©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Targeted Disruption of a B Bradykinin Receptor Gene in Mice Eliminates Bradykinin Action in Smooth Muscle and Neurons (*)

Joseph A. Borkowski (1), Richard W. Ransom (3), Guy R. Seabrook (4), Myrna Trumbauer (2), Howard Chen (2), Ray G. Hill (4), Catherine D. Strader (1), J. Fred Hess (1)(§)

From the (1) Department of Molecular Pharmacology & Biochemistry and the (2) Department of Animal Biochemistry & Molecular Biology, Merck Research Laboratories, Rahway, New Jersey 07065, the (3) Department of New Lead Pharmacology, Merck Research Laboratories, West Point, Pennsylvania 19586, and the (4) Department of Pharmacology, Merck Sharp & Dohme Research Laboratories, Terlings Park, Harlow, Essex, CM20 2QR, United Kingdom

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Mice that are homozygous for the targeted disruption of the gene encoding the B bradykinin receptor have been generated. The gene disruption results in a deletion of the entire coding sequence for the B receptor. The disruption of the B receptor gene has been confirmed by genetic, biochemical, and pharmacological analyses. Mice that are homozygous for the disruption of the B receptor gene are fertile and indistinguishable from their littermates by visual inspection. Bradykinin fails to produce responses in pharmacological preparations from ileum, uterus, and the superior cervical ganglia from these mice. Therefore, expression of a single gene appears to be responsible for conferring responsiveness to bradykinin in these tissues.


INTRODUCTION

The nonapeptide hormone bradykinin (BK)() and the decapeptide kallidin ([Lys]BK) are generated by the action of a family of serine proteases, kallikreins, on a large protein precursor, kininogen, present in plasma and tissue (1, 2, 3, 4) . The removal of the C-terminal arginine residue from these ligands by a carboxypeptidase generates a second set of active hormones (5, 6) . Collectively, these kinins are generated at sites of tissue damage and are rapidly degraded by the angiotensin converting enzyme and other proteases. BK and kallidin are algesic and inflammatory agents. BK acts on afferent sensory neurons to elicit pain and to release additional mediators of pain and inflammation. BK is also a very potent vasoactive hormone that appears to act in a paracrine rather than a systemic fashion. Vasodilation mediated by BK in the airways may contribute to the pathogenesis of asthma (7) . In contrast to this potentially deleterious effect, the vasodilatory properties of BK may be beneficial following an ischemic event (8, 9) .

The physiological actions of the kinins are a consequence of their interaction with transmembrane receptors. Two distinct bradykinin receptor subtypes, B and B, have been proposed by pharmacological analyses (5) and shown to exist by the molecular cloning of cDNAs encoding G-protein-coupled receptors with the appropriate pharmacological profiles (10, 11) . Whereas the B bradykinin receptor subtype is found in healthy smooth muscle and neurons, the B receptor subtype is only detected following tissue injury (12, 13, 14) . The actions of bradykinin and kallidin reside with the B receptor which possesses a high affinity for these ligands but has a dramatically reduced affinity for their carboxypeptidase-generated metabolites (11, 15) . By contrast, the B bradykinin receptor is preferentially activated by [des-Arg]kallidin and possesses a low affinity for bradykinin (5, 10, 16) . The potential for transformation of the selectivity of a peptide ligand from the B to the B receptor by carboxypeptidase action has been observed with both agonists and antagonists (17, 18) . When activated, both receptor subtypes interact with G-proteins to initiate a signal transduction cascade that stimulates phospholipid metabolism. The B receptor subtype activates both phospholipase C and phospholipase A, resulting in the mobilization of Ca and prostaglandin synthesis (19, 20, 21) . Similarly, activation of the B receptor subtype has been recently shown to stimulate phosphatidylinositol metabolism and arachidonic acid release (16, 22) .

