Overexpression of kinin B1 receptors induces hypertensive response to des-Arg9-bradykinin and susceptibility to inflammation.

We demonstrated that rat kinin B(1) receptors displayed a ligand-independent constitutive activity, assessed through inositol phosphate production in transiently or stably transfected human embryonic kidney 293A cells. Substitution of Ala for Asn(130) in the third transmembrane domain resulted in additional constitutive activation of the B(1) receptor. The constitutively active mutant N130A receptor could be further activated by the B(1) receptor agonist des-Arg(9)-bradykinin. To gain insights into the physiological function of the B(1) receptor, we have generated transgenic mice overexpressing wild-type and constitutively active mutant receptors under the control of human cytomegalovirus immediately early gene enhancer/promoter. The rat B(1) receptor transgene expression was detected in the aorta, brain, heart, lung, liver, kidney, uterus, and prostate of transgenic mice by reverse transcription-polymerase chain reaction/Southern blot analysis. Transgenic mice were fertile and normotensive. Overexpression of B(1) receptors exacerbated paw edema induced by carrageenan and rendered transgenic mice more susceptible to lipopolysaccharide-induced endotoxic shock. Interestingly, the hemodynamic response to kinins was altered in transgenic mice, with des-Arg(9)-bradykinin inducing blood pressure increase when intravenously administered. Our study supports an important role for B(1) receptors in modulating inflammatory responses and for the first time demonstrates that B(1) receptors mediate a hypertensive response to des-Arg(9)-bradykinin.

Lys-BK or kallidin in humans (2). In contrast, the B 1 receptor is expressed at very low levels in normal tissues in most animal species but is induced under the influence of inflammation or exposure of tissues to noxious stimuli, mediating the effects of the carboxypeptidase metabolites of intact kinins, des-Arg 9 -BK (DABK), and des-Arg 10 -kallidin (2). The cellular responses of kinin receptors to agonists are transduced primarily via coupling to either G q protein, which in turn activates phospholipase C to stimulate inositol phosphate production, or the G i protein, acting through phospholipase A 2 to stimulate arachidonic acid pathway (5,6).
Over the past years, transgenic and gene-targeting technologies associated with molecular biology tools have provided important knowledge concerning the role of kinin receptors in vivo. Transgenic mice expressing the human B 2 receptor under the control of the Rous sarcoma virus 3Ј-long terminal repeat promoter were hypotensive compared with control littermates (7). Administration of the B 2 receptor antagonist Hoe-140 blunted the blood pressure-lowering effect of the transgene, whereas intra-arterial bolus injection of BK produced more pronounced blood pressure reduction (7). In contrast, deletion of the B 2 receptor in mice produced an unaltered blood pressure phenotype (8) but led to salt-sensitive hypertension and altered nociception (9,10). Using specific antagonists, the B 1 receptor has been implicated in toxic shock, inflammation, and nociception (11). Studies of mice lacking the B 1 receptor provided support to these observations. B 1 receptor knockout animals were healthy, fertile, and normotensive and exhibited hypoalgesia and reduced inflammatory response (12).
Although much has been learned about the physiological role of the B 1 receptor, most studies are about the lipopolysaccharide (LPS)-induced B 1 receptors, because the B 1 receptor is expressed at very low levels, if at all, in normal tissue. In such experimental set-ups, the animals are under systemic inflammation conditions, which preclude the direct study of the function of the B 1 receptor. Therefore, the precise physiological and pathophysiological roles of the B 1 receptor remain elusive. To extend our understanding of the physiological function of the B 1 receptor, we have created a constitutively active mutant of the rat B 1 receptor and generated transgenic mice that overexpress the wild-type B 1 receptor and the mutant receptor under the control of human cytomegalovirus immediately early gene enhancer/promoter. The transgenic mice were characterized, and the findings are reported here.

