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(Hypertension. 1997;29:488.)
© 1997 American Heart Association, Inc.

State-of-the-Art-Lecture

Hypotension in Transgenic Mice Overexpressing Human Bradykinin B₂ Receptor

Dan-zhao Wang; Lee Chao; Julie Chao

From the Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston

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

	Abstract

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Bradykinin binds to its receptor at target organs and exerts a wide spectrum of biological activities including vasodilation, smooth muscle contraction and relaxation, pain, and inflammation. To gain a better insight into the physiological function of this potent vasoactive peptide, we created transgenic mice that harbor the human bradykinin B₂ receptor transgene under the control of the Rous sarcoma virus 3'-LTR promoter (RSV-cHBKR). Expression of HBKR in these transgenic mice was identified in the aorta, brain, heart, lung, liver, kidney, uterus, and prostate gland by reverse transcription-polymerase chain reaction Southern blot analysis. Two transgenic mouse lines expressing the human B₂ receptor resulted in a significant reduction of blood pressure (84.2±0.6 mm Hg, n=28; 76.9±0.8 mm Hg, n=24; P<.001) compared with the control littermates (96.9±0.4 mm Hg, n=52). Administration of Hoe 140, a bradykinin B₂ receptor antagonist, restored the blood pressure of the transgenic mice to normal levels within 1 hour, and the effect diminished within 4 hours. The transgenic mice displayed enhanced blood pressure-lowering effect induced by a bolus intra-aortic injection of kinin and showed increased response in kinin-induced uterine smooth muscle contractility compared with control littermates. These studies show that overexpression of human bradykinin B₂ receptor causes a sustained reduction of blood pressure in transgenic mice. They also suggest that the B₂ receptor-mediated signal transduction pathway plays a role in blood pressure regulation.

Key Words: human bradykinin B₂ receptor • transgenic mice • hypotension • Hoe 140 • uterine contraction

Abbreviations: BK = bradykinin • cHBKR = human BK B₂ receptor cDNA • LBK = Lys-BK • PBS = phosphate-buffered saline • RSV-LTR = Rous sarcoma virus long terminal repeat • RT-PCR = reverse transcription polymerase chain reaction

	Introduction

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Tissue kallikrein is a serine proteinase which is capable of cleaving kininogen to release bioactive kinin peptides.¹ On the binding of kinins to BK receptors, a broad spectrum of biological effects are generated. These include vasodilation, vasoconstriction, smooth muscle contraction and relaxation, ion transport, and glucose metabolism.^1–3 There are two distinct BK receptor subtypes, namely, B₁ and B₂. The B₂ receptor is widely distributed throughout mammalian tissues and has a greater affinity for intact BK and kallidin (LBK),^2,3 while the B₁ receptor has a greater affinity for kinins’ metabolites, Des-Arg⁹-BK or Des-Arg¹⁰-LBK. The B₁ receptor appears in certain pathological processes. Most of the kinins’ actions are mediated by B₂ receptors.²

BK is a potent vasoactive peptide.² Intravascular injection of BK can cause an immediate dilation of the arterial vessels with a concomitant fall in total peripheral vascular resistance and systemic blood pressure.^2,4 The tissue kallikrein-kinin system has long been implicated in blood pressure regulation. Urinary excretion of tissue kallikrein was reported to be significantly reduced in essential hypertensive patients^5,6 and genetically hypertensive rats.^7,8 Polymorphisms in the kallikrein gene have been shown to cosegregate with high blood pressure in the offspring of spontaneously hypertensive rats and normotensive Brown Norway rats.^9,10 Recently, we have established a direct link between blood pressure regulation and tissue kallikrein gene expression by transgenic animal models and somatic gene delivery strategies.^11–14 We further demonstrated that the administration of Hoe 140, a specific B₂ receptor antagonist, could restore the blood pressure of these transgenic mice to normal levels, indicating the hypotensive phenotype was mediated through BK B₂ receptor in this transgenic animal model.^13,14

The human BK B₂ receptor cDNA was cloned and sequenced from a human lung fibroblast cDNA library.¹⁵ It encodes a 364-amino acid protein with characteristics 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 structure have played a crucial role in analyzing the potential function of the B₂ receptor in blood pressure homeostasis by using transgenic and antisense approaches.^21,22 A BK B₂ receptor-deficient mouse line was generated by homologous recombination. However, these "knockout" mice failed to show any altered blood pressure phenotype.²¹ To determine the role of BK B₂ receptor in blood pressure regulation, we created a transgenic mouse model carrying the human BK B₂ receptor cDNA under control of the RSV 3'-LTR promoter.

