Kallikrein stimulates prostacyclin production in bovine vascular endothelial cells

Cultured endothelial cells isolated from bovine carotid aorta produce prostacyclin (prostaglandin I2) and a small amount of prostaglandin E2. The effects of kallikrein (EC 3.4.21.8) on the release of prostacyclin from the cells were studied with the radioimmunoassay technique. Kallikrein stimulated the release of prostacyclin in a dose-dependent manner. The maximal stimulation reached up to 9.2-fold at 0.1 micrograms/ml of kallikrein. The effect was not associated with the activation of the fatty acid cyclooxygenase, but with the stimulation of arachidonic acid release. But kallikrein itself did not have phospholipase activity. On the other hand, at the same doses, kallikrein failed to induce platelet aggregation or enhance platelet aggregation induced by collagen. Our findings suggest that the vasodilator effect of kallikrein is mediated in part by prostacyclin production. Furthermore, we investigated the possibility that the stimulatory effect of kallikrein on prostacyclin production in endothelial cells is associated with kinin formation. Bradykinin and lysylbradykinin (kallidin) also stimulated the release of prostacyclin, but the effects were far less than that of kallikrein. And the stimulation due to the addition of both kallikrein and bradykinin on prostacyclin and arachidonic acid release was not competitive or additive, but synergistic. Moreover, even if fetal calf serum was incubated with kallikrein, bradykinin was not detected at all. When kallikrein was pre-incubated with aporotinin, which is an inactivator of kallikrein, the effect of kallikrein was completely abolished. These findings suggest that the stimulatory effect of kallikrein on the release of prostacyclin from vascular cells is possibly not due to kinin formation, but to other substance(s) produced by this serine proteinase.     

Kallikrein-thrombolytic and hypotensive action in cats--preliminary results.

An intravenous injection of kallikrein produced hypotensive and thrombolytic effects in anesthetized cats, using the blood superfused tendon technique. The thrombolytic action of kallikrein was mediated by an unstable substance. The generation of this substance was abolished by either acetylsalicylic acid (ASA) or aprotonin and enhanced by captopril. The hypotensive action of kallikrein was only partially inhibited by ASA. It is proposed that both these pharmacological effects of kallikrein are mediated by bradykinin which in turn releases prostacyclin from the endothelium. However, in contrast to the thrombolytic effect of kallikrein which is totally mediated by prostacyclin the hypotensive action of kallikrein depends not only on prostacyclin but also on another endothelium-derived vasorelaxant, e.g. EDRF.     

Renal kallikrein: its localization and possible role in renal function.

The distribution of kallikrein in dog kidneys was studied. It was found that kallikrein decreased from the outer to the inner cortex and that the medulla and papilla had very little kallikrein. The site of kallikrein secretion in the nephron was also studied by performing stop-flow techniques in dogs. The highest kallikrein concentration was found in the fractions with the lowest sodium concentration. It was concluded that kallikrein is secreted into the urine at the level of the distal tubule by either the tubule itself or by a structure related to this part of the nephron. In addition, the possible involvement of the kallikrein-kinin system in the regulation of sodium excretion was investigated. Circulating kinins and urinary kallikrein were increased in saline-loaded dogs. Urinary kallikrein also increased in dogs that have "escaped" the sodium-retaining effect of desoxycorticosterone. Experiments in rats with different sodium intake showed a relationship between water and sodium excretion and urinary kallikrein. These data suggest that the kallikrein-kinin system could participate in the regulation of the renal function at the level of the distal tubule or collecting duct.     

Mechanism of urinary kallikrein excretion

Urinary kallikrein excretion was compared with urea excretion in the rat and the results showed that they were correlated. Like urea excretion is flow-dependent, we conclude that the principal mechanism regulating kallikrein excretion is a wash-out effect on renal kallikrein.     

