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Objective: The present study was designed to examine the involvement of bradykinin in thermal and mechanical hyperalgesia induced by chronic constriction nerve injury (CCI) using B1 and B2 receptor antagonists and mutant kininogen-deficient rats., Methods: Sprague-Dawley (SD) rats and Brown Norway (B/N-) rats given CCI treatment on day 0, were used as a model of neuropathic pain. Either a kinin B1 antagonist des-Arg9-[Leu8]-bradykinin or the receptor B2 antagonist HOE-140 was constantly infused into the left jugular vein of SD rats on days 15 to 22 after CCI. Vehicle-treated rats and sham-operated rats without nerve injury were also prepared as controls. In all rats, we observed pain behavior, and measured the latency period of paw withdrawal from the thermal stimuli and, with von Frey filaments, the mechanical pain threshold, before surgery and on days 14 and 22 after CCI. B/N-Katholiek rats, which congenitally lack plasma kininogen and release no kinin, were also tested for hyperalgesic parameters. Expression of kinin receptor mRNA in the dorsal root ganglia was detected by RT-PCR., Results: Most of the rats (88%) showed some pain behavior, which was reduced to 67% by a B1 antagonist and to 57% by a B2 antagonist infused between days 15 to 22. Thermal hyperalgesia was significantly reduced from 7.25 +/- 0.41 sec (mean +/- SEM) to 8.36 +/- 0.41 sec in paw withdrawal latency on day 22 by a B1 antagonist and from 7.24 +/- 0.19 sec to 8.23 +/- 0.21 sec by a B2 antagonist (P < 0.05). Mechanical hyperalgesia was also ameliorated from 0.02 +/- 0.007 g force to 0.16 +/- 0.08 g force in pain threshold by a B1 antagonist and from 0.03 +/- 0.007 g force to 0.10 +/- 0.003 g force on day 22 by a B2 antagonist. Moreover, deficient B/N-Katholiek rats showed a low incidence of thermal and mechanical hyperalgesia on day 14. Expression of both B1 and B2 receptor mRNAs was detected in the lumbar dorsal ganglia ipsilateral to the site of the nerve injury., Conclusion: These data suggests that kinin were at least partly involved in yielding nociceptor hypersensitivity up to day 14 after CCI. Bradykinin and its B1 and B2 receptors were involved in the maintenance of hyperalgesia.

1 To identify the roles of endogenous kinins in prevention of myocardial infarction (MI), we performed the permanent ligation of coronary artery in rats. 2 The size of MI 12, 24, and 48 h after coronary ligation in kininogen-deficient Brown Norway Katholiek (BN-Ka) rats was significantly larger (49.7+/-0.2%, 49.6+/-2%, and 51.1+/-1%, respectively) than that of kinin-replete Brown Norway Kitasato (BN-Ki) rats (42+/-2%, 38.5+/-4%, and 41.5+/-1%). 3 Hoe140, a bradykinin (BK) B(2) receptor antagonist injected (1.0 mg kg(-1), i.v.) half an hour before, and every 8 h after, coronary ligation, significantly increased the size of MI in Sprague-Dawley rats. Aprotinin, a kallikrein inhibitor, which was infused intravenously (10,000 Units kg(-1) h(-1)) with an osmotic mini-pump, significantly increased the size of an MI 24 h after ligation. 4 When evaluated using microspheres, the regional myocardial blood flow around the necrotic lesion in BN-Ka rats 6 h after ligation was reduced more than that in BN-Ki rats with MI by 41-46%. The same was true in Hoe140-treated BN-Ki rats. 5 FR190997, a nonpeptide B(2) agonist, which was infused (10 microg kg(-1) h(-1)) into the vena cava of BN-Ka rats for 24 h with an osmotic mini-pump, caused significant reduction in the size of MI (38+/-3%), in comparison with the size in vehicle solution-treated rats (51+/-3%). The size of MI in FR190997-treated BN-Ka rats was the same as in BN-Ki rats. 6 These results suggested that endogenous kinin has the capacity to reduce the size of MI via B(2) receptor signalling because of the increase in regional myocardial blood flow around the ischaemic lesion.

