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PubMed1985

Immunochemical relatedness between secretory phospholipase A2 and intracellular phospholipase A2.

The immunochemical relationship between rat pancreatic phospholipase A2 and rat splenic phospholipase A2 was examined with the use of anti-rat pancreatic phospholipase A2 antibody as a probe. The immunoelectrophoretic patterns showed that the antibody cross-reacted with the splenic enzyme. The immuno-crossreactivity was also shown by counter immunoelectrophoresis. The splenic phospholipase A2, whether it was purified from the cytosolic fraction or the microsomal fraction, formed an immunoprecipitin band with the anti-pancreatic phospholipase A2 antibody. The antibody was shown to inhibit the activity of the pancreatic phospholipase A2 as well as that of the splenic phospholipase A2.

Animals↗

PubMed1991

Melittin stimulates phosphoinositide hydrolysis and placental lactogen release: arachidonic acid as a link between phospholipase A2 and phospholipase C signal-transduction pathways.

Previous investigations from this laboratory have implicated both phospholipase A2 and phospholipase C in the regulation of human placental lactogen release from human trophoblast. To study further the role of endogenous phospholipase A2 and the relationship between phospholipase A2 activation and phosphoinositide metabolism, we examined hPL and [3H]-inositol release from trophoblast cells in response to agents that stimulate or inhibit the endogenous enzyme. Melittin (0.5-2.0 micrograms/ml) stimulated rapid, dose-dependent, and reversible increases in the release of hPL, prostaglandin E, and [3H]-inositol. Mepacrine (0.1-0.25 mM) inhibited this stimulation. However, mepacrine had no effect on the stimulation of hPL and [3H]-inositol release by exogenous arachidonic acid (AA). These results indicate that the stimulation by melittin of phosphoinositide metabolism and hPL release is mediated by initial activation of phospholipase A2. Furthermore, the results support the possibility that AA, released as a consequence of phospholipase A2 activation, can act as a second messenger linking the two phospholipase pathways.

Arachidonic Acid↗

PubMed1997

A new phospholipase A2 inhibitor, unrelated to substrate analogues: kinetic characterization of the inhibition of secretory phospholipases A2 by PMS 832.

Starting from a series of compounds which were known to be PAF antagonists, we have synthesized molecules that are good inhibitors of PLA2s of groups I or II, with IC50 in the micromolar range (Binisti et al., 1997). In this report we investigate the mechanism of inhibition of bovine and porcine pancreatic phospholipases A2 (group I), and platelet lysate phospholipase A2 (group II) by one of these compounds, 1-(4'-methoxybenzoyl)-2-n-tridecylpiperazine (PMS 832). We show that PMS 832 behaves as a reversible, competitive inhibitor, with Ki values of 4.1 +/- 1.2 and 1.5 +/- 0.4 microM for porcine pancreatic phospholipase A2 and platelet lysate phospholipase A2, respectively. PMS 832 failed to inhibit platelet activation induced by several agonists and was also found to be inactive towards phospholipase C from Bacillus cereus, indicating a high specificity for phospholipase A2 inactivation. Thus, PMS 832 and its derivatives could serve as interesting tools to investigate the role of extracellular phospholipases A2 in inflammatory processes, and may be useful in the development of new anti-inflammatory agents.

Animals↗

PubMed1984

Role of the N-terminus in the interaction of pancreatic phospholipase A2 with aggregated substrates. Properties and crystal structure of transaminated phospholipase A2.