Vasopressin stimulates urinary kallikrein excretion in the isolated erythrocyte-perfused rat kidney

We have found that arginine vasopressin (AVP) (10 pg/ml) stimulates urinary kallikrein in the isolated erythrocyte perfused rat kidney. (In this model, perfusate flow rate approximates blood flow rates in vivo and morphology is normal.) Urinary kallikrein excretion rose from 6.9 +/- 0.8 to 14.9 +/- 2.4 ng/min 20 min after the addition of AVP to the perfusate, and then fell towards baseline levels over the next 30 min. 1-Desamino-8-D-AVP (8 pg/ml) caused a comparable increase in kallikrein excretion. Prostaglandin synthesis inhibition with indomethacin did not alter the stimulatory effect of AVP on kallikrein excretion. Parathyroid hormone 1-34 (144 ng/ml) and calcitonin (102 ng/ml) also increased urinary kallikrein. Kallikrein excretion rose from 9.1 +/- 2.0 to 24 +/- 4.5 ng/min in response to calcitonin and from 8.3 +/- 1.6 to 43.7 +/- 3.4 ng/min following the addition of parathyroid hormone to the perfusate. Kallikrein was found to accumulate in the perfusate in a linear fashion. Based on the slope of the relationship between perfusate kallikrein and time, the rate of release of kallikrein into the perfusate was estimated to be 0.79 ng/min in control kidneys. The rate of release of kallikrein into the perfusate in kidneys treated with AVP was the same (0.74 ng/min). Thus while kallikrein is released into the perfusate, this process is not influenced by AVP. In conclusion, AVP stimulates release of kallikrein into the urine (but not the perfusate) independently of systemic events. The effect of AVP is not mediated by prostaglandins. This effect of AVP is mediated via stimulation of the V2 receptor and also occurs in response to two other hormones (calcitonin and parathyroid hormone) that are known to stimulate adenyl cyclase in the rat distal nephron.     

Kallikrein/kinin protects against myocardial apoptosis after ischemia/reperfusion via Akt-glycogen synthase kinase-3 and Akt-Bad.14-3-3 signaling pathways

Our previous study has shown that human tissue kallikrein protected against ischemia/reperfusion-induced myocardial injury. In the present study, we investigated the protective role of local kallikrein gene delivery in ischemia/reperfusion-induced cardiomyocyte apoptosis and its signaling mechanisms in promoting cardiomyocyte survival. Adenovirus carrying the human tissue kallikrein gene was delivered locally into the heart using a catheter-based technique. Expression and localization of recombinant human kallikrein in rat myocardium after gene transfer were determined immunohistochemically. Kallikrein gene delivery markedly reduced reperfusion-induced cardiomyocyte apoptosis identified by both in situ nick end-labeling and DNA fragmentation. Delivery of the kallikrein gene increased phosphorylation of Src, Akt, glycogen synthase kinase (GSK)-3beta, and Bad(Ser-136) but reduced caspase-3 activation in rat myocardium after reperfusion. The protective effect of kallikrein on apoptosis and its signaling mediators was blocked by icatibant and dominant-negative Akt, indicating a kinin B2 receptor-Akt-mediated event. Similarly, kinin or transduction of kallikrein in cultured cardiomyocytes promoted cell viability and attenuated apoptosis induced by hypoxia/reoxygenation. The effect of kallikrein on cardiomyocyte survival was blocked by dominant-negative Akt and a constitutively active mutant of GSK-3beta, but it was facilitated by constitutively active Akt, catalytically inactive GSK-3beta, lithium, and caspase-3 inhibitor. Moreover, kallikrein promoted Bad.14-3-3 complex formation and inhibited Akt-GSK-3beta-dependent activation of caspase-3, whereas caspase-3 administration caused reduction of the Bad.14-3-3 complex, indicating an interaction between Akt-GSK-caspase-3 and Akt-Bad.14-3-3 signaling pathways. In conclusion, kallikrein/kinin protects against cardiomyocyte apoptosis in vivo and in vitro via Akt-Bad.14-3-3 and Akt-GSK-3beta-caspase-3 signaling pathways.     

