Bradykinin B2 receptor evoked K+ permeability increase mediates relaxation in the rat duodenum.

We have investigated the receptors and associated coupling mechanisms that mediate the smooth muscle relaxant response to bradykinin (BK) in the rat duodenum in vitro. Relaxation in response to BK seems due to a direct action on the longitudinal smooth muscle since effects were demonstrable in the presence of ibuprofen, mepyramine, atropine, guanethidine (all 1 microM), hexamethonium (10 microM) and TTX (0.3 microM). Receptors involved are of the B2 subtype since agonists and antagonists active at B1 receptors were essentially inactive, and the B2 receptor antagonist Lys,Lys-[Hyp3,Thi5,8,D-Phe7]BK was a potent competitive antagonist of BK-induced relaxation (pKB of 7.2 +/- 0.1). The activity of both BK and the antagonist were unchanged by the presence of peptidase inhibitors including the carboxypeptidase inhibitor DL-2-mercaptomethyl-3-guanidinoethylthiopropanoic acid (mergetpa, 10 microM), which prevents conversion of BK analogues to des-Arg9-B1-active products. In high-K+ solution, BK (0.1-10 microM) produced concentration-related increases in 86Rb efflux. Both this permeability increase in high-K+ solution, and the relaxant responses in Krebs solution, were inhibited by low concentrations (10-100 nM) of apamin, as well as the B2 receptor antagonist Lys,Lys-[Hyp3,Thi5,8,D-Phe7]BK (1 microM). These results are compatable with the proposal that BK-evoked relaxation of the rat duodenum is mediated via a subset of B2 receptors for which the antagonist Lys,Lys-[Hyp3,Thi5,8,D-Phe7]BK has a high affinity, and results from stabilisation of the smooth muscle membrane through the opening of apamin-sensitive 86Rb-permeable calcium-activated K+ channels.     

Selectivity of bradykinin analogues for receptors mediating contraction and relaxation of the rat duodenum.

1. Bradykinin produces a biphasic response in the rat duodenum that consists of a relaxation (pD2 = 8.44) followed by a contraction (pD2 = 6.91). 2. The B1 agonist des-Arg9-bradykinin produced a contraction (pD2 = 7.16) but no relaxation. Des-Arg9-[Leu8]-bradykinin, which is a B1 antagonist in other systems produced contraction (pD2 = 7.65) in the rat duodenum. 3. Four bradykinin analogues that are preferential B2 agonists in other tissues had a biphasic effect with pD2 values in the range 7.22-8.68 for relaxation and 6.26-6.91 for contraction. 4. [Thi5,8,D-Phe7]-bradykinin, which is a B2 antagonist in most other systems produced relaxation in the rat duodenum, with a pD2 of 7.49. 5. It is concluded that the contractile component of the response to bradykinin in rat duodenum may be mediated by a subtype of the B1 receptor and the relaxant component by a receptor of the B2 subtype.     

The actions of kinin antagonists on B1 and B2 receptor systems

The newly discovered bradykinin antagonist [Thi5,8,D-Phe7]Bradykinin, supplied by J.-M. Stewart and three other compounds, [D-Phe7]BK, [Thi5,8,D-Phe7]Bradykinin and [Thi6,9,D-Phe8]Kallidin synthesized in our laboratory, were tested for their ability to antagonize bradykinin in four B2 receptor systems, the guinea-pig ileum, the rabbit jugular vein, the dog carotid artery and the dog urinary bladder as well as against desArg9-bradykinin in the rabbit aorta (a B1 receptor system). [D-Phe7]Bradykinin is a partial agonist, while [Thi5,8,D-Phe7]Bradykinin and [Thi6,9,D-Phe8]Kallidin are pure antagonists, the second one showing little BK-like activity on three of the four preparations. The kallidin analogue is more potent in all preparations than the bradykinin one. The two [Thi5,8,D-Phe7]BK (that supplied by J.-M. Stewart and that prepared in our laboratory) show very similar affinities in all preparations. The bradykinin analogue as well as the kallidin one are also active against desArg9-bradykinin in the rabbit aorta, at concentrations similar to those active on B2 receptor systems. The kinin antagonists are however specific for the kinins, since they do not interfere with the myotropic effects of angiotensin or substance P (SP) in the various preparations.     

Bradykinin B2 receptor-mediated phosphoinositide hydrolysis in bovine cultured tracheal smooth muscle cells.

