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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 ability of bovine corpus luteum plasma membranes to bind 125I-choriogonadotropin has been examined after prior treatment of the membranes with phospholipases A, C, and D. Treatment of the purified membranes with low concentrations of phospholipases A and C resulted in the inhibition of the binding of 125I-choriogonadotropin to its receptors, whereas phospholipase D had no effect. Receptor activity was decreased by low concentrations of phospholipase A from either bee venom, Vipera russelli or Crotalus terrificus terrificus. Similarly, low concentrations of phospholipase C from Clostridium perfringens and Clostridium welchii also inhibited the binding activity while comparatively higher concentrations of phospholipase C from Bacillus cereus were required to achieve comparable inhibition. The time required to produce 50% inhibition of in vitro binding by phospholipases A and C was found to be 6 and 23 min, respectively. Upon either removal or chelation of calcium ions by ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid (EGTA) both enzymes were completely inhibited as evidenced by the complete retention of the membrane binding activity. The decrease in the specific binding of choriogonadotropin to membranes after phospholipase digestion resulted in a decrease in the number of binding sites and was not accompanied by a change in the affinity of the hormone-receptor complex. The rates of association and dissociation of the 125I-choriogonadotropin-receptor complex and the equilibrium dissociation constant (Kd) were nearly identical in untreated and phospholipase-treated membranes. Phospholipases did not have any effect on the preformed hormone-receptor complex or on solubilized receptor. Filtration through Sepharose 6B of solubilized 125I-choriogonadotropin-receptor complex from untreated membranes or membranes which had been pretreated with phospholipase C prior to carrying out hormone binding did not alter the profile (Kav 0.38). Gel filtration of membranes treated with phospholipase A showed two peaks of bound radioactivity with distribution coefficients (Kav) of 0.08 and 0.35, respectively.
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PMID:Gonadotropin receptors in plasma membranes of bovine corpus luteum. I. Effect of phospholipases on the binding of 125I-choriogonadotropin by membrane-associated and solubilized receptors. 18 85

DDT1-MF2 smooth muscle cells demonstrated a robust phospholipase C response to norepinephrine, as detected by inositol phosphate accumulation. A selective A1-adenosine receptor agonist, cyclopentyladenosine, caused only a minor stimulation of phospholipase C, which was eliminated in the absence of added extracellular calcium. The simultaneous addition of norepinephrine and cyclopentyladenosine resulted in a synergistic increase in phosphoinositide hydrolysis either in the absence or in the presence of external calcium. In the presence of external calcium and a calcium ionophore, and adenosine agonist caused a significant stimulation of phosphoinositide hydrolysis without the addition of norepinephrine. Influx of extracellular calcium through voltage-sensitive calcium channels did not appear to be required to observe an effect of cyclopentyladenosine, because neither calcium channel antagonists (nifedipine, verapamil, and LaCl3) nor a chelator of extracellular calcium (ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid) were able to alter the degree of potentiation of norepinephrine-stimulated phosphoinositide hydrolysis due to the adenosine agonist. On the other hand, buffering of intracellular calcium concentration with the membrane-permeant calcium chelator quin2 blocked the potentiation. This blockade of potentiation by quin2 was reversed by the addition of extracellular calcium. Agents that stimulated cAMP production or membrane-permeable analogues of cAMP also blocked the action of the adenosine agonist to potentiate norepinephrine-stimulated phosphoinositide hydrolysis. This effect of cAMP was less pronounced in the presence of elevated extracellular calcium and was abolished in the presence of a calcium ionophore. When norepinephrine-stimulated calcium transients were quantitated using fura-2 fluorescence, a reduction in the amplitude of the calcium response was observed in the presence of forskolin. Conversely, both the amplitude and the duration of the calcium response were enhanced by the addition of the adenosine agonist. The results of these studies suggest that the mechanism by which adenosine receptors enhance the stimulation of phosphoinositide hydrolysis is dependent upon a rise in intracellular Ca2+ concentration resulting from the simultaneous activation of alpha 1-adrenergic receptors. The results further suggest that cAMP inhibits this mechanism by decreasing the norepinephrine-stimulated rise in intracellular Ca2+ concentration.
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PMID:Competitive regulation of phospholipase C responses by cAMP and calcium. 131 17

