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)

Isolated gastric smooth muscle cells were prepared from the stomach of Bufo marinus by successive incubation in collagenase without added trypsin. Contraction was determined by image-splitting micrometry and expressed as the mean percentage decrease in cell length from control. Peak contractile response was attained within 30 s. Dose-response curves constructed from peak responses showed that the maximal responses to CCK-OP (37.2 +/- 3.8%), acetylcholine (35.3 +/- 2.5%), and Ca2+ (42.3 +/- 0.9%) were similar. The D50s for octapeptide of cholecystokinin (CCK-OP) and acetylcholine were around 10(-12) M and 10(-11) M, respectively. The response to a combination of submaximal concentrations of acetylcholine and CCK-OP exceeded the individual responses but did not exceed the maximal response to either agent alone. A low concentration of atropine (5 X 10(-10) M) inhibited specifically the maximal response to acetylcholine. A high concentration of atropine (5 X 10(-8) M) inhibited partially the maximal response to CCK-OP but had no effect on the maximal response to Ca2+. It was concluded that 1) dispersed gastric smooth muscle cells are highly sensitive to stimulation; 2) CCK-OP has a direct (myogenic) contractile effect on gastric smooth muscle; and 3) the effect of CCK-OP and acetylcholine are mediated by separate receptors.
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PMID:Interaction of acetylcholine and cholecystokinin with dispersed smooth muscle cells. 11 64

Isolated pancreatic acini were prepared by a new method from mouse and rat pancreases by digestion with purified collagenase and chymotrypsin followed by mechanical shearing. Acini were structurally similar to those of the intact pancreas, having a normal luminal structure but with the basal acinar cell membranes exposed to the incubation medium. Amylase release in response to both cholinergic analogues and the cholecystokinin analogues caerulein and pentagastrin was comparable to that of the intact pancreas, but was much greater than previously reported for isolated acinar cells. Cholinergic-stimulated release was inhibited by atropine with a Ki value of 1.4 nM which is comparable to other muscarinic receptors. All agonists tested, when added at supramaximal concentrations, produced a submaximal release of amylase even though ATP levels and the release of slowly exchanging 45Ca2+ were normal or increased. Acini releasing amylase submaximally after being exposed to supramaximal concentrations of carbachol failed to respond to a maximal amount of caerulein or to the Ca2+ ionophore A23187. It is concluded that the decreased response (desensitization) is a postreceptor phenomenon and possibly mediated by Ca2+ itself.
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PMID:Action of secretagogues on a new preparation of functionally intact, isolated pancreatic acini. 21 42

For special studies on pancreatic diseases a parameter is needed to record alterations of the cellular energy metabolism. In the in vitro model of isolated pancreatic acini, we investigated whether or not at standardized cholecystokinin stimulation the energy-consuming process of enzyme secretion can be used to monitor changes of the energy-supplying capacity. Rat pancreatic acini were isolated via collagenase digestion and characterized by basal and stimulated release of amylase and trypsin, oxygen uptake under resting and maximally uncoupled conditions and by their ability to accumulate actively rhodamine-6G, as a measure of the mitochondrial membrane potential. The stimulation of enzyme release did not find a measurable reflection in rhodamine-6G accumulation and in the respiratory rat. Stepwise uncoupling of oxidative phosphorylation by 2,4-dinitrophenol (DNP) and temporary anoxia were used to simulate disturbances of the pancreatic energy metabolism in vitro. With increasing DNP concentration the enzyme release was significantly reduced. While after 30 min anoxia the enzyme release still exceeded that of unstimulated control, after 60 min anoxia there was no further response to hormonal stimulation. At standardized stimulation and after suitable calibration the enzyme release by acini may be used to monitor alterations of the pancreatic energy metabolism in vitro.
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PMID:Influence of stepwise uncoupling and temporary anoxia on pancreatic enzyme secretion by isolated rat acini. 138 Mar 65

The Authors describe an optimized procedure for the isolation of rat pancreatic acini and the preliminary results concerning the functional characterization of the cells. Isolation is carried out by two sequential digestive steps in a KREBS modified medium containing collagenase, separated by an intermediate step in which acini separation is fostered by incubation in a Ca++ free medium containing the Ca++ chelator EDTA. Final separation is obtained through the application of mechanical forces by aspirating the suspension in plastic pipettes. The choice of the collagenase, the duration and the entity of the mechanical dissociation strength are the main modifications to the classic procedure and allow to obtain a very high yield of cells maintaining their ability to secrete enzymes for a long period (6-7 hours). Functional characterization is completed mainly by the determination of the amylase release stimulated by increasing doses of cholecystokinin.
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PMID:Isolation and functional characterization of rat pancreatic acini. 170 46