	Methods

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Transgene Construction and DNA Preparation
The human BK B₂ receptor cDNA (1.3 kb) containing the entire coding region was amplified from the total RNA of human renal proximal tubular cells²³ by RT-PCR. The sense primer used was 5'-CCATGCCGCTTGCTCCG-3' and antisense primer was 5'-GGAATGCCAAGGAGACA-3'.¹⁶ It was then cloned into pREP8 vector (Invitrogen) at the HindIII site downstream from the RSV-LTR enhancer/promoter. The final construct pREP8-cHBKR was confirmed by restriction mapping and DNA sequencing.

The plasmid pREP8-cHBKR was extracted by the alkaline lysis method and purified by cesium chloride-ethidium bromide gradient centrifugation.²⁴ The purified plasmid DNA was digested with Sal I to release a 2.4-kb transgene fragment, which contains the RSV-LTR enhancer/promoter, human BK B₂ receptor cDNA, and an SV40 polyA segment. This fragment was separated from the vector fragment by sucrose gradient fractionation,²⁵ and dialyzed against the injection buffer (10 mmol/L Tris-HCl/0.25 mmol/L EDTA, pH 7.5). The DNA concentration was measured by absorbance at 260 nm and confirmed by comparing with DNA after agarose gel electrophoresis. It was diluted to 4 µg/mL with the injection buffer and centrifuged at 10 000g for 1 hour to remove dust particles before injection.

Generation and Identification of Transgenic Mice
The purified RSV-cHBKR-SV40 polyA DNA was microinjected into F₂ embryos (C57BL/6xDBA/2), which were allowed to develop to term in the uteri of pseudopregnant females. Offspring were screened by Southern blot analysis to identify transgenic mice. Mouse genomic DNA was extracted from the tail biopsies and digested by Apa I followed by Southern blot analysis using an [³²P]dATP nick-translated human B₂ receptor cDNA probe. Two transgenic founders, No. 8905 and No. 9830, were identified. These founder mice were bred separately with C57BL/6 to establish two independent transgenic lines. The control mice were nontransgenic littermates from the matings. All experimental protocols were approved by the Institutional Animal Research Committee of the Medical University of South Carolina and were carried out according to the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health.

Expression of Human BK B₂ Receptor Transgene
Human B₂ receptor mRNA was identified in transgenic mice by RT-PCR Southern blot analysis. Fresh tissues (aorta, brain, heart, lung, liver, kidney, and uterus or prostate) of male and female transgenic and control mice were homogenized in guanidine isothiocyanate buffer and total RNA was purified by cesium chloride gradient centrifugation.²⁴ One microgram of total RNA was subjected to RT-PCR Southern blot analysis as previously described¹² using the transgene-specific primers and an internal probe. The sequence of the 5' primer is 5'-TTCCTGGATACGCTGCATCG-3'; the 3' primer, 5'-CTCATCAATGTATCTTATCA-3'; and the internal probe, 5'-TCACTGCATTCTAGTTGTGG-3'. The blotted membrane was washed twice in 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 line was transfected with 20 µg of plasmid DNA of RSV-cHBKR by electroporation using a BTX-600 electric cell manipulator (BTX). At 72 hours after transfection, the cells were harvested and lysed in lysis buffer (5 mmol/L Tris-HCl, pH 7.4, with 5 mmol/L EDTA and 5 mmol/L EGTA) and microcentrifuged at 16 000g at 4°C for 10 minutes. The pellet was resuspended in binding assay buffer.²⁶ Ligand-binding assays were performed in duplicate.

