488

Hypotension in Transgenic Mice Overexpressing Human Bradykinin B2 Receptor

Dan-zhao Wang, Lee Chao, Juhe Chao

Abstract Bradykmm binds to its receptor at target organs and exerts a wide spectrum of biological actlvlties including va- sodllation, smooth muscle contraction and relaxation, pain, and mflammatlon To gam a better mslght mto the physlologlcal func- tion of this potent vasoactlve peptlde, we created transgenic mice that harbor the human bradykmm Bz receptor transgene under the control of the Rous sarcoma virus 3’-LTR promoter (RSV- cHBKR) Expression of HBKR m these transgemc mice was identified m the aorta, brain, heart, lung, liver, ludney, uterus, and prostate gland by reverse transcnptlon-polymerase chain reaction Southern blot analysis. Two transgemc mouse lines expressing the human Bz receptor resulted m a significant reductron of blood pressure (84 220 6 mm Hg, n=28, 76 920 8 mm Hg, n=24, P< 001) compared with the control httermates (96 920 4 mm Hg,

n=52) Admmistration of Hoe 140, a bradykmm B2 receptor an- tagonist, restored the blood pressure of the transgemc mice to normal levels wlthm 1 hour, and the effect dlmmlshed wlthm 4 hours The transgemc mice displayed enhanced blood pressure- lowering effect induced by a bolus mtra-aortlc mJechon of kmm and showed increased response in kunn-induced uterine smooth muscle contract&y compared with control littermates These studies show that overexpresslon of human bradykmm B2 recep- tor causes a sustained reduction of blood pressure m transgemc mice. They also suggest that the B, receptor-mediated signal transduction pathway plays a role m blood pressure regulation (Hypertension. 1997;29[part 2]:488-493.)

Key Words l human bradykmm B2 receptor l transgemc mice l hypotenslon l Hoe 140 l uterme contraction

T issue kalhkrem 1s a serme protemase which 1s ca- pable of cleaving kimnogen to release bioactlve kimn peptldes 1 On the bmdmg of kinins to BK

receptors, a broad spectrum of biological effects are gen- erated These include vasodllahon, vasoconstnction, smooth muscle contraction and relaxation, ion transport, and glucose metabohsm I-3 There are two distinct BK recep- tor subtypes, namely, B, and B2 The B2 receptor is widely distributed throughout mammalian tissues and has a greater affinity for intact BK and kalhdin (LBK),2,3 while the B1 receptor has a greater affinity for kmms’ metabohtes, Des-Arg”-BK or Des-Arg lo-LBK. The B, re- ceptor appears m certain pathological processes. Most of the kmms’ actions are mediated by B2 receptors.2

BK 1s a potent vasoactive peptlde 2 Intravascular in- Jectlon of BK can cause an immediate Qlatlon of the ar- terlal vessels with a concormtant fall m total penpheral vascular resistance and systemic blood pressure 2.4 The tissue kallikrein-kmm system has long been implicated m blood pressure regulation. Urinary excretion of tissue kal- l&rem was reported to be significantly reduced m essen- tial hypertensive patlents5.6 and genetically hypertensive rats.7,* Polymorphisms m the kalhkrein gene have been shown to cosegregate with high blood pressure m the off- spring of spontaneously hypertensive rats and normoten- slve Brown Norway rats.9~10 Recently, we have estab- lished a direct link between blood pressure regulation and tissue kalhkrem gene expression by transgemc animal models and somatic gene delivery strategies 11-14 We fur- ther demonstrated that the admmlstratlon of Hoe 140, a specific B2 receptor antagonist, could restore the blood

From the Department of Blochemlstry and Molecular Biology, Medical Umverslty of South Carolina, Charleston

Correspondence to Juhe Chao, PhD, Department of Blochermstry and Molecular Biology, Medical University of South Carolina, 171 Ashley Ave, Charleston, SC 29425

0 1997 Amencan Heart Assoclatlon, Inc

pressure of these transgemc mice to normal levels, mdl- catmg the hypotensive phenotype was mediated through BK B2 receptor m this transgemc animal model 13,14

