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)

We investigated the effects of an exogenous Type I phospholipase C (PLC) from clostridium perfringens on arachidonic acid release and prostaglandin synthesis from gastric mucosa by determining PGE2 release from organ cultured rabbit mucosal biopsies as well as PGE2 synthesis and substrate-dependent inactivation of the prostaglandin cyclooxygenase from endogenously released arachidonic acid in mucosal homogenate. PLC dose dependently stimulated PGE2 secretion from organ cultured mucosa to 145% and 245% at 0.1 and 1.0 U/ml during a 60 minute culture period. This effect was not affected by the calmodulin antagonist N-(6-aminohexyl)-1-5-chloro-1-naphthalene-sulfonamide (W-7) or the intracellular calcium chelator 1,2-bis-(2-aminophenoxy)ethane-N,N,N',N',-tetraacetic acid-acetoxymethyl ester (BAPTA-AM). PLC could not be substituted by phorbol-12-myristate 13-acetate (PMA), an analogue of the diacylglycerol second messenger functions. During a 15 minute preincubation of mucosal homogenate at 37 degrees C, 1mM CaCl2 stimulated PGE2 synthesis from endogenous arachidonic acid about 5-fold compared to an EDTA-control. In contrast, the residual prostaglandin synthesizing capacity, determined by incubation with excess 14C-labelled arachidonic acid, was reduced by CaCl2 to 37% of the EDTA-value. Quinacrine, an inhibitor of arachidonic acid release from phosphatidylethanolamine, reduced both the stimulation of PGE2 synthesis and the inactivation of prostaglandin cyclooxygenase. Therefore we conclude, that this Ca(2+)-effect reflects activation of the Ca-dependent phospholipase A2 (PLA2) and, as a consequence, substrate-induced inactivation of the prostaglandin cyclooxygenase.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Activation of PGE2-secretion from gastric mucosa by a type I phospholipase C is mediated by a direct release of arachidonic acid. 130 34

Extracellular ATP and UTP caused increases in the concentration of cytoplasmic free calcium ([Ca2+]i) and the intracellular level of inositol 1,4,5-trisphosphate (IP3), a second messenger for calcium mobilization, prior to the release of prostacyclin (PGI2) from cultured bovine pulmonary artery endothelial (BPAE) cells. The agonist specificity and dose-dependence were similar for nucleotide-mediated increases in IP3 levels, [Ca2+]i and PGI2 release. An increase in [Ca2+]; and PGI2 release was observed after addition of ionomycin, a calcium ionophore, to BPAE cells incubated in a calcium-free medium. The addition of ATP to the ionomycin-treated cells caused no further increase in [Ca2+]i or PGI2 release. The inability of ATP to cause an increase in [Ca2+]i or PGI2 release in ionomycin-treated cells was apparently due to the ionomycin-dependent depletion of intracellular calcium stores since the subsequent addition of extracellular calcium caused a significant increase in both [Ca2+]i and PGI2 release. Introduction of BAPTA, a calcium buffer, into BPAE cells inhibited ATP-mediated increases in [Ca2+]i and PGI2 release, further evidence that PGI2 release is dependent upon an increase in [Ca2+]i. The increase in [Ca2+]i elicited by ATP apparently caused the activation of a calmodulin-dependent phospholipase A2 since trifluoperazine, an inhibitor of calmodulin, and quinacrine, an inhibitor of phospholipase A2, prevented the stimulation of PGI2 release by ATP. Furthermore, ATP caused the specific hydrolysis of [14C]arachidonyl-labeled phosphatidylcholine and the generation of free arachidonic acid, the rate-limiting substrate for PGI2 synthesis, prior to the release of PGI2 from BPAE cells. These findings suggest that the increase in PGI2 release elicited by ATP and UTP is at least partially dependent upon a phospholipase C-mediated increase in [Ca2+]i and the subsequent activation of a phosphatidylcholine-specific phospholipase A2. ATP analogs modified in the adenine base or phosphate moiety caused PGI2 release with a rank order of agonist potency of adenosine 5'-O-(2-thiodiphosphate) (ADP beta S) greater than 2-methylthioATP (2-MeSATP) greater than ATP, whereas alpha, beta methyleneATP and beta, gamma methyleneATP had no effect on PGI2 release.
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PMID:Mechanisms by which extracellular ATP and UTP stimulate the release of prostacyclin from bovine pulmonary artery endothelial cells. 131 59

