Bradykinin stimulates production of inositol (1,4,5) trisphosphate in cultured mesangial cells of the rat via a BK2-kinin receptor.

1. Using [125I-Tyr0]-BK, as radiolabelled ligand, and various agonists and antagonists of bradykinin (BK) we identified a single class of specific BK2-binding sites in mesangial cell membranes (Bmax = 73 fmol mg-1 protein and Kd = 3.7 nM). 2. Following the addition of 0.1 microM BK, inositol (1,4,5) trisphosphate (IP3) formation increased within 20 s from a basal level of 64 to a maximal value of 175 pmol mg-1 protein. 3. Incubation in a Ca(2+)-free medium did not change IP3 production but a 5 min preincubation with 1 mM EGTA completely prevented the BK-induced IP3 formation, suggesting that IP3 formation is partly dependent on extracellular calcium. 4. The BK2 antagonist D-Arg-Hyp3-D-Phe7-BK (10 microM) but not the BK1 antagonist (des-Arg9-Leu8-BK) abolished IP3 production in response to 0.1 microM BK. Pretreatment of mesangial cells with pertussis toxin was without effect on BK-induced IP3 formation, whereas phorbol 12-myristate 13-acetate significantly enhanced (by 25%) BK-induced IP3 formation. 5. The present data demonstrate that inositol phosphate breakdown in rat mesangial cells can be mediated via activation of a BK2-kinin receptor and is under negative control of protein-kinase C.     

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.     

Effects of kinins upon cytosolic calcium concentrations in mouse mesangial cells

Bradykinin (BK) induces increases in cytosolic calcium concentration [Ca++]i in several cell lines. Because the role of BK in the renal system, particularly in mesangial cell (MC), is not clear, we investigated the effects of kinins on [Ca++]i in mouse-immortalized MC. [Ca++]i was evaluated by spectrofluorometry and expressed as a ratio between the obtained and basal [Ca++]i. BK (0.1 microM) induced a non-sustained increase in [Ca++]i (4.70 +/- 0.27; N = 28). A similar effect was observed with the B2 receptor agonist, Tyr8-BK (0.1 microM, 3.34 +/- 0.48; N = 7), while B1 receptor agonists, des-Arg10-Kallidin (Kal) (1 microM, N = 11) and des-Arg9-BK (1 microM, N = 8), exhibited only discrete responses (1.45 +/- 0.08 and 1.12 +/- 0.04, respectively). Cross-desensitization was seen between BK and Tyr8-BK, but not between BK and des-Arg10-Kal. The BK response was decreased (5.09 +/- 0.30, N = 6 to 1.57 +/- 0.12, N = 7, P < 0.001) by the B2 receptor antagonist HOE 140 (0.1 microM, 15 min), while the B1 receptor antagonist des-Arg9-[Leu8]-BK (1 microM, 15 min) had no effect on BK or des-Arg10-Kal actions. Incubation of cells with Escherichia coli lipopolysaccharide (100 microg/ml, 24 h) alone or in association with tumor necrosis factor-alpha (TNF-alpha) (10 ng/ml, N = 6) did not enhance B1 agonist responses. BK was inhibited by repeated cell washouts in zero Ca++ solution (2.04 +/- 0.19, N = 6 P < 0.001), and the residual response was almost abolished by thapsigargin (Thaps) a sarcoplasmic reticulum (SR) calcium-ATPase inhibitor (1 microM) (1.18 +/- 0.08, N = 5 P < 0.001). Additionally, BK was not inhibited by verapamil (50 microM), nifedipine (30 microM), Ni++ (300 microM) or La (10 microM). In conclusion, BK induces [Ca++]i in mouse MC mainly by B2 receptor activation. B1 receptors have a minor role in this phenomenon even in the presence of known B1 receptor synthesis inducers. Finally, BK mobilizes extracellular calcium sources and, to a lesser extent, intracellular Thaps-sensitive calcium stores. The ion channels involved in calcium influx remain to be detected.     

