Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.24.3 (collagenase)
18,340 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The effects of 7-ketocholesterol on rat hepatocytes prepared by collagenase perfusion were examined. The viability of cells incubated with 100 microM 7-ketocholesterol was significantly lower than those with cholesterol, although the LDH activity in the cultured medium remained unchanged during the incubation. Hepatocytes treated with 7-ketocholesterol produced large amounts of .NO and O2- in the early stage of incubation. Treatment of the hepatocytes with Carboxy-PTIO, which selectively scavenged .NO, or with L-NMMA, an inhibitor of .NO synthase, increased the cell viability. The addition of 7-ketocholesterol to the culture medium tended to increase the ratio of total sterol to phospholipid of the hepatocytes in a time-dependent manner without changing the content of phospholipid. No lipid peroxidation or oxidation of the cellular SH groups, protein SH and glutathione, was apparent. Vitamin E added 1 h before the addition of 7-ketocholesterol prevented the hepatocytes from cell death by suppressing the incorporation of 7-ketocholesterol into the hepatocytes and by scavenging O2-.
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PMID:Cytotoxicity of 7-ketocholesterol toward cultured rat hepatocytes and the effect of vitamin E. 898 32

Nitric oxide (NO) is synthesized in wounds, but its exact role and cellular source are not known. Wound fibroblasts (WF) are phenotypically characterized by increased collagen synthesis and contractility. We hypothesized that WF may be also phenotypically altered during wound healing to synthesize NO. WF were isolated from polyvinyl alcohol sponges implanted in male Lewis rats and harvested 10 days later. Proliferation in response to 10% fetal bovine serum was assessed by [3H]thymidine incorporation in a microculture system. A fibroblast-populated collagen lattice was used for assaying contractility. Collagen synthesis was determined by measuring the collagenase-sensitive fraction of protein-incorporated [3H]proline. Fibroblasts were incubated in the presence or the absence of 0.5 mM S-methyl-isothio-uronium or 0.5 mM N-monomethyl-L-arginine, both competitive inhibitors of NO synthase. WF spontaneously synthesize and release NO (4.60 +/- 0.29 nmol nitrite/microg DNA/48 h). Normal dermal fibroblasts do not synthesize NO. WF NO synthesis was limited to the first and second passages postharvest and was inhibitable by S-methyl-isothio-uronium (96%) and N-monomethyl-L-arginine (84%). In vivo iNOS expression by WF was confirmed by in situ hybridization and immunohistochemistry. Inhibition of endogenous NO synthesis had no effect on fibroblast proliferation. However, fibroblast-mediated collagen contraction was enhanced (p < 0.01), and collagen synthesis was significantly decreased (p < 0.05) by inhibiting NO synthase. The data show that WF are phenotypically altered during the healing process to synthesize NO, which, in turn, regulates their collagen synthetic and contractile activities.
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PMID:Nitric oxide, an autocrine regulator of wound fibroblast synthetic function. 903 87

Although intra-abdominal sepsis is known to impair colon healing by inhibiting anastomotic collagen synthesis, the effect of systemic sepsis on this process is unknown. Endotoxins and cytokines associated with sepsis induce nitric oxide synthesis both systemically and locally within colonic tissue. We hypothesized that systemic sepsis impairs colonic healing and examined a possible correlation with nitric oxide expression. Male Sprague-Dawley rats received intraperitoneal injections of either saline (sham group) or Escherichia coli endotoxin (lipopolysaccharide 1 mg/100 g body weight) at Times -24 and -12 hr (LPS group). All animals underwent laparotomy and left colonic anastomosis at Time 0. At 24 and 96 hr postlaparotomy rats were sacrificed, the anastomoses excised, and [3H]-proline incorporation into protein measured as an index of total new protein synthesis (TNP). Digestion with purified collagenase yielded incorporation into the collagen fraction (CDP). Additional sham and LPS-treated rats were sacrificed at 24, 72, and 120 hr, the anastomoses excised, and nitric oxide synthase activity in the tissue measured by the conversion of [3H]-arginine to [3H]citrulline in an ex vivo culture system. Finally, sham and LPS rats were sacrificed at 120 hr for measurement of colon anastomotic bursting pressure. Systemic sepsis significantly impaired new collagen synthesis in anastomotic tissue at 24 hr compared to control samples (P < 0.02). No difference was noted at 96 hr. TNP synthesis was similar in both groups at 24 or 96 hr. Northern blot analysis confirmed a significant decrease in Type I and Type III collagen mRNA expression at 24 hr in septic rats. Anastomotic bursting pressure was also decreased in the septic group (P < 0.003). Sepsis elevated nitric oxide synthase activity in anastomotic tissue 24 hr postanastomosis, when compared to sham tissue (P < 0.0001). These data suggest that systemic endotoxin induces nitric oxide synthesis at the anastomotic site. The simultaneous dysregulation of collagen gene expression and synthesis with decreased anastomotic strength suggests a possible regulatory role for nitric oxide in gastrointestinal healing.
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PMID:Sepsis impairs anastomotic collagen gene expression and synthesis: a possible role for nitric oxide. 920 51