Physiological roles of angiotensin II type 2 receptor (AT(2)) are not well defined. This study was designed to investigate the mechanisms of AT(2)-dependent vascular relaxation by studying vasodilation in pressurized and perfused rat mesenteric arterial segments. Perfusion of angiotensin II in the presence of AT(1) antagonist elicited vascular relaxation, which was completely dependent on AT(2) receptors on endothelium. FR173657 (>1 microM), a bradykinin (BK) B(2)-specific antagonist, significantly suppressed AT(2)-dependent vasodilation (maximum inhibition: 68.5% at 10 microM). Kininogen-deficient Brown Norway Katholiek rats showed a significant reduction in AT(2)-mediated vasodilatory response compared with normal wild-type Brown Norway rats. Indomethacin (>1 microM), aprotinin (10 microM) and soybean trypsin inhibitor (10 microM) also reduced AT(2)-dependent vasodilation. Our results demonstrated that stimulation of AT(2) receptors caused a significant vasodilation through local production of BK in resistant arteries of rat mesentery in a flow-dependent manner. Such vasodilation counterbalances AT(1)-dependent vasoconstriction to regulate the vascular tone.

Bradykinin (BK) is involved in tumor angiogenesis. To elucidate the mechanism underlying BK-induced angiogenesis, we evaluated the roles of BK in tumor-associated vascular permeability and angiogenesis in the different phases of tumor development in mice bearing sarcoma 180 cells. The vascular permeability was significantly enhanced in the early growth phase (which peaked at day 5), and was thereafter markedly reduced. By contrast, tumor angiogenesis increased gradually over a 20-day experimental period. Oral administration of a B2 receptor antagonist, FR173657 (30 mg/kg/day), significantly suppressed the vascular permeability, but a B1 antagonist, desArg10-Hoe140 (1 mg/kg/day) did not. An immunohistochemical study revealed the presence of immunoreactive B2 receptor in the endothelial cells in the early phase, whereas B2 receptors were also observed in the stromal fibroblasts in the late phase. We also found that VEGF was detected exclusively in the stromal fibroblasts only in the late phase. Furthermore, VEGF immunoreactivity was attenuated by the treatment with FR173657. Tumor angiogenesis was significantly reduced by treating the tumor tissues with FR173657 both in the early phase (days 1-6, 30 mg/kg/day, oral administration) and in the late phase (days 7-12, 30 mg/kg/day, oral administration), whereas it was inhibited by neutralization with anti-VEGF antibody (1 microg/site/day, local injection) only in the late phase. These results suggest that BK would promote angiogenesis by increasing vascular permeability in the early phase via B2 receptor in the endothelial cells and by promoting up-regulation of VEGF via B2 receptor in the stromal fibroblasts in the late phase.

1. The involvement of bradykinin (BK) B(2) receptor in acute pancreatitis induced by pancreaticobiliary duct ligation was investigated in rats. 2. The activities of amylase and lipase in the serum, the water content of the pancreas, and vacuolization of the acinar cells were significantly increased 2 h after obstruction of the duct in Sprague-Dawley rats. 3. Elevated serum amylase activity, increased pancreatic oedema, and damage of the pancreatic tissue were significantly less marked in plasma kininogen-deficient, B/N-Katholiek rats than in the normal strain, B/N-Kitasato rats 2 h after the ligation. 4. Obstruction of the pancreaticobiliary duct augmented the level of (1-5)-BK (Arg(1)-Pro(2)-Pro(3)-Gly(4)-Phe(5)), a stable BK metabolite, in the blood from 73.0+/-21.7 pg ml(-1) at 0 h to 149.8+/-38.0 pg ml(-1) at 2 h after the induction of pancreatitis in SD rats. 5. Administration of a BK B(2) receptor antagonist, FR173657 (100 mg kg(-1), p.o.) or Hoe140 (100 nmol kg(-1), s.c.), reduced the elevation of amylase and lipase activities in the serum and of pancreatic water content in a dose-dependent manner. The effective attenuation of oedema formation and vacuolization by the antagonists was also confirmed light-microscopically. In contrast, treatment with gabexate mesilate or indomethacin did not cause significant suppression of the pancreatitis. 6. These findings suggest a possible involvement of kinin B(2) receptor in the present pancreatitis model. Furthermore, they point to the potential usefulness of the B(2) receptor in clinical acute pancreatitis.

