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)

Long-term ethanol exposure is known to inhibit bradykinin-stimulated phosphoinositide hydrolysis in cultures of neuroblastoma x glioma 108-15 cells. In the present study, [3H]bradykinin binding, GTP-binding protein function, and phospholipase C activity were assayed in cells grown for 4 days in 100 mM ethanol with the aim of elucidating the molecular target of ethanol on signal transduction coupled to inositol trisphosphate and diacylglycerol formation. Ethanol exposure reduced guanosine 5'-O-(3-thiotriphosphate) [GTP(S)]- and, to a lesser extent, NaF/AlCl3-stimulated phosphoinositide hydrolysis, whereas it had no effect on the enzymatic activity of a phosphatidylinositol 4,5-bisphosphate-specific phospholipase C. [3H]Bradykinin binding in the absence of GTP(S) was not influenced by ethanol exposure. However, the reduction in [3H]bradykinin binding seen in control cells after addition of GTP analogue was inhibited in cells grown in ethanol-containing medium. The results indicate that long-term ethanol exposure exerts its effects on receptor-stimulated phosphoinositide hydrolysis primarily at the level of the GTP-binding protein.
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PMID:G proteins coupled to phospholipase C: molecular targets of long-term ethanol exposure. 185 Dec 10

The influence of acute and chronic ethanol exposures on the coupling of neurotensin and bradykinin receptors to phospholipase C was determined in intact N1E-115 cells. Phospholipase C was monitored by the formation of total [3H]inositol phosphates in the presence of lithium in cells prelabeled with [3H]inositol. Acute exposure to ethanol over a range of 50 to 200 mM inhibited the stimulation of [3H]inositol phosphate formation elicited by neurotensin and bradykinin. In cells chronically exposed to 100 mM ethanol for 7 days, neither basal- nor neurotensin-stimulated [3H]inositol phosphate formation differed significantly from those of control (untreated) cells. In contrast, the [3H]inositol response to bradykinin was significantly inhibited in cells chronically exposed to ethanol. Because chronic ethanol exposure had no parallel effects on either the specific binding of [3H]bradykinin or the stimulation of [3H]inositol phosphate formation by the stable GTP analog, guanine 5'-(y-thiotriphosphate), it is suggested that chronic ethanol impairs the ability of bradykinin receptors to activate the guanine nucleotide binding protein associated with phospholipase C. In addition, because chronic ethanol had no effect on the inositol phosphate response to neurotensin, it is proposed that certain types of receptor-guanine nucleotide binding protein interactions are more vulnerable than are others to disruption by chronic ethanol treatment.
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PMID:Selective effects of acute and chronic ethanol exposure on neuropeptide and guanine nucleotide stimulated phospholipase C activity in intact N1E-115 neuroblastoma. 190 59

12-O-Tetradecanoylphorbol-13-acetate (TPA) stimulated the release of [3H]ethanolamine from HeLa cells prelabeled with [3H]ethanolamine within 2 min, and of [3H]choline from cells prelabeled with [3H]choline after a lag of 10-20 min. This result suggests that TPA activates phospholipase D. Propranolol alone or propranolol plus TPA stimulated phosphatidic acid (PA) labeling in cells prelabeled with [3H]hexadecanol. In the presence of ethanol, TPA stimulated the accumulation of labeled phosphatidylethanol (PEth); no PEth was formed in the absence of TPA. TPA-dependent PEth accumulation was not observed in cells pretreated with TPA to down-regulate protein kinase C, whereas propranolol-induced accumulation of PA was unaffected by TPA pretreatment. Incubation of prelabeled cells with propranolol alone caused a rapid loss of label and phospholipid mass from both phosphatidylethanolamine and phosphatidylcholine (PC) together with an accumulation of PA and phosphatidylinositol plus phosphatidylserine. When [3H]hexadecanol-prelabeled cells were pulse labeled with 32P to label nucleotide pools, propranolol induced the accumulation of both 3H- and 32P-labeled PA. When cells were prelabeled with lyso-PC double labeled with 3H and 32P, and incubated with propranolol, only 3H-labeled PA accumulated, indicating that the pathways involved in the basal turnover of PC resulted in the loss of 32P from the lipid. These results suggest that the basal turnover of phosphatidylethanolamine and PC involves the sequential actions of phospholipase C, diglyceride kinase, and PA phosphohydrolase.
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PMID:Phorbol ester-stimulated hydrolysis of phosphatidylcholine and phosphatidylethanolamine by phospholipase D in HeLa cells. Evidence that the basal turnover of phosphoglycerides does not involve phospholipase D. 193 84

