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)

Evidence for the modulation of the P2z-purinoceptor for extracellular ATP in dissociated rat parotid cells is presented in studies using compounds that inhibit protein kinases. Preincubation of acinar cells with the protein kinase catalytic-site inhibitors K-252a and staurosporine, as well as with the regulatory-domain inhibitor sphingosine, specifically potentiates the elevation in cytosolic Ca2+ concentration ([Ca2+]i) mediated by extracellular ATP, but has no effect on the [Ca2+]i elevation mediated by muscarinic receptors through phospholipase C activation. Phorbol dibutyrate (PDBu), which activates protein kinase C (PKC), has no modulatory effect on ATP-mediated [Ca2+]i elevation. Further, pretreatment with PDBu does not reverse or block the effects of K-252a or sphinogosine, arguing against the involvement of PKC. Other pharmacological manipulations indicate that neither calmodulin-dependent nor cyclic-AMP-dependent kinases are involved. Neither the peak intracellular Ca2+ mobilization nor the sustained Ca2+ entry in response to carbachol or to a Ca2+ ionophore (4-bromo-A23187) is altered by the kinase inhibitors that potentiate the [Ca2+]i response to ATP, indicating that effects on the ATP response are not due to non-specific permeability changes, nor to decreased Ca2+ removal from the cytosol. ATP-mediated influx of Mn2+ as well as ATP-induced membrane depolarization are potentiated in cells preincubated with K-252a, directly demonstrating that cation influx is enhanced through a P2z-specific route. These results show that P2z responses (or purinoceptors) can be modulated and suggest that phosphorylation events are involved.
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PMID:Modulation of extracellular ATP-induced Ca2+ responses: role of protein kinases. 821 26

Direct intracellular effects of the divalent cations Mn2+, Cd2+, Co2+, Ni2+, and Zn2+ on pituitary hormone exocytosis in a permeabilized cell system have not previously been investigated. We examined the effects of these ions on LH exocytosis in primary sheep pituitary cells permeabilized with Staphylococcal alpha-toxin. Mn2+ ions strongly stimulated LH release from permeabilized, but not intact, cells. Mn(2+)-stimulated LH release was ATP-dependent and sensitive to N-ethyl maleimide, indicating that it represents true exocytosis. Hormone release triggered by Ca2+ was inhibited by Cd2+, Zn2+, Co2+, and Ni2+ ions. Half-maximal inhibition of Ca(2+)-stimulated LH release was observed with 10 microM Cd2+, 30 microM Zn2+, 0.3 mM Co2+, and 1 mM Ni2+. With the same order of potency these ions inhibited LH release stimulated by Ba2+, Mn2+, or phorbol ester plus cAMP, suggesting that they inhibit exocytosis at an intracellular site common to all these stimuli.
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PMID:Stimulation by Mn2+ and inhibition by Cd2+, Zn2+, Ni2+, and Co2+ ions of luteinizing hormone exocytosis at an intracellular site. 838 85

Extracellular ATP caused a dose-dependent accumulation of inositol phosphates and a rise in cytosolic free Ca2+ ([Ca2+]i) in C6 glioma cells with an EC50 of 60 +/- 4 and 10 +/- 5 microM, respectively. The threshold concentration of ATP (3 microM) for increasing [Ca2+]i was approximately 10-fold less than that for stimulating phosphoinositide (PI) turnover. The PI response showed a preference for ATP; ADP was about 3-fold less potent than ATP but had a comparable maximal stimulation (11-fold of the control). AMP and adenosine were without effect at concentrations up to 1 mM. ATP-stimulated PI metabolism was found to be partially dependent on extracellular Ca2+ and Na+ but was resistant to tetrodotoxin, saxitoxin, amiloride, ouabain, and inorganic blockers of Ca2+ channels (Co2+, Mn2+, La3+, or Cd2+). In Ca(2+)-free medium, ATP caused only a transient increase in [Ca2+]i as opposed to a sustained [Ca2+]i increase in normal medium. The ATP-induced elevation of [Ca2+]i was resistant to Na+ depletion and treatment with saxitoxin, verapamil and nisoldipine, but was attenuated by La3+. The differences in the characteristics of ATP-caused P1 hydrolysis and [Ca2+]i rise suggest that ATP receptors are independently coupled to phospholipase C and receptor-gated Ca2+ channels. Because of the robust effect of ATP in stimulating PI turnover and the apparent absence of P1-purinergic receptors, the C6 glioma cell line provides a useful model for investigating the transmembrane signalling pathway induced by extracellular ATP. The mechanisms underlying the unexpected finding of [Na+]o dependency for ATP-induced PI turnover require further investigation.
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PMID:Extracellular ATP stimulates inositol phospholipid turnover and calcium influx in C6 glioma cells. 838 91

