Bradykinin initiates cytokine-mediated inflammatory hyperalgesia.

1. The hyperalgesic activities in rats of bradykinin, carrageenin and lipopolysaccharide (LPS) were investigated in a model of mechanical hyperalgesia. 2. Bradykinin and carrageenin evoked dose-dependent hyperalgesia with maximum responses of similar magnitude to responses to LPS (1 and 5 micrograms). 3. Hoe 140, an antagonist of BK2 receptors, inhibited in a dose-dependent manner hyperalgesic responses to bradykinin, carrageenin and LPS (1 microgram) but not responses to LPS (5 micrograms), prostaglandin E2, dopamine, tumour necrosis factor alpha (TNF alpha), IL-1, IL-6 and IL-8. 4. Responses to bradykinin and LPS (1 and 5 micrograms) were inhibited by the cyclo-oxygenase inhibitor, indomethacin and by the beta-adrenoceptor antagonist, atenolol. The effects of indomethacin and atenolol were additive: their combination abolished responses to bradykinin and LPS (1 microgram) and markedly attenuated the response to LPS (5 micrograms). 5. Antiserum neutralizing endogenous TNF alpha abolished the response to bradykinin whereas antisera neutralizing endogenous IL-1 beta, IL-6 and IL-8 each partially inhibited the response. The combination of antisera neutralizing endogenous IL-1 beta+IL-8 or IL-6+IL-8 abolished the response to bradykinin. 6. Antisera neutralizing endogenous TNF alpha, IL-1 beta, IL-6 and IL-8 each partially inhibited responses to LPS (1 and 5 micrograms). Increasing the dose of antiserum to TNF alpha or giving a combination of antisera to IL-1 beta+IL-8 or IL-6+IL-8 further inhibited responses to LPS (1 and 5 micrograms). 7. These data show that bradykinin can initiate the cascade of cytokine release that mediates hyperalgesic responses to carrageenin and endotoxin (1 microgram).(ABSTRACT TRUNCATED AT 250 WORDS)     

The use of kinin B1 and B2 receptor knockout mice and selective antagonists to characterize the nociceptive responses caused by kinins at the spinal level

The mechanisms by which kinins induce hyperalgesia in the spinal cord were investigated by using B(1) or B(2) knockout mice in conjunction with kinin selective agonists and antagonists. The i.t. administration of the kinin B(2) receptor agonists, bradykinin (BK) or Tyr(8)-BK produced dose-related thermal hyperalgesia evaluated in the hot-plate test. BK-induced hyperalgesia was abolished by the B(2) receptor antagonist Hoe 140. The i.t. injection of the kinin B(1) receptor agonists, des-Arg(9)-bradykinin (DABK) or des-Arg(10)-kallidin (DAKD) also caused dose-related thermal hyperalgesia. Different from the B(2) agonists, the i.t. injection of DABK or DAKD caused a weak, but prolonged hyperalgesia, an effect that was blocked by the B(1) receptor antagonist des-Arg(9)-[Leu(8)]-bradykinin (DALBK). The i.t. injection of BK caused thermal hyperalgesia in wild-type mice (WT) and in the B(1) receptor knockout mice (B(1)R KO), but not in the B(2) receptor knockout mice (B(2)R KO). Similarly, the i.t. injection of DABK elicited thermal hyperalgesia in WT mice, but not in B(1)R KO mice. However, DABK-induced hyperalgesia was more pronounced in the B(2)R KO mice when compared with the WT mice. The i.t. injection of Hoe 140 or DALBK inhibited the second phase of formalin (F)-induced nociception. Furthermore, i.t. Hoe 140, but not DALBK, also inhibits the first phase of F response. Finally, the i.t. injection of DALBK, but not of Hoe 140, inhibits the long-term thermal hyperalgesia observed in the ipsilateral and in contralateral paws after intraplantar injection with complete Freund's adjuvant. These findings provide evidence that kinins acting at both B(1) and B(2) receptors at the spinal level exert a critical role in controlling the nociceptive processing mechanisms. Therefore, selective kinin antagonists against both receptors are of potential interest drugs to treat some pain states.     