Preparation of Iodinated Ligand and Binding Assay
Tyr⁰-BK (Sigma), the synthetic analog of BK, was used as radioligand for a BK binding assay. It was iodinated by using the chloramine-T method and purified by high-pressure liquid chromatography.²⁷ The specific radioactivity was 167.35 Ci/mmol. Crude membrane protein was prepared, and ligand binding assays were performed following the procedure described previously.²⁶ The concentration of membrane protein was determined by the Lowry assay.²⁸ For saturation studies, ¹²⁵I-Tyr⁰-BK was incubated in duplicate with 100 µg of membrane protein at 25°C in a volume of 250 µL for 60 minutes. Using a Millipore filter (Hoefer Scientific), the assay was terminated by filtration over a Whatman GF/C glass fiber filter presoaked with 0.1% aqueous polyethylenimine 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 in a 1261 Multigamma counter (Pharmacia). Saturable binding was calculated by subtracting the nonspecific binding in the presence of 10 µmol/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 Five readings were taken for each animal. Hoe 140 was diluted in PBS and injected intraperitoneally into both transgenic and control mice at 2 µg per mouse per injection. The blood pressure was measured before and at 1.0, 2.0, and 4.0 hours after Hoe 140 administration. The control group was carried out by injection of PBS into transgenic mice or by injection of Hoe 140 into nontransgenic control mice.

Blood Pressure Measurement by Arterial Cannulation and BK Injection
Mice were anesthetized by intraperitoneal injection of 2,2,2-tribromoethanol in tert-amyl alcohol (Avertin, 20 mg/mL, 0.4 mL/25 g body weight). The right femoral artery and the left carotid artery were cannulated with PE-10 catheters (Clay Adams) for direct arterial pressure recording and drug administration. The distal end of the cannula was connected to a physiological pressure transducer (Statham Laboratories) coupled with a model 7E polygraph (Grass Instrument Co). Blood pressure of the anesthetized mice was measured directly via the intra-arterial route. The mice were given a bolus intra-aortic injection of BK (Sigma). BK was serially diluted and administered at doses from 16 to 500 ng in a volume of 50µL saline per mouse.

Uterine Contraction Assay
The entire uterus was isolated and a 1-cm portion from each horn was hung in 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 FT03C and recorded on a model 7E polygraph (Grass Instrument Co). BK was applied to the tissue bath at a concentration of 0.5 µmol/L. As control, 25 mmol/L of KCl was added to the tissue bath 30 minutes after stimulation by BK.

Statistical Analysis
Group data are reported as mean±SEM. Comparisons of parameters between control and transgenic mouse groups were performed by two-way ANOVA. Differences were considered to be significant at a value of P<.05.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Generation of Transgenic Mice
Fig 1 shows the scheme for preparing the RSV-cHBKR gene fusion construct. The transgene contains the RSV-LTR enhancer-promoter element, the full-length human B₂ receptor cDNA, and a portion of the SV40 intron. 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 transcriptional level. The SV40 intron functions as heterologous 3' untranslated sequence to increase the stability of the transcripts.

View larger version (30K): [in this window] [in a new window]   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 B2 receptor cDNA was cloned into pREP8 at a HindIII restriction site and the transgene was released from pREP8 at a SalI restriction site, as indicated.

Two transgenic founders, No. 8905 (female) and No. 9830 (female), were identified from the 64 initial progenies by Southern blot analysis. The human transgene was detected as a 1.1-kb band in addition to the background of a 3.3-kb mouse endogenous B₂ receptor gene band (data not shown). A comparison of band intensity in Southern blot suggested that line No. 9830 had a higher copy number of the transgene than line No. 8905 (data not shown). There is no evidence of developmental defects associated with the RSV-cHBKR transgene as indicated by the normal morphology and litter size of both founder lines.

