Human bradykinin B(2) receptor is activated by kallikrein and other serine proteases

Bradykinin (BK) and kallidin (Lys-BK), liberated from kininogens by kallikreins, are ligands of the BK B(2) receptor. We investigated whether kallikreins, besides releasing peptide agonist, could also activate the receptor directly. We studied the effect of porcine and human recombinant tissue kallikrein and plasma kallikrein on [Ca(2+)](i) mobilization and [(3)H]arachidonic acid release from cultured cells stably transfected to express human BK B(2) receptor (CHO/B(2), MDCK/B(2), HEK/B(2)), and endothelial cells were used as control cells. As with BK, the actions of kallikrein were blocked by the B(2) antagonist, HOE 140. Kallikrein was inactive on cells lacking B(2) receptor. Kallikrein and BK desensitized the receptor homologously but there was no cross-desensitization. Furthermore, 50 nM human cathepsin G and 50 nM trypsin also activated the receptor; this also was blocked by HOE 140. Experiments excluded a putative kinin release by proteases. [(3)H]AA release by BK was reduced by 40% by added kininase I (carboxypeptidase M); however, receptor activation by tissue kallikrein, trypsin, or cathepsin G was not affected. Prokallikrein and inhibited kallikrein were inactive, suggesting cleavage of a peptide bond in the receptor. Kallikreins were active on mutated B(2) receptor missing the 19 N-terminal amino acids, suggesting a type of activation different from that of thrombin receptor. Paradoxically, tissue kallikreins decreased the [(3)H]BK binding to the receptor with a low K(D) (3 nM) and inhibited it 78%. Thus, kallikreins and some other proteases activate human BK B(2) receptor directly, independent of BK release. The BK B(2) receptor may belong to a new group of serine protease-activated receptors.     

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

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

Kallikreins when activating bradykinin B2 receptor induce its redistribution on plasma membrane

The bradykinin (BK) B2 receptor (R) is directly activated by kallikreins and other serine proteases independent of BK release. Both the Galpha(i) and Galpha(q) proteins are involved, shown by the release of arachidonic acid and [Ca2+]i elevation. Site-directed mutagenesis of the receptor and the lack of heterogeneous desensitization of the human B2R by the BK and kallikrein emphasize among others the differences between activation by the proteases and the peptide. To characterize further the mechanism thereby kallikreins activate and desensitize the B2R we investigated the distribution of the human B2R tagged with the green fluorescent protein (B2-GFP(Ct)) on the plasma membrane of stably transfected Chinese hamster ovary (CHO) cells. We visualized the movement of B2-GFP(Ct) R with confocal fluorescence microscopy after activation by BK or a by serine protease. Continued exposure of the cells to BK led to B2R internalization within 15-20 min. Porcine pancreatic and human recombinant tissue kallikreins induced a rapid definite redistribution of receptors on the plasma membrane within 5 min, prior to internalization. These effects of kallikrein were blocked by the B2R antagonist HOE 140 and by the kallikrein inhibitor, aprotinin. The B2R was also activated by endoproteinases LysC and ArgC and trypsin, but these enzymes did not induce redistribution, only internalization. In control experiments, kallikrein had no effect on cells transfected to stably express the angiotensin-converting enzyme-green fluorescent protein (GFP). Thus, kallikreins when activating the BK B2R also trigger its redistribution on plasma membrane.     

Repertaxin, a novel inhibitor of rat CXCR2 function, inhibits inflammatory responses that follow intestinal ischaemia and reperfusion injury