EXPERIMENTAL PROCEDURES
Materials-Human embryonic kidney 293A cells were obtained from Quantum Biotechnologies. LPS (Salmonella enteritidis, LD 50 : 7.20 mg/ kg) was from Difco Laboratories. myo-[ 3 H]Inositol was from PerkinElmer Life Sciences. LipofectAMINE, culture medium, restriction enzymes, and fetal calf serum were bought from Invitrogen. Puro-* This work was supported by National Institutes of Health Grants HL 29397 and HL52196. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Site-directed Mutagenesis-The mutations were created by using the QuikChange site-directed mutagenesis kit (Stratagene), and the previously cloned wild-type rat B 1 cDNA in pcDNA3 (Invitrogen) was used as a template (14). The following oligonucleotides were used as forward primers: 5Ј-GGC CTC TTG GGG GCC CTT TTA GTC TTG TC-3Ј for the  preparation of clone N54A (nucleotide changes underlined), 5Ј-GTC  ATC AAG GCC GCC CTG TTT GTC AG-3Ј for clone N130A, 5Ј-GCT  ATC AGT CAG CAA CGC TAC AGG CTC-3Ј for clone D144Q, and  5Ј-GCT ATC AGT CAG ACC CGC TAC AGG CTC-3Ј for clone D144T. The reverse primers were complementary to the forward primers described. All of the mutations were confirmed by DNA sequencing. The NruI/NotI fragments in the resultant plasmids were released and inserted at NruI/NotI sites in pIRESpuro (Clontech Lab), leading to vectors for stable transfection.
Transfection and Selection-For transient transfection, 293A cells were seeded into 12-well trays and left to adhere overnight. pcDNA3derived expression vectors were transfected with LipofectAMINE as previously described (14). Transfection mixtures were left on cells for 5 h, and then the cells were treated with a change of standard growth medium for 48 h before functional studies.
For stable transfection, 293A cells were transfected with pIRESpuroderived expression vectors. After 16 h, the medium was changed with complete medium containing puromycin (2 g/ml) to start the selection of stably transfected cells. The medium was changed every 3 days, and after about 12 days, the colonies surviving selection were lifted into 12-well plates, expanded with a maintenance concentration of 2 g/ml puromycin, and screened for ligand specific binding. All of the stock cultures were kept under constant selection pressure of 2 g/ml puromycin, whereas cells seeded in dishes/wells were maintained without puromycin and used within 2-3 days.
Total Inositol Phosphate Measurement-Monolayers of transfected 293A cells grown in 12-well trays were labeled for 24 h with 2 Ci of [ 3 H]inositol in 0.5 ml of inositol-free Dulbecco's modified Eagles' medium supplemented with 0.05% bovine serum albumin and penicillin/ streptomycin. After equilibrated in prewarmed Dulbecco's modified Eagles' medium containing 140 g/ml bacitracin, 100 M Captopril, and 25 mM LiCl for 15-30 min, the cells were stimulated with various ligands at the indicated concentrations for 20 min at 37°C. The released total inositol phosphates (IP) were isolated using Bio-Rad AG1-X8 anion exchange columns (1-ml volume) and quantified as described (15,16).
Radioligand Binding to Intact Cells-Transfected 293A cell monolayers in 12-well plates were washed twice with Dulbecco's modified Eagles' medium and incubated at 4°C with radioligand [ 125 I]Sar-Tyr-⑀Ahx-Lys-des-Arg 9 -bradykinin (14) in the presence or absence of 5 M of the unlabeled ligand in 0.3 ml of Dulbecco's phosphate-buffered saline supplemented with 140 g/ml bacitracin, 1 mg/ml bovine serum albumin, 1 mM 1,10-phenanthroline, and 100 M Captopril. The incubation lasted at least 3 h under gentle agitation. The cells were then rinsed twice with ice-cold phosphate-buffered saline with 0.3% bovine serum albumin followed by solubilization in 0.5 ml of 0.1 M NaOH. The radioactivity of the sample was quantified with a 1261 Mutigamma counter (Pharmacia Corp.). The cell number was determined in parallel wells.
cGMP and cAMP Assays-Stably transfected 293A cells grown in 6-well plates were preincubated for 15 min with Dulbecco's modified Eagles' medium containing 1 mM 3-isobutyl-1-methylxanthine and 100 M Captopril and then stimulated with 1 M DABK for 15 min. The stimulation was terminated by exchanging the incubation medium for 0.5 ml of ice-cold 0.1 M HCl. The cGMP and cAMP productions were determined by radioimmunoassys as described (17,18).