1. Bovine tracheal smooth muscle cells were established in culture to study agonist-induced phosphoinositide (PI) hydrolysis in this tissue. 2. Bradykinin (0.1 nM-10 microM) evoked a concentration-dependent increase (log EC50 (M) = -9.4 +/- 0.2; n = 8) in the accumulation of total [3H]-inositol phosphates in cultured tracheal smooth muscle cells whereas the selective B1 receptor agonist des-Arg9-bradykinin (10 microM) was significantly less effective (16% of bradykinin maximal response; relative potency = 0.2 with respect to bradykinin = 100). 3. The bradykinin-induced increase in PI hydrolysis was unaffected by the B1 receptor antagonist des-Arg9[Leu8]-bradykinin (1 nM-1 microM) but showed marked attenuation in the presence of the B2 receptor antagonists D-Arg,[Hyp3,D-Phe7]-bradykinin (10 nM-10 microM) or D-Arg[Hyp3,Thi5,8,D-Phe7]-bradykinin (10 nM-10 microM). The estimated KB values obtained for these two compounds, assuming competitive antagonism, were 40 +/- 14 nM and 8.6 +/- 2.8 nM for D-Arg,[Hyp3,D-Phe7]-bradykinin and D-Arg[Hyp3,Thi5,8,D-Phe7]-bradykinin respectively. 4. We conclude that bradykinin B2 receptors are expressed in cultured bovine tracheal smooth muscle cells and are coupled to PI hydrolysis mechanisms.     

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.     

Ca(2+)-dependent and -independent mechanism of cyclic-AMP reduction: mediation by bradykinin B2 receptors.

1. Bradykinin caused a transient reduction of about 25% in the cyclic AMP level in forskolin prestimulated DDT1 MF-2 smooth muscle cells (IC50: 36.4 +/- 4.9 nM) and a pronounced, sustained inhibition (40%) of the isoprenaline-stimulated cyclic AMP level (IC50: 37.5 +/- 1.1 nM). 2. The Ca2+ ionophore, ionomycin, mimicked both the bradykinin-induced transient reduction in the forskolin-stimulated cyclic AMP level and the sustained reduction in the isoprenaline-stimulated cyclic AMP level. 3. The Ca(2+)-dependent effect on cyclic AMP induced by bradykinin was mediated solely by Ca2+ release from internal stores, since inhibition of Ca2+ entry with LaCl3 did not reduce the response to bradykinin. 4. The involvement of calmodulin-dependent enzyme activities, protein kinase C or an inhibitory GTP binding protein in the bradykinin-induced responses was excluded since a calmodulin inhibitor, calmidazolium, a PKC inhibitor, staurosporine and pertussis toxin, respectively did not affect the decline in the cyclic AMP level. 5. Bradykinin enhanced the rate of cyclic AMP breakdown in intact cells, which effect was not mimicked by ionomycin. This suggested a Ca(2+)-independent activation of phosphodiesterase activity by bradykinin in DDT1 MF-2 cells. 6. The bradykinin B1 receptor agonist, desArg9-bradykinin, did not affect cyclic AMP formation in isoprenaline prestimulated cells, while the bradykinin B2 receptor antagonists, Hoe 140 (D-Arg[Hyp3, Thi5, D-Tic7, Oic8]-BK) and D-Arg[Hyp3, Thi5,8, D-Phe7]-BK completely abolished the bradykinin response in both forskolin and isoprenaline prestimulated cells. 7. Bradykinin caused an increase in intracellular Ca2+, which was antagonized by the bradykinin B2 receptor antagonists, Hoe 140 and D-Arg[Hyp3, Thi5,8, D-Phe7]-BK.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characteristics of the bradykinin-induced changes in intracellular calcium ion concentration of single bovine tracheal smooth muscle cells.

