Effect of enalaprilat on bradykinin and des-Arg9-bradykinin release following reperfusion of the ischaemic rat heart.

1. The release of bradykinin (BK) and its metabolite, des-Arg9-bradykinin (des-Arg9-BK), was studied following reperfusion of a globally ischaemic rat heart. 2. BK-like immunoreactivity increased from 13 +/- 3 (preischaemic value) to 48 +/- 12 fmol min-1 g-1 (P < 0.05, n = 14) 30 s after reperfusion. No difference in BK release was found between control hearts and hearts pretreated with the angiotensin converting enzyme (ACE or kininase II) inhibitor, enalaprilat (50 ng ml-1). 3. No significant change in des-Arg9-BK-like immunoreactivity during reperfusion was observed in control hearts. In contrast, des-Arg9-BK-like immunoreactivity rose from 44 +/- 15 to 177 +/- 61 fmol min-1 g-1 (P < 0.05, n = 7) 30 s after reperfusion in enalaprilat-treated hearts. 4. In conclusion, BK is released upon reperfusion of the globally ischaemic rat heart. ACE inhibitors, through the inhibition of kininase II, increase the formation of the active metabolite, des-Arg9-BK.     

Metabolism of bradykinin agonists and antagonists by plasma aminopeptidase P.

In addition to angiotensin I converting enzyme (ACE; EC 3.4.15.1) and carboxypeptidase N (CPN; EC 3.4.17.3), other peptidases contribute to bradykinin (BK) degradation in plasma. Rat plasma degraded BK by hydrolysis of the N-terminal Arg1-Pro2 bond, and the characteristics of hydrolysis are consistent with identification of aminopeptidase P (APP; EC 3.4.11.9) as the responsible enzyme. BK and BK[1-5] N-terminal hydrolysis was optimal at neutral pH, was inhibited by 2-mercaptoethanol, dithiothreitol, o-phenanthroline and EDTA, but was unaffected by the aminopeptidase inhibitors amastatin, puromycin and diprotin A, the endopeptidase-24.11 inhibitors phosphoramidon and ZINCOV, and the ACE and CPN inhibitors captopril and D,L-mercapto-methyl-3-guanidinoethylthiopropanoic acid (MERGETPA), respectively. Although kallidin (Lys-BK) was not metabolized directly by APP, conversion to BK by plasma aminopeptidase M (EC 3.4.11.2) resulted in subsequent degradation by APP. BK analogs containing N-terminal Arg1-Pro2 bonds, including [Tyr8-(OMe)] BK and [Phe8 psi(CH2NH)Arg9]BK (B2 agonists), des-Arg9-BK and [D-Phe8]des-Arg9-BK (B1 agonists), and [Leu8]des-Arg9-BK (B1 antagonist), were degraded by APP with Km and Vmax values comparable to those found for BK (Km = 19.7 +/- 2.6 microM; Vmax = 12.1 +/- 1.2 nmol/min/mL). In contrast, B2 antagonists containing D-Arg0 N-termini, including D-Arg[Hyp3,Thi5.8,D-Phe7]BK and D-Arg[Hyp3,D-Phe7,Phe8 psi(CH2NH)Arg9]BK, were resistant to APP-mediated hydrolysis. These data support a role for plasma aminopeptidase P in the degradation of circulating kinins, and a variety of B2 and B1 kinin agonists and antagonists. However, APP does not participate in the degradation of D-Arg0-containing antagonists.     

Evidence for BK1 bradykinin-receptor-mediated prostaglandin formation in osteoblasts and subsequent enhancement of bone resorption.

