B1 Receptor Antagonist des-Arg9 Research Articles

Excitatory effect of bradykinin on intrinsic neurons of the rat heart

The heart receives sympathetic and parasympathetic innervation through the intrinsic cardiac nervous system. Although bradykinin (BK) has negative inotropic and chronotropic properties of cardiac contraction, the direct effect of BK on the intrinsic neural network of the heart is still unclear. In the present study, the effect of BK on the intracardiac ganglion neurons isolated from rats was investigated using the perforated patch-clamp technique.Under current-clamp conditions, application of 0.1 μM BK depolarized the membrane, accompanied by repetitive firing of action potentials. When BK was applied repeatedly, the second responses were considerably less intense than the first application. The BK action was fully inhibited by the B2 receptor antagonist Hoe-140, but not by the B1 receptor antagonist des-Arg9-[Leu8]-BK. The BK response was mimicked by the B2 agonist [Hyp3]-BK. The BK-induced depolarization was inhibited by the phospholipase C inhibitor U-73122. BK evoked inward currents under voltage-clamp conditions at a holding potential of −60 mV. Removal of extracellular Ca2+ markedly increased the BK-induced currents, suggesting an involvement of Ca2+-permeable non-selective cation channels. The muscarinic agonist oxotremorine-M (OxoM) also elicited the extracellular Ca2+-sensitive cationic currents. The OxoM response did not exhibit rundown with repeated agonist application. The amplitude of current evoked by 1 μM OxoM was comparable to that induced by 0.1 μM BK. Co-application of 0.1 μM BK and 1 μM OxoM elicited the current whose peak amplitude was almost the same as that elicited by OxoM alone, suggesting that BK and OxoM activate same cation channels. BK also reduced the amplitude of M-current, while the M-current inhibitor XE-991 affected neither resting membrane potential nor the BK-induced depolarization. From these results, we suggest that BK regulates excitability of intrinsic cardiac neurons by both an activation of non-selective cation channels and an inhibition of M-type K+ channels through B2 receptors.

Muscle hyperalgesia induced by peripheral P2X3 receptors is modulated by inflammatory mediators

ATP, via activation of P2X3 receptors, has been highlighted as a key target in inflammatory hyperalgesia. Therefore, the aim of this study was to confirm whether the activation of P2X3 receptors in the gastrocnemius muscle of rats induces mechanical muscle hyperalgesia and, if so, to analyze the involvement of the classical inflammatory mediators (bradykinin, prostaglandins, sympathetic amines, pro-inflammatory cytokines and neutrophil migration) in this response. Intramuscular administration of the non-selective P2X3 receptor agonist α,β-meATP in the gastrocnemius muscle of rats induced mechanical muscle hyperalgesia, which, in turn, was prevented by the selective P2X3 and P2X2/3 receptors antagonist A-317491, the selective bradykinin B1-receptor antagonist Des-Arg9-[Leu8]-BK (DALBK), the cyclooxygenase inhibitor indomethacin, the β1- or β2-adrenoceptor antagonist atenolol and ICI 118,551, respectively. Also, the nonspecific selectin inhibitor fucoidan. α,β-meATP induced increases in the local concentration of the pro-inflammatory cytokines tumor necrosis factor-α (TNF-α) and interleukin 1β (IL-1β), which were reduced by bradykinin antagonist. Finally, α,β-meATP also induced neutrophil migration. Together, these findings suggest that α,β-meATP induced mechanical hyperalgesia in the gastrocnemius muscle of rats via activation of peripheral P2X3 receptors, which involves bradykinin, prostaglandins, sympathetic amines, pro-inflammatory cytokines release and neutrophil migration. It is also indicated that bradykinin is the key modulator of the mechanical muscle hyperalgesia induced by P2X3 receptors. Therefore, we suggest that P2X3 receptors are important targets to control muscle inflammatory pain.

Role of Bradykinin, Nitric Oxide, and Angiotensin II Type 2 Receptor in Imidapril-Induced Angiogenesis

