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)

In this study we used the bovine thoracic aorta endothelial cell line AG 4762 and primary bovine aortic endothelial cells to investigate the formation of phosphatidic acid (PA) in response to activation of P2-purinergic receptors. 2-Methylthio ATP (2MeSATP) stimulated the formation of [32P]-PA in bovine aortic endothelial cells labelled with 32P(i) for 2.5 hr. A comparison of the response to other ATP analogues suggests that this was mediated via a P2Y-purinergic receptor. Using various agonists at 30 microM there was a correlation between the formation of [32P]PA and of total inositol phosphates in the presence of lithium. The 2MeSATP-stimulated accumulation of [32P]PA showed an initial high rate, followed by a more sustained slower rate. The initial response was independent of extracellular calcium while the later response was dependent on calcium influx. The protein kinase C stimulator phorbol myristate acetate (PMA) produced only a very small enhancement of [32P]PA accumulation compared to 2MeSATP. The 2MeSATP stimulation of both inositol phosphates and [32P]PA was almost eliminated by the presence of PMA. Using cells prelabelled with [3H]methylcholine 2MeSATP produced only a small non-significant enhancement of [3H]choline formation; PMA by contrast formed a much larger amount of [3H]choline. There was no evidence of a change in [3H]phosphocholine. The dissociation between phospholipase D (PLD) activation and [32P]PA accumulation and the correlation between stimulation of [32P]PA accumulation and phospholipase C (PLC) activation all suggest that, using this protocol for labelling cells, the principle route of the stimulation of formation of [32P]PA is via the activation of PLC followed by metabolism of diacylglycerol (DAG) by DAG kinase. These results show that activation of P2Y-purinergic receptors on aortic endothelial cells leads to the formation of phosphatidic acid and that both PLD and PLC pathways are likely to contribute to this response.
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PMID:Stimulation of phosphatidic acid synthesis in bovine aortic endothelial cells in response to activation of P2-purinergic receptors. 156 76

Phosphatidylcholine (PC) hydrolysis has been shown to occur in hormone-stimulated cells and represents a potential metabolic source, in addition to phosphoinositides, for the generation of diradylglycerols (DG). We performed studies in order to quantify the importance of this pathway in DG formation. We incubated murine peritoneal macrophages with platelet-activating factor (PAF), ionomycin, phorbol myristate acetate (PMA) or no stimulus in a series of timed incubations ranging from 15 s to 20 min. We quantified the profiles of the molecular species in the accumulated DG after extraction, specific radiolabelling to give [32P]phosphatidic acid by DG kinase, and conversion to the dimethyl derivative. We used two independent methods for molecular species analysis: (1) reversed-phase h.p.l.c. separation with in-line beta-radiation detection of peaks, and (2) an argentation-t.l.c. separation with scintillation counting of bands. Our results showed a clearly biphasic sequence in the composition of accumulated DG. The molecular species composition of early DG (up to 1 min stimulation time) was very similar to that of unstimulated DG, whereas the proportions of the species present in later DG were substantially altered. In the same experiments, we extracted native phospholipids from unstimulated macrophages, separated phosphatidylinositol (PI), PC, phosphatidylethanolamine (PE) and phosphatidylserine (PS), converted them to the corresponding DGs by using phospholipase C, and determined their molecular species compositions as above. In comparison with the diradyl compositions of stimulated DG, the diradyl composition of PI closely matched that of early DG, the differences between the PC and PI compositions matched the differences between early and late DG very closely, and the compositions of PE and PS were unique and unrelated. We quantified these relationships more precisely by multilinear regression analysis to calculate the theoretical best mix of five molecular species compositions (PI, PC, PE, PS and unstimulated DG) that would most closely replicate the early and late accumulated DG compositions. We found that by both h.p.l.c. and t.l.c. analyses, 15-30% (PAF) or 25-50% (ionomycin and PMA) of the later DG could be accounted for by PC hydrolysis. These results represent quantifications of phospholipid class contributions to stimulated DG formation, and demonstrate the potential importance of PC hydrolysis in phagocytic leucocytes.
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PMID:Quantification of contributions of phospholipid precursors to diradylglycerols in stimulated mononuclear phagocytes. 159 20