A free N-terminal alpha-NH3+ group is absolutely required for full catalytic activity of phospholipase A2 on aggregated substrates. To elucidate how this alpha-NH3+ group triggers catalytic activity, we specifically transaminated this group in various pancreatic phospholipases A2. Porcine, porcine iso-, equine, human, ovine, and bovine phospholipases A2 all loose catalytic activity on micellar substrates due to the inability of the transaminated proteins to bind to neutral micellar substrate analogues, as was found for the zymogens. Loss of activity is pseudo first order, the rate constants being different for the enzymes studied. The transaminated phospholipases A2 have an intact active site, as catalytic activities on monomeric substrates are comparable to those of the respective zymogens. The X-ray structure of transaminated bovine phospholipase A2 at 2.1-A resolution shows that the N-terminal region and the sequence 63-72 in this protein are more flexible than in the native enzyme. Also, in this respect, the transaminated enzyme very much resembles the zymogen structure. In good agreement with this, it was found by photochemically induced dynamic nuclear polarization 1H NMR that aromatic resonances of Trp-3 and Tyr-69 are affected by transamination. In addition, fluorescence spectroscopy of the unique Trp-3 in transaminated bovine phospholipase A2 revealed a red shift of the emission maximum indicative of a more polar environment of Trp-3 in the transaminated phospholipase A2 as compared to the enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1988

Site-specific epsilon-NH2 monoacylation of pancreatic phospholipase A2. 2. Transformation of soluble phospholipase A2 into a highly penetrating "membrane-bound" form.

Long-chain lecithins present in bilayer structures like vesicles or membranes are only very poor substrates for pancreatic phospholipases A2. This is probably due to the fact that pancreatic phospholipases A2 cannot penetrate into the densely packed bilayer structures. To improve the weak penetrating properties of pancreatic phospholipases A2, we prepared and characterized a number of pancreatic phospholipase A2 mutants that have various long acyl chains linked covalently to Lys116 in porcine and to Lys10 in bovine phospholipase A2 [Van der Wiele, F.C., Atsma, W., Dijkman, R., Schreurs, A.M.M., Slotboom, A.J., & De Haas, G.H. (1988) Biochemistry (preceding paper in this issue)]. When monomolecular surface layers of L- and D-didecanoyllecithin were used, it was found that the introduction of caprinic, lauric, palmitic, and oleic acid at Lys116 in the porcine enzyme increases its penetrating power from 13 to about 17, 20, 32, and 22 dyn/cm, respectively, before long lag periods were obtained. Incorporation of a palmitoyl moiety at Lys10 in the bovine enzyme shifted the penetrating power from 11 to about 25 dyn/cm. Only the best penetrating mutant, viz., porcine phospholipase A2 having a palmitoyl moiety at Lys116, was able to cause complete leakage of 6-carboxyfluorescein entrapped in small unilamellar vesicles of egg lecithin under nonhydrolytic conditions. Similarly, only this latter palmitoylphospholipase A2 completely hydrolyzed all lecithin in the outer monolayer of the human erythrocyte at a rate much faster than Naja naja phospholipase A2, the most powerful penetrating snake venom enzyme presently known.

Acylation↗

PubMed1988

Tumour necrosis factor (cachectin) induces phospholipase A2 activity and synthesis of a phospholipase A2-activating protein in endothelial cells.

Tumour necrosis factor (TNF) is an important mediator of endotoxin-induced vascular collapse and other inflammatory reactions. Eicosanoids have been implicated in the pathogeensis of these responses. In order to explore further the potential interactions between TNF and eicosanoid metabolism in eliciting vascular responses, we studied the effects of TNF on the bovine endothelial cell line CPAE. TNF induced cellular retraction observed by light microscope. This morphological change was monitored by the passage of iodinated protein A between adjacent cells and by release of [3H]arachidonic acid metabolites from cells. Both the morphological and functional responses were abrogated by inhibition of eicosanoid synthesis with BW755c. The release of [3H]arachidonic acid metabolites appeared to be mediated by a transient increase in phospholipase A2 activity. Phospholipase C activity was not affected by TNF. The maximal increase in phospholipase A2 activity occurred at 5 min following the addition of TNF. Phospholipase A2 activation, [3H]arachidonic acid-metabolite synthesis and passage of iodinated protein A, required both RNA and protein synthesis and were associated with an increase in the synthesis of a recently described phospholipase A2-activating protein. The Bordetella pertussis toxin, islet-activating protein, also inhibited the increase in phospholipase A2 activity, the release of [3H]arachidonic acid metabolites and the passage of iodinated protein A, suggesting that the TNF receptor-ligand interaction resulting in cellular retraction, phospholipase A2 activation and eicosanoid synthesis, is coupled through the Ni guanine nucleotide regulatory protein in these cells.