The angiotensin II (Ang II)-Ang II type 1 receptor pathway is proangiogenic, whereas studies showed that some angiotensin-converting enzyme inhibitors also stimulate angiogenesis in the setting of tissue ischemia, leaving a controversy of Ang II-mediated angiogenesis. We investigated whether an angiotensin-converting enzyme inhibitor imidapril-induced angiogenesis might be mediated via the tissue bradykinin pathway. To rule out the conventional effects of Ang II on angiogenesis, we used Ang II type 1a receptor knockout (AT1aKO) mice. We examined the effects of the angiotensin-converting enzyme inhibitor imidapril on angiogenesis in a hindlimb ischemia model using AT1aKO mice. After induction of hindlimb ischemia, AT1aKO mice were treated with or without imidapril (1.0 or 0.1 mg/kg per day for 21 days). Angiogenesis was quantified by laser Doppler blood flowmetry and capillary density. Angiogenesis was reduced in AT1aKO mice compared with wild-type mice. Imidapril with either low or high doses enhanced angiogenesis in AT1aKO mice (P<0.01). Ang II type 2 receptor antagonist (PD123319; 30 mg/kg per day) and B1 receptor antagonist (DesArg9-[Leu8]-bradykinin; 50 nmol/kg per day) suppressed the imidapril-induced angiogenesis in AT1aKO mice to an extent even lower than that of nontreated AT1aKO mice. B2 receptor antagonist (Hoechst 140; 100 microg/kg/d) and NO synthase inhibitor (N(G)-nitro-L-arginine methyl ester; 20 mg/kg per day) moderately attenuated the imidapril-mediated angiogenesis. RT-PCR revealed that vascular endothelial growth factor receptor 2 mRNA was reduced with PD123319, DesArg9-[Leu8]-bradykinin, or Hoechst 140, and vascular endothelial growth factor mRNA abundance was suppressed with PD123319 or DesArg9-[Leu8]-bradykinin. In conclusion, imidapril elicited angiogenesis in the setting of tissue ischemia in AT1aKO mice. This angiogenic effect might involve the Ang II-Ang II type 2 receptor pathway in addition to the bradykinin-B1 and bradykinin-B2 receptor/NO-dependent pathways.

A Novel Inflammatory Pathway Involved in Leukocyte Recruitment: Role for the Kinin B1 Receptor and the Chemokine CXCL5

The kinin B1 receptor is an inducible receptor not normally expressed but induced by inflammatory stimuli and plays a major role in neutrophil recruitment, particularly in response to the cytokine IL-1beta. However, the exact mechanism involved in this response is unclear. The aim of this study was to dissect the molecular mechanism involved, in particular to determine whether specific ELR-CXCL chemokines (specific neutrophil chemoattractants) played a role. Using intravital microscopy, we demonstrated that IL-1beta-induced leukocyte rolling, adherence, and emigration in mesenteric venules of wild-type (WT) mice, associated with an increase in B1 receptor mRNA expression, were substantially attenuated (>80%) in B1 receptor knockout mice (B1KO). This effect in B1KO mice was correlated with a selective down-regulation of IL-1beta-induced CXCL5 mRNA and protein expression compared with WT mice. Furthermore a selective neutralizing CXCL5 Ab caused profound suppression of leukocyte emigration in IL-1beta-treated WT mice. Finally, treatment of human endothelial cells with IL-1beta enhanced mRNA expression of the B1 receptor and the human (h) CXCL5 homologues (hCXCL5 and hCXCL6). This response was suppressed by approximately 50% when cells were pretreated with the B1 receptor antagonist des-Arg9-[Leu8]-bradykinin while treatment with des-Arg9-bradykinin, the B1 receptor agonist, caused a concentration-dependent increase in hCXCL5 and hCXCL6 mRNA expression. This study unveils a proinflammatory pathway centered on kinin B1 receptor activation of CXCL5 leading to leukocyte trafficking and highlights the B1 receptor as a potential target in the therapeutics of inflammatory disease.

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 μM) 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 μM, 3.34±0.48; N=7), while B1 receptor agonists, des-Arg10-Kallidin (Kal) (1 μM, N=11) and des-Arg9-BK (1 μM, 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 μM, 15 min), while the B1 receptor antagonist des-Arg9-[Leu8]-BK (1 μM, 15 min) had no effect on BK or des-Arg10-Kal actions. Incubation of cells with Escherichia coli lipopolysaccharide (100 μg/ml, 24 h) alone or in association with tumor necrosis factor-α (TNF-α) (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 μM) (1.18±0.08, N=5 P<0.001). Additionally, BK was not inhibited by verapamil (50 μM), nifedipine (30 μM), Ni++ (300 μM) or La+++ (10 μM). 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.

Inducible expression of the kinin B1 receptor in the endotoxemic heart: mechanisms of des-Arg9bradykinin-induced coronary vasodilation.

