Caspase I-related protease inhibition retards the execution of okadaic acid- and camptothecin-induced apoptosis and PAI-2 cleavage, but not commitment to cell death in HL-60 cells.
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We have previously reported that the putative cytoprotective protease inhibitor, plasminogen activator inhibitor type 2 (PAI-2), is specifically cleaved during okadaic acid-induced apoptosis in a myeloid leukaemic cell line (Br J Cancer (1994) 70: 834-840). HL-60 cells exposed to okadaic acid and camptothecin underwent morphological and biochemical changes typical of apoptosis, including internucleosomal DNA fragmentation and PAI-2 cleavage. Significant endogenous PAI-2 cleavage was observed 9 h after exposure to okadaic acid; thus correlating with other signs of macromolecular degradation, like internucleosomal DNA fragmentation. In camptothecin-treated cells, PAI-2 cleavage was an early event, detectable after 2 h of treatment, and preceding internucleosomal DNA fragmentation. The caspase I selective protease inhibitor, YVAD-cmk, inhibited internucleosomal DNA fragmentation and PAI-2 cleavage of okadaic acid and camptothecin-induced apoptotic cells. YVAD-cmk rather sensitively and non-toxically inhibited camptothecin-induced morphology, but not okadaic acid-induced morphology. In in vitro experiments recombinant PAI-2 was not found to be a substrate for caspase I. The results suggest that caspase I selective protease inhibition could antagonize parameters coupled to the execution phase of okadaic acid- and camptothecin-induced apoptosis, but not the commitment to cell death. (+info)
Kallidin- and bradykinin-degrading pathways in human heart: degradation of kallidin by aminopeptidase M-like activity and bradykinin by neutral endopeptidase.
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BACKGROUND: Since kinins kallidin (KD) and bradykinin (BK) appear to have cardioprotective effects ranging from improved hemodynamics to antiproliferative effects, inhibition of kinin-degrading enzymes should potentiate such effects. Indeed, it is believed that this mechanism is partly responsible for the beneficial effects of angiotensin-converting enzyme (ACE) inhibitors. In the heart, enzymes other than ACE may contribute to local degradation of kinins. The purpose of this study was to investigate which enzymes are responsible for the degradation of KD and BK in human heart tissue. METHODS AND RESULTS: Cardiac membranes were prepared from the left ventricles of normal (n=5) and failing (n=10) hearts. The patients had end-stage congestive heart failure as the result of coronary heart disease or idiopathic dilated cardiomyopathy. Heart tissue was incubated with KD or BK in the presence or absence of enzyme inhibitors. We found no difference in the enzymes responsible for kinin metabolism or their activities between normal and failing hearts. Thus KD was mostly converted into BK by the aminopeptidase M-like activity. When BK was used as substrate, it was converted into an inactive metabolite BK-(1-7) mostly (80% to 90%) by the neutral endopeptidase (NEP) activity, with ACE unexpectedly playing only a minor role. The low enzymatic activity of ACE in the cardiac membranes, compared with that of NEP, was not due to chronic ACE inhibitor therapy, because the cardiac ACE activities of patients, whether receiving ACE inhibitors or not, and of normal subjects were all equal. CONCLUSIONS: The present in vitro study shows that in human cardiac membranes, the most critical step in kinin metabolism, that is, inactivation of BK, appears to be mediated mostly by NEP. This observation suggests a role for NEP in the local control of BK concentration in heart tissue. Thus inhibition of cardiac NEP activity could be cardioprotective by elevating the local concentration of BK in the heart. (+info)
Clearance of human brain natriuretic peptide in rabbits; effect of the kidney, the natriuretic peptide clearance receptor, and peptidase activity.
