Receptors for kinins in the human isolated umbilical vein.

1. The human umbilical vein has been found to contract in response to bradykinin (BK) and desArg9BK. 2. The rank order of potency of agonists, in the presence of the B1 receptor antagonist Lys[Leu8]desArg9BK, is as follows: [Hyp3, Tyr(Me)8]BK (pD2 8.88) = [Hyp3]BK (pD2 8.86) = LysBK (pD2 8.81) > or = BK (pD2 8.60) >> [Aib7]BK (pD2 6.38) >> desArg9BK and LysdesArg9BK (inactive). 3. Hoe 140 (pA2 8.42) inhibits the effects of BK while other B2 receptor peptide antagonists are very weak and WIN 64338 is practically inactive. 4. Venoconstrictor responses to desArg9BK of fresh tissues increase with time during the in vitro incubation and reach a maximum after 4-6 h. The activity of Hoe 140 (pA2 5.48) is negligible against B1 receptor agonists. 5. When measured in the presence of the selective B2 receptor antagonist Hoe 140 (400 nM), the order of potency of kinin related peptides on the B1 receptor is Lys[desArg9]BK (pD2 8.60) > desArg9BK (pD2 6.69). BK, LysBK, [Hyp3]BK and other B2 receptor agonists are inactive. 6. The B1 receptor antagonist, Lys[Leu8]desArg9BK (pA2 7.99), inhibits the response of the human vein to B1 receptor agonists (LysdesArg9BK or desArg9BK), but do not alter the effect of BK. 7. The results summarized in this paper indicate that the human isolated umbilical vein is a sensitive preparation containing both B1 and B2 receptors. The human B2 receptor shows some similarity with that of the rabbit (at least for agonist potencies) and differs from the B2 receptor of the guinea-pig. Compared to the rabbit B1 receptor, the human B1 receptor shows low sensitivity to peptides that lack the N-terminal Lys.     

Autoregulation of bradykinin receptors: agonists in the presence of interleukin-1beta shift the repertoire of receptor subtypes from B2 to B1 in human lung fibroblasts.

Elevated formation of bradykinin (BK) and Lys-BK or kallidin (KD) and their carboxypeptidase metabolites desArg(9)BK and desArg(10)KD is evident at sites of inflammation. Moreover, B2 receptors (B2R), which mediate the action of BK and KD, participates in the acute stage of the inflammatory and pain response, whereas B1 receptors (B1R), through which desArg(9)BK and desArg(10)KD act, partake in the chronic stage. We hypothesized that kinins autoregulate B2R and B1R expression in favor of B1R. Incubation of IMR-90 cells with BK (100 nM) led to a loss (89%) of B2R with a half-life (T(1/2)) of 7.0 min. Concomitantly, BK increased B1R (2- to 3-fold) with a T(1/2) of 120 min. DesArg(10)KD (100 nM) had no effect on B2R but increased B1R (3- to 4-fold) with the same rate as BK. Interleukin-1beta (IL-1beta; 500 pg/ml) also increased B1R (4- to 6-fold). Although both desArg(10)KD and BK increased the level of IL-1beta mRNA, IL-1beta receptor antagonist inhibited the increase in B1R only in response to BK. DesArg(10)KD and BK synergistically increased B1R (9-fold), which was further increased by inclusion of IL-1beta (36-fold). Therefore, kinin metabolism and kinin-stimulated production of cytokines may play a pivotal role in shifting the repertoire of kinin receptor subtypes in favor of B1R during inflammation.     

In vitro and in vivo effects of kinin B(1) and B(2) receptor agonists and antagonists in inbred control and cardiomyopathic hamsters

