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 early biochemical events that link interleukin-1 (IL-1) receptor occupancy to neutral proteinase production in synovial cells were studied. Addition of human r-IL-1 to human synovial cells in culture stimulated phospholipase A2 (PLA2) activity, inositol triphosphate production and plasminogen activator (PA) activity in a dose dependent manner with similar EC50 values (0.1-0.5 nM). These results, coupled with time courses and other studies, suggest that the IL-1 modulation of PA involves both products of PLA2 and phospholipase C (PLC) activation. On the other hand, the IL-1 induction of collagenase may primarily involve PLC and protein kinase C activation.
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PMID:Interleukin-1 mediated signal transduction associated with synovial cell activation. 267 53

Intrapancreatic activation of proteases is believed to play a major role in the pathogenesis of acute necrotizing pancreatitis. Several authors have questioned, however, the central role of trypsin in autodigestion of the pancreas. To clarify the direct effects of pancreatic enzymes and other related factors on acinar cells, we used the model of isolated pancreatic acini. Acini were prepared from male Wistar rats by collagenase digestion. Protein synthesis was measured by incubation of acini with [35S]methionine. Acini were resuspended thereafter in fresh buffer and further incubated for 30-90 min under various conditions [e.g., with pancreatic homogenates, ascites (from rats with pancreatitis induced by sodium taurocholate), pure pancreatic enzymes, and other factors]. The percentage of release of newly synthesized proteins into the culture medium was regarded as a biochemical parameter of cellular integrity. A morphologic score of cellular integrity was obtained via light microscopic evaluation of acini at the end of the various incubations by measuring the degree of cell lysis, loss of cell granules, ballooning, formation of vacuoles, and karyopyknosis. When normal [35S]methionine-labeled pancreatic acini were incubated with various factors, the percentage of release of labeled proteins into the medium was as follows: incubation with HEPES/Ringer's buffer, 1.8%; hemorrhagic pancreatic ascites, 3.8%; pancreatic homogenates, 2.0%; lipase, 1.8%; phospholipase A2, 3.0%; phospholipase A2 + lecithin, 3.2%; trypsin, 2.5%; 5% olive oil, 1.8%; ascites + olive oil, 78.3%; ascites + homogenized epididymal fat, 79.9%; lipase + olive oil, 32.0%; pancreatic homogenates + olive oil, 28.0%; diolein, 2.65%; and oleic acid, 62.9%. The cellular release of radiolabeled proteins showed an inverse correlation with cellular integrity as shown by light microscopy. We postulate that interstitial release of degradation products from triglycerides by lipase causes cellular disruption. Whereas phospholipase A2 and proteases do not seem to be very harmful in the early phases of cellular damage, lipase may play a major role in acute necrotizing pancreatitis.
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PMID:Role of pancreatic enzymes and their substrates in autodigestion of the pancreas. In vitro studies with isolated rat pancreatic acini. 291 45

Macrophages, which produce the collagenolytic enzyme collagenase, are commonly found at sites of connective tissue destruction in chronic inflammatory lesions. Since tissue macrophages are derived from circulating peripheral blood monocytes, we used these less-differentiated, more readily available cells to examine the production and regulation of collagenase. Human monocytes, isolated in large quantities by counterflow centrifugal elutriation, were shown to produce substantial amounts of collagenase when stimulated by concanavalin A (Con A) and to a lesser extent with lipopolysaccharide, while unstimulated monocyte cultures produced negligible collagenase. Collagenase was detected in the culture media within the first 24 hr of culture after activation with peak production at 48 hr. Analysis of the intracellular regulation of collagenase revealed that synthesis of this enzyme required a prostaglandin (PGE2)-dependent step since indomethacin-inhibited enzyme synthesis was reversed by PGE2. Additionally, dibutyryladenosine cyclic monophosphate (dBcAMP) restored collagenase synthesis in indomethacin-blocked cultures, indicating a PGE2-dependent generation of cAMP requirement for collagenase production similar to that demonstrated in experimental animals systems. In additional studies, anti-inflammatory drugs which are known to modulate connective tissue destruction were analyzed for their influence on monocyte-derived collagenase. Dexamethasone, colchicine or retinoic acid all inhibited collagenase synthesis by monocytes in a dose-dependent manner although the effect of these drugs on monocyte PGE2 synthesis differed. Dexamethasone inhibited PGE2 synthesis, which resulted in the suppression of collagenase. However, PGE2 production was unaffected by colchicine whereas retinoic acid caused a significant increase in PGE2 levels. Inhibition of collagenase synthesis by dexamethasone, but not colchicine or retinoic acid, could be reversed by PGE2 or phospholipase A2. These findings provide insight into the intracellular events regulating monocyte collagenase synthesis and also implicate monocytes as a target of anti-inflammatory agents which ameliorate connective tissue degradation associated with chronic inflammatory lesions.
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PMID:Regulation of human peripheral blood monocyte collagenase by prostaglandins and anti-inflammatory drugs. 303 63