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Although the synthetic version of the cardiac peptide human brain natriuretic peptide (hBNP) has demonstrated beneficial cardiovascular effects in clinical studies, little is known about mechanisms governing its elimination from the blood. This study measured the role of the kidney, the natriuretic peptide clearance (NP-C) receptor, and peptidase digestion on the elimination of synthetic hBNP from the plasma compartment of rabbits. The estimated plasma steady state resulting from a continuous i.v. infusion was achieved within 50 min and was related in a linear manner with the infusion rate of the drug. Complete restriction of kidney blood flow by bilateral suture-ligation of the renal arteries compared with sham-treated animals reduced the clearance of hBNP by approximately half (24 +/- 9 ml/min versus 47 +/- 14 ml/min, respectively, p <. 007). Pharmacological blockade of the NP-C receptor with a clearance receptor-specific analog of atrial natriuretic peptide increased in a statistically significant and dose-related manner the plasma steady-state level of hBNP during continuous i.v. infusion of hBNP (maximum effect of 1.9 +/- 0.3-fold, p <.01). The peptidase inhibitor phosphoramidon increased in a dose-related manner the plasma steady-state level of hBNP 1.7 +/- 0.4-fold during continuous i.v. infusion of hBNP in rabbits. These data suggest that the kidney, the NP-C receptor, and peptidases are all important in the elimination of hBNP from the plasma compartment. (+info)
Endothelin-1 is induced by cytokines in human vascular smooth muscle cells: evidence for intracellular endothelin-converting enzyme.
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Endothelin-1 (ET-1) is the predominant endothelin isopeptide generated by the vascular wall and therefore appears to be the most important peptide involved in regulation of cardiovascular events. Many pathologic conditions are associated with elevations of ET-1 in the blood vessel wall. Because these conditions are often cytokine driven, we examined the effects of a mixture of cytokines on ET-1 production in human vascular smooth muscle cells (VSMCs) derived from internal mammary artery and saphenous vein (SV). Incubation of IMA and SV VSMCs with tumor necrosis factor-alpha (10 ng/ml) and interferon-gamma (1000 U/ml) in combination for up to 48 h markedly elevated the expression of mRNA for prepro-ET-1 and the release of ET-1 into the culture medium. This cytokine-stimulated release of ET-1 was inhibited by a series of dual endothelin-converting enzyme (ECE)/neutral endopeptidase inhibitors, phosphoramidon, CGS 26303, and CGS 26393, with an accompanying increase in big ET-1 release but with no effect on expression of mRNA for prepro-ET-1. These same compounds were 10 times more potent at inhibiting the conversion of exogenously applied big ET-1 to ET-1. ECE-1b/c mRNA is present in SV VSMCs, however no ECE-1a is present in these cells. Thus VSMCs most probably contain, like endothelial cells, an intracellular ECE responsible for the endogenous synthesis of ET-1. Under the influence of pro-inflammatory mediators the vascular smooth muscle can therefore become an important site of ET-1 production, as has already been established for the dilator mediators nitric oxide, prostaglandin I2, and prostaglandin E2. (+info)
Inhibition of the activities of matrix metalloproteinases 2, 8, and 9 by chlorhexidine.
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Matrix metalloproteinases (MMPs) are a host cell-derived proteolytic enzyme family which plays a major role in tissue-destructive inflammatory diseases such as periodontitis. The aim of the present study was to evaluate the inhibitory effect of chlorhexidine (CHX) on MMP-2 (gelatinase A), MMP-9 (gelatinase B), and MMP-8 (collagenase 2) activity. Heat-denatured type I collagen (gelatin) was incubated with pure human MMP-2 or -9 activated with p-aminophenylmercuric acetate (APMA), and the proteolytic degradation of gelatin was monitored by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Coomassie blue staining. The effect of CHX on MMP-8 activity was also studied with a cellular model addressing the ability of phorbol myristate acetate (PMA)-triggered human peripheral blood neutrophils (polymorphonuclear leukocytes [PMNs]) to degrade native type I collagen. CHX inhibited the activities of both gelatinases (A and B), but MMP-2 appeared to be more sensitive than MMP-9. Adding calcium chloride to the assay mixtures almost completely prevented the inhibition of MMP-9 activity by CHX, while the inhibition of MMP-2 activity could be reversed only when CHX was used at a low concentration. This observation suggests that CHX may act via a cation-chelating mechanism. CHX dose-dependently inhibited collagenolytic activity of MMP-8 released by PMA-triggered PMNs. MMP-8 without APMA activation was inhibited clearly more efficiently than APMA-activated MMP-8. Our study suggests that the direct inhibition of the MMPs' activities by CHX may represent a new valuable effect of this antimicrobial agent and explains, at least in part, the beneficial effects of CHX in the treatment of periodontitis. (+info)
Tissue inhibitor of metalloproteinase-2 stimulates mesenchymal growth and regulates epithelial branching during morphogenesis of the rat metanephros.