1. Single bovine tracheal smooth muscle (BTSM) cells were cultured and used to measure bradykinin-induced changes in [Ca2+]i by dynamic video imaging. 2. Bradykinin (10 pM-10 microM)-induced an increase in [Ca2+]i over basal levels (69 +/- 2 nM; n = 353) which was concentration-dependent (log EC50 = -8.7 M) in the presence of extracellular calcium ions (2 mM). The bradykinin B2 receptor antagonist, D-Arg[Hyp3,Thi5,8,D-Phe7]- bradykinin, produced a parallel shift to the right of the bradykinin concentration-response curve (log EC50 = -7.1 M and -5.8 M in the presence of 1 microM and 10 microM antagonist respectively) yielding an apparent KD of 26 nM. 3. In the absence of extracellular calcium ions (with 0.1 mM EGTA), bradykinin (10 pM-10 microM) produced a uniform increase in [Ca2+]i from a basal level of 33 +/- 2 nM (n = 140) to approximately 180 nM in BTSM cells indicating an 'all-or-nothing' release of intracellular calcium ions. In the presence of 10 microM D-Arg[Hyp3,Thi5,8,D-Phe7]-bradykinin no responses could be induced by bradykinin at concentrations below 100 nM. However, at 100 nM and 1 microM bradykinin there was no change in the uniform increase in [Ca2+]i in these cells previously observed. 4. In both the absence or presence of D-Arg[Hyp3,Thi5,8,D-Phe7]-bradykinin, there was a concentration-dependent increase in the percentage of cells responding to bradykinin (frequency) under calcium-rich or calcium-free conditions. Individual cells also demonstrated a difference in the sensitivity to any particular concentration of bradykinin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mechanism of contractile responses to bradykinin in canine basilar arteries.

This study was undertaken to investigate the role of endothelium in the modulation of vascular responses to bradykinin and to elucidate the receptor types and mechanism of action of bradykinin in isolated basilar artery. The results showed a contractile response to bradykinin in basilar artery. This contractile response to bradykinin was partially modulated by endothelium in a dose-dependent manner. In addition, both des-Arg9-[Leu8]bradykinin and [d-Arg0,Hyp3,Thi5,8,D-Phe7]bradykinin significantly antagonized the responses to bradykinin. However, the blocking effect and the apparent affinity of [d-Arg0,Hyp3,Thi5,8,D-Phe7]bradykinin (pA2 = 9.6 +/- 0.4) were greater than those with des-Arg9-[Leu8]bradykinin (pA.2 = 7.8 +/- 0.3). These results suggest that two apparently distinct types of BK receptors may exist in basilar arteries. Furthermore, the contractile response to bradykinin in basilar artery was significantly inhibited by 2-nitro-4-carboxyphenyl-N,N-diphenylcarbamate (10(-5) M), H-7 (10(-5) M) and TMB-8 (10(-5) M), but not by indomethacin (10(-5) M) or nordihydroquariaretic acid (10(-5) M). On the other hand, nifedipine, Ca(2+)-free medium, EGTA and Ca(2+)-free medium/EGTA significantly reduced the bradykinin-induced contraction, indicating that part of the contractile response of basilar artery to bradykinin is dependent on extracellular Ca2+. In conclusion, the mechanism of the contractile responses to bradykinin in basilar artery may involve increased intracellular Ca2+ levels acting on the BK1 and BK2 receptor, followed by activation of the phosphoinositide pathway and receptor-mediated Ca2+ channel.     

Bradykinin receptors in the guinea-pig taenia caeci are similar to proposed BK3 receptors in the guinea-pig trachea, and are blocked by HOE 140.

1. Bradykinin (BK) receptors of the guinea-pig taenia caeci were compared with those of the guinea-pig trachea, a preparation proposed to possess novel BK3 receptors. 2. Bradykinin-evoked contractile responses were unaffected in both preparations by the selective BK1 receptor antagonist [des-Arg9,Leu8]-BK (1 microM-10 microM). The BK2 receptor antagonists, D-Arg-[Hyp3,D-Phe7]-BK and D-Arg-[Hyp3,Thi5,8,D-Phe7]-BK, both had low affinities (apparent pKB estimates less than 6) which did not differ significantly between the two preparations (P greater than 0.05). In contrast, the novel bradykinin receptor antagonist D-Arg-[Hyp3,Thi5,D-Tic7,Oic8]-BK (HOE 140) potently antagonized responses to bradykinin with relatively high affinity (apparent pKB = 8.42 +/- 0.15 and 8.94 +/- 0.16 in the taenia caeci, and trachea, respectively). 3. We conclude that the bradykinin receptors in the guinea-pig taenia caeci have similar recognition properties to those present in the guinea-pig trachea, and in this respect the taenia caeci represents a useful preparation for the further study of proposed novel BK3 receptors.     

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.     

Characterization of a novel binding site for 125I-Tyr-D-Arg-[Hyp3,D-Phe7,Leu8]bradykinin on epithelial membranes of guinea pig ileum.