The aims of this study were to examine the possible alterations occurring in the effects of kinins on isolated aortae of inbred control (CHF 148) and cardiomyopathic (CHF 146) hamsters of 150 - 175 and 350 - 375 days of age. Bradykinin (BK) and desArg(9)BK contracted isolated aortae (with or without endothelium) of hamsters of both strains and ages. After tissue equilibration (90 min), responses elicited by both kinin agonists were stable over the time of experiments. The patterns of isometric contractions of BK and desArg(9)BK were however found to be different; desArg(9)BK had a slower onset and a longer duration of action than BK. Potencies (pEC(50) values) of BK in all groups of hamsters were significantly increased by preincubating the tissues with captopril (10(-5) M). No differences in the pEC(50) values and the E(max) values for BK or desArg(9)BK were seen between isolated vessels from inbred control and cardiomyopathic hamsters. The myotropic effect of BK was inhibited by the selective non peptide antagonist, FR 173657 (pIC(50) 7.25+/-0.12 at the bradykinin B(2) receptor subtype (B(2) receptor)). Those of desArg(9)BK, at the bradykinin B(1) receptor subtype (B(1) receptor) were abolished by either R 715 (pIC(50) of 7. 55+/-0.05; alpha(E) = 0), Lys[Leu(8)]desArg(9)BK (pIC(50) of 7.21+/-0. 01; alpha(E) = 0.22) or [Leu(8)]desArg(9)BK (pIC(50) of 7.25+/-0.02; alpha(E) = 0.18). FR 173657 had no agonistic activity, exerted a non competitive type of antagonism and was poorly reversible (lasting more than 5 h) from B(2) receptor. In vivo, FR 173657 (given per os at 1 and 5 mg kg(-1), 1 h before the experiment) antagonized the acute hypotensive effect of BK in anaesthetized hamsters. It is concluded that aging and/or the presence of a congenital cardiovascular disorder in hamsters are not associated with changes in the in vitro aortic responses to either BK or desArg(9)BK.     

Vascular kinin B(1) and B(2) receptor-mediated effects in the rat isolated perfused kidney - differential regulations

1. Bradydykinin (BK) and analogs acting preferentially at kinin B(1) or B(2) receptors were tested on the rat isolated perfused kidney. Kidneys were perfused in an open circuit with Tyrode's solution. Kidneys preconstricted with prostaglandin F(2alpha) were used for the analysis of vasodilator responses. 2. BK induced a concentration-dependent renal relaxation (pD(2)=8.9+/-0.4); this vasodilator response was reproduced by a selective B(2) receptor agonist, Tyr(Me)(8)-BK (pD(2)=9.0+/-0.1) with a higher maximum effect (E(max)=78.9+/-6.6 and 55.8+/-4.3% of ACh-induced relaxation respectively, n=6 and 19, P<0.02). Icatibant (10 nM), a selective B(2) receptor antagonist, abolished BK-elicited relaxation. Tachyphylaxis of kinin B(2) receptors appeared when repeatedly stimulated at 10 min intervals. 3. Des-Arg(9)-BK, a selective B(1) receptor agonist, induced concentration-dependent vasoconstriction at micromolar concentration. Maximum response was enhanced in the presence of lisinopril (1 microM) and inhibited by R 715 (8 microM), a selective B(1) receptor antagonist. Des-Arg(9)-[Leu(8)]-BK behaved as an agonist. 4. A contractile response to des-Arg(9)-BK occurred after 1 of perfusion and increased with time by a factor of about three over a 3 h perfusion. This post-isolation sensitization to des-Arg(9)-BK was abolished by dexamethasone (DEX, 30 mg kg(-1) i.p., 3 h before the start of the experiment and 10 microM in perfusate) and actinomycin D (2 microM). Acute exposure to DEX (10 microM) had no effect on sensitized des-Arg(9)-BK response, in contrast to indomethacin (30 microM) that abolished it. DEX pretreatment however had no effect on BK-induced renal vasodilation. 5. Present results indicate that the main renal vascular response to BK consists of relaxation linked to the activation of kinin B(2) receptors which rapidly desensitize. Renal B(1) receptors are also present and are time-dependently sensitized during the in vitro perfusion of the rat kidneys.     

Kinin B2 receptor is not involved in enalapril-induced apoptosis and regression of hypertrophy in spontaneously hypertensive rat aorta: possible role of B1 receptor