Transient Transfection of Human Embryonic Kidney 293 Cell Line
Expression of the functional B₂ receptor was analyzed in human 293 cells transfected with the RSV-cHBKR plasmid DNA. The results of the BK binding assay show the presence of specific B₂ receptor binding sites in the membrane protein preparation from the transfected cells. The saturation 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 nmol/L and Bmax of 1.01 pmol/mg protein as determined by analyzing saturation data with the equilibrium binding data analysis program.²⁹

View larger version (19K): [in this window] [in a new window]   FIG 2. Representative saturation binding curve and Scatchard plot analysis of human BK B2 receptor in transfected 293 cells. The specific binding of BK (y axis) to membrane proteins from 293 cells transfected with the BK B2 receptor cDNA is shown with increasing concentrations of 125I-Tyr0-BK (x axis). As control, the binding assay was performed on the membranes prepared from the nontransfected 293 cells.

Expression and Tissue Distribution of the Human BK B₂ 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, kidney, and also in the prostate of males and the uterus of females. The expression was not detected in the corresponding tissues of the control mice, indicating the specificity of the assay.

View larger version (68K): [in this window] [in a new window]   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 µg 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.

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 density 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 1.8-fold and 1.4-fold higher, respectively, than those in the control mice (Fig 4).

View larger version (44K): [in this window] [in a new window]   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).

Blood Pressure Analysis of Transgenic Mice
Both lines of transgenic mice showed significant reductions in blood pressure compared with normal controls. The systolic blood pressure measured by the tail-cuff method was 84.2±0.6 mm Hg for line No. 8905 (mean ±SEM, n=28) and 76.9±0.8 mm Hg for line No. 9830 (mean±SEM, n=24). The value for the negative siblings was 96.9±0.4 mm Hg (mean±SEM, n=52) with P<.001 (Fig 5). The values of blood pressure obtained from the indirect tail-cuff method and direct intra-arterial cannulation were compared for accuracy.

View larger version (47K): [in this window] [in a new window]   FIG 5. Systolic blood pressure of RSV-cHBKR transgenic mice vs normal mice. The values are expressed as mean±SEM. Bars represent standard errors. *P<.001 between RSV-cHBKR transgenic mouse lines vs control mice.

To determine if B₂ receptor is responsible for the hypotensive effect, Hoe 140, a specific BK B₂ receptor antagonist, was injected intraperitoneally at a dose of 2 µg 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 transgenic mice. Administration of Hoe 140 had no effect on the blood pressure of control littermates (data not shown).

View larger version (36K): [in this window] [in a new window]   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 µg per mouse per injection. The systolic blood pressure readings were taken at 1.0, 2.0, and 4.0 hours after injection. The values are expressed as mean±SEM. Bars represent standard errors. *P<.05 between Hoe 140-treated vs PBS-treated RSV-cHBKR transgenic mice at 1 hour after injection.

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 pressure 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<.05).

View larger version (15K): [in this window] [in a new window]   FIG 7. Vasodepressor effect of intra-aortic bolus injection of BK on arterial blood pressure of transgenic and control mice. Mean blood pressure was measured via direct intra-arterial cannula after bolus injection of different doses of BK in 50 µL saline, respectively. MBP indicates mean blood pressure. *P<.05 between RSV-cHBKR transgenic group vs control group after injection (n=8).

Uterine Contraction Assay
To directly assess the effect of B₂ receptor overexpression on myometrium inotrophy, we determined isometric tension induced by BK in the isolated tissue bath. In the RSV-cHBKR transgenic mice, the uterine smooth muscle contraction induced by 0.5 µmol/L BK was threefold higher than that of the control mice (n=3, P<.05) (Fig 8). When KCl (25 mmol/L) was applied in the tissue bath after stimulation with BK, no significant difference between the transgenic 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.

View larger version (15K): [in this window] [in a new window]   FIG 8. Effect of BK on mouse uterus preparation. The individual uteri were removed from the mice and prepared as described under "Methods." The tension of the uterine contraction was indicated by the addition of 0.5 µmol/L BK in RSV-cHBKR transgenic mice and control mice. KCl (25 mmol/L) was applied for the control. *P<.05 between RSV-cHBKR transgenic group vs control group induced by BK (n=3).

	Discussion

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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 average blood pressure of one line was 12.7 mm Hg lower and the second line was 20.0 mm Hg lower than their nontrans-genic littermates. It is likely that the more pronounced blood pressure reduction in line No. 9830 is a reflection of the higher transgene copy number in this line. These studies, using transgenic approaches, indicate that BK B₂ receptor contributes to blood pressure homeostasis in vivo.