Animals↗

PubMed1989

Purification and characterization of five variants of phospholipase A2 and complete primary structure of the main phospholipase A2 variant in Heloderma suspectum (Gila monster) venom.

1. Five increasingly anionic variants (Pa1-Pa5) of Ca2+-dependent phospholipase A2 were purified to homogeneity from the venom of the lizard Heloderma suspectum (Gila monster). The purification procedure was based on semi-preparative reverse-phase HPLC followed by anion-exchange HPLC and analytical reverse-phase HPLC. 2. Their Mr were 17,000-18,000, as deduced by SDS/PAGE. Specific activities tested by the capacity to hydrolyze phosphatidylcholines at pH 8.5 decreased as follows: Pa3 greater than Pa5 greater than Pa4 greater than Pa1 greater than Pa2. These activities showed the same optimum pH (9.0), were mainly of the phospholipase A2 type and were lost upon p-bromophenacyl bromide treatment. 3. All five phospholipases efficiently stimulated amylase release from dispersed rat pancreatic acini at pH 7.4, their potency decreasing as follows: Pa2 greater than Pa1 approximately equal to Pa4 greater than Pa3 approximately equal to Pa5. No deleterious effect was apparent based on the lack of lactate dehydrogenase release. 4. The five variants, Pa1-Pa5, differed significantly in amino acid composition and this, together with distinct antigenic properties of Pa2 and Pa5, establishes the subheterogeneity of this new type of phospholipase A2, despite the fact that the N-terminal amino acid sequence (31 residues) of Pa1-Pa5 was exactly the same. 5. The full sequence of the major variant, Pa5, showed that this 142-amino-acid protein exhibited greater similarity to the bee venom enzyme than to any class I or class II secretory phospholipase A2 from snake venom and mammalian pancreas. While Pa5 displayed the highly conserved region between Asp30 and Cys39 (the essential active site of all phospholipases A2), its salient original points included 10 half-cystine residues only, an incomplete N-terminal sequence, large changes in the putative calcium loop, several alterations after the active site and a C-terminal extension never seen in other phospholipases A2, with the only exception being bee venom.

Amino Acid Sequence↗

PubMed1987

Sequence homology between phospholipase and its inhibitor in snake venom. The primary structure of phospholipase A2 of vipoxin from the venom of the Bulgarian viper (Vipera ammodytes ammodytes, Serpentes).

The amino-acid sequence of phospholipase A2 from the neurotoxin vipoxin of the Bulgarian Viper (Vipera ammodytes ammodytes, Serpentes) is presented. The enzyme consists of 122 amino-acid residues including 7 disulfide bonds and thus belongs to phospholipases A2 group IIA. The sequence was determined by automatic Edman degradation of the intact chain and of the peptides obtained after tryptic hydrolysis of the oxidized chain. The short cleavage time of 30 min and another limited tryptic digestion of the oxidized and citraconylated chain provided overlapping peptides. Sequencing was done with liquid- and gas-phase sequenators. The complete alignment of all peptides was facilitated by the high degree of homology with known viperid venom phospholipases A2. In common with mammalian phospholipases, the tryptophan residue in position 30 (essential for enzymatic activity) as well as the histidine in position 47 in the active site are present. Vipoxin phospholipase A2 shows 53.3% homology with another phospholipase A2 from Vipera ammodytes ammodytes venom (Ammodytoxin B), whereas 62% homology was found between both subunits of vipoxin phospholipase A2 and its inhibitor. This high degree of identity can be accounted for in terms of a common origin by gene duplication.

Amino Acid Sequence↗

PubMed1996

Role of phospholipase C and phospholipase A2 in the nitric oxide-independent vasodilator effect of bradykinin in the rat perfused heart.