Guinea-pig gastric mucosal cells isolated by collagenase and pronase digestion were used to study the release of prostanoids prostaglandin I2 (PGI2; measured as 6-keto PGF1 alpha), PGE2, PGF2 alpha and thromboxane A2 (TXA2; measured as TXB2). Lysophosphatide acyltransferase (LAT) and phospholipase A2 (PLA2) were measured in the microsomal fraction of isolated but not separated gastric cells and isolated and enriched parietal and mucous cells. In all cell preparations PLA2 activity was approximately 5 times higher than that of LAT. Acid-activated omeprazole inhibited LAT in a concentration-dependent manner with similar IC50 values in gastric, parietal and mucous cells. It had no effect on PLA2. Gastric cells constantly produced PGI2, PGE2, PGF2 alpha and TXA2. The main prostaglandins released were PGI2 and PGE2. PGF2 alpha and TXA2 were released in smaller quantities. Omeprazole dissolved in polyethylene glycol 400 (PEG) pH 2 inhibited spontaneous PGI2 release in a concentration-dependent manner with an IC50 of 14.3 +/- 4.8 microM. Only concentrations as high as 100 microM produced a significant reduction in PGE2 release by 60%. No significant changes could be detected in the spontaneous release of PGF2 alpha and TXA2. Omeprazole dissolved in PEG pH 7 had no effect on PGI2 release except at 100 microM which led to an insignificant decrease by 40%. These data suggest that omeprazole beyond its inhibitory effect on parietal cell K+/H+-ATPase also affects gastric mucosal prostanoid formation and release. The inhibitory effect on PGI2 does not support the view that omeprazole protects the gastric mucosa by increasing prostanoid formation.
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PMID:Effect of omeprazole on eicosanoid formation in and release from guinea-pig gastric mucosal cells. 329 28

Hepatocyte isolated by collagenase perfusion of livers of male Fischer-344 rats, and treated with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) (50 microM for 30 min at 37 degrees C) to inhibit glutathione reductase, were significantly more vulnerable to cytotoxicity of the bipyridyl herbicide diquat than similarly treated cells of Sprague-Dawley rats. Without compromise of cell defenses by BCNU, diquat was not cytotoxic to hepatocytes from either strain. Microsomal enzyme induction with phenobarbital (80 mg/kg ip for 3 days before hepatocyte isolation) did not potentiate killing of Fischer hepatocytes by diquat. Specific activities of NADPH-cytochrome P-450 reductase in isolated Fischer and Sprague-Dawley rat liver microsomes utilizing 1 mM diquat as acceptor were 0.085 +/- 0.017 and 0.076 +/- 0.028 mumol/mg.min (mean +/- SEM, N = 5), respectively, indicating the capacity for very active redox cycling of diquat by this route in both strains. The serine protease inhibitor, phenylmethylsulfonyl fluoride (100 microM), had no effect on diquat cytotoxicity, but both leupeptin (100 micrograms/ml) and antipain (50 or 100 microM) were able to delay, through not completely prevent, diquat-induced cell death. The phospholipase inhibitors, chlorpromazine (50 or 100 microM) and dibucaine (50 or 100 microM), similarly delayed but did not prevent cell death. Diquat increased the rate of hepatocyte phospholipid hydrolysis, measured as release into the suspending medium of [14C]arachidonic acid previously incorporated into hepatocyte lipids, but although chlorpromazine decreased phospholipid hydrolysis to the control rate, only partial protection against diquat cytotoxicity was seen. These data suggest that activation of phospholipase A2 and proteases by elevation of cytosolic free Ca2+ cannot account entirely for the loss of cell viability observed in the presence of cytotoxic concentrations of diquat.
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PMID:Lethal injury by diquat redox cycling in an isolated hepatocyte model. 342 16

