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.     

BK1 and BK2 bradykinin receptors in the rat duodenum smooth muscle.

1. The dual action of bradykinin (relaxation and contraction) on the rat duodenum was investigated by studying its effect on adenosine 3':5'-cyclic monophosphate (cyclic AMP) levels in cultured duodenal smooth muscle cells, and the effects of apamin on the isolated muscle responses to agonists and antagonists of BK1 and BK2 receptors. 2. No change was observed in the cyclic AMP content of cultured cells incubated with up to 100 nM bradykinin. 3. Apamin (100-500 nM) inhibited the relaxant component and enhanced the contractile component of the responses to bradykinin and to the BK2-specific analogue [Thi5,8,D-Phe7]-bradykinin. 4. Apamin (100-500 nM) did not affect the contractile response of stretched duodenum preparation to the BK1-specific agonist des-Arg9-bradykinin. 5. The BK2 antagonist, [D-Arg0Hyp3Thi5,8,D-Phe7]-bradykinin, at a concentration which completely inhibited the relaxant response to bradykinin and to [Thi5,8,D-Phe7]-bradykinin, also prevented the contraction in response to either agonist in the presence of apamin. 6. Our results demonstrate two populations of bradykinin receptors in rat duodenum: a BK2 subtype responsible for the biphasic response of the non-stretched duodenum, and a BK1 subtype responsible for the contractile effect on the stretched tissue.     

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.     

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)     

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)     

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.     

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.     

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).     

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.     

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.     

Specific receptors for bradykinin-induced cardiac sympathetic chemoreflex in the dog

Bradykinin (BK, 0.03-1 microgram), capsaicin (1 microgram) or potassium chloride (KCl, 13 mumol) applied to the epicardium of the left ventricle of anaesthetized, open-chest dogs, caused reflex tachycardia and pressor effects, whereas des-Arg9-BK (1-100 micrograms), a selective bradykinin B1-receptor agonist, failed to produce any cardiovascular response. Superfusion of the epicardium with a selective B1-receptor antagonist, des-Arg9-[Leu8]BK (50-100 micrograms/min) had no effect on reflex responses to epicardial BK (0.03-0.1 microgram). However, the selective B2-receptor antagonist, D-Arg-[Hyp3,Thi5,8,D-Phe7]BK (10-25 micrograms/min) abolished the reflex effects of 0.03 and 0.1 microgram BK and reduced by 50 to 70% the responses to 1 microgram BK. Another B2-receptor antagonist [Thi6,9,D-Phe8]kallidin (10-50 micrograms/min) also reduced (30-70%) responses to 1 microgram BK. The antagonism was reversible and specific for BK since reflex responses to epicardial application of either capsaicin or KCl were not affected. The results indicate that BK interacts with B2-receptors, probably located on terminals and/or axons of sympathetic afferents supplying the dog heart, to activate a cardiac sympathetic chemoreflex.     

High-affinity bradykinin B2 binding sites sensitive to guanine nucleotides in bovine aortic endothelial cells.

Bradykinin (BK) binding sites were studied in membranes from bovine aorta. [3H]BK specifically bound to one high-affinity binding site (KD = 152 pM; Bmax = 4.6 fmol.mg-1) and was displaced by unlabeled BK (Ki = 121 pM). The B2-agonist kallidin and B2-antagonists D-Arg0[Hyp3,Leu5,8,Gly6,D-Phe7]BK, D-Arg0[Hyp3,D-Phe7]BK, [D-Phe7]BK, and [Thi5,8,D-Phe7]BK inhibited [3H]BK binding with respective Ki values of 101, 282, 678, 2000 and 6000 pM. The B1-antagonist des-Arg9[Leu8]BK had no effect. GTP, GTP gamma S, GDP, and GDP beta S but not 5'-GMP, guanosine, cyclic 3',5'-GMP, ATP, ADP, 5'-AMP, nor adenosine, inhibited [3H]BK binding with an IC50 of 1-3 microM for GTP and GDP and an IC50 of 0.1-0.3 microM for GTP gamma S and GDP beta S. GTP and GDP at 3 microM decreased the Bmax value by 30-70%. Millimolar concentrations of Ca2+ and Mg2+ ions increased [3H]BK binding and counteracted the effect of guanine nucleotides. This study demonstrates the existence of a specific high-affinity B2 BK binding site in bovine aortic endothelial cells. It suggests that this site is located on a G protein-interacting receptor.     