Primary cultures of mouse embryo palate mesenchyme (MEPM) cells incubated with 1-O-[3H]alkyl-2-lyso-sn-glycero-3-phosphocholine ([3H])lyso-PAF) incorporated radiolabel into 1-radyl-2-acyl-sn-glycero-3-phosphocholine (PC) and -phosphoethanolamine (PE). The radiolabeled PC was insensitive to hydrolysis with HCl fumes, whereas at least 82% of the 3H found in the PE was hydrolyzed to 3H-aldehydes by such treatment. Treatment of the PC with Vitride produced [3H]alkylglycerol; similar treatment of the PE produced [3H]alk-1-enylglycerol. None of the radiolabeled products yielded fatty alcohol upon reduction with Vitride. These findings indicate the radiolabeled PC was 1-O-alkyl-linked whereas the PE contained predominantly 1-O-alk-1'-enyl species with smaller amounts of 1-O-alkyl species. Homogenates of MEPM cells which had been prelabeled with [3H]lyso-PAF and [14C]arachidonic acid produced 14C-fatty acid, [3H]lyso-PC, and [3H]alkylglycerol when incubated at selected values of pH and concentrations of calcium. There was no accumulation of [3H]lyso-PE in the various incubation mixtures. Stimulation of MEPM cells with the ionophore A23187 in the presence of calcium and [3H]acetate resulted in the production of 3H-platelet-activating factor (PAF), identified by its migration with authentic PAF and its conversion to 1-O-[3H]alkyl-2,3-diacetylglycerol upon treatment with phospholipase C and acetic anhydride. These studies demonstrate that: (i) MEPM cells are able to incorporate [3H]lyso-PAF into 1-O-alkyl-2-acyl-sn-glycero-3-phosphocholine, the storage form of PAF, and into 1-O-alk-1'-enyl-2-acyl-sn-glycero-3-phosphoethanolamine (PE plasmalogen); (ii) endogenous 1-O-[3H]alkyl-2-acyl-sn-glycero-3-phosphocholine can serve as a substrate for phospholipase A2 in homogenates; and (iii) MEPM cells have the ability to synthesize PAF, thus raising the possibility that this compound may play a role in modulating the physiology of these embryonic cells.
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PMID:Synthesis of platelet activating factor and metabolism of related lipids in embryonic cells. 162 22

Human blood monocytes were activated by bacterial lipopolysaccharide endotoxin (LPS) (10 ng/ml) for cytotoxicity of WEHI-164 mouse fibrosarcoma cells, determined by release of 51Cr from WEHI-164 tumour cells incubated with monocyte supernatants. The chemotactic peptide N-formylmethionyl-leucyl-phenylalanine (FMLP) augmented LPS-induced cytotoxicity but had no effect alone. FMLP but not LPS stimulated phospholipase C (PLC), determined by the release of [3H]inositol phosphates. Addition of tumour promoter and protein kinase C stimulant, phorbol-12-myristate-13-acetate (PMA) at concentrations of 3 x 10(-10) M to 3 x 10(-9) M, resulted in an augmentation of 30-200% in LPS-evoked cytotoxicity. The effects of FMLP and PMA, like the effect of LPS alone, were completely blocked by antibody to recombinant human tumour necrosis factor-alpha (TNF-alpha), indicating that cytotoxicity induced by LPS, FMLP, and PMA were due solely to TNF release. Concentrations of PMA greater than 3 x 10(-9) M caused inhibition of TNF release. Okadaic acid (20 ng/ml), an inhibitor of phosphatases I and IIa, augmented the effects of LPS and the stimulatory effects of low levels of PMA, suggesting that phosphorylation was important in the actions of both LPS and PMA. The effects of LPS and of low levels of PMA were augmented by the protein kinase C (PKC) inhibitors H-7 (10-30 microM), staurosporine (2-10 nM) and calphostin C (0.1 microM). Higher concentrations of the inhibitors prevented LPS-evoked TNF release and its augmentation by low levels of PMA. However, they did not prevent the inhibition by high levels of PMA. One possible explanation for the results is that different isozymes of PKC may mediate the stimulatory as compared to the inhibitory effects of PKC on TNF production.
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PMID:Paradoxical stimulation and inhibition by protein kinase C modulating agents of lipopolysaccharide evoked production of tumour necrosis factor in human monocytes. 162

