Kallikrein inhibitors in rat plasma.

The kallikrein inhibitor contents of human and animal plasma were determined with glandular kallikreins [EC 3.4.21.8]. One ml of plasma could inactivate 20-700 kallikrein units (KU). Rat plasma was the most potent and inactivated 230-700 KU. However, no enzyme capable of inactivating kallikrein could be found in this plasma. Two fractions which inhibited hog pancreatic kallikrein, a fraction corresponding to alpha2-macroglobulin and a fraction which was eluted prior to albumin, were separated from rat plasma by Sephadex G-200 gel filtration. The former inhibitor could inhibit hog pancreatic kallikrein action on Nalpha-benzoyl-L-arginine ethyl ester (BAEE) as well as in the dog vasodilator assay. The other inhibitor was partially purified from rat plasma. One mg of the preparation inhibited 67 KU and the hydrolysis of 5.8 micronmoles/min of BAEE by hog pancreatic kallikrein [EC 3.4.21.8]. The inhibitor also inhibited other glandular and plasma kallikreins, trypsin [EC 3.4.21.4], alpha-chymotrypsin [EC 3.4.21.1], etc. The optimal pH of the inhibitor was 7.5-8. The inhibitor was unstable below pH 5, and was destroyed by heating at temperature above 60 degrees. The isoelectric point of the inhibitor was determined by Ampholine focusing to be 4.4, and its molecular weight was estimated to be 73,000 by Sephadex G-100 and G-150 filtrations. Several experimental results suggested that this inhibitor differed from alpha1-antitrypsin.     

T-kinin release from T-kininogen by rat-submaxillary-gland endopeptidase K

Submaxillary gland extracts have been fractionated to characterize the enzyme responsible for the T-kininogenase activity previously reported in this tissue [Damas, J. & Adam, A. (1985) Mol. Physiol 8, 307-316] and to know whether this activity could be of physiological relevance, since no enzyme reacting in catalytic amounts has been described so far to be able to release a vasoactive peptide from T-kininogen. The purified enzyme, provisionally called endopeptidase K, has an apparent Mr of 27,000 when not reduced prior to analysis but 21,000 after reduction and an acidic pI of 4.3 +/- 0.1. Antigenically, it is not related to tissue kallikrein. Upon incubation with purified T-kininogen it may induce a complete liberation of T-kinin from the precursor provided it is added in stoichiometric amounts. However, in parallel with the liberation of immunoreactive kinin, a proteolysis of T-kininogen is observed which is not restricted to the site of insertion of T-kinin as would be expected using a specific kininogenase. In agreement with these results, no change of the mean blood pressure was observed upon injection of endopeptidase K into the circulation of normal rats even if the amount of injected enzyme was up to ten times that required for tissue kallikrein to induce a significant fall in blood pressure. However, in spite of the large proteolysis induced by incubation with stoichiometric amounts of endopeptidase K, the total papain inhibiting capacity of T-kininogen as well as the value of the apparent inhibition constant, Ki, with this proteinase remained unchanged. Proteolytic fragments which retain cysteine-proteinase-inhibiting activity may therefore be released from T-kininogen by endopeptidase K more easily than immunoreactive kinin, thus emphasizing a prominent function of proteinase inhibitor or of proteinase inhibitor precursor for this molecule.     

Tissue kallikrein and kinin infusion promotes neovascularization in limb ischemia

Adenovirus-mediated kallikrein delivery has been shown to promote blood vessel growth in the limb under both ischemic and normoperfused conditions. Here we investigated whether a continuous supply of kallikrein and kinin peptide can induce neovascularization in a rat model of hindlimb ischemia. Rats underwent femoral artery ligation and localized injection of tissue kallikrein, bradykinin or B1 receptor agonist, followed by infusion of proteins by osmotic minipump. Regional blood flow was monitored weekly by laser Doppler perfusion imaging. Three weeks after surgery, rats receiving kallikrein and kinins showed a significant increase in the perfusion ratio of ischemic vs. normoperfused limb compared to control rats. Similarly, a microsphere assay showed that kallikrein and kinins significantly increased regional blood flow without altering blood pressure. Moreover, kallikrein and kinins significantly augmented capillary and arteriole densities, as quantified by immunostaining with CD-31 and smooth muscle alpha-actin. Both tissue kallikrein and bradykinin increased hemoglobin content in Matrigel implants in mice, providing further evidence of the angiogenic properties. Kinins, when delivered subcutaneously via Matrigel in rats, also increased regional perfusion. This is the first demonstration that local application of tissue kallikrein protein or kinin peptide has therapeutic value in the treatment of ischemic disease by promoting neovascularization.     