Bradykinin-induced vascular responses in dog isolated lingual artery.

1. Kinin-induced vascular responses were studied and kinin receptor subtypes were characterized in canine isolated and preconstricted lingual arteries. 2. A low dose of bradykinin (BK; < 3 x 10(-14) mol) induced only vasodilation, while a higher dose of BK (> 3 x 10(-13) mol) frequently induced a biphasic response: a transient constriction followed by dilation. 3. The BK-induced vasodilation was mostly endothelium dependent but was also partly endothelium independent because although the dilation response was greatly reduced after removal of the endothelium, it was not completely abolished. 4. The dilation response to BK was significantly inhibited by the B2 kinin receptor antagonist HOE 140 and was partly reduced by indomethacin (10 mumol/L) (P < 0.05). 5. Bradykinin-induced vasoconstriction was enhanced in endothelium-denuded preparations. The constriction was significantly inhibited by HOE 140 (10(-10) mol/L). The BK-induced responses were not affected by the B1 kinin receptor antagonist des-Arg9-[Leu8]-BK (3 x 10(-11) mol/L). 6. The B1 kinin receptor agonist des-Arg9-BK (> 10(-12) mol/L) produced vasodilation in 60% of endothelium-intact preparations. In 20% of the endothelium-intact preparations des-Arg9-BK produced a biphasic response: weak vasoconstriction followed by weak vasodilation. The des-Arg9-BK-induced dilation and constriction were significantly inhibited by des-Arg9-[Leu8]-BK (3 x 10(-11) mol/L), but were not affected by HOE 140 (10(-10) mol/L). 7. In conclusion, it appears that both B1 and B2 kinin receptors are present in the dog lingual artery. Both receptor subtypes mediate either vasodilation or vasoconstriction and BK-induced vasodilation is mostly endothelium dependent, although it may also be partially prostaglandin dependent.     

Activation of osteoblastic functions by a mediator of pain, bradykinin

We investigated the effects of bradykinin (BK) on the production of interleukin (IL)-6 and prostaglandin PGE(2), whose molecules are capable of stimulating the development of osteoclasts from their hematopoietic precursors as well as the signal transduction systems involved, in human osteoblasts (SaM-1 cells). BK receptors B1 (B1R) and B2 (B2R) were expressed in SaM-1 and osteosarcoma (SaOS-2, HOS, and MG-63) cells. Treatment of SaM-1 cells with BK increased the synthesis of both IL-6 and PGE(2) and the increase in both was blocked by HOE140 (B2R antagonist), but not by Des-Arg(9)-[Leu(8)]-BK (B1R antagonist). U-73122, a phospholipase C (PLC) inhibitor, suppressed BK-induced IL-6 and PGE(2) synthesis in SaM-1 cells. In addition, BK caused an increase in the intracellular Ca(2+) concentration ([Ca(2+)]i), which was inhibited by pretreatment with HOE140 or 2-aminoethoxydiphenyl borate (2-APB), an inositol 1,4,5-trisphosphate (IP(3)) receptor (IP(3)R) blocker. Furthermore, both SB203580 (an inhibitor of p38 mitogen-activated protein kinase [MAPK]) and PD98059 (an inhibitor of MEK, upstream of ERK) attenuated the BK-induced IL-6 and PGE(2) synthesis. BK treatment resulted in the phosphorylation of p38 MAPK and extracellular signal-regulated kinase (ERK)1/2, and 2-APB could suppress BK-induced phosphorylation of ERK1/2. These findings suggest that BK increased both IL-6 and PGE(2) synthesis in osteoblastic cells via B2R and that PLC, IP(3)-induced [Ca(2+)]i, MEK, and MAPKs were involved in the signal transduction in these cells.     

Bradykinin stimulation of phosphoinositide hydrolysis in guinea-pig ileum longitudinal muscle.