We studied the relationship between phosphoinositide hydrolysis, phosphatidylcholine hydrolysis, and sn-1,2-diacylglycerol (DAG) formation in response to carbachol stimulation in rat parotid acinar cells. Previously, we demonstrated that DAG formation stimulated with 1 microM carbachol was biphasic: the first peak occurred at 5 min and the second one at 20 min. It was also demonstrated that the second peak was regulated in part by a calmodulin/protein kinase C-dependent mechanism. Based on the kinetic analysis of DAG formation and [32P]phosphoinositide breakdown, the first peak of carbachol (1 microM)-stimulated DAG accumulation was found to be related to the breakdown of [32P]phosphatidylinositol 4-monophosphate ([32P]PIP) and [32P]phosphatidylinositol 4,5-bisphosphate ([32P]PIP2). The second peak was found to be related to [32P]PIP2 breakdown. Carbachol stimulated the release of [3H]phosphocholine into the medium, indicating that the predominant pathway for phosphatidylcholine hydrolysis was via phospholipase C. Moreover, carbachol stimulated the release of [3H]choline metabolites in a time- and dose-dependent manner. This agonist slightly stimulated the release of [3H]ethanolamine metabolites. A calmodulin/protein kinase C-dependent mechanism was also studied and was found to be involved in carbachol-stimulated phosphatidylcholine hydrolysis; W-7, a calmodulin inhibitor and staurosporine, a protein kinase C inhibitor, inhibited the carbachol (1-microM)-induced release of [3H]choline metabolites at 20 min in a dose-dependent manner, but did not have inhibitory effects at 5 min. These results suggest that the first peak of DAG accumulation induced by carbachol is predominantly associated with the breakdown [32P]PIP and of [32P]PIP2 and that the second peak is predominantly associated with [32P]PIP2 breakdown and phosphatidylcholine hydrolysis.
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PMID:Mechanism of carbachol-stimulated diacylglycerol formation in rat parotid acinar cells. 132 65

In the dog iris sphincter, muscarinic acetylcholine receptors are coupled either to the stimulation of phospholipase C and muscle contraction or to the stimulation of adenylate cyclase and muscle relaxation, this was found to be dependent upon the concentration of the muscarinic agonist. In contrast to the dog, muscarinic receptors in iris sphincters from different mammalian species were found to be coupled to phospholipase C and contraction at all concentrations of carbachol investigated (1-100 microM). In the dog sphincter, lower concentrations (less than 5 microM) of carbachol stimulated myo-inositol 1,4,5-trisphosphate (IP3) production, inhibited cAMP formation and induced contraction, and higher concentrations (greater than 5 microM) enhanced cAMP formation, inhibited IP3 production and induced relaxation. The mechanisms for the stimulatory effects on cAMP formation through muscarinic receptors were investigated. Carbachol (25 microM) increased both basal and isoproterenol- and forskolin-stimulated cAMP levels. Atropine inhibited the carbachol-stimulated increase in cAMP levels in a dose-dependent manner with an IC50 of 9 nM. Intracellular Ca2+, derived from IP3-induced Ca2+ release and/or from muscarinic receptor-operated Ca2+ influx, and protein kinase C may mediate the muscarinic receptor-linked rise in intracellular cAMP. This conclusion is supported by the following findings. (1) At short time intervals (less than 1 min) carbachol (25 microM) increased IP3 production and contraction and this was followed (between 1 and 20 min) by cAMP formation and muscle relaxation. (2) Carbachol-stimulated IP3 production was detected at a concentration of the agonist 26-fold lower than that required for cAMP formation, and it was completely blocked by the phorbol ester, phorbol 12,13-dibutyrate (50 nM). (3) A Ca(2+)-calmodulin stimulated adenylate cyclase was demonstrated in membranes from dog iris sphincter but not in that from rabbit and bovine. (4) Trifluoperazine (0.1 microM), a calmodulin antagonist, inhibited the carbachol-stimulated cAMP accumulation. (5) The Ca2+ ionophore A23187 and the phorbol ester increased cAMP production in a dose-dependent manner. A23187 potentiated cAMP production induced by either carbachol or by the phorbol ester. (6) Muscarinic stimulation of cAMP production persisted even after the tissue was pretreated with the phorbol ester or staurosporine. (7) Nifedipine (0.01-0.5 microM), a Ca2+ channel antagonist, inhibited carbachol stimulation of cAMP production, suggesting the presence of a muscarinic receptor-operated Ca2+ influx pathway in this tissue.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Carbachol stimulates adenylate cyclase and phospholipase C and muscle contraction-relaxation in a reciprocal manner in dog iris sphincter smooth muscle. 132 47