The tissue kallikrein-kinin system protects against cardiovascular and renal diseases and ischemic stroke independently of blood pressure reduction

Tissue kallikrein (hK1) cleaves low-molecular-weight kininogen to produce kinin peptide, which binds to kinin receptors and triggers a wide spectrum of biological effects. Tissue kallikrein levels are reduced in humans and in animal models with hypertension, cardiovascular and renal diseases. Transgenic mice or rats over-expressing human tissue kallikrein or kinin B2 receptor are permanently hypotensive, and somatic kallikrein gene delivery reduces blood pressure in several hypertensive rat models. Moreover, kallikrein gene delivery or kallikrein protein infusion can directly improve cardiac, renal and neurological function without blood pressure reduction. Kallikrein has pleiotropic effects in inhibiting apoptosis, inflammation, proliferation, hypertrophy and fibrosis, and promoting angiogenesis and neurogenesis in different experimental animal models. Kallikrein's effects can be blocked by kinin B2 receptor antagonists. Mechanistically, tissue kallikrein/kinin leads to increased nitric oxide levels and Akt activation, and reduced reactive oxygen species formation, TGF-beta1 expression, MAPK and nuclear factor-kappaB activation. Our studies indicate that tissue kallikrein, through the kinin B2 receptor and nitric oxide formation, can protect against oxidative damage in cardiovascular and renal diseases and ischemic stroke. These novel findings suggest that kallikrein/kinin may serve as new drug targets for the prevention and treatment of heart failure, renal disease and stroke in humans.     

Generation, in activation and level of blood kinins during tourniquet shock in rabbits

Traumatic shock was induced by the tourniquet method compressing one thigh during 10 hours. Venous blood samples were taken from control animals, as well as twice in the nervous phase of shock - after application and before removal of the tourniquet, and in the humoral-toxic phase - 1, 3 and 5 hours after tourniquet removal, in groups of 10 animals. Determinations included blood kinin level, and plasma kininogen level, and the activity of kallikreins and kininases in the plasma. It was found that during tourniquet shock a significant change occurred in the whole blood kinin system. Proportionally to the severity of shock the level of free kinins and kallikrein activity increased 3-4, times and the level of kininogen and the activity of kininases decreased, especially 3 hours after tourniquet removal.     

Purification and chemical studies on rat urinary kallikrein.

A highly purified kallikrein was obtained from rat urine by chromatography on DE-32 cellulose, affinity chromatography on Bio-gel P-200-Aprotinin and gel filtration over Sephadex G-100 coarse and superfine. A molecular weight of 32,000 by sodium dodecyl sulfate polyacrylamide disc gel electrophoresis was estimated. The aminoacid composition and the esterase activity of the purified material were determined. Biological characterization of the purified kallikrein was tested by liberation of a kinin from rat plasma kininogen, by direct action on the isolated rat uterus and by the lowering of rat arterial pressure after intravenous injection of the enzyme. The preparation of insoluble derivative of Aprotinin is described herein. The polymer used as insoluble support (Bio-gel P-200) was before changed to its corresponding azide, which reacts with Aprotinin; the product maintained the binding property of the Aprotinin with urinary kallikrein.     