1. Bradykinin (BK)-induced contraction of ileal smooth muscle is assumed to be due to phosphoinositide hydrolysis but this has never been reported. We have investigated whether BK receptors are linked to this transduction mechanism in guinea-pig ileum longitudinal muscle and determined whether these receptors are equivalent to those labelled in [3H]-BK binding assays. 2. In membranes prepared from longitudinal muscle, [3H]-BK bound to a single class of sites with high affinity. Characterization of the binding with BK analogues indicated that the radioligand selectivity labelled a B2 type receptor. 3. BK significantly elevated tissue levels of [3H]-inositol phosphates in longitudinal muscle slices preincubated with [3H]-myo-inositol. The agonists potencies of BK, Lys-BK, Met-Lys-BK, Tyr5-BK and Tyr8-BK were in agreement with their relative potencies in the binding assay. The B1 receptor agonist des-Arg9-BK, did not stimulate inositol phosphate production. The response to BK was blocked by known B2 receptor antagonists but not by the B1 antagonist des-Arg9, Leu8-BK. 4. BK-induced phosphoinositide hydrolysis was unaffected by exposure of muscle slices to either atropine or indomethacin. 5. The results indicate that the B2 receptors linked to phosphoinositide turnover in ileal longitudinal muscle exhibit properties similar to those involved in contractile responses. Also, the receptor mediating the phosphoinositide response is likely to be that labelled in the [3H]-BK binding studies.     

Stimulation of bradykinin B2-receptors on endothelial cells induces relaxation and contraction in porcine basilar artery in vitro.

1. The aim of the present study was to characterize the subtypes of bradykinin (BK) receptors that evoke the relaxation and contraction induced by BK and to identify the main contracting and relaxing factors in isolated porcine basilar artery by measuring changes in isometric tension and a thromboxane (TX) metabolite. 2. Endothelial denudation completely abolished both responses. [Thi5,8, D-Phe7]-BK (a B2-receptor antagonist) inhibited the BK-induced relaxation and contraction, whereas des-Arg9, [Leu8]-BK (a B1-receptor antagonist) had no effect. 3. L-nitro-arginine (L-NA, a nitric oxide synthase inhibitor) completely inhibited BK-induced relaxation. Indomethacin (a cyclo-oxygenase inhibitor) completely and ONO-3708 (a TXA2/prostaglandin H2 receptor antagonist) partially inhibited BK-induced contraction, whereas OKY-046 (a TXA2 synthase inhibitor) and nordihydroguaiaretic acid (a lipoxygenase inhibitor) did not. 4. In the presence of L-NA, the contractile response to BK was inhibited by indomethacin or ONO-3708 and was competitively antagonized by [Thi5,8, D-Phe7]-BK (pA2=7.50). In the presence of indomethacin, the relaxant response to BK was inhibited by L-NA and was competitively antagonized by [Thi5,8, D-Phe7]-BK (pA2=7.59). 5. TXA2 release was not induced by BK-stimulation. 6. These results suggest that the endothelium-dependent relaxation and contraction to BK in the porcine basilar artery is mediated via activation of endothelial B2-receptors. The main relaxing factor may be NO and the main contracting factor may be prostaglandin H2.     

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)     

Kinin receptors mediating the effects of bradykinin on the coronary circulation in anaesthetized greyhounds.