Both insulin and glucocorticosteroid (GS) deficiency causes a reduction of amylase synthesis and changes in the dose-response curve of cholecystokinin (CCK) stimulated enzyme secretion in rats. Since we found a reduction of plasma insulin in adrenalectomized rats, we now tested the hypothesis that the regulation of amylase synthesis by insulin may be mediated by GS. Three groups of male rats were investigated: controls, streptozotocin induced diabetics, and diabetics treated with GS. Animals were sacrificed 10-14 days after injection of streptozotocin and isolated pancreatic acini prepared by collagenase digestion. Protein synthesis was measured on the translational level by incubation of acini with 35S-methionine followed by lysis of cells and separation of proteins by SDS-PAGE. In addition, protein synthesis was measured on the transcriptional level by isolation of mRNA from pancreatic acini and translation of proteins using the rabbit reticulocyte lysate system. The loss of insulin in diabetic rats was associated with a 70-90% decrease in amylase synthesis and increases of synthesis of various proteases. This was due to a specific decrease in mRNA coding for amylase and increase in mRNA coding for proteases. Furthermore, the known rightward shift of the dose response curves of CCK stimulated amylase secretion was seen in diabetic animals. Treatment of diabetic rats with GS did deteriorate the catabolic status seen in diabetes with increases in mortality as compared to diabetes alone. However, neither the overall pattern of enzyme synthesis seen in diabetic rats nor the alterations in CCK stimulated enzyme secretion were changed by treatment with GS. We conclude that the regulation of amylase synthesis and enzyme secretion by insulin is not mediated via GS.
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PMID:Pancreatic enzyme synthesis and secretion are independently regulated by insulin and glucocorticosteroids. 170 71

This study examines the influence of ovariectomy and administration of a pharmacologic dose of estradiol on amylase release from isolated-dispersed rat pancreatic acini and cholecystokinin receptors on rat acinar cell membranes. Rats were sham ovariectomized (intact) or ovariectomized (Ovx) and 21 day timed release pellets containing either estradiol (2.5 mg) or vehicle, were implanted subcutaneously. Eighteen days later, pancreatic acini were isolated from rats by collagenase digestion and differential centrifugation. Total cellular amylase, basal and cholecystokinin octapeptide (CCK8) stimulated amylase release and CCK membrane receptors were measured. Acini isolated from estradiol treated Ovx rats had significantly greater total cellular amylase, compared to acini isolated from either intact or Ovx rats. The amplitude of both total stimulated amylase release and percent total stimulated amylase release were significantly greater for acini isolated from vehicle treated Ovx rats, than acini isolated from either intact or estradiol treated Ovx rats. The magnitude of percent total amylase release of acini isolated from estradiol treated Ovx rats was significantly lower than that of acini isolated from intact rats. Cholecystokinin receptor concentration was significantly greater on membranes prepared from vehicle treated Ovx rats, compared to membranes prepared from either intact or estradiol treated Ovx rats. These data indicate that ovariectomy is associated with increased responsiveness of pancreatic acini to CCK stimulation, while chronic estradiol treatment of ovariectomized rats is associated with increased total cellular amylase and decreased acinar cell responsiveness to CCK8. Estrogen mediated alterations in acinar cell amylase content and amylase release may play a role in estrogen related pancreatitis.
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PMID:Estrogens influence cholecystokinin stimulated pancreatic amylase release and acinar cell membrane cholecystokinin receptors in rat. 170 70

The use of pancreatic lobule preparations is one of the well-established approaches to study stimulus-secretion coupling in the exocrine pancreas in vitro. We have developed a kinetic system for the perfusion and intermittent incubation of rat pancreatic lobules. This model allows repeated hormone stimulation for up to 3 h while permitting rapid changes of the cellular environment with no accumulation of secretory or metabolic products. Tissue viability could be demonstrated over 6 h by in vivo toluidine blue exclusion, histology and electron microscopy. Lactate dehydrogenase leakage from cells over 6 h was only 2.4% of total content. No activated trypsin was detected in the perfusion medium. A biphasic dose response was established for cholecystokinin stimulation with a maximal response at 10(-8) M. We conclude that kinetic perfusion and incubation are technically feasible with rat pancreatic lobules. This in vitro model appears particularly suited for the investigation of pharmacologic and metabolic effects on the pancreatic acinar cell when rapid changes of the cellular environment are required and when the accumulation of secretory and metabolic products must be avoided. The technique described requires neither protease inhibition in the medium nor collagenase treatment of the cells.
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PMID:In vitro perfusion and incubation of rat pancreatic lobules in a kinetic system. 172 99