Effect of norepinephrine and vasopressin on renal kallikrein excretion in rats

The purpose of this study was to investigate the effect of norepinephrine and vasopressin on urinary kallikrein excretion in the rat. Two studies were undertaken: (a) acute experiments in which the rats were infused with 30% dextrose in water with the addition of norepinephrine or vasopressin, (b) chronic experiments in which the drugs were infused during seven days through an osmotic minipump. In acute experiments, urinary kallikrein excretion increased without modification in urinary flow and glomerular filtration rate. In chronic experiments, urinary kallikrein excretion was not modified in norepinephrine-treated rats and decreased in vasopressin-infused animals. This decrease followed the modifications of the urine flow. In chronic experiments the dextrose infusion increased urinary kallikrein excretion. In all the groups studied a positive correlation between urine flow and urinary kallikrein excretion was observed. It is concluded that norepinephrine and vasopressin are important stimulators of the urinary kallikrein excretion only in those circumstances where it is necessary to eliminate an excess of water.     

Surface-dependent activation of human factor XII (Hageman factor) by kallikrein and its light chain

In this paper we report the effect of sulfatides on the rate constants of factor XII activation by kallikrein and its isolated light chain (the domain of kallikrein that contains the active site of the enzyme). In the absence of sulfatides, kallikrein and the light chain were equally effective in factor XII activation (k1 = 1.57 X 10(3) M-1 s-1 at pH 7.0). The pH optima were the same (pH 7.0) and the reaction was not affected by variation of the ionic strength. Sulfatides strongly increased the rate constants of factor XIIa formation. In the presence of sulfatides kallikrein was, however, much more active than its light chain. At 330 microM sulfatides, pH 7.0 and 100 mM NaCl the rate constants of factor XII activation were 5.34 X 10(6) M-1 s-1 and 4.17 X 10(4) M-1 s-1 for kallikrein and its light chain, respectively. The pH optimum of factor XII activation by kallikrein in the presence of sulfatides was shifted to pH 6.3, and the reaction became highly ionic-strength-dependent. The rate constant increased considerably at decreasing NaCl concentrations. The optimum pH for light-chain-dependent factor XII activation in the presence of sulfatides remained unaltered and the reaction was not affected by the ionic strength. Binding studies revealed that both kallikrein and factor XII bind to the sulfatide surface, whereas no binding of the light chain of kallikrein was detectable. The isolated heavy chain of kallikrein had the same binding properties as kallikrein, which indicates that the heavy-chain domain contains the functional information for kallikrein binding to sulfatides. Since the effects of pH and ionic strength on the rate constants of kallikrein-dependent factor XII activation in the presence of sulfatides correlated with effects on the binding of kallikrein, it is concluded that under these conditions surface-bound factor XII is activated by surface-bound kallikrein. Our data suggest that sulfatides stimulate kallikrein-dependent factor XII activation by two distinct mechanisms: by making factor XII more susceptible to peptide bond cleavage by kallikrein and by promoting the formation of the enzyme-substrate complex through surface binding of kallikrein and factor XII.     

Urinary kallikrein excretion in normotensive aging female rats

The effect of aging on the intrarenal kallikrein-kinin system activity was investigated in normotensive 3-, 10-, 20-, and 30-month-old female Wistar rats. Urinary kallikrein excretion was measured by three independent assays (immunoreactive concentration, kininogenase, and amidolytic activities) and was found to decrease progressively from 10 to 30 months. In the 30-month-old rats the urinary immunoreactive kallikrein excretion represented 40-44% of the level detected in 3-month-old rats. Active and total kallikrein exhibited the same magnitude of reduction. Furthermore, the active to inactive kallikrein ratio remained unchanged throughout the life period studied. The level of urinary kallikrein inhibitor was studied by measuring the recovery of purified rat urinary kallikrein added in the samples; no change was observed with aging. None of the factors known at present to influence kallikrein excretion could be evoked to explain this age-related decrease. It is therefore suggested that this decrease may reflect a progressive impairment of the intrarenal endocrine function or an alteration in the secretion of the enzyme.     

