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)

ATP promoted biphasic effects on both basal and fMLP-stimulated arachidonic acid (AA) release in neutrophil-like HL60 cells: stimulation in the micromolar range (EC50 = 3.2 +/- 0.9 microM) and inhibition at higher concentrations (EC50 = 90 +/- 11 microM). ATP also inhibited UTP- and platelet activating factor-stimulated AA release. Only stimulatory effects of ATP on basal or fMLP-stimulated phospholipase C were observed. The inhibitory effect of ATP on AA release was not due to reacylation of released AA, chelation of extracellular Ca2+, cell permeabilization, or changes in the rise of [Ca2+]i induced by agonist. The inhibition was rapid, being detected within 5-15 s. The inhibitory effect of ATP on fMLP-stimulated AA release could be desensitized by pretreatment of the cells with 2 mM ATP, but not 20 microM ATP, the concentration that resulted in maximal release of AA and inositol phosphates. The inhibition by ATP was neither dependent on generation of adenosine by ATP hydrolysis nor the result of direct interaction of ATP with P1 purinergic receptors. Among other nucleotides tested (CTP, GTP, ITP, TTP, XTP, adenosine 5'-(beta,gamma-methylene)triphosphate (AMP-PCP), adenyl-5'-yl imidodiphosphate (AMP-P(NH)P), ADP, adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S), and UTP), only UTP and ATP gamma S displayed biphasic effects with potencies and efficacies almost identical to those of ATP. The other nucleotides only exhibited stimulatory effects (EC50 = 60-300 microM). The results are consistent with a model of dual regulation of AA release by two distinct subtypes of P2U receptors in HL60 cells.
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PMID:Dual regulation of arachidonic acid release by P2U purinergic receptors in dibutyryl cyclic AMP-differentiated HL60 cells. 131 16

Perturbation of the T-cell receptor (TCR) complex is followed by the rapid hydrolysis of inositol phospholipids (InsPL) by phospholipase C (PLC), producing diacylglycerol and inositol phosphates, which act as second messengers in signal transduction. The mechanism coupling the TCR to InsPL hydrolysis is not clearly defined, and no information is available on this mechanism in the CD4+ helper subset of T-lymphocytes (Th). We have tested the hypothesis that guanine-nucleotide-binding proteins (G-proteins) may couple the TCR to PLC in a murine Th type II (Th2) cell clone. Cell permeabilization with streptolysin O (SLO) or tetanolysin (TL) was used to allow membrane-impermeable nucleotides access to intracellular sites of action. Exposure of permeabilized Th2 cells to guanosine 5'-[gamma-thio]triphosphate (GTP gamma S), a non-hydrolysable GTP analogue, resulted in a 2.1-2.5-fold increase in inositol phosphate generation. Similarly, perturbation of the TCR with the monoclonal antibody 145.2C11 (directed against the epsilon-chain of the CD3 component of the TCR) resulted in a 3.1-4.2-fold increase in InsPL hydrolysis by permeabilized cells. Both lysins were similarly effective in allowing GTP gamma S induction of InsPL hydrolysis, but TL-permeabilized cells responded better to TCR perturbation than SLO-treated cells. A role for G-proteins in TCR coupling to PLC was further supported by the inhibition of TCR-induced InsPL hydrolysis by guanosine 5'-[beta-thio]diphosphate (GDP beta S), a guanine nucleotide analogue that inhibits G-protein function. ATP was required for TCR-mediated InsPL hydrolysis, and potentiated GTP gamma S-induced hydrolysis. Other nucleotides (i.e. CTP, GDP, GTP, ITP) did not affect the response. These data indicate that G-proteins may contribute to the regulation of PLC activation in Th2 cells, coupling it to the TCR.
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PMID:A role for guanine-nucleotide-binding proteins in mediating T-cell-receptor coupling to inositol phospholipid hydrolysis in a murine T-helper (type II) lymphocyte clone. 164 19