Construction of Wild-type and N130A Mutant Rat B 1 Receptor Transgenes-The bovine growth hormone poly(A) sequence in pcDNA3 was released with ApaI and PvuII and inserted at ApaI/EcoRV sites in pBluescript KS II (Stratagene). The ApaI/SmaI bovine growth hormone poly(A) fragment was then released and inserted at ApaI/SmaI sites in the pcDNA3-derived wild-type and N130A receptor expression vectors described above. The human 4F2 enhancer was amplified by PCR from genomic DNA with the primers 5Ј-TAC TCG AGT GCA GCG CGC CCC CG-3Ј and 5Ј-CTG GGC CCT TCA CCT TCA GAG AGC-3Ј (19). After being cut with XbaI and ApaI, the 4F2 enhancer fragment was inserted at XbaI/ApaI sites, resulting in final transgene vectors. The vectors were cut with NruI and SmaI, and the linear transgenes were separated from the unneeded fragments with agarose gel and prepared for injection by Qiaquick gel extraction columns (Qiagen).
Generation of B 1 Receptor Transgenic Mice-Transgenic mice were created by the Transgenic Facility at the Medical University of South Carolina and the Transgenic Facility of University of Ohio at Cincinnati. Linear transgene was injected into the pronuclei of one-cell mouse embryos, which were then surgically implanted into pseudopregnant female mice. Transgenic founder mice were identified by Southern blot analysis of genomic DNA isolated from tail biopsies. Positive founders identified from each line were bred with normal mice, and then F1 littermates were crossed between themselves. Tail DNA was digested with restriction enzyme KpnI, run on 0.7% agarose gels containing ethidium bromide, and transferred to Immobilon-N membrane by capillary action with 10ϫ SSC overnight, and the blots were hybridized to a rat kinin B 1 receptor cDNA probe as described previously (14,20). Expression of B 1 Receptor Transgene-Total RNA was extracted from mouse tissues using the RNeasy TM columns (Qiagen). Reverse transcription-PCR/Southern blot analysis was performed using the transgene-specific primers and internal probe as previously described (21). The upstream primer is 5Ј-ATG GCG TCC GAG GTC TT-3Ј; the downstream primer is 5Ј-GAC AAA CAC CAG ATC GG-3Ј; and the internal probe is 5Ј-TGG CAG CAA CGA CAG AG-3Ј.
Membrane Preparation and Radioligand Binding Assays-The mice were sacrificed by cervical dislocation. The kidney was removed, and the wet weight was determined. The tissue was homogenized using a Polytron in ϳ20 volumes of ice-cold 20 mM HEPES, pH 7.4. The membranes from the tissue were prepared, and binding assays were performed as described previously (7,14).
Blood Pressure Measurements by Tail Cuff-Systolic blood pressure was measured using a computer system RTBP2000 (Kent Scientific) according to the manufacturer's instruction. Briefly, the mice were placed into the prewarmed harness (38°C). The tail was placed in the occlusion cuff/piezoelectric pulse sensor and distention caused by arterial blood pulses was detected by the sensor and read out onto the computer system. Pressure in the cuff was increased until the pulse was lost. Actual blood pressure was measured as the pressure at which a pulse was detected during cuff depressurization. Ten readings were taken for each animal.
Blood Pressure Measurements by Arterial Cannulation-The mice were initially anesthetized with 2,2,2-tribromoethanol in tert-amyl alcohol (Avertin, 20 mg/ml, 0.4 ml/25 g of body weight) and placed on a heated table to maintain body temperature. The right jugular vein and the left carotid artery were cannulated with PE-l0 catheters (Clay Adams). After the animals were allowed to recover, blood pressure (in the carotid artery) and heart rate were recorded using a computer system MP100 (Biopac systems Inc). The mice were given a bolus injection of BK or DABK from right jugular vein. BK or DABK was serially diluted and administered at doses of 75, 150, and 300 ng in a volume of 50 l of saline/mouse.