We have recently shown that (a) [125I-Tyr8]bradykinin (BK) recognized bradykinin binding sites in guinea pig epithelium membranes with a Kd value of 1.6 nM and a Bmax of 156 fmol/mg protein, and (b) B2 agonists and some B2 antagonists, such as D-Arg-[Hyp3,D-Phe7,Leu8]BK, inhibited this specific binding with a Ki value of 32 nM. In the present study, we have radioiodinated the B2 antagonist Tyr-D-Arg-[Hyp3,D-Phe7,Leu8]BK and have performed a full characterization of the binding properties of this tracer in the same membrane preparation. Equilibrium experiments performed in the absence or presence of an excess of BK (10(-5) M) showed that 125I-Tyr-D-Arg-[Hyp3,D-Phe7,Leu8]BK specifically labelled two different sites. One of these is the same as the site labelled by [125I-Tyr8]BK, and this indicates that 125I-Tyr-D-Arg-[Hyp3,D-Phe7,Leu8]BK interacts specifically with kinin B2 receptors. Equilibrium experiment performed in the presence of an excess of BK (10(-5) M) indicated that specific binding of 125I-Tyr-D-Arg-[Hyp3,D-Phe7,Leu8]BK to the second site is also saturable and Scatchard analysis showed that the site is of high affinity with a Kd of 16.8 nM and a Bmax of 2.08 pmol/mg protein. Surprisingly, unlabelled B2 agonists such as bradykinin, [Tyr8]BK, [Leu8]BK, [Hyp3,Tyr8(OMe)]BK, D-Arg-[Hyp3]BK and kallidin were found to be inactive on this second site. A series of B2 receptor antagonists, Tyr-D-Arg-[Hyp3,D-Phe7,Leu8]BK, D-Arg-[Hyp3,D-Phe7,Leu8]BK, D-Arg-[Hyp3,Leu5,8,D-Phe7]BK, D-Arg-[Hyp3,Gly6,D-Phe7,Leu8]BK and D-Arg-[Hyp3,Thi5,8,D-Phe7]BK inhibited 125I-Tyr-D-Arg-[Hyp3,D-Phe7,Leu8]BK binding with Ki values of 25.0, 20.9, 15.8, 64.6 and 6606.9 nM respectively. On the other hand, [Thi5,8,D-Phe7]BK did not interfere with 125I-Tyr-D-Arg-[Hyp3,D-Phe7,Leu8]BK but was found to be a potent inhibitor of [125I-Tyr8]BK binding (Ki = 53.7 nM). As expected, B1 receptor agonists, antagonists and peptides non-related to BK such as substance P, neurokinin A, neurokinin B, angiotensin II, bombesin, vasopressin and the calcitonin gene related peptide were unable to compete with 125I-Tyr-D-Arg-[Hyp3,D-Phe7,Leu8]BK. The results show that 125I-Tyr-D-Arg-[Hyp3,D-Phe7,Leu8]BK is interacting with two distinct binding sites in the guinea pig epithelium: one is the well known bradykinin B2 receptor and the other is a new, non-characterized binding site that interacts exclusively with some bradykinin receptor antagonists.     

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.     

Multiple mechanisms in the motor responses of the guinea-pig isolated urinary bladder to bradykinin.