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-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.     

The pivotal role of tumour necrosis factor alpha in the development of inflammatory hyperalgesia.

1. The hyperalgesic activities in rats of interleukin-1 beta (IL-1 beta), IL-6, IL-8, tumour necrosis factor alpha (TNF alpha) and carrageenin were investigated. 2. IL-6 activated the previously delineated IL-1/prostaglandin hyperalgesic pathway but not the IL-8/sympathetic mediated hyperalgesic pathway. 3. TNF alpha and carrageenin activated both pathways. 4. Antiserum neutralizing endogenous TNF alpha abolished the response to carrageenin whereas antisera neutralizing endogenous IL-1 beta, IL-6 and IL-8 each partially inhibited the response. 5. The combination of antisera neutralizing endogenous IL-1 beta + IL-8 or IL-6 + IL-8 abolished the response to carrageenin. 6. These results show that TNF alpha has an early and crucial role in the development of inflammatory hyperalgesia. 7. The delineation of the role of TNF alpha, IL-1 beta, IL-6 and IL-8 in the development of inflammatory hyperalgesia taken together with the finding that the production of these cytokines is inhibited by steroidal anti-inflammatory drugs provides a mechanism of action for these drugs in the treatment of inflammatory hyperalgesia.     

5-hydroxytryptamine release from skin mast cells in vivo induced by peptide but not by nonpeptide ligands for bradykinin receptors

A number of biologically active peptides including bradykinin (BK) are known to elicit an unspecific, non-receptor-mediated release of mediators from mast cells. We have investigated whether novel, nonpeptide BK B2 receptor ligands, i.e., the antagonists N-[N-[3-[(3-bromo-2-methylimidazo(1,2-a]pyridin-8-yl)oxymethyl]-2, 4-dichlorophenyl]-N-methylaminocarbonylmethyl]-4-(dimethylamino carbonyl)cinnamylamide hydrochloride (FR167344) and (E)-3-(6-acetamido-3-pyridyl)-N-[N-(2,4-dichloro-3-?(2-methyl-8-quinolin yl)oxymethyl? phenyl]-N-methylaminocarbonylmethyl]acryamide (FR173657), and the agonist 8-[2,6-dichloro-3-[N-[(E)-4-(N-methylcarbamoyl)cinnamidoacetyl+ ++]-N-methylamino] benzyloxy]-2-methyl-4-(2-pyridylmethoxy)quinoline (FR190997), would be devoid of this unspecific action. Paw oedema in anaesthetized rats was observed following subplantar injection of BK, FR190997, the B2 antagonist D-Arg-[Hyp3, Thi5, D-Tic7, Oic8]-BK (icatibant, previously Hoe-140), the B1 agonist des-Arg9-BK (DABK) and the B1 antagonist des-Arg9 [Leu8]-BK (DALBK). The effect of BK was inhibited by systemic pretreatments with icatibant and methysergide in an additive manner, whereas the local effect of icatibant was only sensitive to methysergide. Oedema induced by DABK and DALBK was also attenuated by methysergide. Mepyramine pretreatment was ineffective for all oedema-producing agents. Effects of FR190997 were abolished by pretreatment with icatibant. FR167344 and FR173657 did not induce oedema formation. Depletion of mast cells by compound 48/80 mimicked the effects of methysergide on BK and icatibant and had no effect on FR190997. It is concluded that the novel nonpeptide receptor ligands are devoid of the unspecific mast cell-degranulating action observed with the peptide ligands.     

