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.

Inactivation of C3a and C5a octapeptides by carboxypeptidase R and carboxypeptidase N.

Pro-carboxypeptidase R (proCPR), also known as thrombin-activatable fibrinolysis inhibitor (TAFI), precursor of carboxypeptidase U and plasma carboxypeptidase B is present in plasma and following activation by thrombin/thrombomodulin and/or plasmin can remove arginine from the carboxyterminal of C3a and C5a. We have shown that this enzyme can remove terminal arginine from the C5a octapeptide much more efficiently than the classical anaphylatoxin inactivator, carboxypeptidase N (CPN). Since we have previously demonstrated that proCPR is significantly upregulated in the inflammatory state, this enzyme would appear to significantly contribute to the inactivation of C5a, the most potent of the complement derived anaphylatoxins.

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.

A comparison of C3a and C5a-mediated stable adhesion of rolling eosinophils in postcapillary venules and transendothelial migration in vitro and in vivo.

The comparative ability of the complement anaphylatoxins C3a and C5a to mediate leukocyte adhesion and transendothelial migration in vivo and in vitro was investigated. Superfusion of IL-1beta-stimulated rabbit mesentery with C3a resulted in a rapid and stable adhesion of rolling eosinophils, but not neutrophils, to postcapillary venules. However, C3a failed to evoke subsequent transmigration of the adherent eosinophils. In contrast, C5a induced both the rapid activation-dependent firm adhesion and transmigration of eosinophils and neutrophils through venular endothelium. C3a induced selective shedding of L-selectin and an increase in alphaMbeta2 integrin expression on eosinophils but not neutrophils, while C5a induced shedding of L-selectin and up-regulation of alphaMbeta2 integrin on both eosinophils and neutrophils. Both C3a- and C5a-dependent adhesion to venular endothelium was blocked by ex vivo treatment of eosinophils with anti-alpha4 and anti-beta2 integrin mAbs. In vitro, both C3a (but not C3a(desArg)) and C5a (including C5a(desArg))-dependent transmigration of eosinophils across IL-1beta-stimulated endothelial monolayer was mediated by alpha4beta1 and alphaMbeta2 integrins. Overall these studies suggest that C3a is eosinophil-specific chemotactic mediator that influences selectively eosinophil adhesion but not transmigration in vivo. C5a in contrast is a complete activator of integrin-dependent adhesion as well as transmigration of eosinophils and neutrophils.

Inhibition of plasma kallikrein. Kininase and kinin-like activities of preparations from the medicinal leeches.

The medicinal leech salivary gland secretion deprived of hirudin antithrombin activity inhibits amidolytic (substrate S-2302) and kininogenase (substrate kininogen) activities of plasma kallikrein, the main component of the intrinsic mechanism of blood coagulation. It therefore possesses high anticoagulant properties. Kininase (substrate bradykinin) activity of leech saliva and extracts from the medicinal leeches, as well as kinin-like effects of extracts heated at 100 degrees C have been detected. The last one is correlated with the hyperalgetic property of the heated extract. An analgetic effect was observed with the unheated extract but not with leech saliva after intranasal administration to rats.

Activation of the plasma kallikrein-kinin system by Candida albicans proteinase.

An extracellular carboxyl proteinase produced by the yeast Candida albicans enhanced vascular permeability when injected into the dorsal skin of guinea pigs. The character and mechanism of the permeability-enhancing reaction were studied in vivo and in vitro. Permeability was not enhanced when the C. albicans proteinase was heat treated (100 degrees C, 5 min) or when it was treated with pepstatin, a specific carboxyl proteinase inhibitor. The permeability reaction induced by the C. albicans proteinase was not affected by pretreatment with antihistamine but was greatly augmented by simultaneous injection of a kinin potentiator, carboxypeptidase N inhibitor. However, the simultaneous injection of a kinin-degrading enzyme, carboxypeptidase B, interfered with the reaction. Furthermore, in vitro conversion of plasma prekallikrein to kallikrein by the C. albicans proteinase was observed, and the reaction was inhibited by corn trypsin inhibitor, an inhibitor of activated Hageman factor, and soybean trypsin inhibitor, a well-known inhibitor of plasma kallikrein. These results indicate that C. albicans proteinase enhances vascular permeability through activation of the plasma kallikrein-kinin system, which generates bradykinin.

Growth-promoting activity in serum-free medium of kallikreinlike arginylesteropeptidases from rat submaxillary gland.

