Tripeptidyl carboxypeptidase activity of kininase II (angiotensin-converting enzyme)

The degradation of des-Arg9-brady kinin and its analogues by highly purified preparations of hog lung and kidney kininase II (angiotensin-converting enzyme; peptidyldipeptide hydrolase, EC 3.4.15.1) was studied. The degradative peptides fragments were separated and isolated by high performance liquid chromatography and identified by amino acid analysis. Both enzymes released C-terminal tripeptides from des-Arg9-bradykinin, des-Arg9-(Leu8)-bradykinin, Pro-Pro-Gly-Phe-Ser-Pro-Phe, Pro-Gly-Phe-Ser-Pro-Phe, Gly-Phe-Ser-Pro-Phe, Bz-Gly-Ser-pro-Phe and Bz-Gly-Ala-Pro-Phe. Hydrolysis of Phe-Ser-Pro-Phe, Bz-Gly-His-Pro-Phe, Bz-Gly-Phe-Pro-Phe and Bz-Gly-Gly-Pro-Phe by both enzymes was negligible. These data indicate that kininase II can release C-terminal tripeptides of substrates having a proline residue in the penultimate position such as des-Arg9-bradykinin and its analogues, and that this enzyme is able not only to act as a dipeptidyl carboxypeptidase but also acts as a tripeptidyl carboxy-peptidase. The tripeptidyl carboxypeptidase enzyme was sensitive to inhibition by kininase II inhibitors.     

Some Characteristics of a Peptidyl Dipeptidase (Kininase II) from Rat CSF: Differential Effects of NaC1 on the Sequential Degradation Steps of Bradykinin

Incubation of various authentic peptides with rat CSF in vitro and analysis of their products by HPLC demonstrated the presence in CSF of a peptidyl dipeptidase [peptidyl dipeptide hydrolase; angiotensin I converting enzyme (ACE); kininase II; EC 3.4.15.1] which sequentially degraded bradykinin (BK) by liberating the carboxy-terminal dipeptides and converted angiotensin I to angiotensin II. This CSF enzyme was gel-chromatographed by means of HPLC, and the molecular weight was estimated. The susceptibility to various peptidase inhibitors of the rat CSF enzyme, as well as the effect of NaCl on the degradation of BK and Hip-His-Leu catalyzed by it, was also determined. These properties were compared with those of ACE or kininase II from brain or other tissues, as described in the literature. NaCl was shown to exert specific and concentration-dependent effects on each step of the sequential degradation of BK, via BK(1-7) to BK(1-5), catalyzed by the enzyme. In addition, the enzyme system for metabolism of BK appears to differ between rat CSF and blood, the former containing exclusively kininase II, whereas the latter contains both kininase I (carboxypeptidase N; EC 3.4.12.7) and kininase II.     

Pathways of bradykinin degradation in blood and plasma of normotensive and hypertensive rats

Kinins are vasoactive peptide hormones that can confer protection against the development of hypertension. Because their efficacy is greatly influenced by the rate of enzymatic degradation, the activities of various kininases in plasma and blood of spontaneously hypertensive rats (SHR) were compared with those in normotensive Wistar-Kyoto rats (WKY) to identify pathogenic alterations. Either plasma or whole blood was incubated with bradykinin (10 microM). Bradykinin and kinin metabolites were measured by high-performance liquid chromatography. Kininase activities were determined by cumulative inhibition of angiotensin I-converting enzyme (ACE), carboxypeptidase N (CPN), and aminopeptidase P (APP), using selective inhibitors. Plasma of WKY rats degraded bradykinin at a rate of 13.3 +/- 0.94 micromol x min(-1) x l(-1). The enzymes ACE, APP, and CPN represented 92% of this kininase activity, with relative contributions of 52, 25, and 16%, respectively. Inclusion of blood cells at physiological concentrations did not extend the activities of these plasma kininases further. No differences of kinin degradation were found between WKY and SHR. The identical conditions of kinin degradation in WKY and SHR suggest no pathogenic role of kininases in the SHR model of genetic hypertension.     