Pharmacological studies of bradykinin receptors have led to the proposal that additional bradykinin receptor subtypes exist (23, 24, 25, 26, 27, 28, 29, 30) . Several of these studies are complicated by the fact that they compared the action of synthetic peptide antagonists on bradykinin receptors in tissues isolated from different species (23, 26) . However, other studies describe differences in the pharmacological properties of bradykinin receptors from different tissues within the same species (24, 25, 27-30). From these analyses, separate smooth muscle and neuronal subtypes of the B bradykinin receptor have been proposed (24, 25) . In addition, a B bradykinin receptor subtype has been proposed based on the differential activity of peptide and non-peptide antagonists in blocking BK-induced contraction of the guinea pig trachea (27, 28) .

Much has been learned about the physiological role of these receptors from the development and use of potent B and B bradykinin receptor antagonists (18, 31, 32, 33) , but these reagents are limited by their pharmacokinetic properties. Disruption of a gene by homologous recombination has become a valuable tool in discerning the physiological roles of gene products (34) . We describe here the generation and initial characterization of mice in which the gene encoding the B bradykinin receptor is disrupted by gene targeting. Smooth muscle and neuronal preparations from Bk2r mice have been examined for their ability to respond to BK. The genetic ablation of the B bradykinin receptor will complement pharmacological analyses to define the physiological role of this receptor. In addition, the Bk2r mouse will allow the question of additional genes encoding B bradykinin receptor subtypes to be answered unambiguously.


MATERIALS AND METHODS

Generation of Bk2rMice-Gene targeting was done in the embryonic stem (ES) cell line AB 2.1 (35) derived from J129 Sv mice. The mouse genomic DNA utilized to construct the targeting vector was obtained from a cosmid clone isolated from a library constructed by Dr. John Mudgett (Merck Research Laboratory, Rahway, NJ) from an ES cell line, J1, derived from J129 Sv/Ev mice. ES cell clones containing the targeted disruption of the Bk2r gene were separated from SNL feeder cells by treating the cell culture with trypsin, allowing the feeder cells to reattach for 30-45 min, and removing the unattached ES cells. Two Bk2r-targeted ES clones, KO-5 and KO-24, were injected into C57Bl/6J recipient 3-day-old blastocysts in separate experiments using established techniques (36) . The injected C57Bl/6J recipient blastocysts were reimplanted into day 3 pseudopregnant Tac:SW(fBR) mice and allowed to develop to term. Progeny were screened initially by coat color chimerism, the agouti color being an indicator of ES cell contribution.

Genomic DNA was prepared from a tail biopsy collected by snipping approximately 1.5 cm from the end of the tail of the mouse. Tails were digested overnight at 55 °C by 0.3 µg of Proteinase K in 0.7 ml of buffer containing 50 mM Tris, pH 8.0, 100 mM EDTA, and 0.5% SDS. The DNA was extracted twice with 0.5 ml of phenol/chloroform and once with chloroform. Genomic DNA was precipitated with an equal volume of ethanol, and the DNA clump was transferred using a pipette tip to a 70% ethanol wash and then transferred to and resuspended in TE (10 mM Tris, pH 7.4, 1 mM EDTA).

Genomic Southern blots were performed by modifications of established procedures. Approximately 10 µg of genomic DNA was digested overnight with the appropriate restriction enzyme. The digest was extracted with phenol/chloroform, and chloroform, then ethanol-precipitated. The pellet was resuspended in TE, and the DNA was separated by electrophoresis on an 0.8% agarose gel. DNA on the gel was transferred to a Zeta-Probe GT membrane (Bio-Rad) with a PosiBlot apparatus (Stratagene) and then UV-cross-linked with a Stratalinker (Stratagene). The DNA on the membrane was incubated overnight at 65 °C with >1 10 cpm/ml radiolabeled probe in a solution containing 0.25 M NaHPO, pH 7.2, 6.5% SDS, and 10% dextran sulfate. Probes were radiolabeled with [-P]dCTP (3000 Ci/mmol, Amersham) using a random priming kit (Boehringer Mannheim). Hybridized filters were washed at 60 °C with 0.1 SSC, 0.1% SDS twice for 30 min and then subjected to autoradiography.