Quantification of kininogen in human renal medulla

Renal kininogen was detected in human medullary tissue as well as human medullary tubule suspensions. After treatment with pig pancreatic kallikrein or human renal cortical homogenate liberated kinin was measured by bradykinin radioimmunoassay. In the absence of inhibitors kinins were degraded by kininases located in the same part of the kidney. Several known inhibitors of kininase I and II did not inhibit this activity. Endogenous medullary kininase was inhibited by preincubation of homogenates at 56 degrees C for one hour or by addition of 0.25 mmol/l HgCl2. Under these conditions endogenous medullary kinin release amounted to 9-26 nmol/g protein. The action of renal cortical kininogenase on kinin formation from papillary kininogen was completely inhibited by addition of 1 mumol/l aprotinin. Kininogen examined in renal tubule suspensions revealed an increase in amount per g protein compared to homogenates, confirming the tubular localization of renal kininogen.     

Effect of captopril on renal vascular resistance, renin, prostaglandins and kinin in the isolated perfused kidney

Vasodilatory and natriuretic effects of captopril were studied in the isolated hog kidney perfused with modified Krebs-Ringer solution. Renal arterial infusion of captopril caused increases in releases of renin, prostaglandins (PGE2, 6-keto-PGF1 alpha and PGF2 alpha) and kinin, and was accompanied by a decrease in the renal vascular resistance and an increase in urinary sodium excretion. Indomethacin administered with captopril diminished the saluretic effect of captopril and evoked an increase in kinin, but was associated with a marked decrease in prostaglandin and renin releases, while renal vascular resistance remained decreased. Indomethacin alone did not alter vascular resistance and kinin; however, renin and prostaglandin releases were decreased. Aprotinin administered with captopril showed a decrease in releases of prostaglandins, renin and kinin without any change in vascular resistance. These results suggest that increased release of kinin induced by captopril contributes to a reduction in renal vascular resistance. Increased prostaglandin release after captopril administration may be caused by an increase in kinin without direct involvement of captopril in prostaglandin synthesis. Renal prostaglandins may enhance sodium excretion and mediate renin secretion in captopril perfusion.     

A possible alternative mechanism of kinin generation in vivo by cathepsin L

We investigated the ability of cathepsin L to induce a hypotensive effect after intravenous injection in rats and correlated this decrease in blood pressure with kinin generation. Simultaneously with blood pressure decrease, we detected plasma kininogen depletion in the treated rats. The effect observed in vivo was abolished by pre-incubation of cathepsin L with the cysteine peptidase-specific inhibitor E-64 (1 microM) or by previous administration of the bradykinin B2 receptor antagonist JE049 (4 mg/kg). A potentiation of the hypotensive effect caused by cathepsin L was observed by previous administration of the angiotensin I-converting enzyme inhibitor captopril (5 mg/kg). In vitro studies indicated that cathepsin L excised bradykinin from the synthetic fluorogenic peptide Abz-MTSVIRRPPGFSPFRAPRV-NH2, based on the Met375-Val393 sequence of rat kininogen (Abz = o-aminobenzoic acid). In conclusion, our data indicate that in vivo cathepsin L releases a kinin-related peptide, and in vitro experiments suggest that the kinin generated is bradykinin. Although it is well known that cysteine proteases are strongly inhibited by kininogen, cathepsin L could represent an alternative pathway for kinin production in pathological processes.     

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.     

Potential role of kinins in the effects of taurine in high-fructose-fed rats

The present work investigates the involvement of kinins in the effects of taurine in fructose-fed hypertensive rats. The effects of taurine on blood pressure, plasma glucose, insulin, and the insulin sensitivity index were determined. Angiotensin-converting enzyme (ACE) activity and nitrite content in plasma, plasma and tissue kallikrein activity, and taurine content were also investigated. The blood pressure changes in response to the coadministration of inhibitors of the synthesis of nitric oxide (NO), prostaglandins (PGs), or a kinin receptor blocker along with taurine was also evaluated. Fructose-fed rats had higher blood pressure and elevated plasma levels of glucose and insulin. Kallikrein activity, taurine, and nitrite contents were significantly lower in fructose-fed rats as compared with controls. The increases in systolic blood pressure, hyperglycemia, and hyperinsulinemia were controlled by taurine administration in fructose-fed rats. ACE activity was lower, while nitrite and taurine content and kallikrein activity were higher, in taurine-supplemented rats as compared with fructose-fed rats. A significant increase in blood pressure was observed in rats cotreated with the inhibitors Hoe 140 (a kinin receptor blocker), L-NAME (a NO synthase inhibitor), or indomethacin (a PG synthesis inhibitor) with taurine for 1 week as compared with taurine-treated fructose-fed rats. This suggests that the antihypertensive effect of taurine in fructose-fed rats was blocked by the inhibitors. Augmented kallikrein activity and, hence, increased kinin availability may be implicated in the effects of taurine in fructose-fed hypertensive rats.     