1. Treatment with enalapril induces smooth muscle cell apoptosis and regression of aortic hypertrophy in spontaneously hypertensive rats (SHRs), whereas combined blockade of angiotensin II AT(1) and AT(2) receptors does not. We postulated that vascular apoptosis with enalapril involves enhanced half-life of bradykinin (BK) and kinin B(2) receptor stimulation. 2. SHR, 11-weeks old, were treated for 4 weeks with enalapril (30 mg kg(-1) day(-1)), Hoe 140 (500 microg kg(-1) day(-1); B(2) receptor antagonist), alone or in combination. Controls received vehicle. 3. The half-life of hypotensive responses to intra-arterial bolus injections of BK were significantly increased in SHR anesthetized after 4 weeks of enalapril, an effect prevented by Hoe 140. The magnitude of BK-induced hypotension was significantly attenuated in all rats treated with Hoe 140. 4. As compared to placebo, enalapril treatment significantly reduced blood pressure (-34+/-2%), aortic hypertrophy (-20+/-3%), hyperplasia (-37+/-5%) and DNA synthesis (-61+/-8%), while it increased aortic DNA fragmentation by two-fold. Hoe 140 given alone or in combination with enalapril affected none of these parameters. 5. As a possible alternative mechanism, aortae isolated during the second week of enalapril treatment showed a transient upregulation of contractile responses to des-Arg(9)BK (EC(50)<1 nM), which were significantly reduced by [Leu(8)]des-Arg(9)BK (10 microM). Moreover, in vitro receptor autoradiography revealed an increase in expression of B(1) and B(2) receptor binding sites by 8-11 days of enalapril treatment. 6. Aortic apoptosis induction and hypertrophy regression with enalapril do not involve kinin B(2) receptors in SHR. Kinins acting via B(1) receptors remains a candidate mechanism.     

Non-competitive pharmacological antagonism at the rabbit B(1) receptor

The B(1) receptor for kinins, stimulated by kinin metabolites without the C-terminal Arg residue (e.g., des-Arg(9)-bradykinin (BK) and Lys-des-Arg(9)-BK), is an increasingly recognized molecular target for the development of analgesic and anti-inflammatory drugs. Recently developed antagonists of this receptor were compared to a conventional antagonist, Ac-Lys-[Leu(8)]-des-Arg(9)-BK, in pharmacological assays based on the rabbit B(1) receptor. B-9858 (Lys-Lys-[Hyp(3), Igl(5), D-Igl(7), Oic(8)]des-Arg(9)-BK) and three other analogues possessing the alpha-2-indanylglycine(5) (Igl(5)) residue (order of potency B-9858 approximately B-10146>B-10148>B-10050) were partially insurmountable antagonists of des-Arg(9)-BK in the contractility assay based on rabbit aortic rings. B-9858-induced depression of the maximal effect was more pronounced in tissues treated with the protein synthesis inhibitor cycloheximide to block the spontaneous increase of response attributed to the post-isolation formation of B(1) receptors, and only partly reversible on washing. By comparison, Ac-Lys-[Leu(8)]des-Arg(9)-BK was a surmountable antagonist (pA(2) 7. 5), even in cycloheximide-treated tissues. B-9958 (Lys-[Hyp(3), CpG(5), D-Tic(7), CpG(8)]des-Arg(9)-BK) was also surmountable (pA(2) 8.5). The binding of [(3)H]-Lys-des-Arg(9)-BK to recombinant rabbit B(1) receptors expressed in COS-1 cells was influenced by two of the antagonists: while Ac-Lys-[Leu(8)]des-Arg(9)-BK competed for the radioligand binding without affecting the B(max), B-9858 decreased the B(max) in a time-dependent and washout-resistant manner. B-9858 and analogues possessing Igl(5) are the first reported non-competitive, non-equilibrium antagonists of the kinin B(1) receptor.     

Actions of ZD0947, a novel ATP-sensitive K+ channel opener, on membrane currents in human detrusor myocytes

