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)

As part of an ongoing investigation of human mast cell heterogeneity, we have isolated, partially purified, and characterized the uterine mast cell and compared it with mast cells isolated from other organs. The average histamine content of myometrium and leiomyofibroma obtained from hysterectomies was 2.1 +/- 0.3 (mean +/- SEM) microgram/g of tissue (n = 10), and the histamine content of the two tissues did not differ significantly. A mild collagenase, hyaluronidase, and DNase digestion was used to disperse the uterine mast cells, with an average yield of 9.5% (range, 0 to 21%). The average histamine/uterine mast cell was 2.1 +/- 0.2 pg (n = 3), and 61 +/- 7% (n= 3) of the uterine mast cells survived overnight culture. Early purification efforts with Percoll gradients have yielded up to 80% pure uterine mast cells, with an average of 27 +/- 10% (n = 5). Uterine mast cells released histamine in response to the secretogogues anti-IgE and A23187 but did not respond to substance P or to the basophil secretogogues FMLP, C5a, and 12-O-tetradecanoylphorbol-13-acetate. After 1 microgram/ml anti-IgE stimulation, the uterine mast cell appeared to make significant quantities of PGD2 (89 +/- 26 ng/10(6) cells, n = 6) (p less than 0.05), as assayed by RIA. Simultaneously, leukotriene C4 release was 45 +/- 15 ng/10(6) cells, (n = 6) (p less than 0.05), as assayed by RIA. Combined gas-chromatography mass spectroscopy analysis of anti-IgE-stimulated cell supernatants confirmed the production of PGD2. In pharmacologic studies, isobutyl-methylxanthine and isoproterenol blocked anti-IgE-induced histamine release. The uterine mast cell is similar to the lung mast cell in terms of response to secretogogues and release of arachidonic acid metabolites. Ultrastructurally, the uterine mast cell contains scroll granules, crystal granules, combined granules, homogeneously dense granules, and large lipid bodies, many with focal lucencies within them. Particle granules, most frequently present in gut mast cells of mucosal origin, were absent from uterine mast cells. Although certain features are analogous to the ultrastructure of skin or lung mast cells, the combination of structures is distinctive for uterine mast cells.
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PMID:Human uterine mast cells. Isolation, purification, characterization, ultrastructure, and pharmacology. 171 65

A deficiency of luteal cell prostaglandin F2 alpha (PGF2 alpha) receptors might help explain the well documented refractoriness of pig corpora lutea to the luteolytic effects of PGF2 alpha administered in vivo before day 12 of the estrous cycle. Accordingly, experiments were conducted to measure the levels of [3H] PGF2 alpha-binding sites/receptors on collagenase-dispersed pig luteal cells taken at different stages of the estrous cycle. Pig corpora lutea were obtained surgically at various stages of the estrous cycle and dissociated with collagenase in medium 199. Dissociated mixed luteal cells (approximately 5-15 x 10(4) large luteal cells/tube) were assayed for [3H]PGF2 alpha-binding activity by saturation (Scatchard) analysis. In preliminary experiments it was determined that PGF2 alpha binding was maximal after incubation for 45 min at 30 C in assay buffer of pH 5.75. Additionally, it was determined that [3H]PGF2 alpha binding was displaceable by PGF2 alpha = PGD2 greater than PGE2 greater than 13,14-dihydro-15-keto-PGF2 alpha. Other eicosanoids did not inhibit [3H]PGF2 alpha binding. Two distinct classes of binding sites (high affinity Kd = 19-64 nM; low affinity Kd = 262-3103 nM) were observed at all stages of the estrous cycle. From studies using enriched (by elutriation) large (greater than 30 microns) and small (10-20 microns) luteal cells it appeared that the high affinity binding site was largely confined to large luteal cells, whereas the low affinity binding site was found on both large and small luteal cells. The concentrations (number per large luteal cell) of high affinity PGF2 alpha-binding sites of mixed (unelutriated) luteal cell preparations was low on days 6-8 (0.6 x 10(6) sites/cell) and increased gradually up to 1.4 x 10(6) sites/cell on day 12. The concentrations of binding sites were increased approximately 3-fold on day 13 (4.6 x 10(6) sites/cell; P less than 0.05 vs days 6-12) and remained elevated on days 14 and 16-17 (approximately 3 x 10(6) sites/cell). In summary, these results indicate the existence of a high affinity PGF2 alpha-binding site in pig (large) luteal cells, which is probably the luteal PGF2 alpha receptor. The numbers of these putative PGF2 alpha receptors are low during the early luteal phase (before day 12), but increase thereafter (days 13, 14, and 16-17). This may provide one explanation for the observed refractoriness in vivo of pig corpora lutea to PGF2 alpha before, and increased sensitivity to PGF2 alpha after, day 12 of the estrous cycle.
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PMID:Prostaglandin F2 alpha receptors on enzyme-dissociated pig luteal cells throughout the estrous cycle. 215 25