1. The mechanism of gastroprotective action of an antiulcer drug, sucralfate, was investigated. Studies in vivo were conducted with groups of rats with and without indomethacin pretreatment, and the animals received sucralfate followed by ethanol. In the in vitro system, gastric mucosa was cultured in the presence of sucralfate with and without indomethacin. 2. The in vivo experiments revealed that ethanol caused extensive gastric lesions which were significantly reduced following sucralfate pretreatment. Furthermore, sucralfate was also capable of preventing the detrimental effect of indomethacin on gastric mucus gel dimension and its mucin content. 3. The data with gastric mucosal culture showed that the sucralfate elicited increase in mucin was accompanied by the enhanced turnover of mucosal phosphoinositides. 4. Regardless of the inclusion of indomethacin, sucralfate evoked 23% reduction in phosphatidylinositol, 24% increase in inositol-1-phosphate and 3.4-fold increase in inositol-1,4,5-trisphosphate, thus indicating the activation of phosphoinositide-specific phospholipase C. 5. The results demonstrate that the gastric mucosal protective action of sucralfate is not mediated by endogenous prostaglandins, but appears to involve the metabolism of phosphoinositide-derived messenger molecules.
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PMID:Gastric mucosal protection by sucralfate involves phosphoinositides participation. 196 55

The enzymatic pathways for formation of 1,2-diradylglyceride in response to epidermal growth factor in human dermal fibroblasts have been investigated. 1,2-Diradylglyceride mass was elevated 2-fold within one minute of addition of EGF. Maximal accumulation (4-fold) occurred at 5 minutes. Since both diacyl and ether-linked diglyceride species occur naturally and may accumulate following agonist activation, we developed a novel method to determine separately the alterations in diacyl and ether-linked diglycerides following stimulation of fibroblasts with EGF. Utilizing this method, it was found that approximately 80% of the total cellular 1,2-diradylglyceride was diacyl, the remaining 20% being ether-linked. Addition of EGF caused accumulation of 1,2-diacylglyceride without alteration in the level of ether-linked diglyceride. Thus, the observed induction of 1,2-diradylglyceride by EGF was due exclusively to increased formation of 1,2-diacylglyceride. In cells labelled with [3H]choline, the water soluble phosphatidylcholine hydrolysis products, phosphorylcholine and choline, were increased 2-fold within 5 minutes of addition of EGF. No hydrolysis of phosphatidylethanolamine, phosphatidylserine, or phosphatidylinositol was observed. Quantitation by radiolabel and mass revealed equivalent elevations in phosphorylcholine and choline, suggesting stimulation of both phospholipase C and phospholipase D activities. To identify the presence of EGF-induced phospholipase D activity, cells were labelled with exogenous [3H]1-0-hexadecyl, 2-acyl phosphatidylcholine and its conversion to phosphatidic acid in response to EGF determined. Radiolabelled phosphatidic acid was detectable in 15 seconds after addition of EGF and was maximal (3-fold) at 30 seconds. Consistent with the presence of EGF-induced phospholipase D activity, treatment of cells with EGF, in the presence of [14C]ethanol, resulted in the rapid formation of [14C]phosphatidylethanol, the product of phospholipase D-catalyzed transphosphatidylation. The formation of phosphatidylethanol, which competes for the formation of phosphatidic acid by phospholipase D, did not diminish the induction of 1,2-diglyceride by EGF. These data suggest that the phosphatidic acid formed by phospholipase D-catalyzed hydrolysis of phosphatidylcholine is not a major precursor of the observed increased 1,2-diglyceride. Thus, the induction of 1,2-diacylglycerol by EGF may occur primarily via phospholipase C-catalyzed hydrolysis of phosphatidylcholine.
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PMID:Epidermal growth factor-induced hydrolysis of phosphatidylcholine by phospholipase D and phospholipase C in human dermal fibroblasts. 199 79