Activated phagocytes produce large amounts of reactive oxygen intermediates, including peroxides. In addition to their microbicidal effect, it has recently been suggested that reactive oxygen species play a role as intracellular messengers. The mechanism of action remains unknown, but peroxides have been reported to increase tyrosine phosphorylation, an effect potentiated by vanadate. In this report we studied the effects of a combination of H2O2 and vanadate on Ca2+ homeostasis in granulocytic HL60 cells. The peroxides induced a transient elevation of cytosolic [Ca2+] associated with release from internal stores. Ca2+ mobilization was accompanied by increased generation of inositol 1,4,5-trisphosphate, implicating phospholipase C (PLC). A sizable increase in phosphotyrosine accumulation by several polypeptides in the M(r) 20,000 to 250,000 range preceded the [Ca2+] changes. We therefore considered the possibility that tyrosine phosphorylation of a phospholipase mediates the observed effects. Differentiated (granulocytic) HL60 cells did not have detectable levels of PLC gamma 1 but had substantial PLC gamma 2. Immunoprecipitation and immunoblotting experiments demonstrated that PLC gamma 2 becomes tyrosine-phosphorylated upon treatment of the cells with peroxides of vanadate. If associated with activation, such phosphorylation of PLC gamma 2 can account for the rise in [Ca2+]. Although capable of mobilizing internal Ca2+ stores, the peroxides failed to produce the sustained [Ca2+] increase predicted by the "capacitative" model. Mn2+ influx determinations indicated that this is due to impairment of divalent cation entry by the peroxides, uncoupling the plasma membrane from the internal stores. Changes in [Ca2+] homeostasis could mediate some of the messenger actions of reactive oxygen species.
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PMID:Cytosolic [Ca2+] homeostasis and tyrosine phosphorylation of phospholipase C gamma 2 in HL60 granulocytes. 842 12

A new cytolytic toxin, designated as S-Hemolysin, was found in the culture filtrate of Streptomyces sp. strain No. A-6288, isolated from a soil sample. The molecular weight of S-Hemolysin was estimated to be 10,000 by SDS-polyacrylamide gel electrophoresis and to be 20,000 by Sephadex G-100. S-Hemolysin is a glycoprotein that is composed of 102 amino acid residues with 11.6% glucose, and the isoelectric point is around pH 5.8. The phospholipase C activity of S-Hemolysin was specific for the following substrates in this order: sphingomyelin > lysophosphatidylethanolamine > lysophosphatidylcholine > phosphatidylethanolamine > phosphatidylcholine. S-Hemolysin had hemolytic activity against rabbit, human, and sheep erythrocytes, but did not cause aggregation of human platelets. These activities were accelerated with Mg2+, Mn2+, and Co2+ ions and inhibited by the addition of Ca2+, Cu2+, and Zn2+ ions. This enzyme was shown to be different from the known bacterial phospholipase C.
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PMID:Purification and some properties of S-Hemolysin produced by Streptomyces sp. strain no. A-6288. 854 46

Metal selectivity of exocytosis was analyzed by comparing the effects of polyvalent metal cations Ca2+, Ba2+, Sr2+, Pb2+, La3+, Cd2+, Co2+, Tb3+, Mn2+, and Zn2+ on the release of norepinephrine (NE) from staphylococcal alpha-toxin-permeabilized bovine chromaffin cells. Pb2+, La3+, Cd2+, Sr2+, and Ba2+ activated NE secretion accompanied by the release of intragranular dopamine beta-hydroxylase but not cytosolic lactate dehydrogenase, indicating the activation of the mechanism of exocytosis. The release triggered by saturating concentrations of Pb2+, La3+, Cd2+, and Sr2+ was nonadditive with Ca2+, indicating a common site of action. In contrast, the Ba2(+)-evoked NE release was additive with Ca2+ and the Ca2+ agonists Pb2+, La3+, Cd2+, and Sr2+, suggesting that Ba2+ activates secretion at a site distinct from the Ca2+ receptor. In distinction to the NE release evoked by Pb2+, La3+, Cd2+, and Ba2+, the Sr(2+)-evoked NE release was associated with a significant elevation of Ca2+ concentration in the medium and abolished by Ca2+ chelation. This indicates that the secretagogue effect of Sr2+ was indirect and secondary to the displacement of bound Ca2+, Co2+ and Mn2+ inhibited the NE release evoked by Ca2+, Sr2+, Pb2+, La3+, and Cd2+ but had no effect on the Ba(2+)-dependent secretion. Tb3+ and Zn2+ were without effect on exocytosis.
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PMID:Metal selectivity of exocytosis in alpha-toxin-permeabilized bovine chromaffin cells. 859 35