The cytochrome P450-dependent component of the coronary vasodilator action of bradykinin which requires activation of K+ channels was examined in terms of the contribution of phospholipases in the rat Langendorff heart preparation. This component was isolated by inhibition of nitric oxide synthase with nitroarginine and cyclooxygenase with indomethacin, neither of which affects the coronary vasodilator action of bradykinin. However, nitroarginine elevated coronary perfusion pressure from approximately 40 to 130 mm Hg. The phospholipase C inhibitor, U73122 {1-(6-((17 beta-3-methoxyestra-1,3,5(10)-trien-17-yl) amino)hexyl)-1H-pyrrole-2,5-dione}, reduced coronary vasodilator responses to bradykinin by greater than 80%. U73122 also diminished the coronary vasodilator action of cromakalim which activates ATP-sensitive K+ channels. The maleimide moiety of U73122 that has the capacity to affect K+ channels inhibited cromakalim-induced coronary vasodilation, but did not affect that to bradykinin. Inhibition of diacylglycerol lipase with RHC 80267 {1,6-bis-(cyclohexyloximinocarbonylamino)-hexane} was without an overall effect on coronary vasodilator responses to bradykinin. The cytosolic phospholipase A2 inhibitor, AACOCF3 {arachidonyl trifluoromethyl ketone¿} decreased responses to bradykinin by up to 90% whereas inhibitors of the secretory form of phospholipase A2 oleyloxyethyl phosphorylcholine and ONO-RS-082 {2-(p-amylcinnamoyl)amino-4-chlorobenzoic acid} were less effective than either AACOCF3 or U73122. The phospholipase inhibitors demonstrated selectivity as they did not affect the coronary vasodilator responses to nitroprusside. We obtained additional evidence for the antiphospholipase activity of the inhibitors by demonstrating their capacity to suppress bradykinin-stimulated increases in the release of prostacyclin, measured as 6-keto prostaglandin F1 alpha. The phospholipase inhibitors did not affect cyclooxygenase activity as the ability of arachidonic acid to stimulate prostaglandin formation was unimpaired. These results indicate that the coronary vasodilator action of bradykinin is linked to the activities of both phospholipase C and A2.

Animals↗

PubMed1992

Spontaneous domain formation of phospholipase A2 at interfaces: fluorescence microscopy of the interaction of phospholipase A2 with mixed monolayers of lecithin, lysolecithin and fatty acid.

Fluorescence microscopy has recently been proven to be an ideal tool to investigate the specific interaction of phospholipase A2 with oriented substrate monolayers. Using a dual labeling technique, it could be shown that phospholipase A2 can specifically attack and hydrolyze solid analogous L-alpha-DPPC domains. After a critical extent of monolayer hydrolysis the enzyme itself starts to aggregate forming regular shaped protein domains (Grainger et al. (1990) Biochim. Biophys. Acta 1023, 365-379). In order to confirm that the existence of hydrolysis products in the monolayer is necessary for the observed aggregation of phospholipase A2, mixed monolayers of D- and L-alpha-DPPC, L-alpha-lysoPPC and palmitic acid in different ratios were examined. The phase behavior and the interaction of these films with phospholipase A2 were directly visualized with an epifluorescence microscope. Above a certain critical concentration of lysolecithin and palmitic acid in the monolayer, compression of these mixed films leads to phase separation and formation of mixed domains of unknown composition. Their high negative charge density is evidenced by preferential binding of a cationic dye to these phase-separated areas. Introduction of fluorescence-labeled phospholipase A2 underneath these mixed domains results in rapid binding of the protein to the domains without visible hydrolytic activity, regardless of whether the L-form or the D-form of the DPPC were used. In binary mixtures, only those with DPPC/palmitic acid show formation of phase-separated areas which can be specifically targeted by phospholipase A2 leading to a rapid formation (within 2 min) of protein domains. Experiments with pyrenedecanoic acid containing monolayers give the first direct evidence that acid is located above the enzyme domains. These results show that a locally high negative charge density of the phase-separated domains is one of the prerequisites for the binding of phospholipase A2. In addition, however, small amounts of D- or L-alpha-DPPC headgroups within the domains of the monolayer seem to be necessary for recognition followed by fast binding of the protein to the domains. This is confirmed by experiments with mixed monolayers of diacetylene carboxylic acid and D-alpha-DPPC. The acid--immiscible with lecithin--forms well defined pure acid domains in the monolayer. While the cationic dye can be docked rapidly to these phase separated areas, no preferential enzyme binding and thus no protein domain formation below these acid domains can be induced.