BACKGROUND AND PURPOSE: ATP-sensitive K+ channels (K(ATP) channels) play important roles in regulating the resting membrane potential of detrusor smooth muscle. Actions of ZD0947, a novel KATP channel opener, on both carbachol (CCh)-induced detrusor contractions and membrane currents in human urinary bladder myocytes were investigated. EXPERIMENTAL APPROACH: Tension measurements and patch-clamp techniques were utilized to study the effects of ZD0947 in segments of human urinary bladder. Immunohistochemistry was also performed to detect the expression of the sulphonylurea receptor 1 (SUR1) and the SUR2B antigens in human detrusor muscle. KEY RESULTS: ZD0947 (> or = 0.1 microM) caused a concentration-dependent relaxation of the CCh-induced contraction of human detrusor, which was reversed by glibenclamide. The rank order of the potency to relax the CCh-induced contraction was pinacidil > ZD0947 > diazoxide. In conventional whole-cell configuration, ZD0947 (> or = 1 microM) caused a concentration-dependent inward K+ current which was suppressed by glibenclamide at -60 mV. When 1 mM ATP was included in the pipette solution, application of pinacidil or ZD0947 caused no inward K+ current at -60 mV. Gliclazide (< or =1 microM), a selective SUR1 blocker, inhibited the ZD0947-induced currents (Ki = 4.0 microM) and the diazoxide-induced currents (high-affinity site, Ki1 = 42.4 nM; low-affinity site, Ki2 = 84.5 microM) at -60 mV. Immunohistochemical studies indicated the presence of SUR1 and SUR2B proteins, which are constituents of KATP channels, in the bundles of human detrusor smooth muscle. CONCLUSIONS AND IMPLICATIONS: These results suggest that ZD0947 caused a glibenclamide-sensitive detrusor relaxation through activation of glibenclamide-sensitive KATP channels in human urinary bladder.     

The use of kinin B1 and B2 receptor knockout mice and selective antagonists to characterize the nociceptive responses caused by kinins at the spinal level

The mechanisms by which kinins induce hyperalgesia in the spinal cord were investigated by using B(1) or B(2) knockout mice in conjunction with kinin selective agonists and antagonists. The i.t. administration of the kinin B(2) receptor agonists, bradykinin (BK) or Tyr(8)-BK produced dose-related thermal hyperalgesia evaluated in the hot-plate test. BK-induced hyperalgesia was abolished by the B(2) receptor antagonist Hoe 140. The i.t. injection of the kinin B(1) receptor agonists, des-Arg(9)-bradykinin (DABK) or des-Arg(10)-kallidin (DAKD) also caused dose-related thermal hyperalgesia. Different from the B(2) agonists, the i.t. injection of DABK or DAKD caused a weak, but prolonged hyperalgesia, an effect that was blocked by the B(1) receptor antagonist des-Arg(9)-[Leu(8)]-bradykinin (DALBK). The i.t. injection of BK caused thermal hyperalgesia in wild-type mice (WT) and in the B(1) receptor knockout mice (B(1)R KO), but not in the B(2) receptor knockout mice (B(2)R KO). Similarly, the i.t. injection of DABK elicited thermal hyperalgesia in WT mice, but not in B(1)R KO mice. However, DABK-induced hyperalgesia was more pronounced in the B(2)R KO mice when compared with the WT mice. The i.t. injection of Hoe 140 or DALBK inhibited the second phase of formalin (F)-induced nociception. Furthermore, i.t. Hoe 140, but not DALBK, also inhibits the first phase of F response. Finally, the i.t. injection of DALBK, but not of Hoe 140, inhibits the long-term thermal hyperalgesia observed in the ipsilateral and in contralateral paws after intraplantar injection with complete Freund's adjuvant. These findings provide evidence that kinins acting at both B(1) and B(2) receptors at the spinal level exert a critical role in controlling the nociceptive processing mechanisms. Therefore, selective kinin antagonists against both receptors are of potential interest drugs to treat some pain states.     

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.     

Detection of bradykinin B1 receptors in rat aortic smooth muscle cells

The tritiated bradykinin B1 receptor agonist [3H]des-Arg(10)-kallidin bound to a single class of high-affinity binding sites (K(d) = 0.5 +/- 0.16 nM; B(max) = 15,000 +/- 8,000 sites/cell) on cultured rat aortic smooth muscle cells. [3H]Des-Arg(10)-kallidin association and dissociation kinetics were monoexponential, making it possible to determine the association and dissociation rate constants (k(+1) = 1.5 10(5) M(-1) sec(-1); k(-1) = 4.2 10(-5) sec(-1)). [3H]Des-Arg(10)-kallidin binding was inhibited by specific ligands of bradykinin B1 and B2 receptors with a rank order of potency consistent with that known for bradykinin B1 receptors in other species (des-Arg(9)-[Leu(8)]bradykinin = des-Arg(10)-kallidin = des-Arg(9)-bradykinin = des-Arg(10)-[Leu(9)]kallidin > des-Arg(10)-HOE-140 > bradykinin > HOE-140). Bradykinin B1 receptor mRNA was also detected in these cells. Des-Arg(10)-kallidin increased cytosolic free Ca2+ levels, phosphoinositide turnover, and arachidonic acid release at nanomolar concentrations (respective EC(50) values: 16 +/- 2, 4 +/- 2.7, 6 +/- 2 nM). These functional effects of des-Arg(10)-kallidin could be blocked by the bradykinin B1 receptor antagonist des-Arg(9)-[Leu(8)]bradykinin, but were not sensitive to bradykinin B2 receptor antagonists. These results therefore show that rat aortic smooth muscle cells in culture express functional bradykinin B1 receptors.     

