Autonomic Neuroscience: Basic and Clinical 150 (2009) 38–44

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Autonomic Neuroscience: Basic and Clinical

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Nitric oxide synthesis blockade reduced the baroreflex sensitivity in trained rats

Hugo C.D. Souza a,⁎, João E. De Araújo a, Marli C. Martins-Pinge b, Izabela C. Cozza a, Daniel P. Martins-Dias a

a Exercise Physiology Laboratory of the Department of Biomechanics, Medicine and Rehabilitation, School of Medicine of Ribeirão Preto, University of São Paulo, Brazilb Department of Physiological Sciences of the University of Londrina, Brazil

⁎ Corresponding author. Department of BiomechanicMedicine of Ribeirão Preto, University of São Paulo,14049Fax: +55 16 3602 4413.

E-mail address: hugocds@fmrp.usp.br (H.C.D. Souza

1566-0702/$ – see front matter © 2009 Elsevier B.V. Aldoi:10.1016/j.autneu.2009.04.007

a b s t r a c t

a r t i c l e i n f o

Article history:

Received 12 September 2008Received in revised form 16 March 2009Accepted 20 April 2009

Keywords:Cardiovascular autonomic controlHypertensionBaroreflexPhysical trainingNitric oxide

Objective: The present study has investigated the effect of blockade of nitric oxide synthesis on cardiovascularautonomic adaptations induced by aerobic physical training using different approaches: 1) double blockadewith methylatropine and propranolol; 2) systolic arterial pressure (SAP) and heart rate variability (HRV) bymeans of spectral analysis; and 3) baroreflex sensitivity.Methods: Male Wistar rats were divided into four groups: sedentary rats (SR); sedentary rats treated withNω-nitro-L-arginine methyl ester (L-NAME) for one week (SRL); rats trained for eight weeks (TR); and ratstrained for eight weeks and treated with L-NAME in the last week (TRL).Results: Hypertension and tachycardia were observed in SRL group. Previous physical training attenuated thehypertension in L-NAME-treated rats. Bradycardiawas seen in TR and TRL groups, although such a conditionwasmore prominent in the latter. All trained rats had lower intrinsic heart rates. Pharmacological evaluation of cardiac

autonomic tonus showed sympathetic predominance in SRL group, differently thanother groups. Spectral analysisof HRV showed smaller low frequency oscillations (LF: 0.2–0.75 Hz) in SRL group compared to other groups. Ratstreated with L-NAME presented greater LF oscillations in the SAP compared to non-treated rats, but oscillationswere found to be smaller in TRL group. Nitric oxide synthesis inhibition with L-NAME reduced the baroreflexsensitivity in sedentary and trained animals.Conclusion: Our results showed that nitric oxide synthesis blockade impaired the cardiovascular autonomicadaptations induced by previous aerobic physical training in rats that might be, at least in part, ascribed to adecreased baroreflex sensitivity.

© 2009 Elsevier B.V. All rights reserved.

1. Introduction

Nitric oxide (NO) plays an important role in the control of vasculartonus and arterial pressure (AP) (Furchgott and Zawadzki, 1980).Endogenous reduction of NO synthesis is related to several physio-pathological disorders or associated conditions, such as increase invascular tonus, increase in platelet adhesion, decrease in endothelial-dependent vasodilatation, hypercholesterolemia, diabetes, and arter-iosclerosis (Moncada and Higgs, 1991).

In fact, nitric oxide plays an important role in regulating peripheralblood circulation (Palmer et al., 1987; Klimaschewski et al., 1992; Todaet al., 1993). However, there are studies demonstrating the NOinvolvement in the regulation of cardiac and vascular autonomic controlby means of modulation, particularly the central sites of cardiovascularautonomic neural integration (Matsuda et al., 1995; Kantzides andBadoer, 2005;Martins-Pinge et al., 2007).Wehave reported in aprevious

s and Rehabilitation, School of-900, Ribeirão Preto, SP, Brazil.