Cardiac kinin level in experimental diabetes mellitus: role of kininases

Diabetes mellitus impairs the cardiac kallikrein-kinin system by reducing cardiac kallikrein (KLK) and kininogen levels, a mechanism that may contribute to the deleterious outcome of cardiac ischemia in this disease. We studied left ventricular (LV) function and bradykinin (BK) coronary outflow in buffer-perfused, isolated working hearts (n = 7) of controls and streptozotocin (STZ)-induced diabetic rats before and after global ischemia. With the use of selective kininase inhibitors, the activities of angiotensin I-converting enzyme, aminopeptidase P, and neutral endopeptidase were determined by analyzing the degradation kinetics of exogenously administered BK during sequential coronary passages. Basal LV function and coronary flow were impaired in STZ-induced diabetic rats. Neither basal nor postischemic coronary BK outflow differed between control and diabetic hearts. Reperfusion after 15 min of ischemia induced a peak in coronary BK outflow that was of the same extent and duration in both groups. In diabetic hearts, total cardiac kininase activity was reduced by 41.4% with an unchanged relative kininase contribution compared with controls. In conclusion, despite reduced cardiac KLK synthesis, STZ-induced diabetic hearts are able to maintain kinin liberation under basal and ischemic conditions because of a primary impairment or a secondary downregulation of kinin-degrading enzymes.     

High flaxseed (linseed) diet restores endothelial function in the mesenteric arterial bed of spontaneously hypertensive rats.

The effect of high flaxseed diet (HFD) on blood pressure (BP) and mesenteric arterial bed (MAB) reactivity was studied in spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto (WKY) rats. HFD did not affect BP in either SHR (control, 157 +/- 3; HFD, 153 +/- 3 mmHg) or WKY (control, 114 +/- 2; HFD, 117 +/- 2 mmHg) rats. Increases in perfusion pressure of the endothelium-intact MAB to phenylephrine and norepinephrine were higher (p < 0.05) in SHR than in WKY rats and the HFD failed to alter these responses. Vasorelaxant responses to acetylcholine (ACh) and bradykinin (BK) were greater (p < 0.05) in SHR maintained on HFD compared to SHR on control diet. While HFD also enhanced ACh responses in WKY, the effect was less than in SHR. Responses to sodium nitroprusside (SNP), were similar in all groups. Since ACh and BK-induced responses of the MAB were augmented in SHR on HFD, with no changes in BP, it is suggested that HFD improves endothelial vasorelaxant function through a pressure-independent mechanism.     

Metabolism of bradykinin analogs by angiotensin I converting enzyme and carboxypeptidase N.

Bradykinin (BK) analogs such as Lys-Lys-BK, des-Arg9-BK and [Leu8]des-Arg9-BK were poor substrates for angiotensin I converting enzyme (ACE), and analogs containing D-Phe7 residues, or a pseudopeptide C-terminal bond, were completely resistant. However, many of these analogs were metabolized by carboxypeptidase N (CPN) including Lys-Lys-BK, [Tyr8(OMe)]BK and D-Phe7-containing analogs, with Km and Vmax values comparable to those for BK. The only analogs completely resistant to both ACE and CPN were the B2 agonist [Phe8 psi(CH2NH)Arg9]BK, the B2 agonist D-Arg[Hyp3,D-Phe7,Phe8 psi(CH2NH)Arg9]BK, and the B1 agonist [D-Phe8]des-Arg9-BK. These data indicate an important role for plasma CPN and vascular CPN-like activity in the metabolism of the widely used ACE-resistant/D-Phe7-containing antagonists of B2 kinin receptors.     