1. Neutrophils are thought to play a major role in the mediation of reperfusion injury. CXC chemokines are known inducers of neutrophil recruitment. Here, we assessed the effects of Repertaxin, a novel low molecular weight inhibitor of human CXCL8 receptor activation, on the local, remote and systemic injuries following intestinal ischaemia and reperfusion (I/R) in the rat. 2. Pre-incubation of rat neutrophils with Repertaxin (10(-11)-10(-6) m) inhibited the chemotaxis of neutrophils induced by human CXCL8 or rat CINC-1, but not that induced by fMLP, PAF or LTB(4), in a concentration-dependent manner. Repertaxin also prevented CXCL8-induced calcium influx but not CXCL8 binding to purified rat neutrophils. 2. In a model of mild I/R injury (30 min of ischaemia and 30 min of reperfusion), Repertaxin dose-dependently (3-30 mg kg(-1)) inhibited the increase in vascular permeability and neutrophil influx. Maximal inhibition occurred at 30 mg kg(-1). 4. Following severe I/R injury (120 min of ischaemia and 120 min of reperfusion), Repertaxin (30 mg kg(-1)) markedly prevented neutrophil influx, the increase in vascular permeability both in the intestine and the lungs. Moreover, there was prevention of haemorrhage in the intestine of reperfused animals. 5. Repertaxin effectively suppressed the increase in tissue (intestine and lungs) and serum concentrations of TNF-alpha and the reperfusion-associated lethality. 6. For comparison, we also evaluated the effects of an anti-CINC-1 antibody in the model of severe I/R injury. Overall, the antibody effectively prevented tissue injury, systemic inflammation and lethality. However, the effects of the antibody were in general of lower magnitude than those of Repertaxin. 7. In conclusion, CINC-1 and possibly other CXC chemokines, acting on CXCR2, have an important role during I/R injury. Thus, drugs, such as Repertaxin, developed to block the function of the CXCR2 receptor may be effective at preventing reperfusion injury in relevant clinical situations.     

Role of tachykinin NK receptors on the local and remote injuries following ischaemia and reperfusion of the superior mesenteric artery in the rat

Neuropeptides acting on tachykinin NK receptors play an important role in the amplification of inflammatory responses. We have assessed the effects of tachykinin NK receptor blockade on the injuries following intestinal ischaemia and reperfusion (I/R) in rats. The tachykinin NK(1) receptor antagonist SR140333 dose-dependently (0.05 to 0.5 mg kg(-1)) suppressed the local (intestine) and remote (lung) increases in vascular permeability and neutrophil recruitment following mild I/R injury. A structurally-distinct NK(1) receptor antagonist, CP99,994, but not tachykinin NK(2) or NK(3) receptor antagonists also suppressed mild I/R injury. Neonatal pretreatment with capsaicin effectively depleted sensory neurons and abrogated the injuries following mild I/R. Treatment with SR140333 (0.5 mg kg(-1)) significantly reversed severe reperfusion-induced local and remote increases in vascular permeability, neutrophil recruitment, intestinal haemorrhage and blood neutropaenia, but did not prevent the lethality associated with severe I/R. Post-ischaemic treatment with SR140333 significantly inhibited the elevations of TNF-alpha in the intestine and lung, but not serum, following severe I/R. The increase in the concentrations of IL-10 in the lung and serum were also suppressed. Post-ischaemic blockade of tachykinin NK(1) receptors markedly inhibited the local and remote injuries, but not lethality, following reperfusion of the SMA in rats. Neuropeptides, possibly substance P, released from sensory nerves appear to account for the activation of these tachykinin NK(1) receptors. Antagonists of the tachykinin NK(1) receptor may be useful adjuncts in the treatment of the injuries which occur following reperfusion of an ischaemic vascular territory.     

Effects of inhibition of PDE4 and TNF-alpha on local and remote injuries following ischaemia and reperfusion injury

1. The effects of phosphodiesterase (PDE)4 and TNF-alpha inhibition were assessed on the local and remote injuries following intestinal ischaemia and reperfusion (I/R) injury in rats. 2. The PDE4 inhibitor rolipram dose-dependently (1 - 10 mg kg(-1)) suppressed the local (intestine) and remote (lung) increases in vascular permeability and neutrophil recruitment following mild I/R injury. SB207499 (ariflo), a structurally-distinct PDE4 inhibitor, also suppressed the injuries following mild I/R injury. 3. In a severe model of I/R injury, treatment with rolipram (10 mg kg(-1)) partially reversed the local and remote increases in vascular permeability, neutrophil recruitment, intestinal haemorrhage and intestinal LTB(4) concentrations. The anti-TNF-alpha anti-serum was more effective than rolipram at inhibiting local and remote injuries and prevented the lethality associated with severe I/R. 4. Rolipram and anti-TNF-alpha prevented the increase in the concentrations of TNF-alpha in the lung and intestine, but rolipram only partially inhibited the elevation of this cytokine in serum. Rolipram had little effect on the increases of IL-1 beta concentrations in lung and serum, whereas treatment with anti-TNF-alpha markedly increased the concentration of this cytokine. Concentrations of IL-10 rose significantly in the lung and serum and these increases were blocked by rolipram or anti-TNF-alpha. 5. The capacity of PDE4 inhibitors to block the recruitment of neutrophils into tissues, the production of LTB(4) and of the pro-inflammatory cytokines TNF-alpha, IL-1 beta and IL-6 appear to underlie their anti-inflammatory effects in our model of I/R injury. Overall, PDE4 inhibition was less effective than inhibition of TNF-alpha for protection against I/R injury.     