1 We have investigated the role of kinin B1 receptor induction in the endotoxemic rat heart and elucidated the mechanisms underlying B1 receptor-mediated coronary vasodilation. We also investigated the role of these receptors in endotoxin-induced hypotension. 2 Endotoxin treatment induced cardiac B1 receptor mRNA expression and promoted a coronary vasodilation response to des-Arg9bradykinin (DABK; ED50 = 149.4 pmol, n = 9) ex vivo peaking at 6 h. The B1 receptor antagonist des-Arg9-[Leu8]-BK (DALBK, 30 nM) significantly (P<0.05) inhibited the DABK-induced response (pA2 = 8.4, n = 5) whilst HOE140 (B2 receptor antagonist, 10 nM) was inactive (n = 4). 3 Removal of the endothelium or infusion with indomethacin (5 microM), but not L-NAME (300 microM) or ODQ (1 microM), inhibited (>85%, P<0.05, n = 5) the DABK-induced response. DABK caused a dose-dependent release of the prostacyclin metabolite, 6-keto-PGF1a (Emax = 0.3 ng ml-1, n = 6). 4 In vitro perfusion of hearts with endotoxin (1 microg ml-1, n = 6) or interleukin-1beta (5 ng ml-1, n = 6) induced B1 receptor mRNA expression and promoted a time-dependent vasodilation response to DABK. 5 Endotoxin treatment (6 h) in vivo promoted a hypotensive response to DABK (ED50 = 29.7 nmol kg-1, n = 10) which was antagonised by DALBK (3-6 nmol kg-1 min-1, P<0.05, n = 7). DALBK (3 nmol kg-1 min-1) and des-Arg10HOE140 (B1 receptor antagonist, 30 nmol kg-1 min-1) produced a 5.3% (n = 6, P<0.05) and 8.8% (n = 5, P<0.05) reversal, respectively, of endotoxin-induced hypotension. 6 In summary, we have shown that in endotoxemia activation of B1 receptors causes coronary vasodilation via endothelial prostacyclin release. Additionally, B1 receptor antagonists partially reversed endotoxin-induced hypotension. Therefore activation of B1 receptors may have a role to play in the vascular changes associated with endotoxemia.

Bradykinin down-regulates LPS-induced eosinophil accumulation in the pleural cavity of mice through type 2-kinin receptor activation: a role for prostaglandins.

1. The role of both exogenously administered and endogenously generated bradykinin (BK) on LPS-induced eosinophil accumulation in the mice pleural cavity was investigated by means of treatment with BK selective receptor agonists/antagonists and captopril. 2. Intrathoracic (i.t.) injection of LPS (250 ng cavity(-1)) induced eosinophil influx at 24 h as previously described (Bozza et al., 1993). Pretreatment with the B1 receptor antagonist des-Arg9-[leu-8]BK (0.025 and 0.25 nmol cavity(-1)) showed no effect on this phenomenon, whereas pretreatment with the B2 receptor antagonists, NPC 17731 (0.025 and 0.25 nmol cavity(-1)) or HOE 140 (2.5 nmol cavity(-1)), increased LPS-induced eosinophil influx. Accordingly, pretreatment with captopril at 10 mg kg(-1) i.p., inhibited eosinophil infiltration induced by LPS in the pleural cavity, suggesting that endogenous BK is down-regulating LPS-induced eosinophil accumulation. 3. BK administered at 15 and 25 nmol cavity(-1), i.t. or i.p. also inhibited LPS-induced eosinophil accumulation. BK alone had no effect on the basal number of leucocytes in the pleural or peritoneal cavity in doses up to 25 nmol cavity(-1). Nevertheless, when injected at doses of 50 and 100 nmol cavity(-1) BK induced leucocyte influx characterized by neutrophil and eosinophil accumulation at 24 h. 4. Similarly to what was observed with BK, a specific B2 receptor agonist, Tyr8BK, administered at 0.25 nmol cavity(-1) i.p., significantly inhibited the eosinophil influx induced by LPS. 5. The mechanism by which B2 receptor agonists inhibit LPS-induced eosinophil accumulation was investigated by pretreating the animals with indomethacin or a selective cyclooxygenase-2 inhibitor, NS-398. Pretreatment with either indomethacin or NS-398 had no effect on eosinophil influx induced by LPS alone, but those drugs were able to restore the LPS-induced eosinophil influx in Tyr8BK (0.25 nmol cavity(-1)) injected mice. 6. In conclusion, endogenously generated bradykinin seems to modulate, through activation of B2 receptors, eosinphil accumulation induced by LPS via a mechanism dependent on prostanoid synthesis.

Response of fetal rabbit ductus arteriosus to bradykinin: role of nitric oxide, prostaglandins, and bradykinin receptors.