).

l rights reserved.

study that NO synthesis blockade with Nω-nitro-L-argininemethyl ester(L-NAME)promoted an increase in cardiac sympathetic influenceaswellas a decrease in baroreflex sensitivity (Souza et al., 2001).

On the other hand, aerobic training physics in either experimentalanimals or human beings can promote important adaptations forheart and cardiovascular autonomic control, often including reductionin mean AP. Aerobic physical exercises are also usually employed forpreventive purposes and combined with therapies aimed at control-ling chronic-degenerative diseases in order to attenuate the effects ofcardiovascular risk factors (Arakawa, 1993; Kelley and McClellan,1994; Tsatsoulis and Fountoulakis, 2006; Warburton et al., 2006). Themechanisms involved in cardiovascular adaptations induced byphysical exercises are not fully understood. It has been suggestedthe NO participation, either directly by improving the endothelialfunction, or indirectly by influencing cardiac autonomic adaptationscharacterized by increased cardiac vagal tonus and decreasedcardiovascular sympathetic tonus (Danson and Paterson, 2003; Kuruet al., 2002; Laterza et al., 2007).

Therefore, the objective of the present study was to investigate theeffect of NO synthesis blockade on cardiovascular autonomic adapta-tions induced by previous physical training in conscious rats. In orderto do so, we have used different approaches: 1) pharmacological

Table 1Baseline values (mean±SEM) of heart rate (HR, mean arterial pressure (AP) andautonomic pharmacological blockade in sedentary and trained conscious rats afternitric oxide blockade with L-NAME.

Sedentary Trained

SR SRL TR TRL

(n=14) (n=14) (n=14) (n=14)

Baseline valuesHeart Rate, bpm 346±5 403±15⁎ 314±6⁎+ 290±8⁎+#

Mean AP, mmHg 98±3 162±4⁎ 102±2 133±4⁎+#

Tonic autonomic controlMethylatropine, bpm 458±10 434±8 414±12⁎ 399±11⁎+

Propranolol, bpm 331±9 356±10 290±8⁎+ 269±9⁎+

Intrinsic Heart Rate, bpm 392±6 379±7 332±4⁎+ 323±9⁎+

Sedentary rats (SR); sedentary rats L-NAME (SRL); trained rats (TR) and trained ratsL-NAME (TRL). Values are mean±SEM. ⁎Pb0.05 compared to SR; +Pb0.05 comparedto SRL; #Pb0.05 compared to TR.

39H.C.D. Souza et al. / Autonomic Neuroscience: Basic and Clinical 150 (2009) 38–44

evaluation of cardiac autonomic tonus by means of double blockadeusing methylatropine and propranolol; 2) evaluation of heart ratevariability and systolic arterial pressure in terms of frequency usingspectral analysis; and 3) evaluation of baroreflex cardiac sensitivity.

2. Methods

All experimental procedures involved in this study were approvedby the ethics committee for animal experiments of the University ofSão Paulo, Ribeirão Preto School of Medicine.

2.1. Animals

Male Wistar rats (150–180 g) were kept in individual cages underconditions of controlled temperature (21 °C) and a dark/light cycleof 12 h. The animals were divided into four experimental groups:1) Sedentary rats treated with feed and water ad libitum for 8 weeks(SR Group; n=14). 2) Sedentary rats treated with feed and water adlibitum for 8weeks and L-NAME dissolved in drinkingwater (70mg/kg)during the last week (SRL Group; n=14). 3) Trained rats submitted toswimming exercise for 8 weeks (TR Group; n=14). 4) Trained ratssubmitted to swimming exercise for 8 weeks and treated with L-NAMEduring the lastweek (TRLGroup;n=14). The inhibition of NO synthesisduring seven dayswas chosen because inprevious studyhas shown thatthis model promotes important hypertension and damage in cardio-vascular autonomic control (Souza et al., 2001).

