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:3.1.4.3 (phospholipase C)
18,461 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The phosphorylation of the lipocortin-related protein, p68, found in Ca2+-dependent association with the submembranous cytoskeleton has been studied using isolated human placental syncytiotrophoblast plasma membrane vesicles. p68 undergoes rapid, cation-independent phosphorylation in unstimulated membrane vesicles which was inhibited, in a dose-dependent manner, by insulin, platelet-derived growth factor, macrophage colony stimulating factor, protein kinase C-activating phorbol esters and phosphatidylinositol-specific phospholipase C. Epidermal growth factor had no effect on overall p68 phosphorylation. Transferrin induced an increase in p68 phosphorylation. However, phosphotyrosine was detected in p68 after treatment with epidermal growth factor, macrophage colony stimulating factor or transferrin, whereas a reduction in p68 phosphorylation appeared to be restricted to serine. cAMP and both cholera and pertussis toxins inhibited p68 phosphorylation. Both toxins were synergistic with the effects of insulin and platelet-derived growth factor whilst being antagonistic to the effect of transferrin. Epidermal growth factor and both human and equine immunoglobulin G, all of which alone did not affect overall p68 phosphorylation, reduced cholera or pertussis toxin-induced inhibition of p68 phosphorylation. Several phosphatase inhibitors failed to prevent macrophage colony stimulating factor-induced reduction of p68 phosphorylation. These results indicate that (i) p68 is a potential substrate of receptor tyrosyl kinases, (ii) p68 is not phosphorylated by protein kinase C or cAMP-dependent kinase and (iii) p68 phosphorylation is inhibited by activation of multiple pathways including those employing diacylglycerol or cAMP as second messengers.
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PMID:The phosphorylation of p68, a calcium-binding protein associated with the human syncytiotrophoblast submembranous cytoskeleton, is modulated by growth factors, activators of protein kinase C and cyclic AMP. 255 24

Phosphoinositide-specific phospholipase C (PI-PLC) activity was determined in homogenates of adipocytes treated with maximal concentrations of insulin. PI-PLC activity measured using exogenous [3H]phosphatidylinositol [( 3H]PI) and exogenous [3H]phosphatidylinositol 4,5-bisphosphate [( 3H]PIP2) was not altered by prior exposure of adipocytes to insulin. It was possible to see oxytocin-induced breakdown of phosphoinositides but no effect of insulin was seen in intact adipocytes.
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PMID:Insulin does not activate a phosphoinositide-specific phospholipase C in adipocytes. 255 35

To evaluate the regulation and effects of pancreatic islet lipoxygenase, adult rat islets were permeabilized, using digitonin or staphylococcal alpha-toxin, and then were studied in a medium simulating an intracellular milieu at fixed ambient concentrations of Ca2+. Permeabilized islets retained 12-lipoxygenase activity, as indicated by conversion of tritiated arachidonic acid to a predominant peak of [3H]12-hydroxyeicosatetraenoic acid (12-HETE); this activity was inhibited (89-98%) by the lipoxygenase blockers nordihydroguaiaretic acid (35 microM), BW755c (250 microM) or ETYA (35 microM). Lesser amounts of compounds coeluting with 15- and 11-HETE (but little or no 5-HETE) were formed; however, 11-HETE (and possibly some 15-HETE) was probably synthesized (at least in part) via cyclooxygenase, as suggested by the partial synthesis blockade induced by 50 microM ibuprofen. The production of 12-HETE did not require the presence of Ca2+, Mg2+ or ATP; it also was not stimulated by addition of cyclic AMP, a phorbol ester, or calmodulin. However, it was augmented modestly by provision of a basal cytosolic free Ca2+ concentration of 60-80 nM, with no further increase at physiologically elevated levels of 260-530 nM. Elevations in cytosolic free Ca2+ concentrations induced insulin release which was inhibited by cooling, epinephrine or protein kinase inhibitors and, therefore, was exocytotic in nature. Lipoxygenase inhibitors blocked this insulinotropic effect of calcium at submaximal or saturating Ca2+ concentrations (with or without its potentiation by 12-O-tetradecanoylphorbol-13-acetate, an activator of protein kinase C) by 53-82%. However, they did not reduce the Ca2+-independent secretory effects (at subnanomolar Ca2+ concentrations) of the phorbol ester alone. Similar results were seen using dibutyryl cyclic AMP to activate protein kinase A. The alpha 2-adrenergic agonists epinephrine or clonidine inhibited Ca2+-, TPA- or cyclic AMP-induced insulin release without reducing HETE formation. We conclude that (1) islet lipoxygenase is constitutively expressed and is not physiologically regulated by alpha 2-adrenergic agonism, Ca2+ or protein kinases; (2) lipoxygenase modulates insulin release; HETE production is not merely an epiphenomenon reflecting the activation (or inhibition) of exocytotic secretion; (3) islet lipoxygenase inhibitors reduce insulin secretion, at least in part, by blocking the direct effects of Ca2+ on exocytosis and/or its synergism with Ca2+-binding proteins such as protein kinase C; and (4) these same inhibitors do not directly poison protein kinase C or A, or the exocytotic apparatus.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Blockade by lipoxygenase inhibitors of Ca2+-dependent insulin secretion from permeabilized rat islets. A molecular mechanism distinct from that of alpha 2-adrenergic agonists. 256 95

