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)

Platelet-activating factor (PAF) is an unusually potent phospholipid known to be produced by neuronal cells and to modulate cerebral blood flow and metabolism. In previous studies with NCB-20 cells, we reported that PAF induced a significant mobilization of intracellular free Ca2+ ([Ca2+]i), which was inhibited by PAF antagonists. The increase was the result of release from intracellular stores and influx from extracellular sources. The present study was designed to characterize further PAF receptor-mediated cellular signal-transduction mechanisms in myo-[3H]inositol-labeled cells. PAF induced a concentration-dependent increase in phosphatidylinositol (Pl) metabolism, with EC50 values of 1.96 +/- 0.62 nM and 1.12 +/- 0.50 nM for inositol trisphosphate (IP3) and inositol monophosphate (IP1) formation, respectively (four experiments). The maximal production of IP3 and IP1 induced by 50 nM PAF was 254 +/- 34% and 178 +/- 25% over the basal, respectively (four experiments). PAF-induced Pl metabolism was concentration-dependently inhibited by the PAF antagonist BN50739, with an IC50 value of 6.48 +/- 0.52 nM (four experiments). The protein kinase C (PKC) activator phorbol 12,13-dibutyrate concentration-dependently inhibited PAF-induced Pl metabolism and [Ca2+]i mobilization in NCB-20 cells, of NCB-20 cells with pertussis toxin (PTX) resulted in a concentration-dependent inhibition of PAF-induced IP3 production and intracellular Ca2+ release, with a maximal reduction of 66.9 +/- 3.5% and 63 +/- 6.1%, respectively, at 300 ng/ml PTX. PTX in the presence of [32P]NAD specifically [32P]ADP-ribosylated a 38-kDa protein in membranes prepared from NCB-20 cells. Pretreatment of the cells with PTX resulted in a concentration-dependent inhibition of subsequent 32P-labeling of the toxin substrate in the membranes and correlated with the uncoupling of PAF-induced IP3 formation. PAF (0.01-10 nM) elicited a concentration-related stimulation in guanosine 5'-O-(3-[35S]) triphosphate ([35S]GTP gamma S) binding to G alpha i(1,2) proteins, which was inhibited by the PAF antagonist BN50739. PAF at 10 nM also increased [35S]GTP gamma S binding to G alpha s and G alpha o. PAF-evoked activation of G alpha i(1,2) and G alpha o was reduced by preincubation with PTX. Our results reveal that neuronal cells possess PAF receptors linked through guanine nucleotide-binding proteins to phospholipase C and receptor-operated Ca2+ channels that are regulated by PKC. Both PTX-sensitive and -insensitive guanine nucleotide-binding proteins appear to couple the PAF receptor to activation of phospholipase C and the increase in [Ca2+]i. These results contribute to the further understanding of the mechanisms behind PAF actions on neuronal cells.
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PMID:Platelet-activating factor stimulates phosphoinositide turnover in neurohybrid NCB-20 cells: involvement of pertussis toxin-sensitive guanine nucleotide-binding proteins and inhibition by protein kinase C. 131 8

The protein kinase C (PKC) activator, phorbol 12, 13-dibutyrate (PDBu) dose-dependently inhibited platelet-activating factor (PAF)-induced [Ca2+]i elevation and inositol monophosphate (IP1) accumulation in neurohybrid NG108-15 cells with IC50 values of 162 nM and 35 nM, respectively. Pretreatment of NG108-15 cells with PKC inhibitor H-7 partially prevented the inhibitory effect of PDBu on PAF-induced [Ca2+]i elevation as well as PI metabolism in NG108-15 cells. Pretreatment of the cells with pertussis toxin (PTX) resulted in a dose-dependent inhibition of PAF-induced IP1 and IP3 accumulation but only slightly affected PAF-induced [Ca2+]i elevation in NG108-15 cells. The results reveal that PAF receptor-mediated Ca2+ mobilization and PI metabolism in NG108-15 cells are regulated by PKC while a PTX-sensitive G protein is coupled to PAF receptor for inducing activation of phospholipase C.
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PMID:Protein kinase C activator phorbol 12, 13-dibutyrate inhibits platelet activating factor-stimulated Ca2+ mobilization and phosphoinositide turnover in neurohybrid NG108-15 cells. 132 41