Nitric oxide plays a major role in vascular tone control. Increased blood levels of bradykinin (BK), which stimulates nitric oxide biosynthesis, occur at birth. BK effects on ductus arteriosus (DA) tone were investigated in fetal rabbit under fetal (2.5% O2 "low PO2") and neonatal (30% O2 "high PO2") conditions using in vitro isometric tension studies. Intact and endothelium-denuded DA, contracted with norepinephrine (ED75-90), showed a biphasic response to BK, with relaxation at 10(-9) to 10(-7) M BK and contraction at 10(-6) to 10(-5) M BK. BK (10(-6) to 10(-5) M) contracted intact DA from baseline tension, with greater contraction under high PO2. The B2-receptor antagonist D-Arg-[Hyp3,Thi5,D-Tic7,Oic8]-BK (Hoe-140, 10(-7) M) abolished relaxation, but not contraction, to BK in intact and denuded DA. The B1-receptor antagonist des-Arg9-[Leu5]-BK (10(-7) M) reduced BK-induced contraction but not relaxation in intact DA only. Nitric oxide synthase inhibitors, N(omega)-nitro-L-arginine methyl ester (10(-4) M) and N(omega)-monomethyl-L-arginine (10(-4) M) partially inhibited relaxation to BK in intact DA, with L-arginine (3 x 10(-4) M) reversing N(omega)-monomethyl-L-arginine inhibition. N(omega)-nitro-L-arginine methyl ester (10(-4) M) caused a small but significant inhibition of relaxation to BK in denuded DA. Indomethacin (2.8 x 10(-6) M), a cyclooxygenase inhibitor, abolished relaxation but not contraction to BK in intact and denuded DA. BK-induced relaxation of the DA acts through B2-receptors, releasing both nitric oxide and prostaglandins, whereas endothelial B1-receptors may mediate contraction. BK action on isolated DA changes from relaxation to contraction as its concentration increases, with greater contraction at neonatal PO2. Thus increased BK levels at birth may aid functional closure of the DA.

Characterisation of kinin receptors on the human isolated umbilical artery.

The aim of this study was to determine the kinin responses on human umbilical artery (HUA) and to characterise the kinin receptors present on this tissue. The HUA was found to constrict in response to both the B2 receptor agonist, bradykinin (BK), and the B1 receptor agonist, des-Arg9-BK. The presence of indomethacin (2.79 microM) was found to have no significant effect on the responses to BK and des-Arg9-BK. The presence of the B2 receptor antagonist, HOE-140 (+2.79 microM indomethacin), resulted in a concentration-related rightward displacement of the concentration-effect curves to BK. The antagonism of the constrictor responses to BK by HOE-140 was found to be competitive (pA2 = 7.5). The responses to BK were not significantly affected by the B1 receptor antagonist des-Arg9(leu8)BK. The presence of des-Arg9(leu8)BK (+2.79 microM indomethacin) resulted in a concentration-related rightward displacement of the concentration-effect curve to des-Arg9-BK. The antagonism of the response to des-Arg9-BK by des-Arg9(leu8)BK was found to be competitive (pA2 = 5.9). The responses to des-Arg9-BK were not significantly affected by HOE-140. The results of the present study suggest that the products of the cyclooxygenase pathway are not involved in the kinin constrictor response on HUA and indicate the presence of B1 and B2 receptors on this tissue.

Biphasic response to bradykinin in isolated porcine iliac arteries is mediated by bradykinin B1 and B2 receptors.

Bradykinin-induced responses were studied in isolated porcine iliac arteries. Relaxation was endothelium dependent and seen at low concentrations (10(-10)-10(-8) M) of bradykinin. It was inhibited by the bradykinin B2-receptor antagonist icatibant (HOE-140) and by the nitric oxide synthase inhibitor Nomega-nitro-L-arginine. Bradykinin-induced relaxation was significantly potentiated by the kininase I carboxypeptidase inhibitor mergepta (10(-6) M). Bradykinin (>10(-7) M) elicited contraction of preparations with or without endothelium. The contraction was abolished by indomethacin but was not affected by the thromboxane A2/prostaglandin H2-receptor antagonist SQ 29,548. Icatibant and the bradykinin B1-receptor antagonist desArg9[Leu8]bradykinin significantly decreased bradykinin-induced contraction regardless of endothelial function. The contraction also was decreased by treatment with mergepta. The bradykinin B1-receptor agonist desArg9-bradykinin contracted endothelium-denuded arterial strips. This contraction was significantly decreased by desArg9[Leu8]bradykinin but not by icatibant. The desArg9-bradykinin-induced contraction also was inhibited by the protein-synthesis inhibitor cycloheximide. Neither bradykinin-induced relaxation nor contraction was affected by the ACE inhibitors enalaprilat or cilazaprilat. In conclusion, bradykinin-induced relaxation of isolated porcine iliac arteries was mediated by endothelial bradykinin B2 receptors and mainly nitric oxide. Bradykinin-induced contraction was endothelium independent, indomethacin sensitive, and probably mediated by bradykinin B1 (inducible) and B2 receptors located in the vascular smooth-muscle layer. Kininase I carboxypeptidase, and not ACE, is the main enzyme responsible for bradykinin degradation in these vessels.