Membrane Binding Assay

Mice were sacrificed by cervical dislocation. The uterus and ileum were removed and the wet weight was determined. The tissues were homogenized, using a Polytron, in approximately 20 volumes of ice-cold 20 mM HEPES, pH 7.4. Membranes were prepared, and binding assays were performed as described previously (37) .

Uterine Contraction Assay

The entire uterus was removed, and the two horns were separated. The uterus horns were placed in buffer (154 mM NaCl, 5.6 mM KCl, 0.45 mM CaCl, 5.9 mM NaHCO, 2.6 mM glucose) bubbled with 95% O/5% CO, and connective tissue and fat were removed. A 2-cm portion was removed from each horn and attached to a glass tissue holder with 3-0 surgical silk and placed in a tissue bath containing buffer maintained at 30 °C and bubbled with O/CO as above. One gram of tension was applied to the tissue, and tension was measured by a Gould Statham force transducer and recorded on a HP 7758B system. Tissues were washed four times over a 1-h equilibration period prior to the addition of pharmacological agents. The tension was then readjusted and compounds were added; following the addition of each compound, the preparation was washed for 30-45 min before the addition of the subsequent compound.

Superior Cervical Ganglia Assay

Superior cervical ganglia were removed and dissected as described previously (38) . Ganglia from control and Bk2r mice were desheathed and placed in a grease-gap recording chamber. The chamber was perfused with buffer (125 mM NaCl, 5 mM KCl, 1 mM KHPO, 2.5 mM CaCl, 1 mM MgSO, 25 mM NaHCO, 10 mM glucose, and 0.1 µM tetrodotoxin) maintained at 25 °C and bubbled with gas as described above. The potential difference between the ganglion cell body and the postganglionic trunk was measured using Ag/AgCl electrode connected by a DC amplifier to a chart recorder. Between the addition of each pharmacological agent, the preparation was washed for 40-60 min.


RESULTS

Embryonic stem (ES) cells (AB 2.1) derived from mouse strain J129 Sv/Ev were transfected with a targeting vector designed to disrupt the Bk2r gene encoding the B bradykinin receptor. The coding sequence for the mouse B bradykinin receptor lies on a single exon (39, 40) which corresponds to exon 4 in the structure of the rat B bradykinin receptor gene (41) . The pharmacological profile of this mouse bradykinin receptor expressed in mammalian cells is consistent with the B subtype classification (40) . The positive-negative selection (34) targeting vector constructed contains 1 kb of mouse genomic DNA immediately upstream of the coding sequence of the mouse B bradykinin receptor, a neomycin resistance gene which replaces the coding sequence of the B bradykinin receptor, 5.4 kb of mouse genomic DNA downstream of the B receptor coding sequence, and a tk gene (Fig. 1A). Homologous recombination of this vector with the mouse chromosome should result in the replacement of the entire coding sequence of the mouse B bradykinin receptor with the neomycin resistance gene. The drug (1-(2-deoxy 2-fluoro-B-D-arabinofluranosyl)-5-iodouracil) (FIAU) was utilized to enrich for ES cell clones containing a homologous recombination event by selecting against the presence of the tk gene (34) . Genomic Southern analysis was utilized to identify three positive ES cell clones (42) . Two of these clones were injected into mouse blastocysts isolated from C57Bl/6 mice; one produced four highly chimeric male mice. Each of these four chimeric mice was bred to C57Bl/6 wild type mice; three of the mice were found to transmit the agouti coat color marker associated with the embryonic stem cells to offspring, indicating germ line transmission. Genomic DNA was isolated from the agouti offspring, digested with DraI, and probed with a 1.6-kb fragment that lies downstream of the DNA contained in the targeting vector. The presence of the disrupted allele was monitored by the shift of the DraI fragment from 9.0 kb in wild type to 7.5 kb (Fig. 1). Approximately 50% of these F mice were found to be Bk2r. Bk2r siblings were interbred to obtain mice homozygous for the disruption. The progeny of these matings generated the expected genotypes: homozygous wild type, heterozygotes, and homozygous knockouts (Fig. 1B).