We have investigated the coupling of muscarinic acetylcholine receptors (mAChR) to phospholipid hydrolysis in a human neuroblastoma cell line, LA-N-2, by measuring the formation of 3H-inositol phosphates (3H-IP) and of [3H]phosphatidylethanol ([3H]PEt) in cells prelabeled with [3H]inositol and [3H]oleic acid. The muscarinic agonist carbachol (CCh) stimulated the phospholipase C (PLC)-mediated formation of 3H-IP in a time- and dose-dependent manner (EC50 = 40-55 microM). In addition, in the presence of ethanol (170-300 mM), CCh elevated levels of [3H]PEt [which is regarded as a specific indicator of phospholipase D (PLD) activity] by three- to sixfold. The effect of CCh on PEt formation also was dose dependent (EC50 = 50 microM). Both effects of CCh were antagonized by atropine, indicating that they were mediated by mAChR. Incubation of LA-N-2 cells with the phorbol ester phorbol 12-myristate 13-acetate (PMA, 0.1 microM; 10 min) increased [3H]PEt levels by up to 10-fold. This effect was inhibited by the protein kinase C (PKC) inhibitor staurosporine (1 microM) or by pretreatment for 24 h with 0.1 microM PMA, by 74% and 65%, respectively. In contrast, the effect of CCh on PEt accumulation was attenuated by only 28% in the presence of staurosporine (1 microM). In summary, these results suggest that, in LA-N-2 neuroblastoma cells, mAChR are coupled both to phosphoinositide-specific PLC and to PLD. PKC is capable of stimulating PLD activity in these cells; however, it is not required for stimulation of the enzyme by mAChR activation.
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PMID:Stimulation of phospholipase D activity in human neuroblastoma (LA-N-2) cells by activation of muscarinic acetylcholine receptors or by phorbol esters: relationship to phosphoinositide turnover. 200 44

Muscarinic receptor-induced changes in the activities of phospholipase D (PLD) and of phosphoinositide-phospholipase C (PI-PLC) were investigated in human embryonic kidney (HEK) cells transfected with, and stably expressing, the human m1, m2, m3, and m4 mAChR subtypes, respectively. PLD and PI-PLC activities in these four transfected cell lines as well as in nontransfected cells were measured by the formation of [3H]phosphatidylethanol [( 3H]PEt) and [3H]inositol phosphates [( 3H]IP) after labeling cellular phospholipids with [3H]oleic acid and [3H]inositol. The muscarinic receptor agonist carbachol had no significant effects on [3H]PEt and [3H]IP formation in nontransfected HEK cells. In cells expressing the m1 or m3 receptors carbachol (1 mM; in the presence of 400 mM ethanol and 10 mM lithium chloride) caused the formation of [3H]PEt of about 12,000 cpm/mg protein (basal PEt formation was not measurable) and increased [3H]IP formation by 20,000-30,000 cpm/mg (a 7-10-fold increase over basal levels). The EC50 values (0.3-1.5 microM) were similar for both effects and both mAChR subtypes. In contrast, in cells expressing m2 or m4 receptor subtypes the magnitude of [3H]PEt (about 4,000 cpm/mg protein) or [3H]IP (3,000-4,000 cpm/mg) formation was much smaller and the EC50 values (20-40 microM) much higher than for the m1 and m3 receptors. Neomycin (1 mM) inhibited the m1 and m3 receptor-mediated production of IP by 50%, whereas the PEt formation was attenuated by 20% in the same cells. We conclude that activation of all of the four mAChR subtypes, although with different efficiencies, can stimulate PLD. The m1 and m3 receptor-mediated stimulation of the PLD may be at least partially independent of the PI-PLC stimulation.
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PMID:Coupling of transfected muscarinic acetylcholine receptor subtypes to phospholipase D. 200 63