Activation of osteoblastic functions by a mediator of pain, bradykinin

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

Bradykinin B1 and B2 receptors, tumour necrosis factor alpha and inflammatory hyperalgesia

The effects of BK agonists and antagonists, and other hyperalgesic/antihyperalgesic drugs were measured (3 h after injection of hyperalgesic drugs) in a model of mechanical hyperalgesia (the end-point of which was indicated by a brief apnoea, the retraction of the head and forepaws, and muscular tremor). DALBK inhibited responses to carrageenin, bradykinin, DABK, and kallidin. Responses to kallidin and DABK were inhibited by indomethacin or atenolol and abolished by the combination of indomethacin + atenolol. DALBK or HOE 140, given 30 min before, but not 2 h after, carrageenin, BK, DABK and kallidin reduced hyperalgesic responses to these agents. A small dose of DABK+ a small dose of BK evoked a response similar to the response to a much larger dose of DABK or BK, given alone. Responses to BK were antagonized by HOE 140 whereas DALBK antagonized only responses to larger doses of BK. The combination of a small dose of DALBK with a small dose of HOE 140 abolished the response to BK. The hyperalgesic response to LPS (1 microg) was inhibited by DALBK or HOE 140 and abolished by DALBK + HOE 140. The hyperalgesic response to LPS (5 microg) was not antagonized by DALBK + HOE 140. These data suggest: (a) a predominant role for B2 receptors in mediating hyperalgesic responses to BK and to drugs that stimulate BK release, and (b) activation of the hyperalgesic cytokine cascade independently of both B1 and B2 receptors if the hyperalgesic stimulus is of sufficient magnitude.     

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.     

The myostimulating effect of tissue kallikrein on rat uterus

The mechanism of the myostimulating activity of rat tissue kallikrein on rat uterus was re-examined using uterus from kininogen-deficient rats and HOE 140 (D-Arg[Hyp³, This, D-Tic⁷, Oic⁸]bradykinin), a specific bradykinin receptor-B₂ antagonist. The uterus from kininogen-deficient rats was 50 times less sensitive to rat kallikrein than that from normal rats. HOE 140 (6 to 60 nM) inhibited the contracting effects of bradykinin and of rat kallikrein. Porcine kallikrein had no effect on rat uterus. Bradykinin and rat kallikrein induced a relaxation of rat duodenum. The duodenum from kininogen-deficient rats was 100 times less sensitive to rat kallikrein than the duodenum from normal rats. HOE 140 (0.6 to 3 nM) inhibited the relaxing effects of bradykinin and of kallikrein. Preincubation of rat kallikrein with aprotinin (Trasylol) abolished the effects of kallikrein on smooth muscles. HOE 140 inhibited the amidolytic activity of tissue kallikrein with a K_i value of 220 W. HOE 140, at micromolar concentrations, suppressed the kininogenase activity of tissue kallikrein. Plasma of deficient rats contained 0.7% of the normal levels of kininogens. After washing the blood vessels with saline, kininogens were present in uterine homogenates but not in duodenal homogenates from both rat strains. Uterine kininogens from deficient rats amounted to 19% of the kininogen content of the uterus of normal rats. The blood pressure of anaesthetized rats was lowered by rat tissue kallikrein but not by porcine kallikrein.We conclude that the myostimulating activity of kallikrein mostly depends on kinin formation from kininogens present in the tissue. These kininogens arise from blood contamination in the duodenum and also from a local store in the uterus. The presence of a significant amount of kininogens in the uterus from deficient rats might suggest a local synthesis of kininogens. HOE 140 inhibits the enzymatic activity of kallikrein at micromolar concentrations.     