The human BK B2 receptor cDNA was cloned and se- quenced from a human lung fibroblast cDNA library 15 It encodes a 364-amino acid protein with charactenstlcs of the G-protein-coupled receptor superfamily The genes encoding the human, mouse, and rat B2 receptors have also been cloned and sequenced 16-20 Molecular cloning of the B2 receptor gene and elucidation of the gene struc- ture have played a crucial role m analyzing the potential function of the B2 receptor m blood pressure homeostasls by usmg transgemc and antisense approaches 21.22 A BK Bz receptor-deficient mouse line was generated by ho- mologous recombmatlon However, these “knockout” mice failed to show any altered blood pressure pheno- type.21 To determine the role of BK B2 receptor m blood pressure regulation, we created a transgemc mouse model carrying the human BK B2 receptor cDNA under control of the RSV 3’-LTR promoter

Methods Transgene Construction and DNA Preparation

The human BK Bz receptor cDNA (1 3 kb) containing the en- tire coding region was amplified from the total RNA of human renal proximal tubular cells*3 by RT-PCR The sense pnmer used was 5’-CCATGCCGCTTGCTCCG-3’ and antisense primer was 5’-GGAATGCCAAGGAGACA-3’ 16 It was then cloned mto pREP8 vector (Invltrogen) at the HlndIII site downstream from the RSV-LTR enhancer/promoter The final construct pREP8- cHBKR was confirmed by restriction mapping and DNA sequencmg

The plasmld pREP%cHBKR was extracted by the alkaline ly- SIS method and purified by cesmm chlonde-etludmm bromide gradient centrlfugatlon 24 The purified plasmid DNA was dl- gested with Sal I to release a 2 4-kb transgene fragment, which contams the RSV-LTR enhancer/promoter, human BK B2 recep- tor cDNA, and an SV40 polyA segment This fragment was sep- arated from the vector fragment by sucrose gradlent fractlona- tlon,25 and dialyzed against the mJectlon buffer (10 mmol/L

by guest on June 16, 2018http://hyper.ahajournals.org/

Dow

nloaded from

Wang et al Human Bradykinin B2 Receptor Transgenic Mice 489

Selected Abbreviations and Acronyms

BK = bradykmm cHBKR = human BK Bz receptor cDNA

I LBK = Lys-BK PBS = ohosuhate-buffered saline I

RSV-LTR = kous’ sarcoma virus long terminal repeat RT-PCR = reverse transcription polymerase chain

reaction

Tns-HCI/O 25 mmol/L EDTA, pH 7 5) The DNA concentration was measured by absorbance at 260 nm and confirmed by com- paring with X DNA after agarose gel electrophoresls It was ch- luted to 4 pg/mL with the mJection buffer and centrifuged at 10 OOOg for 1 hour to remove dust particles before mjection

Generation and Identification of Transgenic Mice The purified RSV-cHBKR-SV40 polyA DNA was mlcrom-

Jetted mto Fz embryos (C57BL/6XDBA/2), which were allowed to develop to term m the uteri of pseudopregnant females Off- sprmg were screened by Southern blot analysis to identify trans- gemc mice Mouse genonuc DNA was extracted from the tail biopsies and digested by Apa I followed by Southern blot analysis using an [a3’P]dATP nick-translated human Bz receptor cDNA probe Two transgemc founders, No 8905 and No 9830, were ldentlfied These founder mice were bred separately with C57BL/6 to establish two independent transgemc hnes The control mice were nontransgemc httermates from the matings All expenmen- tal protocols were approved by the Institutional Animal Research Committee of the Medical University of South Carohna and were carried out according to the Guide for the Care and Use of Lab- oratory Anzm& of the National Institutes of Health

Expression of Human BK Bz Receptor Transgene Human Bz receptor mRNA was identified in transgemc mice

by RT-PCR Southern blot analysis Fresh tissues (aorta, brain, heart, lung, hver, kidney, and uterus or prostate) of male and female transgemc and control mice were homogemzed m gua- nichne lsothlocyanate buffer and total RNA was purified by ce- slum chloride gradient centrifugation 24 One microgram of total RNA was subjected to RT-PCR Southern blot analysis as previ- ously described12 using the transgene-specific primers and an m- temal probe The sequence of the 5’ primer IS 5’-TTCCTGGAT ACGCTGCATCG-3’, the 3’ primer, 5’-CTCATCAATGTATCT TATCA3’, and the internal probe, 5’-TCACTGCATTCTAGT TGTGG-3’ The blotted membrane was washed twice m 6X SSC at 55°C and exposed to Kodak X-OMAT film at -80°C