Paw Edema-Inflammation of one hind paw of mice was induced by intraplantar injection of 20 l of 1% carrageenan (dissolved in saline), 3 g of capsaicin in 10 l (dissolved in 5% ethanol, 5% Tween 80 and 90% saline), or DABK (50 or 300 nmol in saline), whereas the contralateral paw received the same volume of vehicle. Thirty min post injection of capsaicin and DABK or 3 h after carrageenan administration, the mice were sacrificed, both hind paws were cut off at the ankle, and the difference between their weights, representing paw edema, was calculated.
Response to Endotoxic Shock-LPS was dissolved in sterile 0.9% NaCl. The mice were injected intraperitoneally with a single dose of LPS (24 mg/kg body weight), and the percentage of survivors was determined at 12-h intervals. Both of the control groups were injected with 0.9% NaCl.
Statistical Analysis-The group data are expressed as the means Ϯ S.E. The data were compared between experimental groups by one-way analysis of variance. Differences between groups were further evaluated by Fisher's protected least squares differences. Differences were considered significant at a value of p Ͻ 0.05.

Generation and Characterization of Rat B 1 Receptor
Mutants-To generate the constitutively active mutants of B 1 receptors, site-directed mutagenesis was directed at the Asn 54 , Asn 130 , and Asp 144 residues in the rat B 1 receptor (Fig. 1). The amino acid replacements were N54A, N130A, D144Q, and D144T. The receptor expression vectors were transiently transfected into 293A cells for assessing constitutive activity by determining agonist-independent IP production. At optimal transfection conditions, all of the mutants were expressed at a comparable level but significantly lower than the wild-type receptor (data not shown). As shown in Table I, the wild-type B 1 receptor displayed a marked ligand-independent, spontaneous activity (104% above control levels of the mock-transfected cells), and the N54A mutant showed an impaired basal activity compared with the wild-type receptor. Substitution of Ala for Asn 130 resulted in significantly constitutive activation of the rat B 1 receptor (409% above control levels). In contrast, the agonist-independent IP accumulations in 293A cells expressing D144Q and D144T mutants were similar to that of mock transfected cells, and the constitutive activity of the wild-type receptor was abolished by the mutations. Interestingly, the maximal extent of DABK stimulation of IP production was significantly reduced for all mutants compared with the wild-type receptor.
To better characterize the N130A mutant receptor, stable expression of the N130A mutant in 293A cells was established. For comparison, stable expression of the wild-type rat B 1 receptor in 293A cells was also established. Such stably transfected 293A cells were analyzed for receptor density and their affinity for radioligand [ 125 I]Sar-Tyr-⑀Ahx-Lys-des-Arg 9 -bradykinin and used in functional assays. The N130A receptor was expressed at a level of about 25% of the wild-type receptor in the stably transfected 293A cells (Table II) To assess the mode of coupling between the B 1 receptor and adenylate cyclase and guanylate cyclase and the effect of the mutation of Asn 130 into Ala on the DABK-induced cAMP and cGMP production, the intracellular cAMP and cGMP levels were measured in the stably transfected 293A cells. As shown in Table II, the basal cAMP and cGMP levels in the 293A cells stably expressing the wild-type receptor were similar to those in the nontransfected 293A cells, whereas in the 293A cells stably expressing the N130A receptor, the basal cAMP and cGMP levels were elevated. DABK challenge increased intracellular cAMP and cGMP production in the 293A cells stably expressing the wild-type and N130A receptors: 116-fold increase in cAMP levels and 5.2-fold increase in cGMP levels for the wild-type receptor versus 14-fold increase in cAMP levels and 1.7-fold increase in cGMP levels for the N130A mutant.
Using the stably transfected 293A cells, the dose-dependent DABK stimulation of IP production for the wild-type and N130A receptor was investigated. As shown in Fig. 2, the N130A mutant could be further activated by over 5-fold by saturation doses of DABK, whereas the wild-type receptor could be amplified to a even higher degree (ϳ16-fold).