Human tissue kallikrein. I. Isolation and characterization of human pancreatic kallikrein from duodenal juice

Human pancreatic kallikrein was purified from duodenal juice by ion exchange chromatography on DEAE-Sepharose and immunoaffinity chromatography. Thus, an enzyme preparation with a specific activity (using Ac-Phe-Arg-OEt as substrate) of 1 000 U/mg protein was obtained. A specific biological activity of 1310 KE/mg protein was measured in the dog blood pressure assay and of 0.361 HMW kininogen-U/mg, corresponding to the liberation of 383 micrograms bradykinin-equivalents per mg enzyme per min from HMW kininogen in the rat uterus assay. In dodecyl sulfate gel electrophoresis one protein band corresponding to a molecular mass of 27 kDa was obtained. Using gel filtration on Ultrogel AcA-44 a molecular mass of 40 kDa was measured. The amino-acid composition was determined and isoleucine and alanine were identified as the only N-terminal amino-acid residues. On isoelectric focusing four protein bands with isoelectric points of 5.60, 5.65, 5.70 and 5.85 were separated. The bimolecular velocity constant for the inhibition by diisopropyl fluoro phosphate was determined as 10.5 l x mol-1 x min-1. The dissociation constant Ki of the human pancreatic kallikrein-aprotinin complex was calculated to be 1.5 x 10(-10)M. The kinetic constants for the kallikrein-catalysed hydrolysis of Ac-Phe-Arg-OEt and D Val-Leu-Arg-Nan were determined. Immunological studies showed a close relationship between the human pancreatic kallikrein and other human tissue kallikreins, especially with human urinary kallikrein. Detergents such as Triton X-100, Tween 20 and lysolecithin, as well as human serum albumin, activated the human pancreatic kallikrein preparation.     

Bradykinin and kininogens in bovine milk

Two peptides exhibiting kinin activity in an isolated rat uterus assay were purified from pasteurized skim bovine milk. The amino acid sequence of the more prominent peptide was found to be that of bradykinin. Partially purified kinin preparations were also obtained from N-tosyl-L-phenylalanyl chloromethyl ketone-treated trypsin digests of non-fat dry milk and insoluble lactalbumin. The application of fast atom bombardment/mass spectrometry permitted detection of the bradykinin protonated molecular ion in each of these samples. Collision-activated decomposition of the ion of m/z 1061 confirmed it to be the protonated molecular ion of bradykinin. Fast atom bombardment/mass spectrometry analysis further confirmed the occurrence of bradykinin in a pancreatic kallikrein digest of a partially purified bovine milk kininogen preparation. In apparent contrast with bovine plasma kininogens, the forms of kininogen which occur in milk include a high Mr kininogen (Mr greater than 68,000) and a low Mr kininogen (Mr 16,000-17,000). Kinin formation from the high Mr kininogen is catalyzed by porcine pancreatic kallikrein or trypsin.     

Characterization and purification of a kallikrein from human pancreatic juice and immunological comparison with other kallikreins.

A prekallikrein has been demonstrated in human pancreatic juice and the active enzyme has been purified from this material. The purification procedure included filtration on Sephadex G-100, chromatography on DEAE-cellulose and affinity chromatography on trypsin-inhibitor Sepharose. The purified kallikrein appeared to be homogeneous by polyacrylamide gel electrophoresis at pH 8.3 and by immunoelectrophoresis. Human pancreatic kallikrein is immunologically different from human plasma kallikrein and from pancreatic kallikreins of other species (hog, cat, rat and dog). Human pancreatic kallikrein has common antigenic determinants with human urinary and submandibular kallikreins but probably not with parotid kallikrein.     