Study of the effect of high molecular weight kininogen upon the fluid-phase inactivation of kallikrein by C1 inhibitor

Plasma kallikrein and factor XIa circulate bound to high molecular weight kininogen, and such binding has been reported to protect these enzymes from inactivation by their respective inhibitors. However, this observation is controversial, and the effect of high molecular weight kininogen upon the interaction between kallikrein and C1 inhibitor (C1-INH) has been questioned. We have re-evaluated this reaction and studied the rate of inhibition of kallikrein by C1-INH in the presence and absence of high molecular weight kininogen. The second-order rate constant of inhibition of kallikrein by C1-INH was unaffected by saturating concentrations of high molecular weight kininogen. Our results suggest that although high molecular weight kininogen clearly augments the rate of formation of kallikrein and other enzymes of the contact activation pathway, it has no effect on the rate of enzyme inhibition by C1-INH.     

Kallikrein stimulates arachidonic acid release and production of prostaglandins from TEA3A1 endocrine thymic epithelial cells.

Using TEA3A1 rat endocrine thymic epithelial cells, we demonstrated that kallikrein (EC 3.4.21.35) not only stimulated the release of arachidonic acid (AA) and its metabolites from TEA3A1 cells but also enhanced the intracellular synthesis of prostaglandin E2 (PGE2) and thromboxane B2 (TXB2) by approx. 2-fold. The stimulatory effect of kallikrein was dose- and time-dependent and could be blocked by aprotinin, a kallikrein inhibitor. It was found that the phospholipase A inhibitors ONO RS082 [2-(p-amylcinnamoyl)amino-4-chlorobenzoic acid], and mepacrine (6-chloro-9-[(4-dimethylamino)-1-methyl)]amino-2-methoxyacridine; quinacrine) also inhibited the kallikrein-stimulated release of AA and its metabolites. It is suggested that the kallikrein-induced stimulatory effect might be mediated through a phospholipase A2 pathway. The effect of bradykinin was studied and no significant stimulation was observed, even at a high dose (10 micrograms/ml). This suggested that the formation of kinin does not have a role in the kallikrein-induced stimulation of AA release from TEA3A1 cells. Furthermore, the effect of kallikrein was also totally abolished by adding pepstatin A, a known inhibitor of renin, pepsin and cathepsin D which does not inhibit kallikrein itself. This indicates that kallikrein did not act on the phospholipase-like enzyme directly. There is at least one more enzyme, a pepstatin A-inhibitable proteinase, that acts as a mediator for kallikrein-induced regulation of AA release.     

Active-site labeling of rat and human urinary kallikrein by chloro(N alpha-p-tosyllysyl)methane

This is the first report to demonstrate that chloro(N alpha-p-tosyllysyl)methane (TosLys-CH2Cl) inhibits mammalian glandular kallikrein activities. The inhibitory effect of TosLysCH2Cl on purified rat urinary kallikrein was carried out with three assay methods: 1) Tos-Arg-OMe hydrolysis activity measured by a radiochemical method; 2) kininogenase activity using purified bovine low molecular weight kininogen as substrate and the released kinins subsequently measured by radioimmunoassay; 3) bioassay using isolated rat uterus preparation. Purified rat urinary kallikrein was inhibited by TolLysCH2Cl in a dose and time-dependent manner with all three methods used. The inhibition of purified human urinary kallikrein esterase and kinin-releasing activities were also demonstrated. The results indicate that TosLysCH2Cl inactivates kallikrein activity and support the notion that reactive histidine residue(s) participates in the active center of Kallikrein for catalysis.     

Purification and characterization of a kallikrein from human submaxillary glands

A tissue kallikrein was purified over 1500-fold from the postmicrosomal supernatant of human submaxillary glands. The purified enzyme gave a single band, corresponding to an apparent molecular weight of 42,000 on SDS-polyacrylamide gel electrophoresis. This enzyme cross-reacted with the anti-human urinary kallikrein antiserum. The purified enzyme was characterized in comparison with the purest human urinary kallikrein preparation. Both enzymes hydrolyzed the synthetic substrate, Ac-Phe-Arg-OMe, most effectively. Aprotinin, TLCK, and PMSF suppressed the enzyme activities, while SBTI, LBTI, and alpha 1-antitrypsin had no effect at all. The purified enzyme generated kinin from the natural substrate, kininogen. It was concluded therefore that the purified enzyme is a typical tissue kallikrein.     