Generation of Transgenic Mice-Using the wild-type and N130A cDNAs, we have constructed two transgenes for development of transgenic mice. The transgene consists of the cytomegalovirus immediately early gene enhancer/promoter, the wild-type or N130A rat B 1 receptor cDNA, the human 4F2 enhancer, and the bovine growth hormone poly(A) sequence (Fig. 4). Three transmitting founder lines, including one wildtype line, WT2510, and two N130A lines, N130A58 and N130A2592, were identified by Southern blot analysis of genomic DNA. Heterozygous wild-type and N130A transgenic  mice showed no gross phenotypic abnormalities. All of the transgenic mice were fertile. However, mating F1 generation heterozygotes of both N130A lines produced smaller litters compared with the nontransgenic control and WT2510 line, and a non-Mendelian ratio against N130A mice was observed in the offspring. In the following studies heterozygous transgenic mice were used. Expression of B 1 Receptor Transgene-The distribution of transgene mRNA expression in F1 and F2 generation heterozygous mice was determined by reverse transcription-PCR/ Southern blot analysis. As expected, both male and female transgenic mice showed significant overexpression of the transgene mRNA in the aorta, kidney, liver, heart, brain, and lung and in the prostate of males and the uterus of females. Fig. 5 shows the result from line N130A58. Using specific B 1 receptor radioligand [ 125 I]Sar-Tyr-⑀Ahx-Lys-des-Arg 9 -BK (14), strong B 1 receptor binding activity was detected in membranes prepared from the kidneys of all transgenic lines (data not shown). In contrast, neither transgene mRNA expression nor B 1 receptor (including endogenous) binding activity could be detected in the corresponding tissues of the nontransgenic control mice.
Blood Pressure-All of the heterozygous transgenic mice were normotensive. The systolic blood pressures of transgenic mice 10 weeks old were 78.7 Ϯ 8.5 mmHg (n ϭ 11) for line WT2510, 75.0 Ϯ 7.6 mmHg (n ϭ 14) for line N130A58, and 76.2 Ϯ 6.3 mmHg (n ϭ 11) for line N130A2592 versus 80.9 Ϯ 3.9 mmHg (n ϭ 11) for age-matched nontransgenic control littermates. Intravenous injection of B 1 receptor agonist DABK via Reverse transcription-PCR Southern blot revealed the expression of mutant N130A receptor mRNA in mouse tissues. 2 g of total RNA was reversely transcribed, and one-twentieth of the resultant cDNA was then subjected to PCR for 30 cycles, followed by Southern blot analysis. ϩ represents N130A58 mice, and Ϫ represents nontransgenic mice. F indicates female, and M indicates male.

TABLE II
Characterization of the wild-type and N130A mutant receptors 293A cells stably expressing the wild-type (WT) and the N130A mutant (N130A) rat kinin B 1 receptor were established, and the receptor binding sites (B max ) and dissociation constant (K d ) were determined by using radioligand [ 125 I]Sar-Tyr-Ahx-Lys-des-Arg 9 -bradykinin as described under "Experimental Procedures." The basal (Basal) and 1 M DABK-stimulated (Maximal) intracellular cAMP and cGMP levels were also measured. The results are the means Ϯ S.E. of three experiments.  the jugular vein produced a transient increase of mean arterial blood pressure (MABP) in anesthetized transgenic mice but not in nontransgenic control littermates (Fig. 6A). The duration of MABP increase lasted over 5 min. 75 ng of DABK led to an increase of blood pressure by up to 15 mmHg. In contrast, intravenous injection of B 2 receptor agonist BK into transgenic mice caused a remarkable primary MABP reduction, followed by a blood pressure bounce-back going beyond basal level, whereas in nontransgenic mice the blood pressure just returned to basal level, after a similar primary MABP reduction (Fig. 6B). Single or subsequent injections of higher doses of DABK did not result in a further increase of blood pressure in transgenic mice (data not shown).