The insulinotropic action of glucose, the most potent physiologic insulin secretagogue, involves its metabolism. However, no glucose metabolite has ever been identified as a key intermediate. We tested the abilities of a number of glucose metabolites to stimulate insulin release from pancreatic islets. Of all of these metabolites, glyceraldehyde 3-phosphate was the most potent insulin secretagogue. In numerous experiments over 3 years, insulin release by 4 mM glyceraldehyde phosphate ranged from 50 to 200% of that initiated by 16.7 mM glucose--a near-maximal insulin stimulus. At concentrations of 1 and 4 mM, glyceraldehyde phosphate was even more potent than the known secretagogues glucose and glyceraldehyde. Glucose metabolites were also tested for their ability to stimulate inositol tris-, bis-, and monophosphate formation by permeabilized islets. Only glyceraldehyde phosphate stimulated inositol phosphate formation and this stimulation occurred at concentrations of glyceraldehyde phosphate which could be present in the beta cell under physiologic conditions (K0.5 = 25 microM). The current results are consistent with the idea that glyceraldehyde phosphate is a key insulinotropic glucose metabolite that might act directly (or rather directly via a receptor) on the phospholipase C that forms inositol trisphosphate in the plasma membrane.
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PMID:Glyceraldehyde phosphate: an insulin secretagogue with possible effects on inositol phosphate formation in pancreatic islets. 264 93

The effect of interference with diacylglycerol metabolism was investigated in pancreatic mouse islets. In the presence of the diacylglycerol lipase inhibitor RHC 80,267, glucose-induced insulin secretion was reduced 50-60%; whereas carbacholin-induced insulin secretion was unaffected. Addition of the diacylglycerol kinase inhibitor R 59,022 did not change glucose-stimulated insulin secretion but abolished the inhibition seen in the presence of RHC 80,267. RHC 80,267 increased islet glucose utilisation, measured as formation of tritiated water from 5-[3H]-glucose, 3-fold but did not affect glucose oxidation to CO2, lactate production or islet ATP levels. Glucose utilisation in leucocytes and hepatocytes was not increased by addition of RHC 80,267. Islet lipid production from glucose was augmented 4-fold in the presence of RHC 80,267 but only accounted for about 5% of the increase in glucose utilisation. The activity of adenylate cyclase and phosphoinositide-specific phospholipase C was unaffected by RHC 80,267. Concentrations of RHC 80,267 below 35 mumol/l did not alter the activity of phospholipase A2; whereas higher concentrations of the drug inhibited phospholipase A2 activity approx 25%. The data support the hypothesis that production of arachidonic acid from diacylglycerol may be involved in regulation of insulin secretion.
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PMID:Effect of diacylglycerol lipase inhibitor RHC 80267 on pancreatic mouse islet metabolism and insulin secretion. 265 50

