Substrate specificity and inhibition of human kallikrein-related peptidase 3 (KLK3 or PSA) activated with sodium citrate and glycosaminoglycans.

We report the enzymatic properties and substrate specificity of human recombinant KLK3 in the presence of glycosaminoglycans (GAGs) and sodium citrate. This salt is highly concentrated in prostate and in its presence KLK3 had a similar hydrolytic efficiency as chymotrypsin. In contrast to the latter peptidase, KLK3 activated by sodium citrate efficiently hydrolyzed substrates containing R, H and P at the P1 position. Activated KLK3 also cleaved peptides derived from the bradykinin domain of human kininogen at the same sites as human kallikrein KLK1, but presented low kininogenase activity. Angiotensin I has several sites for hydrolysis by KLK3; however, it was cleaved only at the Y-I bond (DRVY downward arrowIHPFHL). Sodium citrate modulated KLK3 conformation as observed by alterations to the intrinsic fluorescence of phenylalanines and tryptophans. Activated KLK3 was reversibly inhibited by Z-Pro-Prolinal and competitively inhibited by ortho-phenantroline. Together, these are noteworthy observations for the future design of specific non-peptide inhibitors of KLK3 and to find natural substrates.

Expression of endogenous nuclear bradykinin B2 receptors mediating signaling in immediate early gene activation.

Bradykinin (BK) represents a pro-inflammatory mediator that partakes in many inflammatory diseases. The mechanism of action of BK is thought to be primarily mediated by specific cell surface membrane B2 receptors (B2Rs). Some evidence has suggested, however, the existence of an intracellular/nuclear B2R population. Whether these receptors are functional and contribute to BK signaling remains to be determined. In this study, by mean of Western blotting, 3D-confocal microscopy, receptor autoradiography and radioligand binding analysis, we showed that plasma membrane and highly purified nuclei from isolated rat hepatocytes contain specific B2R that bind BK. The results depicting B2R nuclear expression in isolated nuclear organelles were reproduced in situ on hepatic sections by immunogold labeling and transmission electron microscopy. Functional tests on single nuclei, by means of confocal microscopy and the calcium-sensitive probe fluo-4AM, showed that BK induces concentration-dependent transitory mobilization of nucleoplasmic calcium; these responses were blocked by B2R antagonist HOE 140, not by the B1R antagonist R954 and, were also found in wild-type C57/Bl6 mice, but not in B2R-KO mice. In isolated nuclei, BK elicited activation/phosphorylation of Akt, acetylation of histone H3 and ensuing pro-inflammatory iNOS gene induction as determined by Western blot and RT-PCR. ChIP assay confirmed binding of acetylated-histone H3 complexes, but not B2R, to promoter region of iNOS gene suggesting that B2R-mediated gene expression is bridged with accessory downstream effectors. This study discloses a previously undescribed mechanism in BK-induced transcriptional events, via intracrine B2R-mediated signaling, occurring in rat autologous hepatic cells.

Rat tissue kallikrein releases a kallidin-like peptide from rat low-molecular-weight kininogen.

The kallikrein-kinin system is subdivided into the plasma and tissue-kallikrein-kinin system, with bradykinin (BK) and kallidin (KAL) (Lys(0)-bradykinin) as functional peptides. This occurs in both humans and other mammals. Both peptides are released by plasma and tissue-kallikrein. BK, but not KAL, has been detected in rats until now. One can explain this observation by the structural differences found in the sequence of rat high- and low-molecular kininogen containing an Arg-residue instead of a Lys-residue in front of the N-terminus of the BK sequence. Nevertheless, we were able to measure a kallidin-like peptide (KLP), in rat plasma and urine, using a specific KAL antiserum. In order to confirm our data, we isolated low-molecular-weight kininogen from rat plasma and incubated it with purified rat glandular kallikrein. The generated peptide was retained on a high-pressure liquid chromatography column and displaced by an excess of angiotensin I. The KLP-containing fraction was identified with the KLP radioimmunoassay. A specific ion signal with a mass to charge ratio (m/z) of 1216.73 was detected with matrix-assisted laser desorption/ionization mass spectrometry. As proposed earlier, the structure of this peptide is Arg(1)-KAL, instead of Lys(1)-KAL. The structural similarity between the Lys- and the Arg-residue explains the high crossreactivity (80%) of KLP with the specific KAL antibody. The incubation of KLP with angiotensin-converting enzyme yields two molecules with masses of 913.4 and 729.3 containing the sequence H-Arg-Arg-Pro-Pro-Gly-Phe-Ser-Pro-OH and H-Arg-Arg-Pro-Pro-Gly-Phe-OH. The enzymatic cleavage could be inhibited by captopril. The data suggest that in rats, as in other mammals, the tissue kallikrein-kinin system mediates its physiological effects via a kallidin-like peptide, which is Arg(1)-kallidin (Arg(0)-bradykinin).