1. The effects of the BK1 bradykinin (BK)-receptor agonist des-Arg9-BK on bone resorption and prostaglandin formation in osteoblasts have been studied. 2. Des-Arg9-BK (1 microM) stimulated the release of 45Ca from prelabelled neonatal mouse calvarial bones and the formation of prostaglandin E2 (PGE2) in calvarial bones. The stimulatory effect on bone resorption and PGE2 formation could be totally inhibited by indomethacin, flurbiprofen and hydrocortisone. 3. The BK1 receptor antagonist des-Arg9-Leu8-BK (10 microM) inhibited des-Arg9-BK (0.01-0.1 microM)-induced release of 45Ca from prelabelled neonatal mouse calvarial bones, while leaving BK (0.1-1 microM)-induced 45Ca release unaffected. 4. In isolated osteoblast-like cells from neonatal mouse calvarial bones, des-Arg9-BK (1 microM) induced a slowly developing increase in PGE2 formation that was significantly different from untreated controls after 24 h. Treatment with BK caused a rapid burst (within minutes) of PGE2 formation. 5. Des-Arg9-Leu8-BK (10 microM) selectively inhibited des-Arg9-BK (1 microM)-induced PGE2 and prostacyclin formation in isolated osteoblast-like cells incubated for 72 h. Des-Arg9-Leu8-BK did not affect BK and Lys-BK (1 microM)-induced PGE2 and prostacyclin formation in isolated osteoblast-like cells incubated for 72 h. 6. These data indicate that osteoblasts are equipped with BK1-receptors mediating enhanced prostaglandin formation and subsequent bone resorption.     

Haemodynamic and cardiac effects of kinin B1 and B2 receptor stimulation in conscious instrumented dogs.

1. Mongrel dogs were chronically instrumented with an intra-aortic catheter, a Königsberg intraventricular pressure transducer and a Döppler flow probe around the left coronary artery. After ganglionic blockade with hexamethonium, the cardiovascular effects of bradykinin B1 and B2 receptor agonists, des-Arg9-bradykinin and bradykinin (BK), were investigated in the presence and absence of specific antagonists. The contribution of nitric oxide (NO) and prostanoids to the cardiovascular effects of kinins was also examined. 2. BK (1 microgram kg-1 min-1) and des-Arg9-BK (1 microgram kg-1 min-1) both given as a 2 min i.v. infusion, produced a significant decrease in mean arterial pressure (MAP, -34 +/- 4% for BK and -45 +/- 2% for des-Arg9-BK) and coronary vascular resistance (CVR, -37 +/- 5% for BK and -50 +/- 2% for des-Arg9-BK), without affecting cardiac contractility, left ventricular end diastolic pressure, and coronary velocity. BK caused a significantly greater decrease in MAP and CVR than des-Arg9-BK (P < 0.05). 3. Pretreatment with the B1 receptor antagonist, des-Arg9-[Leu8]-BK (25 micrograms kg-1) significantly inhibited the decrease in MAP and CVR produced by des-Arg9-BK but not by BK. Infusion of des-Arg9-[Leu8]-BK alone also induced a significant decrease in MAP and CVR (P < 0.05). In the presence of the B2 receptor antagonist, Hoe 140 (25 micrograms kg-1), only the decreases in MAP and CVR caused by BK were significantly reduced (P < 0.05). 4. Inhibition of NO synthase with N omega-nitro-L-arginine (L-NOARG, 45 mg kg-1) significantly (P < 0.05) prevented the decrease in CVR but not MAP induced by des-Arg9-BK, whilst responses to BK were not affected by L-NOARG pretreatment. Inhibition of prostanoid synthesis with indomethacin (25 mg kg-1) did not affect the reductions in MAP and CVR induced by des-Arg9-BK or BK. 5. In conclusion, i.v. des-Arg9-BK and BK administration induced reductions in MAP and CVR suggesting that in conscious instrumented dogs both B1 and B2 receptors are present and can affect systemic blood pressure and coronary resistance regulation. Our results also suggest that prostanoids are not involved in the vascular response to kinins and that coronary vascular B1 receptors are at least in part coupled to the release of NO.     