Platelet activating factor (PAF) stimulated aggregation and [32P]-phosphatidic acid (PA) production was compared in normal and diabetic human subjects in platelet rich plasma. The concentration of PAF for half maximal (50%) aggregation of normal and diabetic platelets was 50 nM and 8 nM, respectively. PAF stimulated [32P]-PA production (a metabolite of phospholipase C pathway) was also greater in the platelets from diabetic subjects. This [32P]-PA production was inhibited by the PAF receptor antagonists SRI-63441 and SRI-63675. When the levels of glycosylated hemoglobin (HbA1c) were compared with the PAF stimulated [32P]-PA production a significant relationship was observed. These studies have demonstrated for the first time that diabetic human platelets show hypersensitivity to PAF in both aggregation and [32P]-PA production compared to normal subjects. This may be a result of some modification in phospholipid turnover mechanism and is receptor mediated. Further, the relationship of the degree of aggregation and [32P]-PA production to the level of HbA1c suggest that the insulin deficiency may contribute to these effects.
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PMID:Hypersensitivity of diabetic human platelets to platelet activating factor. 132 54

Human preimplantation embryos secrete platelet-activating factor (PAF), which stimulates prostaglandin E2 synthesis from secretory endometrium. This study investigated the action of PAF on phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2)-specific phospholipase C activity in human endometrium. Slices of normal endometrium were incubated with 5 microCi/ml myo-[2-3H] inositol for 3 h at 37 degrees C in 95% O2 and 5% CO2 to label tissue phosphoinositides. Inositol phosphates were extracted using trichloroacetic acid precipitation and diethylether neutralization and production was measured using Dowex 1-X8 anion-exchange column chromatography. PAF induced rapid and concentration-dependent accumulation of inositol phosphates (IP) from secretory endometrium, but had no effect on endometrium removed in the proliferative phase of the menstrual cycle. The IP3 fraction was significantly elevated from a median value of 14.0 c.p.m. mg-1 dry wt [range: 8-41 c.p.m. mg-1 dry wt] to 28.0 c.p.m. mg-1 dry wt [range: 11-87 c.p.m. mg-1 dry wt, P less than 0.002] following 1 min exposure of secretory endometrium to PAF-acether, in the presence of 10 mM LiCl. PAF-induced hydrolysis of PtdIns(4,5)P2 was inhibited by the specific PAF receptor antagonist WEB 2086, in a dose-dependent manner (P less than 0.02), indicating that in human endometrium PtdIns(4,5)P2 hydrolysis is mediated via a PAF receptor. These results indicate that PAF receptor coupling activates endometrial PtdIns(4,5)P2-specific phospholipase C only in the secretory phase of the menstrual cycle, suggesting that the PAF response may be under ovarian steroid regulation. It is proposed that the ability of the endometrium to respond to PAF appears to be a feature of the preparation of this tissue for implantation and that the second messengers generated may play a role in cellular processes involved in the maternal recognition of very early human pregnancy.
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PMID:Platelet-activating factor stimulates phospholipase C activity in human endometrium. 161 19

Platelet-activating factor (PAF) is a proinflammatory lipid that has platelet-stimulating property. PAF receptor-coupled activation of phosphoinositide-specific phospholipase C (PLC) and phosphorylation of several proteins has already been established in our laboratory. To investigate further the molecular mechanism and relationship between activation of PLC and protein phosphorylation, we have used Genistein (a putative inhibitor of tyrosine-specific protein kinases), phosphotyrosine antibody, and phosphoamino acid analysis to probe the involvement of tyrosine kinase in this process. Washed rabbit platelets were loaded with myo-[2-3H]inositol and challenged with PAF (100 nM) after pretreatment with Genistein. PLC-mediated production of radioactive inositol monophosphate, inositol diphosphate, and inositol triphosphate was monitored. PAF alone caused stimulation of PLC activity [( 3H]inositol triphosphate production), whereas pretreatment with Genistein (0.5 mM) diminished PAF-stimulated PLC activity to basal level. Genistein also blocked PAF-stimulated platelet aggregation at this dose. In contrast to Genistein, staurosporine which inhibits protein kinase C, potentiated PAF-stimulated [3H]inositol triphosphate production. Genistein substantially inhibited the combined effects of staurosporine and PAF on inositol triphosphate production. Genistein also reduced PAF-induced phosphorylation of Mr 20,000 and 50,000 proteins. Phorbol 12-myristate 13-acetate-induced Mr 40,000 protein phosphorylation was also affected by Genistein. The above results suggested that Genistein inhibited tyrosine kinase at an early stage of signal transduction by inhibiting PLC. This, in turn, decreased the activation of protein kinase C and, therefore, caused a reduction in Mr 40,000 protein phosphorylation. The inhibition of PLC by Genistein raised the possibility of involvement of tyrosine kinase in PAF receptor-coupled PLC activation. Western blot analysis using monoclonal antibody to phosphotyrosine demonstrated that PAF stimulated the tyrosine phosphorylation of two major proteins of 50,000 and 60,000 molecular weight. When platelets were challenged with PAF after treatment with either Genistein or CV-6209 (a PAF receptor antagonist), the reactivity of these proteins to monoclonal antibody was inhibited. Phosphoamino acid analysis of Mr 50,000 and 60,000 proteins confirmed that PAF increased the phosphorylation of tyrosine residues in both Mr 50,000 and 60,000 proteins and that this was inhibited by Genistein. Thus, PAF caused a receptor-dependent phosphorylation of tyrosine residues on Mr 50,000 and 60,000 proteins. Based on these observations, it is concluded that tyrosine kinase is involved in the PAF receptor-coupled PLC activation and signal transduction mechanism.
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PMID:Platelet-activating factor stimulation of tyrosine kinase and its relationship to phospholipase C in rabbit platelets: studies with genistein and monoclonal antibody to phosphotyrosine. 169 37