Angiotensin processing is partially carried out by carboxypeptidases in the rat mesenteric arterial bed perfusate

Here we investigated the possible association between the carboxypeptidase A (CPA)-like activity of the rat mesenteric arterial bed (MAB) perfusate and the ability of this fluid of forming angiotensin (Ang) 1-9 and Ang 1-7 upon incubation with Ang I and Ang II, respectively. Initially, we observed that anion exchange chromatography of the perfusate would consistently split the characteristic Z-Val-Phe-hydrolyzing activity of CPA-like enzymes into five distinct peaks, whose proteolytic activities were then determined using also Ang I and Ang II as substrates. The resulting proteolytic profile for each peak indicated that rat MAB perfusate contains a complex mixture of carboxypeptidases; tentatively, five carboxypeptidases were distinguished based on their substrate preferences toward Z-Val-Phe, Ang I and Ang II. The respective reactions, namely, Z-Val-Phe cleavage, Ang I to Ang 1-9 conversion and Ang II to Ang 1-7 conversion, were inhibited by 1,10-phenanthroline and nearly fully blocked by potato carboxypeptidase inhibitor. Also, all the CPA-like activity peaks prepared by anion exchange chromatography were tested negative for contaminating Ang I-converting enzyme-2, cathepsin A and prolylcarboxypeptidase. Overall, our results indicate that rat MAB perfusate contains a multiplicity of Ang I and Ang II-processing CPA-like enzymes whose proteolytic specificities suggest they might perform peculiar regulatory roles in the local renin-angiotensin system.     

Kinetic characterization and inhibition of the rat MAB elastase-2, an angiotensin I-converting serine protease

An elastase-2 has been recently described as the major angiotensin (Ang) II-forming enzyme of the rat mesenteric arterial bed (MAB) perfusate. Here, we have investigated the interaction of affinity-purified rat MAB elastase-2 with some substrates and inhibitors of both pancreatic elastases-2 and Ang II-forming chymases. The Ang II precursor [Pro 11 -D-Ala 12]-Ang I was converted into Ang II by the rat MAB elastase-2 with catalytic efficiency of 8.6 min-1 microM-1, and the chromogenic substrates N-succinyl-Ala-Ala-Pro-Leu-p-nitroanilide and N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide were hydrolyzed by the enzyme with catalytic efficiencies of 10.6 min-1 microM-1 and 7.6 min-1 microM-1, respectively. The non-cleavable peptide inhibitor CH-5450 inhibited the rat MAB elastase-2 activities toward the substrates Ang I (IC50 = 49 microM) and N-succinly-Ala-Ala-Pro-Phe-p-nitroanilide (IC 50 = 4.8 microM), whereas N-acetyl-Ala-Ala-Pro-Leu-chloromethylketone, an effective active site-directed inhibitor of pancreatic elastase-2, efficiently blocked the Ang II-generating activity of the rat MAB enzyme (IC 50 = 4.5 microM). Altogether, the data presented here confirm and extend the enzymological similarities between pancreatic elastase-2 and its rat MAB counterpart. Moreover, the thus far unrealized interaction of elastase-2 with [Pro 11-D-Ala 12]-Ang I and CH-5450, both regarded as selective for chymases, suggests that evidence for the in vivo formation of Ang II by chymases may have been overestimated in previous investigations of Ang II-forming pathways.     

Functional role,cellular source,and tissue distribution of rat elastase-2, an angiotensin II-forming enzyme

We recently described a chymostatin-sensitive elastase-2 as the major angiotensin (ANG) II-forming enzyme in the perfusate of the rat mesenteric arterial bed (MAB) with the same cDNA sequence as rat pancreatic elastase-2. The role of this enzyme in generating ANG II was examined in the rat isolated and perfused MAB. The vasoconstrictor effect elicited by ANG I and the renin substrate tetradecapeptide was only partially inhibited by captopril but abolished by the combination of captopril and chymostatin or N-acetyl-Ala-Ala-Pro-Leu-chloromethylketone (Ac-AAPL-CK; inhibitor originally developed for human elastase-2). The effect induced by [Pro11,d-Ala12]-ANG I, an ANG I-converting enzyme (ACE)-resistant biologically inactive precursor of ANG II, was blocked by chymostatin or Ac-AAPL-CK. It was also demonstrated that cultured rat mesenteric endothelial cells synthesize elastase-2 and that mRNA for this enzyme can be detected in different rat tissues such as the pancreas, MAB, lung, heart, kidney, liver, and spleen. In conclusion, the demonstration of a functional alternative pathway to ACE for ANG II generation in the rat MAB and the fact that cultured MAB endothelial cells are capable of producing and secreting elastase-2 represent strong evidence of a physiological role for this enzyme in the rat vasculature.     