Endotoxin sensitization to kinin B(1) receptor agonist in a non-human primate model: haemodynamic and pro-inflammatory effects

1. Although endotoxaemia induces kinin B(1) receptors in several animal models, this condition is not documented in primates. This study examined the up-regulation of haemodynamic and pro-inflammatory responses to the B(1) agonist des-Arg(10)-kallidin (dKD) in a non-human primate model. 2. Green monkeys (Cercopithecus aethiops St Kitts) received lipopolysaccharide (LPS; 90 microg kg(-1)) or saline intravenously. After 4 h, anaesthetized monkeys were cannulated via the carotid artery to monitor blood pressure changes following intra-arterial injections of dKD or the B(2) agonist bradykinin (BK). Oedema induced by subcutaneous kinin administration was evaluated as the increase in ventral skin folds in anaesthetized monkeys injected with captopril at 4 h to 56 days post-LPS. 3. LPS increased rectal temperature but did not affect blood pressure after 4 h. dKD reduced blood pressure (E(max): 27+/-4 mmHg; EC(50): 130 pmol kg(-1)) and increased heart rate (E(max): 33 b.p.m.) only after LPS. In contrast, the dose-dependent fall in blood pressure with BK was comparable in all groups. The selective B(1) antagonist [Leu(9)]dKD (75 ng kg(-1) min(-1), intravenously) abolished responses to dKD but not BK. 4. dKD injection induced oedema dose-dependently (2.4+/-0.1 mm at 150 nmol) only following LPS (at 4 h to 12 days but not 56 days). In contrast, BK-induced oedema was present and stable in all monkeys. Co-administration of [Leu(9)]dKD (150 nmol) significantly reduced oedema induced by dKD (50 nmol). 5. These results suggest LPS up-regulation of B(1) receptor effects in green monkeys. This non-human primate model may be suitable for testing new, selective B(1) antagonists with therapeutic potential as anti-inflammatory agents.     

Modulatory effect of bradykinin on the release of noradrenaline from rat isolated atria.

1. We investigated the modulation by bradykinin (BK) of electrically induced noradrenaline release in rat isolated atria preincubated with [3H]-noradrenaline. 2. BK (1-100 nM) enhanced significantly the stimulation-induced outflow of radioactivity in a concentration-dependent manner with a calculated EC50 of 0.58 nM. 3. Des-Arg9-BK (0.1-100 nM), a selective B1 receptor agonist, did not modify the stimulation-induced outflow of radioactivity. Hoe 140 (10 nM), a selective B2 receptor antagonist, but not [Leu8]-des-Arg9-BK (100 nM), a selective B1 receptor antagonist, blocked the facilitatory effect of BK. 4. The effect of BK was not affected by diclofenac (1 microM), a cyclo-oxygenase inhibitor. Bisindolylmaleimide (1 microM), a protein kinase C inhibitor, significantly reduced the facilitatory effect of BK (10 nM), angiotensin II (0.3 microM) and phorbol dibutyrate (0.1 and 1 microM) but not of fenoterol (1 microM). 5. The results suggest that BK enhances noradrenaline release via a prejunctional B2 kinin receptor in the rat atrium. The effect appears to involve protein kinase C as a second messenger.     

Mediators involved in the febrile response induced by Tityus serrulatus scorpion venom in rats.