Lipopolysaccharide (LPS) plays a key role in the pathogenesis of sepsis. Cardiac function and the inotropic response to beta-adrenergic stimulation are impaired in sepsis. We hypothesized that LPS, in clinically relevant levels (1 ng/mL), directly depresses contractility and beta-adrenergic responses in cardiac myocytes. Cardiac myocytes were isolated from the left ventricle of adult rabbits using digestive enzymes (collagenase and protease). We depyrogenated the enzymes (LPS contamination lowered from 100 to 300 ng/mL to < 0.7 ng/mL) to minimize development of LPS tolerance during cell isolation. After 6 hours of incubation with 1 ng/mL LPS, there was a decrease in the extent of active cell shortening with no change in Ca2+ transients (measured with indo 1 fluorescence), indicating decreased myofilament responsiveness to Ca2+. This was related to NO pathways, since cGMP (a second messenger of NO) increased in cardiac myocytes and LPS effects were completely reversed with a 1 mmol/L NG-monomethyl-L-arginine (L-NMMA, a NO synthase inhibitor). LPS did not alter the intracellular Ca2+ response to beta-adrenergic stimulation with isoproterenol but attenuated the contractile response (maximal cell shortening, 15.5 +/- 1.0% versus 23.3 +/- 1.1% in control myocytes; P < .001). LPS attenuation of the contractile response to isoproterenol was restored completely by L-NMMA and almost completely restored (to 86% of the control response) by an inhibitor of cGMP-dependent protein kinase. We conclude that LPS depresses cardiac contractility and the contractile response to beta-adrenergic stimulation by a NO-cGMP-mediated decrease in myofilament responsiveness to Ca2+. The direct effects of low levels of LPS on cardiac myocytes may contribute to cardiac depression and hemodynamic decompensation during sepsis.
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PMID:Lipopolysaccharide depresses cardiac contractility and beta-adrenergic contractile response by decreasing myofilament response to Ca2+ in cardiac myocytes. 940 Mar 82

We studied the expression of candidate molecules for tissue injury in vasculitic neuropathies immunohistochemically, using samples obtained by nerve biopsy from seven patients with necrotizing angitis. In the involved vessels of all samples, numerous infiltrating cells were positive for perforin, nitric oxide synthase (m-NOS), cyclooxygenase-2 (COX-2), or matrix metalloproteinase-1 (MMP-1). Cell-mediated cytotoxicity may be involved in the pathogenesis of small vessel injury in vasculitic neuropathies. In the endoneurium following axonal degeneration, scattered and phagocytosing macrophages showed immunostaining for m-NOS and MMP-1, but COX-2 was all but restricted to phagocytosing macrophages. This suggests a role for prostaglandins in nerve damage.
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PMID:Mechanisms of tissue injury in vasculitic neuropathies. 948 78

Interleukin-1 beta (IL-1) is implicated in cartilage destruction in arthritis through promotion of matrix metalloproteinase production. Upregulation of collagenase gene expression by IL-1 is known to require the transactivators Fos and Jun. Recently, reactive oxygen species (ROS) have been suggested to act as intracellular signaling molecules mediating the biological effects of cytokines. Here, we demonstrated ROS production by IL-1-stimulated bovine chondrocytes and that neutralizing ROS activity by the potent antioxidant, N-acetylcysteine, or inhibiting endogenous ROS production by diphenyleneiodonium (DPI), significantly attenuated IL-1-induced c-fos and collagenase gene expression. The inhibitory effect of DPI implicates enzymes such as NADPH oxidase in the endogenous production of ROS. Chondrocytes were also found to produce nitric oxide (NO) upon IL-1 stimulation. That NO may mediate part of the inducing effects of IL-1 was supported by the observation that L-NG-monomethylarginine, a NO synthase inhibitor, partially inhibited IL-1-regulated collagenase expression. Moreover, treatment of chondrocytes with the NO-producing agent, S-nitroso-N-acetylpenicillamine, was sufficient to induce collagenase mRNA levels. In summary, our results suggest that ROS released in response to IL-1 may function as second messengers transducing extracellular stimuli to their targets in the nucleus, leading to augmentation of gene expression.
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PMID:Interleukin-1 beta induction of c-fos and collagenase expression in articular chondrocytes: involvement of reactive oxygen species. 951 43

Nitric oxide (NO) has been shown to be a mediator of hypoxic injury in rat renal proximal tubules (PT). However, the role of NO in hypoxic injury to mouse. PT has not been examined. The aim of the present study was to determine the effect of knockout of nitric oxide synthase (NOS) isoforms on hypoxic injury in mouse PT. Mouse PTs were isolated by collagenase digestion and Percoll centrifugation. The nonselective NOS inhibitor, N-nitro-L-arginine methyl ester (L-NAME, 10 mM), but not its inactive stereoisomer D-NAME, protected against hypoxic injury as assessed by LDH release. Carboxy-imidazolineoxyl N-oxide (carboxy-PTIO, 100 microM), a stable NO scavenger, also afforded cytoprotection against hypoxic injury. To determine the role of the different NOS isoforms in the hypoxic injury, we examined the effect of hypoxia on PT isolated from knockout mice in which either the inducible NOS (iNOS) endothelial NOS (eNOS) or neuronal NOS (nNOS) gene was lacking. PT isolated from iNOS knockout mice were resistant to hypoxic injury compared to wild-type controls. In contrast, PT isolated from both nNOS and eNOS knockout mice were not protected against hypoxic injury. In conclusion, the present study demonstrates that NO is a mediator of hypoxic PT injury in the mouse and that knockout of the iNOS gene is cytoprotective against this hypoxic PT injury.
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PMID:Effect of hypoxia on proximal tubules isolated from nitric oxide synthase knockout mice. 960 95