1,2-Dipalmitoylphosphatidylcholine↗

PubMed1998

U73122 and U73343 inhibit receptor-mediated phospholipase D activation downstream of phospholipase C in CHO cells.

The aminosteroid 1-(6-¿[17beta-3-methoxyestra- 1,3,5(10)-trien- 17-yl]-amino¿hexyl)- 1H-pyrrole-2,5-dione (U73122) and its inactive analogue 1-(6-¿[17beta-3-methoxyestra-1,3,5(10)-trien- 17-yl]-amino¿hexyl-2,5-pyrrolidine-dione (U73343) are widely used to study the involvement of G protein-coupled 1-phosphatidylinositol-phosphodiesterase, or phospholipase C, in receptor-mediated cell activation. The present work shows that both aminosteroids inhibit cholecystokinin-(26-33)-peptide amide (CCK-8)-induced phospholipase D activation equipotently in Chinese hamster ovary cells expressing the cholecystokinin-A receptor (CHO-CCK(A) cells). In addition, the two aminosteroids virtually completely inhibited thapsigargin- and 12-O-tetradecanoylphorbol 13-acetate (TPA)-induced phospholipase D activation. Since the latter two drugs mimic inositol 1,4,5-trisphosphate-mediated Ca2+ mobilisation and 1,2-diacylglycerol-mediated protein kinase C activation. respectively, this suggests that both U73122 and U73343 act downstream of phospholipase C to inhibit receptor-mediated phospholipase D activation. U73122, but not U73343. effectively inhibited both TPA/Ca2+-stimulated phospholipase D activation and TPA/phosphatidylserine-stimulated protein kinase C activation in a homogenate of CHO-CCK(A) cells. The data presented suggest that U73122 may act at the level of protein kinase C to inhibit activation of phospholipase D. The exact site of action of U73343 is presently unknown.

Animals↗

PubMed1993

Evidence for a nucleotide receptor on adrenal medullary endothelial cells linked to phospholipase C and phospholipase D.

1. We have investigated whether the 'atypical' P2-purinoceptor previously described on adrenal microvasculature endothelial cells is a nucleotide receptor (responds to pyrimidines and purines) and is linked to phospholipase D as well as phospholipase C. 2. Cultured bovine adrenal medullary endothelial (BAME) cells responded to the pyrimidine UTP, as well as the purines. The total [3H]-inositol phosphate responses were with a rank order of UTP > ATP- = adenosine 5'-O-(3-thio-triphosphate) (ATP gamma S) >> 2MeSATP. The selective P2x agonist beta, gamma-methylene ATP was inactive. 3. Construction of dose-response curves to ATP, ATP gamma S and UTP in the presence and absence of additional agonists showed that responses to ATP gamma S and UTP were not additive, nor were those to UTP and ATP. This suggests that purines and pyrmidines acted via a common nucleotide receptor. 4. 32P-labelled BAME cells, in the presence of butanol, produced [32P]-phosphatidylbutanol (PBut) when stimulated with ATP gamma S or the protein kinase C activator, tetradecanoyl phorbol acetate (TPA). 5. Cells labelled with [3H]-palmitate and stimulated in the presence of butanol generated [3H]-PBut with the same order of agonist potencies seen for inositol phosphate responses. 6. The protein kinase C inhibitor, Ro 31-8220, abolished TPA and agonist stimulation of [3H]-PBut production. 7. These observations, and our related studies on bovine aortic endothelial cells, provide the first demonstration of a phospholipase C linked nucleotide receptor on vascular endothelial cells. It is concluded that BAME cells express a nucleotide receptor linked to phospholipase C and phospholipase D, but that activation of phospholipase D is probably down-stream of phospholipase C.