Bradykinin B2-receptor-mediated Stimulation of Exocytotic Noradrenaline Release from Cardiac Sympathetic Neurons

Myocardial ischemia, as well as angiotensin-converting-enzyme-inhibitors, increase cardiac concentrations of the non-apeptide bradykinin. Cardiac effects of bradykinin are potentially mediated by modulation of sympathoadrenergic neurotransmission. Accordingly, the present study was designed to examine the influence of bradykinin on exocytotic noradrenaline release from rat isolated perfused heart. Exocytotic noradrenaline release was induced by electrical field stimulation (1 min, 5 V, 6 Hz) twice to compare the effect of intervention (S2) with respective control stimulation (S1). The overflow of endogenous noradrenaline was determined by high pressure liquid chromatography and electrochemical detection. The results are expressed as the mean S2/S1ratio±s.e.m.Bradykinin (1μmol/l) evoked a significant increase in noradrenaline release (S2/S1: 1.60±0.12;P<0.01), which was even more pronounced after inhibition of neuronal reuptake of noradrenaline by desipramine (0.1μmol/l: S2/S1: 1.83±0.15;P<0.01) excluding interference of bradykinin with the noradrenaline uptake1carrier. The concentration–response curve for bradykinin (0.1 nmol/l to 10μmol/l) revealed a maximum effect at 1μmol/l and an EC50-value of 7.5 nmol/l. The effect of bradykinin was unaltered by the B1-receptor antagonist des-Arg9(Leu8)-bradykinin (1μmol/l; S2/S1: 1.69±0.17), whereas it was reduced significantly by the B2-receptor antagonist Hoe 140 (1μmol/l; S2/S1: 1.14±0.11;P<0.05). Des-Arg9-bradykinin (1μmol/l), a specific B1-agonist, had no effect on stimulation-induced noradrenaline release (S2/S1: 0.94±0.08). Utilizing pharmacological interventions, we attempted to characterize the intraneuronal signal transduction pathway mediating the effect of bradykinin on exocytosis. Neither inhibition of cyclooxygenase nor blockade of nitric oxide synthesis affected bradykinin-induced stimulation of noradrenaline release. Likewise, inhibition of protein kinase C by bisindolylmaleimide (1μmol/l) or tyrosine kinase by genistein (10μmol/l) had no effect on the promoting action of bradykinin. In contrast, inhibition of cytosolic phospholipase A2activity by the specific inhibitor AACOCF3(1μmol/l) prevented bradykinin-induced increase in noradrenaline release (S2/S1: 1.09±0.15;P<0.01). In conclusion, bradykinin increases exocytotic release of endogenous noradrenaline from cardiac sympathetic neurons via activation of presynaptic B2-receptors. Intraneuronal coupling of B2-receptors to phospholipase A2appears to mediate the facilitatory effect of bradykinin on noradrenaline release in rat heart.

Vascular action of bradykinin is mediated by B2-subtype receptors in the canine lingual artery.

The mechanisms underlying the responses of the canine lingual artery to bradykinin (BK) have been examined. In endothelium-intact lingual artery ring preparations contracted to a stable plateau tension by the addition of norepinephrine (NE), BK produced B2-receptor antagonist [D-Phe7]-BK-sensitive and endothelium-dependent relaxations. Theobserved effect was significantly inhibited by nitric oxide (NO) inactivator hemoglobin, methylene blue, a good inhibitor of soluble guanylate cyclase, and by NG-monomethyl-L-arginine, a NO synthase inhibitor, but not by B1-receptor antagonist des-Arg9-[Leu8]-BK, suggesting endothelial production of the vasodilator agent NO by B2-receptors activation. In endotheliumdenuded rings in the absence of active tension caused by NE, BK increased muscle tension and cytosolic Ca2+ level; these were inhibited by [D-Phe7]-BK. The concentration-dependent contraction produced by BK of endothelium-denuded rings was also attenuated by [D-Phe7]-BK; protein kinase C inhibitor H-7 and phospholipase C inhibitor neomycin B reduced the BK-induced contraction. Indomethacin was without effect. Hence, it can be concluded that BK can cause relaxation and contraction in the canine lingual artery via B2-receptors activation. The relaxation response to BK might be mediated by endothelial production of No.BK has the potential to be a vasoconstrictor by exerting an effect on vascular smooth muscle cells, but not on endothelial cells, via B2-receptors activation. It is also postulated that the contractile response to BK may be due to the production of second messengers derived from the action of phospholipase C, but not due to contractile prostanoids, when B2-receptors are activated.

Involvement of bradykinin B1 receptors in the polymorphonuclear leukocyte accumulation induced by IL-1 beta in vivo in the mouse.

