischWeekblad vol 7 no 4 pp 167ndash168 1985
[6] A L Piccinelli S Arana A Caceres R D Di Villa BiancaR Sorrentino and L Rastrelli ldquoNew lignans from the rootsof Valeriana prionophylla with antioxidative and vasorelaxant
activitiesrdquo Journal of Natural Products vol 67 no 7 pp 1135ndash1140 2004
[7] G M Massanet E Pando F Rodriguez-Luis and E ZubialdquoLignans a reviewrdquo Fitoterapia vol 60 no 1 pp 3ndash35 1989
[8] E LGhisalberti ldquoCardiovascular activity of naturally occurringlignansrdquo Phytomedicine vol 4 no 2 pp 151ndash166 1997
[9] A M Gurney ldquoMechanisms of drug-induced vasodilationrdquoJournal of Pharmacy and Pharmacology vol 46 no 4 pp 242ndash251 1994
[10] D F Silva I G A Araujo J G F Albuquerque et alldquoRotundifolone-induced relaxation is mediated by BKCa chan-nel activation and Ca
119907channel inactivationrdquo Basic and Clinical
Pharmacology and Toxicology vol 109 no 6 pp 465ndash475 2011[11] B M Altura and B T Altura ldquoDifferential effects of substrate
depletion on drug-induced contractions of rabbit aortardquo TheAmerican Journal of Physiology vol 219 no 6 pp 1698ndash17051970
[12] W-J Zang C W Balke and W G Wier ldquoGraded 1205721-adrenoceptor activation of arteries involves recruitment ofsmooth muscle cells to produce ldquoall or nonerdquo Ca2+ signalsrdquo CellCalcium vol 29 no 5 pp 327ndash334 2001
[13] R F Furchgott and J V Zawadzki ldquoThe obligatory role ofendothelial cells in the relaxation of arterial smooth muscle byacetylcholinerdquo Nature vol 288 no 5789 pp 373ndash376 1980
[14] M Feletou R Kohler and P M Vanhoutte ldquoNitric oxideorchestrator of endothelium-dependent responsesrdquo Annals ofMedicine vol 44 no 7 pp 694ndash716 2012
[15] Y Tanaka Y Mochizuki H Tanaka and K Shigenobu ldquoSig-nificant role of neuronal non-N-type calcium channels in thesympathetic neurogenic contraction of rat mesenteric arteryrdquoBritish Journal of Pharmacology vol 128 no 7 pp 1602ndash16081999
[16] T Godfraind and A Kaba ldquoBlockade or reversal of the contrac-tion induced by calcium and adrenaline in depolarized arterialsmooth musclerdquo British Journal of Pharmacology vol 36 no 3pp 549ndash560 1969
[17] S Kobayashi H Kanaide and M Nakamura ldquoK+-depolarization induces a direct release of Ca2+ fromintracellular storage sites in cultured vascular smooth musclecells from rat aortardquo Biochemical and Biophysical ResearchCommunications vol 129 no 3 pp 877ndash884 1985
[18] B Ay Y S Prakash C M Pabelick and G C Sieck ldquoStore-operated Ca2+ entry in porcine airway smooth musclerdquo Ameri-can Journal of Physiology vol 286 no 5 pp L909ndashL917 2004
[19] G M Dick and J D Tune ldquoRole of potassium channels incoronary vasodilationrdquo Experimental Biology andMedicine vol235 no 1 pp 10ndash22 2010
[20] EAKo JHan I D Jung andW S Park ldquoPhysiological roles ofK+ channels in vascular smoothmuscle cellsrdquo Journal of SmoothMuscle Research vol 44 no 2 pp 65ndash81 2008
[21] K Lawson ldquoPotassiumchannel openers as potential therapeuticweapons in ion channel diseaserdquo Kidney International vol 57no 3 pp 838ndash845 2000
[22] D X P Brochet and P D Langton ldquoDual effect of initial [K]on vascular tone in rat mesenteric arteriesrdquo Pflugers ArchivEuropean Journal of Physiology vol 453 no 1 pp 33ndash41 2006
[23] F Jiang C G Li and M J Rand ldquoRole of potassium channelsin the nitrergic nerve stimulation-induced vasodilatation in theguinea-pig isolated basilar arteryrdquo British Journal of Pharmacol-ogy vol 123 no 1 pp 106ndash112 1998
10 Evidence-Based Complementary and Alternative Medicine
[24] N B Standen and J M Quayle ldquoK+ channel modulation inarterial smooth musclerdquo Acta Physiologica Scandinavica vol164 no 4 pp 549ndash557 1998