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.     

Bradykinin receptor antagonists used to characterize the heterogeneity of bradykinin-induced responses in rat vas deferens

The application of bradykinin to the isolated, transmurally stimulated rat vas deferens caused two effects: increase of the basal tension of the tissue and potentiation of the magnitude of electrically driven twitches. These bradykinin responses were not evenly distributed along the ductus. The direct contractile action of bradykinin was found to be stronger in the epididymal half of the tissue while the potentiation of the muscle twitches was more pronounced in the prostatic half of the rat ductus. Bradykinin is more potent to potentiate the electrically driven twitches than to act as a postjunctional agonist. Tyr-bradykinin, [Tyr5]bradykinin and [Tyr8]bradykinin exhibited significant differences in the potency ratio to produce each of these responses. [Thi5,8,D-Phe7]bradykinin is a weak postjunctional agonist but was a full agonist to potentiate the electrically induced twitches. Furthermore, this compound antagonized the bradykinin-induced contractions. [Hyp3,Thi5,8,D-Phe7]bradykinin was devoid of agonist activity at either pre- or postjunctional sites; it behaved as a pure antagonist and was more than potent its non-hydroxylated analog. The addition of a D-Arg residue at the amino terminal increased the antagonist potency significantly. The pA2 of D-Arg-[Hyp3,Thi5,8,D-Phe7]bradykinin to antagonize the postjunctional effect of bradykinin was 6.35, a value that differed significantly from the value of 6.93 required to block the prejunctional effect of the peptide. The bradykinin receptor antagonists did not modify significantly the magnitude of the contractile responses caused by angiotensin II, norepinephrine or 5-hydroxy-tryptamine.     

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.     

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.     

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.     

Metabolism of bradykinin agonists and antagonists by plasma aminopeptidase P.

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

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.     

Cardiovascular effects of intrathecally administered bradykinin in the rat: characterization of receptors with antagonists.

1. The effects of intrathecal (i.t.) pretreatment with selective B1 or B2 kinin receptor antagonists were studied on the cardiovascular response to i.t. injection of bradykinin (BK) in conscious freely moving rats. 2. BK (81 pmol) produced an increase in mean arterial pressure (MAP: 9-13 mmHg) and decrease in heart rate (HR: 20-30 beats min-1) that reached a maximum 2 min after injection. 3. The BK-induced cardiovascular responses were dose-dependently and reversibly reduced by four antagonists with the following rank order of potency: Tyr, D-Arg[Hyp3,D-Phe7,Leu8]-BK = D-Arg[Tyr3,D-Phe7,Leu8]-BK = D- Arg[Hyp3,D-Phe7,Leu8]-BK > D-Arg[Hyp3,Thi5,D-Tic7,Oic8]-BK (Hoe 140). These compounds failed to alter the cardiovascular response to i.t. injection of 8.1 nmol of substance P. 4. Other compounds acting on the B2 receptor, namely D-Arg[Hyp3,Gly6,Leu8]-BK, D-Arg[Hyp3,D-Phe7]-BK, D-Arg[Hyp2,Thi5,8,D-Phe7]-BK and D-Arg[Hyp3,Gly6,D-Phe7,Leu8]-BK or on the B1 receptor, [Leu8]-desArg9-BK, did not influence the cardiovascular responses to BK at doses devoid of intrinsic activity on MAP and HR. 5. None of the kinin receptor antagonists caused motor impairment, respiratory arrest or persisting cardiovascular changes. 6. These results confirm that the cardiovascular effects induced by i.t. BK are mediated by the activation of a B2 receptor in the rat spinal cord. However, the rank order of potency of antagonists does not conform to the classical B2 functional site characterized in peripheral tissues.