The expression of the human B₂ receptor transgene was detected at the transcriptional level in a wide variety of tissues including the aorta, brain, heart, liver, lung, kidney, uterus, and prostate gland. The expression pattern is similar to that of the B₂ receptor in human tissues.^16,17 The B₂ receptor transgene expression was further analyzed at the protein level by a ligand-receptor binding assay, which revealed an increase in density of BK-binding sites. This increase in B₂ receptor density in the transgenic mice was confirmed by a uterine contraction assay. The uterus has the most abundant B₂ receptor transcripts,³⁰ and BK stimulates the contraction of the uterus smooth muscle via B₂ receptors.² Our results showed that the contractility in response to BK was markedly enhanced in the isolated uterine horn of the transgenic mice. Moreover, the transgenic mice also displayed an enhanced blood pressure-lowering effect by intra-arterial bolus injection of BK. These findings demonstrate that B₂ receptors are overexpressed in the transgenic mice.

The hypotensive phenotype of these transgenic mice was abolished by Hoe 140, a specific B₂ receptor antagonist, demonstrating that the reduction in blood pressure in these mice was mediated by BK B₂ receptors (Fig 6). Our previous studies showed that the systolic blood pressure of transgenic mice expressing the human tissue kallikrein 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 administration.^11,13,14 These results demonstrated that the hypotensive effect was achieved through the B₂ receptors in the transgenic mice overexpressing the kallikrein transgene. These findings suggest that the B₂ receptors in normal mice are not fully occupied and that high levels of kinin in the kallikrein transgenic mice caused a higher level of B₂ receptor occupancy and thus produced the hypotensive phenotype. This would imply that the BK B₂ receptors are present in excess with respect to local kinin levels and a further increase in the B₂ receptor level should not affect the blood pressure of these mice. Contrary to this prediction, the results of this study show that the blood pressure was significantly reduced in mice overproducing the B₂ receptor. An interpretation for this finding is that both kinins and B₂ receptors are not utilized efficiently in vivo. Kinins have a very short half-life, and B₂ receptor may not capture kinins effectively before its degradation by kininases.³¹ In the kallikrein transgenic mice, a high level of kinins may enhance the chances of their binding to the BK B₂ receptor. Alternatively, a high level of BK B₂ receptor in the B₂ transgenic mice may enable the receptor to capture the free kinins more rapidly. This may in part explain the findings that overexpression of either tissue kallikrein or B₂ receptor results in a hypotensive phenotype.

It is also possible that a small fraction of the total B₂ receptor pool exists in the activated conformation even in the absence of its agonist, thus the endogenous G-protein-coupled receptors exhibit spontaneous activity in their natural environment in the absence of agonist occupancy.^32,33 When receptor expression is high, a small fraction of the receptors in the activated conformation could be sufficient to activate its intracellular effecters to conduct its signal transduction pathways. Overexpression of B₂ receptor may therefore cause a significant increase in the number of spontaneously activated B₂ receptors in the transgenic mice in the absence of BK and thus produce an increase in cellular concentration of second messengers to maintain an enhanced physiological response. An example in support of this possibility is provided by the studies employing transgenic mice overexpressing ß₂-adrenergic receptor.³⁴

The transgenic animal model overexpressing BK B₂ receptor could be useful for studying the physiological function of the B₂ receptor in blood pressure regulation and inflammation. Recently, another animal model was generated in which the mouse BK B₂ receptor gene was disrupted by homologous recombination.²¹ It is interesting to note that the B₂ receptor-deficient mice did not show an altered blood pressure phenotype, but the contractility of uterine smooth muscle in response to BK was eliminated in these mice.²¹ These findings demonstrate the unique features and usefulness of each animal model in studying receptor function. It is likely that both the transgenic and knockout animal models could be useful for dissecting the functional roles of BK B₂ receptor in blood pressure regulation.

	Acknowledgments

 
This work was supported by National Institutes of Health grants HL 29397 and HL 44083.

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