2.2. Physical training

The 8-week swimming training programwas conducted in a glassaquarium (100 cm long, 80 cm wide and 80 cm high) with heatedwater (30 °C). Before beginning the training program, the animalswere submitted to a two-week adaptation program, that is, the waterexercises lasted 10 min initially and then were gradually increased to45min. After the adaptation program, the animals were trained for 1 ha day, 6 days a week. The sedentary animals were submitted to waterstress for 2 min a day during the training program.

2.3. Experimental protocol

On the sixth day of the last week, under tribromoethanolanaesthesia (250 mg/kg, i.p.), the animals were instrumented withfemoral venous and arterial catheters (PE-50 fused to PE-10) filledwith heparinised saline (500 IU/mL) and exteriorized through theanimal's back. Twenty-four hours after the surgical procedures, APwas measured in conscious rats kept in a quiet environment. AP wasrecorded with a pressure transducer (ADInstruments — MLT0380)and the amplified signal (ADInstruments — ML110) was fed to acomputer acquisition system (ADInstruments — PowerLab 8/30).Mean arterial pressure (MAP) and heart rate (HR) were calculatedfrom arterial pulse pressure.

2.4. Sympatho-vagal tonus and intrinsic heart rate

Methylatropine (4mg/kg) and propranolol (5mg/kg)were used toblock the parasympathetic and sympathetic influences on HR in SR(n=14), SRL (n=14), TR (n=14), and TRL (n=14) groups. After thebasal period (30 min), half of the rats were injected with methyla-tropine, and the HR was recorded during the next 15 min to evaluatethe parasympathetic effect on HR. Propranolol was then injected andHR was recorded for another 12 min to determine the intrinsic heartrate. The other half of the rats received the same drugs but in areversed sequence (propranolol/methylatropine) during the sameperiod of time (12/15min) in order to assess the sympathetic effect onHR and also to determine the intrinsic heart rate. Data from themethylatropine/propranolol (SR, n=07; SRL, n=07; TR, n=07; TRL,

n=07) and propranolol/methylatropine (SR, n=07; SRL, n=07; TR,n=07; TRL, n=07) sequences were pooled to provide basal HR(before any blockade) and intrinsic heart rate.

2.5. Power spectral analysis of heart rate and arterial pressure variability

The baseline AP and HR recorded during a 30-min period wereprocessed by a customised computer software which applies analgorithm to detect cycle-to-cycle inflection points in the pulsatile APsignal, thus determining beat-by-beat values of systolic and diastolicpressures. Beat-by-beat pulse interval series from pulsatile AP signalwere also generated by measuring the length of time betweenadjacent systolic waves. From the baseline 30-min recording period,the time series of pulse interval (PI) and systolic arterial pressure(SAP) were divided into contiguous segments of 300 beats, over-lapped by half. After calculating mean value and variance of eachsegment, they were submitted to a model-based autoregressivespectral analysis as described elsewhere (Malliani et al., 1991; Rubiniet al., 1993; Task Force, 1996). Briefly, a modelling of the oscillatorycomponents presented in stationary segments of beat-by-beat timeseries of PI and SAP was calculated based on Levinson–Durbinrecursion, with themodel order chosen according to Akaike's criterion(Malliani et al., 1991). This procedure allows an automatic quantifica-tion of the centre frequency and power of each relevant oscillatorycomponent present in the time series. The oscillatory componentswere labelled as very low (VLF: 0.01–0.20 Hz), low (LF: 0.20–0.75 Hz)or high frequency (HF: 0.75–2.50 Hz). The power of LF and HFcomponents of PI variability was also expressed in normalised units,obtained by calculating the percentage of the LF and HF variabilitywith respect to the total power after subtracting the power of the VLFcomponent (frequenciesb0.20 Hz). The normalisation proceduretends to minimise the effect of the changes in total power on theabsolute values of LF and HF variabilities (Malliani et al., 1991; Rubiniet al., 1993; Task Force, 1996).