The characterization and purification of the growth-promoting activity present in rat submaxillary gland extracts, known to be required for the proliferation of adipose precursor cells in serum-free medium, have been undertaken. Fractionation of the extracts by ion-exchange chromatography, gel filtration, and affinity chromatography on immobilized benzamidine allowed the copurification of the mitogenic activity with two distinct arginylesteropeptidases of apparent molecular weight 25,000; one of these enzymes has been purified to homogeneity and shown to be immunologically related to tonin, a well-characterized kallikreinlike protease from submaxillary gland. The specificity of both enzymes was similar to that of plasma and glandular kallikreins, as indicated by the relative rates of hydrolysis of peptide p-nitroanilide substrates. Prior treatment of the kallikreinlike proteases with phenylmethylsulfonylfluoride or aprotinin abolished completely both mitogenic and arginylesteropeptidase activities, indicating that enzymatic activity was essential for the manifestation of their growth-promoting ability. The kallikreinlike proteases from rat submaxillary gland were able to replace thrombin to support the proliferation of Chinese hamster lung fibroblasts in serum-free medium. These results underline the role of proteases in controlling cell growth and are discussed in light of adipose tissue development.

Hyperoxia-induced converting enzyme insufficiency in conscious rat: cardiovascular effects.

The effects of exposing rats to hyperoxia (100 percent O2) at normal atmospheric pressure for periods of 24-48 hours on components of the renin-angiotensin system and on blood pressure control were examined. Intrapulmonary conversion of angiotensin I (AI) to angiotensin (AII) was assessed using an isolated lung preparation perfused at constant flow. Exposure of rats to hyperoxia for 44-48 hours reduced single pass conversion of AI to AII in the pulmonary circulation from control levels of 82 +/- 4 to 29 +/- 5 percent (p less than 0.001). AII levels in trunk blood of 44-48 hour O2 exposed animals were 5.2 +/- 1.9 pg/ml, compared to 37.9 +/- 10.0 pg/ml in controls (p less than 0.001). Mean arterial pressure decreased significantly from 117 +/- 4.3 to 103 +/- 6.7 mmHg (p less than 0.05) in the O2 exposed group despite a threefold increase in plasma renin activity. The pressor response to exogenous AI was significantly diminished by O2 exposure, while the pressor response to exogenous AII remained unchanged from control. Pulmonary angiotensin-converting enzyme activity fell to approximately 50 percent of control in O2 exposed animals, but circulating converting enzyme activity was not change in this group. None of these alterations was apparent following 24 hours of hyperoxic exposure. These data suggest that O2 induced impairment in activity of angiotensin-converting enzyme at the endothelial membrane level has functionally significant effects on cardiovascular homeostasis, probably via reduced generation of endogenous AII.

Studies of the digestion of bradykinin, lysyl bradykinin, and kinin-degradation products by carboxypeptidases A, B, and N.

We have compared the digestion of bradykinin, lysyl bradykinin, and kinin degradation products by carboxypeptidases N, B and A (CPN, CPB and CPA). Carboxypeptidase N removed the C-terminal arginine from bradykinin or lysyl bradykinin to leave the des-Arg derivative of each, and no further degradation occurred regardless of enzyme concentration or time of incubation. However, both CPB and CPA degraded the des-Arg derivatives to remove the C-terminal phenylalanine. The inhibitory effect of phosphate ions upon this activity of CPB (but not CPA) suggests that CPA may be responsible for the formation of free phenylalanine seen upon degradation of kinins in plasma or serum. However, angiotensin converting enzyme degraded des-Arg9-bradykinin in plasma or serum prior to such Phe removal to yield the pentapeptide Arg-Pro-Pro-Gly-Phe and the tripeptide Ser-Pro-Phe. We demonstrated that CPB degraded Arg-Pro-Pro-Gly-Phe but not Ser-Pro-Phe; this reaction was also inhibited by phosphate ions. Carboxypeptidase A, on the other hand, liberated Phe from both peptides in phosphate-buffered saline and accounted, at least in part, for the free phenylalanine detected. Carboxypeptidase N did not digest the aforementioned pentapeptide or tripeptide. It is clear that carboxypeptidase B and carboxypeptidase A had overlapping activities, depending upon the substrate tested, and were distinguished by the effects of different ionic environments. We further suggest a role for carboxypeptidases other than CPN in the degradation of kinins in human plasma or serum.

Hydrolysis of opioid hexapeptides by carboxypeptidase N. Presence of carboxypeptidase in cell membranes.