Electrically permeabilized RINm5F cells were used to assess the factors required for activation of protein kinase C (PKC) and insulin secretion. PKC was activated either by phorbol 12-myristate 13-acetate (PMA) or by the generation of endogenous diacylglycerol in response to the nonhydrolyzable guanine nucleotide analog guanosine 5'-O-(thiotriphosphate) (GTP gamma S). As shown previously, both PMA and GTP gamma S elicit Ca2+-independent insulin secretion. This effect was mimicked by guanyl-5'-yl imidodiphosphate (Gpp(NH)p) but not by guanosine 5'-O-(3-fluorotriphosphate) and guanosine 5'-O-(3-phenyltriphosphate) possessing only one negative charge in the gamma-phosphate group. The action of PMA was mediated by PKC, since the agent caused both phosphorylation of specific protein substrates and association of the enzyme with cellular membranes. This translocation was independent of the Ca2+ concentration employed. In contrast, GTP gamma S only promoted association of PKC with membranes at 10(-6) and 10(-5) M Ca2+ and failed to alter significantly protein phosphorylation in the absence of Ca2+. Neither Gpp(NH)p, which stimulates insulin release, nor the other two GTP analogs, increased the proportion of PKC associated with membranes. To verify that the Ca2+-dependent effect of GTP gamma S on PKC is due to activation of phospholipase C, we measured the generation of diacylglycerol. GTP gamma S indeed stimulated diacylglycerol production in the leaky cells by about 50% at Ca2+ concentrations between 10(-7) and 10(-5) M, an effect which was almost abolished in the absence of Ca2+. Thus, at 10(-7) M Ca2+, the concentration found in resting intact cells, the generated diacylglycerol was not sufficient to cause PKC insertion into the membrane, demonstrating that both elevated Ca2+ and diacylglycerol are necessary for translocation to occur. It is concluded that while PKC activation by PMA elicits Ca2+-independent insulin secretion, the kinase seems not to mediate the stimulatory action of GTP analogs in the absence of Ca2+.
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PMID:Different requirements for protein kinase C activation and Ca2+-independent insulin secretion in response to guanine nucleotides. Endogenously generated diacylglycerol requires elevated Ca2+ for kinase C insertion into membranes. 265 16

Pancreatic islet beta-cells and insulin-producing RINm5F cells were electroporated in the presence of the c-Ha-ras oncogene, to assess the possible involvement of the encoded product in coupling extracellular receptors to phospholipase C. After two days the c-Ha-ras-transfected cells increased their expression of c-Ha-ras mRNA. These cells were also found to contain more [3H]InsP3, suggesting an increased basal (non-ligand-activated) phospholipase C activity. In addition, the transfected cells were unable to respond to ligand (bombesin) activation of phospholipase C. The ras-transfected insulin-producing cells showed enhanced phosphorylation of a 200 kDa substrate crossreacting with an antibody to an 80 kDa protein kinase C substrate. The phorbol ester 12-O-tetradecanoyl 13-acetate and bombesin also induced phosphorylation of the 200 kDa substrate. All of these changes occurred without changes in the rates of [3H]thymidine incorporation. The results suggest that the mutated c-Ha-ras oncogene directly or indirectly stimulates the basal phospholipase C activity of these cells.
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PMID:Transfection of insulin-producing cells with a transforming c-Ha-ras oncogene stimulates phospholipase C activity. 265 77