Role of the renal kallikrein-kinin system in the development of salt-sensitive hypertension

The role of the renal kallikrein-kinin system in the development of salt-sensitive hypertension was studied using mutant kininogen-deficient Brown-Norway Katholiek (BN-Ka) rats, which generate no kinin in their urine, and other hypertensive rat models. It was found that ingestion of a low sodium diet or infusion of NaCl in doses slightly above 0.15 M caused hypertension and sodium accumulation in erythrocytes and the cerebrospinal fluid of kininogen-deficient BN-Ka rats. Development of hypertension in the deoxycorticosterone-acetate-salt model was completely prevented by administration of a newly discovered inhibitor, ebelactone B, of carboxypeptidase Y-like exopeptidase (an urinary kininase). The urinary kallikrein excretion of spontaneously hypertensive rats was lower than that of Wistar Kyoto rats at 4 weeks of age and did not increase by administration of furosemide, a diuretic agent, although approximately 50% of the diuretic action of this agent was dependent upon the renal kallikrein-kinin system in normal rats. In conclusion, the renal kallikrein-kinin system works as a safety valve for excess sodium intake.     

Effects of the vasopeptidase inhibitor omapatrilat on cardiac endogenous kinins in rats with acute myocardial infarction

The purposes of this study were to evaluate and to compare the effects of simultaneous angiotensin-converting enzyme (ACE) and neutral endopeptidase 24.11 (NEP) inhibition by the vasopeptidase inhibitor omapatrilat (1 mg. kg(-1). day(-1)) with those of the selective ACE inhibitor enalapril (1 mg. kg(-1). day(-1)) on survival, cardiac hemodynamics, and bradykinin (BK) and des-Arg(9)-BK levels in cardiac tissues 24 h after myocardial infarction (MI) in rats. The effect of the co-administration of both B(1) and B(2) kinin receptor antagonists (2.5 mg. kg(-1). day(-1) each) with metallopeptidase inhibitors was also evaluated. The pharmacological treatments were infused subcutaneously using micro-osmotic pumps for 5 days starting 4 days before the ligation of the left coronary artery. Immunoreactive kinins were quantified by highly sensitive and specific competitive enzyme immunoassays. The post-MI mortality of untreated rats with a large MI was high; 74% of rats dying prior to the hemodynamic study. Mortality in the other MI groups was not significantly different from that of the untreated MI rats. Cardiac BK levels were not significantly different in the MI vehicle-treated group compared with the sham-operated rats. Both omapatrilat and enalapril treatments of MI rats significantly increased cardiac BK concentrations compared with the sham-operated group (P < 0.05). However, cardiac BK levels were significantly increased only in the MI omapatrilat-treated rats compared with the MI vehicle-treated group (P < 0.01). Cardiac des-Arg(9)-BK concentrations were not significantly modified by MI, and MI with omapatrilat or enalapril treatment compared with the sham-operated group. The co-administration of both kinin receptor antagonists with MI omapatrilat- and enalapril-treated rats had no significant effect on cardiac BK and des-Arg(9)-BK levels. Thus, the significant increase of cardiac BK concentrations by omapatrilat could be related to a biochemical or a cardiac hemodynamic parameter on early (24 h) post-MI state.     

