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)

Pulmonary arterial microvascular smooth muscle function governs many aspects of lung physiology and pathophysiology. Acutely, microvascular smooth muscle cells (SMC) modulate pulmonary vascular resistance; chronically, they contribute to vascular remodeling. Recent work has also suggested a possible immune function for pulmonary smooth muscle through cytokine-stimulated nitric oxide production. To facilitate study of the mechanisms underlying these functions, we have developed methods for isolating pulmonary arterial microvessels from the rat and culturing SMC from these vessels. The pulmonary arterial circulation was filled with a suspension of iron oxide in agar, and a subpleural tissue sample was obtained. The vessels were cleared of surrounding lung parenchyma by partial collagenase digestion, and the iron-containing arteries were separated magnetically. The diameter of the harvested arteries confirmed an intraacinar origin, and the cultured cells expressed smooth muscle isoforms of alpha-actin and myosin but did not take up acetylated low density lipoprotein. To assess a possible immune effector role for these cells, confluent monolayers were stimulated with cytokines and endotoxin. At 24 h, immunofluorescent staining for inducible nitric oxide synthase was prominent within these cells. Nitric oxide production, as measured by nitrite levels in the cell-conditioned medium, was also markedly elevated but reduced by adding NG-monomethyl-L-arginine. We conclude that rat pulmonary arterial microvascular SMC can be obtained by the iron oxide infusion method and that these cells express an inducible nitric oxide synthase after cytokine stimulation.
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PMID:Culture of pulmonary microvascular smooth muscle cells from intraacinar arteries of the rat: characterization and inducible production of nitric oxide. 751 71

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

Nitric oxide is a highly reactive mediator released in the liver by hepatocytes, Kupffer cells and endothelial cells during endotoxin-induced inflammation. In this study we determined whether Ito cells also produce nitric oxide after exposure to endotoxin. For induction of endotoxemia, rats were injected intravenously with Escherichia coli lipopolysaccharide (2.5 mg/kg). Ito cells were isolated from the animals 48 hr later by means of in situ perfusion of the liver with protease and collagenase followed by purification on an arabinogalactan gradient. Ito cells from untreated and endotoxemic rats were found to produce low levels of nitric oxide in response to interferon-gamma. In both cell types, this response depended on L-arginine and was blocked by NG-monomethyl-L-arginine, a specific nitric oxide synthase inhibitor. Cells from rats treated with endotoxin produced significantly more nitric oxide than did cells from untreated animals; this was due, at least in part, to increased expression of protein for an inducible form of nitric oxide synthase. These cells also responded to stimulation with lipopolysaccharide in vitro, as well as the combination of interferon-gamma and lipopolysaccharide, which was synergistic in stimulating nitric oxide production. Tumor necrosis factor-alpha and macrophage colony-stimulating factor were also found to stimulate nitric oxide production by Ito cells from endotoxemic rats. In addition, in these cells, tumor necrosis factor-alpha synergized with interferon-gamma in inducing nitric oxide production. The combination of interferon-gamma and lipopolysaccharide was also found to inhibit Ito cell DNA synthesis, as measured on the basis of [3H]-thymidine uptake.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Induction of hepatic Ito cell nitric oxide production after acute endotoxemia. 752 4

Hepatic macrophages and endothelial cells play an important role in the clearance of endotoxin from the portal circulation. These cells are activated by endotoxin to release reactive mediators including superoxide anion, hydrogen peroxide, and nitric oxide, which have been implicated in hepatic inflammation and tissue injury. In the present studies we analyzed mechanisms regulating the production of nitric oxide by hepatic macrophages and endothelial cells following in vivo exposure to endotoxin. Rats were injected intravenously with Escherichia coli lipopolysaccharide (LPS, 5 mg/kg). Cells were isolated from the animals 48 h later by in situ perfusion of the liver with collagenase and pronase followed by differential centrifugation and centrifugal elutriation. We found that macrophages and endothelial cells from both untreated and endotoxin-treated rats readily synthesized nitric oxide following in vitro stimulation with interferon-gamma (IFN-gamma) and LPS alone and in combination. This response was dependent on l-arginine and was blocked by two nitric oxide synthase inhibitors, NG-monomethyl-l-arginine and l-canavanine. Macrophages produced more nitric oxide in response to LPS or LPS plus IFN-gamma than endothelial cells. In addition, nitric oxide production by both cell types in response to LPS plus IFN-gamma was increased after treatment of rats with endotoxin. Macrophages appeared to be more sensitive than endothelial cells to the in vivo effects of this inflammatory stimulus. Northern and Western blot analysis demonstrated that nitric oxide production by macrophages and endothelial cells in response to LPS plus IFN-gamma was due to increased expression of an inducible form of nitric oxide synthase (iNOS) mRNA and protein. Using fluorescence image analysis, iNOS protein was found to be localized in the cytoplasm of the cells. Treatment of rats with endotoxin was associated with increased expression of iNOS protein in the macrophages. The phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA) also stimulated nitric oxide production by macrophages and endothelial cells from endotoxin-treated rats, although not as effectively as LPS and IFN-gamma. Macrophages were more responsive than endothelial cells to TPA. Furthermore, depletion of the cells of glutathione using buthionine sulfoximine had no major effect on nitric oxide production by macrophages but resulted in small but significant inhibition in endothelial cells. This suggests that this sulfhydryl-containing tripeptide does not regulate intracellular levels of reactive nitrogen intermediates in activated macrophages.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Distinct patterns of nitric oxide production in hepatic macrophages and endothelial cells following acute exposure of rats to endotoxin. 752 31