The epigenetic basis for the aberrant expression of kallikreins in human cancers

The tissue kallikrein gene family consists of 15 genes tandemly arranged on human chromosome 19q13.4. Most kallikrein genes are characterized by aberrant expression patterns in various human cancers, a feature that makes them ideal cancer biomarkers. In the present study, we investigated the effect of the epigenetic drug compound 5-aza-2'-deoxycytidine on the expression of downregulated kallikrein genes in prostate, breast, and ovarian cancer cell lines. Reactivation of multiple kallikrein genes was observed, although some of these genes do not contain CpG islands in their genomic sequence. Epigenetic regulation provides a new mechanism for the pharmacological modulation of kallikreins in human cancers with putative therapeutic implications.     

Protein-protein interactions in contact activation of blood coagulation. Binding of high molecular weight kininogen and the 5-(iodoacetamido) fluorescein-labeled kininogen light chain to prekallikrein, kallikrein, and the separated kallikrein heavy and light chains

Binding of the 5-(iodoacetamido)fluorescein (IAF)-labeled high molecular weight (HMW) kininogen light chain to prekallikrein and D-Phe-Phe-Arg-CH2Cl-inactivated kallikrein was monitored by a 0.040 +/- 0.002 increase in fluorescence anisotropy. Indistinguishable average dissociation constants and stoichiometries of 14 +/- 3 nM and 1.1 +/- 0.1 mol of prekallikrein/mol of IAF-light chain and 17 +/- 3 nM and 0.9 +/- 0.1 mol of kallikrein/mol of IAF-light chain were determined for these interactions at pH 7.4, mu 0.14 and 22 degrees C. Prekallikrein which had been reduced and alkylated in 6 M guanidine HCl lost the ability to increase the fluorescence anisotropy of the IAF-kininogen light chain, suggesting that the native tertiary structure was required for tight binding. The kallikrein heavy and light chains were separated on the basis of the affinity of the heavy chain for HMW-kininogen-Sepharose, after mild reduction and alkylation of kallikrein under nondenaturing conditions. Under these conditions, alkylation with iodo [14C]acetamide demonstrated that only limited chemical modification had occurred. Binding of the IAF-kininogen light chain to the isolated alkylated kallikrein heavy chain, when compared to prekallikrein and kallikrein, was characterized by an indistinguishable increase in fluorescence anisotropy, average dissociation constant of 14 +/- 3 nM, and stoichiometry of 1.2 +/- 0.1 mol of kallikrein heavy chain/mol of IAF-light chain. In contrast, no binding of the D-Phe-Phe-Arg-CH2Cl-inactivated kallikrein light chain was detected at concentrations up to 500 nM. Furthermore, 300 nM kallikrein light chain did not affect IAF-kininogen light chain binding to prekallikrein, kallikrein, or the kallikrein heavy chain. The binding of monomeric single chain HMW-kininogen to prekallikrein, kallikrein, and the kallikrein heavy and light chains was studied using the IAF-kininogen light chain as a probe. Analysis of the competitive binding of HMW-kininogen gave average dissociation constants and stoichiometries of 12 +/- 2 nM and 1.2 +/- 0.1 mol of prekallikrein/mol of HMW-kininogen, 15 +/- 2 nM and 1.3 +/- 0.1 mol of kallikrein/mol of HMW-kininogen, 14 +/- 3 nM and 1.4 +/- 0.2 mol of kallikrein heavy chain/mol of HMW-kininogen, and no detectable effect of 300 nM kallikrein light chain on these interactions. We conclude that a specific, nonenzymatic interaction between sites located exclusively on the light chain of HMW-kininogen and the heavy chain of kallikrein or prekallikrein is responsible for the formation of 1:1 noncovalent complexes between these proteins.     