Interaction of bradykinin and des-Arg9-bradykinin with isolated pig coronary arteries: mechanical and electrophysiological events

This paper describes the effect of bradykinin (BK) and des-Arg9-BK on the isometric tension and smooth muscle membrane potential of transverse strips of pig coronary artery. BK causes a relaxation of contracted muscle. This effect is particularly evident in muscle which has previously been contracted by acetylcholine. The relaxation is accompanied by a transient hyperpolarization of the vascular smooth muscle. Des-Arg9-BK, in contrast, causes a contraction of the muscle which is not accompanied by a significant change of transmembrane potential. The relaxing action of BK depends on the presence of the endothelium. In a "cascade" experiment, evidence is presented that a relaxing factor is released by the endothelium in response to BK. Thus the perfusate from a BK-stimulated intact artery can cause the relaxation of a pre-contracted de-endothelialized artery. We conclude that the endothelium has B2-receptors which cause the release of a humoral factor which hyperpolarizes and relaxes the muscle. The contracting action of des-Arg9-BK does not depend on the endothelium and appears to be mediated through B1-receptors directly on smooth muscle by pharmacomechanical coupling.     

Generation and characterization of a highly stable form of activated thrombin-activable fibrinolysis inhibitor

Activated thrombin-activable fibrinolysis inhibitor (TAFIa) is a carboxypeptidase B that can down-regulate fibrinolysis. TAFIa is a labile enzyme that can be inactivated by conformational instability or proteolysis. TAFI is approximately 40% identical to pancreatic carboxypeptidase B (CPB). In contrast to TAFIa, pancreatic CPB is a stable protease. We hypothesized that regions or residues that are not conserved in TAFIa compared with pancreatic CPB play a role in the conformational instability of TAFIa and that replacement of these non-conserved residues with residues of pancreatic CPB would lead to a TAFIa molecule with an increased stability. Therefore, we have expressed, purified, and characterized two TAFI-CPB chimeras: TAFI-CPB-(293-333) and TAFI-CPB-(293-401). TAFI-CPB-(293-333) could be activated by thrombin-thrombomodulin, but not as efficiently as wild-type TAFI. After activation, this mutant was unstable and was hardly able to prolong clot lysis of TAFI-deficient plasma. Binding of TAFI-CPB-(293-333) to both plasminogen and fibrinogen was normal compared with wild-type TAFI. TAFI-CPB-(293-401) could be activated by thrombin-thrombomodulin, although at a lower rate compared with wild-type TAFI. The activated mutant displayed a markedly prolonged half-life of 1.5 h. Plasmin could both activate and inactivate this chimera. Interestingly, this chimera did not bind to plasminogen or fibrinogen. TAFI-CPB-(293-401) could prolong the clot lysis time in TAFI-deficient plasma, although not as efficiently as wild-type TAFI. In conclusion, by replacing a region in TAFI with the corresponding region in pancreatic CPB, we were able to generate a TAFIa form with a highly stable activity.     

Carboxypeptidase M in Madin-Darby canine kidney cells. Evidence that carboxypeptidase M has a phosphatidylinositol glycan anchor

Carboxypeptidase M, a plasma membrane-bound enzyme, is present in many human organs and differs from other carboxypeptidase that cleave basic COOH-terminal amino acids. Cultured Madin-Darby canine kidney (MDCK) distal tubular cells contain a kininase I-type enzyme that inactivates bradykinin by releasing Arg9. We found the properties of this kininase to be identical with carboxypeptidase M. In fractionated cells, carboxypeptidase activity sediments with membranes; and detergents, trypsin, and phosphatidylinositol-specific phospholipase C solubilize it, similar to results with human placental carboxypeptidase M. Ten microM 2-mercaptomethyl-3-guanidinoethylthiopropanoic acid and 1 mM o-phenanthroline inhibit, whereas 1.0 mM CoCl2 activates the enzyme. It has a neutral pH optimum and cleaves COOH-terminal Arg or Lys in bradykinin and in shorter peptides. The relative hydrolysis rates of peptides in the presence or absence of 1 mM CoCl2 were similar to those obtained with human carboxypeptidase M. The carboxypeptidase in MDCK cells (54 kDa) cross-reacts with antibodies to human carboxypeptidase M in Western blotting, but not with antibodies to plasma carboxypeptidase N. The enzyme is a glycoprotein; chemical deglycosylation reduced the size to 48 kDa. The presence of the enzyme on the cell membrane of MDCK cells was also shown with transmission electron microscopy using immunogold, which indicated that the enzyme is on the apical side. In addition, MDCK cells contain neutral endopeptidase 24.11 (enkephalinase) and prolylcarboxypeptidase (angiotensinase C) activities. Partitioning of solubilized carboxypeptidase M into Triton X-114 and water indicates that trypsin and phospholipase C remove a hydrophobic tail, while detergent solubilization leaves the hydrophobic moiety intact. Labeling of MDCK cells with [3H]ethanolamine resulted in the synthesis of radiolabeled carboxypeptidase M as determined by immunoprecipitation and fluorography. Thus, MDCK cells contain membrane-bound carboxypeptidase M, which is anchored to the plasma membrane via phosphatidylinositol-glycan. As a major kininase of the distal tubules, it may regulate salt and water excretion.     