Undifferentiated and differentiated HL-60 leukemic cells possess nucleotide receptors which functionally couple to phospholipase C via pertussis toxin-sensitive guanine nucleotide-binding proteins (G-proteins). We investigated the role of extracellular nucleotides in the regulation of beta-glucuronidase release in HL-60 cells. In dibutyryl cyclic AMP (Bt2cAMP)-differentiated HL-60 cells, the chemotactic peptide, N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMet-Leu-Phe), the phosphorothioate analogue of ATP, adenosine 5'-O-[3-thio]triphosphate (ATP[gamma S]), and UTP increased cytosolic Ca2+ from 100 nM up to 1.2 microM with EC50 values of 4 nM, 1 microM and 100 nM, respectively. In these cells, ATP[gamma S] induced exocytosis with an EC50 of 4 microM and an effectiveness amounting to 50-70% of that of fMet-Leu-Phe. ATP, ITP, UTP, CTP, and uridine 5'-O-[2-thio]diphosphate activated exocytosis as well. Phorbol myristate acetate (PMA) induced exocytosis with an EC50 of 115 ng/ml and an effectiveness similar to that of ATP[gamma S]. Cytochalasin B (CB) differently potentiated exocytosis induced by ATP[gamma S], fMet-Leu-Phe and PMA. Treatment of Bt2cAMP-differentiated HL-60 cells with pertussis toxin (500 ng/ml) for 24 h resulted in ADP-ribosylation of more than 97.5% of the G-proteins. Under these conditions, pertussis toxin almost completely inhibited the increase in cytosolic Ca2+ and beta-glucuronidase release induced by fMet-Leu-Phe but only partially inhibited the effects of ATP[gamma S] and UTP. fMet-Leu-Phe at a non-stimulatory concentration (1 nM) potentiated ATP[gamma S]-induced beta-glucuronidase release in the presence but not in the absence of CB. In contrast, ATP[gamma S] and fMet-Leu-Phe synergistically activated superoxide formation in the absence of CB. PMA potentiated superoxide formation induced by ATP[gamma S] or fMet-Leu-Phe and did not affect exocytosis induced by ATP[gamma S] or fMet-Leu-Phe. In undifferentiated HL-60 cells, fMet-Leu-Phe, ATP[gamma S], UTP and PMA did not induce beta-glucuronidase release. fMet-Leu-Phe did not increase cytosolic Ca2+ in undifferentiated HL-60 cells, whereas ATP[gamma S] and UTP were similarly potent and effective as in Bt2cAMP-differentiated cells. In differentiated HL-60 cells, fMet-Leu-Phe induced aggregation, and ATP[gamma S] induced a transient shape change. Our results show (I) that exocytosis in HL-60 cells does not obligatorily depend on CB. (II) Purine and pyrimidine nucleotides activate exocytosis via pertussis toxin-sensitive and -insensitive signal transduction pathways.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Nucleotide-, chemotactic peptide- and phorbol ester-induced exocytosis in HL-60 leukemic cells. 196 23

Porcine brain dolichol kinase activity is effectively solubilized by extracting salt-washed microsomes with 1% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS). When the detergent-solubilized activity is chromatographed on Sepharose CL-6B, a low amount of dolichol kinase activity is recovered in the void volume, and a dolichol kinase activator (DKA) is eluted (Ve/Vo = 1.9-2.2) with the bulk of the membrane phospholipids. Although only approximately 20% of the activity applied to the Sepharose CL-6B column is detected in the column fractions, virtually all of the original activity is restored when the Vo fraction is recombined with DKA. Endogenous DKA, isolated from brain microsomes, is heat-stable, is extractable with CHCl3/CH3OH (2:1), and has the chemical and chromatographic properties of phosphatidylethanolamine (PE) and phosphatidylcholine (PC). Moreover, approximately 50% of the stimulatory activity is lost when the PC present in the DKA fraction is degraded by purified phospholipase C from Clostridium perfringens. Also consistent with a phospholipid co-factor requirement, the dolichol kinase activity recovered in the partially phospholipid-depleted fraction (Vo) is markedly stimulated by various molecular species of exogenous purified PC or PE, but not by phosphatidylinositol, phosphatidic acid, phosphatidylserine, phosphatidylglycerol, or sphingomyelin. A comparison of defined molecular species shows that PCs containing oleoyl or linoleoyl groups in the 1 and 2 positions are the most stimulatory, suggesting that the fatty acyl moieties are involved in the enzyme-phospholipid interaction. Kinetic analyses indicate that PC enhances the interaction between dolichol kinase and dolichol, the lipophilic substrate, but does not alter the apparent Km for CTP.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Separation of brain dolichol kinase from endogenous activating factors: evidence that phospholipid enhances the interaction between enzyme and dolichol. 215 52