Synergic effects of kallikrein-kinin and prostaglandins on renin release on infusion of isolated hog kidney with aldosterone

The effects of infusion of a large amount of aldosterone into the renal artery of isolated perfused hog kidney on the release of renin, prostaglandins (PG) and kinin and the excretion of urinary kallikrein were investigated. Infusion of aldosterone at a rate of 100 ng/min (100 to 800 ng/ml of perfusate) resulted in significant releases of renin, PG (PGE₂, 6-0-PGF_1α), and kinin and increase in urinary kallikrein. Infusion of aldosterone and an inhibitor of kallikrein, aprotinin, decreased the releases of renin, PG and kinin and infusion of aldosterone with indomethacin decreased the release of PG but increased that of kinin and urinary kallikrein without significant change in renin releases. These findings suggest that the release of renin by aldosterone may result from synergic effects of renal PG and the kallkrein-kinin system.     

Isolation of an enzymatically active tissue kallikrein from human seminal plasma by immunoaffinity chromatography

A tissue kallikrein from human seminal plasma was isolated by immunoaffinity chromatography and characterized. Its molecular mass was determined by gel filtration to be approximately 40000 Da. The enzyme preparation liberates kinin from human HMW kininogen (specific activity: 0.594 HMW kininogen-U/mg), lowers the blood pressure of dogs after intravenous injection (specific activity: 1740 biol. kallikrein unit/mg) and is strongly inhibited by aprotinin but not by soybean trypsin inhibitor. N alpha-Acetyl-L-phenylalanyl-L-arginine ethyl ester, D-valyl-L-leucyl-L-agrine ethyl ester and N-benzyloxycarbonyl-L-tyrosine p-nitrophenyl ester are cleaved with identical rates by the enzyme from human seminal plasma and human urinary kallikrein.     

Bradykinin (B2 independent effect of captopril on the development of pressure overload cardiac hypertrophy

Besides the reduction of angiotensin II formation, locally increased kinins may play a role in the cardiovascular action of angiotensin converting enzyme (ACE) inhibitors.To characterize the contribution of bradykinin to the effects of ACE inhibition by captopril on the development of pressure overload hypertrophy, sham-operated rats and rats with ascending aortic constriction were treated with captopril (80 mg/kg/day) or captopril and B₂-kinin receptor antagonist HOE 140 (0.5 mg/kg/day) for 7 weeks. Left ventricular mass and geometry, hydroxyproline concentration and myosin isozymes (marker of a fetal phenotype) were assessed. Rats with aortic constriction exhibited a marked increase in left ventricular weight and diastolic pressure-volume relationship was shifted to smaller volumes. Signs of congestive heart failure were not apparent. The hydroxyproline concentration remained unaltered. However, the proportion of isomyosin V3 was increased (p < 0.05). Administration of captopril reduced (p < 0.05) systolic blood pressure, body and cardiac weight in all treated rats. The reduction of left ventricular weight was disproportionally higher in pressure overloaded rats, thus the relative left ventricular weight decreased by 15% (p < 0.05). Captopril augmented the isomyosin V1 expression (p < 0.05) in sham operated as well as pressure overloaded rats. The isomyosin V1 percentage was inversely related to the relative left ventricular weight. Two different (p < 0.05) correlation lines were detected for untreated and captopril treated rats. None of captopril associated effects were removed by simultaneously administered B₂ kinin receptor antagonist HOE 140.Thus, stimulation of bradykinin B2 receptor appears not to mediate the effects of captopril on cardiac growth and contractile proteins during the development of pressure overload hypertrophy.     