Characterization of non-peptide bradykinin B2 receptor agonist (FR 190997) and antagonist (FR 173657)

Pharmacologic parameters for a novel non-peptide bradykinin (BK)-B2 receptor agonist, 8-[2,6-dichloro-3-[N-[(E)-4-(N-methylcarbamoylcinnamidoacetyl]-N-+ ++methylano] benzyloxy]-2-methyl-4-(2-pyridylmethoxy)quinoline (FR 190997) (pEC50, ED50 values) and for the antagonist (E)-3-(6-acetamido-3-pyridyl)-[N-[2,4-dichloro-3-[(2-methyl-8-quinolinyl ) oxymethyl] phenyl]-N-methylaminocarbonylmethyl] acrylamide (FR 173657) (pIC50, ID50 values) were measured using conventional contractile B2 receptor bioassays from rabbit, guinea pig and rat tissues and by mean of animal blood pressure models performed on anesthetized animals in the same species. In vitro assays (on the rabbit jugular vein and the guinea pig ileum) demonstrated that both the onset and duration of action of FR 190997 are prolonged compared to BK. These in vitro effects of FR 190997 strongly desensitized upon repeated tissue applications. Similar pEC50 values (7.7) were measured on the rabbit and the guinea pig tissues. In vivo, when injected intraarterially, FR 190997 produced hypotensive responses in rabbits and guinea pigs with ED50 values of 3.7 +/- 0.5 and 8.9 +/- 3.6 nmol/kg, respectively. Both the contractile and the hypotensive effects of FR 190997 were abolished by pretreating tissues (1 microM) or animals (0.1-0.5 micromol/kg) with D-Arg-[Hyp3,Thi5,D-Tic7,Oic8]BK (HOE 140) or FR 173657. FR 173657 (pIC550 approximately 8.40), as well as other known antagonists (e.g., HOE 140, D-Arg-[Hyp3,D-Phe7,Leu8]BK), inhibited the in vitro myotropic effects of BK on the rabbit, guinea pig and rat tissues. FR 173657 also abrogated the in vivo hypotensive responses elicited by BK in the rabbit (ID50 57 +/- 9 nmol/kg), the guinea pig (ID50 215 +/- 56 nmol/kg) and the rat (ID50 187 +/- 50 nmol/kg). The in vivo duration of action of FR 173657 was significantly lower in the rabbit (= 20 min) than in the guinea pig and the rat (> 90 min). It is concluded that the non-peptides FR 190997 and FR 173657 enable efficient activation and antagonism of rabbit and guinea pig B2 receptors. These non-peptide molecules represent a marked progress in medicinal chemistry and may be useful to define the role played by the kallikrein/kinin system in vivo.     

Kinins are involved in the antiproteinuric effect of angiotensin-converting enzyme inhibition in experimental diabetic nephropathy

The present study examined non-insulin-treated streptozotocin (STZ)-induced diabetic rats to determine the role of kinins in diabetic nephropathy. Their involvement in the renoprotective effect of the angiotensin-converting enzyme inhibitor (ACEI) ramipril was investigated using the bradykinin (BK) B(2)-receptor antagonist, icatibant (HOE 140), or a combination of the two drugs.Although, none of the treatments prevented the decline of the glomerular filtration rate (GFR) in diabetic rats, ramipril (3 mg/kg/day), but not icatibant (HOE 140; 500 microg/kg/day), prevented proteinuria in these animals. However, the antiproteinuric effect of ramipril was reduced by 45% when combined with icatibant. To explore whether the renal kallikrein-kinin system (KKS) belongs to the underlying mechanisms of these findings, we also determined urinary BK levels, renal kallikrein (KLK) and angiotensin-converting enzyme (ACE) activity as well as renal cortical mRNA levels of neutral endopeptidase 24.11 (NEP) and low-molecular weight (LMW) kininogen. STZ led to a reduction of renal KLK and ACE activity and NEP expression and to a three-fold increase of urinary BK excretion and renal kininogen expression. Icatibant given alone had no effect on these parameters. In contrast, ramipril treatment normalized urinary protein and BK excretion as well as kininogen mRNA expression without affecting NEP mRNA expression or KLK and ACE activity.Our data demonstrate that renal BK is increased in severe STZ-induced diabetes mellitus, but may affect glomerular regulation only to a minor degree under this condition. However, kinins are partly involved in the antiproteinuric action of ACEI at this stage of diabetic nephropathy.     