Platelet-activating factor (PAF) activates human platelets by binding to a putative PAF receptor which evokes the rapid formation of inositol-1,4,5-trisphosphate (IP3) by phospholipase C mediated phosphatidylinositol-4,5-bisphosphate (PIP2) hydrolysis. Stimulation of [3H]inositol-labeled human platelets by PAF (1 nM-1 microM) resulted in a concentration-dependent increase of intracellular IP3, IP2 and inositolmonophosphate (IP1). IP1 levels increased up to three-fold upon maximum stimulation by 100 nM PAF. The EC50 concentration for PAF was 1.2 +/- 0.3 nM. Addition of the hetrazepinoic PAF antagonist, WEB 2086, inhibited PAF stimulated hydrolysis of PIP2 in a dose-dependent manner. WEB 2086 (100 microM) blocked inositol-1,4,5-trisphosphate formation down to baseline levels (IC50 = 33 +/- 12 microM WEB 2086). In thrombin and ADP stimulated platelets, inositol phosphate (IP) generation was not influenced by WEB 2086. It is concluded that WEB 2086 selectively antagonizes PAF-induced increases in IP and does not interfere directly with intracellular signal transduction. Instead, WEB 2086, which has been shown to bind specifically and with high affinity (Ki 15 nM) to human platelets, acts as a competitive antagonist at the PAF receptor level.
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PMID:Inhibition by the PAF antagonist WEB 2086 of PAF induced inositol-1,4,5-trisphosphate production in human platelets. 181 88

Platelet-activating factor (PAF), a unique phospholipid mediator, possesses potent proinflammatory, smooth-muscle contractile and hypotensive activities, and appears to be crucial in the pathogenesis of bronchial asthma and in the lethality of endotoxin and anaphylactic shock. Despite this, little is known of the molecular properties of the PAF receptor and related signal transduction systems. Although several lines of evidence suggest that activation of the PAF receptor stimulates phospholipase C and subsequent inositol trisphosphate formation through G protein(s), the PAF receptor and calcium channel are reported to show a close relation. As a first approach to cloning lipid autacoid receptors, we have isolated complementary DNA for the PAF receptors. Our strategy involved gene expression in Xenopus laevis oocytes and electrophysiological detection of PAF-induced responses. Sequence analysis indicates that the receptor belongs to the superfamily of G protein-coupled receptors.
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PMID:Cloning by functional expression of platelet-activating factor receptor from guinea-pig lung. 184 31