Figure 1: A, schematic of the targeted disruption of the Bk2r gene. The targeting vector (top), the endogenous Bk2r gene (middle) with the restriction sites, and probes were utilized to detect restriction fragments indicative of the wild type chromosome. Restriction sites are BamHI (B), DraI (D), EcoRI (R), and NsiI (N). The initiation of the putative coding sequence for the B bradykinin receptor is indicated by ATG and the stop by TGA. The targeted Bk2r allele (bottom) with the restriction sites and probes were utilized to discern restriction fragments diagnostic of the disrupted chromosome. B, genomic Southern blot of DNA isolated from the progeny of mating heterozygous Bk2r mice. The genomic DNA was digested with DraI and probed with the 1.6-kb 3` probe. Representatives of the three genotypes are shown. Lane 1, Bk2r; lane 2, Bk2r; lane 3, Bk2r.



The deletion of the coding sequence for the B bradykinin receptor in the Bk2r mice was confirmed by genomic Southern analysis. The single band of 9 kb that was detected in both genomic DNA from Bk2r and Bk2r mice by an 818-bp probe containing the DNA encoding the putative seven transmembrane helices of the receptor was absent in Bk2r mice (Fig. 2). To confirm that the expected gene targeting occurred upstream of the B receptor coding sequence genomic DNA extracted from 26 Bk2r mice was digested with EcoRI and probed with a 600-bp fragment upstream of the DNA contained in the targeting vector. A single band of 4 kb indicative of the disrupted allele was detected in each of the Bk2r mice, whereas the wild type band of 6 kb was detected in controls (data not shown). Taken together, these results indicate that the desired gene targeting event disrupted the B bradykinin receptor gene in the expected manner which resulted in the deletion of the coding sequence for the receptor.


Figure 2: Genomic Southern blot of DNA extracted from Bk2r (1), Bk2r (2), Bk2r (3), and Bk2r (4). The genomic DNA was digested with DraI and probed with the 3` probe (A). The blot was stripped and reprobed (B) with an 818-bp probe generated by polymerase chain reaction using the primers 5`-TTCCTCTGGGTGCTGTTCGT-3` and 5`-CACGATCACGTACACCAGTG-3` using the cloned mouse B bradykinin receptor as a template. These polymerase chain reaction primers are based on the human B bradykinin receptor DNA sequence and correspond to sequences encoding putative transmembrane domain 1 and transmembrane domain 7 of the B bradykinin receptor.



A total of 158 F progeny were generated by breeding Bk2r siblings. The genotype distribution of the progeny was 34 Bk2r, 76 Bk2r, and 48 Bk2r. analysis indicates that these results do not differ significantly from the predicted 1:2:1 Mendelian ratio for the transmission of the disrupted allele (p = 0.26). Thus, the disruption of the B receptor gene does not appear to have any gross effects on mouse development and viability. Adult and juvenile Bk2r mice are indistinguishable from heterozygous or homozygous wild type littermates by visual inspection. Clinical pathology was performed on six Bk2r mice (three male and three female) and four control mice (two Bk2r and two Bk2r mice; one of each sex). There were no differences in serum sodium, potassium, calcium, chloride, or phosphorus between control and knockout animals. In addition, no abnormalities were detected in either the gross pathology or the histopathology of the Bk2r mice.

The proposed role of BK receptors in reproduction was examined by establishing five mating pairs of Bk2r mice. All five of the mating pairs generated offspring. Four of the mating pairs produced offspring approximately 3 weeks after mating, with an average litter size of nine pups. The fifth mating pair produced offspring after a delay of 3 months. These results indicate that the B bradykinin receptor is not critical for fertility.

Previous studies have indicated that a high level of B receptor expression is found in the ileum and uterus (11, 40) , tissues that have been used extensively in characterizing BK receptor pharmacology and function. Membranes were prepared from ileum and uterus from six control (either Bk2r or Bk2r) and six Bk2r mice and tested for their ability to bind 1 nM [H]BK (Fig. 3). The ilea from individual control animals exhibited 5.9 ± 1.1 pmol/g of tissue (wet weight) of specific BK binding sites. By contrast, no BK binding sites were detected in membranes prepared from the ilea of Bk2r mice. Similarly, specific BK binding sites were detected in uterine membranes from control mice but not from Bk2r mice. Therefore, the disruption of the gene encoding the B bradykinin receptor eliminates the [H]BK binding site in these two smooth muscle preparations.