Local application of the bradykinin B1 receptor antagonist desArg9[Leu8]BK, but not the B2 receptor antagonist Hoe 140, attenuated by approximately 50% the polymorphonuclear leukocyte accumulation into 6-day-old air pouches observed in response to application of murine IL-1 beta (5 ng). The selective appearance of a chemotactic response to the bradykinin B1 receptor agonist desArg9BK only in air pouches pretreated (4 h) with IL-1 beta indicated the involvement of this receptor type during this acute inflammation model. The pro-migratory action of desArg9BK was magnified when the effect of IL-1 on polymorphonuclear leukocyte accumulation had subsided (i.e., 24 h post-IL-1). desArg9[Leu8]BK, but not Hoe 140, antagonized the effect of desArg9BK, indicating an action on B1 receptors, but not B2 receptors. The cellular infiltration observed following application of desArg9BK in IL-1-sensitized air pouches was due to the release of neuropeptides from C fibers, as indicated by the inhibitory action of the substance P antagonist, RP 67,580, and the calcitonin-gene related peptide antagonist, CGRP-(8-37). In conclusion, this study provides evidence that activation of C fibers, which is necessary for the achievement of a full chemotactic action of IL-1, is due to up-regulation (or induction) of bradykinin B1 receptors and describes, for the first time, a relationship between these receptors and polymorphonuclear leukocyte recruitment.

Involvement of B1 and B2 receptors in bradykinin-induced rat paw oedema.

1. The mechanisms involved in bradykinin (BK)-induced oedema in the rat paw as well as the interactions between BK and several inflammatory mediators, have been investigated. 2. Intraplantar injection of BK (1 nmol/paw) in rats pretreated with captopril (5 mg kg-1, s.c.) caused a small amount of oedema formation (0.17 +/- 0.05 ml). Des-Arg9-BK (DABK, a selective B1 receptor agonist) up to 300 nmol/paw caused minimal oedema (0.03 +/- 0.01 ml). 3. Co-administration of prostaglandin E2 (PGE2), prostaglandin I2 (PGI2), calcitonin gene-related peptide (CGRP), 5-hydroxytryptamine (5-HT), substance P (SP) or platelet activating factor (PAF) (1 pmol-1 nmol/paw) with BK (1 nmol/paw) dose-dependently potentiated BK-induced paw oedema. The rank order of potency (mean ED50, pmol/paw) for this effect was: SP (8.1) > PAF (13.7) > PGI2 (20.5) > 5-HT (23.8) > CGRP (25.7) > PGE2 (52.0). Co-administration of BK with the various inflammatory mediators resulted in maximal paw oedemas (ml) of: PGE2 (0.71 +/- 0.02); PGI2 (0.66 +/- 0.02); 5-HT (0.65 +/- 0.01); SP (0.63 +/- 0.05); CGRP (0.60 +/- 0.05) and PAF (0.47 +/- 0.02) ml. Histamine (up to 1 nmol/paw) was ineffective in potentiating the response to BK. 4. Hoe 140 or NPC 17731 (two selective B2 receptor antagonists, 0.1-3 nmol/paw) produced dose-dependent inhibition of paw oedema potentiation induced by co-injection of BK with other mediators with the following mean ID50s (nmol/paw): Hoe 140-1.4; 1.3; 1.5 and 1.1 and NPC 17731-1.0; 1.0; 0.9 and 0.7; in the presence of PGE2, PGI2, CGRP and SP, respectively. The selective B1 receptor antagonist des-Arg9 [Leu8]-BK (DALBK, up to 300 nmol/paw) had no effect.5. Daily intraplantar injections of BK (10 nmol/paw) once a day for 7 consecutive days caused a progressive and complete desensitization of the paw oedema, which was specific for BK, since paw oedema induced by PAF, PGE2, SP or histamine was not affected. In addition, the oedema caused by BK in the paw desensitized to the peptide was almost completely reversed if BK was co-injected with PGE2, PGI2 or SP (1 nmol/paw). Injection of PGE2 or SP (10 nmol/paw) together with the first BK injection (1O nmol/paw), partially prevented BK-induced desensitization.6. When animals were completely desensitized to BK, DABK (100nmol/paw) caused paw oedema(0.25 +/- 0.03 ml) which was consistently blocked by the B1 receptor antagonist, DALBK (100 nmol/paw).7. Treatment of animals with dexamethasone (0.5 mg kg-1, s.c., 24 h previously) antagonized paw oedema induced by DABK (100 nmol/paw) in desensitized paws, but not that induced by BK (3 nmol/paw) in naive paws. The steroid also prevented the recovery of oedema seen after co-injection of BK with PGE2 or PGI2 (1 nmol/paw) in desensitized paws.8. These results suggest that both B, and B2 receptors are involved in BK-induced rat paw oedema. The B2 receptors are constitutive, but induction of expression of B, receptors seems to occur only after complete desensitization of the paw to BK. In addition, very low doses of inflammatory mediators markedly potentiate BK-induced paw oedema and can attenuate BK-induced paw oedema desensitization.Such mechanisms may be relevant for the manifestation of acute and chronic inflammatory processes.