Bradykinin (BK, 0.05 micrograms/kg) or glyceryl trinitrite (5 micrograms/kg) injected into the left circumflex coronary artery of anaesthetized, open-chest greyhounds, caused pronounced increases in large coronary artery diameter (CD) and coronary blood flow (CBF), whereas des-Arg9-BK (0.05-0.3 micrograms/kg), a selective bradykinin B1 agonist, dose dependently elevated CBF but had little effect on CD. BK-induced increases in CD and CBF were not affected by the intracoronary infusion of a selective B1 receptor antagonist, des-Arg9-[Leu8]BK (40 micrograms/min), but were significantly reduced by the infusion of a selective B2 receptor antagonist, D-Arg0-[Hyp3,Thi5,8,D-Phe7] BK (10-12 micrograms/min). The antagonism was reversible and specific for BK since responses to glyceryl trinitrate were not affected. Bilateral vagotomy (n = 3) or autonomic blockade with atropine (0.1 mg/kg i.v.) and propranolol (1 mg/kg i.v.) (n = 5) resulted in significant attenuation of BK-induced increases in CBF but not that of CD. It is concluded that BK is a potent dilator of both conductance and resistance coronary vessels in anaesthetized greyhounds. The dilatation of conductance vessels appears to involve a selective interaction with B2 receptors, while BK-induced increase in CBF may be mediated by both B1 and B2 receptors and involve participation of neuroreflex mechanisms.     

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.     

Correlation between brain bradykinin receptor binding sites and cardiovascular function in young and adult spontaneously hypertensive rats

Intracerebroventricular (i.c.v.) effects of bradykinin (BK) B(1) and B(2) receptor agonists and antagonists were assessed on mean arterial blood pressure (MAP) and heart rate (HR) in awake unrestrained spontaneously hypertensive rats (SHR, aged of 8 and 16 weeks) and age-matched Wistar Kyoto rats (WKY). Quantitative in vitro autoradiographic studies were also performed on the brain of both strains with specific radioligands for B(2) receptors [(125)I]HPP-Hoe 140 and B(1) receptors [(125)I]HPP-des-Arg(10) and Hoe140. MAP increased linearly with doses of BK (81-8100 pmol) and the amplitudes were significantly greater in SHR, particularly at 16 weeks. While BK evoked a negative linear trend on HR (bradycardia) in WKY, a positive one (tachycardia) was observed in adult SHR. In both strains, BK-induced pressor response was blocked by equimolar doses of B(2) receptor antagonist, D-Arg-[Hyp(3), Thi(5), D-Tic(7), Oic(8)]-BK (Hoe 140), but not by B(1) receptor antagonist, AcLys[D-betaNal(7), Ile(8)]des-Arg(9)-BK (R-715). B(1) receptor agonists (Sar-[D-Phe(8)]-des-Arg(9)-BK, des-Arg(9)-BK, des-Arg(10)-Kallidin) and antagonist (R-715 alone or with Hoe 140) had no or marginal effect on MAP and HR at doses up to 8100 pmol in SHR and WKY. Higher densities of specific [(125)I]HPP-Hoe 140 labelling were found in discrete brain areas of SHR, especially in regions associated with cardiovascular function. Low levels of [(125)I]HPP-[des-Arg(10)]-Hoe140 binding sites were seen in WKY and SHR, yet densities were significantly greater in midbrain and cortical regions of SHR aged of 16 weeks. Contrary to SHR, ageing caused a downregulation of B(2) and B(1) receptor binding sites in specific brain nuclei in WKY. It is concluded that the hypersensitivity of the pressor response to i.c.v. BK in SHR occurs during both the early and established phases of hypertension in parallel with the enhancement of B(2) receptor binding sites in various cardiovascular brain centres. In contrast, brain B(1) receptors do not seem to participate in the central pressor effects of kinins nor in the maintenance of hypertension in SHR.     

The role of bradykinin B1 receptors in the maintenance of intra-articular plasma extravasation in chronic antigen-induced arthritis.