Intraluteal infusion of the prostaglandin (PG) synthesis inhibitor, sodium meclofenamate (Mec) causes premature luteolysis in rhesus monkeys. To evaluate further the actions of PG synthesis inhibitors in primate luteal function, we examined the in vitro effects of Mec and another inhibitor, flurbiprofen (Flur), on PG, cAMP, and progesterone (P) production by macaque luteal tissue obtained at midluteal phase of the menstrual cycle. First, collagenase dispersed luteal cells were incubated with 0-100 microM Mec or Flur, either alone or in the presence of 10 microM arachidonic acid (AA) to assess PGF2 alpha and PGE2 synthesis. Levels of both PGF2 alpha and PGE2 were stimulated (P less than 0.05) by AA (3.3- and 5.8-fold, respectively). Maximal suppression (P less than 0.01) of basal and AA-stimulated PGF2 alpha and PGE2 synthesis was elicited by 1 microM Mec and Flur. Second, adenylate cyclase activity, measured by the conversion of alpha 32P-ATP to alpha 32P-cAMP, was monitored in luteal homogenates exposed to increasing doses of Mec and Flur either alone or with maximal stimulatory doses of hCG, PGE2, or PGI2. Mec elicited a dose-dependent reduction (P less than 0.01) in control activity (incubated with 50 microM GTP), as well as inhibiting hCG- and PG-stimulated activity. The presence of 100 microM Mec suppressed (P less than 0.01) hCG-, PGE2- and PGI2-stimulated activity to control levels, but had no effect on activity stimulated by GMP-P(NH)P or forskolin. In contrast, Flur at any dose did not alter control activity or that stimulated by hormonal or nonhormonal activators. Third, P production by dispersed luteal cells was quantified during exposure to 0, 1, and 100 microM Mec or Flur alone or with maximal stimulatory doses of hCG, PGE2, PGD2, 6 beta PGI1, PGA2, or dibutyryl cAMP (dbcAMP). All hormones and dbcAMP stimulated (P less than 0.01) P synthesis 2-3 fold over basal levels, except PGA2, which had no effect. The presence of 100 microM Mec reduced (P less than 0.01) basal P production by 62% and abolished (P less than 0.05) hCG-, PG-, and dbcAMP-induced stimulation. Conversely, neither 1 microM Mec nor either dose of Flur affected P synthesis in the absence or presence of hormones or dbcAMP. These data indicate that: 1) Mec and Flur are potent inhibitors of PG synthesis in primate luteal cells in vitro and 2) higher doses of Mec suppress PG- and gonadotropin-sensitive adenylate cyclase activity and P production.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Disparate effects of the prostaglandin synthesis inhibitors, meclofenamate, and flurbiprofen on monkey luteal tissue in vitro. 230 10

The developmental profile of prostaglandin (PG)-synthesizing enzymes in liver was investigated in rats from the fetus to 2 years old. In the neonatal period, the activities of PGD2-(2.7 nmol/min/mg protein) and PGE2-(2.2 nmol/min/mg protein) synthesizing enzymes were predominant, whereas PGE2-synthesizing enzyme alone further increased in activity during adult to old ages (5.2-6.1 nmol/min/mg protein). In order to determine the sites of PGs production in rat liver, we prepared hepatocytes and non-hepatocytes by a collagenase digestion method. Regardless of the ages we examined, the major PG produced in the hepatocytes was proved to be PGE2, on the other hand, PGD2 was almost exclusively produced in the non-hepatocytes. These results suggest that each PG may have individual roles in the development of rat liver.
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PMID:Prostaglandin synthesizing enzyme activities of hepatocytes and non-hepatocytes in rat liver as a function of age. 381 60