Prostanoid involvement in the relaxation of in vitro mesenteric artery by bradykinin and des-Arg9-bradykinin

Bradykinin (BK), des-Arg9-BK, prostaglandin E2, prostacyclin and angiotensin II all produced concentration-related relaxation of rabbit in vitro de-endothelialized superior mesenteric arterial ring preparations precontracted with phenylephrine. Responses were reproducible at time 1 h and 4 h after setting up the preparations. The cyclo-oxygenase inhibitor indomethacin (2.8 x 10(-6) M) and the protein synthesis inhibitor cycloheximide (7.2 x 10(-5) M) introduced at t = 0 h inhibited relaxant responses to BK, des-Arg9-BK and angiotensin II, but not prostaglandin E2 or prostacyclin at t = 1 h and t = 4 h. Cycloheximide and indomethacin applied to the tissues at t = 3 h inhibited relaxant responses to BK, des-Arg9-BK and angiotensin II (but not prostaglandin E2 or prostacyclin) at t = 4 h. It is concluded that BK, des-Arg9-BK and angiotensin II (but not prostaglandin E2 or prostacyclin) induced relaxant responses of the in vitro rabbit mesenteric artery are dependent upon the generation of a relaxant prostanoid from the tissue and that cycloheximide produces its effect in this tissue by a mechanism similar to that seen with indomethacin.     

Mediation by B1 and B2 receptors of vasodepressor responses to intravenously administered kinins in anaesthetized dogs.

1. Vasodepressor responses to intravenous (i.v.) injection of bradykinin (BK) and des-Arg9-BK, a selective B1 kinin receptor agonist, were characterized following i.v. pretreatment with selective B1 ([Leu8]-des-Arg9-BK) and B2 (Hoe 140) kinin receptor antagonists in anaesthetized dogs. 2. Des-Arg9-BK (0.05-3.3 nmol kg-1) produced dose-dependent decreases in mean arterial blood pressure with a ED50 0.4 nmol kg-1. The vasodepressor effects evoked by des-Arg9-BK (0.6 nmol kg-1) and BK (0.2 nmol kg-1) were greater after i.v. and i.a. injections, respectively. 3. The vasodepressor response to BK (0.6 nmol kg-1) but not to des-Arg9-BK (0.6 nmol kg-1) was significantly (P < 0.001) blocked by pretreatment with the B2 receptor antagonist, Hoe 140. 4. The vasodepressor response to des-Arg9-BK (0.6 nmol kg-1) but not to BK (0.6 nmol kg-1) was significantly (P < 0.001) reduced by pretreatment with the selective B1 receptor antagonist, [Leu8]-des-Arg9-BK. Although both B1 and B2 receptor antagonists caused a transient fall in blood pressure, their inhibitory action was unlikely to be related to a desensitization mechanism. 5. Inhibition of prostaglandin synthesis with indomethacin prevented the vasodepressor response induced by arachidonic acid (1 mg kg-1, i.v.) but not that to BK or des-Arg9-BK (0.6 nmol kg-1). 6. These results suggest, firstly, that the vasodepressor responses to i.v. BK and des-Arg9-BK are mediated by the activation of B2 and B1 receptors, respectively; secondly, that prostaglandins are not involved in the vasodepressor responses to kinins.(ABSTRACT TRUNCATED AT 250 WORDS)     

Angiotensin converting enzyme inhibitors and expression of des-Arg9-BK (kinin B1) receptors in vivo

The effect of the selective kinin B1 receptor agonist des-Arg9-BK was studied on blood pressure and on the in vitro aorta of rabbits pretreated 18 h earlier with lipopolysaccharide from E. coli, an infusion of bradykinin or with one of three angiotensin converting enzyme inhibitors captopril, enalapril or teprotide. The hypotensive response in vivo and contractile response seen on the in vitro aorta was selectively increased to des-Arg9-BK in all pretreated groups compared to controls, effects which were blocked by the selective competitive kinin B1 receptor antagonist des-Arg9-[Leu8]BK. Dexamethasone given to lipopolysaccharide pretreated rabbits had no effect on the increased hypotensive response seen with des-Arg9-BK. The skin vascular permeability response to des-Arg9-BK, bradykinin and histamine remained unchanged in the groups pretreated with lipopolysaccharide or captopril compared to controls. The possible mechanism(s) whereby angiotensin converting enzyme inhibitors produce this effect and the possible relevance to the inflammatory side-effects seen with this group of drugs is discussed.     

