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)

Although serotonin (5-HT) release from enterochromaffin (EC) cells is considered to be regulated by multiple receptor-mediated mechanisms, little is known about the signal transduction. Here, we introduce the methods to isolate the mouse ileal crypts, which consist of various types of cells including EC cells, and to analyze the intracellular calcium dynamics. Ileal tissue was inserted with a plastic straw and the smooth muscle layers were peeled off. The mucosa were digested with collagenase and then suspended by moderate pipetting. Ileal crypts were separated by stepwise filtrations through 2 different nylon-meshes. The isolated crypt contained 0-3 EC cells as identified by immunostaining using anti-5-HT antibody followed by confocal microscopy. Isolated crypts were attached to a coverglass by an adhesive material (Cell-Tak) and loaded with fura-2/AM. Intracellular calcium dynamics in EC cells were obtained by digital video-imaging analysis of fura-2 fluorescence followed by the identification of EC cells with immunostaining of 5-HT granules. By these methods, it was suggested that norepinephrine elicited a transient elevation of intracellular calcium concentration in EC cells via alpha 2-adrenoceptors. These methods could be also useful to analyze the signal transduction system in intestinal endocrine cells that contain various intestinal hormones such as gastrin, cholecystokinin or secretin.
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PMID:[Analysis of intracellular calcium dynamics in enterochromaffin cells of small intestine]. 1067 96

Disruption or absence of hepatocyte keratins 8 and 18 is associated with chronic hepatitis, marked hepatocyte fragility, and a significant predisposition to stress-induced liver injury. In contrast, pancreatic keratin disruption in transgenic mice that express keratin 18 Arg89 --> Cys (K18C) is not associated with an obvious pancreatic pathology. We compared the effects of keratin filament disruption on pancreatic acini or acinar cell viability, and on cholecystokinin (CCK)-stimulated secretion, in transgenic mice that overexpress wild-type keratin 18 and harbor normal extended keratin filaments (TG2) and K18C mice. We also compared the response of these mice to pancreatitis induced by a choline-deficient ethionine-supplemented diet or by caerulein. Despite extensive cytoplasmic keratin filament disruption, the apicolateral keratin filament bundles appear intact in the acinar pancreas of K18C mice, as determined ultrastructurally and by light microscopy. No significant pancreatitis-associated histologic, serologic, or F-actin/keratin apicolateral redistribution differences were noted between TG2 and K18C mice. Acinar cell viability and yield after collagenase digestion were lower in K18C than in TG2 mice, but the yields of intact acini and their (125)I-CCK uptake and responses to CCK-stimulated secretion were similar. Our results indicate that keratin filament reorganization is a normal physiologic response to pancreatic cell injury, but an intact keratin cytoplasmic filament network is not as essential in protection from cell injury as in the liver. These findings raise the possibility that the abundant apicolateral acinar keratin filaments, which are not as evident in hepatocytes, may play the cytoprotective role that is seen in liver and other tissues. Alternatively, identical keratins may function differently in different tissues.
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PMID:Effects of keratin filament disruption on exocrine pancreas-stimulated secretion and susceptibility to injury. 1069 32

This study investigates the effects of dephostatin, a new tyrosine phosphatase inhibitor, on intracellular free calcium concentration ([Ca2+]i) and amylase secretion in collagenase dispersed rat pancreatic acinar cells. Dephostatin evoked a sustained elevation in [Ca2+]i by mobilizing calcium from intracellular calcium stores in either the absence of extracellular calcium or the presence of lanthanium chloride (LaCl3). Pretreatment of acinar cells with dephostatin prevented cholecystokinin-octapeptide (CCK-8)-induced signal of [Ca2+]i and inhibited the oscillatory pattern initiated by aluminium fluoride (AlF4), whereas co-incubation with CCK-8 enhances the plateau phase of calcium response to CCK-8 without modifying the transient calcium spike. The effects of dephostatin on calcium mobilization were reversed by the presence of the sulfhydryl reducing agent, dithiothreitol. Stimulation of acinar cells with thapsigargin in the absence of extracellular Ca2+ resulted in a transient rise in [Ca2+]i. Application of dephostatin in the continuous presence of thapsigargin caused a small but sustained elevation in [Ca2+]i. These results suggest that dephostatin can mobilize Ca2+ from both a thapsigargin-sensitive and thapsigargin-insensitive intracellular stores in pancreatic acinar cells. In addition, dephostatin can stimulate the release of amylase from pancreatic acinar cells and moreover, reduce the secretory response to CCK-8. The results indicate that dephostatin can release calcium from intracellular calcium pools and consequently induces amylase secretion in pancreatic acinar cells. These effects are likely due to the oxidizing effects of this compound.
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PMID:Effect of dephostatin on intracellular free calcium concentration and amylase secretion in isolated rat pancreatic acinar cells. 1082 34