Two B1 and B2 bradykinin receptor antagonists fail to inhibit the Ca2+ response elicited by bradykinin in human skin fibroblasts

The elevation of intracellular [Ca2+] induced by bradykinin (Bk) was monitored with fura-2 fluorescence in human skin fibroblasts. Neither [des-Arg10][Leu9]kallidin nor D-Arg[Hyp3,Thi5,D-Tic7,Oic8]bradykinin (HOE140) inhibited the Ca2+ response stimulated by Bk. Moreover, each behaved as a partial agonist causing the elevation of intracellular [Ca2+].     

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.     

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.     

Upregulation of B1 receptor mediating des-Arg9-BK-induced rat paw oedema by systemic treatment with bacterial endotoxin.

1. The effect of pretreatment with bacterial endotoxin (LPS, 10 micrograms, i.v., 24 h) on the bradykinin B1 and B2 receptor-induced oedema in the rat paw, and the interaction of B1-mediated responses with other inflammatory mediators, was investigated. 2. Intraplantar (i.pl.) injection of the selective B1 agonist, des-Arg9-BK (DABK, 100 nmol) in naive animals pretreated with the angiotensin converting enzyme inhibitor, captopril caused a small increase in paw volume (0.04 +/- 0.003 ml, mean +/- s.e. mean, n = 6), while the B2-selective agonist, tyrosine8-bradykinin (T-BK, 3 nmol) induced marked oedema (0.36 +/- 0.02 ml). However, i.pl. injection of DABK (3-300 nmol) in rats pretreated with LPS (24 h beforehand) resulted in a marked dose- and time-related increase in paw volume, with mean ED50 of 24.1 nmol. In contrast, oedema caused by T-BK (3 nmol) was reduced by 79 +/- 4% in animals treated with LPS when compared with naive animals. 3. Oedema caused by prostaglandin E2 (PGE2, 10 nmol) was unaffected by LPS treatment, while oedema induced by histamine (100 nmol), 5-hydroxytryptamine (5-HT, 10 nmol) and substance P (SP, 3 nmol) was reduced (P < 0.05). 4. The selective B1 antagonist, des-Arg9[Leu8]-BK (100-300 nmol), produced dose-dependent inhibition of DABK (100 nmol)-induced paw oedema in LPS-treated animals with mean IC50 of 134 nmol, while the selective B2 antagonists, Hoe 140 and NPC 17731 (each 10 nmol), had no effect. 5. Treatment of animals with dexamethasone (0.5 mg kg-1, s.c.) 24 or 48 h prior to LPS injection resulted in a graded inhibition of DABK (100 nmol)-induced oedema formation (58 +/- 3 and 82 +/- 2%, respectively), and almost reversed to control value oedema formation induced by T-BK (3 nmol) in LPS-pretreated rats. Cycloheximide (1 mg kg-1, s.c.) or indomethacin (2 mg kg-1, i.p.) pretreatment 24 and 1 h prior to LPS injection, respectively, markedly inhibited DABK (100 nmol)-induced paw oedema (98 +/- 2 and 50 +/- 4%, respectively). 6. Intraplantar injection of submaximal dose of DABK (10 nmol) in LPS-treated rats produced modest paw oedema (0.09 +/- 0.03 ml). However, i.pl. injections of PGE2, prostacyclin (PGI2), calcitonin-gene-related peptide (CGRP), SP, 5-HT, or platelet activating factor (PAF) (each 1 nmol), which alone caused little or no paw oedema, resulted in a potentiation of the DABK-induced oedema. The increases in paw volume (in ml) were: PGE2 + DABK (0.31 +/- 0.03), PGI2 + DABK (0.39 +/- 0.02), CGRP+DABK (0.35 +/- 0.04), DABK+SP (0.33 +/- 0.04), DABK + 5-HT (0.40 +/- 0.02) and DABK+PAF (0.38 +/- 0.016) ml. In contrast, histamine (1 nmol) was ineffective in potentiating the response to DABK. 7. The selective B1 receptor antagonist, DALBK (100-300 nmol), produced dose-dependent inhibition of paw oedema potentiation induced by co-injection of DABK and other mediators with mean ID50S (nmol) of: 180, 160, 139 and 135 in the presence of PGE2, PGI2, SP and 5-HT, respectively. 8. These results demonstrate that DABK-induced increase in paw volume in LPS-treated rats is probably mediated by induction of B1 receptors, associated with downregulation of B2 receptors. The induction of B1 receptors by LPS is sensitive to dexamethasone and cycloheximide treatment and requires activation of cyclo-oxygenase pathway. In addition, B1 receptors, when upregulated following LPS treatment, can interact in a synergistic manner with several inflammatory mediators such as PGI2, PGE2, CGRP, PAF and 5-HT. Such results indicate that induction of the B1 receptor might have a significant pathophysiological role in modulating chronic inflammatory diseases.     