Oxidants may play a central role in the pathogenesis of adult respiratory distress syndrome, and phospholipase activation is a potential mechanism of oxidant-induced injury of alveolar epithelial cells. Studies were performed in rat alveolar type II epithelial cells (RAEC) after 3 days in culture. As measured by 51Cr and lactate dehydrogenase release, H2O2 caused time- and dose-dependent cytotoxicity to RAEC. RAEC phospholipids labeled with [14C]-stearic acid ([14C]SA) and [3H]arachidonic acid ([3H]AA) released free fatty acids in response to H2O2 in a manner that closely paralleled the cytotoxicity indexes. Analysis of phospholipid subclasses indicated that phosphatidylcholine was preferentially affected. Analysis for putative products of phospholipase activity revealed significant increases in diacylglycerol and phosphorylcholine, expected products of phospholipase C, as well as significant increases in L-alpha-lysophosphatidylcholine and L-alpha-glycerophosphocholine, expected products of phospholipase A2. Increases in phospholipase D activity were not detected. To determine whether H2O2-stimulated phospholipase activity might be Ca2+ stimulated, RAEC were loaded with fura-2/AM, and changes in intracellular Ca2+ concentrations ([Ca2+]i) were monitored by epifluorescent microscopy. Exposure to H2O2 caused elevations in [Ca2+]i, and the time and dose relationships were consistent with the hypothesis that the release of [14C]SA and [3H]AA is related to changes in cellular Ca2+ concentrations. Additionally, pretreatment with MAPTAM, an intracellular chelator of calcium, partially blocked H2O2-mediated [3H]AA liberation. However, experiments in saponin-permeabilized RAEC, in which [Ca2+]i was strongly buffered by ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid, indicate that H2O2-induced phospholipase activity also has a Ca(2+)-independent component.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:H2O2 injury causes Ca(2+)-dependent and -independent hydrolysis of phosphatidylcholine in alveolar epithelial cells. 141 20

The effects of endothelin, a novel vasoconstrictive peptide, on the delayed rectifier K+ current (IK) were examined in single dialyzed cells from guinea pig ventricles. Either big endothelin or endothelin-1 enhanced IK at a dissociation constant of 2 nM with L-type Ca2+ current being unaffected. Under intracellular perfusion with pCa 7.6 solution, 3 nM big endothelin increased IK by 55 +/- 38.5%. Either pretreatment with 10 microM 1-(5-isoquinolinylsulfonyl)-2-methyl-piperazine (H 7) or a low Ca2+ [10 mM ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) and minus CaCl2] internal solution diminished the enhancement. Preceding stimulation of protein kinase C (PKC) by 10-20 nM 12-O-tetradecanoylphorbol-13-acetate also reduced the degree of enhancement. When Na+ was eliminated from the solutions, endothelin increased IK distinctively in cells internally dialyzed with a low Ca2+ solution. This enhancement was not abolished by either pretreatment with H 7 or by removal of Ca2+ from the external perfusate but by increasing the internal EGTA concentration to 40 mM. Preincubation with ryanodine or internal perfusion with heparin also reduced the IK enhancement under Na(+)-free conditions. Intracellular application of 200 microM guanosine 5'-O-(3-thiotriphosphate) effectively attenuated the effect of endothelin. It is concluded that endothelin enhances IK via phospholipase C-mediated PKC activation and intracellular Ca2+ mobilization. GTP-binding protein is involved in these reactions.
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PMID:Endothelin enhances delayed potassium current via phospholipase C in guinea pig ventricular myocytes. 153 93