We have investigated whether the cytochrome P450 system is involved in Ca(2+) signalling in rat pancreatic acinar cells. Intracellular free [Ca(2+)] ([Ca(2+)](i)) was measured in collagenase-isolated cells using fura-2 microspectrofluorimetry and imaging. The imidazole P450 inhibitor ketoconazole (5 - 50 microM) inhibited [Ca(2+)](i) oscillations induced by cholecystokinin octapeptide (CCK). However, ketoconazole also raised baseline [Ca(2+)](i) when applied in the absence of CCK. These effects were mimicked by 5 - 50 microM SKF96365, an imidazole widely used as an inhibitor of Ca(2+) entry. The non-imidazole P450 inhibitor proadifen (SKF525A) inhibited CCK-induced [Ca(2+)](i) oscillations at a concentration of 10 - 50 microM. Proadifen alone caused intracellular Ca(2+) release at 25 or 50 microM, but not at 10 microM. Octadecynoic acid and 1-aminobenzotriazole, structurally-unrelated non-imidazole P450 inhibitors, did not alter baseline [Ca(2+)](i) or CCK-evoked oscillations. We compared cumulative CCK dose-response relationship in control cells and in cells where P450 had been induced by prior injection of animals with beta-naphthoflavone. Only minor differences were apparent, with induced cells showing some decrease in responsiveness at moderate and higher concentration of CCK (30 pM - 3 nM). Direct assessment of depletion-activated Ca(2+) entry showed no clear differences between control and induced cells. In conclusion, we could find no compelling evidence for a role of P450 in controlling Ca(2+) signalling generally, or Ca(2+) entry in particular, in pancreatic acinar cells. Induction of P450 is therefore probably toxic to acinar cells via a Ca(2+)-independent mechanism.
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PMID:Pharmacological evaluation of the role of cytochrome P450 in intracellular calcium signalling in rat pancreatic acinar cells. 1103 Jul 26

We have demonstrated by the use of isolated rat pancreatic acini that exogenous prostaglandins of the E type inhibit secretagogue-stimulated amylase secretion. We here studied whether the pancreas is a source of prostaglandin synthesis and whether prostaglandins mediate regulation of pancreatic enzyme secretion by various diets. Prostaglandin E2 was measured by enzyme immunoassay in pancreatic acini from either normal animals or after 10 days of feeding with different diets. Acini were prepared by collagenase digestion. Amylase secretion was measured after stimulation with cholecystokinin in the presence or absence of indomethacin, an inhibitor of prostaglandin synthesis. Prostaglandin E2 concentration in pancreatic acini was comparable to other organs such as kidney and liver. Feeding a diet enriched in proteins caused an increase of cholecystokinin-stimulated maximal amylase secretion and a decrease of prostaglandin E2 concentration. Incubation of acini with indomethacin caused a decrease in prostaglandin E2 concentration and an increase in cholecystokinin stimulated amylase secretion. We conclude that regulation of pancreatic enzyme secretion by diets may be mediated by prostaglandins.
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PMID:Role of prostaglandins in regulation of pancreatic enzyme secretion by various diets. 1128 Nov 77

The objective of this study was to examine the effects of 10% food restriction on body weight, plasma cholecystokinin (CCK) levels, and exocrine pancreatic function in male Sprague-Dawley rats. A matched group of rats with unrestricted access to food served as controls. After ingesting the diets for 32 da, the rats were killed and blood obtained for plasma cholecystokinin, glucose, and insulin determinations. To evaluate pancreatic function, the pancreases were removed, weighed, and digested with collagenase to isolate pancreatic acini, which were incubated with maximal stimulating dose of CCK. The fraction of amylase that was released into the medium was measured. To explore the role of membrane receptors in exocrine pancreatic secretion, CCK receptor affinity and CCK receptor capacity were determined by radioligand binding assays in isolated, purified membranes from pancreatic acini. Compared to the control group, rats with 10% food restriction showed (a) reduced body weight gain, (b) increased pancreatic weight, (c) increased plasma CCK level, and (d) no significant changes in plasma glucose or insulin levels. The food-restricted group showed a reduction of pancreatic function, assessed by measuring amylase release in response to maximal CCK stimulation; the amylase release was diminished by 35% in the food-restricted group. In isolated acinar cell membranes from food-restricted rats, CCK receptor affinity and capacity were reduced by 23% and 16%, respectively, compared to controls. These results indicate that consumption of less food than normal affects pancreatic function by a mechanism that evidently involves CCK release and downregulation of CCK receptors. The data suggest that CCK plays an important physiological role in the adaptation to eating less food, and thereby to the lowering of body weight in rats and, possibly, in other animals.
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PMID:Effect of food restriction on plasma cholecystokinin levels and exocrine pancreatic function in rats. 1168 49