Vascular mode of action of kinin B1 receptors and development of a cellular model for the investigation of these receptors.

1. Kinins exert a contractile effect on rabbit aortic rings via the stimulation of B1 receptors. Des-Arg9-bradykinin (BK) is more potent than BK on this receptor type. The mode of action of des-Arg9-BK on rabbit aortic tissue has been studied by both the aortic ring contractility assay and a cellular model using cultured aortic smooth muscle cells (SMCs). 2. The des-Arg9-BK-induced contractions in rabbit aortic rings were unaffected by pretreatments with nifedipine, indomethacin, REV-5901 (a 5-lipoxygenase blocker) and LY-83583 (a guanylyl cyclase inhibitor); however, the protein kinase inhibitors H-7 and H-9 significantly reduced the maximal effect of des-Arg9-BK. 3. The contractile responses to des-Arg9-BK in calcium-free Krebs solution were slightly but not significantly attenuated in amplitude, as compared to paired control tissues bathed in Krebs solution, and sustained plateaus of contraction were observed in the absence of Ca2+. However, Ca2+ replenishment further increased the kinin-induced contraction measured in Ca(2+)-free bathing fluid. 4. Despite the lack of evidence of a mediating role for prostaglandin in the mechanical response to des-Arg9-BK, the kinin stimulated the release of prostacyclin from rabbit aorta rings measured as immunoreactive 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha).(ABSTRACT TRUNCATED AT 250 WORDS)     

Pharmacological and functional characterization of bradykinin receptors in canine cultured tracheal epithelial cells.

1. A direct [3H]-bradykinin ([3H]-BK) binding assay has been used to characterize the BK receptors in canine cultured tracheal epithelial cells (TECs). Based on receptor binding assay, TECs have specific, saturable, high-affinity binding sites for [3H]-BK. 2. The specific [3H]-BK binding was time- and temperature-dependent. Equilibrium of association of [3H]-BK with the BK receptors was attained within 30 min at room temperature and 1 h at 4 degrees C, respectively. 3. Analysis of binding isotherms yielded an apparent equilibrium dissociation constant (KD) of 1.5 +/- 0.2 nM and a maximum receptor density (Bmax) of 53.2 +/- 5.2 fmol mg-1 protein. The Hill coefficient for [3H]-BK binding was 1.00 +/- 0.02. The association (K1) and dissociation (K-1) rate constants were (7.6 +/- 1.1) x 10(6) M-1 min-1 and (9.2 +/- 1.5) x 10 M-3 min-1, respectively. KD, calculated from the ratio of K-1 and K1, was 1.2 +/- 0.3 nM, a value close to that calculated from Scatchard plots of binding isotherms. 4. Neither a B1 receptor selective agonist (des-Arg9-BK, 0.1 nM - 10 microM) nor antagonist ([Leu8, des-Arg9]-BK, 0.1 nM - 10 microM) significantly inhibited [3H]-BK binding to TECs, which excludes the presence of B1 receptors in canine TECs. 5. The specific binding of [3H]-BK to canine TECs was inhibited by the B2 receptor selective antagonists ([D-Arg0, Hyp3, Thi5, D-Tic7, Oic8]-BK (Hoe 140, 0.1 nM-10 microM) and [D-Arg0, Hyp3, Thi5.8, D-Phe7]-BK, 0.1 nM - 10 microM) and agonists (BK and kallidin, 0.1 nM-10 microM) with a best fit by a one-binding site model. The order of potency for the inhibition of [3H]-BK binding was kallidin = BK = Hoe 140 > [D-Arg0, Hyp3, Thi5,8, D-Phe7]-BK. 6. BK and kallidin significantly induced concentration-dependent accumulation of IPs with a half-maximal response (EC50) at 17.6 +/- 3.5 and 26.6 +/- 5.3 nM, respectively, while the B1-selective agonist, des-Arg9-BK did not stimulate IPs accumulation and the B1-selective antagonist [Leu8, des-Arg9]-BK did not inhibit BK-induced IPs accumulation. Two B2-selective antagonists, Hoe 140 and [D-Arg0, Hyp3, Thi5,8, D-Phe7]-BK, inhibited BK-stimulated IPs accumulation with apparent pKB values of 8.8 +/- 0.3 and 7.0 +/- 0.3, respectively. 7. It is concluded that the pharmacological characteristics of the BK receptors in canine cultured TECs are primarily of the B2 receptor subtype which might regulate the function of tracheal epithelium through the activation of this receptor subtype coupling to PI hydrolysis.     