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

The agonist-induced change in Ca2+ sensitivity of smooth muscle myofilaments was investigated in intact and permeabilized vascular preparations isolated from the rat and the rabbit. In intact rat mesenteric artery, membrane depolarization by 80 mM K+ solution or alpha-adrenergic stimulation by norepinephrine (NE) increased tension monotonically with increasing extracellular Ca2+ concentration ([Ca2+]e). The [Ca2+]e-tension curve generated during activation by NE was located to the left of that during activation by high K+. The protein kinase C (PKC) activator 12-O-tetradecanoylphorbol-13-acetate (TPA) shifted the high K+ [Ca2+]e-tension curve to the left but did not affect the NE curve. In rat mesenteric artery permeabilized by alpha-toxin, tension was measured while the intracellular free Ca2+ concentration ([Ca2+]i) was controlled using 2 mM ethylene glycol-bis(beta-aminoethyl ether)-N,N,N'N'-tetraacetic acid and Ca2+ buffer solutions. The alpha-toxin-permeabilized fibers developed tension as a function of Ca2+ concentration. TPA and guanosine 5'-[gamma-thio]triphosphate (GTP gamma S, a nonhydrolyzable GTP analogue) significantly shifted the pCa-tension curve to the left. In intact rabbit inferior vena cava, tension was recorded simultaneously with [Ca2+]i as measured by fura-2. TPA caused a gradual increase in tension without change in [Ca2+]i. In rabbit mesenteric artery permeabilized by alpha-toxin, the tissue still responded to NE, indicating that alpha-adrenergic receptors remained intact. The response to NE was augmented by GTP and inhibited by guanosine 5'-[beta-thio]diphosphate (GDP beta S, a nonhydrolyzable GDP analogue) suggesting that a G protein is coupled with the alpha-adrenergic receptor.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Evidence for increased myofilament Ca2+ sensitivity in norepinephrine-activated vascular smooth muscle. 211 42

The influence of Ca2+ on the activity of the taurine transport system was investigated in rabbit small intestinal brush-border membrane vesicles. Preincubation of the brush-border membrane vesicles with Ca2+ prepared by the Mg2(+)-aggregation method markedly decreased the NaCl gradient-dependent uptake of taurine in these vesicles. Uptake of glucose and alanine, both dependent on a Na+ gradient, were also decreased by Ca2(+)-treatment, but their reduction was very small compared with that of taurine uptake. The inhibitory effect of Ca2+ was dose- and time-dependent. The inhibition was reduced by the presence of ethylene glycol-bis(beta-amino ethyl ether)-N,N,N'-N'-tetraacetic acid during treatment of the membrane vesicles with Ca2+. Neomycin partially protected the taurine transporter activity from the Ca2(+)-induced inhibition, but indomethacin did not. 5-Nitro-2-(3-phenylpropylamino)benzoate, a Cl(-)-channel blocker, did not increase taurine uptake in the Ca2(+)-treated membrane vesicles. It is concluded that the Ca2(+)-induced inhibition of taurine uptake in rabbit intestinal brush-border membrane vesicles is not due to accelerated dissipation of the ion gradient driving forces across the membrane but rather to a direct effect on the transporter, most likely mediated by the activation of the membrane-bound phospholipase C.
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PMID:Calcium-induced inhibition of taurine transport in brush-border membrane vesicles from rabbit small intestine. 212 7

