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 vasoconstrictor effect, the binding, and the response of inositol phosphates to endothelin-1 (ET-1) were investigated in blood vessels of deoxycorticosterone acetate (DOCA)-salt hypertensive rats within 2 weeks of development of hypertension and in uninephrectomized control rats. In DOCA-salt and uninephrectomized rats, plasma levels of endothelin were similar (1.2 +/- 0.1 fmol/ml). Thoracic aorta and mesenteric artery rings devoid of endothelium presented significantly decreased responses to increasing concentrations of ET-1. Binding of ET-1 to mesenteric artery membranes was significantly lower in DOCA-salt rats (106 +/- 22 fmol/mg protein) than in uninephrectomized rats (172 +/- 19 fmol/mg protein, p less than 0.05), whereas affinity was similar. Phosphoinositide metabolism was examined in aorta and mesenteric arteries after incubation with [3H]myoinositol. Inositol phosphates were separated by high-performance liquid chromatography. In response to 100 nmol/l ET-1, accumulation of inositol 1,4,5-trisphosphate after 20 seconds and of inositol monophosphate, inositol bisphosphate, and inositol 1,3,4-triphosphate after 30 minutes (in the presence of 25 mmol/l LiCl) were significantly lower in DOCA-salt hypertensive than in uninephrectomized control rats, in both aorta and mesenteric arteries. In conclusion, decreased density of ET-1 receptors in DOCA-salt hypertensive rats results in decreased activation of phospholipase C and, consequently, reduced vasoconstriction induced by ET-1. Because the decrease in vasoconstrictor effects of ET-1 is found in the absence of endothelium, it is likely that receptor downregulation rather than prior receptor occupancy underlies these findings.
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PMID:Endothelin vascular receptors and responses in deoxycorticosterone acetate-salt hypertensive rats. 131 Apr 86

The accumulation of both Inositol-(1,4,5)-trisphosphate (IP3) and Inositol-(1,3,4,5)-tetrakisphosphate (IP4) after hormonal stimulation has a physiological role, possibly in altering Ca2+ levels in cardiac tissue. However, the accumulation of inositol polyphosphate under pathophysiological conditions has not been studied. In our experiments the metabolism of phatidylinositol and IP3 in cardiac myocytes as investigated. It was shown that basal levels of cytosolic phosphatidylinositol specific phospholipase C (PI-PLC), phosphatidylinositol-(4,5)-bisphosphate specific phospholipase C (PIP2-PLC) activities markedly increased in stroke-prone spontaneously hypertensive rats (SHRSP) with age compared with age matched Wistar Kyoto rats (WKY). IP3 kinase and IP3 phosphatase activities also increased in SHRSP hearts with age. Their activities increased in WKY, but to a lesser extent than in SHRSPs. These data suggest that a PI turnover pathway such as the phosphatidylinositol 4,5-bisphosphate-IP3-Ca2+ pathway or the diacylglyceride-protein kinase C pathway may have an important role in the development of hypertrophy in SHRSP heart.
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PMID:Phosphatidylinositol and inositolphosphatide metabolism in hypertrophied rat heart. 131 48

[3H]Inositol ([3H]Ins) labeling of phosphoinositides was studied in rat brain cortical membranes. [3H]Ins was incorporated into a common lipid pool through both CMP-dependent and independent mechanisms. These are as follows: (1) a reverse reaction catalyzed by phosphatidyl-inositol (PtdIns) synthase, and (2) the reaction performed by the PtdIns headgroup exchange enzyme, respectively. Membrane phosphoinositides prelabeled in either CMP-dependent or independent fashions were hydrolyzed by guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S)- and carbachol-stimulated phospholipase C. Unlike CMP-dependent labeling, however, CMP-independent incorporation of [3H]Ins into lipids was inhibited by 1 mM (0.04%) sodium deoxycholate. Thus, when PtdIns labeling and phospholipase C stimulation were studied in a concerted fashion, [3H]Ins was incorporated into lipids primarily through the PtdIns synthase-catalyzed reaction because of the presence of deoxycholate required to observe carbachol-stimulation of phospholipase C. Little direct breakdown of [3H]PtdIns was detected because production of myo-[3H]inositol 1-monophosphate was minimal and myo-[3H]inositol 1,4-bisphosphate was the predominant product. Although PtdIns labeling and 3H-polyphosphoinositide formation were unaffected by GTP gamma S and carbachol and had no or little lag period, GTP gamma S- and carbachol-stimulated appearance of 3H-Ins phosphates exhibited an appreciable lag (10 min). Also, flux of label from [3H]Ins to 3H-Ins phosphates was restricted to a narrow range of free calcium concentrations (10-300 nM). These results show the concerted activities of PtdIns synthase, PtdIns 4-kinase, and phospholipase C, and constitute a simple assay for guanine nucleotide-dependent agonist stimulation of phospholipase C in a brain membrane system using [3H]Ins as labeled precursor.
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PMID:Concerted CMP-dependent [3H]inositol labeling of phosphoinositides and agonist activation of phospholipase C in rat brain cortical membranes. 131 77