The phospholipid platelet-activating factor (PAF) (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) stimulated the accumulation of inositol phosphates in cultures of rat and bovine anterior pituitary cells. In response to PAF, inositol 1,4-bisphosphate showed the largest percent increase of the inositol phosphates in the presence of lithium chloride. PAF induced an increase of inositol 1,4,5-trisphosphate, the biologically active isomer responsible for mobilization of intracellular calcium. A characterization of the PAF response indicated that PAF, but not its biologically inactive enantiomer, induced the accumulation of inositol phosphates in the rat anterior pituitary. Further, the PAF receptor antagonist L652731 reduced PAF stimulation. The ED50 for PAF-induced inositol 1,4-bisphosphate accumulation was 0.4 nM. PAF induced a rapid response that did not persist beyond 20 min. While PAF treatment of anterior pituitary cells did not alter TRH-induced inositol phosphate accumulation, it did prevent a second exposure of PAF from inducing inositol phosphate accumulation. These data suggest that PAF induces a rapid stimulation of phospholipase C causing the hydrolysis of phosphatidylinositol 4,5-bisphosphate and the generation of the second messengers, inositol 1,4,5-trisphosphate and diglyceride, in anterior pituitary tissue. This action is transient probably due to PAF receptor desensitization. The action of PAF on generation of inositol phosphates may account, in part, for PAF-induced secretion of PRL and GH.
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PMID:Platelet activating factor induces inositol phosphate accumulation in cultures of rat and bovine anterior pituitary cells. 216 2

Human monocytic leukemic U-937 cells, when differentiated with dimethylsulfoxide to macrophage-like state, express receptors for platelet-activating factor (PAF). In the differentiated U-937 cells, PAF induced hydrolysis of phosphoinositides and synthesis of inositol phosphates. PAF-induced production of inositol phosphates was rapid, concentration-dependent and was inhibited by a receptor antagonist CV3988, indicating that it was mediated via a specific receptor. In fura-2-loaded, differentiated U-937 cells, PAF induced immediate and concentration-dependent calcium mobilization [( Ca++]i) that was inhibited by CV3988, but not by calcium channel blockers. Addition of an increasing concentration of calcium chelator, ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid, to the medium inhibited a large fraction (approximately 75%) of PAF receptor-induced [Ca++]i mobilization thus suggesting the majority of [Ca++]i mobilization was originated from extracellular milieu and a small portion (approximately 25%) was originated from intracellular sources. The inositol phosphate production induced by PAF, however, was independent from the extracellular calcium and was not inhibited by the addition of ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid. Neither [Ca++]i mobilization or phosphoinositide metabolism in U-937 cells was sensitive to treatment of pertussis toxin, but both types of effects were sensitive to treatment by an inhibitor of phospholipase C, manoalide. These results suggest that in differentiated U-937 cells PAF receptor is coupled through a pertussis toxin-insensitive guanine nucleotide binding protein to a phosphoinositide specific phospholipase C. Inositol-trisphosphate, and possibly diacylglycerol, could be the intracellular messengers for PAF receptor in U-937 cells.
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PMID:Platelet-activating factor-induced phosphoinositide metabolism in differentiated U-937 cells in culture. 253 1

Treatment of 32P-labeled rabbit platelets with platelet-activating factor (PAF) caused a time- and dose-dependent phosphorylation of several proteins including five major phosphorylated proteins with apparent molecular weights of 20,000, 35,000, 40,000, 65,000, and 150,000. Both PAF and thrombin caused a rapid increase followed by a decrease in phosphorylation of proteins, indicating the occurrence of a phosphorylation-dephosphorylation process. Four separate PAF receptor antagonists, CV-3988, CV-6209, SRI-63-441, and SRI-63-675 drastically reduced the PAF-stimulated protein phosphorylation. The order of potency was SRI-63675 greater than SRI-63441 greater than or equal to CV-6209 greater than CV-3988. These antagonists had no effect on thrombin-stimulated protein phosphorylation. Pretreatment of platelets with PAF (0.1 nM) completely abolished any further protein phosphorylation by the same concentration of PAF. PAF pretreatment shifted the dose response of protein phosphorylation by about 2 log units, to the right. When platelets were treated with PAF (10 nM) for 10 min, this abolished phosphorylation of proteins by any concentration of PAF. These studies indicated a homologous desensitization of protein phosphorylation. Interestingly, PAF-pretreated platelets still exhibited phosphorylation of proteins by thrombin. On the other hand, a lack of protein phosphorylation by PAF or thrombin was observed in platelets preexposed to thrombin and this demonstrated a heterologous desensitization. It is concluded that phosphorylation of proteins by PAF is a PAF receptor-coupled event and that this process is desensitized in platelets preexposed to PAF. The fact that both the activation of phosphoinositide-specific phospholipase C and the phosphorylation of proteins are desensitized in PAF-pretreated platelets suggests that a close "regulatory" intercommunication between these processes exists.
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PMID:Desensitization of platelet-activating factor-stimulated protein phosphorylation in platelets. 253 56


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