Phosphatidylinositol 4-phosphate (PIP) kinase (E.C. 2.7.1.68) has been purified about 1200-fold from rat liver plasma membranes, taking advantage of affinity chromatography on quercetin-Sepharose as a novel step. The purified PIP kinase showed no contamination by the following enzyme activities: phosphatidylinositol (PI) kinase (EC 2.7.1.67), protein kinase C (EC 2.7.1.-), diacylglycerol kinase (EC 2.7.1.-), phospholipase C (EC 3.1.4.11), protein-tyrosine kinase (EC 2.7.1.112), alkaline phosphatase (EC 3.1.3.1), triphosphoinositide phosphomonoesterase (EC 3.1.3.36), adenylate kinase (EC 2.7.4.3) and cAMP-dependent protein kinase (EC 2.7.1.37). The liver membrane enzyme requires high Mg2+ concentrations with a KM value of 10 mM. Ca2+ or Mn2+ could replace Mg2+ to a certain, though small, extent. Apparent KM values with respect to PIP and ATP were 10 and 65 microM, respectively. GTP was slightly utilized by the kinase as phosphate donor while CTP was not. Quercetin inhibited the enzyme with Ki = 34 microM. Extending our previous observations (Urumow, T. and Wieland, O.H. (1986) FEBS Lett. 207, 253-257 and Urumow, T. and Wieland, O.H. (1988) Biochim. Biophys. Acta 972, 232-238) [gamma S]pppG still stimulated the PIP kinase in extracts of solubilized liver membranes. 20-40% (NH4)2SO4 precipitation of the membrane extracts yielded a fraction that contained the bulk of enzyme activity but did not respond to stimulation by [gamma S]pppG any longer. This was restored by recombination with a protein fraction collected at 40-70% (NH4)2SO4 saturation, presumably containing a GTP binding protein and/or some other factor separated from the PIP kinase. In the reconstituted system [gamma S]pppG stimulated PIP kinase in a concentration dependent manner with maximal activation at 5 microM. This effect was not mimicked by [gamma S]pppA and was blocked by [beta S]ppG. These results strongly support our view that in liver membranes PIP kinase is regulated by a G-protein.
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PMID:Purification and partial characterization of phosphatidylinositol-4-phosphate kinase from rat liver plasma membranes. Further evidence for a stimulatory G-protein. 215 97

We have previously determined that human neutrophils and monocytes, as well as neutrophil/monocyte progenitor cells, express a subtype of P2-purinergic receptors (for ATP) which activate the inositol phospholipid signalling system. In the present study, membranes prepared from HL-60 promyelocytic leukemia cells were used to examine the mechanism by which these ATP receptors activate phosphatidylinositol-specific phospholipase C (PI-PLC) under defined in vitro conditions. Micromolar concentrations of the receptor agonists ATP, UTP, and ATP gamma S stimulated the GTP-dependent formation of inositol bisphosphate (IP2) and inositol trisphosphate (IP3) in washed membranes prepared from undifferentiated HL-60 cells prelabeled with [3H]inositol. The stimulatory effects of these nucleotides on PI-PLC appeared to be mediated through a GTP binding protein since minimal inositol polyphosphate accumulation was observed in the absence of guanine nucleotides. The increased inositol polyphosphate formation triggered by these nucleotide receptor agonists did not result from inhibition of GTP breakdown. Neither was it a consequence of increased [3H]polyphosphatidylinositol levels resulting from enhanced activity of membrane-associated PI- or PIP-kinases. Instead, the stimulated phospholipase activity was apparently receptor-mediated. The rank order of potency observed in these in vitro membrane assays (ATP = UTP greater than ATP gamma S much greater than TTP greater than CTP much greater than beta, gamma-CH-ATP) was similar to that observed with intact HL-60 cells. This order of potency appears to distinguish the P2-purinergic receptors expressed by human phagocytic leukocytes from the P2 gamma-purinergic receptors which activate PI-PLC in turkey erythrocyte membranes.
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PMID:P2-purinergic receptors activate a guanine nucleotide-dependent phospholipase C in membranes from HL-60 cells. 216 87