Transient Transfection of Human Embryonic Kidney 293 Cells

A human embryonic kidney 293 cell hne was transfected with 20 pg of plasrmd DNA of RSV-cHBKR by electroporatlon using a BTX-600 electric cell manipulator (BTX) At 72 hours after transfectlon, the cells were harvested and lysed m lysis buffer (5 mmol/L Tns-HCl, pH 7 4, with 5 mmol/L EDTA and 5 mmol/L EGTA) and tmcrocentnfuged at 16 OOOg at 4°C for 10 minutes The pellet was resuspended m bmclmg assay buffer 26 Ligand-bmdmg assays were performed m duplicate

Preparation of Iodinated Ligand and Binding Assay Tyr’-BK (Sigma), the synthetic analog of BK, was used as

radiohgand for a BK bmdmg assay It was lodmated by using the chloramme-T method and purified by high-pressure liquid chro- matography 27 The specific radioactivity was 167 35 Cl/mmol Crude membrane protein was prepared, and hgand bmdmg assays were performed following the procedure described previously 26 The concentration of membrane protein was determined by the Lowry assay ** For saturation studies, I*?-Tyr’-BK was mcu- bated m duplicate with 100 pg of membrane protein at 25°C m

a volume of 250 @L for 60 minutes Using a M&pore filter (Hoefer Scientific), the assay was terminated by filtration over a Whatman GF/C glass fiber filter presoaked with 0 1% aqueous polyethylemmme for 3 hours The tubes and filters were rinsed three times with 4 mL of ice-cold 25 mmol/L TES buffer, pH 6.8, and the filters were counted m a 1261 Multigamma counter (Pharmacia) Saturable bmdmg was calculated by subtracting the nonspecific bmdmg m the presence of 10 pmol/L unlabeled BK

Blood Pressure Measurement by the Tail-Cuff Method

Systolic blood pressure was measured with the tail-cuff method as previously described 11 Rve readmgs were taken for each an- imal Hoe 140 was diluted m PBS and injected mtraperitoneally mto both transgemc and control mice at 2 pg per mouse per mJection The blood pressure was measured before and at I 0, 2 0, and 4 0 hours after Hoe 140 admmlstratlon The control group was carried out by mjection of PBS mto transgemc mice or by inJection of Hoe 140 mto nontransgemc control mice

Blood Pressure Measurement by Arterial Cannulation and BK Injection

Mice were anesthetized by mtraperitoneal inJection of 2,2,2- tnbromoethanol m tert-amyl alcohol (Avertm, 20 mg/mL, 0 4 mW25 g body weight) The right femoral artery and the left ca- rotid artery were cannulated with PE- 10 catheters (Clay Adams) for direct arterial pressure recordmg and drug admmistration The chstal end of the cannula was connected to a physlologlcal pres- sure transducer (Statham Laboratories) coupled with a model 7E polygraph (Grass Instrument Co) Blood pressure of the anesthe- tized mice was measured directly vta the mtra-arterial route The mice were given a bolus mtra-aortlc mJectlon of BK (Sigma) BK was serially diluted and adtmmstered at doses from 16 to 500 ng in a volume of 50 PL saline per mouse

Uterine Contraction Assay The entire uterus was isolated and a l-cm portion from each

horn was hung m a tissue bath as previously described 21 One gram of tension was applied to the uterine horn, and the tension was measured by a force-displacement transducer FTO3C and recorded on a model 7E polygraph (Grass Instrument Co) BK was applied to the tissue bath at a concentration of 0.5 pmol/L As control, 25 mmol/L of KC1 was added to the tissue bath 30 mmutes after stlmulatlon by BK

Statistical Analysis Group data are reported as mean?SEM Compansons of pa-

rameters between control and transgemc mouse groups were per- formed by two-way ANOVA Differences were considered to be significant at a value of P< 05

Results Generation of Transgenic Mice

Fig I shows the scheme for preparing the RSV-cHBKR gene fusion construct. The transgene contams the RSV- LTR enhancer-promoter element, the full-length human Bz receptor cDNA, and a portion of the SV40 mtron The heterologous RSV enhancer-promoter element directs a high level of expression in a wide variety of tissues and reduces the negative feedback control at the transcnp- tlonal level The SV40 mtron functions as heterologous 3’ untranslated sequence to increase the stability of the transcripts