1. Bradykinin (1 nm-1 microM) produced a contraction of bladder strips excised from the dome of the guinea-pig urinary bladder, an effect which was greatly enhanced by removal of the mucosal layer or by thiorphan (10 microM). All subsequent experiments were performed in mucosa-free strips and in the presence of thiorphan. 2. In carbachol (5 microM)-contracted strips, bradykinin produced a concentration (1 nm-1 microM)-dependent transient relaxation. 3. Kallidin was slightly more potent than bradykinin in producing a contraction and a relaxation of the carbachol-induced tone. By contrast, [des-Arg9]-bradykinin, a selective B1 receptor agonist was barely effective up to 1 microM. 4. The contractile response to bradykinin was: (a) unaffected by either tetrodotoxin (1 microM), in vitro capsaicin desensitization (10 microM for 30 min) or apamin (0.1 microM); (b) antagonized by indomethacin (5 microM), the prostaglandin receptor antagonist SC-19220 (100 microM) or the B2 receptor antagonist [D-Arg0, Hyp3, Thi5,8, Phe7]-bradykinin (10 micron) and (c) almost abolished by nifedipine (1 microM). 5. The antagonism of the contractile response to bradykinin produced by indomethacin and SC-19220 was non-additive while that produced by indomethacin and the B2 receptor antagonist was additive. 6. The relaxant response to bradykinin was unaffected by tetrodotoxin, in vitro capsaicin desensitization or indomethacin but antagonized in a competitive manner by the B2 receptor antagonist. Further, this response was abolished by apamin (0.1 microM) but unaffected by glibenclamide (1 microM). 7. Bradykinin (10 microM) produced a consistent release of calcitonin gene-related peptide-like immunoreactivity (CGRP-LI) but not substance P-LI from the guinea-pig bladder muscle. CGRP-LI release by bradykinin was greatly reduced in bladders exposed to indomethacin. [des-Arg9]-bradykinin (10 microM) was ineffective. 8. We conclude that: (a) bradykinin-induced contraction involves activation of both B2 receptors and prostanoid synthesis, via distinct mechanisms which act by inducing calcium influx via nifedipine-sensitive channels; (b) bradykinin-induced relaxation involves activation of B2 receptors and opening of apamin-sensitive potassium channels; (c) bradykinin stimulates sensory nerves in this tissue largely via prostanoid production.     

BK2 but not BK1 receptors mediating contractile response in human umbilical arteries: role of thromboxane A2

Bradykinin receptors have been divided into B1 and B2 subtypes. The aim of this study on human umbilical arteries was: i) to compare the recognition properties of the mediating contractions of bradykinin receptors; and ii) to assess the possible role of thromboxane A2 in a bradykinin-induced contraction of smooth muscle. Umbilical arteries were dissected and mounted in organ baths for isometric measurement of force. Our results showed that the B1 agonist [Sar1dPhe8desArg9]-bradykinin had no effect on the concentration-response 10(-9)-3 x 10(-5) mM. Cumulative additions of bradykinin (10(-9)-3 x 10(-5) mM) and of the B2 agonist [Hyp3TyrMe8]-bradykinin (10(-9)-3 x 10(-5) mM) produced dose-dependent contractions. Dose response curves to bradykinin (10(-9)-3 x 10(-5) mM) were not significantly altered by the presence of B1 selective antagonist [des-Arg9, Leu8]-bradykinin (10(-5) mM), or by the selective B2 antagonist [Thi(5,8), D-Phe7]-bradykinin (10(-5) mM). However, Hoe 140 D-Arg-[Hyp3, Thi5,D-Tic7, Oic8]-bradykinin, an antagonist of B2 responses, significantly inhibited bradykinin-induced contraction. The responses to bradykinin were unaffected by indomethacin (10(-4) mM), dazoxiben (10(-5) mM) or even by nordihydroguaiaretic acid (10(-5) mM). However, bradykinin contractions were antagonized in a noncompetitive manner by quinacrine (10(-5) mM). These results showed that bradykinin contracts human umbilical arteries essentially through B2 receptors. Moreover, the responses to bradykinin are unlikely to be mediated by the cyclooxygenase/lipooxygenase pathway. The inhibitory effects of quinacrine may be due to a specific or nonspecific effect at a cellular level on smooth muscle contractility, or due to a direct action to block Ca2+ influx at membrane level.     

Histamine release from rat peritoneal mast cells by kinin antagonists

Kinins are known to be potent releasers of histamine. In the present study, the B2 antagonist analogues of bradykinin (BK) and kallidin (KD): [( Thi6,9,D-Phe8]KD, [Thi5,8,D-Phe7]BK) were also found to be potent releasers of histamine from rat mast cells and not to exert any antagonistic effect. Similarly, two B1 receptor antagonists, des-Arg10-[Leu9]KD and des-Arg9-[Leu8]BK, were shown to promote histamine release and acted only as agonists. The order of potency of these analogues was the following: [Thi6,9,D-Phe8]KD greater than [Thi5,8,D-Phe7]BK greater than des-Arg10- [Leu9]KD greater than des-Arg9-[Leu8]BK. The high activity of some of these compounds suggests that (1) the potency of kinins for histamine release from rat mast cells not only increases in parallel with the number of positively charged amino acids (Lys, Arg) but also, because of others factors such as peptide conformation, receptor binding affinity or receptor accessibility, (2) the histamine release by kinins is either a non-specific effect not mediated by receptors or, if receptors are involved, these sites may be different from other kinin receptors (i.e. B1 or B2).     