炎症介质在内毒素引起大鼠肠系膜血管床降钙素基因相关肽释放中的作用

本研究在离体大鼠肠系膜血管床灌流模型上,采用特异放免法测定灌流液中的降钙素基因相关肽（ＣＧＲＰ）,观察几种常见炎症介质在内毒素引起ＣＧＲＰ释放中的作用。结果显示：前列腺素合成酶抑制剂地塞米松、布洛芬与消炎痛,缓激肽受体Ｂ２拮抗剂ＨＯＥ１４０,血小板激活因子拮抗剂ＷＥＢ２０８６,组胺Ｈ１受体拮抗剂苯海拉明以及和组胺Ｈ２受体拮抗剂西咪替丁,均能明显抑剂制内毒素引起的ＣＧＲＰ释放,５羟色胺受体拮抗剂ＩＣＳ２０５９３０却无明显作用。从而提示：内毒素引起ＣＧＲＰ释放是通过炎症介质前列腺素、缓激肽、血小板激活因子和组胺介导的,阻断或减少炎症介质的产生可望减轻内毒素血症时ＣＧＲＰ的过量释放。     

Des-Arg9-bradykinin metabolism in patients who presented hypersensitivity reactions during hemodialysis: role of serum ACE and aminopeptidase P.

Bradykinin (BK) has been proposed as the principal mediator of hypersensitivity reactions (HSR) in patients dialyzed using negatively charged membranes and concomitantly treated with angiotensin-converting enzyme (ACE) inhibitors. We investigated the metabolism of exogenous BK added to the sera of 13 patients dialyzed on an AN69 membrane with a history of HSR (HSR+ patients) and 10 others who did not present such a reaction (HSR- patients) while dialyzed under the same conditions. No significant difference in the t1/2 of BK was found between the patient groups. However, the t1/2 of generated des-Arg9-BK was significantly increased (2.2-fold) in HSR+ patients compared to HSR-subjects. Preincubation of the sera with an ACE inhibitor (enalaprilat) significantly increased the t1/2 of both BK and des-Arg9-BK in both groups. There was no significant difference between the groups with respect to the t1/2 of BK, but there was a significantly greater increase (3.8-fold) in the t1/2 of des-Arg9-BK in HSR+ patients compared to HSR-subjects. The level of serum aminopeptidase P (APP) activity showed a significant decrease in the HSR+ sera when compared to HSR-samples. In HSR- and HSR+ patients, a significant inverse relation (r2 = 0.6271; P < 0.00005) could be calculated between APP activity and des-Arg9-BK t1/2. In conclusion, HSR in hemodialyzed patients who are concomitantly treated with a negatively charged membrane and an ACE inhibitor can be considered as a multifactorial disease in that a decreased APP activity resulting in reduced degradation of des-Arg9-BK may lead to the accumulation of this B1 agonist that could be responsible, at least in part, for the signs and symptoms of HSR.     

The kininases of Bothrops jararaca plasma

Evidence is presented to suggest that kininase activity of Bothrops jararaca plasma is due to the presence of at least three distinct enzymes: a carboxypeptidase B type enzyme, similar to that found in human plasma in that its activity is enhanced by Co2+ (1 X 10(-4) M); a carboxypeptidase B type enzyme whose activity is unaffected by Co2+, and an enzyme which cleaves bradykinin to liberate Phe-Arg as the major peptide fragment formed. The latter enzyme is responsible for the major kininase activity of this snake plasma and is identified as a dipeptide hydrolase.     