Two transgemc founders, No 8905 (female) and No 9830 (female), were identified from the 64 mltlal proge- nies by Southern blot analysis. The human transgene was detected as a 1.1 -kb band m addition to the background of a 3.3-kb mouse endogenous B2 receptor gene band (data

by guest on June 16, 2018http://hyper.ahajournals.org/

Dow

nloaded from

490 Hypertension Vol29, No 1, Purt 2 January 1997

TGA

ATG + Released by Sal I

TGA 1 1)

p RSV HBKR cDNA SV40 pA

FIG 1. Diagram of the human BK B2 receptor transgene DNA construct. The solid bar represents a full-length human BK B2 receptor cDNA (1.3 kb). pRSV denotes the Rous sarcoma virus 3’-LTR promoter and SV40 pA represents the polyadenylation signal of the SV40 gene. The human BK Ba receptor cDNA was cloned into pREP8 at a HindIll restriction site and the transgene was released from pREP8 at a Sal I restriction site, as indicated.

not shown). A comparison of band intensity in Southern blot suggested that line No. 9830 had a higher copy num- ber of the transgeoe than line No. 8905 (data not shown). There is no evidence of developmental defects associated with the RSV-cHBKR transgene as indicated by the nor- mal morphology and litter size of both founder lines.

Transient Transfection of Human Embryonic Kidney 293 Cell Line

Expression of the functional Bz receptor was analyzed in human 293 cells transfected with the RSV-cHBKR plas- mid DNA. The results of the BK binding assay show the presence of specific B2 receptor binding sites in the mem- brane protein preparation from the transfected cells. The sat-

25000 1 0 Transfected cells 0 Non-transfected cells

0 100 200 300 ‘iw Bound (PM)

0.00 1.00 2.00 3.00 4.00 1251-Tyro-BK (nM)

FIG 2. Representative saturation binding curve and Scatchard plot analysis of human BK BZ receptor in transfected 293 cells. The specific binding of BK o/ axis) to membrane proteins from 293 cells transfected with the BK B2 receptor cDNA is shown with increasing concentrations of i25i-Tyro-BK (x axis). As control, the binding assay was performed on the membranes prepared from the nontransfected 293 cells.

Aorta Heart Liver Uterus F F M FMFMFMFMFF -++---I-+--++-+

! i ** /

ii i i Brain Lung Kidney Prostate

FFMFMFMFMFMMM -++--++--++-+

’ -*A 9 i . ’ :a* , i : i

\

FIG 3. Expression of human BK B2 receptor mRNA in transgenic mice. RT-PCR Southern blot shows the expression of human BK B2 receptor cDNA in transgenic mice. RT-PCR was performed using 1 pg of total RNA. A pair of oligonucleotide primers specific to the human B2 receptor transgene were used. The RT-PCR products were hybridized with a nested probe specific to the transgene in the Southern blot analysis.

uration curves demonstrated that the transfected cells, but not the nontransfected cells, have a high density of binding sites (Fig 2). Scatchard plot analysis revealed that the transfected cell membrane contained specific BK binding site with a kD of 0.93 nmolL and Bmax of 1 .Ol pmol/mg protein as deter- mined by analyzing saturation data with the equilibrium bind- ing data analysis program.29

Expression and Tissue Distribution of the Human BK Bz Receptor Transgene

The expression of the human BK B2 receptor mRNA in the transgenic mice was analyzed by RT-PCR Southern blot analysis. The analysis was specific for the transgene since the 3’ primer and the internal probe were located in the SV40 portion of the transcript. Fig 3 shows that both male and female transgenic mice express the human B2 receptor mRNA in the aorta, brain, heart, lung, liver, kid- ney, and also in the prostate of males and the uterus of females. The expression was not detected in the corre- sponding tissues of the control mice, indicating the speci- ficity of the assay.

BK Binding Assay To analyze the transgene expression at the protein level,

membranes were prepared from the kidney, uterus, and brain of both transgenic and control mice (n=6). The den- sity of BK binding sites in the kidney of transgenic mice was approximately five times higher than that of the control mice (Fig 4). The density of BK binding sites in the uterus and brain of transgenic mice was 1%fold and l.Cfold higher, respectively, than those in the control mice (Fig 4).