Adenosine Triphosphate↗

PubMed2001

Thyroxine signal transduction in liver cells involves phospholipase C and phospholipase D activation. Genomic independent action of thyroid hormone.

BACKGROUND: Numerous investigations demonstrate a novel role of thyroid hormone as a modulator of signal transduction. Protein kinase C (PKC) is critical to the mechanism by which thyroid hormones potentiate both the antiviral and immunomodulatory actions of IFNgamma in different cells and regulate the exchange of signalling phospholipids in hepatocytes. Because nothing is known about accumulation of PKC modulator - diacylglycerol in cells treated with T4, we examined the nongenomic effect of thyroid hormones on DAG formation and phospholipase activation in liver cells. RESULTS: The results obtained provide the first demonstration of phospholipase C, phospholipase D and protein kinase C nongenomic activation and diacylglycerol (DAG) accumulation by L-T4 in liver cells. The experiments were performed in either the [14C]CH3COOH-labeled rat liver slices or isolated hepatocytes pre-labeled by [14C]oleic acid. L-T4 activates the DAG production in a concentration- and time-dependent manner. DAG formation in stimulated cells is biphasic and short-lived event: there is an initial, rapid rise in DAG concentration and then a slower accumulation that can be sustained for a few minutes. The early phase of L-T4 generated DAG only is accompanied by phosphatidylinositol 4,5-bisphosphate level decrease and inositol 1,4,5-trisphosphate formation while the second phase is abolished by PKC inhibitor l,(5-isoquinolinesulphonyl)2methylpiperasine dihydrochloride (H7) and propranolol. The second phase of DAG production is accompanied by free choline release, phosphatidylcholine content drop and phosphatidylethanol (Peth) formation. Inhibitor of phospholipase-C-dependent phosphoinositide hydrolysis, neomycin sulfate, reduced the Peth as well as the DAG response to L-T4. CONCLUSIONS: The present data have indicated the DAG signaling in thyroid hormone-stimulated liver cells. L-thyroxine activates a dual phospholipase pathway in a sequential and synchronized manner: phospholipase C initiates the DAG formation, and PKC mediates the integration of phospholipase D into the signaling response during the sustained phase of agonist stimulation.

Animals↗

PubMed1992

Interleukin 1-stimulated prostacyclin synthesis in endothelium: lack of phospholipase C, phospholipase D, or protein kinase C involvement in early signal transduction.