2.6. Baroreflex sensitivity

Baroreflex sensitivity was determined by the method of Head andMccarty (1987). Changes in MAP were elicited by alternating bolusinjections of phenylephrine (0.1 to 16.0 µg/kg) and sodium nitroprus-side (0.1 to 32.0 µg/kg). MAP and HR were measured before andimmediately after injection of phenylephrine (or sodium nitroprus-side) when the arterial pressure achieved a new steady-state level.The two parameters were then allowed to return to baseline, afterwhich the next injectionwas given. A total of at least six increases andsix decreases in MAP of different degrees were elicited in each rat.

Fig. 1. Graphic representation of basal heart rate (black line), intrinsic heart rate (dashedline) with respective percentual variations of heart rate following parasympatheticblockade with methylatropine (upper part of the gray rectangle) and sympatheticblockade with propranolol (lower part of the dark gray rectangle) for sedentary rats(SR), sedentary rats L-NAME (SRL), trained rats (TR), trained rats L-NAME (TRL).⁎Pb0.05vs. sedentary rats; +Pb0.05 vs. sedentary rats L-NAME; #Pb0.05 vs. trained rats.

40 H.C.D. Souza et al. / Autonomic Neuroscience: Basic and Clinical 150 (2009) 38–44

Baroreflex sensitivity was quantified by the slope of the regression lineobtained by the best-fit points showing changes in HR and AP inrelation to baseline values (Coleman, 1980).

2.7. Statistical analysis

Data are reported as mean±SEM. The results of pharmacologicalblockade, spectral analysis and baroreflex sensibility were assessed by

Fig. 2. Examples of representative autoregressive spectra calculate from series of pulse intesedentary rats L-NAME (SRL), trained rats (TR) and trained rats L-NAME (TRL).

two-way ANOVA followed by the post-hoc Tukey test. Significantdifferences were considered when pb0.05.

3. Results

Table 1 shows basal MAP and HR values of all groups studied. SRLgroup had hypertensive and tachycardic animals compared to othergroups. TRL group also had hypertensive rats, but in a numbersignificantly smaller than SRL group. Trained rats presented brady-cardia compared to controls (SR group), although such a conditionwas found to be significantly lower in TRL group than in TR group.

Fig. 1 and Table 1 show the results regarding the pharmacologicalevaluation of cardiac autonomic tonus and intrinsic heart rate (IHR)with respective percentual variations following intravenous adminis-tration of methylatropine and propranolol (Fig. 1). SRL grouppresented sympathetic predominance in the determination of basalHR, differently than other groups presenting vagal predominance.

Fig. 2 shows representative spectra of PI and SAP for all groupsstudied. Fig. 3 and Table 2 show partial results regarding the temporalvariability analysis of PI series by means of spectral analysis usingautoregressive method. TRL group showed an increase in totalvariance compared to SRL and TR groups. In addition, total variancewas found to be lower in SRL group compared to SR group. In absolutevalues, these both groups had less predominant LF oscillations in PIcompared to SR and TRL groups. In normalised units, SRL groupshowed reduced LF oscillations in relation to other groups. Withregard to HF oscillations in PI (absolute values), SRL group showedreduction compared to SR and TRL groups. In normalized units, therats submitted to physical training (TR and TRL groups) had greater HFoscillations in relation to sedentary animals (SR and SRL groups).

Fig. 4 and Table 2 also show the results regarding the variabilityanalysis of SAP. Groups inwhich the animals were treatedwith L-NAMEhadgreater total variance andgreater LFoscillations, althoughSRLgrouphad the greatest values.

Fig. 5 shows the results regarding the pharmacologic analysis ofbaroreflex sensitivity by means of multiple dosages of phenylephrine

rval (ms2, top) and systolic arterial pressure (mmHg2, bottom) of sedentary rats (SR),

Fig. 4. Spectral power density of systolic arterial pressure in low (LF) and high frequency(HF) bands in sedentary rats (SR), sedentary rats L-NAME (SRL), trained rats(TR), trained rats L-NAME (TRL).⁎Pb0.05 vs. sedentary rats; +Pb0.05 vs. sedentaryrats L-NAME; #Pb0.05 vs. trained rats.