We have isolated, partially purified, and characterized the mast cells from human heart tissue. The histamine content of left and right ventricles and septum of hearts obtained from 25 patients undergoing heart transplantation was 5.4 +/- 0.6, 5.3 +/- 0.5, and 5.6 +/- 0.5 micrograms/g of wet tissue, respectively. Ultrastructural study of cardiac mast cells revealed scroll, crystal, and mixed granules, homogeneously dense granules, and lipid bodies in the cytoplasm. A mild collagenase digestion was used to disperse the heart mast cells; the average yield was 3.2 +/- 0.6% (range: 0.8 to 13.6%). The average histamine and tryptase content/heart mast cells was 3.3 +/- 0.2 pg (n = 25) and 24.2 +/- 4.3 micrograms/10(6) cells (n = 11), respectively. Survival of cardiac mast cells after overnight culture was 71.9 +/- 5.4% (n = 23). The purification of human heart mast cells can be brought from less than 0.1 to 12% by a combination of low-speed centrifugation over albumin (2%) solution and Percoll gradient. Viability as shown by trypan blue exclusion was greater than 90%. Heart mast cells released histamine in response to immunologic (anti-IgE, anti-Fc epsilon RI, and C5a) and nonimmunologic stimuli (recombinant human stem cell factor, A23187, and compound 48/80) but did not respond to substance P, FMLP, 12-O-tetradecanoylphorbol-13-acetate, or acetylcholine. There was a linear correlation between the percentage of release caused by anti-IgE and anti-Fc epsilon RI, whereas there was no correlation between the release caused by C5a and anti-IgE-mediated stimuli. Cross-linking with anti-IgE of IgE on heart mast cells induced the release of tryptase (10.1 +/- 2.1 micrograms/10(7) cells; n = 10) and the de novo synthesis of PGD2 (17.3 +/- 4.3 ng/10(6) cells; n = 10) and of leukotriene C4 (19.1 +/- 4.5 ng/10(6) cells; n = 10). There was a linear correlation between the percentage of histamine secretion and tryptase release (r = 0.67; p < 0.001) induced by cross-linking of Fc epsilon RI. similarly, there was a significant correlation between percentage of histamine secretion and PGD2 (r = 0.63; p < 0.001) and LTC4 (r = 0.64; p < 0.001) release. Immunoelectron microscopy demonstrated the presence of chymase in cardiac mast cells. Mast cells isolated from human heart can be a useful model with which to study the role of these cells and their mediators in cardiac anaphylaxis and cardiovascular diseases.
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PMID:Human heart mast cells. Isolation, purification, ultrastructure, and immunologic characterization. 753 85

Hepatocytes isolated from rats by the collagenase perfusion method were cultured as monolayers at concentrations of 0.4-1.1 x 10(6) attached cells/dish (9 cm2) for 1-3 days and the effect of prostaglandins on their glycogenolysis was studied. By use of [14C]glycogen-labeled cells, prostaglandin E2 (PGE2) was found to have a stimulatory effect on glycogen degradation at high cell density (more than 0.8 x 10(6) cells/dish) in 1-day cultures. PGE2 was maximally effective at 10(-7) M, increasing [14C]release from cellular [14C]glycogen to 2-3 times the basal level after 1 h incubation, and to plateau level within 2 h. PGE1, 16,16-dimethyl PGE2 and PGF2 alpha had similar effects, but PGD2 and dinor-PGE1 (a metabolite of PGE1 and PGE2 in hepatocytes) had no effect. This prostaglandin-induced glycogen degradation was observed in 1-day cultures, with a maximum between 20-30 h, but not in 2-day and later cultures. Treatment of hepatocytes with pertussis toxin potentiated PGE2-stimulated glycogen degradation, indicating that the effect involves a different pathway from that for inhibition of glucagon- and epinephrine-stimulated glycogenolysis by E series prostaglandins reported previously.
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PMID:Stimulation of glycogen degradation by prostaglandin E2 in primary cultured rat hepatocytes. 832 15