Blood Pressure Analysis of Transgenic Mice Both lines of transgenic mice showed significant reduc-

tions in blood pressure compared with normal controls. The systolic blood pressure measured by the tail-cuff method was 84.2kO.6 mm Hg for line No. 8905 (mean ?SEM, n=28) and 76.920.8 mm Hg for line No. 9830 (mean%SEM, n=24). The value for the negative siblings waLy 96.9+0.4 mm Hg (mean-+SEM, n=S2) with P<.OOl (Fig 5). The values of blood pressure obtained from the

by guest on June 16, 2018http://hyper.ahajournals.org/

Dow

nloaded from

Wcmg et nl Human Bradykinin Bz Receptor Transgenic Mice 491

Kidney Uterus Brain

FIG 4. Determination of transgene expression by radioligand binding assay. The BK ligand binding assay was performed using membrane proteins prepared from the kidney, uterus, and brains of six mice 4 to 5 months old. Bar graph represents the fold increase of the BK-binding sites in kidney, uterus, and brain of transgenic and control mice (n=6).

indirect tail-cuff method and direct intraarterial cannula- tion were compared for accuracy.

To determine if Bz receptor is responsible for the hy- potensive effect, Hoe 140, a specific BK B2 receptor an- tagonist, was injected intraperitoneally at a dose of 2 pg per mouse (n=3). Fig 6 shows that Hoe 140 administration restored the blood pressure of the transgenic mice to a level close to that of the control mice in 1 hour and the effect of Hoe 140 diminished within 4 hours while vehicle buffer (PBS) had no effect on the blood pressure of trans- genie mice. Administration of Hoe 140 had no effect on the blood pressure of control littermates (data not shown).

Bolus intra-aortic injections of BK resulted in a transient decrease of mean blood pressure in both transgenic and nontransgenic control mice in a dose-dependent manner (n=8) (Fig 7). The sensitivity to BK-induced blood pres- sure reduction between the transgenic and control mice differs significantly. The vasodepressor effect of BK is more pronounced in transgenic mice than in control mice when the same dose is applied (P<.O5).

Uterine Contraction Assay To directly assess the effect of B2 receptor overexpres-

sion on myometrium inotrophy, we determined isometric

I Control

t1=3

Line 8905 Line 9830

FIG 5. Systolic blood pressure of RSV-cHBKR transgenic mice vs normal mice. The values are expressed as meaniSEM. Bars represent standard errors. *P<.OOl between WV-cHBKR trans- genie mouse lines vs control mice.

0 1 2 4

Time (Hours)

FIG 6. Systolic blood pressure of RSV-cHBKR transgenic mice vs normal mice before and after Hoe 140 administration. Hoe 140 was administered intraperitoneally at 2 pg per mouse per injection. The systolic blood pressure readings were taken at 1 .O, 2.0, and 4.0 hours after injection. The values are expressed as mean+SEM. Bars represent standard errors. *I%.05 between Hoe 140-treated vs PBS-treated RSV-cHBKR transgenic mice at 1 hour after injection.

tension induced by BK in the isolated tissue bath. In the RSV-cHBKR transgenic mice, the uterine smooth muscle contraction induced by 0.5 pmol/L BK was threefold higher than that of the control mice (n=3, P<.O5) (Fig 8). When KC1 (25 mmol/L) was applied in the tissue bath after stim- ulation with BK, no significant difference between the trans- genie and control groups on the KCl-induced contraction was observed (Fig 8). There was no significant difference in uterine weight between transgenic and control mice.

Discussion This study shows that transgenic mice overexpressing

human BK B2 receptor have a hypotensive phenotype. Two mouse lines carrying the human B2 receptor transgene were independently isolated and characterized. The aver- age blood pressure of one line was 12.7 mm Hg lower and the second line was 20.0 mm Hg lower than their nontrans-

I - I , , I . , ,

16 32 62.5 125 250 500

Bradykinin (ng)

FIG 7. Vasodepressor effect of intra-aortic bolus Injection of BK on arterial blood pressure of transgenrc and control mice. Mean blood pressure was measured via direct intra-arterial cannula after bolus injection of different doses of BK In 50 PL saline, respectively. MBP indicates mean blood pressure. *P<.O5 be- tween RSV-cHBKR transgenic group vs control group after in- jection (n=8).

by guest on June 16, 2018http://hyper.ahajournals.org/

Dow

nloaded from

Hypertension Vol29, No I, Part 2 January 1997

*P<OM 0 Control n Transgemc l-

Bradykinin (0.5 PM) KCI (25 mM)

FIG 8 Effect of BK on mouse uterus preparatron The rndrvrdual uteri were removed from the mace and prepared as described under “Methods ’ The tension of the uterine contraction was indicated by the addrtron of 0 5 pmol/L BK In RSV-cHBKR trans- genie mice and control mice KCI (25 mmol/L) was applied for the control *PC 05 between WV-cHBKR transgenrc group vs control group induced by BK (n=3)

gemc littermates. It is hkely that the more pronounced blood pressure reduction m lme No 9830 is a reflection of the higher transgene copy number m this line. These studies, using transgemc approaches, indicate that BK Bz receptor contributes to blood pressure homeostasis m VIVO.