Fig. 3. Spectral power density of pulse interval in low (LF) and high frequency (HF)bands in absolute (ms2) and normalizes units (nu) calculated from time series insedentary rats (SR), sedentary rats L-NAME (SRL), trained rats (TR), trained rats L-NAME(TRL).⁎Pb0.05 vs. sedentary rats; +Pb0.05 vs. sedentary rats L-NAME; #Pb0.05 vs.trained rats.

41H.C.D. Souza et al. / Autonomic Neuroscience: Basic and Clinical 150 (2009) 38–44

and sodium nitroprusside. TR group had an increased gain in reflextachycardia, whereas groups whose animals were treated with L-NAME(SRL and TRL group) had a decrease compared to control animals (SRgroup). When the reflex tachycardic response was compared between

Table 2Spectral parameters of pulse interval (PI) and systolic arterial pressure (SAP) calculatedfrom time series using autoregressive spectral analysis in sedentary rats (SR), sedentaryrats L-NAME (SRL), trained rats (TR) and trained rats L-NAME (TRL).

Sedentary Trained

SR SRL TR TRL

(n=14) (n=14) (n=14) (n=14)

Baseline valuesPI, ms 0.17±0.002 0.15±0.005⁎ 0.19±0.003⁎+ 0.21±0.005⁎+#

SAP, mmHg 117±5 184±5⁎ 123±3+ 156±4⁎+#

Spectral parameters; PIVariance, ms2 13.9±1.6 6.8±1.9⁎ 10.6±3.5 19.4±1.8+#

LF, ms2 1.1±0.1 0.4±0.1⁎ 0.5±0.1⁎ 1.4±0.2+#

LF, nu 10.5±0.9 4.8±0.9⁎ 8.5±1.3+ 10.1±1.4+

HF, ms2 7.8±1.2 3.2±0.9⁎ 5.9±1.9 11.6±1.3+#

HF, nu 73.2±3.2 63.9±4.7 85.3±2.7⁎+ 85.4±1.8⁎+

LF/HF 0.18±0.02 0.19±0.09 0.15±0.05 0.14±0.03Spectral parameters; SAPVariance, mmHg2 12.1±2.2 45.1±6.1⁎ 13.9±2.6+ 23.2±2.2⁎+#

LF, mmHg2 8.6±1.7 37.9±4.8⁎ 9.6±2.2+ 18.3±2.4⁎+#

HF, mmHg2 2.3±0.3 3.8±0.4 2.4±0.4 2.4±0.4

Values are mean±SEM. ⁎Pb0.05 compared to SR; +Pb0.05 compared to SRL; #Pb0.05compared to TR.

animals treated with L-NAME, those from SRL group showed less gain.With regard to the reflex bradycardic response, rats treatedwith L-NAME(SRL and TRL groups) also had less gains compared to non-treatedanimals (SR and TR groups). When L-NAME-treated animals werecompared, TRL group showed the least gain.

Fig. 5. Average baroreflex linear curves obtained by means changes in heart rate (HR)due to induced changes in mean arterial pressure (MAP) in sedentary rats (SR),sedentary rats L-NAME (SRL), trained rats (TR) and trained rats L-NAME (TRL). Linesrepresent the linear regressions between changes in heart rate andmean arterial pressure.The slope of linear regression was used as the index (gain) of the baroreflex sensitivity(bpm/mmHg). ⁎Pb0.05 vs. sedentary rats; +Pb0.05 vs. sedentary rats L-NAME; #Pb0.05vs. trained rats.