Inflammation-Intraplantar injection of carrageenan resulted in a marked inflammation seen by paw swelling in normal and transgenic mice (Fig. 7). But the paw edema induced in transgenic mice was more severe. The percentage of the weight increase of the carrageenan-injected paw over the contralateral vehicle-injected paw was 38.4 Ϯ 6.7% for line WT2510, 42.1 Ϯ 5.0% for line N130A58, and 37.4 Ϯ 6.5% for line N130A2592 versus only 21.7 Ϯ 5.8% for nontransgenic control mice. However, there was no significant difference in paw weight increase between transgenic mice and nontransgenic mice after induction by either capsaicin or DABK (data not shown). We then evaluated the response of transgenic mice to the lethal effects of endotoxic shock. To this end, the mice were injected with a high dose of LPS (24 mg/kg of body weight). Fig. 8 shows the percentage of survivors after LPS injection. Within the first 36 h about 82% of transgenic mice from lines WT2510 and N130A58, but only 45% of nontransgenic mice died. After 3 days, the mortality rate of WT2510 and N130A58 mice reached 87 and 99%, respectively, whereas nontransgenic mice reached only 76%. In contrast, mock injection of vehicle did not cause death in either group. DISCUSSION In the absence of agonist, GPCRs spontaneously isomerize between the inactive and active conformations, with the equilibrium shifted toward the predominantly inactive conformation (24,25). Some receptor mutations induce an agonist-independent shift in isomerization equilibrium toward the active conformation and evoke second messenger responses in the absence of agonist. Such constitutive activations of GPCRs are generally believed to result from an increase in receptor conformational flexibility caused by the loss of intramolecular constraints (26). Our present study shows that the wild-type rat kinin B 1 receptor displayed a marked constitutive activity in the transiently or stably transfected 293A cells. During the preparation of this manuscript, Leeb-Lundberg et al. (27) reported that the human counterpart B 1 receptor also exhibited a high level of constitutive activity in transiently transfected 293 cells and so did the rabbit wild-type B 1 receptor, suggesting that a high constitutive activity might be a common characteristic of the B 1 receptor.
Using single amino acid replacements based on findings from other GPCRs, we generated four mutants of rat B 1 receptors. Asn 54 in TMD I is highly conserved in the superfamily of seven-TMD GPCRs (28). It was thought to be part of a highly conserved transmembrane "polar pocket," involved in receptor activation (29). Substitution of the homologous Asn residue with Ala induced a moderate constitutive activation of the ␣ 1〉 -adrenergic receptor (29). However, in the case of the rat B 1 receptor, similar mutation did not result in enhancement of constitutive activation. Asp 144 is part of the highly conserved DRY motif (triplet of amino acids: Asp-Arg-Tyr) located at the boundary of TMD III and the second intracellular loop (Fig. 1). The DRY motif has played a pivotal role in the signal transduction pathway of GPCRs (30 -32). Mutations of the aspartate residue are reported to lead to constitutive activity for some GPCRs (31)(32)(33). Similar mutation in the homologous position in the rat B 1 receptor failed to induce detectable constitutive activity. However, this result is not unprecedented. For some adrenergic and muscarinic receptors, mutations in the aspartate residue of the DRY motif did not result in agonist-independent constitutive activity (34,35). Asn 130 in the rat B 1 receptor is 14 residues N-terminal to the DRY motif. Mutations in this homologous position such as Cys 128 in the ␣ 1B -adrenergic receptor (36), Cys 116 in the ␤ 2 -adrenergic receptor (37), and Asn 111 in the angiotensin AT 1 receptor (38) led to constitutive activity, suggesting that this particular amino acid position may function as a switch that regulates transition between distinct receptor conformations (37). Based on molecular modeling of the AT 1 receptor and their finding that mutation of Asn 111 to Ala led to constitutive activation of the AT 1 receptor, Bonnafous and co-workers (38) proposed that Asp 74 , Asn 111 , and Trp 253 were involved in the AT 1 receptor activation. They found that these residues were conserved in the kinin B 2 receptor and that mutation of Asn 113 to Ala in the homologous position resulted in a high constitutive activation of the human kinin B 2 receptor (39). Interestingly, these residues were also conserved in the B 1 receptor (i.e. Asp 93 , Asn 130 , and Trp 273 for the rat B 1 receptor). Here we show that mutation of the homologous Asn 130 to Ala in the rat B 1 receptor induced a marked constitutive activation. Leeb-Lundberg et al. (27) reported that mutation of homologous Asn 121 to Ala in the human B 1 receptor also caused a further increase in constitutive activity, indicating that this Asn is indeed involved in constraining the B 1 receptor in an inactive state. Our findings suggest that the molecular events associated to their activation processes are probably conserved between kinin B 1 and B 2 receptors.