The effects of diadenosine pentaphosphate (AP5A), and diadenosine hexaphosphate (AP6A) on the cytosolic-free Ca2+ concentration ([Ca2+]i) were evaluated in cultured human fibroblast cells (HF cells) using the fluorescent dye technique. AP5A, and AP6A concentration-dependently increased [Ca2+]i in HF cells. The addition of 10 mumol/1 AP5A and AP6A significantly increased [Ca2+]i in HF cells from 71 +/- 3 nmol/1 (n = 184) to 241 +/- 39 nmol/1 (n = 11; P < 0.001 compared to resting value) and to 227 +/- 26 nmol/1 (n = 23; P < 0.001), respectively. The purinoceptor P2 blockers, suramin and pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS), inhibited the diadenosine polyphophate-induced [Ca2+]i increase, whereas the P2y purinoceptor blocker, reactive blue, had no effect. Adenosinetriphosphate (ATP) and the P2x agonist, alpha 1 beta-methylene-ATP also significantly increased [Ca2+]i in HF cells, whereas the P2y agonist methylthio-ATP showed only a small [Ca2+]i response. Diadenosine polyphosphates mainly induced transplasmamembrane Ca2+ influx as was confirmed by experiments in the absence of extracellular Ca2+ or by manganese quenching studies. Organic (verapamil) and inorganic Ca2+ channel blockers (NiCI2) significantly reduced the AP6A induced transplasmamembrane Ca2+ influx. The inhibitor of phosphatidylcholine-specific phospholipase C, D609, significantly reduced the effect of diadenosine polyphosphates on [Ca2+]i in HF cells. It is concluded that diadenosine polyphosphates regulate transplasmamembrane Ca2+ influx after occupation of P2x receptors via activation of phosphatidylcholine-specific phospholipase C and hence of voltage-operated Ca2+ channels.
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PMID:Diadenosine polyphosphates regulate cytosolic calcium in human fibroblast cells by interaction with P2x purinoceptors coupled to phospholipase C. 867 37

Pig kidney aminopeptidase P (AP-P; EC 3.4.11.9) has been purified to homogeneity after its solubilisation from brush border membranes by phosphatidylinositol-specific phospholipase C. The effects of various activators and inhibitors of AP-P activity have been examined with a number of different substrates for the enzyme. The hydrolysis of bradykinin and ArgProPro is inhibited at Mn2+ concentrations above 10(-5) M, whereas the hydrolysis of other substrates (GlyProHyp, beta-casomorphin, substance P) is substantially activated, with 4-10 mM Mn2+ being optimal. The thiol reagent, p-chloromercuriphenylsulphonic acid, inhibits the hydrolysis of GlyProHyp but markedly activates the hydrolysis of bradykinin. A number of inhibitors of angiotensin converting enzyme (ACE; EC 3.4.15.1), previously reported to inhibit the hydrolysis of GlyProHyp, have no effect on the hydrolysis of bradykinin except in the presence of Mn2+. Differences were also observed in the degree of inhibition of GlyProHyp and bradykinin hydrolysis by EDTA and their reactivation by divalent cations. The hydrolysis of GlyProHyp follows Michaelis-Menten kinetics with a Km value of 2.7 mM. Bradykinin inhibits GlyProHyp hydrolysis with an I50 of 1.4 microM. The hydrolysis of bradykinin by AP-P reveals anomalous nonlinear kinetics indicative of negative cooperativity or the presence of more than one active site for this substrate. These results indicate that substrates for AP-P can be divided into 2 groups based on their responses to inhibitors and cation activators.
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PMID:Inhibition and metal ion activation of pig kidney aminopeptidase P. Dependence on nature of substrate. 869 47