The biological activity of platelet-activating factor (PAF) is comprised by a few molecular species of phosphatidylcholine which contain a fatty alcohol connected by an ether linkage to the sn-1 position of the glycerol backbone and an acetate ester at the sn-2 position. The various molecular species of PAF differ in chain length and degree of unsaturation in the fatty alcohol residue side-chain. PAF is rapidly hydrolyzed to lyso-PAF by an acetylhydrolase enzyme which is quite active in a number of cells that synthesize PAF. We describe a method for quantitation of lyso-PAF which involves conversion to its propionate derivative in the presence of an internal standard (deuterium-labelled PAF), digestion to the diglyceride with Bacillus cereus phospholipase C, conversion to the pentafluorobenzoate derivative and capillary column gas chromatographic-negative-ion methane chemical ionization mass spectrometric analysis. Distinct molecular species of lyso-PAF can be individually quantitated at levels of 1 ng or less. These methods are applied to the demonstration of lyso-PAF accumulation in renal tissue from transplanted allografts undergoing acute rejection, in renal tissue from kidneys subjected to cold storage and autotransplantation, and in intestinal mucosa subjected to warm ischemia and reperfusion.
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PMID:Quantification of distinct molecular species of the 2-lyso metabolite of platelet-activating factor by gas chromatography-negative-ion chemical ionization mass spectrometry. 162 94

The effects of phorbol 12-myristate 13-acetate (TPA) or ATP on phosphatidylcholine (PC) hydrolysis were investigated in cultured type II pneumocytes prelabeled with [3H]choline or 1-O-[3H]octadecyl-sn-glycero-3-phosphocholine ([3H]lyso-PAF). In cells prelabeled with [3H]choline, TPA or ATP stimulated an increase in [3H]choline, [3H]phosphocholine, and [3H]glycerophosphocholine. The formation of these choline metabolites was associated with a concomitant loss of [3H]PC but not from disaturated PC or phosphatidylinositol. In cells prelabeled with [3H]lyso-PAF, the formation of [3H]phosphatidic acid (PA) and then [3H]1,2-DG was stimulated by TPA or ATP and was associated with a loss of 3H from PC but not from disaturated PC or phosphatidylinositol. There was a concentration-dependent formation of [3H]1,2-DG and [3H]PA in response to ATP. Downregulation of protein kinase C with TPA abolished the stimulation of PC hydrolysis. In addition to the generation of metabolites indicative of phospholipase C and/or D activity, [3H]lyso-PC, a product of phospholipase A2, was also generated in response to TPA. These findings suggest an important role for PC breakdown in signal transduction in type II pneumocytes.
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PMID:Stimulation of phosphatidylcholine hydrolysis in type II alveolar epithelial cells. 163 29

Extracellular ATP (10(-3) M) stimulated [3H]phosphatidylcholine secretion approximately 3.4-fold in rat type II pneumocytes prelabeled overnight with [3H]choline. The same concentration of ATP caused a rapid increase in [3H]inositol trisphosphate (IP3) and a decrease in [3H]phosphatidylinositol bisphosphate (PIP2) in [3H]inositol-prelabeled cells. ATP also caused a biphasic increase in 1,2-[3H]diacylglycerol in cells prelabeled with [3H]arachidonic acid: a rapid increase that peaked at 10 s followed by a larger increase that peaked at 5-10 min. The first peak in diacylglycerol and the increase in IP3 are consistent with phospholipase C action on PIP2 and generation of second messengers that promote mobilization of intracellular Ca2+ and activation of protein kinase C. However, at the level of phosphatidylcholine secretion the stimulatory effects of ATP and of direct activators of protein kinase C, 12-O-tetradecanoylphorbol-13-acetate (TPA) and 1,2-dioctanoyl-sn-glycerol, were at least additive, suggesting that activation of protein kinase C may not be the major signal transduction mechanism in ATP action or alternatively that ATP activates a different isoform of protein kinase C. Pretreatment of type II cells with TPA for 30 min led to a subsequent 40% diminution in the stimulatory effects of ATP on both phosphatidylcholine secretion and IP3 generation.
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PMID:ATP-stimulated inositol phospholipid metabolism and surfactant secretion in rat type II pneumocytes. 164 84