Angiogenesis is an important event in tumor growth. We evaluated the contribution of endogenous bradykinin to tumor-associated angiogenesis and tumor growth using pharmacological approaches in mice bearing sarcoma 180 cells. The weight of implanted tumors increased in parallel with increased hemoglobin contents (a parameter to evaluate angiogenesis) over a 20-day experimental period. Daily administration of bradykinin B2-receptor antagonists, Hoe140 (0.1 and 1 mg/kg per day, local injection) or FR173657 (30 mg/kg per day, p.o.), significantly suppressed the increment in angiogenesis and tumor weight, but a B1-receptor antagonist, desArg10-Hoe140 (1 mg/kgperday), did not. Administration of a plasma kallikrein inhibitor, soybean trypsin inhibitor (3 mg/site per day), significantly suppressed angiogenesis and tumor growth. In contrast, bradykinin-degrading enzyme inhibitors, captopril and phosphoramidon (500 microg/site per day), enhanced angiogenesis and increased tumor weight. Our results suggest that bradykinin, produced by plasma kallikrein or plasma kallikrein-like enzymes, promote tumor-associated angiogenesis and tumor growth in vivo.

To evaluate whether neutral endopeptidase (NEP) inhibitors have adverse respiratory effects, the influence of a NEP inhibitor on bradykinin (BK)-induced bronchoconstriction was investigated. In anesthetized and artificially ventilated guinea pigs, changes in airway opening pressure (Pao) were measured as an index of bronchoconstriction. An infusion of phosphoramidon (3 mg kg(-1) h(-1)), a NEP inhibitor, significantly enhanced the bronchoconstriction induced by high-dose BK (30 nmol kg(-1), i.v.). Capsaicin (0.1 mg kg(-1), i.v.) and SR48968 (0.3 mg kg(-1), i.v.), an NK2 receptor antagonist, significantly inhibited the phosphoramidon-induced enhancement of BK-induced bronchoconstriction, although FK888 (3 mg kg(-1), i.v.), an NK1 receptor antagonist, did not. Both neurokinin A (NKA) (0.1-3 nmol kg(-1), i.v.) and substance P (SP) (0.1-3 nmol kg(-1), i.v.) induced dose-dependent bronchoconstriction which was enhanced by phosphoramidon infusion, although these enhancements were more prominent in the NKA series. Phosphoramidon partially inhibited BK degradation in lung homogenate, and both NKA and SP degradation in the lung homogenate were significantly suppressed by phosphoramidon. In bronchoalveolar lavage fluid (BALF), levels of NKA and SP were significantly elevated after a bolus of BK with a phosphoramidon infusion. These results suggest that NEP inhibitors may have adverse respiratory effects resulting from inhibition of the degradation of neurokinins, but mainly of NKA, when a large amount of BK is generated.

1. To determine whether the antihypertensive response in deoxycorticosterone acetate (DOCA) salt-treated rats was mediated by kinins on the luminal side of renal tubules or in the circulation, selective urinary kininase inhibitors were administered to normal Brown Norway Kitasato (BN-Ki) rats and kininogen-deficient Brown Norway Katholiek (BN-Ka) rats. 2. Kinins were degraded by neutral endopeptidase (NEP) and carboxypeptidase Y-like kininase (CPY) in urine, but were inactivated mainly by angiotensin-converting enzyme (ACE) in the plasma. 3. Ebelactone B inhibited CPY, while poststatin inhibited CPY and NEP. 4. Daily administration of poststatin (5 mg/kg per day, s.c.) for 3 days reduced blood pressure (BP) in DOCA salt-treated BN-Ki rats, but not in BN-Ka rats. 5. Ebelactone B (5 mg/kg per day, s.c.) also reduced BP in BN-Ki rats, which was accompanied by increased urinary sodium excretion, but had no effect on BP in BN-Ka rats. 6. Lisinopril (5 mg/kg per day, s.c.) had no effect on BP in either rat strain. 7. Arterial kinin levels in BN-Ki rats increased significantly (2.2-4.6 pg/mL) with captopril (10 mg/kg, s.c.). However, arterial kinin levels that induced hypotension following the infusion of bradykinin (1000 ng/kg per min, i.v.) were 110-fold higher than endogenous arterial kinin levels attained following captopril. 8. These results suggest that inhibition of kinin degradation on the luminal side of the renal tubules may effectively attenuate hypertension.