1. The role of bradykinin B1 and B2 receptors in bradykinin- and des-Arg9-bradykinin-induced plasma extravasation in normal and inflamed rat knee joints was investigated by use of an antigen-induced model of chronic arthritis. A modification of an Evans blue extraction technique allowed the unstimulated (basal) plasma extravasation to be assessed in this model. The contributions of bradykinin B1 and B2 receptors towards basal synovial plasma extravasation were determined. 2. In normal knees, intra-articular injection of bradykinin (BK) induced plasma extravasation in a potent, dose-dependent manner with a threshold of 0.01 nmol and an ED50 of 0.1 nmol. In day 5 arthritic knees, basal plasma extravasation was substantially enhanced. Lower doses of BK had no demonstrable effect and increases above basal extravasation were first observed at 0.1 nmol. Thereafter the dose-response mirrored the response in normal knees and the maximal response was unaltered. 3. The B1 agonist, des-Arg9-BK, induced slight but significant plasma extravasation in normal knees but was less potent than bradykinin. This response was inhibited by the B1 receptor antagonist, des-Arg9, [Leu8]-BK. Lower doses of des-Arg9-BK bradykinin did not significantly increase basal extravasation in day 5 arthritic knees but, in contrast to BK, the maximal response was significantly enhanced. 4. The B2 antagonist, Hoe 140, inhibited BK-induced plasma extravasation in normal joints over a dose-range of 0.1-1.0 nmol but was relatively inactive in day 5 inflamed knees.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Bradykinin-induced phosphoinositide hydrolysis and Ca2+ mobilization in canine cultured tracheal epithelial cells

1. Experiments were designed to differentiate the mechanisms and subtype of kinin receptors mediating the changes in intracellular Ca2+ concentration ([Ca2+]i) induced by bradykinin (BK) in canine cultured tracheal epithelial cells (TECs). 2. BK and Lys-BK caused an initial transient peak of [Ca2+]i in a concentration-dependent manner, with half-maximal stimulation (pEC50) obtained at 7.70 and 7.23, respectively. 3. Kinin B2 antagonists Hoe 140 (10 nM) and [D-Arg0, Hyp3, Thi5,8, D-Phe7]-BK (1 microM) had high affinity in antagonizing BK-induced Ca2+ response with pKB values of 8.90 and 6.99, respectively. 4. Pretreatment of TECs with pertussis toxin (100 ng ml(-1)) or cholera toxin (10 microg ml(-1)) for 24 h did not affect the BK-induced IP accumulation and [Ca2+]i changes in TECs. 5. Removal of Ca2+ by the addition of EGTA or application of Ca2+-channel blockers, verapamil, diltiazem, and Ni2+, inhibited the BK-induced IP accumulation and Ca2+ mobilization, indicating that Ca2+ influx was required for the BK-induced responses. 6. Addition of thapsigargin (TG), which is known to deplete intracellular Ca2+ stores, transiently increased [Ca2+]i in Ca2+-free buffer and subsequently induced Ca2+ influx when Ca2+ was re-added to this buffer. Pretreatment of TECs with TG completely abolished BK-induced initial transient [Ca2+]i, but had slight effect on BK-induced Ca2+ influx. 7. Pretreatment of TECs with SKF96365 and U73122 inhibited the BK-induced Ca2+ influx and Ca2+ release, consistent with the inhibition of receptor-gated Ca2+-channels and phospholipase C in TECs, respectively. 8. These results demonstrate that BK directly stimulates kinin B2 receptors and subsequently phospholipase C-mediated IP accumulation and Ca2+ mobilization via a pertussis toxin-insensitive G protein in canine TECs. These results also suggest that BK-induced Ca2+ influx into the cells is not due to depletion of these Ca2+ stores, as prior depletion of these pools by TG has no effect on the BK-induced Ca2+ influx that is dependent on extracellular Ca2+ in TECs.     

Des-Arg9-bradykinin increases intracellular Ca2+ in bronchoalveolar eosinophils from ovalbumin-sensitized and -challenged mice