Canine jejunal epithelial cells were isolated and maintained in short-term culture to study cholecystokinin (CCK) release. Sequential digestion of jejunal mucosa with collagenase and ethylenediaminetetraacetic acid was followed by counterflow elutriation to enrich CCK-containing cells. After 40 hours in culture on collagen-coated plates, 8.4% of the initially seeded cells were attached; 8.7% of them stained positive with a C-terminal CCK/gastrin antibody and 2.5% stained positive with a gastrin-specific antibody. Basal release of CCK into the culture medium amounted to 1.3% of total cell content over 105 minutes. Receptor-independent stimulation of protein kinase C by the phorbol ester beta-phorbol-12-myristate-13-acetate caused significant CCK release. The inactive form, 4 alpha-phorbol-12-myristate-13-acetate, had no effect. Activation of adenylate cyclase by 10(-5) mol/L forskolin evoked a 2.5-fold increase in CCK concentrations, which was completely abolished by 10(-8) mol/L somatostatin. L-phenylalanine stimulated CCK release at 20 and 50 mmol/L, whereas D-phenylalanine caused significant hormone output only at 50 mmol/L. L-tryptophan had no effect. Cholecystokinin release stimulated by L-phenylalanine was not influenced by the addition of either somatostatin or somatostatin antibody. In conclusion, a system of isolated canine jejunal epithelial cells was developed in short-term culture. This preparation proved suitable for the study of CCK release on a cellular basis.
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PMID:Cholecystokinin release from isolated canine epithelial cells in short-term culture. 172 60

Dispersed canine antral mucosal cells were prepared by sequential steps of collagenase digestion and EDTA treatment. Cell preparations enriched in gastrin cells were made by centrifugal elutriation followed by step density gradient centrifugation. Specific, saturable, and reversible binding of 125I-[Tyr4]-bombesin was found in all preparations. This saturable binding was time, temperature, and cell number dependent. In both velocity (elutriator) and density cell separation experiments, saturable binding of bombesin correlated with the distribution of cells containing gastrin- but not somatostatin-like immunoreactivity. Maximal specific binding to gastrin (G) cell-enriched fractions was reached in 45 min at 37 degrees C and constituted 90% of total binding. Addition of 100 nM nonradioactive bombesin to cells incubated with 50 pM 125I-[Tyr4]-bombesin for 45 min resulted in time-dependent dissociation of specifically bound tracer to about 40% of the maximal equilibrium binding. Analysis of saturable equilibrium binding yielded a best fit to a one-site model of high affinity binding sites with an apparent Kd of 85 +/- 14 pM and a Bmax of 231,000 +/- 71,000 receptors/gastrin cell. Nonradioactive [Tyr4]-bombesin and related analogs inhibited the specific binding of the tracer in a dose-related manner. The rank order of potency, determined at the IC50, of [Tyr4]-bombesin and related analogs for inhibition of specific binding was bombesin greater than [Tyr4]-bombesin = hGRP-27 greater than GRP-10 greater than ranatensin much greater than neuromedin B. Cholecystokinin, somatostatin, substance K, and kassinin each tested at a concentration of 1 microM did not inhibit bombesin binding.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Characterization of bombesin receptors on canine antral gastrin cells. 197 73

The neuropeptide galanin is present in intrapancreatic nerve fibers and is known to affect the secretion of the islet hormones. Its most potent effect is thereby the inhibition of insulin secretion. In the present study, we investigated whether galanin influences amylase secretion from isolated rat pancreatic acini. Acini were isolated by the collagenase digestion technique and incubated for 45 min in a Krebs-Henseleit medium with or without addition of the cholinergic agonist carbachol or the C-terminal octapeptide of cholecystokinin (CCK-8) in the presence or absence of galanin. Carbachol, at its optimal concentration (10(-5) M), stimulated amylase secretion to 11.8 +/- 0.5% of total amylase content compared to 4.3 +/- 0.3% in controls (p less than 0.001). Galanin, (10(-8)-10(-9) M), reduced the carbachol-induced amylase secretion to 10.4 +/- 0.3% (p less than 0.01). Galanin at concentration levels below 10(-10) M had no significant effect. At 10(-8) M, CCK-8 stimulated amylase secretion to 9.7 +/- 0.6% compared with 5.2 +/- 0.3% in controls (p less than 0.01). Galanin (10(-7) M) reduced this stimulation to 8.0 +/- 0.4% (p less than 0.05). Galanin did not affect basal amylase secretion. It is concluded that the intrapancreatic neuropeptide galanin weakly inhibits carbachol- and CCK-8-induced amylase secretion from isolated rat pancreatic acini. Thus, galanin has the capability to directly affect not only endocrine but also exocrine pancreatic secretion although its effect of inhibiting amylase secretion seems weak.
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PMID:Galanin inhibits amylase secretion from isolated rat pancreatic acini. 246 Aug 54


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