Kallidin-like peptide mediates the cardioprotective effect of the ACE inhibitor captopril against ischaemic reperfusion injury of rat heart

1. The potential cardioprotective effect of ACE inhibitors has been attributed to the inhibition of bradykinin degradation. Recent data in rats documented a kallidin-like peptide, which mimics the cardioprotective effect of ischaemic preconditioning. This study investigates in isolated Langendorff rat heart the effect of the ACE inhibitor captopril, the role of bradykinin, kallidin-like peptide, and nitric oxide (NO). 2. The bradykinin level in the effluent of the control group was 14.6 pg ml(-1) and was not affected by captopril in the presence or absence of kinin B2-receptor antagonist, HOE140. 3. The kallidin-like peptide levels were approximately six-fold higher (89.8 pg ml(-1)) and increased significantly by treatment with captopril (144 pg ml(-1)), and simultaneous treatment with captopril and HOE140 (197 pg ml(-1)). 4. Following 30 min ischaemia in the control group, the creatine kinase activity increased from 0.4 to 53.4 U l(-1). In the captopril group and in the captopril+L-NAME group, the creatine kinase activity was significantly lower (18.5 and 22.8 U l(-1)). This beneficial effect of captopril was completely abolished by the kinin B2-receptor antagonist, HOE140, as well as by the kallidin antiserum. 5. Perfusion of the hearts with kallidin before the 30 min ischaemia, but not with bradykinin, yielded an approximately 50% reduction in creatine kinase activity after reperfusion. 6. Pretreatment with L-NAME alone and simultaneously with captopril, and with kallidin, respectively, suggests a kinin-independent action of NO before the 30 min ischaemia on coronary flow and a kinin-dependent action after ischaemia. 7. These data show that captopril increases kallidin-like peptide in the effluent. Kallidin-like peptide via kinin B2 receptor seems to be the physiological mediator of cardioprotective actions of captopril against ischaemic reperfusion injury. HOE140 as well as the kallidin antiserum abolished the cardioprotective effects of captopril.     

Mechanisms underlying the relaxation response induced by bradykinin in the epithelium-intact guinea-pig trachea in vitro

In this study, we investigated some of the signalling pathways involved in bradykinin (BK)-induced relaxation in epithelium-intact strips of the guinea-pig trachea (GPT + E). BK induced time- and concentration-dependent relaxation of GPT + E. Similar responses were observed for prostaglandin E2 (PGE2) or the combination of subthreshold concentrations of BK plus PGE2. The nonselective cyclooxygenase (COX) inhibitors indomethacin or pyroxicam, or the selective COX-2 inhibitors DFU, NS 398 or rofecoxib, but not the selective COX-1 inhibitor SC 560, all abolished BK-induced relaxation. The tyrosine kinase inhibitors herbimycin A and AG 490 also abolished BK-induced relaxation in GPT + E. The nonselective nitric oxide synthase (NOS) inhibitor 7-NINA concentration-dependently inhibited BK effects. BK-induced relaxation was prevented by the selective antagonists for EP3 (L 826266), but not by EP1 (SC 19221), EP1/EP2 (AH 6809) or EP4 (L161982) receptor antagonists. Otherwise, the selective inhibitors of protein kinases A, G and C, mitogen-activated protein kinases, phospholipases C and A2, nuclear factor-kappaB or potassium channels all failed to significantly interfere with BK-mediated relaxation.BK caused a marked increase in PGE2 levels, an effect that was prevented by NS 398, HOE 140 or AG 490. COX-2 expression did not differ in preparations with or without epithelium, and it was not changed by BK stimulation. However, incubation with BK significantly increased the endothelial NOS (eNOS) and neuronal NOS (nNOS) expression, independent of the epithelium integrity. Our results indicate that BK-induced relaxation in GPT + E depends on prostanoids (probably PGE2 acting via EP3 receptors) and NO release and seems to involve complex interactions between kinin B2 receptors, COX-2, nNOS, eNOS and tyrosine kinases.     