We have identified a Ca(2+)-dependent polyphosphoinositide-specific phospholipase C activity in Dictyostelium discoideum. Addition of Ca2+ (20 microM) results in the rapid formation of Ins(1,4,5)P3 within 5 s and leads to sustained inositol phosphate production for up to 40 min in membranes prepared from [3H]inositol-labelled cells. The phospholipase C activity is primarily membrane-bound under the conditions used to lyse the cells. In addition to this activity we also identified a family of Ca(2+)-regulated phospholipase activities active on a range of phospholipid substrates, using [3H]palmitate labelling. Inositol-specific phospholipase C activity is highest in vegetatively growing cells and in starved cells during the first 6 h in development, during which time Ca2+ elicited a 5-fold stimulation of inositol phosphate formation. After this time, total activity decreased progressively until 15 h, after which the activity remained constant up until 24 h. During this period, Ca2+ was able to stimulate a 2-fold increase in inositol phosphates.
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PMID:Characterization of phospholipase activity in Dictyostelium discoideum. Identification of a Ca(2+)-dependent polyphosphoinositide-specific phospholipase C. 131 14

The present studies were conducted to evaluate the effects of protein kinase C activators on the inositol phospholipid-phospholipase C second messenger system in isolated bovine luteal cells. This report describes the effects of phorbol esters on inositol phosphate accumulation in LH- and prostaglandin F2 alpha (PGF2 alpha)-stimulated bovine luteal cells. Corpora lutea of early pregnancy were dispersed with collagenase and luteal cells were prelabelled for 3 h with [3H]inositol. Inositol phosphates produced in response to LH or PGF2 alpha were analyzed by ion exchange column chromatography. The tumor promoter and protein kinase C activator 12-O-tetradecanolyphorbol 13-acetate (TPA) had no effect on basal levels of inositol phosphates but inhibited LH-stimulated accumulation of inositol mono-, bis-, and trisphosphates by 72%, 68%, and 65%, respectively. TPA reduced the response to maximally effective concentrations of LH and tripled the concentrations of LH required to evoke half-maximal accumulation of inositol mono-, bis-, trisphosphates. The inhibitory effects of TPA were rapid (5 min) whether added before or after treatment with LH. Treatment with TPA also reduced (58%) the initial phase of intracellular calcium mobilization in LH-treated cells. The inhibitory effects of TPA were not associated with acute reductions in [3H]inositol incorporation, [3H]inositol phospholipid levels, cAMP levels, or progesterone accumulation in control or LH-stimulated luteal cells. The effects of phorbol esters were concentration dependent and specific for active tumor promoters with 10-50 nM TPA producing maximal inhibitory effects. A synthetic diacylglycerol, 1-oleyl-2-acetylglycerol, mimicked the inhibitory effects of TPA. In contrast, pretreatment with a physiological activator of protein kinase C, PGF2 alpha, had no effect on LH-stimulated inositol phosphate accumulation. The inhibitory effects of TPA could not be explained by a generalized inhibition of phospholipase C or G-proteins since the accumulation of inositol phosphates in PGF2 alpha- and NaF-treated cells was not inhibited by TPA. These results demonstrate that tumor promoting phorbol esters modulate the inositol phospholipid-phospholipase C transmembrane signaling system in LH-stimulated bovine luteal cells. The results suggest that phorbol esters may alter the coupling of the LH-receptor complex to phospholipase C. These findings implicate protein kinase C in the regulation of transmembrane signaling in the bovine corpus luteum.
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PMID:Modulation of luteinizing hormone-stimulated inositol phosphate accumulation by phorbol esters in bovine luteal cells. 132 81