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Development of the embryonic kidney results from reciprocal signaling between the ureteric bud and the metanephric mesenchyme. To identify the signaling molecules, we developed an assay in which metanephric mesenchymes are rescued from apoptosis by factors secreted from ureteric bud cells (UB cells). Purification and sequencing of one such factor identified the tissue inhibitor of metalloproteinase-2 (TIMP-2) as a metanephric mesenchymal growth factor. Growth activity was unlikely due to TIMP-2 inhibition of matrix metalloproteinases because ilomastat, a synthetic inhibitor of these enzymes, had no mesenchymal growth action. TIMP-2 was also involved in morphogenesis of the ureteric bud, inhibiting its branching and changing the deposition of its basement membrane; these effects were due to TIMP-2 inhibition of matrix metalloproteinases, as they were reproduced by ilomastat. Thus, TIMP-2 regulates kidney development by at least 2 distinct mechanisms. In addition, TIMP-2 was secreted from UB cells by mesenchymal factors that are essential for ureteric bud development. Hence, the mesenchyme synchronizes its own growth with ureteric morphogenesis by stimulating the secretion of TIMP-2 from the ureteric bud. (+info)
Reactive oxygen species activate a Ca2+-dependent cell death pathway in the unicellular organism Trypanosoma brucei brucei.
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Here we examine a cell death process induced by reactive oxygen species (ROS) in the haemoflagellate Trypanosoma brucei brucei. Ca2+ distribution in cellular compartments was measured with stable transformants expressing aequorin targeted to the cytosol, nucleus or mitochondrion. Within 1.5 h of ROS production, mitochondrial Ca2+ transport was impaired and the Ca2+ barrier between the nuclear envelope and cytosol was disrupted. Consequently the mitochondrion did not accumulate Ca2+ efficiently in response to an extracellular stimulus, and excess Ca2+ accumulated in the nucleus. The terminal transferase deoxytidyl uridine end labelling assay revealed that, 5 h after treatment with ROS, extensive fragmentation of nuclear DNA occurred in over 90% of the cells. Permeability changes in the plasma membrane did not occur until an additional 2 h had elapsed. The intracellular Ca2+ buffer, EGTA acetoxymethyl ester, prevented DNA fragmentation and prolonged the onset of changes in cell permeability. Despite some similarities to apoptosis, nuclease activation was not a consequence of caspase 3, caspase 1, calpain, serine protease, cysteine protease or proteasome activity. Moreover, trypanosomes expressing mouse Bcl-2 were not protected from ROS even though protection from mitochondrial dysfunction and ROS have been reported for mammalian cells. Overall, these results demonstrate that Ca2+ pathways can induce pathology in trypanosomes, although the specific proteins involved might be distinct from those in metazoans. (+info)
The pathway regulating MDM2 protein degradation can be altered in human leukemic cells.
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The MDM2 protein regulates the functional activity of the p53 tumor suppressor through direct physical association. Signals that control MDM2 expression are poorly understood but are likely to play an important role in the regulation of p53 activity. We show here that the half-life of MDM2 protein is shorter in proliferating than in quiescent peripheral blood mononuclear cells. We also demonstrate that MDM2 protein half-life is extended in some, but not all, p53 mutant human leukemic cell lines. In at least one of these p53 mutant lines, increased MDM2 protein stability is associated with higher amounts of MDM2 protein. Moreover, we demonstrate that MDM2 protein accumulates to a much greater extent in proteasome inhibitor-treated cells containing unstable MDM2 than in cells possessing stable MDM2. These results demonstrate that MDM2 expression is regulated by events that control the stability of the protein and suggest that the normal regulation of MDM2 turnover can be altered in tumor cell lines. (+info)