Activation of the 92 kDa type IV collagenase by tissue kallikrein

Type IV collagenases are secreted as latent 92 and 72 kDa proenzymes which are then activated extracellularly. The mechanisms by which they are activated in vivo are not clear. We have studied the activation of porcine endothelial cell type IV collagenases by tissue and plasma kallikrein, and found that tissue kallikrein was a very efficient activator of the 92 kDa type IV collagenase. Enzyme cleavage was observed at concentrations of tissue kallikrein as low as 0.1 μg/ml. Plasma kallikrein had no effect. By comparison, plasmin, which has been proposed to be the physiological activator of interstitial collagenase and stromelysin, and elastase were much less effective, and high concentrations (plasmin at 100–200 μg/ml and elastase at 20 μg/ml) were required to cause only a limited cleavage which was not associated with an increase in activity, as observed by the gelatin-gel lysis assay. In addition tissue kallikrein was found by immunohistochemistry to be present in the extracellular matrix of the intima of porcine aortic vessel wall. These findings suggest that tissue kallikrein can be a potential activator of the 92 kDa type IV collagenase in vivo. © 1993 Wiley-Liss, Inc.     

Changes in pressor and depressor reactions following partial pancreatectomy]

Experiments were conducted on 39 mongrel pubertal dogs. The course of pressor and depressor reactions under conditions of normal vital activity and after partial corporocaudal resection of the pancreas was studied. The principal indices characterizing the changes of pressor reaction to noradrenaline, and of the depressor one -- to bradykinin and kallikrein before and 10 to 12 days after the corporocaudal resection of the pancreas were determined. Resection of the corporo-caudal part of the pancreas intensified the pressor reactions to noradrenaline, and depressor ones to bradykinin and kallikrein; it increased the initial negative chronotropic effect of catecholamines on the heart, and also enhanced the positive chronotropic effect of noradrenaline, kallikrein and bradykinin on the heart.     

Tissue kallikrein synthesis and its modification by testosterone or low dietary sodium.

A method has been developed to measure the relative rate of rat tissue kallikrein synthesis which employs a specific antiserum raised against a purified rat urinary kallikrein. Incorporation of [35S]methionine into kallikrein and protein 20 min after intraperitoneal injection was measured in submaxillary gland, pancreas, kidney and descending colon. Kallikrein content was measured with a direct radioimmunoassay, and kallikrein-specific incorporation of [35S]methionine measured after immunoprecipitation. Kallikrein specific radioactivity (c.p.m./mg of enzyme) was about 100-fold greater than that in total protein in both kidney and colon. In contrast, in pancreas the incorporation into the enzyme was only 5-fold higher than into protein, and in submaxillary gland the incorporation was equivalent. Measured as kallikrein-specific radioactivity relative to total protein radioactivity incorporated in 20 min, kallikrein represents 0.18% of total protein synthesis in the kidney, 0.34% in the pancreas, 0.41% in the colon, but 7.29% in the submaxillary gland. Dietary Na+ restriction increased the relative rate of kallikrein synthesis 1.8-fold in the kidney without a comparable effect in submaxillary gland. In contrast, testosterone increased the relative rate of synthesis 2.3-fold in submaxillary gland, but decreased it in kidney. The data show that endogenous kallikrein synthesis differs markedly in various tissues, and that interventions which are known to change kallikrein content or excretion also change the relative rate of enzyme synthesis.     

Inhibition of proteases in coagulation, kinin-forming and complement systems by alpha2-plasmin inhibitor.

Inhibitory activities of alpha2-plasmin inhibitor against various proteases were investigated. The inhibitor promptly inhibited the esterolytic activity of alpha-chymotrypsin and progressively inhibited the esterolytic or amidolytic activities of bovine plasma kallikrein, bovine thrombin and bovine activated factor X. Heparin had no effect on the reaction of the inhibitor with thrombin or activated factor X. However, the inhibitor had no effect on the activities of human C-1-esterase, papain and snake venom kininogenase. On the basis of its rapid inhibition of kallikrein, alpha2-plasmin inhibitor is considered to exert some regulating effect on kallikrein activity in plasma.