Figure 3: Binding of 1 nM [H]BK to membranes from smooth muscle tissues of control and Bk2r mice. Membranes were prepared from the ileum and uterus of Bk2r and Bk2r control (CON) mice and Bk2r ``knockout'' (KO) mice. The ileum membranes were prepared from ilea removed from individual animals and assayed separately. Whereas uterine tissue was removed from the six control animals and pooled prior to preparing membranes, likewise the uterine tissue removed from six Bk2r mice was pooled. Binding assays were performed as described under ``Materials and Methods.''



The action of bradykinin on the uterus is to elicit a smooth muscle contraction (5) . In control mice, 1 uM BK contracted uterine muscle strips as did 1 µM oxytocin and 50 mM KCl (Fig. 4). By contrast, 1 µM BK was unable to produce a contraction in Bk2r mice (n = 11). The uterine muscle from the Bk2r mice was capable of contracting as demonstrated by the ability of oxytocin or KCl to stimulate a contraction to the same degree as the controls. Thus, the disruption of the gene encoding the B bradykinin receptor destroys not only the BK binding site in the uterus but also the functional response to BK. The B receptor agonist [des-Arg]kallidin was inactive on the uteri from control and Bk2r mice (data not shown). Taken together, these results strongly indicate that the actions of BK in these smooth muscle preparations are mediated through a single B bradykinin receptor subtype.


Figure 4: Uterine contraction assay. The individual uteri were removed from the mice and prepared as described under ``Materials and Methods.'' The tension of the uterine contraction elicited by the addition, in order, of 1 µM bradykinin (BK), 1 µM oxytocin (OT), and 50 mM KCl was analyzed in Bk2r and Bk2r control (C) mice and Bk2r ``knockout'' (KO) mice. Between the addition of each agent, the preparation was washed for 30-40 min.



Previous studies have suggested the existence of smooth muscle and neuronal subtypes of the B bradykinin receptor (24, 25) . To address this issue, the effect of the disruption of the B bradykinin receptor gene on a BK response in a neuronal preparation was analyzed. BK caused a dose-dependent depolarization of the isolated superior cervical ganglia from Bk2r control mice with a maximum response at 3 µM of 168 ± 21% relative to 1 µM muscarine (n = 6) (data not shown). In contrast, BK was totally inactive on the superior cervical ganglia from Bk2r mice (n = 8), whereas the ability of either muscarine or substance P methyl ester (23 ± 8%, n = 4) to depolarize the ganglia was unaltered (Fig. 5). Thus, disruption of the B receptor gene eliminates BK-mediated actions in both neuronal and smooth muscle preparations, supporting the idea that a single gene encodes the B bradykinin receptor subtype in mammals.


Figure 5: Depolarization of the superior cervical ganglia from control and Bk2r mice. The superior cervical ganglia was isolated and prepared as described under ``Materials and Methods.'' Depolarizations to agents administered at the time indicated by the arrow were analyzed in Bk2r mice (A) and Bk2r mice (B). The depolarization was elicited by 1 µM muscarine, 3 µM bradykinin, and 1 µM substance P methyl ester (SP-O-Me). The agents were added in the order indicated, and the preparation was washed 40-60 min between each agent. The time and voltage scale is indicated by the inset.




DISCUSSION

We have generated a mouse in which the B bradykinin receptor is disrupted by gene targeting, resulting in a deletion of the entire coding sequence for this receptor. Molecular genetic analysis indicates that the disruption occurred in the predicted manner. The disruption of the B receptor has no visible phenotype. However, the loss of B receptor function can be readily demonstrated in several tissues, indicating that the gene encoding the receptor has indeed been disrupted.