Tityus serrulatus venom (Tsv) was intraperitoneally (ip) injected at doses of 75, 150 and 300mug/kg and IL-1beta (2.0 microg/kg) was given intravenously (iv) to male Wistar rats. Rectal temperature was measured by radiotelemetry. Vagotomy was performed according to Bluthe et al. [1994. Lipopolysaccharide induces sickness behaviour in rats by a vagal mediated mechanism. C R Acad. Sci. 317(6), 499-503]. Cerebrospinal fluid (CSF) and peritoneal fluid (PF) levels of bradykinin (BK) were measured by ELISA. B(1) (des-Arg(9)-[Leu(8)]-BK; DALBK) and B(2) kinin receptor (icatibant) antagonists (1.0 mg/kg each), the induced nitric oxide synthase inhibitor aminoguanidine (50.0 mg/kg), the neuronal nitric oxide synthase inhibitor 7-nitroindazole (30.0 mg/kg), the dual cyclooxygenase inhibitor ibuprofen (10.0 mg/kg), the selective interleukin-1 receptor antagonist IL-ra (2.0 mg/kg) and dipyrone (120 mg/kg) were given ip. Celecoxib (5 mg/kg) was given per os (po). Tsv at doses of 75 microg/kg evoked no change in rectal temperature while at doses of 150 and 300 microg/kg it promoted long-lasting fever (2 degrees C+/-0.1). Tsv (150 microg/kg) increased by nearly 3 and 5 times, respectively BK concentration in the CSF and in the PF. Subdiaphragmatic vagotomy or 7-nitroindazole reduced, icatibant, DALBK, IL-1ra, aminoguanidine and dipyrone abolished, while ibuprofen and celecoxib failed to affect Tsv-induced fever. These results suggest that PGs do not play a relevant role, whereas, kinins via their B(1) and B(2) receptors, IL-1, nitric oxide and vagal neurotransmission are involved in Tsv-induced fever.     

DesArg9-D-Arg[Hyp3,Thi5,D-Tic7,Oic8]bradykinin (desArg10-[Hoe140]) is a potent bradykinin B1 receptor antagonist.

DesArg9-D-Arg[Hyp3,Thi5,D-Tic7,Oic8]BK is a potent and stable B1 bradykinin (BK) receptor antagonist which was one order of magnitude more potent (IC50 1.2 x 10(-8) M) in the isolated rabbit aorta than the known selective B1 BK receptor antagonist, desArg9-[Leu8]BK (IC50 1.1 x 10(-7) M). DesArg9-D-Arg[Hyp3,Thi5,D-Tic7,Oic8]BK is the desArg10 derivative of Hoe140, a new, potent, stable, selective and long-acting B2 BK receptor antagonist. In B2 organ preparation it was three orders of magnitude less potent than Hoe140. Since it is potent and stable it could contribute to the investigation of B1 BK receptor function.     

Involvement of bradykinin B1 and B2 receptors in pulmonary leukocyte accumulation induced by Sephadex beads in guinea pigs.

The effects of selected bradykinin receptor antagonists on leukocyte infiltration into the lungs were studied in a model of guinea pig lung inflammation induced by the intravenous injection of Sephadex beads. The bradykinin B1 receptor antagonist, [Leu8]desArg9-BK (40 mg kg(-1) 24 h(-1)) and the bradykinin B2 receptor antagonist, DArg[Hyp3,Thi5,DTic7,Oic8]BK (code name HOE 140; 4 mg kg(-1) 24 h(-1)), administered intravenously by osmotic pumps, significantly reduced eosinophil counts by 33% and 42% in bronchoalveolar fluid, respectively. HOE 140 decreased neutrophil counts by 35%. LysLys[Hyp3,Igl5,D-Igl7,Oic8]desArg9BK+ ++ (code name B 9858), a newly described bradykinin B1 receptor antagonist, administered intraperitoneally (1 mg kg(-1)), decreased eosinophil and neutrophil counts by 45% in bronchoalveolar fluid. D-Arg[Hyp3,Igl5,D-Igl7,Oic8]BK (code name B 9430), a non-selective bradykinin B1/B2 receptor antagonist, also administered intraperitoneally (1 mg kg(-1)), decreased eosinophil and macrophage counts by 62% and 80% in bronchoalveolar fluid. These results suggest that bradykinin B1 and B2 receptors are involved in leukocyte recruitment in our model of lung inflammation.     