The basal IP production by the N130A receptor in the stably transfected 293A cells was about 3-fold higher than that by the wild-type receptor in the stably transfected 293A cells. But if one considers that the expression level of the N130A receptor is about 25% of that of the wild-type B 1 receptor and that increasing the expression of receptors could increase signal to phospholipase C as demonstrated by other researchers with other GPCRs (27,31,39), the N130A receptor would have a up to 12-fold increase in basal activity compared with the wild-type receptor.
Constitutively active mutants have been shown to be responsible for several hereditary and acquired diseases and have been used to produce transgenic mice serving as unique experimental models (40). Because the wild-type rat B 1 receptor has a high basal activity and the N130A mutant is highly constitutively active, we expect that overexpressing these receptors in mice would allow the emergence of any pathophysiological consequences associated with B 1 receptors. B 1 receptor-mediated hypotensive responses have been documented in rabbit, rat, pig, and dog (11). Unexpectedly, we found that all three B 1 receptor transgenic lines were normotensive. One explanation for this finding is that some strong unknown compensatory in vivo mechanisms exist and that any such compen-sation might more likely dilute the manifestation of altered blood pressure phenotype. Alternatively, the B 1 receptor may not be as important in the normal modulation of hemodynamics, which is compatible with the observation that B 1deficient mice are normotensive (12). It should be noted that the B 1 receptor-mediated hypotensive effect is generally only observed following an inflammatory stimulus, such as endotoxin treatment (11).
Surprisingly, intravenous administration of DABK produced a MABP increase in transgenic mice, and a subsequent injection of DABK was almost as potent as the first injection, which can be explained by the lack of desensitization of B 1 receptors (41). Intravenous injection of BK initially caused blood pressure reduction in transgenic mice, and then the blood pressure bounced back and went beyond the basal level, whereas in the control littermates BK only produced a transient blood pressure reduction. This discrepancy could be due to the conversion of some of the injected BK by kininase I into DABK in vivo (42), which then acted on the constitutively expressed B 1 receptor, causing a blood pressure increase in transgenic mice. B 1 receptor ligands have been reported to cause vasoconstriction of a range of blood vessels from several species (43). The DABKmediated hypertensive effect in B 1 receptor transgenic mice probably resulted from B 1 receptor-induced peripheral resistance. However, the central role of B 1 receptors in hypertension cannot be ruled out at this time. Alvarez et al. (44) reported that brain B 1 receptor blockade lowers blood pressure in spontaneously hypertensive rats but not in normotensive rats. Similarly, we demonstrated that intracerebroventricular administration of B 1 receptor agonists increases blood pressure in both Wistar Kyoto rats and spontaneously hypertensive rats, whereas B 1 receptor blockade with antisense oligonucleotides reduced blood pressure in spontaneously hypertensive rats but not in Wistar Kyoto rats (45).
In B 1 receptor transgenic mice, we found that carrageenaninduced paw edema was significantly enhanced compared with that in nontransgenic control littermates. It was reported that carrageenan, a water-extractable polysaccharide obtained from various seaweeds, could activate kinin release and induced the B 1 receptor (46,47). This enhancement in B 1 receptor transgenic mice is probably because released kinins could immediately activate the constitutively expressed B 1 receptors without time delay for the B 1 receptor induction as in nontransgenic control. Overexpression of B 1 receptors appears to have no significant effects on DABK or capsaicin-induced acute edema. However, this finding is in line with the observation that the acute edema produced by kinins injection into rat paw showed mediation by a form of B 2 receptor, without a significant involvement of B 1 receptors (48), even if the animal was pretreated with LPS (49). Compared with nontransgenic control mice, line WT2510 was prone to endotoxic shock, but N130A lines were more susceptible. The reason is not clear for the time being. But these findings clearly support the notion that B 1 receptors play an important role in modulating inflammatory responses.