We have developed an ELISA for BK-(1-5) (Arg1-Pro2-Pro3-Gly4-Phe5). In rat carrageenin-induced pleurisy, in which a plasma exudation peak was observed 5 h after carrageenin, BK levels in the exudates were negligible (< 60 pg/rat). BK-(1-7) (des-Phe8-Arg9-BK) was detectable (900-400 pg/rat) over the entire course of the inflammation. However, a larger amount of BK-(1-5) was detectable in association with the increase in plasma exudation, showing a peak (8800 +/- 1200 pg/rat) 3 h after carrageenin. Bromelain (10 mg/kg, i.v.) and soy bean trypsin inhibitor (0.3 mg/rat, intra-pleural) significantly reduced BK-(1-5) levels (by 60-93%, 3, 7 and 19 h after carrageenin) and plasma exudation rates (by 61-74%, 3 and 7 h after carrageenin). Dexamethasone (0.3 mg/kg, i.p.) reduced BK-(1-5) levels (by 78%) and decreased plasma exudation (by 70%) 3 h after carrageenin. In nasal allergy patients, antigen challenge of nasal mucosa elevated BK-(1-5) levels and active kallikrein levels in nasal washes. These results verify that BK-(1-5) determined by ELISA is a good indicator for release of kinins in vivo.

Administration of human recombinant granulocyte colony-stimulating factor (G-CSF, 100 micrograms/kg/day, s.c) to rats for 4 days significantly increased circulating neutrophil counts (by 1130%), together with an increase in mononuclear leukocyte counts (by 119%). Infiltrated pleural neutrophil counts in G-CSF-treated rats (G-CSF-r) 5 h after the intrapleural injection of zymosan-activated serum were significantly higher (by 155%) than those in control rats (Vehicle-r). In carrageenin-induced pleurisy, counts of infiltrated pleural neutrophils in G-CSF-r 5 and 7 h after carrageenin were significantly higher (by 119% and 116%) than those in Vehicle-r. G-CSF treatment increased the volume of pleural exudate and the plasma exudation rate by 122% and 226%, compared to values in Vehicle-r 5 h after carrageenin. Cobra venom factor (75 micrograms/kg, i.v.) significantly reduced pleural neutrophil migration in G-CSF-r (by 53%) and Vehicle-r (by 49%). Bromelain (10 mg/kg, i.v.) and aspirin (100 mg/kg, p.o.) reduced pleural neutrophil migration and reduced exudate volume and plasma exudation. Intrapleural bradykinin-(1-5) and prostaglandin E2 levels were significantly higher in G-CSF-r than in Vehicle-r. The increased neutrophil migration in G-CSF-r may be attributed to enhanced activation of the complement system facilitated by increased plasma exudation due to bradykinin and prostaglandins.

Captopril (10 mg/kg, i.p.) increased the arterial bradykinin (BK) level (Art-BK) of non-treated Sprague-Dawley rats (SD), determined by an ELISA, from 10.8 +/- 3.2 pg/ml to 32.9 +/- 5.4 pg/ml significantly (p < 0.05, n = 6). Intravenous infusion of BK (100-3000 ng/kg/min) dose-dependently increased heart rate (HR) and decreased mean blood pressure (MBP), the former at lower doses than the latter, and the hypotensive response became significant at 3000 ng/kg/min. Art-BK determined during infusion of the lowest dose of BK (100 ng/kg/min) was 12 times the endogenous Art-BK after captopril administration. In spontaneously hypertensive rats, Wistar Kyoto rats, and deoxycorticosterone acetate-salt treated hypertensive rats, Art-BK (450-1280 pg/ml) determined during intravenous BK-infusion (1000-3000 ng/kg/min), which induced significant hypotension, was 20 to 100 times the endogenous Art-BK (4.5-64 pg/ml) with captopril treatment. These results suggest that the increased Art-BK due to inhibition of kinin degradation by captopril could not account for the hypotension due to this angiotensin converting enzyme inhibitor in normotensive and hypertensive rats.