Evidence for participation of B1 and B2 kinin receptors in formalin-induced nociceptive response in the mouse.

1. This study was designed to investigate the role of bradykinin (BK), as well as the subtype of BK receptors involved, in formalin-induced hindpaw pain in the mouse by use of selective B1 and B2 receptor antagonists. In addition, we have analysed whether or not BK may be involved in formalin-induced hindpaw oedema in the mouse. 2. The pretreatment of animals with captopril (2 and 5 mg kg-1, s.c.) significantly increase the first and the second phases of formalin-induced pain. 3. Co-injection of the selective B1 receptor antagonist des-Arg9[Leu8]-BK (0.2-0.4 nmol/paw), together with formalin, caused graded and similar inhibitions of both phases of formalin-induced pain. Similar results were obtained with the B2 antagonists NPC 349 (D-Arg[Hyp3,Thi5,8-D-Phe7]-BK) and NPC 567 (D-Arg[Hyp3, D-Phe7]-BK) (0.2 and 0.6 nmol/paw). Higher concentrations of these antagonists (1 nmol/paw) failed to antagonize formalin-induced pain. 4. The new potent and selective B2 receptor antagonists, Hoe 140 (D-Arg[Hyp3,Thi5,D-Tic7,Oic8]-BK), NPC 17731 (D-Arg[Hyp3, trans-4-propoxy-D-proline (transpropyl)7, Oic8]-BK), and NPC 17761 (D-Arg[Hyp3, trans-4-propoxy-D-proline (trans thiophenyl)7, Oic8]-BK) (0.02 to 1.0 nmol/paw), also caused significant inhibitions of both phases of formalin-induced pain. When Hoe 140 was injected subcutaneously 30 min before formalin injection (9.9 and 99 nmol kg-1), it significantly attenuated both phases of formalin-induced pain. The putative non-peptide BK antagonist, MV 8612 (1.6 to 9.6 nmol/paw), but not MV 8608 (5.5 to 33 nmol/paw), caused a graded inhibition of both phases of formalin induced pain, being, however, more active against the first phase.5. The pretreatment of animals with morphine (2.6 to 13 micromol kg-1, s.c.) caused dose-dependent and equipotent inhibitions of both phases of formalin-induced pain. In contrast, in domethacin (2.7 to 27 micromol kg-1) antagonized only the second phase of formalin-induced pain.6. The B2 receptor antagonists, Hoe 140, NPC 17731, NPC 17761, NPC 349 and NPC 567, all caused a significant inhibition of formalin-induced hindpaw oedema. A similar inhibition was also observed within domethacin but not with captopril or morphine.7. Our results provide strong evidence for the important role of endogenous BK, acting through both B1 and B2 receptors, in the genesis of both phases of formalin-induced persistent pain in the mouse. In addition, the current results also demonstrate that the inflammatory oedema associated with the later phase of formalin-induced pain seems to be mediated by endogenous BK, via activation of B2 receptors.

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.

Kinin-induced relaxations of the rat duodenum.

Both bradykinin (BK) and des-Arg9-BK induced relaxations of the isolated longitudinal smooth muscles of the rat duodenum. No contractile effects were observed with both peptides at concentrations up to 1 mumol/l. Des-Arg9-BK was about 1000 times less potent than BK. The novel B2 antagonist HOE 140 (D-Arg-[Hyp3, Thi5, D-Phe7, Oic8]-BK) potently inhibited the BK-induced relaxations, but did not affect the relaxations induced by des-Arg9-BK. Conversely, the B1 receptor antagonist des-Arg9-[Leu8]-BK only inhibited des-Arg9-BK, but did not affect BK-induced relaxations. The relaxations induced by BK and by des-Arg9-BK were inhibited by apamin (1 mumol/l) demonstrating that apamin-sensitive K+ channels are involved. In contrast, tetraethylammonium (1 mmol/l) did not inhibit the relaxations. BK-induced relaxations were reduced by about 25% in the presence of indomethacin (10 mumol/l) although the concentration-response curve to BK was not shifted to the left. Prostaglandin E1 caused relaxations with a pD2 value of 9.2. It is concluded that both BK and des-Arg9-BK can elicit relaxations of the rat duodenum via pharmacologically distinct kinin receptor subtypes, but via similar effector mechanisms.

Bradykinin-induced oedema formation proceeds from B2 receptor stimulation and is potentiated by concomitantly released prostaglandins.