Rat intestinal alkaline phosphatase (IAP) is unique among the brush-border membrane enzymes in that it is released bidirectionally (lumen and blood) and exists in either soluble (serum) or particulate (cellular) form. To elucidate the mechanism of membrane release, we examined the effects of phosphatidylinositol-specific phospholipase C (PtdIns-PLC) and serum anchor-specific phospholipase D (PLD) on the solubility of the various tissue forms of IAP. The "solubility" of cytosol IAP could be explained in part by intracellular PtdIns-PLC activity, detected by production of acidic IAP isomers, and by ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA)-sensitive PtdIns hydrolysis. Contamination with serum (abundant with anchor-specific PLD) was responsible for the complete or partial solubilization of IAP that was found during processing of light mucosal scrapings. Anchor-specific PLD activity was increased after fat feeding, and the IAP released did not react with antiserum that recognizes the PtdIns-PLC-released phospholipid portion of trypanosomal variable surface glycoprotein. These data are consistent with the hypothesis that, after secretion from the enterocyte bound to a phospholipid-rich membranous particle, IAP release into serum is mediated by serum anchor-specific PLD. The soluble forms of IAP in the lumen and the cytosol fraction appear to be due to a combination of endogenous PtdIns-PLC activity and anchor-specific PLD contamination that occurs during cell fractionation.
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PMID:Both tissue and serum phospholipases release rat intestinal alkaline phosphatase. 222 Oct 72

1. Phospholipases have been proposed to play a key role in sperm acrosome reaction. To examine the activation mechanism of phospholipases and subsequently sperm fertilizing capacity. Ca2+ fluxes and phospholipid turnover (breakdown and synthesis) were investigated in golden hamster spermatozoa during acrosome reaction. 2. Upon exposure of the spermatozoa to 1.7 mM Ca2+, a net uptake by the cells occurred in two distinguishable phases. 3. Depletion of extracellular Ca2+ by ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) at a time that an initial Ca2+ uptake was observed to reach almost steady-state, prevented the secondary Ca2+ uptake and acrosome reaction. 4. The time course of an initial Ca2+ uptake seemed to precede that of the acrosome reaction. 5. Incubation of the spermatozoa with Ca2+ in the presence of [3H]glycerol induced a rapid increase in labeling of phosphatidic acid, a key intermediate of phosphinositide turnover initiated by the action of phospholipase C, which appeared to parallel the time course of a first phase of Ca2+. 6. Phospholipase A2 activation, detected by lysophospholipid formation, slightly delayed the initial events of first Ca2+ uptake and phosphatidic acid production. 7. It is concluded that first Ca2+ entry into the cells, associated with phosphatidic acid production, activates a phospholipase A2, leading to the production of substances, like lysophospholipids and fatty acids, which may contribute to acrosome reaction.
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PMID:Evidence for tight coupling of phospholipase activation and Ca2+ influx during acrosome reaction of golden hamster spermatozoa. 233 83

In rat membranous nephropathy, formation of the C5b-9 membrane attack complex (MAC) leads to proteinuria in association with glomerular visceral epithelial cell (GEC) injury. These alterations in GEC function and morphology might result from changes in intracellular free Ca2+ concentration [( Ca2+]i) and activation of phospholipases. We demonstrate that in cultured rat GEC, antibody-directed formation of noncytolytic amounts of the MAC induced a rapid and sustained increase in [Ca2+]i that was partly inhibited by ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA). The MAC elevated levels of inositol bis- (IP2) and trisphosphate (IP3), as well as 1,2-diacylglycerol (DAG) and phosphatidic acid (PA). In permeabilized GEC, IP3 released Ca2+ from intracellular stores. Cellular 45Ca2+ uptake was also increased by the MAC. Thus, in GEC, the MAC induced Ca2+ mobilization from intracellular stores secondary to activation of phospholipase C and production of IP3, as well as enhanced Ca2+ influx. In addition, C5b-9 stimulated release of arachidonic acid (AA), prostaglandin F2 alpha, and thromboxane A2. Indomethacin partially inhibited the increase in DAG levels observed with the MAC, whereas the prostaglandin H2/thromboxane A2 analogue U46619 elevated DAG, suggesting that an eicosanoid product of MAC-induced AA release may enhance the activation of phospholipase C. Activation of phospholipases by the MAC may lead to altered GEC function and thereby contribute to the pathophysiological changes that characterize complement-dependent rat membranous nephropathy.
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PMID:Complement C5b-9 complex activates phospholipases in glomerular epithelial cells. 251 64


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