Effects of the treatment with glibenclamide, an ATP-sensitive potassium channel blocker, on intestinal ischemia and reperfusion injury

Intestinal ischemia and reperfusion injury is dependent on the recruitment and activation of neutrophils. Glibenclamide, an ATP-sensitive potassium channel (K(ATP)) blocker, has been shown to suppress neutrophil migration and chemotaxis during acute inflammatory responses by a mechanism dependent on its K(ATP) channel blocking activity. In the present study, we evaluated whether the treatment with glibenclamide prevented local, remote and systemic injury following reperfusion of the ischemic superior mesenteric artery in rats. The artery was made ischemic for a period of 30 or 120 min followed by 30 (mild I/R) or 120 (severe I/R) min of reperfusion, respectively. Glibenclamide (0.8 to 20 mg/kg) or vehicle was administered subcutaneously 40 min prior to the reperfusion. Glibenclamide dose-dependently inhibited the reperfusion-associated increase in vascular permeability and neutrophil accumulation in mild I/R. In the severe injury model, glibenclamide inhibited inflammatory parameters, as assessed by Evans blue extravasation, neutrophil influx and haemoglobin content, and the increase in TNF-alpha (tumor necrose factor-alpha) and IL (interleukin)-6 levels in the intestine and lung. The drug did not affect the increase in IL-1beta and IL-10 levels. TEA, a nonselective potassium channel blocker, also inhibited reperfusion injury in both intestine and lungs of animals submitted to mild and severe I/R. Our experiments suggest a role for K(ATP) channels in mediating neutrophil influx and consequent reperfusion-associated injury in rats. The lack of effect of these drugs on the reperfusion-associated hypotension and lethality may limit their usefulness after severe reperfusion injury.     

Effects of combined neutral endopeptidase 24-11 and angiotensin-converting enzyme inhibition on femoral vascular conductance in streptozotocin-induced diabetic rats

1. The successive effects of the angiotensin-converting enzyme inhibitor captopril (CAP, 2 mg kg(-1)+1 mg kg(-1) 30 min(-1) infusion) and the neutral endopeptidase 24-11 inhibitor retrothiorphan (RT, 25 mg kg(-1)+12.5 mg kg(-1) 30 min(-1) infusion) were studied on femoral vascular conductance (FVC) in streptozotocin-induced diabetic (STZ-SD) and control Sprague-Dawley (C-SD) rats. The role of the kinin-nitric oxide (NO) pathway was assessed by (1) using pre-treatments: a bradykinin (BK) B2 receptor antagonist (Hoe-140, 300 microg kg(-1)), a NO-synthase inhibitor (N(omega)-nitro-L-arginine methyl ester, L-NAME, 10 mg kg(-1)), a kininase I inhibitor (DL-2-mercaptomethyl-3-guanidinoethylthiopropanoic acid, MGTA, 10 mg kg(-1)+20 mg kg(-1) 20 min(-1) infusion) and (2) comparing the effects in STZ-induced diabetic (STZ-BN) and control Brown-Norway kininogen-deficient (C-BN) rats. 2. In C-SDs, CAP and CAP+RT increased FVC similarly. In STZ-SDs, FVC and FBF were decreased compared to C-SDs. CAP+RT increased them more effectively than CAP alone. 3. In both C-SDs and STZ-SDs, the femoral bed vasodilatation elicited by CAP was inhibited by Hoe-140 and L-NAME. The FVC increase elicited by CAP+RT was not significantly reduced by Hoe-140 but was inhibited by L-NAME and Hoe-140+MGTA. 4. In C-BNs, the vasodilatator responses to CAP and CAP+RT were abolished and highly reduced, respectively. In STZ-BNs, these responses were abolished. 5. These results show that in STZ-SDs, CAP+RT improve FBF and FVC more effectively than CAP alone. These effects are linked to an increased activation of the kinin-NO pathway. BK could lead to NO production by BK B2 receptor activation and another pathway in which kininase I may be involved.     