42 H.C.D. Souza et al. / Autonomic Neuroscience: Basic and Clinical 150 (2009) 38–44

4. Discussion

The results obtained in the present study showed that chronicblockade of NO synthesis in sedentary animals caused abnormalities inthe cardiovascular autonomic control. These abnormalities werecharacterized by prominent basal tachycardia, predominance of cardiacsympathetic tonus, impairment inHRandAPmodulation, and reductionin baroreflex sensitivity. In turn, animals submitted only to physicaltraining showed basal bradycardia as well as improvement of cardiacautonomic control,whichwas characterized by increasedHFoscillationsin heart rate and baroreflex sensitivity. On the other hand, the blockadeof NO synthesis with 8-week aerobic training attenuated the improve-ment in some autonomic parameters evaluated.

Aerobic physical training is widely employed as a non-pharmaco-logical anti-hypertensive therapy, attenuating AP values, reducing therisk of metabolic and cardiovascular diseases often associated withendothelial dysfunction, and decreasing the activity of eNOS (Carteret al., 2003; Maiorana et al., 2003; Husain, 2002). In our study,previous physical training was shown to be effective in attenuating APincrease induced by treatment with L-NAME. According to somestudies, the decreased AP observed in trained rats submitted to thismodel of experimental hypertension would be related to increasedcapillarity blood flow, optimal efficacious tissue oxygen consumption,and improvement of the systemic peripheral vasodilatation due to theendothelial recovery resulting from physical training (Kuru et al.,2002; Husain, 2002).

Physical training also preventedbasal tachycardia inducedby L-NAMEtreatment. This tachycardia seems to be the result of abnormalities inautonomic regulation of HR involving reduction in baroreflex sensitivityassociated with high sympatho-excitation, thus changing the cardiacautonomic balance. These abnormalities resulted in predominancesympathetic in the determination of basal HR (Souza et al., 2001; Scroginet al.,1998). Other hypothesis is based on increased activation of humoralendogenous factors (endothelins, angiotensin II, and others) promotedby the blockade of NO synthesis, thus influencing positively the cardiacchronotropism (Dzau, 1993; Masullo et al., 2000). Nevertheless, thishypothesis was not corroborated by our study because the doublepharmacological blockadewithmethylatropine and propranolol showedthat intrinsic HR of sedentary animals treated with L-NAME was notdifferent from that of controls.

In contrast, physical training promoted basal bradycardia in allanimals studied. The mechanisms accounting for this bradycardiahave not been entirely elucidated. Some hypotheses have beensuggested, including participation of NO. Studies showed that NOseems to increase the HR response to vagal stimulation both in vitro(Sears et al., 1999) and in vivo (Elvan et al., 1997). This response wouldinvolve the NO-GMPc pathway, facilitating cholinergic cardiactransmission and consequently inducing basal bradycardia throughphysical training (Mohan et al., 2002). Further study demonstratedthat gene transfer of neuronal NOS (nNOS) into the atrial wallcorresponded to vagal phenotype induced by physical training, thussuggesting that nNOS could be crucial for this increased cardiac vagalactivity (Danson and Paterson, 2003). Contrary to this hypothesis,however, another study showed that physical training inducedchanges in sinus node automaticity and impulse conduction observedin endurance athletes could be attributed to the intrinsic physiology ofthe heart instead of autonomic influences (Stein et al., 2002). Ourresults showed that basal bradycardia found in trained animals wasthe result of reduced intrinsic pacemaker rate. In addition, the trainedanimals submitted to blockade of NO synthesis presented a lowerbasal bradycardia. As the reason for such prominent reduction in basalHR is not entirely known, further investigation is warranted. Never-theless, alterations in cardiac tissue induced by blockade of NOsynthesis could contribute to reduce the basal HR, since it wasobserved a prominent increase in cardiac fibrosis in trained animalssubmitted to such experiment (Souza et al., 2007).

Autonomic evaluation by using different approaches showed theimportant role played by both NO and physical training regarding theneural control of cardiovascular system. The NO synthesis blockadepromoted a reversion of autonomic balance characterized by sympa-thetic autonomic component predominating over the parasympatheticcomponent in the determination of basal HR. Animals submitted toprevious physical training and treated with L-NAME did not show thisreversion, keeping thepredominance of theparasympathetic autonomiccomponent over the sympathetic one.