Previous studies have shown that vertebrate rod outer segments (ROS) have a light activated phospholipase C which hydrolyzes phosphatidylinositol-4,5-bisphosphonate (PIP2). Three different experimental approaches have been used to test the hypothesis that the phosphatidylinositol (PI) biosynthetic cycle is present in ROS and that PIP2 can be regenerated from DG independent of rod inner segments. In the first study, enzyme activities of the PI cycle were assayed simultaneously in the presence of CTP, myo-inositol and [gamma-32P]ATP using endogenous lipids as substrates. Under these conditions, broken (leaky) ROS prepared by continuous sucrose gradient centrifugation showed PI, PIP and DG kinase activities similar to those found in intact ROS and non-ROS membranes, whereas PI synthetase activity was much lower in the leaky ROS than in the other two fractions. The relative distribution of PI synthetase specific activity in the three membrane preparations was similar to that of the microsomal enzyme marker cytochrome c reductase. ROS prepared by discontinuous sucrose gradient centrifugation showed only 2-3% of whole homogenate PI synthetase or phosphatidyl: cytidyl transferase activities, and the distribution of activities was the same as for microsomal and mitochondrial marker enzymes. In the second study, whole retinas were incubated with myo-[2-3H]inositol or [2-3H]glycerol in vitro, and the time course of incorporation of radioactivity into PI and other phospholipids was determined for ROS and three other retinal fractions. Over a 10-hr period, the rate of incorporation of myo-[2-3H]inositol or [2-3H]glycerol into PI in ROS was lowest among the various retinal fractions. In the third study, chemical analysis of the molecular species composition of PI, DG and phosphatidic acid (PA) from ROS shows that PA is substantially different from PI and DG, the latter two being quite similar. These results are consistent with a precursor-product relationship between PI and DG, but not with the conversion of DG to PA or of PA to PI. Taken together, these three studies indicate that ROS do not have PI synthetase or phosphatidyl: cytidyl transferase activities, but do have DG, PI and PIP kinase activities. Thus, the PI in ROS lost through rapid turnover must be replaced with molecules derived from de novo synthesis in the inner segment of the photoreceptor cell.
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PMID:Phosphoinositide metabolism in frog rod outer segments. 216 31

Extracellular ATP and UTP caused a rapid formation of InsP3, with similar kinetics and dose-dependences. ITP also displayed strong agonistic properties in terms of InsP3 production, whereas CTP was almost inactive. Pretreatment of the cells with pertussis toxin attenuated ATP- and UTP-stimulated InsP3 generation to a comparable extent, indicating that both nucleotides couple to phospholipase C by a pertussis-toxin-sensitive G-protein. Short-term (15 min) treatment of the cells with phorbol 12-myristate 13-acetate (PMA) produced a dose-dependent inhibition of ATP- and UTP-induced InsP3 formation. Furthermore, down-regulation of protein kinase C by long-term (24 h) exposure of the cells to PMA resulted in a comparable potentiation of phosphoinositide hydrolysis by both nucleotides. Preincubation of mesangial cells with ATP or UTP caused a pronounced cross-desensitization of subsequent nucleotide-stimulated InsP3 production. ATP and UTP displayed no additivity in terms of InsP3 formation, when used at maximally effective concentrations. In contrast, the peptide hormone angiotensin II interacted in an additive manner with either nucleotide in stimulating phosphoinositide hydrolysis. Reactive Blue 2, a putative P2y-purinoceptor antagonist, caused a rightward shift of both the ATP and UTP dose-response curves. However, since 2-methylthio-ATP was only a partial agonist in stimulating InsP3 formation, the mesangial-cell ATP receptor appears to be different from a classic P2y-receptor. In summary, these results provide no evidence for separate purino- and pyrimidino-ceptors on mesangial cells. In contrast, ATP and UTP may use a common nucleotide receptor for transducing their signals in mesangial cells.
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PMID:Comparison of extracellular ATP and UTP signalling in rat renal mesangial cells. No indications for the involvement of separate purino- and pyrimidino-ceptors. 217 64