Inositol-lipid-specific phospholipase C-delta 1 (PtdIns-PLC delta 1) was expressed in Escherichia coli as a fusion protein containing a short 22-amino-acid lac-Z-derived amino terminus. Under appropriate conditions, the phospholipase constituted approximately 0.2% of the detergent-soluble protein and could be purified to near homogeneity in a simple three step protocol. The catalytic properties of the purified enzyme closely resemble those of the eukaryote-derived protein. The suitability of bacterial expression for the investigation of PtdIns-PLC delta regulation is discussed.
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PMID:Expression, purification and characterisation of a functional phosphatidylinositol-specific phospholipase C-delta 1 protein in Escherichia coli. 133 58

Acrosomal reaction is an essential prerequisite to fertilization. The changes in lipid composition of sperm membranes cause fusion of the plasma and outer acrosomal membranes that results in the exocytosis of acrosomal contents. We report that both bull and rabbit spermatozoa contain a phosphatidylcholine-specific phospholipase C (PC-PLC) that hydrolyzes L-alpha-dipalmitoyl-(choline-methyl-14C-153.0 Ci/mmol and a phosphatidylinositol-specific phospholipase C (PI-PLC) that hydrolyzes L-alpha-(Myo-Inositol-2-3H (N)-5.2 Ci mmol. PI-PLC from bull sperm acrosome has been purified 568 x fold with a specific activity 6.25 +/- 0.6 nmol/min/mg protein, km 0.004 mM, and Vmax 12 nmol/min/mg protein. Both enzymes had optimum at pH 7.5. The activity of PC-PLC remained unaffected by varying concentrations of Ca2+, whereas PI-PLC activity was significantly increased. The bulk of PI-PLC was found to be associated with inner acrosomal membrane of bull and rabbit sperm, while PC-PLC was found in the outer acrosomal membranes in the bull sperm and the plasma membrane of the rabbit sperm. Both enzymes are compartmentalized in sperm cell.
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PMID:Phosphatidylcholine and phosphatidylinositol-specific phospholipases C of bull and rabbit spermatozoa. 133 40

The receptor that interacts with the mammalian bombesin-related peptide neuromedin B (NMB) is ubiquitous in the gastrointestinal tract and central nervous system. However, little is known regarding its cellular mechanisms of action. This receptor has been recently cloned, sequenced, and stably transfected into BALB 3T3 fibroblasts, permitting detailed study of the pharmacology and coupled biological activities of this receptor. In the present study, we compare the ability of transfected receptors to alter cell function with that of receptors natively expressed in small numbers by the rat glioblastoma cell line C6. NMB inhibited binding of 125I-[D-Tyro]NMB with high affinity in transfected cells (Ki = 3.08 +/- 0.14 nM) and in C6 cells (Ki = 1.90 +/- 1.10 nM), whereas the bombesin-related agonists gastrin-releasing peptide (GRP) and [D-Phe6, D-Ala11, Leu14]bombesin(6-16) (GRP analogue) had 100- and 300-fold lower affinities, respectively, for NMB receptors in either cell type. For both cell systems, maximal binding was observed between 5 and 15 min at 22 degrees. Both cell types internalized NMB at similar rates, with > 70% of bound ligand being internalized by 60 min at 22 degrees. The nonhydrolyzable guanosine analogue guanosine 5'-(beta,gamma-imido)triphosphate was equipotent in causing a decrease in binding of 125I-[D-Tyro]NMB due to decreased receptor affinity in both cell types, without a change in receptor number, demonstrating that the NMB receptor remained coupled to a guanine nucleotide-binding protein in both native and transfected cells. In both cell systems, NMB increased inositol monophosphate, inositol bisphosphate, and inositol trisphosphate in a time-dependent fashion. Inositol phosphates were increased in a dose-dependent fashion, with similar half-maximal values being obtained for NMB in both cell types (transfected, 1.01 +/- 0.09 nM; C6, 2.09 +/- 0.15 nM) and for the GRP analogue (transfected, 1855 +/- 140 nM; C6, 2129 +/- 250 nM). NMB mobilized intracellular Ca2+ in both cell systems, and the dose-response curves were superimposible (EC50 for transfected, 0.10 +/- 0.08 nM; C6, 0.11 +/- 0.02 nM). These data demonstrate that activation of the receptor for NMB stimulates phospholipase C and increases intracellular Ca2+. These results also demonstrate that transfected and native NMB receptors behave similarly, suggesting that the transfected cell line will be useful in future studies investigating ligand-receptor interactions, as well as in molecular biological studies of the structure-function relationship of the receptor.
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PMID:Neuromedin B receptors retain functional expression when transfected into BALB 3T3 fibroblasts: analysis of binding, kinetics, stoichiometry, modulation by guanine nucleotide-binding proteins, and signal transduction and comparison with natively expressed receptors. 133 12