By using spectral analysis we showed that the abnormalities inautonomic modulation of HR variability observed in sedentaryanimals treated with L-NAME were characterized by a decrease intotal variance, a finding attributed to reductions in LF and HF oscilla-tions. However, only LF oscillations were kept reduced following valuenormalization. HR oscillations in LF band in non-anaesthetized ratshave been associated with sympathetic and parasympathetic mod-ulation, whereas oscillations in HF have been associated only withparasympathetic modulation (Japundzic et al., 1990; Cerutti et al.,1991). The reduction in LF oscillations does not seem to reflect thepredominance of cardiac sympathetic tonus observed in sedentaryanimals treated with L-NAME. This paradox in which the increasein sympathetic activity promotes decrease in LF oscillations wasobserved in previous observations in rats (Souza et al., 2001). Also,other studies have shown similar results with different models,demonstrating a reduction in these oscillations during maneuversinducing a sharp increase in sympathetic activity, e.g. postural change,exercise and severe cardiac failure (Arai et al., 1989; Van de Borneet al., 1997; Pichon et al., 2004). In situations where physiologicalmechanisms are maximally mobilized in order to preserve homeostasis,the cardiovascular reserve would not be enough to sustain variability. Infact, the reduced LF oscillations observed in this hypertension model isnot knownyet, but it has been suggested that the increase in sympatheticactivity in association with abnormalities in central autonomic modula-tion, impairment regulation of arterial baroreflex, and changes in cardiacsensitivity to catecholamine would account for such reductions inoscillations (Souza et al., 2001; Scrogin et al., 1998; Van de Borne et al.,1997).

On the other hand, physical training prior to L-NAME treatmentwas shown to be effective in preventing the reduction in LFoscillations of HR. This fact seems to be associated with lessersympathetic influence and predominance of parasympathetic com-ponent for determination of basal HR, a finding observed in theanimals by means of double pharmacological blockade. In addition, alltrained animals, including those treatedwith L-NAME, had an increasein HF oscillations of HR in normalized units, thus suggesting a highinfluence on the cardiac parasympathetic autonomic modulation.

With regard to SAP variability, it was demonstrated that blockadeof NO synthesis using L-NAME caused a marked increase in LFoscillations, which were attenuated in animals submitted to previousphysical training. Consequently, the increase in systolic arterial pres-sure LF oscillations can promote activation of mechanical-sensitiveand autocrine pathways, thus promoting concentric cardiac hyper-trophy and higher incidence of cardiovascular dysfunctions (Souzaet al., 2007; Martinka et al., 2005). This abnormality indicates theimportant role played by NO in modulating the AP variability. In thiscase, NO has a buffering action on LF oscillations, opposing thevascular sympathetic modulation (Persson, 1997). This bufferingaction would be triggered particularly by the increased shear stressin endothelial cells, consequently promoting production and releaseof NO. Oscillations induced by sympathetic activation would occurat frequencies ranging from 0.2 to 0.6 Hz (LF band), whereas thebaroreflex control of AP variability would occur at frequencies below0.1 Hz (Nafz et al., 1997). Therefore, our results also provide evidencefor the participation of NO in the modulation of LF oscillations in SAP.With regard to attenuating found on LF oscillations in the animalstrained and treated with L-NAME, one cannot say whether it is due to

43H.C.D. Souza et al. / Autonomic Neuroscience: Basic and Clinical 150 (2009) 38–44

adaptive vascular mechanisms or to a decrease in vascular sympa-thetic autonomic drive.