We have shown previously that angiotensin-II (A-II) controls proto-oncogene (c-fos, jun-B and c-jun) mRNA accumulation in bovine adrenal fasciculata cells (BAC). Since BAC contain both subtypes (AT-1 and AT-2) of the A-II receptor, we have investigated which subtype was involved in the effect of A-II on proto-oncogene mRNA by using a selective antagonist for AT-1 (DUP 753) and for AT-2 (CGP 42112A). DUP 753, but not CGP 42112A, inhibited the stimulatory effect of A-II on proto-oncogene mRNA, with ID50s of 4 x 10(-7) M, 7 x 10(-7) M and 2 x 10(-6) M for c-fos, jun-B and c-jun, respectively. Neither of the two antagonists by themselves had a direct effect on proto-oncogene mRNA. As the A-II AT-1 receptors are coupled to the phospholipase C system in BAC, we have investigated whether the A-II effects on the proto-oncogenes were mediated by protein kinase C (PKC) or by Ca2+ calmodulin. First, activation of PKC by the phorbol ester, PMA, increased the level of three proto-oncogene mRNAs, whereas calcium ionophore had no effect. Second, staurosporine, a specific inhibitor of PKC, reduced the stimulatory action of A-II on proto-oncogene mRNA by 80-90%, whereas trifluoroperazine, an inhibitor of calmodulin, had no significant effect. These results demonstrate that the effects of A-II on proto-oncogene mRNA are mediated by AT1 receptor subtypes, mainly through activation of the PKC pathway.
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PMID:Angiotensin-II-induced expression of proto-oncogene (c-fos, jun-B and c-jun) mRNA in bovine adrenocortical fasciculata cells (BAC) is mediated by AT-1 receptors. 142 67

We describe here and partially characterize a Ca(2+)-independent phospholipase A2 that acts on phosphatidylinositol in normal human peripheral blood neutrophils. Neutrophils incubated with myo-[3H]inositol to form [3H]phosphatidylinositol and then stimulated with the calcium ionophore A23187 produced [3H]lysophosphatidylinositol. This deacylation was further characterized in cell sonicates by the specific release of [3H]arachidonic acid from exogenous [1-14C]stearoyl-2-[3H]arachidonyl-phosphatidylinositol. This phospholipase A2 is Ca2+ independent, retaining full activity in the presence of 10 mM EDTA, and is optimally active at alkaline pH (pH 9). A phosphatidylinositol-hydrolyzing phospholipase C activity was characterized by the production of [3H]-/[14C]-diglycerides. This phospholipase C activity is dependent on the presence of exogenous Ca2+ and is optimally active at neutral pH (pH 7.5). The lipoxygenase/cyclooxygenase inhibitors eicosatetraenoic acid and nordihydroguaiaretic acid and the calmodulin antagonist trifluoperazine were the only compounds tested that showed significant inhibition of phospholipase A2 activity. However, none of these phosphatidylinositol-hydrolyzing phospholipase A2 inhibitory compounds resulted in the accumulation of any radiolabeled diglyceride, monoglyceride, or phosphatidic acid intermediates. Following subcellular fractionation on sucrose density gradients, it was found that the plasma membrane-enriched fractions contained the highest specific activity for phospholipase A2; however, the cytosolic fraction contained a large part of the total phospholipase A2 activity. Furthermore, when neutrophils were first exposed to several agents, including lipopolysaccharide, phorbol myristate acetate, or N-formyl-methionyl-leucyl- phenylalanine, and then subfractionated, there was a significant translocation of the enzyme activity from the cytosolic fraction to the membrane-enriched fractions. These data suggest that this Ca(2+)-independent, phosphatidylinositol-hydrolyzing phospholipase A2 may play an important role in early cell activation, providing free arachidonic acid for subsequent metabolism into biologically active eicosanoids.
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PMID:Phosphatidylinositol hydrolysis by phospholipase A2 and C activities in human peripheral blood neutrophils. 146 38

Maximal calcium-activated tension (Tmax) was measured from saponin-and alpha-toxin permeabilized strips of rat anococcygeus muscle stimulated repetitively. 50 mins after saponin treatment, Tmax had declined to 20% of the initial value. Calmodulin (1 microM) only partially reduced this decline. Lowering the ionic strength of the bathing solution from 0.2M to 0.07M markedly reduced the rate of decline of tension. Addition of calmodulin to the bathing medium of low ionic strength prevented the decline of force still further; 50 mins after permeabilization, Tmax declined to 80% of the original level. Loss of endogenous calmodulin should be prevented by permeabilization with alpha-toxin from Staphylococcus aureus. Nevertheless, Tmax still declined to approximately 50% of the original value after 50mins. However, negligible force reduction was observed if the bathing solution had an ionic strength of 0.07M.
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PMID:Effects of low ionic strength and calmodulin on the decline in maximal calcium-activated force in permeabilized smooth muscle. 152 74