Peripheral kinin B(1) and B(2) receptor-operated mechanisms are implicated in neuropathic nociception induced by spinal nerve ligation in rats

The kinin system can contribute distinctly to the sensory changes associated with different models of nerve injury-induced neuropathic pain. This study examines the roles of kinin B(1) and B(2) receptor-operated mechanisms in alterations in nociceptive responses of rats submitted to unilateral L5/L6 spinal nerve ligation (SNL) injury. Behavioural responses to ipsilateral hind paw stimulation with acetone (evaporation-evoked cooling), radiant heat (Hargreaves method) or von Frey hairs revealed that SNL rats developed long-lasting cold allodynia (from Days 3 to 40 post-surgery, peak on Day 6), heat hyperalgesia (stable peak from Days 9 to 36) and tactile allodynia (stable peak from Days 3 to 51). SNL rats manifested nocifensive responses to intraplantar injections on Day 12 of the selective B(1) receptor agonist des-Arg(9)-bradykinin (DABK) and augmented responses to the selective B(2) receptor agonist bradykinin (BK; each at 0.01-1nmol/paw). Systemic treatment of SNL rats with des-Arg(9)-Leu(8)-BK or HOE 140 (peptidic B(1) and B(2) receptor antagonists, respectively; 0.1-1mumol/kg, i.p.) selectively blocked responses triggered by DABK and BK (1nmol/paw) and alleviated partially and transiently established cold allodynia, heat hyperalgesia and (to a lesser extent) tactile allodynia. Western blot analysis revealed enhanced expression of kinin B(1) and B(2) receptor protein in ipsilateral L4-L6 spinal nerve and hind paw skin samples collected on Day 12 after SNL surgery. These results indicate that peripheral pronociceptive kinin B(1) and B(2) receptor-operated mechanisms contribute significantly to the maintenance of hind paw cold and mechanical allodynia and heat hyperalgesia induced by L5/L6 SNL in rats.     

Role of calcium ions in kinin-induced chloride secretion

Electrogenic ion transport across the epithelium lining the descending colon of male Sprague-Dawley rats has been measured under short-circuit conditions. Responses to kallidin (lysylbradykinin) were inhibited by 70% if calcium was removed from the solution bathing the basolateral aspect of the tissue. Under identical conditions responses to prostaglandin E1 and dibutyryl cyclic adenosine monophosphate were not changed. Forskolin, which directly activates the catalytic subunit of adenylate cyclase, was inhibited by 35% by calcium removal, whereas responses to the phosphodiesterase inhibitor isobutylmethylxanthine were inhibited by 45% by the same procedure. In the absence of calcium, strontium could substitute in promoting the chloride secretory events triggered by kallidin. Magnesium ions antagonized the effects of the kinin in the presence of calcium ions in the bathing solution. The effects of kallidin were partially antagonized by verapamil and trifluoperazine and were potentiated by isobutylmethylxanthine. These results, together with earlier evidence, suggest that kinin elicits a chloride secretory response in this epithelium by stimulating the formation of prostaglandins which then activate adenylate cyclase. Extracellular calcium ions appear to have an important role in the proximal part of this cascade for prostaglandin generation. However, biochemical correlates of these biophysical responses presented in the following paper indicate a more complex role for calcium in the genesis of the kinin response.     