Stimulated human neutrophils form biologically active kinin peptides from high and low molecular weight kininogens.

Human neutrophils play a pivotal role in acute inflammation. However, their capacity to generate bioactive kinin peptides has not been established as yet. We have examined the ability of neutrophil enzymes to release biologically active kinins in vitro from purified human H- and L-kininogens. Neutrophils isolated from human blood were stimulated with f-Met-Leu-Phe, thrombin, or human immunoglobulin G adsorbed to silica particles. Supernatants were incubated with iodinated kininogens, and polyacrylamide gel electrophoresis analyzed aliquots taken after a range of incubation times. A time-course analysis demonstrated that supernatants from stimulated neutrophils caused a rapid hydrolysis of both substrates, resulting in an accumulation of fragments ranging from 20 to less than 10 kDa. Radioimmunoassay (RIA) revealed that all supernatants were able to generate kinins in vitro. High-performance liquid chromatography of the generated peptides indicated that they had a retention time similar to that of bradykinin and Met-Lys-bradykinin, clearly recognized as kinin peptides when the corresponding fractions were tested by RIA. The kinin-immunoreactive fractions produced lowering of blood pressure and a dramatic increase in venular permeability. Biological activity of the neutrophil-generated kinins was completely abolished by the B2 receptor antagonist HOE140, indicating that over the time-course of the experiments, only kinin B2 agonists appeared to have been generated and that cellular actions of these were mediated by kinin B2 receptors. Together, our results demonstrate that human neutrophil proteases can release kinins from both plasma kininogens, suggesting that these peptides may participate actively during acute inflammation.

Lipopolysaccharide injection into the cerebral ventricle evokes kininogen induction in the rat brain.

Kinins, such as bradykinin and Lys-bradykinin, are important mediators in peripheral inflammation. Although the existence of the components necessary for generating kinins has been demonstrated in the brain, a functional role of the kinin-generating system in cerebral inflammation remains to be defined. The aim of the present study was to elucidate whether inflammatory stimuli alter the mRNA levels of components for the kallikrein-kinin system, including kallikreins, kininogens and bradykinin type 2 (B(2)-) receptor in rat brain using the reverse transcription polymerase chain reaction. The intracerebroventricular (i.c.v.) injection of lipopolysaccharide (LPS; 0.25 microg/animal) resulted in the elevation of T-kininogen and high-molecular-weight (H-) kininogen mRNAs in various brain regions within 24 h, prominently in the choroid plexus. The appearance of immunoreactive T-kininogen was demonstrated in the epithelium of the choroid plexus, but not in the matrix and vessels, after i.c.v. injection of LPS. The mRNA levels of kallikreins, such as tissue kallikrein, T-kininogenase and plasma kallikrein, and B(2)-receptor did not change in any brain region following i.c.v. injection of LPS. The levels of cyclooxygenase-2 mRNA in the choroid plexus were increased within 2 h after i.c.v. injection of LPS, and pretreatment with indomethacin (3 microg/animal, i.c.v.) abolished the LPS-induced elevation of T- and H-kininogen mRNAs in the choroid plexus. The i.c.v. injection of prostaglandin E(2) (100 ng/animal) also caused increases in the mRNA levels of T- and H-kininogens in various brain regions, including the choroid plexus. These results suggest that LPS stimulates the induction of kininogens in the brain, especially the choroid plexus, by stimulating the production of arachidonic metabolites such as prostaglandin E(2).