The purpose of this study was to determine the cellular basis for fluoride ion (F-)-induced contractions of isolated aortic rings from both the rat and the rabbit. The F- contractions were not affected by endothelial denudation but were enhanced in the presence of A (0.1 or 1.0 mM) added to the bathing Krebs' solution. The contractile effect of F- also was not modified by bathing with Ca(++)-free + ethylene glycol bis(b-aminoethylether)-N,N-tetracetic acid Krebs' solution or nifedipine (10 microM), but was attenuated by inorganic (Cd++, Co++ and Ni++) Ca++ antagonists in normal and Ca(++)-free Krebs' media. Bis(o-aminophenoxy)-ethane-N-N-N'-N'-tetraacetic acid, ryanodine and intracellular Ca++ modulators, respectively, caused 36.1 +/- 6.1%, 16.4 +/- 6.8% and 52.3 +/- 7.3% inhibition of the contractile response to F- in a Ca(++)-free media while causing near complete inhibition of norepinephrine-induced contractions. F- contractions were also inhibited by the calmodulin antagonists W-7 and calmidazolium (IC50 = 23.0 +/- 7.0 and 45.0 +/- 10.0 microM, respectively). On the other hand, the protein kinase C antagonists staurosporine and H-7 potently (IC50 = 0.016 +/- 0.007 and 1.1 +/- 0.5 microM, respectively) inhibited the fluoride-induced contractions. Aortic rings from the rabbit were similarly potently antagonized by the protein kinase C inhibitors, however, K(+)-induced contractions were also equally sensitive to these agents in both rat and rabbit tissues. The putative phospholipase C inhibitor neomycin was significantly less effective (IC50 = 13.0 +/- 5.0, 0.44 +/- 0.09 and 0.89 +/- 0.40 mM) at inhibiting F- than norepinephrine and KCl contractile effects.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Mechanism of vascular smooth muscle contraction by sodium fluoride in the isolated aorta of rat and rabbit. 164 1

Calmodulin antagonists stimulated phosphatidylinositol-4,5-bisphosphate phospholipase C in soluble and particulate fractions of bovine rod outer segments. Antagonists tested include trifluoperazine, melittin, calmidazolium, compound 48/80, W-13 [N-(4-aminobutyl)-5-chloro-1-naphthalenesulfonamide], and W-7 [N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide]. All were effective, but W-7 was chosen for further characterization of the effect, which was most pronounced in the soluble fraction. Phospholipase C activity in the soluble fraction did not increase linearly with the quality of enzyme assayed, suggesting the presence of an endogenous inhibitor or an inhibitory self-association of the enzyme. W-7 appeared to counteract this inhibition, resulting in a linear activity-quantity relationship. Stimulation by W-7 was therefore largest when large amounts of crude enzyme were assayed and small or nil when small amounts were assayed. The effect of W-7 was also dependent on [Ca2+], with half-maximal stimulation occurring between 0.1 and 1 microM. W-7 and W-13 were much more effective than their nonchlorinated analogues W-5 and W-12 at increasing phospholipase C activity. While this pattern of effectiveness is typical of calmodulin-mediated processes, the absence of any effect by added calmodulin and the retention of W-7 sensitivity by purified CaM-free enzyme argue against regulation by CaM. Octyl glucoside, a nonionic detergent, mimicked some of the effects of CaM antagonists, suggesting that the antagonists act by interfering with protein-protein interactions. It appears likely that CaM antagonists prevent an inhibitory multimerization or aggregation of at least one form of ROS phospholipase C.
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PMID:Activation of bovine rod outer segment phosphatidylinositol-4,5-bisphosphate phospholipase C by calmodulin antagonists does not depend on calmodulin. 165 98

Phosphoinositide-specific phospholipase C (PI-PLC) activity in whole homogenates of mouse pancreatic islets decreased 60-85% when the homogenates were incubated at 37 degrees C for 1 h in the presence of down to micromolar concentrations of Ca2+. Ca(2+)-induced inactivation was augmented by calmodulin, the phorbol ester 12-O-tetradecanoylphorbol 13-acetate in the presence of ATP-Mg, and by Mg2+. Inactivation was inhibited when ATP was removed and completely abolished by trifluoperazine and EGTA. Inactivation was not affected by the non-phosphorylating ATP analogue, AMP-PCP, GMP-PNP, glucose, Zn2+ or a series of protease inhibitors. These observations suggest that PI-PLC in broken cell preparations of pancreatic islets may be inactivated via phosphorylation by Ca(2+)-calmodulin-stimulated protein kinase and/or protein kinase C. Inactivation of PI-PLC was reversible. Reactivation started after approx. 2 h incubation, when the concentration of ATP in the homogenate was below 0.15 x 10(-6) M. PI-PLC activity returned to values approx. 25% higher than the initial values. PI-PLC inactivation via phosphorylation by the mentioned protein kinases may constitute a feedback control on the phosphoinositide response, attenuating subsequent diacylglycerol formation and/or Ca2+ mobilization by inositol trisphosphate.
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PMID:Ca(2+)- and ATP-dependent reversible inactivation of pancreatic islet phosphoinositide-specific phospholipase C activity. 166 65


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