Tissue kallikrein infusion prevents cardiomyocyte apoptosis, inflammation and ventricular remodeling after myocardial infarction

We investigated the effect of tissue kallikrein infusion on cardiac protection at acute and sub-acute phases after myocardial infarction (MI). Immediately after MI, rats were infused with purified tissue kallikrein, with or without icatibant (a kinin B2 receptor antagonist). Intramyocardial injection of kallikrein reduced myocardial infarct size and inhibited cardiomyocyte apoptosis at 1 day after MI associated with increased nitric oxide levels, Akt and glycogen synthase kinase-3beta phosphorylation and decreased caspase-3 activation. Kallikrein infusion for 7 days improved cardiac function, normalized left ventricular wall thickness and decreased monocyte/macrophage infiltration in the infarct heart. Kallikrein treatment reduced NADH oxidase expression and activity, superoxide formation and malondialdehyde levels, and reduced MAPK and Ikappa-Balpha phosphorylation, NF-kappaB activation and MCP-1 and VCAM-1 expression. Kallikrein's effects were all blocked by icatibant. These results indicate that kallikrein through kinin B2 receptor activation prevents apoptosis, inflammation and ventricular remodeling by increased nitric oxide formation and suppression of oxidative stress-mediated signaling pathways.     

Adenovirus-mediated human prostasin gene delivery is linked to increased aldosterone production and hypertension in rats

Prostasin has been demonstrated to be an activator of epithelial sodium channels in cultured renal and bronchial epithelial cells. In this study, we evaluated the effects of adenovirus-mediated gene transfer of human prostasin on blood pressure regulation and sodium reabsorption in Wistar rats. Expression of human prostasin mRNA was identified in rat adrenal gland, liver, kidney, heart, lung, and aorta, and immunoreactive human prostasin was detected in the circulation and urine of rats receiving prostasin gene transfer. A single injection of adenovirus carrying the prostasin gene caused prolonged increases in blood pressure for 3-4 wk. Blood pressure increase was accompanied by elevated plasma aldosterone levels and reduced plasma renin activity. The increase in blood pressure and plasma aldosterone levels as well as the reduction of plasma renin activity correlated with the expression of human prostasin transgene. Elevated plasma aldosterone levels were detected at 3 days after gene transfer before the development of hypertension, indicating that stimulation of mineralocorticoid production is the primary target of prostasin. Prostasin gene transfer significantly reduced urinary K(+) excretion but increased urinary Na(+) and kallikrein excretion. Elevated renal kallikrein levels promote natriuresis, which may lead to sodium escape and prevent further increases of blood pressure after prostasin gene transfer. In summary, these results suggest that prostasin participates in blood pressure and electrolyte homeostasis by regulating the renin-angiotensin-aldosterone and kallikrein-kinin systems.     

Induction of vascular leakage and blood pressure lowering through kinin release by a serine proteinase from Aeromonas sobria

Aeromonas sobria causes septic shock, a condition associated with high mortality. To study the mechanism of septic shock by A. sobria infection, we examined the vascular leakage (VL) activity of A. sobria serine proteinase (ASP), a serine proteinase secreted by this pathogen. Proteolytically active ASP induced VL mainly in a bradykinin (BK) B(2) receptor-, and partially in a histamine-H(1) receptor-dependent manner in guinea pig skin. The ASP VL activity peaked at 10 min to 1.8-fold of the initial activity with an increased BK B(2) receptor dependency, and attenuated almost completely within 30 min. ASP produced VL activity from human plasma apparently through kallikrein/kinin system activation, suggesting that ASP can generate kinin in humans. Consistent with the finding that a major part of the ASP-induced VL was reduced by a potent kallikrein inhibitor, soybean trypsin inhibitor that does not affect ASP enzymatic activity, ASP activated prekallikrein but not factor XII to generate kallikrein in a dose- and incubation time-dependent manner. ASP produced more VL activity directly from human low m.w. kininogen than high m.w. kininogen when both were used at their normal plasma concentrations. Intra-arterial injection of ASP into guinea pigs lowered blood pressure specifically via the BK B(2) receptor. These data suggest that ASP induces VL through prekallikrein activation and direct kinin release from kininogens, which is a previously undescribed mechanism of A. sobria virulence and could be associated with the induction of septic shock by infection with this bacterium. ASP-specific inhibitors, and kinin receptor antagonists, might prove useful for the treatment or prevention of this fatal disease.