In turn, evaluation of baroreflex control of HR has showed that NOblockade in sedentary animals promoted a decrease in baroreflex gain,as evidenced in earlier studies (Souza et al., 2001; Scrogin et al., 1998;Lantelme et al., 1994). Such a decrease is in accordance with NO-deficiency changes found by pharmacological evaluation of cardiacautonomic tonus and analysis of HR variability. The impairment incardiovascular autonomic control due to NO-deficiency, which wasevaluated by different approaches, suggests the presence of abnorm-alities in baroreflex arch involving afference, integrated centre, andefference (Matsuda et al., 1995; Elvan et al., 1997; Jimbo et al., 1994).Nevertheless, changes in neural-cardiovascular integration induced byNO-deficiency may be the main cause of autonomic abnormalitiesfound in this model of experimental hypertension. Neuronal nitricoxide synthase (nNOS) is present in specific brain sites accounting forcardiovascular control (Kantzides and Badoer, 2005; Chang et al.,2003). The results obtained from different animal species showed thatNO can influence the cardiovascular integrating centre, particularlynucleus tractus solitarii (NTS) and rostral ventrolateral medulla(RVLM), thus promoting facilitation and inhibition, respectively(Tagawa et al., 1994; Tseng et al., 1996; Togashi et al., 1992). Withrespect to NTS, NO seems to induce an increase in the neuronaldischarge, thus reducing the sympathetic activity and increasing thevagal activity (Tagawa et al., 1994; Tseng et al., 1996). With respect toRVLM, NO seems to promote reduction in the sympathetic autonomicdrive (Togashi et al., 1992). Also, NO is thought to modulate theneuronal activity in other sites composing the cardiovascular regula-tion, such as caudal ventrolateral medulla (CVLM) and paraventricularnucleus (PVN) (Kantzides and Badoer, 2005).

NO synthesis blockade after physical training attenuated the gainincrease for reflex tachycardic response, thus reducing the baroreflexsensitivity. Additionally, reflex bradycardic responses were muchreduced. This finding may be attributed to a lower basal HR in theseanimals, which impeded further reduction. On the other hand, it wasalso observed that only trained animals had an increase in reflextachycardia gain compared to sedentary ones. This finding corrobo-rates the results of an earlier study showing that such a gain is causedby the improvement of aortic baroreflex sensitivity due to increasedvascular complacency induced by physical exercise (Brum et al.,2000).

The effect of physical exercise on cardiovascular control has beenextensively studied. It was demonstrated that physical trainingpromotes important adaptations, either directly by improving theendothelial function or indirectly by promoting adaptations in cardio-vascular autonomic control, as observed in our study (Kuru et al.,2002; Laterza et al., 2007). With regard to regions accounted forautonomic adaptations, it has been suggested neural remodelling aswell as production and release of endogenous substances in differentsites of reflex arch (Mueller and Hasser, 2006; Felix and Michelini,2007). The exercise appears to reduce tonic GABAAergic inhibition ofNTS neurons involved in HR control attenuating the sympatho-excitation (Mueller and Hasser, 2006). With respect to PVN, studyshowed that (nor)adrenergic innervation undergoes neural remodel-ing induced by exercise (Higa-Taniguchi et al., 2007). Moreover,blockade of PVN adrenoreceptors prevented exercise-mediated incre-ments in norepinephrine and corticosterone plasma levels (Scheurinket al., 1990). With regard to NO, studies demonstrated that physicalexercise might promote an increase in eNOS and nNOS expression(Sessa et al., 1994; DiCarlo et al., 2002; Zheng et al., 2005). In centralregions such as hypothalamus, NTS, and RVLM, NOwas involved in theexercise-dependent adaptations characterized by a decrease in thesympathetic autonomic drive (Higa-Taniguchi et al., 2007; Krameret al., 2000; Zucker et al., 2004).

In conclusion, our results showed that nitric oxide synthesisblockade impaired the cardiovascular autonomic adaptations induced

by previous aerobic physical training in rats that might be, at least inpart, ascribed to decreased baroreflex sensitivity. The mechanismsinvolved in these processes have not been entirely elucidated, butfurther studies should be carried out in order to identify them.

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