Intrathecal bradykinin administration: opposite effects on nociceptive transmission

Injected intrathecally, bradykinin (BK) produced either hyperalgesia (0.15 microg) or antinociception (6.0 microg) in rats when thermal noxious stimuli were used. Similarly, des-Arg(9)-BK at the lower dose (0.15 microg) decreased, whereas at the higher dose (6.0 microg) it increased the threshold to thermal noxious stimuli; however, these effects were less pronounced than those of BK. The antinociception induced by BK was abolished by HOE 140, a B(2) receptor antagonist, injected intrathecally at a dose of 1.3 ng and was markedly attenuated by des-Arg(10)-HOE 140, a B(1) receptor antagonist (1.15 ng i.t.). The results obtained in this study showed that--depending on the dose used--BK and des-Arg(9)-BK could produce pro- as well as antinociceptive actions. Both B(2) and B(1) receptors are involved in the action of intrathecally applied BK.     

The kinin B1 receptor antagonist des-Arg9-[Leu8]bradykinin: an antagonist of the angiotensin AT1 receptor which also binds to the AT2 receptor.

1. Agonists and antagonists of kinin B1 and B2 receptors were evaluated in vitro for their effects against angiotensin II (AII)-induced contractile responses in the rabbit aorta and for their binding properties to angiotensin AT1 and AT2 receptors from purified membrane of rat liver and lamb uterus respectively. 2. In aortic rings, the kinin B1 receptor antagonist, des-Arg9-[Leu8]bradykinin (BK) (3-100 microM) caused a concentration-dependent decrease in sensitivity and a depression of the maximum response to AII. Des-Arg10-[Leu9]kallidin (KD), des-Arg9-BK, des-Arg10-KD, BK or KD at 3 microM had no effect against AII-induced contractions. 3. Des-Arg9-[Leu8]BK (3 or 100 microM) did not affect contractions of aortic rings to histamine, potassium chloride, endothelin-1, 5-hydroxytryptamine, noradrenaline and the thromboxane A2-mimetic, U46619. 4. Des-Arg9-[Leu8]BK displaced [125I]-Sar1-AII binding to the AT1 subtype in rat liver membranes with a Ki value of 1.1 +/- 0.4 microM. Values of Ki for des-Arg9-BK and KD were 45 +/- 13 microM and 25 +/- 22 microM, respectively. The other kinin derivatives des-Arg10-KD, BK and des-Arg10-[Leu9]KD at concentrations up to 100 microM did not bind to the AT1 receptor. 5. All the kinin derivatives except BK bound to AT2 receptors in lamb uterus membranes. Values of Ki for des-Arg9-[Leu8]BK, des-Arg10-[Leu9]KD, des-Arg9-BK, des-Arg10-KD and KD were 0.3 +/- 0.1, 0.7 +/- 0.1, 1.2 +/- 0.3, 1.5 +/- 0.3 and 7.0 +/- 1.6 microM, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

