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)

Nitric oxide (NO) is a messenger molecule that is produced from L-arginine by NO synthase (NOS). Some NOS isoforms are present in cells constitutively, whereas others can be induced by cytokines. Recent evidence suggests that NO inhibits intracellular pH regulation by the vacuolar H(+)-adenosinetriphosphatase (ATPase) in macrophages, which contain an inducible form of NOS. The vacuolar H(+)-ATPase is involved in proton secretion in intercalated cells in the collecting duct. We have therefore examined the effect of NO on bafilomycin-sensitive H(+)-ATPase activity in individual cortical collecting ducts (CCD) microdissected from collagenase-treated kidneys of normal rats using a fluorometric microassay. Incubation of CCD with the NO donors, sodium nitroprusside (0.1 and 1 mM) or 3-morpholino-sydnonimine hydrochloride (SIN-1, 30 microM), caused a dose-dependent decrease in H(+)-ATPase activity. Incubation of CCD with lipopolysaccharide (LPS) and interferon-gamma, which induces NOS in macrophages, decreased H(+)-ATPase activity by 85%. This effect was prevented by simultaneous incubation with N omega-nitro-L-arginine, a competitive inhibitor of NOS, indicating that the decrease in H(+)-ATPase activity was caused by NO production. Incubation with 8-bromo-guanosine 3',5'-cyclic monophosphate (cGMP) also inhibited H(+)-ATPase activity, suggesting that NO may exert its effect in the CCD via activation of guanylyl cyclase and production of cGMP. Immunohistochemistry using antibodies to the macrophage-type NOS revealed strong labeling of intercalated cells in the CCD, confirming the presence of NOS in these cells.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Nitric oxide inhibits bafilomycin-sensitive H(+)-ATPase activity in rat cortical collecting duct. 752 55

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

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

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

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

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

Cytokine-induced expression of inducible nitric oxide synthetase (iNOS) and production of nitric oxide (NO) by pancreatic islet cells has been suggested as one potential mechanism for beta cell destruction. In this study, we investigated the role of iNOS and NO in islet primary non-function. Islets were assessed for their function, viability and expression of iNOS. Adult rat and pig islets isolated by collagenase digestion and fetal pig pancreas (FPP) grafts isolated by collagenase digestion or high oxygen culture were transplanted into C57BL6 mice and nude mice. iNOS protein was detected by immunohistochemistry. iNOS protein was found in normal rat and pig pancreas and adult rat and pig islets that were isolated by collagenase digestion and transplanted into either C57BL6 mice or nude mice. iNOS was not detected in fetal pig islet grafts, regardless of whether collagenase was used in the isolation process. In adult pig islet grafts, the presence of iNOS protein correlated with high levels of islet cell apoptosis and primary non-function. Despite the persistent presence of iNOS in rat islets, there was no evidence that it had a deleterious effect on rat islet viability, or function. Therefore, in isolated adult pig islets, there was a correlation between iNOS expression and apoptosis, suggesting that iNOS activation may be deleterious to the adult pig islets. However, other factors such as the fragility of the islet capsule may be equally important. By contrast, fetal pig islets did not express iNOS and this may be an important reason for their enhanced viability when compared with adult islet tissue.
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PMID:Inducible nitric oxide synthetase is expressed in adult but not fetal pig pancreatic islets. 1102 65

Prostaglandins participate in the regulation of sodium and water renal excretion. They are synthesized by cyclooxygenases (COX): the constitutive isoform and the enzyme regulated by physiological stimuli (COX-2). Our previous immunohistochemical studies have demonstrated the presence of COX-2 in a subset of thick ascending limb (TAL) of Henle cells and its induction during the postnatal period and after adrenalectomy. Previous results suggested that this induction phenomenon proceeds by recruitment of TAL cells from the cortex to the outer medulla. The present work aimed to specifically address these preliminary observations by using immunohistochemical techniques in single microdissected nephron segments. Normal adult rats, adrenalectomized rats, adrenalectomized rats on dexamethasone and 5, 10, and 15 days postnatal age were used (Sprague-Dawley rats, n= 5 each group). Glomeruli and different segments of nephron were microdissected from collagenase-treated kidney tissue. Tubules were immunostained with specific antibodies against COX-2. We confirmed that COX-2 was localized exclusively in TAL segments; it was induced after adrenalectomy and during postnatal age, peaking at 15 days after birth. We provided morphological evidence that the induction of COX-2 along TAL proceeded in a defined pattern by recruitment of cells from the cortical portion close to the glomeruli toward the outer medulla. No COX-2 was observed in the post-macula densa portion of the segments. Our results provide the anatomical basis for the contribution of COX-2 in physiological mechanisms such as renin secretion, tubuloglomerular feedback, and the interaction with neuronal NO synthase at the juxtaglomerular apparatus.
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PMID:Renal cyclooxygenase-2: evidence for recruitment of thick ascending limb of henle cells in microdissected nephron segments. 1156 45

Since uterine cervical ripening is an active biochemical process similar in part to an inflammatory reaction, nitric oxide (NO) has been proposed as a key mediator of this event. However, the mechanism by which NO modulates human cervical ripening has not been fully elucidated. In the present study we investigated the presence of NO synthases in human uterine cervix by immunohistochemistry and reverse transcriptase-polymerase chain reaction analysis. Furthermore, we examined the presence of NO-mediated regulation of matrix metalloproteinase-1 (MMP-1) production in cultured human uterine cervical fibroblast cells using enzyme-linked immunosorbent assay and Northern blot analysis. Endothelial and inducible NO synthases were detected in the form of mRNA and protein expression in pregnant uterine cervix. Interleukin-1alpha (IL-1alpha) increased the expression of inducible NO synthase mRNA in cultured human uterine cervical fibroblast cells. IL-1alpha also increased MMP-1 secretion from the cultured cervical fibroblast cells. This IL-1alpha-augmented MMP-1 secretion was partially but significantly blocked by an NO synthase inhibitor, N(G)-nitro-L-arginine methyl ester. On the other hand, NO donors increased MMP-1 production in the cultured cervical fibroblast cells. These findings together suggest that NO contributes to the process of cervical ripening via enhancement of MMP-1 production, and that IL-1alpha increases MMP-1 secretion from cervical fibroblasts at least in part via NO synthesis.
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PMID:Nitric oxide increases matrix metalloproteinase-1 production in human uterine cervical fibroblast cells. 1157 67


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