Activation of protein kinase C leads to a strong induction of tissue-type plasminogen activator (t-PA) expression in endothelial cells. Using endothelial cells from human umbilical vein (HUVECs) and human aorta (HAECs), we have studied this regulation of t-PA and its inhibitor, plasminogen activator inhibitor-1 (PAI-1), at the mRNA level and have compared their induction with the expression of platelet-derived growth factors A and B (PDGF-A and PDGF-B) and the proto-oncogenes c-jun and c-fos. Treatment of HUVECs with exogenous bacterial phospholipase C or the synthetic diacylglycerol 1-oleoyl-2-acetylglycerol led to a threefold and a twofold increase, respectively, in t-PA concentrations in 24-hour-conditioned medium. Similarly, the more stable protein kinase C activator 4 beta-phorbol-12-myristate-13-acetate (PMA) caused about a 10-fold increase in t-PA antigen levels. This effect of PMA is maximal between 8 and 16 hours at a concentration of 10 nM and is fully accounted for by parallel increases in t-PA mRNA levels. An increase in intracellular cyclic adenosine monophosphate levels by forskolin (10 microM) slightly diminished t-PA expression but further enhanced the PMA-induced increases in t-PA synthesis and mRNA levels by at least twofold. PMA also enhanced the mRNA levels of two other important endothelium-expressed genes, PDGF-A and PDGF-B, with a time profile similar to that of t-PA, with peak values about fivefold higher than control values. Forskolin did not further stimulate this PMA-induced PDGF expression in HUVECs, which suggests a regulatory mechanism different from that of t-PA. Qualitatively very similar induction patterns of t-PA, PDGF-A, and PDGF-B were seen with HAECs. In contrast to t-PA and PDGF, PAI-1 mRNA and antigen levels increased only slightly after PMA treatment of HUVECs or HAECs; forskolin alone or in combination with PMA diminished the expression of PAI-1. The induction of t-PA mRNA by PMA was dependent on protein synthesis and was preceded by a strong transient increase in c-jun and c-fos mRNA levels; the induction of c-fos but not of c-jun was potentiated by forskolin. Because the products of these two proto-oncogenes form dimeric complexes for which specific binding sites are present in the t-PA promoter region, they may mediate the protein kinase C-dependent increase in t-PA gene expression, including the stimulating action of cyclic adenosine monophosphate.
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PMID:Role of protein kinase C and cyclic adenosine monophosphate in the regulation of tissue-type plasminogen activator, plasminogen activator inhibitor-1, and platelet-derived growth factor mRNA levels in human endothelial cells. Possible involvement of proto-oncogenes c-jun and c-fos. 164 85

We studied the effects of changing the intracellular Ca level ([Ca]i) and activating protein kinase C on the cardiac T and L Ca channels in single canine ventricular and Purkinje cells. Lowering [Ca]i increased the L current but decreased the T current, whereas elevating [Ca]i caused opposite changes. In ventricular cells, isoproterenol (1 microM) increased the amplitude of not only the L but also the T currents; the latter effect probably was secondary to a rise in [Ca]i following the augmentation of the L current. 12-O-tetradecanoylphorbol-13-acetate (TPA, 100 nM) decreased the T current but first increased and then decreased the L current. The TPA effects on the T and L currents were not mimicked by a phorbol ester that does not activate PKC (4 alpha-phorbol 12,13-didecanoate) and were prevented by a protein kinase inhibitor (H-8), confirming the involvement of PKC activity in these modulatory processes. We conclude that elevating [Ca]i and activating PKC have opposite effects on the T and L Ca currents in canine cardiac cells. The extent and time course of the changes in these two intracellular messengers will most likely determine the effects on the two cardiac Ca currents of neurotransmitters and hormones that can activate phospholipase C.
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PMID:Different effects of intracellular Ca and protein kinase C on cardiac T and L Ca currents. 165 11

The activation of phospholipase D by platelet-activating factor (PAF) in the human promonocytic cell line U937 has been investigated. In cells prelabeled with [3H]palmitic acid, addition of PAF or phorbol 12-myristate 13-acetate (PMA) induced the synthesis of [3H]phosphatidylethanol, indicating phospholipase D activation. When U937 cells were preincubated for 5 min with PMA, and then stimulated with PAF, formation of phosphatidylethanol was greatly enhanced. In contrast, under the same experimental conditions PMA treatment blocked completely the PAF-induced inositol phosphates formation in cells prelabeled with [3H]inositol. Thus, PMA treatment demonstrates that phospholipase D activation can occur independently from phosphoinositide-specific phospholipase C activation during PAF stimulation in U937 cells. On the other hand, the data herein presented suggest that influx of external calcium is required for phospholipase D activation by PAF, as assessed by complete inhibition of the enzyme activity by chelation of extracellular calcium or by treatment with the calcium channel blocker verapamil. Based on these findings, a hypothetical model for phospholipase D activation is discussed.
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PMID:Platelet-activating factor synergizes with phorbol myristate acetate in activating phospholipase D in the human promonocytic cell line U937. Evidence for different mechanisms of activation. 165 58


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