A role for phospholipase C (PLC) hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) as a mechanism of alpha 1-adrenergic signal transduction in human airway epithelial cells (AEC) was investigated in isolated normal tracheal and cystic fibrosis (CF) nasal epithelial cells grown in in vitro culture and prelabeled with 3 muCi myo-[3H]inositol/ml for 72 h. Breakdown of polyphosphoinositides was measured using thin-layer chromatography to detect phosphatidylinositol, phosphatidylinositol 4-phosphate (PIP), and PIP2. Inositol phosphates were separated by ion-exchange column chromatography. In normal AEC, the addition of the endogenous catecholamine l-epinephrine produced a rapid, transient accumulation of inositol 1,4,5-trisphosphate (IP3) and inositol 1,4-bisphosphate (IP2) and breakdown of PIP and PIP2. IP3 increased 1.7-fold and IP2 1.6-fold after 20 and 40 s, respectively. A maximal decrease of 35% PIP2 and 30% PIP is observed after 20 and 40 s, respectively. The effects of l-epinephrine were not blocked by the beta-adrenergic antagonist dl-propranolol but were mimicked by the alpha 1-adrenergic agonist methoxamine. Prazosin, an alpha 1-adrenergic antagonist, and pertussis toxin (PTX) blocked the effects of l-epinephrine and methoxamine. Addition of l-epinephrine and methoxamine to CF nasal epithelial cells also induced prazosin-sensitive polyphosphoinositide breakdown and inositol phosphate accumulation. A 2.2-fold accumulation of IP3 was observed after 10 s and 2.0-fold increase in IP2 after 20 s. Maximal decreases of 32% PIP2 and 23% PIP levels were observed after 20-s incubation with l-epinephrine. PTX reduced the effects of l-epinephrine and significantly blocked the effects of methoxamine.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Alpha 1-adrenergic signaling in human airway epithelial cells involves inositol lipid and phosphate metabolism. 134

Inositol glycans were prepared from reductively radiomethylated human erythrocyte acetylcholinesterase by sequential treatment with Proteinase K, methanolic KOH, and phosphatidylinositol-specific phospholipase C. Four glycans denoted alpha-delta were resolved by anion exchange high performance liquid chromatography (HPLC). Each glycan was subjected to hydrolysis in 4 M trifluoroacetic acid, and their hexose and hexose phosphate compositions were determined by anion exchange HPLC. The predominant glycan alpha showed a relative stoichiometry of 2 mannoses, 1 mannose 6-phosphate, 1 radiomethylated glucosamine, 1 radiomethylated ethanolamine, and 1 inositol. In contrast, the stoichiometry of glycan beta was 1 mannose, 2 mannose 6-phosphates, 1 radiomethylated glucosamine, 2 radiomethylated ethanolamines, and 1 inositol. Glycans alpha and beta were analyzed by electrospray ionization-mass spectrometry, and respective parent ions of m/z 1266 and 1417 were observed. The fragmentation pattern produced by collision-induced dissociation mass spectrometry of these parent ions was consistent with a common linear core glycan sequence prior to radiomethylation of ethanolamine-phosphate-mannose - mannose - mannose - glucosamine - inositol. Glycan alpha contained a single additional radiomethylated phosphoethanolamine branching from the mannose adjacent to glucosamine, whereas glycan beta contained two additional radiomethylated phosphoethanolamines, one branching from each of the mannoses nearest to glucosamine. Trifluoroacetic acid hydrolysis did not cleave within the N,N-dimethylglucosamine-inositol-phosphate moiety in these glycans, and this component was resolved by anion exchange HPLC and structurally confirmed by mass spectrometry. Dephosphorylation of this component by treatment with 50% HF produced N,N-dimethylglucosamine-inositol, and this conjugate was shown to have a characteristic elution time on cation exchange chromatography in an amino acid analyzer. Both of these fragments involving an intact radiomethylated glucosamine-inositol bond are proposed as new diagnostic indicators in the search for minor glycoinositol phospholipids in cells and tissues.
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PMID:Glycan components in the glycoinositol phospholipid anchor of human erythrocyte acetylcholinesterase. Novel fragments produced by trifluoroacetic acid. 138 56


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