1. Flash photolysis of caged compounds of phenylephrine, inositol 1, 4, 5 trisphosphate (InsP3), GTP gamma S, ATP, and CTP has been successfully used to study excitation-contraction coupling, contractile regulation, and contraction in smooth muscle. Major processes explored with this method were (a) the delay between agonist-receptor interaction and contraction and between the rise in InsP3, Ca2+ release and contraction; (b) the effect of myosin light chain phosphorylation on the rate of force development and the respective contributions of phosphorylation and crossbridge kinetics to differences between phasic and tonic smooth muscles; (c) the kinetics of the crossbridge cycle. We have also reviewed recent results obtained by other methods and bearing on the mechanisms of pharmacomechanical Ca2+ release and modulation of the Ca2+ sensitivity of the regulatory/contractile apparatus. 2. The long delay (1.5 at 22 degrees C) following activation of alpha 1-adrenergic receptors through photolysis of caged phenylephrine and the high Q10 of this process are consistent with the hypothesis that activation of phospholipase C is the major mechanism of alpha-adrenergic pharmacomechanical Ca2+ release. 3. The delay between photolysis of caged InsP3 and Ca2+ release is short: 30 ms or less, while the latency of contraction is significant (0.3-0.5 s at 22 degrees C) and similar to the lag between the rise in [Ca2+]i and force development in intact smooth muscles. The latency of contraction following photolysis of caged ATP in permeabilized muscles in rigor, in the presence of Ca2+ and calmodulin, is similar, about 0.2-0.5 s at 22 degrees C. 4. In muscles in which the myosin light chains are maintained in a phosphorylated state during rigor, photolysis of caged ATP initiates contractions with a short delay (10 ms or less). This result and those summarized above (2 and 3) suggest that the major portion of the delay between agonist-receptor interaction and contraction is due to activation of phospholipase C and InsP3 production, and about 0.2-0.5 s of the delay (22 degrees C) can be ascribed to prephosphorylation reactions between Ca2+, calmodulin, and myosin light chain kinase, and/or to mechanical processes, or to the chemical kinetics of two-step reactions. 5. Force development from rigor, initiated by photolysis of caged ATP in the presence of Ca2(+)-calmodulin, is rate-limited by myosin light chain phosphorylation; it is significantly accelerated if the myosin light chains are already phosphorylated prior to photolysis.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Flash photolysis studies of excitation-contraction coupling, regulation, and contraction in smooth muscle. 218 79

Cultured pituitary cells prelabeled with myo-[2-3H] inositol were permeabilized by ATP4-, exposed to guanine nucleotides and resealed by Mg2+. Addition of guanosine 5'-0-(3-thio triphosphate) (GTP gamma S) to permeabilized cells, or gonadotropin releasing hormone (GnRH) to resealed cells, resulted in enhanced phospholipase C activity as determined by [3H] inositol phosphate (Ins-P) production. The effect was not additive, but the combined effect was partially inhibited by guanosine 5'-0-(2-thiodiphosphate) (GDP beta S) or by neomycin. Surprisingly, addition of GDP beta S (100-600 microM) on its own resulted in a dose-related increase in [3H]Ins-P accumulation. Several nucleoside triphosphates stimulated phospholipase C activity in permeabilized pituitary cells with the following order: UTP greater than GTP gamma S greater than ATP greater than CTP. The stimulatory effect of UTP, ATP and CTP, but not GTP gamma S or GDP beta S, could also be demonstrated in normal pituitary cells suggesting a receptor-activated mechanism. GTP and GTP gamma S decreased the affinity of GnRH binding to pituitary membranes and stimulated LH secretion in permeabilized cells. These results suggest the existence of at least two G-proteins (stimulatory and inhibitory) which are involved in phospholipase C activation and GnRH action in pituitary cells.
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PMID:Effect of guanine nucleotides on phospholipase C activity in permeabilized pituitary cells: possible involvement of an inhibitory GTP-binding protein. 249 87


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