1-[6-[[17 beta-3-Methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1 H-pyrrole-2,5-dione (U-73122) has been proven to be a useful tool in investigation of phospholipase C (PLC)-coupled signal transduction during cell activation. In the present studies, the inhibition by U-73122 of cytosolic free Ca2+ concentration ([Ca2+]i) of neutrophils was investigated. U-73122 suppressed the [Ca2+]i elevation of neutrophils suspended in Ca(2+)-containing medium challenged by N-formyl-Met-Leu-Phe (fMLP), cyclopiazonic acid (CPA) and ionomycin. The concentrations of U-73122 required for inhibition of CPA- and ionomycin-induced changes with IC50 values 4.06 +/- 0.27 microM and 4.04 +/- 0.44 microM, respectively, is almost 10-times that required for inhibition of the fMLP-induced response (IC50 value 0.62 +/- 0.04 microM). U-73122 also reduced the intracellular Ca2+ mobilization of neutrophils suspended in Ca(2+)-free medium stimulated by fMLP and CPA, but not by ionomycin, with IC50 values 0.52 +/- 0.02 microM and 6.82 +/- 0.74 microM, respectively. 1-[6-[[17 beta-3-Methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-2,5-pyrr olidinedione (U-73343), a close analog of U-73122 that does not inhibit PLC activity, suppressed the [Ca2+]i elevation of neutrophils challenged by fMLP in Ca(2+)-containing medium, but not in Ca(2+)-free medium, with IC50 value 22.30 +/- 1.61 microM. In Mn(2+)-quench studies, U-73122 suppressed the Mn2+ influx in CPA-activated neutrophils (IC50 value was 7.16 +/- 0.28 microM) as well as in resting neutrophils (IC50 value was 6.72 +/- 0.30 microM). U-73343 also suppressed the Mn2+ influx in resting neutrophils in a concentration-dependent manner. These results suggest that the inhibitory effect of U-73122 on [Ca2+]i of activated neutrophils is attributed partly to the suppression of Ca2+ release from the intracellular Ca2+ stores through PLC inhibition, and partly to the blockade, especially at higher concentrations, of Ca2+ entry from the extracellular space through PLC-independent processes.
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PMID:U-73122, an aminosteroid phospholipase C inhibitor, may also block Ca2+ influx through phospholipase C-independent mechanism in neutrophil activation. 873 91

Alkaline phosphatase activity was released up to 100% from the membrane by incubating the rat osseous plate membrane-bound enzyme with phosphatidylinositol-specific phospholipase C. The molecular weight of the released enzyme was 145,000 on Sephacryl S-300 gel filtration and 66,000 on PAGE-SDS, suggesting a dimeric structure. Solubilization of the membrane-bound enzyme with phospholipase C did not destroy its ability to hydrolyse PNPP, ATP and pyrophosphate. The hydrolysis of ATP and PNPP by phosphatidylinositol-specific phospholipase C-released enzyme exhibited 'Michaelian' kinetics with K0.5 = 70 and 979 microM, respectively. For pyrophosphate, K0.5 was 128 microM and site-site interactions were observed (n = 1.4). Magnesium ions were stimulatory (K0.5 = 1.5 mM) and zinc ions were a powerful noncompetitive inhibitor (Ki = 6.2 microM) of phosphatidylinositol-specific phospholipase C-released enzyme. Phosphatidylinositol-specific phospholipase C-released alkaline phosphatase was relatively stable at 40 degrees C. However, with increasing temperature from 40-60 degrees C, the enzyme was inactivated rapidly following first order kinetics and thermal inactivation constants varied from 5.08 x 10(-4) min-1 to 0.684 min-1. Treatment of phosphatydilinositol-specific phospholipase C-released alkaline phosphatase with Chellex 100 depleted to 5% its original PNPPase activity. Magnesium (K0.5 = 29.5 microM), manganese (K0.5 = 5 microM) and cobalt ions (K0.5 = 10.1 microM) restored the activity of Chelex-treated enzyme, demonstrating its metalloenzyme nature. The stimulation of Chelex-treated enzyme by calcium ions (K0.5 = 653 microM) was less effective (only 26%) and occurred with site-site interactions (n = 0.7). Zinc ions had no stimulatory effects. The possibility that the soluble form of the enzyme, detected during endochondral ossification, would arise by the hydrolysis of the Pl-anchored form of osseous plate alkaline phosphatase is discussed.
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PMID:Characterization of the phosphatidylinositol-specific phospholipase C-released form of rat osseous plate alkaline phosphatase and its possible significance on endochondral ossification. 875 Nov 58


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