Carboxypeptidase N, purified to homogeneity from human plasma, rapidly hydrolyzed Lys6- or Arg6-enkephalins when measured by high pressure liquid chromatography. Comparison of the kinetics of hydrolysis of the enkephalin hexapeptides and bradykinin by carboxypeptidase N revealed the following values for the Km and kcat: Arg6-Met5-enkephalin, 49 microM, 1024 min-1; Arg6-Leu5-enkephalin, 57 microM, 375 min-1; Lys6-Met5-enkephalin, 216 microM, 6204 min-1; bradykinin, 19 microM, 58 min-1. Thus, while bradykinin had the lowest Km, the specificity constants (kcat/Km) for all the enkephalin hexapeptides were higher than that of bradykinin due to their high turnover numbers. Preincubation of the enzyme with 0.1 mM CoCl2 increased both the kcat and Km of bradykinin and Arg6-Met5-enkephalin. Similar results were obtained when the above experiments were conducted with the active 48,000 dalton subunit of carboxypeptidase N. Basic carboxypeptidase activity was found in the amniotic fluid, in membrane fractions of various human and bovine tissues, and in cultured cells in the following order of decreasing specific activity: human placental microvilli, human kidney, human amniotic fluid, human lung, bovine lung, bovine pulmonary artery, human foreskin fibroblasts, human pulmonary arterial endothelial cells, and human lung fibroblasts. The membrane-bound carboxypeptidase activity had a neutral pH optimum and behaved similarly to plasma carboxypeptidase N in the presence of various inhibitors and activators. It was different from the carboxypeptidase activity in bovine adrenal chromaffin granules which had an acid pH optimum and was inhibited by sulfhydryl reagents. These studies show that human carboxypeptidase N, an enzyme found in high concentration in blood, readily hydrolyzes Arg6- or Lys6-enkephalins. It could thus control the levels of these peptides if they are released into the circulation from the adrenal gland. In addition, a membrane-bound carboxypeptidase N-like enzyme in various tissues may regulate the local levels of biologically active peptides containing C-terminal basic amino acids such as hexapeptide enkephalins, kinins, anaphylatoxins or fibrinopeptides.

Human plasma inactive renin: purification and activation by proteases.

A new affinity chromatographic procedure was devised to purify inactive renin by using a selective hydrophobic interaction of inactive renin to octyl-Sepharose. Additional extensive purification was accomplished by immunoaffinity chromatography on antihuman renin immunoglobulin G-Sepharose. A trace amount of active renin was removed by chromatography on pepstatin-Sepharose. Human plasma inactive renin purified by this method was free from protease inhibitors and permitted the investigation of protease-mediated activation without the acid treatment which was used previously to remove inhibitors. Human plasma kallikrein, human plasmin, cathepsin B1, and arginine esteropeptidases associated with mouse epidermis growth factor and nerve growth factor were effective activators. Human urinary kallikrein, hog pancreatic kallikrein, and rat urinary esterase A were inefficient activators of low potency. Thrombin, factor Xa, factor XIIa, and urokinase did not activate inactive renin. The in vitro activation of 56,000-dalton inactive renin by these proteases was not accompanied by a recognizable reduction in molecular weight. Activation required plasma albumin, presumably as a protecting substance. These results suggest that human inactive renin can be activated by a minimum change in its molecular size.

Central action of bradykinin (I). Electroencephalogram of bradykinin and its degradation system in rat brain.

Both in vivo and in vitro experiments were performed to elucidate the relationship between the action of bradykinin (BK) and the possibility of release of pharmacologically active fragments from BK in the rat brain. In in vivo experiments, the activities of electroencephalogram (EEG) increased immediately after the intracerebral administration of 5 nmole BK. The effect was prolonged by intracerebral pretreatment of o-phenanthroline which inhibits plasma kininases. In vitro experiments, o-phenanthroline inhibited partially purified enzyme of rat brain which released fragments of BK possessing phenylalanine, serine, proline and/or arginine as N-terminal amino acids. Only arginine and minute amounts of phenylalanine were observed after the incubation with o-phenanthroline. The evidence suggests that the inhibition of the enzyme by o-phenanthroline resulted in a prolongation of the excited phase produced by BK on the EEG and on behavior. Supporting evidence indicates that Ser-Pro containing fragments derived from BK are concerned with the sedative phase of BK which was observed after the excitative stage.

Experimental gingivitis in ODU plaque-susceptible rats. V. The presence of bradykinin in the gingival tissue and the bradykinin inactivating factor in rat dental plaque.

Plaque formation and gingival inflammation were found in ODU plaque-susceptible rats. A study was performed to determine the relationship between the presence of bradykinin in gingival tissue and the inflammatory capacity of rat plaque. Bradykinin content in gingival tissue and the bradykinin inactivating activity of dental plaque of 12-month-old ODU rats were measured. The presence of bradykinin was observed in the gingival tissue of the ODU rat and a bradykinin inactivating factor was found in the rat plaque. The factor was destroyed at 60 degrees C for 30 minutes, at pH 1.5 or by treatment with perchloric acid. The optimum pH of the factor was approximately 8.0. Chelate reagents, such as O-phenanthroline and EDTA . 4Na, strongly inhibited the bradykinin inactivating factor. The protease inhibitor, Trasylol, also inhibited this factor. The SH reagent, L-cysteine, inhibited weakly and Zn++ inhibited less. The investigation suggested that the rat plaque posseses the ability to inactivate bradykinin and plays an important role in modifying the inflammatory response caused by bradykinin in gingival tissue.