The expression of the human B2 receptor transgene was detected at the transcriptional level m a wide vanety of tissues mcludmg the aorta, brain, heart, liver, lung, kidney, uterus, and prostate gland The expression pattern is sim- ilar to that of the B2 receptor m human tissues. 16317 The B2 receptor transgene expression was further analyzed at the protein level by a hgand-receptor binding assay, whtch revealed an increase m density of BK-bmdmg sites This increase m B2 receptor density m the transgemc mice was confirmed by a uterine contraction assay The uterus has the most abundant Bz receptor transcripts,30 and BK sttm- ulates the contraction of the uterus smooth muscle via B2 receptors 2 Our results showed that the contractthty m re- sponse to BK was markedly enhanced m the isolated uter- me horn of the transgemc mice. Moreover, the transgemc mice also displayed an enhanced blood pressure-lowermg effect by mtra-arterial bolus mJection of BK These find- mgs demonstrate that Bz receptors are overexpressed m the transgemc mice.

The hypotensive phenotype of these transgemc mice was abohshed by Hoe 140, a specific B2 receptor antago- nist, demonstrating that the reduction in blood pressure m these mice was mediated by BK B2 receptors (Fig 6) Our prevtous studtes showed that the systohc blood pressure of transgemc mice expressmg the human tissue kalhkrem gene is significantly lower than that of the control mice and the blood pressure of these mice could be restored to normal levels by Hoe 140 admmlstration 11~3~4 These re- sults demonstrated that the hypotensive effect was achieved through the B2 receptors m the transgemc mice overexpressing the kalhkrem transgene These findings suggest that the B2 receptors m normal mice are not fully occupied and that high levels of kmm m the kalhkrem transgenic mice caused a higher level of B2 receptor oc- cupancy and thus produced the hypotensive phenotype This would imply that the BK B1 receptors are present m excess with respect to local hnm levels and a further m- crease m the Bz receptor level should not affect the blood pressure of these mice Contrary to this prediction, the re-

sults of this study show that the blood pressure was stg- mficantly reduced m mice overproducmg the B2 receptor An mterpretation for this finding is that both kmms and Bz receptors are not utilized efficiently m viva Kimns have a very short half-life, and B2 receptor may not capture kmms effectively before its degradation by kmmases 31 In the kalhkrem transgemc mice, a high level of kmms may enhance the chances of then bmdmg to the BK B:! receptor Alternatively, a high level of BK B2 receptor m the B2 transgemc mice may enable the receptor to capture the free kmms more rapidly. This may m part explain the findings that overexpresston of either tissue kalhkrem or B2 recep- tor results m a hypotensive phenotype

It is also possible that a small fraction of the total B2 receptor pool exists m the activated conformation even m the absence of its agonist, thus the endogenous G-protem- coupled receptors exhibit spontaneous activity m their nat- ural envtronment m the absence of agonist occupancy 32 Q When receptor expression is high, a small fraction of the receptors m the activated conformatton could be sufficient to activate its intracellular effecters to conduct its signal transduction pathways Overexpression of B2 receptor may therefore cause a stgmficant mctease m the number of spontaneously activated B2 receptors m the transgenic mice m the absence of BK and thus produce an increase m cellular concentration of second messengers to maintain an enhanced physiological response An example in sup- port of this possibthty is provided by the studies employ- mg transgemc nuce overexpressmg ,&adrenergic receptor s4

The transgemc animal model overexpressmg BK BZ re- ceptor could be useful for studying the physiological func- tion of the BZ receptor m blood pressure regulation and mflammation Recently, another animal model was gen- erated m which the mouse BK B2 receptor gene was dis- rupted by homologous recombmatton 21 It 1s mterestmg to note that the Bz receptor-deficient mice did not show an altered blood pressure phenotype, but the contract&y of utenne smooth muscle m response to BK was ehmmated m these mace 21 These findings demonstrate the unique features and usefulness of each animal model m studying receptor function. It IS likely that both the transgemc and knockout animal models could be useful for dlssectmg the functional roles of BK B2 receptor m blood pressure regulation