The cascade of transmembrane signaling events that follow the occupancy of the interleukin 1 receptor remain poorly defined. We examined potential postreceptor transduction systems involved in human recombinant interleukin 1-beta-stimulated prostacyclin synthesis in human umbilical vein endothelium. Challenge of human umbilical vein endothelium monolayers with recombinant interleukin 1-beta resulted in dose- and time-dependent tritiated arachidonate release and prostacyclin synthesis consistent with phospholipase A2 activation. Prostacyclin synthesis after interleukin 1-beta (10 ng/ml) was detected 4 hours after stimulation and peaked at 16 to 24 hours. To examine whether interleukin 1-beta produced early activation of a phosphoinositide-specific phospholipase C, human umbilical vein endothelium monolayers were labeled with tritiated-2-myoinositol and inositol polyphosphates recovered after interleukin 1-beta stimulation. In contrast to the potent agonist, alpha-thrombin, interleukin 1-beta failed to significantly increase inositol phosphate production when examined for up to 4 hours. The absence of a significant increase in the Cai++ secretagogue, IP3, was confirmed in human umbilical vein endothelium monolayers loaded with the Ca++ photoprotein probe aequorin. Basal aequorin luminescence was unaltered after interleukin 1-beta (0 to 2 hours), whereas both alpha-thrombin and Ca++ ionophore A23187 produced rapid rises in Cai++. The intracellular Ca++ antagonist BAPTA and the extracellular Ca++ chelator EGTA produced significant inhibition of interleukin 1-beta-stimulated prostacyclin generation at 4 to 8 hours, suggesting either an indirect inhibitory effect of these agents on phospholipase A2 activity or that an increase in Ca++ may be a late event in the transduction scheme after interleukin 1 stimulation. Interleukin 1-beta-stimulated protein kinase C, phospholipase D, and adenylyl cyclase activities (0 to 4 hours) were unchanged from controls. Despite the absence of increased plasma membrane protein kinase C activity up to 4 hours after interleukin 1, pretreatment of human umbilical vein endothelium monolayers with staurosporine or phorbol myristate acetate (18 hours) to reduce protein kinase C activities, significantly attenuated the interleukin 1-stimulated prostanoid responses at 16 hours but not at 4 hours. Furthermore, short (5 minute) pretreatment with phorbol myristate acetate dramatically augmented interleukin 1-mediated prostacyclin responses in synergistic fashion, suggesting that protein kinase C may modulate interleukin 1 signal transducing pathways. In summary, these studies suggest that interleukin 1-beta-mediated endothelial cell phospholipase A2 activity and prostacyclin synthesis occur via a novel transducing pathway that does not involve early activation of phospholipase C, phospholipase D, or adenylate cyclase.(ABSTRACT TRUNCATED AT 400 WORDS)

Arachidonic Acid↗

PubMed1982

Organization and role of platelet membrane phospholipids as studied with phospholipases A2 from various venoms and phospholipases C from bacterial origin.

Phospholipases A2 from various snake or bee venoms and phospholipases C secreted as exotoxins by several bacteria have been used to study the transverse distribution of phospholipids in the platelet plasma membrane and their role in platelet activation. An asymmetric distribution was described for phospholipids, characterized by a preferential localization of sphingomyelin and phosphatidylcholine in plasma membrane outer leaflet, whereas the inner half contains almost all of the anionic procoagulant phosphatidylserine and phosphatidylinositol. Such a distribution might explain the latency of procoagulant activity in resting platelets and implies an intracellular localization of arachidonic acid, the precursor of prostaglandins and thromboxanes. The external arachidonic acid is involved in phospholipase A2-induced aggregation, whereas phospholipase C from Clostridium welchii stimulates platelets through a thromboxane-independent pathway. The latter one is directly linked to the formation of phosphatidic and lysophosphatidic acids, which are able to activate cells through calcium mobilization. So, phospholipase C represents an interesting tool for studying the biochemical processes accompanying stimulation, since it is shown that it mimics the effects of an intracellular phospholipase C, the role of which in platelet activation is discussed.

Animals↗

PubMed1992

Action of phospholipases A2 and C on free fatty acid release during complete ischemia in rat neocortex. Effect of phospholipase C inhibitor and N-methyl-D-aspartate antagonist.

The levels of brain free fatty acids rapidly increase after the onset of ischemia. The purpose of this study was to investigate the action of phospholipases A2 and C during complete ischemia based on the effects of a phospholipase C inhibitor (phenylmethylsulfonyl fluoride) and the N-methyl-D-aspartate antagonist MK-801 on the release of free fatty acids in rat neocortex. Complete brain ischemia was induced in rats with cardiac arrest by intracardiac injection of KCl. Free fatty acid levels in the neocortex were measured 0, 2, 4, and 8 minutes after cardiac arrest. Phenylmethylsulfonyl fluoride inhibited the release of free fatty acids primarily from phosphatidylinositol during the first 2 minutes of ischemia and from phosphatidylcholine and phosphatidylethanolamine at 4 to 8 minutes of ischemia. Conversely, MK-801 inhibited free fatty acid release mainly from phosphatidylcholine and phosphatidylethanolamine at 2 to 4 minutes of ischemia. These results indicate that the release of free fatty acids during the first 2 minutes of ischemia can be attributed mostly to the action of phospholipase C, and that the activation of phospholipase C further influences the activation of phospholipase A2 in the subsequent course, while phospholipase A2 predominantly acts after 2 minutes of ischemia.