We have established primary colonic epithelial cell culture from adult rabbits and examined effects of anti-inflammatory drugs on prostaglandin (PG) E2 production. Colonic epithelium of adult rabbits was scraped and minced into small pieces. They were incubated for isolation in Hanks' balanced salt solution with 0.35% collagenase and Earle's solution with 1 mM EDTA. Isolated cells were cultured in Coon's modified Ham's F-12 medium with 10% fetal bovine serum and antibiotics on collagen coated cell wells. The medium was refed twice a week. The production of PGs was assessed by high pressure liquid chromatography (HPLC). PGE2 and PGF2 alpha were measured by radioimmunoassay. Within 24 hours after inoculation, the cell clumps attached to the surface of the wells and cells began to spread out and grow. Monolayer cultures became confluent in 4 days. Phase contrast microscopy showed that these cells consisted of a homogeneous population of epithelial cells with large oval nuclei, polyhedral shape, and organized sheet-like growth pattern. HPLC profile showed synthesis of 6-keto-PGF1 alpha, thromboxane B2, PGF2 alpha, PGE2, and PGD2 by cultured cells. Quantitatively, 117 +/- 7 ng/mg-protein/hour PGE2 and 7.4 +/- 0.7 ng/mg-protein/hour PGF2 alpha were produced. While hydrocortisone (10(-4) - 10(-2) M) did not show a significant effect on PGE2 production, indomethacin (10(-8) - 10(-6) M), and 5-aminosalicylic acid (2X10(-4)-5X10(-3) M) inhibited PGE2 production. We have established relatively convenient procedure for primary culture of colonic epithelial cells from adult rabbits. Different actions of anti-inflammatory drugs on PGE2 synthesis suggest that these cultured cells might be a good tool for the various cellular functional studies of normal colonic epithelial cells.
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PMID:Primary colonic epithelial cell culture of the rabbit producing prostaglandins. 843 Feb 23

1. Prostaglandin E2 (PGE2) is an autacoid that decreases proteoglycan synthesis, increases metalloprotease production by cultured chondrocytes, and can modulate some of the actions of interleukin-1 on cartilage. The objective of the present study was to characterize the subtype of prostaglandin E2 receptor present in bovine chondrocytes in culture. 2. Primary cultures of articular chondrocytes were prepared from slices of bovine carpal cartilage by sequential digestion with type III hyaluronidase, trypsin, type II collagenase, followed by overnight incubation in Dulbecco's Modified Eagle's Medium (DMEM) with type II collagenase, washing, and seeding at a density of 2 x 10(5) cells cm-2 in DMEM with 10% foetal bovine serum. 3. PGE2 and carbaprostacyclin induced dose-dependent increases in intracellular cyclic AMP in bovine chondrocytes in culture. The potencies of these compounds were different, and maximal doses of PGE2 and carbaprostacyclin had an additive effect. PGD2 induced a small increase in intracellular cyclic AMP only at a high concentration (10(-5) M). 4. PGE2 was more potent that the EP2 agonist 11-deoxy-PGE1 at inducing increases in intracellular cyclic AMP. The EP2 agonist butaprost, however, induced only a small increase at a concentration of 10(-5)M. 17-Phenyl-PGE2 (EP1 agonist), sulprostone and MB 28767 (15S-hydroxy-9-oxo-16-phenoxy-omega-tetranorprost-13E-enoic acid) (EP3 agonists) did not induce an increase in intracellular cyclic AMP at concentrations up to 10(-5)M. 5. The EP4 antagonist AH 23848B ([1 alpha(Z),2 beta, 5 alpha]-(+/-) -7-[5-[[(1,1'-biphenyl)-4-yl]methoxyl-2-(4-morpholinyl) -3-oxocyclopentyl]-5-heptenoic acid) antagonized PGE2 but not carbaprostacyclin effects on intracellular cyclic AMP. The Schild plot slope was different from 1 but this could be due to an interaction of PGE2 with IP receptors in high doses. The exact nature of the antagonism by compound AH 23848B could not be definitely established in these experimental conditions. 6. Neither PGE2 nor any of its analogues inhibited the increase in intracellular cyclic AMP induced by forskolin, and pertussis toxin did not alter the response to PGE2, suggesting that no Gi-coupled PGE2 receptors are present in these cells. Stimulation with PGE2 did not induce significant increases in intracellular inositol-trisphosphate levels nor increases in intracellular free calcium as determined by confocal microscopy, suggesting the absence of phospholipase-C-coupled or of calcium channel-coupled PGE2 receptors in bovine chondrocytes in these experimental conditions. 7. These results show for the first time that bovine chondrocytes in culture present a functional PGE2 receptor that has some pharmacological characteristics of an EP4 subtype, as well as an IP receptor.
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PMID:Characterization of the PGE2 receptor subtype in bovine chondrocytes in culture. 884 20