An enriched fraction of human decidual cells that synthesizes and releases human PRL (hPRL) was obtained by isopycnic centrifugation of collagenase- and hyaluronidase-dispersed cells through Percoll. The cells that synthesized and released hPRL banded at a density of 1.017-1.045 g/ml, an area of the gradient comprising only a small percentage of the total decidual DNA. The enriched cells formed distinct colonies in culture and contained hPRL, as evidenced by indirect immunofluorescent staining with anti-PRL serum. Plated at a density of 5.0 x 10(5) cells/well, the cells produced hPRL at a mean rate of 8.1 +/- 1.1 ng/microgram DNA . 24 h (mean +/- SD) for 8 days. Like decidual explants, the enriched cells responded to phospholipase A2 (0.1 U/ml) with a 54% decrease in hPRL release and to placental conditioned medium (0.5 mg protein/ml medium) with a 62% increase. Insulin (8.3 x 10(-7) M), progesterone (10(-5) - 10(-12) M), and estradiol (10(-5) - 10(-12) M) did not affect hPRL release over 6 days. These results indicate that enriched PRL-releasing cells, obtained by the isopycnic centrifugation of collagenase- and hyaluronidase-dispersed cells, are a useful model for the study of the synthesis and release of PRL.
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PMID:Characterization of the synthesis and release of prolactin by an enriched fraction of human decidual cells. 630 Jan 79

An enriched fraction of human placental cells that synthesize and release both placental lactogen (hPL) and hCG was obtained by isopycnic centrifugation of collagenase/hyaluronidase-dispersed cells through a density gradient of 40% Percoll. The enriched cells, which banded at a density of approximately 1.01 g/ml, comprised 10-15% of the total DNA. During the first 24 h after attachment, the cells released 50-250 ng hPL and 4-10 mIU hCG/10(6) cells. Thereafter, the rate of hPL release decreased, while the rate of hCG and [35S]trichloroacetic acid-precipitable protein release remained constant. The enriched cells responded to phospholipase A2, low extracellular calcium, and (Bu)2cAMP in a manner similar to that of placental explants. Phospholipase A2 (0.1 and 1 U/ml) stimulated hPL release by 270% and 568%, respectively, and low extracellular calcium (0-0.18 mM) stimulated hPL release by 48%. (Bu)2 cAMP (1 mM) stimulated hCG release by 42%, but had no effect on hPL. Estradiol (10(-5)-10(-12) M) and progesterone (10(-5)-10(-10) M) had no effect on the synthesis and release of either hPL or hCG over a 6-day period. In addition, insulin (8.3 X 10(-7) M) and changes in medium glucose content (0-5 mg/ml) had no effect on hPL release over a 72-h period. Since the enriched trophoblast cells respond to provocative stimuli in a manner similar to that of explants and placental fragments, this cell population is a useful model system for investigations of the cellular mechanisms of hPL and hCG release.
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PMID:Characterization of the synthesis and release of human placental lactogen and human chorionic gonadotropin by an enriched population of dispersed placental cells. 630 87

Previous studies have demonstrated that exposure of guinea pig macrophages to a primary signal, such as lipopolysaccharide (LPS), stimulates the synthesis of prostaglandin E2 (PGE2) which, in turn, elevates cAMP levels resulting in the production of the enzyme, collagenase. The potential of regulating the biochemical events in this activation sequence was examined with the anti-inflammatory agents dexamethasone and colchicine, which suppress the destructive sequelae in chronic inflammatory lesions associated with the degradation of connective tissue. The addition of dexamethasone with LPS to macrophage cultures resulted in a dose-dependent inhibition of PGE2 and collagenase production, which was reversed by the exogenous addition of phospholipase A2. Collagenase production was also restored in dexamethasone-treated cultures by the addition of products normally produced as a result of phospholipase action, such as arachidonic acid, PGE2 or dibutyryl-cAMP. Since the effect of dexamethasone was thus linked to phospholipase A2 inhibition, mepacrine, a phospholipase inhibitor, was also tested. Mepacrine, like dexamethasone, caused a dose-dependent inhibition of PGE2 and collagenase. In addition to corticosteroid inhibition, colchicine was also found to block collagenase production. However, this anti-inflammatory agent had no effect on PGE2 synthesis. Colchicine was effective only when added at the onset of culture and not 24 h later, implicating a role for microtubules in the transmission of the activation signal rather than enzyme secretion. The failure of lumicolchicine to inhibit collagenase activity provided additional evidence that microtubules are involved in the activation of macrophages. These findings demonstrate that dexamethasone and colchicine act at specific steps in the activation sequence of guinea pig macrophages to regulate collagenase production.
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PMID:Regulation of guinea pig macrophage collagenase production by dexamethasone and colchicine. 632 92