In anesthetized, mechanically ventilated guinea pigs, infusion of captopril (1 mg/kg/h), an angiotensin converting enzyme inhibitor, significantly enhanced bronchoconstriction induced by intravenous injection of bradykinin (BK; 0.1-30 nmol/kg). Pretreatment of guinea pigs with capsaicin (100 mu g/kg) slightly suppressed the bronchoconstriction by BK alone and almost all of the enhancement of BK-induced bronchoconstriction by captopril was suppressed. Intravenous injection of substance P (SP; 0.1-100 nmol/kg), neurokinin A (NKA; 0.1-30 nmol/kg) and neurokinin B (NKB; 0.1-30 nmol/kg) also induced dose-dependent bronchoconstriction but captopril treatment enhanced only the bronchoconstriction induced by SP. BK degradation in bronchoalveolar lavage fluid (BALF) in vitro was significantly suppressed by captopril (p < 0.05). Captopril infusion to guinea pigs significantly increased the levels of BK, SP, and NKA in BALF after BK injection (p < 0.05). FK224, an NK1 and NK2 receptor antagonist and SR 48968, an NK2 receptor antagonist, significantly suppressed the bronchoconstriction induced by BK alone (p < 0.01 and p < 0.05, respectively) as well as the enhancement by captopril (p < 0.01). It can be concluded that the enhancement of BK-induced bronchoconstriction by captopril was attributable to inhibition of the degradation of BK itself and thereby enhanced release of NKA and partly of SP from sensory nerves by BK.

Incubation of bradykinin with human urine resulted in a successive degradation of bradykinin-(1-8), bradykinin-(1-7), bradykinin-(1-6), and bradykinin-(1-5). Although D,L-2-mercaptomethyl-3-guanidinoethylthiopropanoic acid (100 microM) and captopril (100 microM) did not have any significant effect on bradykinin degradation in human urine, the neutral endopeptidase inhibitor phosphoramidon (100 microM), a carboxypeptidase Y-like exopeptidase inhibitor ebelactone B (100 microM), and o-phenanthroline (100 microM) significantly inhibited bradykinin degradation by 36%, 38% and 48% respectively. The combination of phosphoramidon and ebelactone B completely (by 95%) inhibited bradykinin degradation in human urine. At pH 5, bradykinin degradation was performed by carboxypeptidase Y-like exopeptidase; at pH 7, this degradation was performed by neutral endopeptidase in addition to carboxypeptidase Y-like exopeptidase. From these results, it can be concluded that carboxypeptidase Y-like exopeptidase and/or neutral endopeptidase certainly have a role in kinin degradation in human urine under neutral and acid pH conditions.

Incubation of bradykinin with rat urine resulted in the successive degradation of bradykinin to bradykinin-(1-8), bradykinin-(1-7) and bradykinin-(1-6). In contrast, in rat plasma, bradykinin was degraded via either bradykinin-(1-8) or bradykinin-(1-7) to bradykinin-(1-5). Phosphoramidon (1 mM) partially inhibited the degradation of bradykinin by rat urine, as well as the conversion of bradykinin-(1-7) to bradykinin-(1-6). D,L-2-Mercaptomethyl-3-guanidinoethylthiopropanoic acid (1 mM) and captopril (1 mM) did not have a significant effect on any of the degradation steps in rat urine. In contrast, all of the degradation steps in urine, namely, from bradykinin to bradykinin-(1-8), from bradykinin-(1-8) to bradykinin-(1-7) and from bradykinin-(1-7) to bradykinin-(1-6), were markedly inhibited by poststatin (1 mM), even though this compound was reported originally to be a novel inhibitor of post-proline cleaving enzyme. Poststatin (1 mM) did not inhibit the degradation of bradykinin in rat plasma. These results indicate that poststatin is an effective inhibitor of kinin-degrading enzyme in rat urine.