We have employed confocal laser scanning microscopy to investigate how intracellular free calcium concentration ([Ca2+]i) is influenced by hydrogen peroxide (H2O2) in collagenase-dispersed mouse pancreatic acinar cells. In the absence of extracellular calcium, treatment of cells with increasing concentrations of H2O2 resulted in an increase in [Ca2+]i, indicating the release of calcium from intracellular stores. Micromolar concentrations of H2O2 induced an oscillatory pattern, whereas 1 mmol H2O2/L caused a slow and sustained increase in [Ca2+]i. H2O2 abolished the typical calcium release stimulated by thapsigargin or by the physiological agonist cholecystokinin octapeptide (CCK-8). Depletion of either agonist-sensitive or mitochondrial calcium pools was unable to prevent calcium release induced by 1 mmol H2O2/L, but depletion of both stores abolished it. Additionally, lower H2O2 concentrations were able to release calcium only after depletion of mitochondrial calcium stores. Treatment with either the phospholipase C inhibitor U-73122 or the inhibitor of the inositol 1,4,5-trisphosphate (IP3) receptor xestospongin C did not modify calcium release from the agonist-sensitive pool induced by 100 micromol H2O2/L, suggesting the involvement of a mechanism independent of IP3 generation. In addition, H2O2 reduced amylase release stimulated by CCK-8. Finally, either the H2O2-induced calcium mobilization or the inhibitory effect of H2O2 on CCK-8-induced amylase secretion was abolished by dithiothreitol, a sulphydryl reducing agent. We conclude that H2O2 at micromolar concentrations induces calcium release from agonist-sensitive stores, and at millimolar concentrations H2O2 can also evoke calcium release from the mitochondria. The action of H2O2 is mediated by oxidation of sulphydryl groups of calcium ATPases independently of IP3 generation.
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PMID:Dose-dependent effect of hydrogen peroxide on calcium mobilization in mouse pancreatic acinar cells. 1646 88

Information regarding age-induced Ca(2+) signal alterations in nonexcitable cells is limited. In addition, little evidence exists on the ability of melatonin to palliate the effects of aging on Ca(2+) signals and mitochondrial potential, a parameter involved in both Ca(2+) signaling and aging. We studied the ability of melatonin to prevent the effects of aging on intracellular Ca(2+) homeostasis and mitochondrial potential in exocrine cells. Pancreatic acinar cells were obtained from adult (3 months old) and aged (22-24 months old) mice by collagenase dispersion. Ca(2+) signals, in situ mitochondrial potential and in vitro amylase secretion were determined. Secretion in response to increasing levels of the secretagogues, acetylcholine and cholecystokinin (CCK), were impaired in aged pancreatic acini. This decrease was accompanied by an inhibition in the amplitude of the peak response to maximal concentrations of the agonists, and by a decrease in the pattern of Ca(2+) oscillations induced by postprandial levels of CCK. Both the size of the calcium pools, assessed by low levels of ionomycin, and capacitative calcium entry, induced by depletion of the stores with thapsigargin, were diminished in aged cells. These changes in Ca(2+) homeostasis were associated with depolarization of intracellular mitochondria. Oral administration of melatonin for 3 months to aged mice restored the secretory response, the amplitude and frequency of Ca(2+) responses, the size of intracellular calcium pools, the capacitative calcium entry, and the mitochondrial potential. In conclusion, melatonin restores secretory function, Ca(2+) signals and mitochondrial potential of aged exocrine cells.
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PMID:Age-related alterations in Ca2+ signals and mitochondrial membrane potential in exocrine cells are prevented by melatonin. 1831 4

The role of the peptide hormone gastrin in stimulating gastric acid secretion is well established. Mature amidated gastrin is processed from larger peptide precursor forms. Increasingly these processing intermediates, such as glycine-extended gastrin (G-Gly) and progastrin, have been shown to have biological activities of their own, often separate and complementary to gastrin. Although G-Gly is synthesized and secreted by gastric antral G-cells, the physiological functions of this putative mediator are unclear. Gastrin and cholecystokinin (CCK) stimulate the secretion of somatostatin from gastric D-cells as part of the feedback control of gastric acid. In this study the effect of G-Gly and gastrin on the release of somatostatin from rabbit fundic D-cells was examined. D-cells were obtained by collagenase-EDTA digestion and elutriation and cultured for 48 hours. With a 2 hour exposure to the peptides, gastrin but not G-Gly stimulated somatostatin release. Treatment of D-cells for 24 hours with gastrin or G-Gly individually, significantly enhanced subsequent basal as well as CCK- and GLP-1-stimulated somatostatin release. Twenty four hours exposure to gastrin combined with G-Gly synergistically enhanced basal and agonist-stimulated somatostatin release and cellular somatostatin content. Gastrin and G-Gly may be important in the longer term regulation of D-cell function.
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PMID:Glycine-extended gastrin enhances somatostatin release from cultured rabbit fundic D-cells. 2435 82


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