Bradykinin (BK) and phorbol 12-myristate 13-acetate (PMA) both stimulate the hydrolysis of phosphatidylcholine (PC) in human fibroblasts, resulting in the formation of phosphatidic acid (PA) and diacylglycerol (DG) (Van Blitterswijk, W.J., Hilkmann, H., de Widt, J., and Van der Bend, R.L. (1990) J. Biol. Chem. 266, 10337-10343). Stimulation with BK resulted in the rapid and synchronous formation of [3H]choline and [3H]myristoyl-PA from the correspondingly prelabeled PC, indicative of phospholipase D (PLD) activity. In the presence of ethanol or n-butanol, transphosphatidylation by PLD resulted in the formation of [3H]phosphatidylethanol or - butanol, respectively, at the cost of PA and DG formation. This suggests that PC-derived DG is generated via a PLD/PA phosphohydrolase pathway. A more pronounced but delayed formation of these products was observed by PMA stimulation. The Ca2+ ionophore ionomycin also activated PLD and accelerated (synergized) the response to PMA. Both [3H] choline and [3H]phosphocholine were released into the extracellular medium in a time- and stimulus-dependent fashion, without apparent changes in the high intracellular levels of [3H]phosphocholine. The protein kinase C (PKC) inhibitors staurosporin and 1-O-hexadecyl-2-O-methylglycerol inhibited BK- and PMA-induced activation of PLD. Down-regulation of PKC by long-term pretreatment of cells with phorbol ester caused a dramatic drop in background [3H]choline levels, while subsequent stimulation with BK, ionomycin, or PMA failed to increase these levels and failed to induce transphosphatidylation. From these results we conclude that PLD activation is entirely mediated by (downstream of) PKC. Unexpectedly, however, BK stimulation of these PKC-depleted cells caused a marked generation of DG from PC within 15 s, which was not seen in BK-stimulated control cells, suggesting PC breakdown by a phospholipase C (PLCc). We conclude that cells stimulated with BK generate DG via both the PLCc and the PLD/PA hydrolase pathway, whereas PMA stimulates mainly the latter pathway. BK stimulation of normal cells leads to activation of PKC and, by consequence, to attenuation of the level of PLCc-generated DG and to stimulation of the PLD pathway, whereas the reverse occurs in PKC-down-regulated cells.
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PMID:Phospholipid metabolism in bradykinin-stimulated human fibroblasts. II. Phosphatidylcholine breakdown by phospholipases C and D; involvement of protein kinase C. 203 86

Normodense human eosinophils have been labeled in 1-0-alkyl-phosphatidylcholine (alkyl-PC) with 32P by incubating isolated cells with alkyl-[32P]lysoPC. Stimulation of these 32P-labeled cells with C5a, A23187 or PMA in the presence of 0.5% ethanol resulted in time- and dose-dependent formation of alkyl-[32P]phosphatidic acid (alkyl-[32P]PA) and alkyl-[32P]phosphatidylethanol (alkyl-[32P]PEt). Because cellular ATP does not contain 32P, alkyl-[32P]PA must have been formed by the hydrolytic action of phospholipase D (PLD) and not by the combined actions of phospholipase C and DG kinase. Regardless of the stimulating agent, alkyl-[32P]PEt formation paralleled that of alkyl-[32P]PA, suggesting that alkyl-PEt was the result of a PLD-catalyzed transphosphatidylation reaction between alkyl-PC and ethanol. These data provide the first definitive proof of receptor- and nonreceptor-mediated activation of PLD in normodense eosinophils derived from human blood.
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PMID:Activation of phospholipase D in normodense human eosinophils. 211 91

Ethanol is known to inhibit the activation of platelets in response to several physiological agonists, but the mechanism of this action is unclear. The addition of physiologically relevant concentrations of ethanol (25-150 mM) to suspensions of washed human platelets resulted in the inhibition of thrombin-induced secretion of 5-hydroxy[14C]tryptamine. Indomethacin was included in the incubation buffer to prevent feedback amplification by arachidonic acid metabolites. Ethanol had no effect on the activation of phospholipase C by thrombin, as determined by the formation of inositol phosphates and the mobilization of intracellular Ca2+. Moreover, ethanol did not interfere with the thrombin-induced formation of diacylglycerol or phosphatidic acid. Stimulation of platelets with phorbol ester (5-50 nM) resulted in 5-hydroxy[14C]tryptamine release comparable with those with threshold doses of thrombin. However, ethanol did not inhibit phorbol-ester-induced secretion. Ethanol also did not interfere with thrombin- or phorbol-ester-induced phosphorylation of myosin light chain (20 kDa) or a 47 kDa protein, a known substrate for protein kinase C. By electron microscopy, ethanol had no effect on thrombin-induced shape change and pseudopod formation, but prevented granule centralization and fusion. The results indicate that ethanol does not inhibit platelet secretion by interfering with the activation of phosphoinositide-specific phospholipase C or protein kinase C by thrombin. Rather, the data demonstrate an inhibition of a Ca2(+)-mediated event such as granule centralization.
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PMID:Ethanol inhibits thrombin-induced secretion by human platelets at a site distinct from phospholipase C or protein kinase C. 211 42


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