The Relaxing Effect of ��-Defensin 1 on the Adrenergic Responses of Rat Bladder

Defensins, cysteine-rich cationic polypeptides released from neutrophils, are known to have powerful antimicrobial properties. In this study, we sacrificed 30 rats to investigate the effects of α-defensin 1 on detrusor muscle contractions in isolated rat bladder. From the experiments we found relaxing effects of α-defensin 1 on the contractions induced by phenylephrine (PE) but not by bethanechol (BCh) in the detrusor smooth muscles. To determine the mechanisms of the effects of α-defensin 1, the changes of effects on PE-induced contraction by α-defensin 1 pretreatment were observed after pretreatment of Rho kinase inhibitor (Y-27632), protein kinase C (PKC) inhibitor (Calphostin C), potent activator of PKC (PDBu; phorbol 12,13-dibutyrate), and NF-κB inhibitors (PDTC; pyrrolidinedithiocarbamate and sulfasalazine). The contractile responses of PE (10^-9~10^-4 M) were significantly decreased in some concentrations of α-defensin 1 (5×10^-9 and 5×10^-8 M). When strips were pretreated with NF-κB inhibitors (PDTC and sulfasalazine; 10^-7~10^-6 M), the relaxing responses by α-defensin 1 pretreatment were disappeared. The present study demonstrated that α-defensin 1 has relaxing effects on the contractions of rat detrusor muscles, through NF-κB pathway. Further studies in vivo are required to clarify whether α-defensin 1 might be clinically related with bladder dysfunction by inflammation process.     

The pharmacology of T-kinin and des-Arg(11)-T-kinin in primary cultures of rat bladder smooth muscle cells

T-kinin and its putative carboxypeptidase product des-Arg(11)-T-kinin are members of the kinin family that are unique to the rat. Primary cultures of rat bladder smooth muscle cells were used to investigate the pharmacology of these peptides. Calcium imaging experiments showed that rat bladder smooth muscle cells responded to both bradykinin and des-Arg(9)-bradykinin with an increase in [Ca(2+)](i) and responses to both agonists could be observed in the same cell. A more detailed pharmacological characterisation with a range of bradykinin receptor agonists and antagonists using 45Ca(2+) efflux confirmed the presence of both B(1) and B(2) bradykinin receptors. Using this cellular model, we confirm that T-kinin is a bradykinin B(2) receptor agonist and show for the first time that des-Arg(11)-T-kinin is a potent and selective bradykinin B(1) receptor agonist. In addition, using cells expressing the cloned rat and human bradykinin B(2) receptors plus the Ca(2+)-sensitive protein aequorin, T-kinin was shown to be selective for the rat over the human bradykinin B(2) receptor.     

Contribution of B(2) receptors for bradykinin in arthus reaction-induced plasma extravasation in wild-type or B(2) transgenic knockout mice

The aim of the present study was to investigate the contribution of bradykinin (BK) B(1) and B(2) receptors in a model of type III hypersensitivity, the reverse passive Arthus reaction (RPA), in wild-type mice and transgenic B(2) knockout littermates. BK (10 microg mouse(-1)) or bovine serum albumin (0.5 mg mouse(-1)) induced a sustained Evans blue extravasation for more than 80 min in naive or rabbit anti-bovine serum albumin-treated mice (RPA model), respectively. The response to the two stimuli was prevented by the B(2) receptor antagonist, HOE-140, but not by [Leu(8)]desArg(9)-BK (B(1) receptor antagonist). In contrast to the wild-type littermates, RPA and bradykinin were unable to trigger an increase in plasma extravasation in B(2) knockout mice. Furthermore, endothelin-1 (5 microg mouse(-1)) and a selective NK-1 receptor agonist [Sar(9),Met (O(2))(11)]-SP (20 microg mouse(-1)), triggered a significant increase in peritoneal plasma extravasation in both wild-type and B(2) knockout animals. A pretreatment with indomethacin (200 microg mouse(-1)) significantly reduced the RPA-induced but not the BK-induced increase in Evans blue extravasation. Furthermore, RPA, but not BK, triggered a significant indomethacin-sensitive increase in peritoneal prostaglandin E(2) content. Our results suggest a pivotal role for B(2) receptors in the mechanism of plasma extravasation which occurs during the reverse passive Arthus reaction in the mouse. Moreover, our results suggest an important contribution of prostanoids in the plasma leakage mechanisms triggered by RPA but not by bradykinin.