Active digestive enzymes are involved in the pathophysiology of acute pancreatitis. Previous studies have mainly focused on the role of trypsin in the autodigestive process. The present study compares the noxious potential of different pancreatic enzymes to damage acinar cells. Acinar cells were isolated from rat pancreas by collagenase digestion. Cell viability was studied by (1) exclusion of trypan blue, (2) release of lactate dehydrogenase, and (3) release of newly synthesized proteins identified with methionine labeled with sulfur 35. Cells were then incubated in oxygenated N-2-hydroxyethylpiperazine-N-'-2-ethanesulfonic acid-Ringer solution containing different concentrations of various active digestive enzymes. Uptake of trypan blue was the most sensitive and reliable test of cell damage when compared with release of lactate dehydrogenase or radiolabeled newly synthesized proteins. All active digestive enzymes studied caused dose-dependent cell damage. The noxious potential, however, was strikingly different for the various enzymes. Pancreatic elastase in nanomolar concentrations caused marked cell damage after 45 to 90 minutes of incubation. Lipase and chymotrypsin caused a similar damage only at micromolar concentrations, whereas even millimolar concentrations of trypsin failed to cause significant damage. The present results confirmed recent work showing that lipase and phospholipase A2 probably cause cell damage through release of free fatty acids and lysolecithin. Although activation of trypsin might be the trigger to start the activation cascade in acute pancreatitis, trypsin itself is markedly less noxious to acinar cells when compared with other digestive enzymes. Elastase by far had the greatest noxious potential of all enzymes evaluated. Studies analyzing therapeutic effects of protease inhibitors should evaluate not only the inhibitory potential against trypsin but also that against other digestive enzymes, particularly elastase.
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PMID:Active pancreatic digestive enzymes show striking differences in their potential to damage isolated rat pancreatic acinar cells. 784 75

Lysophosphatidylcholine (LPC) increases extracellularly during ischemia in vivo in both animals and man as judged by measurements from venous effluents, but more recent studies have shown little or no increase in buffer-perfused, isolated heart preparations. The appearance of LPC in blood and lymph in animals and in venous effluents in man in response to ischemia suggests a vascular site for the production of LPC. The present study was performed to assess whether thrombin could stimulate phospholipase A2 in endothelial cells and whether this would evoke an increase in and release of LPC. Endothelial cells were disassociated from canine aortas by incubating with 0.1% collagenase for 20 min. Cells were plated and allowed to grow to confluence. Measurement of LPC was performed using Bligh and Dyer extraction of lipids, high performance liquid chromatography separation, and quantification of LPC using a recently developed radiometric assay employing [3H]acetic anhydride. Incubation of endothelial cells with thrombin (0.05 unit/ml) resulted in a 2.5-fold increase in LPC to 2.3 +/- 0.1 nmol/mg of protein at 2 min (p < 0.01) and returned to control levels within 20 min. The increase in LPC induced by thrombin exhibited a concentration-dependent response with an ED50 = 0.04 unit/ml. A concentration-dependent increase in LPC was also elicited by stimulation with the peptide portion of the thrombin receptor's tethered ligand SFLLRNPNDKYEPF with an ED50 = 8 microM. The LPC produced was rapidly and completely released into the surrounding media. Hirudin completely blocked the thrombin-induced increase in LPC. Dansylarginine N-(3-ethyl-1,5-pentanediyl)amide (0.1 microM), which rapidly inactivates thrombin's proteolytic activity in situ without impairing binding, or phenyl-prolyl-arginyl-chloromethyl ketone (PPACK, 5 nM), which inactivates thrombin due to chemical alteration of the proteolytic site, each prevented the increase in LPC in response to thrombin. Stimulation of protein kinase C with phorbol 12-myristate-13-acetate (PMA, 1 microM) enhanced the response to thrombin. In contrast, staurosporine (100 nM), H7 (15 microM), or chronic treatment with PMA for 20 h to down-regulate protein kinase C completely prevented the increase in LPC in response to thrombin. Thus, thrombin stimulation of endothelial cells in vivo during ischemia may be a primary mechanism contributing to the marked increase in LPC extracellularly during ischemia.
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PMID:Thrombin-induced release of lysophosphatidylcholine from endothelial cells. 839 49


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