The ability of des-Arg9-bradykinin to relax rabbit isolated mesenteric arteries is acquired during in vitro incubation

Strips of rabbit mesenteric arteries precontracted with phenylephrine relaxed when exposed to des-Arg9-bradykinin (BK), a typical B1 receptor agonist. We showed that the ability of des-Arg9-BK to relax this preparation was acquired during in vitro incubation and was abolished by cycloheximide treatment. In this preparation intimal damage reduced but did not abolish the relaxant effect of des-Arg9-BK, and indomethacin uncovered a contractile effect of des-Arg9-BK also acquired during the in vitro incubation.     

Endothelium-dependent relaxations mediated by inducible B1 and constitutive B2 kinin receptors in the bovine isolated coronary artery.

1. Rings of bovine left anterior descending coronary artery (LAD) were contracted with the thromboxane A2-mimetic, U46619 (1-30 nM), to approximately 40% of their maximum contraction to 125 mM KCl Krebs solution (KPSSmax) for comparison of responses to the B1 and B2 kinin receptor agonists, des-Arg9-bradykinin (des-Arg9-BK) and bradykinin (BK), respectively. Relaxation responses were normalized as percentages of the initial U46619-induced contraction level, while contractile responses were expressed as percentages of KPSSmax. 2. After 6 h of in vitro incubation in Krebs solution at 37 degrees C, des-Arg9-BK (pEC50, 8.00 +/- 0.08; maximum response (Rmax), 93.9 +/- 1.9%) and BK (pEC50, 9.75 +/- 0.07; Rmax, 100.1 +/- 0.7%) caused endothelium-dependent relaxations in precontracted rings of bovine LAD which were competitively and selectively antagonized by the B1 receptor antagonist, des-Arg9-[Leu8]-BK (pA2, 6.27 +/- 0.11) and the B2 receptor antagonist Hoc-140 (pA2, 9.63 +/- 0.14), respectively. 3. At 3 h of in vitro incubation, the sensitivity (pEC50, 7.45 +/- 0.10) and Rmax (84.6 +/- 3.3%) to des-Arg9-BK were significantly less than those obtained in the same tissues at 6 h (pEC50, 7.94 +/- 0.06; Rmax, 91.4 +/- 2.5%), whereas endothelium-dependent relaxations to BK and ACh were unaffected by incubation time. 4. Relaxation responses to des-ARg9-BK, but not BK, at both 3 h and 6 h were significantly attenuated by the protein synthesis inhibitors, cycloheximide (30 and 100 microM) and actinomycin D (2 microM). 5. At 6 h, the nitric oxide (NO) synthase inhibitor, NG-nitro-L-arginine (L-NOARG, 100 microM), caused a significant 2 fold decrease in pEC50 (9.58 +/- 0.03) but had no effect on Rmax for BK. For des-Arg9-BK, L-NOARG (100 microM) caused a marked and significant decrease in both the pEC50 and Rmax and revealed contractions to low concentrations of des-Arg9-BK. In both cases, L-NOARG inhibition was reversed in the presence of L-arginine (10 mM). 6. At 6 h removal of the endothelium abolished relaxation responses to des-Arg9-BK and BK, and for des-Arg9-BK, but not BK, unmasked concentration-dependent contractions (pEC50, 7.57 +/- 0.09; Rmax, 83.4 +/- 9.1%). The sensitivity of contractions to des-Arg9-BK increased slightly from 3 h (pEC50, 7.37 +/- 0.08) to 6 h (pEC50, 7.62 +/- 0.12) of in vitro incubation; however, there was a small but significant depression in the maximum response over this time (Rmax, 126.8 +/- 8.5% and 103.3 +/- 8.6% for 3 h and 6 h of incubation respectively). 7. In conclusion, the bovine LAD contains inducible B1 and constitutive B2 endothelial cell kinin receptors, both of which mediate endothelium-dependent relaxation partly via the release of NO. B1 receptors were also present on the smooth muscle layer of the bovine LAD.     