Involvement of bradykinin, cytokines, sympathetic amines and prostaglandins in formalin-induced orofacial nociception in rats

1. This study characterises some of the mechanisms and mediators involved in the orofacial nociception triggered by injection of formalin into the upper lip of the rat, by assessing the influence of various treatments on behavioural nociceptive responses (duration of facial rubbing) elicited either by a low subthreshold (i.e. non-nociceptive; 0.63%) or a higher concentration of the algogen (2.5%). 2. The kininase II inhibitor captopril (5 mg kg(-1), s.c.) and prostaglandin(PG) E(2) (100 ng lip(-1)) potentiated both phases of the response to 0.63% formalin, whereas tumour necrosis factor (TNF alpha; 5 pg lip(-1)), interleukin(IL)-1 beta (0.5 pg lip(-1)), IL-6 (2 ng lip(-1)) and IL-8 (200 pg lip(-1)), or the indirectly acting sympathomimetic drug tyramine (200 microg lip(-1)), each augmented only the second phase of nociception. 3. Conversely, both phases of nociception induced by 2.5% formalin were inhibited by the bradykinin (BK) B(2) receptor antagonist HOE140 (5 microg lip(-1)) or the selective beta(1)-adrenoceptor antagonist atenolol (100 microg lip(-1)). However, the BK B(1) receptor antagonist des-Arg(9)-Leu(8)-BK (1 and 2 microg lip(-1)), antibody and/or antiserum against each of the cytokines, the adrenergic neurone blocker guanethidine (30 mg kg(-1) day(-1), s.c., for 3 days) and the cyclooxygenase(COX)-2 inhibitor celecoxib (50 and 200 microg lip(-1), s.c.; or 1 and 3 mg kg(-1), i.p.) reduced only the second phase of the response. The nonselective COX inhibitor indomethacin and the 5-lipoxygenase activating protein inhibitor MK886 did not change formalin-induced nociception. 4. Our results indicate that BK, TNF-alpha, IL-1 beta, IL-6, IL-8, sympathetic amines and PGs (but not leukotrienes) contribute significantly to formalin-induced orofacial nociception in the rat and the response seems to be more susceptible to inhibition by B(2) receptor antagonist and selective COX-2 inhibitor than by B(1) receptor antagonist or nonselective COX inhibitor.     

Bradykinin-induced vascular responses in dog isolated lingual artery.

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

Pharmacological characterization of rabbit corpus cavernosum relaxation mediated by the tissue kallikrein-kinin system.

1. The roles of the tissue kallikrein-kinin system and nitric oxide (NO) release in Phoneutria nigriventer venom-induced relaxations of rabbit corpus cavernosum (RbCC) smooth muscle have been investigated by use of a bioassay cascade. 2. Phoneutria nigriventer venom (10-30 micrograms), porcine pancreatic kallikrein (100 mu), rabbit urinary kallikrein (10 mu), bradykinin (BK, 0.3-3 nmol), acetylcholine (ACh, 0.3-30 nmol) and glyceryl trinitrate (GTN, 0.5-10 nmol) caused relaxations of the RbCC strips. Captopril (1 microM) substantially potentiated Phoneutria nigriventer venom- and BK-induced RbCC relaxations without affecting those elicited by GTN. 3. The bradykinin B2 receptor antagonist, Hoe 140 (D-Arg-[Hyp3,Thi5,D- Tic7,Oic8]-BK, 50 nM), aprotinin (10 micrograms ml-1) and the tissue kallikrein inhibitor, Pro-Phe-Aph-Ser-Val- Gln-NH2 (KIZD-06, 1.3 microM) significantly inhibited Phoneutria nigriventer venom-induced RbCC relaxations, without affecting those provoked by GTN and ACh. The B1 receptor antagonist, [Leu9]des Arg10BK (0.5 microM) and soybean trypsin inhibitor (SBTI, 10 micrograms ml-1) had no effect on Phoneutria nigriventer venom-induced RbCC relaxations. 4. The relaxations induced by Phoneutria nigriventer venom, porcine pancreas kallikrein, BK and ACh were significantly inhibited by N omega-nitro-L-arginine methyl ester (L-NAME, 10 microM) but not by D-NAME (10 microM). L-NAME did not affect GTN-induced relaxations. L-Arginine (300 microM), but not D-arginine (300 microM), significantly reversed the inhibitory effect of L-NAME. 5. Our results indicate that Phoneutria nigriventer venom activates the tissue kallikrein-kininogen-kinin system in RbCC strips leading to NO release and suggest a functional role for this system in penile erection.     