A role of kinins in reproduction has been proposed due to the presence of the components of the kallikrein-kinin system in the uterus and in semen. The uterus contains a relatively high density of B bradykinin receptors. BK has been demonstrated to be a proinflammatory agent, and the implantation of the embryo has been likened to an inflammatory response (43) . This information, coupled with recent work showing an increase in kallikrein levels in the implantation node in rats, has led to the proposal that kinins participate in the implantation of the embryo by regulating local blood flow (43) . The results of other studies indicate that BK may be an important factor in promoting sperm motility (44) . Our results demonstrate that the Bk2r mice are fertile and that they have normal litter sizes. Although these data do not rule out a role for kinins in reproduction, they demonstrate that the B receptor does not play an essential role in this process. It remains possible that the kinin pathway is involved in reproduction by acting through the B bradykinin receptor, which responds to the carboxypeptidase metabolites of kallidin and bradykinin. However, it remains to be determined whether the expression of the B bradykinin receptor is induced during reproductive processes.

Previously, several pharmacological studies have proposed the existence of multiple B bradykinin subtypes, possibly encoded by different genes (23, 24, 25, 26, 29, 30) . A number of these studies were based on pharmacological differences in tissue preparations isolated from different species. More recent studies using the potent B receptor antagonist HOE-140 have clarified some of the previous results and led to the proposal that much of the pharmacological diversity observed was due to differences in the species from which the receptor was derived (45) . This proposal was supported by the demonstration that cloned species homologs of the B bradykinin receptor are pharmacologically distinct (39, 40) . However, pharmacological differences have also been observed in different tissues from the same species. For example, in rat vas deferens [Thi,D-Phe]BK acts as a full agonist in stimulating sympathetic nerves to elicit muscle twitch and as a weak partial agonist in directly stimulating smooth muscle contraction (25) . These observations, coupled with differences in the ability of [D-Arg,Hyp,Thi,D-Phe]BK to antagonize the neurogenic and musculotropic effects of BK (24) , led to the proposal of different smooth muscle and neuronal B bradykinin receptor subtypes (24, 25) . The finding of the present study that BK receptor function is totally eliminated in both the uterus and the sympathetic superior cervical ganglia of Bk2r mice clearly demonstrates that a single gene is responsible for sensitivity to BK in both of these tissues. This observation suggests that the pharmacological differences previously reported between neuronal and smooth muscle preparations are not a consequence of genetic diversity. The cloning of identical B bradykinin receptor cDNAs from cultured neuronal cells and smooth muscle is also consistent with a single B bradykinin receptor gene encoding BK receptors in both smooth muscle and neurons (39, 46, 47) . A recent report has suggested that two different subtypes of B receptors, termed B and B and characterized by differing affinities for BK analogues (K for BK of 2 pM and 910 pM, respectively), are present in guinea pig ileum membranes (30) ; similar findings were previously reported in rat myometrial membranes (29) . The total loss of all specific [H]BK binding sites in membranes prepared from the ileum of Bk2r mice and loss of BK action in the uterus argues that these high and low affinity sites are not due to genetically distinct receptor subtypes. Our results are consistent with a single gene encoding the B bradykinin receptor subtype; however, an exhaustive survey of the effects of BK on intact animals and on tissues isolated from control and Bk2r mice will be necessary to determine whether other genes might encode additional B bradykinin receptor subtypes or the proposed tracheal B receptor subtype (27, 28) .

The demonstration that the Bk2r mice have the phenotype of BK insensitivity in several different tissues indicates that we have produced an animal devoid of the B bradykinin receptor. The Bk2r mice will provide an animal model for the evaluation of the diverse roles of the B bradykinin receptor in the inflammatory response, the cardiovascular system, renal function, and reproduction. The Bk2r mice will also provide an animal model for the exploration of the role of the B receptor in the absence of the B receptor.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence and reprint requests should be addressed.

The abbreviations used are: BK, bradykinin; ES, embryonic stem; kb, kilobase(s); bp, base pair(s).


ACKNOWLEDGEMENTS

We wish to thank Drs. John Mudgett and Lex Van der Ploeg for helpful advice. We thank Dr. Karla Stevens for clinical pathology, Frank Shen for statistical analyses, and B. Bowery for excellent technical assistance.


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