Bradykinin, a stimulator of B1 and B2 receptors, significantly increased PGI2 synthesis and fluid extravasation, as assessed by the increase of organ weight when injected intraarterially into the artificially perfused rabbit hindquarters preparation. Diclofenac pretreatment prevented PGI2 synthesis and significantly reduced fluid extravasation. Oedema formation and PGI2 synthesis were also significantly reduced by a simultaneous infusion of papaverine. Selective stimulation of B1-type receptors with des-Arg9-bradykinin was not accompanied by edema formation nor PGI2 synthesis but by a significant rise in vascular resistance. The combined application of bradykinin and the selective B1 receptor antagonist des-Arg9-[Leu8]-bradykinin also resulted in oedema formation and PGI2 synthesis, the extent, however, being significantly reduced in comparison to the application of bradykinin only. The data suggest that bradykinin-induced oedema proceeds from B2 receptor stimulation and is potentiated by simultaneous stimulation of B1 receptors. The kinin-induced fluid extravasation seems to be augmented by concomitantly generated prostaglandins.

Evidence that Cultured Airway Smooth Muscle Cells Contain Bradykinin B2 and B3 Receptors

We examined bradykinin-induced 45Ca2+ efflux and prostaglandin synthesis in guinea pig tracheal smooth muscle cells maintained in tissue culture. We also studied the effects of a B1 receptor agonist and antagonist, a B2 receptor antagonist, and the cyclooxygenase inhibitor indomethacin. In cultured smooth muscle cells, bradykinin (0.1 nM to 10 microM) stimulated efflux of 45Ca2+ and induced the synthesis of prostaglandin E2 and the prostacyclin metabolite 6-keto-prostaglandin F1 alpha. DesArg9-bradykinin, a B1 receptor agonist, had no effect on 45Ca2+ efflux or prostaglandin synthesis, and no responses to bradykinin were unaffected by the B1 receptor antagonist desArg9-[Leu8]-bradykinin. Indomethacin (1 microM) abolished bradykinin-induced prostaglandin synthesis but was without effect on 45Ca2+ efflux. NPC 567 (DArg[Hyp3,DPhe7]-bradykinin), a B2 receptor antagonist, had no effect on bradykinin-induced 45Ca2+ efflux, but abolished prostaglandin synthesis. Unlike in membranes prepared freshly from guinea pig tracheal smooth muscle, the B2 receptor antagonist inhibited completely (Ki, 12 nM) binding of [3H]-bradykinin to membranes prepared from cultured tracheal smooth cells. These data suggest that tracheal smooth muscle cells, maintained in culture, express B2 receptors that mediate bradykinin-induced prostaglandin synthesis. The observation that bradykinin-induced efflux of calcium ions was unaffected by B1 or B2 antagonists provides further evidence that airway smooth muscle may contain a novel B3 receptor.

Effect of bradykinin on airway ciliary motility and its modulation by neutral endopeptidase.

We studied the effect of bradykinin on ciliary activity and its modulation by peptidases in cultured rabbit tracheal epithelium in vitro. Bradykinin (10(-7) M) elicited a rapid, transient increase in ciliary beat frequency (CBF) from the baseline values of 1,031 +/- 25 to 1,388 +/- 38 beats/min (mean +/- SE, p less than 0.001), followed by a decline to a steady-state value of 1,180 +/- 30 beats/min, which was still greater than the baseline CBF. This ciliostimulation was dose-dependently inhibited by the B2-receptor antagonist (D-Arg,Hyp3,Thi5.8,D-Phe7)-bradykinin but not by the B1-receptor antagonist (Des-Arg9,Leu8)-bradykinin. Nifedipine, Ca2+-free medium, indomethacin, the phospholipase A2 inhibitor mepacrine, and the methyltransferase inhibitor 3-deazaadenosine reduced the change in CBF. Involvement of tachykinins, leukotrienes, prostaglandin D2, or thromboxane A2 was ruled out because bradykinin's action was not affected by (D-Pro2,D-Trp7.9)-substance P, nordihydroguaiaretic acid, or SQ29548, an antagonist for prostaglandin D2 and thromboxane A2. Bradykinin also increased prostaglandin E2 release (p less than 0.01), an effect that was abolished by indomethacin and Ca2+ deficiency. The CBF dose-response curve for bradykinin was shifted to lower concentrations by 1 log U by the neutral endopeptidase inhibitor phosphoramidon (p less than 0.01), whereas the angiotensin-converting enzyme inhibitor captopril was without effect. These results suggest that bradykinin interacts with B2-type receptors and stimulates ciliary activity through Ca2+-dependent prostaglandin E2 release, and that neutral endopeptidase may play a role in modulating the effect of bradykinin on airway mucociliary transport.