We developed an enzyme immunoassay (EIA) specific for Arg1-Pro2-Pro3-Gly4-Phe5 ([1-5]-BK) for determination of the levels of this peptide in biological fluids. Previously developed EIAs for bradykinin (BK) and for des-Phe8-Arg9-BK ([1-7]BK) were also used. Incubation of rat plasma with glass powder resulted in the transient appearance of BK. A degradation product, [1-7]BK, could be detected in the incubation mixture for a longer period of time. When compared with BK and [1-7]BK, a larger amount of [1-5]BK was detectable even longer. In carrageenan-induced pleurisy in rats, which was associated with a peak rate of plasma exudation 5 hr after administration of carrageenan, BK was undetectable (< 160 pg/rat) in the pleural exudates. By contrast, [1-7]BK was detectable over the entire course of the inflammatory response. A larger amount of [1-5]BK was detectable. The peak level of [1-5]BK was 6050 +/- 1050 pg/rat, 5 hr after administration of carrageenan. Inhibition of the generation of BK by intrapleural administration of soy bean trypsin inhibitor (0.3 mg/rat) 30 min before collection of pleural fluid resulted in significant reductions in the levels of both [1-7]BK (by 51-65%) and [1-5]BK (by 63-79%) in the exudates 3, 7 and 19 hr after administration of carrageenan. Intraperitoneal administration of captopril (10 mg/kg) caused a marked reduction (by 98%) in levels of [1-5]BK in exudates 3 hr after administration of carrageenan. The reduction was accompanied by an increase in the level of BK up to 1250% of that in untreated rats. These results indicate that the newly developed EIA for [1-5]BK might be a useful tool for verifying the release of kinin in vivo.

Degradation of bradykinin (BK) in human plasma was investigated by searching for a stable metabolite as a marker of kinin release in vivo. BK, incubated with diluted plasma, was degraded to des-Arg9-BK (des-9-BK), des-Phe8-Arg9-BK (des-8,9-BK) and Arg-Pro-Pro-Gly-Phe ([1-5]BK). Des-9-BK was degraded to [1-5]BK without an increase in des-8,9-BK, and des-8,9-BK was also degraded to [1-5]BK. D,L-2-Mercaptomethyl-3-guanidinoethyl-thiopropanoic acid inhibited the formation of des-9-BK from BK, whereas captopril inhibited the formation of des-8,9-BK from BK as well as that of [1-5]BK from des-9-BK or des-8,9-BK. The half lives of BK, des-9-BK, des-8,9-BK and [1-5]BK under the present experimental conditions were 60 min, 90 min, 14 min and 4.2 hr, respectively. Among the metabolites, [1-5]BK was the most stable one and may be used as a marker for BK production in vivo.

The experimental study was carried out to examine whether intraperitoneal injection of carrageenin or endotoxin activates plasma kallikrein-kinin system to induce plasma exudation in rats. Intraperitoneal injection of 2% lambda-carrageenin induced plasma exudation in the peritoneal cavity by activation of plasma prekallikrein. Intraperitoneal injection of endotoxin (3mg/kg) also resulted in intraperitoneal plasma exudation, but plasma kallikrein-kinin system did not seem to be involved.

In guinea pig plasma, bradykinin (BK) was degraded mainly to des-Arg1-BK by an aminopeptidase-like enzyme, which was inhibited by 2-mercaptoethanol. Besides this degradation, BK was also hydrolyzed by kininase I and kininase II from C-terminal end to des-Arg9-BK, des-Phe8-Arg9-BK and Arg-Pro-Pro-Gly-Phe ([1-5] BK). The formation of des-9-BK was strongly blocked by DL-2-mercaptomethyl-3-guanidinoethylthiopropanoic acid (MGPA) and that of des-8,9-BK and [1-5] BK was inhibited by captopril. When guinea pigs were pretreated with a cocktail of 2-mercaptoethanol, MGPA and captopril, intravenous administration of leukotriene (LT) C4 (10 nmol/kg) caused an increase in the levels of free kinin in the bronchial washings of guinea pigs. This increase was accompanied with the increase in glandular-kallikrein activity, which could be inhibited by aprotinin. As BK is reported to induce both bronchoconstriction and bronchial secretion, the increased free BK induced by LTC4 might enhance the effect of LTC4.

Des-Phe8-Arg9-BK could be detected in the entire course of rat carrageenin pleurisy up to 24 h, together with a reduction of the residual levels of high molecular weight kininogen and prekallikrein. On the basis of this continuous release of bradikinin, prostaglandin E2 was released up to 5 h in the pleural exudate and enhanced the plasma leakage. In rat cardiac infarction, the initial increase in the number of polymorphonuclear leukocytes in the cardiac tissue was accompanied by leukotriene B4 in the tissue and this was followed by the second increase and activation of the complement system.