Induction of kinin B1 receptor-dependent vasoconstriction following balloon catheter injury to the rabbit carotid artery.

1. Balloon catheter injury to the rabbit carotid artery damaged the endothelium and induced neointima formation over 7 days. The area of intima, expressed as a percentage of the media, was 16.2 +/- 4.2% and 8.2 +/- 0.1% in balloon catheter-injured and sham-operated arteries. 2. Seven days after arterial injury, carotid arteries were isolated and set up as ring preparations in organ baths for isometric tension measurements. Balloon catheter-injured arteries first contracted with noradrenaline (0.01-0.1 microM), contracted further in a concentration-dependent manner to bradykinin (BK; pD2, 5.98 +/- 0.22; Emax, 41.3 +/- 5.2% of KCl) and to des-Arg9-BK (pD2, 7.12 +/- 0.36; Emax, 46.0 +/- 9.9% of KCl). In contrast, vessel segments with endothelium either intact or acutely removed were unresponsive to both BK receptor agonists. 3. The concentration-contraction curves for BK and for des-Arg9-BK were shifted to the right by the B1 receptor antagonist, [Leu8]des-Arg9-BK (3 microM), but not by the selective B2 receptor antagonist, Hoe 140 (1 microM). 4. Thus, BK and its metabolite, des-Arg9-BK act as vasoconstrictor agents following balloon catheter injury. These effects appear to be mediated by activation of B1 receptors.     

A stable metabolite, Arg-Pro-Pro-Gly-Phe, of bradykinin in the degradation pathway in human plasma.

Degradation of bradykinin (BK) in human plasma was investigated by searching for a stable metabolite as a marker of kinin release in vivo. BK, incubated with diluted plasma, was degraded to des-Arg9-BK (des-9-BK), des-Phe8-Arg9-BK (des-8,9-BK) and Arg-Pro-Pro-Gly-Phe ([1-5]BK). Des-9-BK was degraded to [1-5]BK without an increase in des-8,9-BK, and des-8,9-BK was also degraded to [1-5]BK. D,L-2-Mercaptomethyl-3-guanidinoethyl-thiopropanoic acid inhibited the formation of des-9-BK from BK, whereas captopril inhibited the formation of des-8,9-BK from BK as well as that of [1-5]BK from des-9-BK or des-8,9-BK. The half lives of BK, des-9-BK, des-8,9-BK and [1-5]BK under the present experimental conditions were 60 min, 90 min, 14 min and 4.2 hr, respectively. Among the metabolites, [1-5]BK was the most stable one and may be used as a marker for BK production in vivo.     

Pharmacological and molecular evidence for kinin B1 receptor expression in urinary bladder of cyclophosphamide-treated rats.

1. In the present study, we developed an experimental model of cystitis induced by cyclophosphamide (CYP). In order to characterize des-Arg9-BK-induced contraction on the urinary bladder (UB) during the development of inflammation and to quantify kinin B1 receptor gene expression using a quantitative RT - PCR technique. 2. In the presence of peptidase inhibitors captopril (10 microM), DL-thiorphan (1 microM) and DL-2-mercaptomethyl-3-guanidino-ethylthiopropanoic acid (MERGEPTA 5 microM), bradykinin (BK) (0.3 - 3,000 nM) evoked a concentration-dependent contraction of rat UB which was not different between the CYP- and vehicle-treated groups. Unlike BK, des-Arg9-BK (0.3 - 100,000 nM) did not contract UB from vehicle-treated rats but contracted vigorously bladder strips from CYP-treated rats 14, 24 and 168 h after treatment. In UB of 24 h treated rat, the pD2 value of des-Arg9-BK was 7.3+/-0.1. 3. The cyclo-oxygenase inhibitor indomethacin (3 microM) reduced by 30% the maximal response of des-Arg9-BK. Both the kinin B1 receptor antagonists des-Arg9-[Leu8]BK (10 microM) and des-Arg10-Hoe 140 (10 microM) produced a rightward shift of the concentration-response curve to des-Arg9-BK yielding pKB values of 6.8+/-0.2 and 7.2+/-0.1, respectively, whilst the kinin B2 receptor antagonist Hoe 140 (1 microM) had no effect. 4. After CYP treatment, mRNA coding for the kinin B1 receptor appeared predominantly in UB. In this organ, the induction was progressive, reaching a maximum 48 h after CYP treatment. 5. In conclusion, the present study provides strong evidence for an induction of kinin B1 receptors in UB of CYP-treated rats. This was associated at a molecular level with an increase in mRNA expression of the gene coding for the kinin B1 receptor. This kinin receptor displayed the whole features of a classical rat kinin B1 receptor.     