Nitric oxide (NO.) is a multifunctional messenger molecule generated by a family of enzymes, collectively termed the nitric oxide synthases. We investigated the role of NO. in the modulation of two metal-dependent proteolytic enzymes (collagenase and stromelysin) which are activated during inflammatory and infective arthritis. The inflammatory mediators interleukin-1 beta (IL-1 beta), tumor necrosis factor-alpha (TNF-alpha) and the bacterial cell wall fragment endotoxin, induced both nitric oxide synthase activity and stromelysin and collagenase activity in whole cell preparations and in conditioned media from explants of bovine and human cartilage. Both NO2- (the stable end-product of NO.) and metalloprotease activity were inhibited by competitive inhibitors of nitric oxide synthase. The NO. donor, S-nitroso-N-acetyl-D,L-penicillamine (SNAP) also induced metalloprotease activity in a dose-dependent fashion. These data provide evidence that NO. plays a regulatory role in the activation of metal-dependent proteases in articular chondrocytes and cartilage.
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PMID:Nitric oxide activates metalloprotease enzymes in articular cartilage. 752 96

Galanin has numerous effects on gastrointestinal motility in different species; however, its cellular basis of action in mediating these effects is unclear. Dispersed gastric smooth muscle cells have been shown to possess high-affinity galanin receptors that increase cAMP and cause relaxation. Recent studies show some smooth muscle relaxants such as VIP cause relaxation by both cAMP-dependent and -independent mechanisms. It is unknown if galanin's cellular basis of relaxation is similar or different from that of VIP. To investigate galanin's relaxant effect and compare it to VIP's effect, dispersed smooth muscle cells from guinea pig stomach were prepared by collagenase digestion. The mean length in resting cells was 110 +/- 2 microns and, with carbachol treatment, contracted to 89 +/- 2 microns. VIP and galanin alone had no effect on cell length, but each caused a dose-dependent inhibition of carbachol-induced contraction and both had an EC50 of 3-7 nM. Galanin (1 microM) and VIP (1 microM) increased cellular cAMP from 118 +/- 10 pmol/10(6) cells in control to 212 +/- 14 and 214 +/- 12 pmol/10(6) cells, respectively. The protein kinase A inhibitor, Rp-cAMPS, at 100 microM, completely inhibited the relaxant effect of an EC50 concentration of galanin (3 nM), but only inhibited that by VIP by 80% (p < 0.05). Adding the nitric oxide inhibitor, L-NNA (NG-nitro-L-arginine), at 100 microM did not alter the length of resting cells or inhibit carbachol-induced contraction. However, L-NNA (100 microM) decreased VIP-induced relaxation by 45%, whereas it had no effect on galanin-induced relaxation.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Galanin-induced relaxation in gastric smooth muscle cells is mediated by cyclic AMP. 753 25

Immunohistochemical staining of endobronchial biopsies has identified increased expression of the 21-amino-acid peptide endothelin (ET) and the inducible form of the enzyme nitric oxide synthase (iNOS) within the airway epithelium in asthma. Elevated concentrations of ET are also recovered in bronchoalveolar lavage fluid from asthma patients. iNOS generates the gas nitric oxide from L-arginine, and elevated levels of NO in exhaled air have been described in asthma. ET is a potent bronchoconstrictor and levels of ET in lavage and resting airflow obstruction are correlated. The effects of ET on bronchomotor tone may be modified by NO as this is a bronchodilator. The relative balance between ET and NOS may thus contribute to resting bronchomotor tone. ET also stimulates fibroblast proliferation, collagen gene expression and through its inhibitory actions on collagenase will promote airway wall collagen deposition and contributes to airway wall thickening which underlies bronchial hyperresponsiveness. The regulation of these epithelial events may thus be important to the control of asthma.
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PMID:Epithelially derived endothelin and nitric oxide in asthma. 754 73

We cultured smooth muscle cells as explants from rat mesenteric arterioles (40-200 microns in diameter) obtained by injecting a suspension of iron oxide intraarterially and magnetically separating the arterioles after collagenase digestion of adventitial tissue. In third-passaged cells we ascertained smooth muscle purity of > 98% by characteristic morphology, contraction responses, and specific immunofluorescence staining. Treatment of growth-arrested (in 0.4% fetal calf serum) cells with platelet-derived growth factor (0.3-7.5 nM) or angiotensin II (0.001-1000 nM) induced 3H-thymidine incorporation and cell proliferation in a dose-dependent manner (P < 0.01). S-nitroso-N acetylpenicillamine (0.05-0.5 mM), a nitric oxide-generating compound, inhibited 10% fetal calf serum-induced 3H-thymidine incorporation (P < 0.05) and cell proliferation (P < 0.01). The antimitogenic effect of S-nitroso-N-acetylpenicillamine was significantly reduced by hemoglobin and potentiated by superoxide dismutase (P < 0.01). In addition to a new technique for culturing mesenteric arteriolar smooth muscle cells, these findings provide evidence that platelet-derived growth factor, angiotensin II, and nitric oxide may be involved in their growth control.
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PMID:Culture of rat mesenteric arteriolar smooth muscle cells: effects of platelet-derived growth factor, angiotensin, and nitric oxide on growth. 811 39