Immunohistochemical distributions of the tissue kallikrein-kinin system in ischemic and non-ischemic mouse heart.

Kinins have been shown to play a cardioprotective role during myocardial ischemia. However, the localization of each of the components of the kallikrein-kinin system in the heart has not been determined in a cell type-specific manner. Recently, mK1 has been identified as the major tissue kallikrein with the strongest bradykinin-forming activity among the products of the mouse tissue kallikrein gene superfamily. In the study presented here, we investigated the localizations of mK1, kininogen and bradykinin B2 receptors (B2Rs) in ischemic and non-ischemic left ventricles by immunohistochemistry. Kininogen, which contains bradykinin as a surface epitope, was detected by an anti-bradykinin antibody. Changes in the amounts of mK1 and B2R were evaluated by Western blot analysis. Myocardial ischemia was induced by ligation of the left anterior descending coronary artery for 60 min followed by reperfusion for 24 h. mK1 and B2Rs were most abundantly expressed in the vascular endothelium and, to a lesser extent, in fibroblasts. No immunohistochemical signal of these molecules was detected in myocytes. Kininogen was localized in the vascular endothelium and the smooth muscle layer. Myocardial ischemia, although it had no effect on the localization of these molecules, increased the amounts of mK1 and B2R. We have obtained immunohistochemical evidence that all components of the tissue kallikrein-kinin system are present in the mouse heart. The coronary artery is the major site of kallikrein-kinin activity both in ischemic and non-ischemic hearts.

Release of a new vascular permeability enhancing peptide from kininogens by human neutrophil elastase.

Stimulated neutrophils produced vascular permeability enhancing (VPE) activity in the presence of high molecular weight kininogen (HMWK), which was inhibited mainly by a neutrophil elastase (NE) inhibitor or a bradykinin (BK) B(2)-receptor antagonist. NE (>3 nM) generated VPE activity from kininogens at normal plasma concentrations with the smaller protein being several fold more responsive than the larger protein, through releasing a new VPE peptide (E-kinin), SLMKRPPGFSPFRSSRI. Synthetic E-kinin, SLMKRPPGFSPFRSS and SLMKRPPGFSPFR had VPE and blood pressure lowering activities, which were comparable to the activities of BK and completely inhibited by B(2)-receptor antagonists. Interestingly, E-kinin and SLMKRPPGFSPFRSS did not induce smooth muscle contraction. These results suggest that E-kinin formed in vivo may be processed at the carboxy-terminus to give a peptide that can bind to the B(2)-receptor. The molecular mechanism for neutrophil-associated VPE may be explained by excision of E-kinin from kininogens by NE, followed by further processing of the peptide.

Cathepsin L, but not cathepsin B, is a potential kininogenase.

Although papain-like enzymes are strongly inhibited by their natural tight-binding inhibitors of the cystatin superfamily, cathepsins B and L may still retain some residual proteolytic activity toward Z-Phe-Arg-AMC in the presence of an excess of kininogen. This activity is abolished by adding E-64 or chicken cystatin. Cathepsins B and L show a single band of gelatinolytic activity when subjected to gelatin-SDS-PAGE. Adding high Mr kininogen, low Mr kininogen, T-kininogen, or chicken cystatin to cathepsin L results in additional intense bands of enzyme activity corresponding to the protease-inhibitor complexes. Cathepsin B does not produce these additional bands. This gelatinolytic activity was inhibited by E-64, but not by EDTA, PMSF or Pefabloc. Cathepsin L also specifically generated kinins from high and low molecular weight kininogens in vitro, but cathepsin B did not. T-kininogen did not release any immunoreactive kinins when complexed with cathepsin L, as previously observed using tissue kallikreins. The ability of cathepsin L to generate vasoactive peptides raises the question of the physiological significance of this mechanism during inflammation.