Pharmacological modulation of the up-regulated responses to des-Arg9-bradykinin in vivo and in vitro

Des-Arg9-bradykinin (BK) is a selective agonist of the B1 type receptors for kinins. The biological responses to des-Arg9-BK are known to be selectively up-regulated following some types of tissue injury. Two models using rabbits were previously characterized. Firstly, in vivo, lipopolysaccharide (LPS) induces a state of sensitivity to des-Arg9-BK, which becomes a hypotensive peptide. Pretreatment of rabbits with dexamethasone sodium phosphate (DEX) did not modify the induction of cardiovascular sensitivity by LPS. Secondly, isolated rabbit aortic strips incubated in vitro exhibit a spontaneous, time-dependent increase in their responsiveness to des-Arg9-BK. This up-regulation process is selectively inhibited by DEX and stimulated by LPS applied in vitro. DEX application prevented the stimulant effect of LPS in vitro. A variety of growth factors and interleukins as well as interferon-gamma, serotonin, bradykinin and the metalloprotease inhibitor 2-mercaptoethanol were ineffective in modulating the spontaneous up-regulation of contractile responses to des-Arg9-BK. Of the known substances which do modulate this system, only epidermal growth factor (EGF) enhanced aortic contractions to des-Arg9-BK following an acute (15 min) exposure near the end of the 6-hour incubation period used in these studies. The possible involvement of des-Arg9-BK and related peptides in immunopathology is discussed.     

Selective labelling of bradykinin receptor subtypes in WI38 human lung fibroblasts.

1. Binding of the B1 bradykinin receptor radioligand, [3H]-des-Arg10-kallidin (-KD) and the B2 receptor radioligand [3H]-bradykinin (-BK) was investigated in membranes prepared from WI38 human foetal lung fibroblasts. 2. One-site analysis of the saturation data for [3H]-des-Arg10-KD gave an equilibrium dissociation constant (KD) value of 0.51 +/- 0.12 nM and a maximum receptor density (Bmax) of 260 +/- 49 fmol mg-1 of protein. [3H]-des-Arg10-KD binding was displaced by ligands in the order: des-Arg10-KD > KD > > des-Arg9[Leu8]-BK > des-Arg9-BK > Hoe 140 > > BK, implying that it was binding selectively to B1 receptors. 3. One-site analysis of the binding of [3H]-BK to W138 membranes indicated that it had a KD value of 0.25 +/- 0.06 nM and a Bmax of 753 +/- 98 fmol mg-1 of protein. The potencies for displacement of [3H]-BK binding were: Hoe 140 > > BK = KD > > > des-Arg10-KD = des-Arg9[Leu8]-BK = des-Arg9-BK, which was consistent with binding to B2 receptors. 4. This is the first characterization of [3H]-des-Arg10-KD binding to include both kinetic and equilibrium data, and demonstrates that [3H]-des-Arg10-KD has a high affinity for human B1 bradykinin receptors and is sufficiently selective to be used as a radioligand for B1 receptors in human cells or tissues expressing an excess of B2 BK receptors.