Acknowledgments This work was supported by National Institutes of Health

grants HL 29397 and HL 44083

References 1 Bhoola KD, Flgueroa CD, Worthy K Blolegulatmn of kmms kal-

Ilkrem\, kmmogens, and kmmdseb Phatnmd Rev 1992,44 l-80 2 Regoh D, Barabe J Pharmacology of bladykmm and related kmms

Pharmacol Rev 1980,32 l-46 3 Hall JM Bradykmm receptors pharmacological properties and blo-

loglcal roles Pharmacol Ther 1992.56 131-190 4 Olmsted F, Page IH Hemodynamic effects of eledolsm, kalhdm II

and bradykmm m unanesthetlzed dogs Am J Phqstol 1962,203 95 l-954

5 Elhot R, Nuzum FR Urmary excretion of a depressor substance (kal- l&rem of Frey and Kraut) in artenal hypertension Endocnnolog~ 1934,18 462-474

6 Mareohus HS, Geller RG. Rsano JJ, Stoerdsma A Altered urmw kal- hkreyn excrenon m human hypertension L.uncer 1971,2 1063-1065

7 Carretero OA, Polomskl C, Hampton A, Sclch AG Urinary kalh- krem. olasma renm and aldosterone m New Zealand aenet&llv hv- perter&e (GH) rats Clan Exp Pharmncol Phyrd 1576.3 55-59 .

8 Ader JL. Tran-Van T, Praddaude FJ Low urmary excretion of actwe and total kalhkrem in young spontaneously hypertenswe rats and

by guest on June 16, 2018http://hyper.ahajournals.org/

Dow

nloaded from

Wang et al Human Bradykinin B2 Receptor Transgenic Mice 493

9

10

11

12

13

14

15

16

17

18

19

20

21.

effect of long-term anglotensm convertmg enzyme mhlbltlon J Hy- perrens 1986,4 151-153 Woodley-Mdler C, Chao J, Chao L Restnctmn fragment length poly- morphism mapped m spontaneously hypertensive rats usmg kalh- krem probes J Hypertens 1989,7 865-871 Pravenec M, Ken V, Kunes .I, Sc~ch G, Carrretero OA, Slmonet LI Cosegregation of blood pressure with a kalhkrem gene family poly- morphism Hypertension 1991.17 242-246 Wang J, Xiong W, Yang Z, Davis T, Dewey MJ, Chao J, Chao L Human tissue kalhkrem Induces hypotenslon m transgemc mice Hy- pertenszon 1994,23 236-243 Wang C, Chao L, Chao J Direct gene dehvery of human tissue kal- hkrem reduces blood pressure m spontaneously hypertensive rats J Clm Invesf 1995,95 1710-1716 Chao J, Chao L Functional analysis of human tissue kalhkrem in transgemc mouse models Hypertenston 1996.27 491-494 Song Q, Chao J, Chao L Hugh level of circulatmg human tissue kalhkrem mduces hypotenslon m a transgemc mouse model C[tn Exp Hypertens In press Hess JF, Borkowski JA, Young GS, Strader CD, Ransom RW Clon- ing and pharmacologlcal characterization of a human bradykmm (BK-2) receptor Btochem Bzophys Res Commun 1992,184 260-268 Ma JX, Wang DZ, Ward DC, Chen LM, Dessal T, Chao J, Chao L Structure and chromosomal locahzatlon of the gene (BDKRB2) en- coding human bradykmm Bz receptor Genomzts 1994.23 362-369 Ma JX, Wang DZ, Chao L, Chao J Cloning, sequence analysis and expression of the gene encoding the mouse bradykmln B2 receptor Gene 1994,149 283-288 Wang DZ, Ma JX, Chao L, Chao J Molecular clonmg and sequence analysis of rat bradykmm Bz receptor gene Etochtm Bzophys Acta 1994,1219 171-174 Pesquero JB, Lindsey CJ, Zeh K, Patva CM, Ganten D, Bader M Molecular structure and expression of rat B2 bradykmm receptor gene J Bml Chem 1994,269 26920-26925 Kdmmerer S, Braun A, Arnold N, Rosc.her AA The human brady- kmm B, receptor gene full length cDNA, genomtc orgamzatlon and ldentlficatlon of the regulatory region Btochem Bcophys Res Com- mun 1995,211 226-233 Borkowskt JA, Ransom RW, Seabrook GR, Trumbauer M, Chen H, Htll RG, Strader CD, Hess JF Targeted dtsruptton of a Bz bradyktnm