Inhibition of rats adjuvant arthritis by a bradykinin antagonist hoe 140 and its influence on kallikreins

• 1.1. This study examines the effect of Hoe 140, a bradykinin (BK) 2 receptor antagonist, indomethacin and prednisolone on chronic adjuvant arthritis of the knee in rats. We also evaluated the influence of Hoe 140 on BK-forming enzymes in the synovial and paw tissues.
• 2.2. Adjuvant arthritis was induced in male Sprague-Dawley rats in the right knee by injecting 0.05 ml of a fine suspension of heat-killed Mycobacterium tubercle bacilli in liquid paraffin (5 mg/ml).
• 3.3. Hoe 140 (1.5 mg/kg i.p.), indomethacin (2.5 mg/kg orally) and prednisolone (3.0 mg/kg orally) administration for 9 days resulted in significant suppression of knee joint swelling. Plasma and tissue kallikrein levels were raised (P<0.01) in the synovial and paw tissues of adjuvant arthritic rats. Hoe 140 treatment reduced (P<0.05) tissue kallikrein but increased (P<0.01) plasma kallikrein levels in synovial tissue.
• 4.4. Hoe 140 treatment did not alter (P>0.05) the raised plasma and tissue kallikrein levels in the paw tissue. The findings indicate that Hoe 140 may be a useful anti-inflammatory agent and BK plays a major role in this adjuvant-induced arthritis model.

     

Oral antinociception and oedema inhibition produced by NPC 18884, a non-peptidic bradykinin B2 receptor antagonist

This study investigates the antinociceptive and the oedema inhibition properties of the novel non-peptide bradykinin (BK) B2 receptor antagonist, NPC 18884. Given by i.p. or p.o. routes NPC 18884 produced graded and long-lasting (at least 2.5h and 5.0h, respectively, for i.p. and p.o. administration) inhibition of acetic acid-induced abdominal constrictions in mice, with mean ID50 values of 8.3 nmol/kg and 439.9 nmol/kg. NPC 18884 also inhibited kaolin-induced abdominal constrictions (44+/-9% and 48+/-3% of inhibition, for i.p. and p.o. routes, respectively). Given by i.p. or p.o. routes NPC 18884 attenuated both phases of formalin-induced licking, as well as formalin-induced oedema formation. At similar doses NPC 18884 produced significant inhibition of capsaicin-induced nociception. NPC 18884, like HOE 140 given i.p., prevented the nociception caused by BK with mean ID50 values of 0.85 nmol/kg and 0.44 nmol/kg, respectively. Given orally NPC 18884, but not HOE 140, caused graded inhibition of BK-induced nociception (mean ID50 value of 50 nmol/kg). In rats, NPC 18884 given i.p. prevented BK and carrageenan-induced hyperalgesia (mean ID50 values of 6 nmol/kg and 13 nmol/kg), without affecting the hyperalgesia induced by des-Arg9-bradykinin (DABK) or by prostaglandin E2 (PGE2). NPC 18884 given i.p. inhibited the mouse paw oedema induced by tyrosine8-bradykinin or by carrageenan, but had no effect on DABK-induced oedema in mice pre-treated with Escherichia coli endotoxin, or that induced by PGE2. Thus, the novel non-peptide BK B2 receptor antagonist NPC 18884 produces rapid onset, potent and relatively long-lasting oral antinociceptive and oedema inhibition properties. The anti-BK actions of NPC 18884 are quite selective towards the BK B2 receptor-mediated responses.