[25] E A Ko W S Park A L Firth N Kim J X-J Yuan and JHan ldquoPathophysiology of voltage-gatedK+ channels in vascularsmooth muscle cells modulation by protein kinasesrdquo Progressin Biophysics and Molecular Biology vol 103 no 1 pp 95ndash1012010
[26] F Sun E Hayama Y Katsube R Matsuoka and T NakanishildquoThe role of the large-conductance voltage-dependent andcalcium-activated potassium (BKCa) channels in the regulationof rat ductus arteriosus tonerdquo Heart and Vessels vol 25 no 6pp 556ndash564 2010
[27] M T Nelson and J M Quayle ldquoPhysiological roles andproperties of potassium channels in arterial smooth musclerdquoAmerican Journal of Physiology vol 268 no 4 pp C799ndashC8221995
[28] T Karkanis S Li J G Pickering and S M Sims ldquoPlasticityof KIR channels in human smooth muscle cells from internalthoracic arteryrdquo American Journal of Physiology vol 284 no 6pp H2325ndashH2334 2003
[29] A Schwab ldquoFunction and spatial distribution of ion channelsand transporters in cell migrationrdquo American Journal of Physi-ology vol 280 no 5 pp F739ndashF747 2001
[30] H Ouadid-Ahidouch M Roudbaraki P Delcourt A Ahi-douchN Joury andN Prevarskaya ldquoFunctional andmolecularidentification of intermediate-conductance Ca2+-activated K+channels in breast cancer cells association with cell cycleprogressionrdquo American Journal of Physiology vol 287 no 1 ppC125ndashC134 2004
[31] J Ledoux M E Werner J E Brayden and M T NelsonldquoCalcium-activated potassium channels and the regulation ofvascular tonerdquo Physiology vol 21 no 1 pp 69ndash78 2006
[32] PD LangtonMTNelson YHuang andN B Standen ldquoBlockof calcium-activated potassium channels inmammalian arterialmyocytes by tetraethylammonium ionsrdquo American Journal ofPhysiology vol 260 no 3 pp H927ndashH934 1991
10 Evidence-Based Complementary and Alternative Medicine
[24] N B Standen and J M Quayle ldquoK+ channel modulation inarterial smooth musclerdquo Acta Physiologica Scandinavica vol164 no 4 pp 549ndash557 1998
[25] E A Ko W S Park A L Firth N Kim J X-J Yuan and JHan ldquoPathophysiology of voltage-gatedK+ channels in vascularsmooth muscle cells modulation by protein kinasesrdquo Progressin Biophysics and Molecular Biology vol 103 no 1 pp 95ndash1012010
[26] F Sun E Hayama Y Katsube R Matsuoka and T NakanishildquoThe role of the large-conductance voltage-dependent andcalcium-activated potassium (BKCa) channels in the regulationof rat ductus arteriosus tonerdquo Heart and Vessels vol 25 no 6pp 556ndash564 2010
[27] M T Nelson and J M Quayle ldquoPhysiological roles andproperties of potassium channels in arterial smooth musclerdquoAmerican Journal of Physiology vol 268 no 4 pp C799ndashC8221995
[28] T Karkanis S Li J G Pickering and S M Sims ldquoPlasticityof KIR channels in human smooth muscle cells from internalthoracic arteryrdquo American Journal of Physiology vol 284 no 6pp H2325ndashH2334 2003
[29] A Schwab ldquoFunction and spatial distribution of ion channelsand transporters in cell migrationrdquo American Journal of Physi-ology vol 280 no 5 pp F739ndashF747 2001
[30] H Ouadid-Ahidouch M Roudbaraki P Delcourt A Ahi-douchN Joury andN Prevarskaya ldquoFunctional andmolecularidentification of intermediate-conductance Ca2+-activated K+channels in breast cancer cells association with cell cycleprogressionrdquo American Journal of Physiology vol 287 no 1 ppC125ndashC134 2004
[31] J Ledoux M E Werner J E Brayden and M T NelsonldquoCalcium-activated potassium channels and the regulation ofvascular tonerdquo Physiology vol 21 no 1 pp 69ndash78 2006
[32] PD LangtonMTNelson YHuang andN B Standen ldquoBlockof calcium-activated potassium channels inmammalian arterialmyocytes by tetraethylammonium ionsrdquo American Journal ofPhysiology vol 260 no 3 pp H927ndashH934 1991