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.     

The spinal and peripheral roles of bradykinin and prostaglandins in nociceptive processing in the rat.

The effect of peripheral and intrathecal administration of the bradykinin B2 receptor antagonist HOE140 and the non-steroidal anti-inflammatory drug indomethacin were studied on the response of dorsal horn nociceptive neurones to formalin in the anaesthetized rat. Peripheral pretreatment with HOE140 reduced both phases of the formalin response whereas subcutaneous administration of indomethacin (5 mg/kg) reduced only the second phase. Pre-treatment with intrathecal HOE140 resulted in a dose-dependent reduction of the second phase whereas a high dose (500 micrograms) intrathecal indomethacin reduced both phases of the response. Bradykinin plays a role in peripheral nociception during the first and second phase of the response whereas the prostaglandins are only involved during the second phase. The converse is true centrally, the prostaglandins appear to be involved in both phases of the formalin response whereas bradykinin only plays a central role during the second phase of the response.     

Bradykinin-induced depolarization of primary afferent nerve terminals in the neonatal rat spinal cord in vitro.

1. Application of bradykinin (BK) to the spinal cord of the neonatal rat evoked depolarizations which could be recorded via either the dorsal or ventral roots. However, responses recorded via the ventral root were abolished by removal of extracellular Ca2+ or the addition of Cd2+, while responses recorded via the dorsal root were unaffected. 2. The response recorded via the ventral root was inhibited by the substance P antagonist spantide, while responses recorded via the dorsal root were unaffected. 3. Depolarizations recorded via the dorsal root were concentration-dependent with an EC50 of 30 nM. These responses were not antagonized by the BK1 selective antagonist Leu8des-Arg9BK, but were antagonized by D-Arg0Hyp3Thi5,8D-Phe7BK with a pA2 of 6.8 +/- 0.6, which is similar to the values determined for other BK2-mediated responses. 4. Application of phorbol dibutyrate (PDBu) to the spinal cord also evoked a depolarization with respect to the dorsal root. This response to PDBu was enhanced by removal of extracellular Ca2+, while the response to BK was unaffected. 5. The potent protein kinase inhibitor staurosporine reduced the response to PDBu, but did not affect the response to BK. 6. These results suggest that BK by acting on BK2 receptors can depolarize the central terminals of primary afferent nerve fibres. This response to BK does not appear to be mediated via the activation of protein kinase C. The depolarization to BK recorded via the ventral root of the spinal cord is indirect and may be secondary to the action of BK on the primary afferent terminals.     

Changes in blood glucose and plasma insulin levels induced by bradykinin in anaesthetized rats.

1. The influence of bradykinin (BK) on blood glucose and plasma insulin levels was investigated in anaesthetized rats. 2. Blood glucose level was dose-dependently increased by intravenous infusion of BK. This effect of BK was enhanced by captopril, an inhibitor of angiotensin-converting enzyme (ACE). Des-Arg9-bradykinin (DABK), a kinin B1 receptor agonist, did not modify blood glucose levels while the effect of BK was inhibited by Hoe-140, a kinin B2 receptor antagonist. 3. The effect of BK was reduced by the NO-synthase inhibitor, N(omega)-nitro-L-arginine methyl ester (L-NAME), and by the cyclo-oxygenase inhibitor, indomethacin. The effect of BK was suppressed by the association of propranolol with phentolamine or phenoxybenzamine. It was also reduced by hexamethonium, a ganglion-blocking drug. In adrenalectomized rats, the infusion of BK slightly decreased blood glucose levels. 4. The hyperglycaemic effect of adrenaline was suppressed by propranolol associated with phentolamine or phenoxybenzamine, but it was not modified by L-NAME. 5. Infusion of BK did not modify plasma insulin levels. However, after phentolamine and propranolol, BK induced a transient 2 fold rise in plasma insulin levels. The release of insulin was dose-dependent and inhibited by Hoe-140. 6. We conclude that infusion of BK induces, via a stimulation of B2 receptors, the release of NO and of prostanoids. The latter agents activate through a reflex pathway the release of catecholamines from the adrenal medulla. This release increases blood glucose levels and reduces plasma insulin levels. After adrenoceptor inhibition, BK induces a secretion of insulin, via the stimulation of B2 receptors.     