Inactivation of bradykinin by placental peptidases.

Normal human placental eluate (0.15 M NaCl) has very high capacity for inactivating synthetic bradykinin (its specific activity is 20--50 times higher than that of normal human serum). In chromatography on DEAE-Sephadex A 50, several fractions with bradykinin-inactivating capacity were recovered. On Cbo-Phe-Arg synthetic substrate, one fraction with kininase activity was identified as carboxypeptidase N. This is not identical with serum (pregnancy) carboxypeptidase N and does not therefore pass into the blood stream. The properties of other fractions with kininase activity were studied. Aminopeptidase activity degrading L-lysine-p-nitranilide and L-arginine-beta-naphthylamide was separate from kininase activity.     

Kininase I-type carboxypeptidases enhance nitric oxide production in endothelial cells by generating bradykinin B1 receptor agonists

Kininase I-type carboxypeptidases convert native kinin agonists for B(2) receptors into B(1) receptor agonists by specifically removing the COOH-terminal Arg residue. The membrane localization of carboxypeptidase M (CPM) and carboxypeptidase D (CPD) make them ideally situated to regulate kinin activity. Nitric oxide (NO) release from human lung microvascular endothelial cells (HLMVEC) was measured directly in real time with a porphyrinic microsensor. Bradykinin (1-100 nM) elicited a transient (5 min) peak of generation of NO that was blocked by the B(2) antagonist HOE 140, whereas B(1) agonist des-Arg(10)-kallidin caused a small linear increase in NO over 20 min. Treatment of HLMVEC with 5 ng/ml interleukin-1beta and 200 U/ml interferon-gamma for 16 h upregulated B(1) receptors as shown by an approximately fourfold increase in prolonged (>20 min) output of NO in response to des-Arg(10)-kallidin, which was blocked by the B(1) antagonist des-Arg(10)-Leu(9)-kallidin. B(2) receptor agonists bradykinin or kallidin also generated prolonged NO production in treated HLMVEC, which was significantly reduced by either a B(1) antagonist or carboxypeptidase inhibitor, and completely abolished with a combination of B(1) and B(2) receptor antagonists. Furthermore, CPM and CPD activities were increased about twofold in membrane fractions of HLMVEC treated with interleukin-1beta and interferon-gamma compared with control cells. Immunostaining localized CPD primarily in a perinuclear/Golgi region, whereas CPM was on the cell membrane. These data show that cellular kininase I-type carboxypeptidases can enhance kinin signaling and NO production by converting B(2) agonists to B(1) agonists, especially in inflammatory conditions.     

Kininase from the Latrodectus tredecimguttatus venom. Characteristics of the enzyme as a thiol endopeptidase hydrolyzing the -Pro7-Phe8- bond of bradykinin

The nature of the bradykinin (BK)-hydrolyzing (kininase) activity of peptidhydrolase isolated from spider (Latr. tredecimguttatus) venom has been studied. It was found that the BKase activity of the enzyme is fully inhibited by organic mercurials (10(-5)-10(-6) M) as well as by 5,5'-dithiobis(2-nitrobenzoic acid) (10(-7) M); the latter blocks three SH-groups within the enzyme molecule. Serine and metalloproteinase inhibitors have no effect on the kininase activity. Thin-layer chromatography on silicagel revealed that the highly purified enzyme hydrolyzes the -Pro7-Phe8- bond of BK liberating the C-terminal dipeptide, HPhe-ArgOH. Besides, the kininase splits off the C-terminal tripeptide from angiotensin I by hydrolyzing its -Pro7-Phe8-bond. The enzyme does not exhibit any exopeptidase activity with free and N-substituted tri- and pentapeptides. The data obtained suggest that the Latr. tredecimguttatus kininase can be related to thiol endopeptidases hydrolyzing the peptide bonds formed by proline carboxyl.