Activation of Gi-like proteins, a receptor-independent effect of kinins in mast cells.

The peptide hormones bradykinin and kallidin (Lys-bradykinin), as well as their analogues [des-Arg9]-bradykinin, a selective B1 agonist, [des-Arg9,Leu8]-bradykinin, a selective B1 antagonist, and [Thi5,8,D-Phe7]-bradykinin and D-Arg0-[Hyp3,D-Phe7]-bradykinin, two selective B2 antagonists, induced rapid histamine release from purified rat peritoneal mast cells. In contrast, the N-terminal fragment bradykinin-(1-5) was inactive. These peptides also activate the GTPase activity of GTP-binding proteins (G proteins) (Go/Gi) purified from calf brain, with an order of potency identical to that observed on mast cells, [Thi5,8,D-Phe7]-bradykinin much greater than kallidin greater than bradykinin greater than D-Arg0-[Hyp3,D-Phe7]-bradykinin greater than [des-Arg9]-bradykinin greater than [des-Arg9,Leu8]-bradykinin greater than bradykinin-(1-5). This correlation suggested that G proteins are the targets of kinins in mast cells. Accordingly, the concomitant increase in inositol trisphosphates and release of histamine elicited by kinins were inhibited by pertussis toxin pretreatment of mast cells. The inhibitory effect of benzalkonium chloride showed that the G proteins involved belong to the Gi type. GTPase activity was measured in the supernatant of homogenized mast cells but not in the membranous fraction. This activity was stimulated by kinins and by the venom peptide mastoparan. The potency of peptides was similar to that observed with purified bovine G proteins. Sodium dodecyl sulfate-gel electrophoresis of mast cell supernatant revealed pertussis toxin-induced ADP-ribosylation of two proteins, in the Mr 41,000 and 40,000 range, i.e., similar to purified alpha-subunits of Gi1 and Gi2 or Gi3 subtypes. The data support the proposal that bradykinin and analogues act like mastoparan, substance P, and compound 48/80, interacting first with sialic acid residues of the cell surface and then with Gi-like proteins, inducing phospholipase C activation and intracellular calcium mobilization.     

Hoe 140 a new potent and long acting bradykinin-antagonist: in vivo studies.

1. The potency, duration of action and tolerability of Hoe 140, a novel and highly potent bradykinin (BK) antagonist in vitro, has been tested in different in vivo models and compared with the well-known BK antagonist D-Arg-[Hyp2, Thi5,8, D-Phe7]BK. 2. Hoe 140 is highly potent and long acting in inhibiting BK-induced hypotensive responses in the rat. Four hours after s.c. administration of 20 nmol kg-1, inhibition still amounted to 60% whereas the effect of 200 nmol kg-1 of D-Arg-[Hyp2, Thi5,8, D-Phe7]BK was not significant. 3. BK-induced bronchoconstriction in guinea-pigs was strongly inhibited by Hoe 140. The magnitude and duration of inhibition confirmed the findings obtained in the blood pressure experiments in the rat. 4. Carrageenin-induced inflammatory oedema of the rat paw was considerably inhibited at i.v. doses between 0.1 and 1 mg kg-1. 5. In conscious dogs, intravenous doses of 0.01 and 0.1 mg kg-1 of Hoe 140 and D-Arg-[Hyp2, Thi5,8, D-Phe7]BK were well tolerated. At doses of 1 mg kg-1 adverse effects occurred that were attributed to the residual BK agonistic activity of both compounds. 6. Hoe 140 has been shown to be a highly potent and long acting BK antagonist in vivo in different animal species and models. This makes it appropriate to investigate further the physiological and pathophysiological role of BK.     

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.     

Hoe 140 a new potent and long acting bradykinin-antagonist: in vitro studies.