Intrathecal bradykinin administration: opposite effects on nociceptive transmission

Injected intrathecally, bradykinin (BK) produced either hyperalgesia (0.15 microg) or antinociception (6.0 microg) in rats when thermal noxious stimuli were used. Similarly, des-Arg(9)-BK at the lower dose (0.15 microg) decreased, whereas at the higher dose (6.0 microg) it increased the threshold to thermal noxious stimuli; however, these effects were less pronounced than those of BK. The antinociception induced by BK was abolished by HOE 140, a B(2) receptor antagonist, injected intrathecally at a dose of 1.3 ng and was markedly attenuated by des-Arg(10)-HOE 140, a B(1) receptor antagonist (1.15 ng i.t.). The results obtained in this study showed that--depending on the dose used--BK and des-Arg(9)-BK could produce pro- as well as antinociceptive actions. Both B(2) and B(1) receptors are involved in the action of intrathecally applied BK.     

Effect of tumour necrosis factor-alpha and interleukin 1beta on endothelium-dependent relaxation in rat mesenteric resistance arteries in vitro

1. Pre-eclampsia is associated with elevated proinflammatory cytokine levels and endothelial dysfunction. This study examined the effect of two cytokines, tumour necrosis factor-alpha (TNF) and interleukin-1beta (IL-1) on endothelium-dependent relaxation in response to acetylcholine (ACH), bradykinin (BK) and histamine (HIS) in rat mesenteric small arteries in vitro. 2. Rat mesenteric arteries were mounted in an isometric myograph. Tone was induced with phenylephrine (PE) or a depolarizing solution containing 80 mM KCl (K(80)). Relaxation was measured in response to ACH, BK, HIS and sodium nitroprusside (SNP), an endothelium-independent relaxant. Inhibition of NO synthase by a combination of N(omega)-monomethyl-L-arginine (L-NMMA) and N(omega)-nitro-L-arginine methyl ester (L-NAME) significantly inhibited relaxation in response to ACH and BK. Addition of an inhibitor of cyclooxygenase, indomethacin, had no additional effect when added to L-NMMA and L-NAME. Inhibition of endothelium-derived hyperpolarizing factor (EDHF) by K(80) partially reduced responses to ACH and BK. Inhibition of HIS-induced relaxation was more marked with K(80). L-NMMA and L-NAME largely abolished the remaining relaxation to ACH, BK and HIS in arteries contracted with K(80). 3. Preincubation with TNF for 30 min caused an inhibition of relaxation in response to ACH and BK in arteries contracted with PE. Responses to HIS and SNP were not affected by TNF under these conditions. TNF also inhibited ACH-induced relaxation in arteries contracted with K(80). IL-1 had no effect on responses to ACH and the combination of TNF and IL-1 was not more effective than TNF alone. 4. The inhibitory effect of TNF on ACH-induced relaxation was abolished by coincubation with superoxide dismutase (SOD) and was not seen if NO synthase was inhibited by L-NMMA and L-NAME. 5. TNF inhibits the NO-dependent component of endothelium-dependent relaxation in response to ACH and BK, but does not inhibit the EDHF-dependent component. This effect may be attributable to the ability of TNF to increase levels of superoxide anions (O(2)(-)) and the ability of O(2)(-) to inactivate NO. This mechanism could contribute to the endothelial dysfunction seen in situations where TNF is elevated, such as pre-eclampsia.