The mechanism by which nitric oxide inhibits the incorporation of [3H]thymidine into rat articular chondrocytes (AC) in culture was studied. First-passage articular chondrocytes, isolated by collagenase digestion of cartilage fragments from humeral and femoral heads of 1- and 18-month-old rats, were used in all experiments. NO-generating compounds, isosorbide dinitrate or sodium nitoprusside, inhibited the incorporation of [3H]thymidine and the release of prostaglandin E2 (PGE2) and stimulated cyclic guanosine monophosphate (cGMP) production by rat AC monolayers in a concentration-dependent manner. The cells from old rats were much less sensitive to NO donors and also produced less PGE2 and cGMP. Blocking the production of endogenous NO with NG-monomethyl-L-arginine (L-NMA), an inhibitor of NO synthase, stimulated DNA synthesis. cGMP was found to be a key mediator of the inhibition of DNA synthesis by NO donors in rat AC. 6-Anilino-5,8-quinolinedione (LY83583), an inhibitor of NO-dependent cGMP release, stimulated [3H]-thymidine incorporation, whereas the cGMP analog, 8- bromo-cGMP, inhibited L-NMA-induced or LY83583-induced stimulation of [3H]thymidine incorporation. NO donors blocked the stimulation of DNA synthesis induced by L-NMA and only marginally blocked that of LY83583. Indomethacin had no effect on the inhibition of DNA synthesis by NO or 8-bromo-cGMP. These results show that NO donors induce inhibition of DNA synthesis probably by elevating cGMP. The relative insensitivity of senescent cells to NO donors may be due, at least in part, to their decreased capacity to produce cGMP.
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PMID:The mechanism of inhibition of DNA synthesis in articular chondrocytes from young and old rats by nitric oxide. 970 83

Isolated hepatocyte suspensions prepared by collagenase perfusion released high levels of nitrite into the extracellular medium during an 8-hr incubation. The release was time dependent, with increases first occurring by 4 hr and continuing throughout the remainder of the incubation period. Nitrite production was inhibited by the nitric oxide synthase (NOS) inhibitors aminoguanidine and N(G)-nitro-L-arginine methyl ester (L-NAME), indicating that the nitrite is derived from nitric oxide (NO) production from NOS activity. Nitrite production was not related to bacterial or Kupffer cell contamination. The protein synthesis inhibitor cycloheximide and the transcription inhibitor actinomycin D also prevented nitrite production by parenchymal hepatocytes. Calcium-independent NOS enzyme activity increased with incubation times, and this increase coincided with the observed increases in nitrite production. Our results suggest that NOS is induced following the isolation of hepatocytes, and this induction results in the formation of high levels of NO.
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PMID:Time-dependent production of nitric oxide by rat hepatocyte suspensions. 1023 Jul 65

Inflammatory cytokines play a major role in cartilage destruction in diseases such as osteoarthritis and rheumatoid arthritis. Because physical therapies such as continuous passive motion yield beneficial effects on inflamed joints, we examined the intracellular mechanisms of mechanical strain-mediated actions in chondrocytes. By simulating the effects of continuous passive motion with cyclic tensile strain (CTS) on chondrocytes in vitro, we show that CTS is a potent antagonist of IL-1 beta actions and acts as both an anti-inflammatory and a reparative signal. Low magnitude CTS suppresses IL-1 beta-induced mRNA expression of multiple proteins involved in catabolic responses, such as inducible NO synthase, cyclo-oxygenase II, and collagenase. CTS also counteracts cartilage degradation by augmenting mRNA expression for tissue inhibitor of metalloproteases and collagen type II that are inhibited by IL-1 beta. Additionally, CTS augments the reparative process via hyperinduction of aggrecan mRNA expression and abrogation of IL-1 beta-induced suppression of proteoglycan synthesis. Nonetheless, the presence of an inflammatory signal is a prerequisite for the observed CTS actions, as exposure of chondrocytes to CTS alone has little effect on these parameters. Functional analysis suggests that CTS-mediated anti-inflammatory actions are not mediated by IL-1R down-regulation. Moreover, as an effective antagonist of IL-1 beta, the actions of CTS may involve disruption/regulation of signal transduction cascade of IL-1 beta upstream of mRNA transcription. These observations are the first to show that CTS directly acts as an anti-inflammatory signal on chondrocytes and provide a molecular basis for its actions.
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PMID:Cyclic tensile strain acts as an antagonist of IL-1 beta actions in chondrocytes. 1086 Oct 84


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