The effects of the selective bradykinin B1 receptor agonist, des-Arg9-bradykinin and the bradykinin B2 receptor agonist, bradykinin were studied on the intracellular free Ca2+ concentration ([Ca2+]i) in murine bronchoalveolar lavage cells from control and ovalbumin-sensitized mice using fura-2 microfluorimetry. The bronchoalveolar lavage cells of control mice, which were predominantly alveolar macrophages, showed an increase in [Ca2+]i in response to bradykinin (1 microM) but not to des-Arg9-bradykinin (1 microM), indicating the presence of functional bradykinin B2 receptors and the absence of B1 receptors. Such elevation in [Ca2+]i induced by bradykinin was totally inhibited by the selective bradykinin B2 receptor antagonist, D-Arg0-Hyp3-Thi5-D-Tic7-Oic8-bradykinin (HOE-140; 10 microM). In contrast, bronchoalveolar lavage cells from ovalbumin-sensitized and -challenged mice significantly responded to both bradykinin and des-Arg9-bradykinin, indicating the presence of both functional bradykinin B1 and B2 receptors. Eosinophils exhibited higher response to des-Arg9-bradykinin (1 microM; 485% increase in [Ca2+]i) compared to bradykinin (1 microM; 163% increase in [Ca2+]i). This des-Arg9-bradykinin-induced [Ca2+]i increase was markedly inhibited by the selective bradykinin B1 receptor antagonist, Ac-Lys-[D-betaNal7, Ile8]des-Arg9-bradykinin (R-715; 10 microM). Des-Arg9-bradykinin neither modified the basal [Ca2+]i in lymphocytes nor in mononuclear cells from ovalbumin-sensitized and challenged mice, while bradykinin produced a [Ca2+]i increase in both cell types. Our results further support the implication of the inducible bradykinin B1 receptors in airway inflammatory response in ovalbumin-sensitized and challenged mice.     

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.     

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.     

D-Arg[Hyp3-Thi5-D-Tic7-Tic8]-bradykinin, a potent antagonist of smooth muscle BK2 receptors and BK3 receptors.

D-Arg[Hyp3-Thi5-D-Tic7-Tic8]-bradykinin (NPC 16731) inhibited bradykinin (BK) binding and BK-induced contraction in guinea-pig ileum, being markedly more potent than D-Phe7-BK analogues as a BK2 receptor antagonist. In isolated trachea NPC 16731, unlike other BK2 antagonists, inhibited BK binding and BK-induced contraction, and 45Ca2+ efflux in tracheal smooth muscle cells. That NPC 16731 potently inhibits BK effects in trachea provides further evidence for the existence of the airway BK3 receptor.     

Des-Arg9-bradykinin-induced increases in intracellular calcium ion concentration in single bovine tracheal smooth muscle cells.

1. Dynamic video imaging was used to measure the des-Arg9-bradykinin-induced changes in the intracellular free calcium ion concentration ([Ca2+]i) of single bovine tracheal smooth muscle (BTSM) cells. 2. In the presence of extracellular calcium ions, des-Arg9-bradykinin (1 nM-10 microM) produced a concentration-dependent increase in the [Ca2+]i over basal levels yielding an EC50 value of 316 nM. The percentage of cells responding to each concentration of des-Arg9-bradykinin also increased in a concentration-dependent manner (from 9% to 100%). 3. The bradykinin B2 receptor antagonist, D-Arg[Hyp3,Thi5,8,D-Phe7]-bradykinin (10 microM), was without effect on the calcium response of the cells when added 2 min prior to des-Arg9-bradykinin (100 nM). However, the B1 receptor antagonist, des-Arg9Leu8-bradykinin (10 microM), completely abolished the des-Arg9-bradykinin-induced response. 4. Under calcium-free conditions, des-Arg9-bradykinin induced an increase in [Ca2+]i at concentrations of 1 microM and 10 microM. The response to 10 microM des-Arg9-bradykinin was reduced by preincubation of either D-Arg[Hyp3, Thi5,8,D-Phe7]-bradykinin (10 microM) or des-Arg9Leu8-bradykinin (10 microM). 5. We conclude that bradykinin B1 receptors are expressed by cultured BTSM cells and mediate the des-Arg9-bradykinin-induced influx of calcium ions at low agonist concentrations (< 1 microM). At higher concentrations, des-Arg9-bradykinin (1 microM and 10 microM) can stimulate both B1 and B2 receptors to effect intracellular calcium release under calcium-free conditions.