We have previously reported that the potent tumor-promoting agent 12-O-tetradecanoylphorbol-13-acetate (TPA) and a factor from fetal calf serum (FCS) markedly enhance the transformation of mouse C3H 10T1/2 and Rat 6 fibroblasts, when added to cultures following transfection with plasmid pT24 DNA that contains an activated c-Ha-ras oncogene. In the present study, we examined possible enhancing or inhibiting effects of various chemicals on the transformation of Rat 6 fibroblasts by T24 DNA when tested in the presence of calf serum, calf serum plus TPA or FCS. We found that, like TPA, the chemicals mezerein, 1-oleoyl-2-acetylglycerol, and phospholipase C increased the yield of T24-induced foci, thus further implicating protein kinase C as a critical constituent in this process. Low concentrations (10(-6)-10(-7)M) of retinoic acid (both trans and 13-cis) also stimulated cell transformation. Several compounds inhibited T24-induced transformation. These included nontoxic concentrations of the calcium ionophore A23187, indomethacin, and epsilon-amino-n-caproic acid. Compounds that failed to exert a significant reproducible effect included vasopressin, vitamin D3, selenium, antipain, Bowman-Birk inhibitor, vitamin B12, epidermal growth factor, platelet-derived growth factor, insulin, and transferrin. These findings suggest that this simple in vitro system might be useful for detecting enhancers and inhibitors of ras oncogene-induced cell transformation and also elucidating their mechanisms of action.
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PMID:Effects of various chemical agents on the transformation of rat fibroblasts by an activated c-Ha-ras oncogene. 266 19

In neonatal rat islet cells prelabelled with [14C-methyl] choline, the phorbol ester 12-O-tetradecanoylphorbol-13-acetate rapidly activated a phospholipase D-like mechanism as suggested by the accumulation in cells and medium of choline (but not of phosphorylcholine or glycerophosphorylcholine, markers for phospholipase C and phospholipase A2 action on phosphatidylcholine). This finding was confirmed by a rise in phosphatidic acid (but not diglyceride or arachidonic acid) in fatty acid-labelled cells. Phospholipase D was also activated by ionomycin or sodium fluoride; however, this was accompanied by parallel increases in diglyceride, monoacylglycerol and arachidonic acid in the absence of phosphorylcholine generation, suggesting that these agents also activated a phospholipase C-diglyceride lipase pathway acting on non-choline-containing phosphoglycerides (presumably phosphoinositides). In conjunction with our recent demonstration of insulinotropic effects of phosphatidic acid (M. Dunlop and R. Larkins, Diabetes, in press), our findings suggest for the first time a possible role for phospholipase D activation in the stimulation of insulin release and may imply a novel site of action for phorbol esters in the regulation of exocytosis.
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PMID:A phospholipase D-like mechanism in pancreatic islet cells: stimulation by calcium ionophore, phorbol ester and sodium fluoride. 267 33

Isolated islets from adult rats or obese hyperglycemic (ob/ob) mice were incubated with human recombinant interleukin 1 beta in order to study whether the acute effects of the cytokine on islet insulin release are associated with changes in islet phospholipase C activity, Ca2+ handling or protein phosphorylation. The cytokine stimulated insulin release both at low and high glucose concentrations during one hour incubations. In short-term incubations (less than 1 min) interleukin 1 beta did not affect the production of inositoltrisphosphate. Addition of interleukin 1 beta affected neither the cytoplasmic free Ca2+ concentration at rest nor that observed subsequent to stimulation with a high concentration of glucose. Furthermore, the endogenous protein kinase C activity, as visualized by immunoprecipitation of a 32P-labelled substrate for this enzyme, was not altered by interleukin 1 beta. Separation of 32P-labelled proteins by means of 2-dimensional gel electrophoresis failed to reveal any specific effects of the cytokine on the total protein phosphorylation activity. These results suggest that the stimulatory effects on insulin release exerted by interleukin 1 beta are not caused by acute activation of phospholipase C and protein kinase C or by an alteration of islet Ca2+ handling of the B-cells.
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PMID:Human interleukin 1 beta stimulates islet insulin release by a mechanism not dependent on changes in phospholipase C and protein kinase C activities or Ca2+ handling. 268 30


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