22

23

24

25

26

27

28

29

30

31

32

33

34

receptor gene m mice ellmmates btadykmm action m smooth muscle and neurons J Bzol Chem 1995,270 13706-13710 Madeddu P, Parpagha PP, Glorloso N, Chao L, Chao J Annsense mhibitlon of the kalhkretn-kmm system in spontaneously hyperten- sive rats Hypertenston In press Detrisac CJ, Sens MA, Garvm AJ, Spicer SS, Sens DA Tissue culture of human kidney eplthehal cells of proximal tubule origin Ktdney Int 1984,25 383-390 Sambrook J, Fntsch EF, Mamatts T Molecular Clonmg A Lobora- tory Manual 2nd ed Cold Spring Harbor, NY Cold Spring Harbor Laboratory Press Btebertch CJ, Utset MF. Awgulewttsch A, Ruddle FH Evidence for posmve and negattve regulat& of the Hox-3 1 gene ProcNatlAcad Set U S A 1990,87 8462-8466 Manmng DC, Vavrek R, Stewart JM, Snyder SH Two bradykmm bmdmg sites with plcomolar affinities J Pharmacol Exp Ther 1986, 237 504-5 12 Emond C, Bascands JL, Cabos-Boutot G, Pecher C, Glroldmt JP Effect of changes in sodium or water intake on glomerular B,-kmm bmdmg sites Am J Physzol 1989,257 F353-F358 Lowry OH Rosebrough NJ, Farr AL, Randall RJ Protein measurement with the Fohn phenol reagent J Blol Chem 1951,193 265-275 McPherson GA Computer-asstqted analysis of complex concentra- tion-response data J Pharmacol Methods 1985,13 125-134 McEachem AE, Shelton ER, Bhakta S, Obernolte R, Bach C, Zuppan P, FUJI&I J, Aldrich RW, Jamagm K Expression clomng of a rat B1 bradykmm receptor Proc Nat1 Acad &I II S A 1991.88 7724-7128 Saameh K, Eskes TKAB Bradykmm and cardiovascular system es- tunatton of half-life Am / Physlol 1962,203 261-265 Leeb-Lundberg LM, Mathis SA, Herzlg MC Antagonists of brady- kmm that stabthze a G-protem-uncoupled state of the Bz receptor act as inverse agonists in rat myometrtal cells J Btol Chem 1994,269 25970-25973 Bond RA, Leff P, Johnson TD, Mllano CA, Rockman HA, McMmn TR, Apparsundaram S, Hyek MF, Kenakm TP, Allen LF Physlolog- lcdl effects of inverse agonists m transgenic mice with myocardldl overexpression of the &adrenoceptor Nature 1995,374 272-276 Mdano CA, Allen LF, Rockman HA, Dolber PC, McMmn TR, Chlen KR, Johnson TD, Bond RA, Lefkowttz RJ Enhanced myoLardla1 function m transgemc mice overexpressmg the &adrenerglc recep- tor Sctence 1994,264 582-586

by guest on June 16, 2018http://hyper.ahajournals.org/

Dow

nloaded from

Dan-zhao Wang, Lee Chao and Julie Chao Receptor2Hypotension in Transgenic Mice Overexpressing Human Bradykinin B

Print ISSN: 0194-911X. Online ISSN: 1524-4563 Copyright © 1997 American Heart Association, Inc. All rights reserved.

is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231Hypertension doi: 10.1161/01.HYP.29.1.488

1997;29:488-493Hypertension. 

http://hyper.ahajournals.org/content/29/1/488the World Wide Web at:

The online version of this article, along with updated information and services, is located on

  http://hyper.ahajournals.org//subscriptions/

is online at: Hypertension Information about subscribing to Subscriptions: 

http://www.lww.com/reprints Information about reprints can be found online at: Reprints:

  document. Permissions and Rights Question and Answer information about this process is available in the

is located, click Request Permissions in the middle column of the Web page under Services. Further requestedthe Editorial Office. Once the online version of the published article for which permission is being

can be obtained via RightsLink, a service of the Copyright Clearance Center, notHypertensionpublished in Requests for permissions to reproduce figures, tables, or portions of articles originallyPermissions:

by guest on June 16, 2018http://hyper.ahajournals.org/

Dow

nloaded from