Animals↗

PubMed1989

Bradykinin-induced activation of phospholipase A2 is independent of the activation of polyphosphoinositide-hydrolyzing phospholipase C.

This study evaluates the role of phosphatidylinositol 4,5-bisphosphate (PIP2) and its metabolites as possible mediators in the activation of phospholipases A2 in porcine aortic endothelial cells. We compared the time courses of bradykinin-induced turnover of phosphoinositides and the appearance of unesterified arachidonic acid (uAA) and eicosanoids. The metabolism of phosphoinositides was examined in cells prelabeled with [3H]inositol, which has a similar distribution as the endogenous inositol lipids. At 37 degrees C, bradykinin induced a rapid rise in lysophosphatidylinositol (lyso-PI) and inositol 1,4,5-trisphosphate (IP3) as well as a decrease in PIP2. Lyso-PI formation was detected at 10 s, as early as PIP2 degradation and IP3 formation. This suggests that the activation of PIP2-hydrolyzing phospholipase C and PI-hydrolyzing phospholipase A2 are simultaneous. However, at 30 degrees C, lyso-PI formation was detected in the absence of an increase in IP3 indicating that the activation of phospholipase A2 does not require the accumulation of IP3. The time course of formation of uAA and eicosanoids were examined in [3H]arachidonic acid-prelabeled cells. The 3H radioactivity was distributed among the phospholipid classes and subclasses the same as the endogenous phospholipids. Bradykinin stimulated the intracellular accumulation of uAA, detectable at 5 s, earlier than that of 1,2-diacylglycerol and phosphatidic acid. Such immediate formation of uAA further supports the notion that activation of phospholipase A2 is a very early event during the interaction of bradykinin with porcine endothelial cells, and that PIP2 hydrolysis is not prerequisite for the initial activation of phospholipase A2.

Animals↗

PubMed2005

Functional analysis of the phospholipase C gene CaPLC1 and two unusual phospholipase C genes, CaPLC2 and CaPLC3, of Candida albicans.

Phospholipases C are known to be important regulators of cellular processes but may also act as virulence factors of pathogenic microbes. At least three genes in the genome of the human-pathogenic fungus Candida albicans encode phospholipases with conserved phospholipase C (Plc) motifs. None of the deduced protein sequences contain N-terminal signal peptides, suggesting that these phospholipases are not secreted. In contrast to its orthologue in Sacharomyces cerevisiae, CaPLC1 seems to be an essential gene. However, a conditional mutant with reduced transcript levels of CaPLC1 had phenotypes similar to Plc1p-deficient mutants in S. cerevisiae, including reduced growth on media causing increased osmotic stress, on media with a non-glucose carbon source, or at elevated or lower temperatures, suggesting that CaPlc1p, like the Plc1p counterpart in S. cerevisiae, may be involved in multiple cellular processes. Furthermore, phenotypic screening of the heterozygous DeltaCaplc1/CaPLC1 mutant showed additional defects in hyphal formation. The loss of CaPLC1 cannot be compensated by two additional PLC genes of C. albicans (CaPLC2 and CaPLC3) encoding two almost identical phospholipases C with no counterpart in S. cerevisiae but containing structural elements found in bacterial phospholipases C. Although the promoter sequences of CaPLC2 and CaPLC3 differed dramatically, the transcriptional pattern of both genes was similar. In contrast to CaPLC1, CaPLC2 and CaPLC3 are not essential. Although Caplc2/3 mutants had reduced abilities to produce hyphae on solid media, these mutants were as virulent as the wild-type in a model of systemic infection. These data suggest that C. albicans contains two different classes of phospholipases C which are involved in cellular processes but which have no specific functions in pathogenicity.

Amino Acid Sequence↗