Treatment of rats with bacterially derived lipopolysaccharide (LPS), a condition that mimics acute endotoxemia, results in a significant increase in the number of endothelial cells and macrophages in the liver. This is correlated with the release of proinflammatory and cytotoxic mediators that induce liver damage. In the present studies, we analyzed the effects of various inflammatory mediators released during the pathogenesis of hepatic injury on proliferation of liver nonparenchymal cells. To induce acute endotoxemia female Sprague-Dawley rats were injected intravenously with 5 mg/kg LPS. Endothelial cells and macrophages were isolated 48 h later by combined collagenase and pronase perfusion of the liver followed by centrifugal elutriation. Interleukin-1 alpha (IL-1 alpha), interleukin-6 (IL-6), and tumor necrosis factor alpha (TNF-alpha) had no effect on proliferation of either endothelial cells or macrophages. In contrast, whereas interleukin-1 beta (IL-1 beta) inhibited the proliferation of endothelial cells from untreated rats, this cytokine stimulated the growth of cells from endotoxemic rats. The colony-stimulating factors, granulocyte-macrophage colony-stimulating factor (GM-CSF) and macrophage colony-stimulating factor (M-CSF), also markedly enhanced the proliferation of endothelial cells, as well as macrophages from endotoxemic rats. Macrophages from endotoxemic rats were more sensitive to the colony-stimulating factors than cells from untreated rats. In contrast, the inflammatory mediators LPS and interferon-gamma (IFN-gamma) inhibited endothelial cell and macrophage growth, an effect that was partially blocked in endothelial cells by the nitric oxide synthase inhibitor NG-monomethyl-L-arginine (L-NMMA). This suggests that growth inhibition in these cells is mediated, in part, by nitric oxide. Interestingly, in both endothelial cells and macrophages from endotoxemic rats, GM-CSF, M-CSF, and IL-1 beta synergized with LPS and IFN-gamma to induce nitric oxide production. This was correlated with a further inhibition of proliferation that was partially reversed by L-NMMA in endothelial cells but not macrophages. Taken together these data demonstrate that endothelial cell and macrophage proliferation in the liver is controlled by a variety of mediators released during endotoxemia; however, the mechanisms regulating growth in the two cell types are distinct.
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PMID:Regulation of hepatic endothelial cell and macrophage proliferation and nitric oxide production by GM-CSF, M-CSF, and IL-1 beta following acute endotoxemia. 814 21

Tumor necrosis factor (TNF alpha) has been shown to inhibit insulin release and it has been postulated to-be an important effector in islet rejection. We studied the effect of cryopreservation on glucose oxidation rate (GOR), lipid synthesis, hormone secretion (insulin, glucagon, somatostatin, thyrotropin-releasing hormone), and cyclic guanosine 3',5'-monophosphate (cGMP) content of human islets, in the presence or absence of TNF alpha, looking for changes that could explain a different susceptibility to rejection for cryopreserved islets. Islets were isolated from multiple organ donor pancreata by collagenase digestion. The islets were then cultured for 7 days, cryopreserved (-0.25 degrees C/min), and stored in liquid N2. After 24 h of culture, thawed islets were cultured for an other 24 h in the presence or absence of TNF alpha. Islets were then washed to remove the cytokine and incubated in Krebs-Ringer bicarbonate (5 or 20 mM glucose), and both the cGMP content of the islets and the hormone concentration in the medium were determined by radio-immunoassay. GOR was measured as the production of 14CO2 from 5 or 20 mM D-[U-14C]glucose, and de novo lipid synthesis was determined as D-[U-14C]glucose incorporation into different lipidic fractions. Cryopreservation did not significantly modify the hormone response to glucose but it partially reversed the TNF alpha-induced inhibitory effect on insulin release in the presence of 20 mM glucose. In addition, the inhibitory effect of TNF alpha on phosphatidylcholine labeling was attenuated in cryopreserved islets compared with noncryopreserved islets. TNF alpha significantly stimulated islet nitrite production and cGMP accumulation, both effects being of a similar magnitude in cryopreserved and noncryopreserved islets. Our results suggest that cryopreservation can modify the metabolic and hormone response of human islets to TNF alpha. This effect is not mediated by changes in the TNF alpha-induced islet nitric oxide production or cGMP accumulation.
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PMID:Influence of cryopreservation on the sensitivity of human islets to tumor necrosis factor. 878 31


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