1. Hoe 140 (D-Arg-[Hyp3, Thi5, D-Tic7, Oic8]bradykinin) is a new bradykinin (BK)-antagonist. It was tested in several in vitro assays and compared with D-Arg-[Hyp2,Thi5,8,D-Phe7]BK. 2. In receptor binding studies in guinea-pig ileum preparations, Hoe 140 showed an IC50 of 1.07 x 10(-9) mol l-1 and a KI value of 7.98 x 10(-10) mol l-1. 3. In isolated organ preparations Hoe 140 and D-Arg-[Hyp2,Thi5,8, D-Phe7]BK inhibited bradykinin-induced contractions concentration dependently, with IC50-values in the guinea-pig ileum preparation of 1.1 x 10(-8) mol l-1 and 3 x 10(-5) mol l-1, respectively. pA2 values in this tissue were 8.42 and 6.18, respectively. In the rat uterus preparation the IC50 value was 4.9 x 10(-9) mol l-1 for Hoe 140. D-Arg-[Hyp2, Thi5,8, D-Phe7]BK showed an IC50 of 4.0 x 10(-6) mol l-1. The IC50 values in the guinea-pig isolated pulmonary artery were 5.4 x 10(-9) mol l-1 and 6.4 x 10(-6) mol l-1, respectively. In the rabbit aorta no inhibitory effects on Des-Arg9-BK induced contractions were observed. 4. In cultured bovine endothelial cells, Hoe 140 antagonized (IC50 = 10(-8) mol l-1) bradykinin-induced endothelium-derived relaxing factor (EDRF) release and the bradykinin-induced increase in cytosolic free calcium (IC50 = 10(-9) mol l-1).(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of the bradykinin receptor in the human nasal airway using the binding of [125I]-Hoe 140.

1. The aim of this study was to characterize the kinin receptor in the human nasal airway using [125I]-Hoe 140 binding to a membrane preparation from human nasal turbinates and to compare Ki values from binding displacement by antagonists with the functional effects of these drugs in vivo. We also investigated the effect of Hoe 140 ([D-Arg0, Hyp3, Thi5, D-Tic7, Oic8]-bradykinin), on bradykinin release into the nasal airway. 2. In a membrane preparation from human nasal turbinates removed during surgery, [125I]-Hoe 140 labelled a single, saturable binding site. The equilibrium dissociation constant (at 20 degrees C) for [125I]-Hoe 140 binding to the receptor was 0.46 +/- 0.08 nM. The Bmax was 0.136 +/- 0.003 pmol mg-1 protein and the Hill coefficient was 1.01 +/- 0.07. 3. The association rate constant for [125I]-Hoe 140 binding to the receptor was 0.20 +/- 0.06 nM-1 min-1 and the dissociation rate constant was 0.14 +/- 0.01 min-1. These values were determined at 4 degrees C. The equilibrium dissociation constant calculated from these rate constants was 0.70 nM. 4. Bradykinin and the B2 receptor antagonists, NPC 567, NPC 17731, NPC 17761, [1-adamantane acetyl-D-Arg0, Hyp3, Thi5,8, D-Phe7]-bradykinin, WIN 64338 and Hoe 140 displaced [125I]-Hoe 140 binding: the Ki values from binding displacement are consistent with values expected from a B2 receptor. The B1 agonist, [des-Arg9]-bradykinin and the B1 antagonist, [des-Arg9]-Hoe 140 failed to displace [125I]-Hoe 140 binding at concentrations up to 1 microM. 5. The bradykinin antagonist, Hoe 140, 10 to 200 micrograms, given by intranasal aerosol, produced a dose-related inhibition of the reduction in minimal nasal cross-sectional area (Amin) induced by bradykinin in normal subjects and by house dust mite antigen in subjects with allergic rhinitis to house dust mite. Hoe 140, 10 to 200 micrograms, also caused a dose-related inhibition of the release of albumin into the nasal cavity following challenge with bradykinin. 6. [1-Adamantane acetyl-D-Arg0, Hyp3, Thi5,8, D-Phe7]-bradykinin, 30 to 200 micrograms, caused a dose-related inhibition of the reduction in Amin and the release of albumin into the nasal cavity induced by bradykinin. NPC 567 ([D-Arg0, Hyp3, D-Phe7]-bradykinin) failed to inhibit the reduction in Amin or the release of albumin into the nasal cavity at a dose of 10 mg. 7. Challenge of allergic subjects with house dust mite antigen caused a significant elevation of the bradykinin concentration in nasal lavage fluid and a reduction in Amin. Hoe 140, 100 micrograms, prevented the antigen-induced reduction in Amin and also abolished the antigen-induced increase of bradykinin in nasal lavage fluid. 8. We conclude that there is a B2 bradykinin receptor in the human nasal airway which mediates nasal blockage and plasma extravasation induced by either bradykinin or antigen challenge. It is possible that Hoe 140 inhibits kallikrein in the human nasal airway as well as blocking the B2 receptor.