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PubMed2005

Group X secretory phospholipase A2 can induce arachidonic acid release and eicosanoid production without activation of cytosolic phospholipase A2 alpha.

Group X secretory phospholipase A2 (sPLA2-X) and cytosolic phospholipase A2 alpha (cPLA2alpha) are involved in the release of arachidonic acid (AA) from membrane phospholipids linked to the eicosanoid production in various pathological states. Recent studies have indicated the presence of various types of cross-talk between sPLA2s and cPLA2alpha resulting in effective AA release. Here we examined the dependence of sPLA2-X-induced potent AA release on the cPLA2alpha activation by using specific cPLA2alpha or sPLA2 inhibitors as well as cPLA2alpha-deficient mice. We found that Pyrrophenone, a cPLA2alpha-specific inhibitor, did not suppress the sPLA2-X-induced potent AA release and prostaglandin E2 formation in mouse spleen cells. Furthermore, the amount of AA released by sPLA2-X from spleen cells was not significantly altered by cPLA2alpha deficiency. These results suggest that sPLA2-X induces potent AA release without activation of cPLA2a, which might be relevant to eicosanoid production in some pathological states where cPLA2a is not activated.

Animals↗

PubMed1999

Targeting of venom phospholipases: the strongly anticoagulant phospholipase A(2) from Naja nigricollis venom binds to coagulation factor Xa to inhibit the prothrombinase complex.

The strongly anticoagulant basic phospholipase A(2) (CM-IV) from Naja nigricollis venom has previously been shown to inhibit the prothrombinase complex of the coagulation cascade by a novel nonenzymatic mechanism (S. Stefansson, R. M. Kini, and H. J. Evans Biochemistry 29, 7742-7746, 1990). That work indicated that CM-IV is a noncompetitive inhibitor and thus it interacts with either factor Va or factor Xa, or both. We further examined the interaction of CM-IV and the protein components of the prothrombinase complex. Isothermal calorimetry studies indicate that CM-IV does not bind to prothrombin or factor Va, but only to factor Xa. CM-IV has no effect on the cleavage of prothrombin by factor Xa in the absence of factor Va. However, in the presence of factor Va, CM-IV inhibits thrombin formation by factor Xa. With a constant amount of CM-IV, raising the concentration of factor Va relieved the inhibition. The phospholipase A(2) enzyme inhibits by competing with factor Va for binding to factor Xa and thus prevents formation of the normal Xa-Va complex or replaces bound factor Va from the complex. Thus factor Xa is the target protein of this anticoagulant phospholipase A(2), which exerts its anticoagulant effect by protein-protein rather than protein-phospholipid interactions.

Animals↗

PubMed1975

Phospholipases. III. Effects of ionic surfactants on the phospholipase-catalyzed hydrolysis of unsonicated egg lecithin liposomes.

Apparent values of Km and Vmax have been measured for catalysis of hydrolysis of unsonicated egg lecithin liposomes, activated through addition of 0.4 M n-hexanol, by phospholipases A2 from bee and snake venoms and by phospholipase C from Clostridium welchii as a function of the concentration of three surfactants: hexadecylamine, hexadecyltrimethylammonium bromide, and dihexadecyl phosphate. For all three enzymes, values of Km and Vmax show little or no dependence on the concentration of these ionic surfactants, demonstrating that the liposomal surface charge is not a crucial factor in determining susceptibility to phospholipase-catalyzed hydrolysis.

Amines↗

PubMed1995

Glycan requirements of glycosylphosphatidylinositol phospholipase C from Trypanosoma brucei. Glucosaminylinositol derivatives inhibit phosphatidylinositol phospholipase C.

Glycosylphosphatidylinositol phospholipase C (GPI-PLC) from Trypanosoma brucei and phosphatidylinositol phospholipase C (PI-PLC) from Bacillus sp. both cleave glycosylphosphatidylinositols (GPIs). However, phosphatidylinositol, which is efficiently cleaved by PI-PLC, is a very poor substrate for GPI-PLC. We examined GPI-PLC substrate requirements using glycoinositol analogs of GPI components as potential inhibitors. Glucosaminyl (alpha 1-->6)-D-myo-inositol (GlcN(alpha 1-->6)Ins), GlcN(alpha 1-->6)Ins 1,2-cyclic phosphate, GlcN(alpha 1-->6)-2-deoxy-Ins, and GlcN(alpha 1-->6)Ins 1-dodecyl phosphonate inhibited GPI-PLC. GlcN(alpha 1-->6)Ins was as effective as Man-(alpha 1-->4)GlcN(alpha 1-->6)Ins; we surmise that GlcN(alpha 1-->6)Ins is the crucial glycan motif for GPI-PLC recognition. Inhibition by GlcN(alpha 1-->6)Ins 1,2-cyclic phosphate suggests product inhibition since GPIs cleaved by GPI-PLC possess a GlcN(alpha 1-->6)Ins 1,2-cyclic phosphate at the terminus of the residual glycan. The effectiveness of GlcN(alpha 1-->6)-2-deoxy-Ins indicates that the D-myo-inositol (Ins) 2-hydroxyl is not required for substrate recognition, although it is probably essential for catalysis. GlcN(alpha 1-->6)-2-deoxy-L-myo-inositol, unlike GlcN(alpha 1-->6)-2- deoxy-Ins, had no effect on GPI-PLC; hence, GPI-PLC can distinguish between the two enantiomers of Ins. Surprisingly, GlcN(alpha 1-->6)Ins 1,2-cyclic phosphate was not a potent inhibitor of Bacillus cereus PI-PLC, and GlcN(alpha 1-->6)Ins had no effect on the enzyme. However, both GlcN(alpha 1-->6)Ins 1-phosphate and GlcN(alpha 1-->6)Ins 1-dodecyl phosphonate were competitive inhibitors of PI-PLC. These observations suggest an important role for a phosphoryl group at the Ins 1-position in PI-PLC recognition of GPIs. Other studies indicate that abstraction of a proton from the Ins 2-hydroxyl is not an early event in PI-PLC cleavage of GPIs. Furthermore, both GlcN(alpha 1-->6)-2-deoxy-Ins 1-phosphate and GlcN(alpha 1-->6)-2-deoxy-L- myo-inositol inhibited PI-PLC without affecting GPI-PLC. Last, the aminoglycoside G418 stimulated PI-PLC, but had no effect on GPI-PLC. Thus, these enzymes represent mechanistic subclasses of GPI phospholipases C, distinguishable by their sensitivity to GlcN(alpha 1-->6)Ins derivatives and aminoglycosides. Possible allosteric regulation of PI-PLC by GlcN(alpha 1-->6)Ins analogs is discussed.

Animals↗

PubMed1989

Phospholipase C and haemolytic activities of Clostridium perfringens alpha-toxin cloned in Escherichia coli: sequence and homology with a Bacillus cereus phospholipase C.

The Clostridium perfringens alpha-toxin (phospholipase C) gene (cpa) has been cloned and expressed in Escherichia coli. The biological activities of the cloned gene product have been analysed and the complete nucleotide sequence of the cpa gene has been determined. The cloned cpa gene product, which is exported to the periplasm in E. coli, possesses both phospholipase C and haemolytic activities. Haemolysis is not apparent when cell extracts are incubated with isotonic suspensions of sheep erythrocytes, but can be detected and quantified readily when dilutions of the same extracts are placed in wells in sheep-blood agar plates. Like other sequenced clostridial genes, the cpa gene has a high AT content (66.4%), exhibits a strong bias for using codons with A or T in the wobble position, and the 350 base pairs upstream from the gene have a significantly higher AT content (79.5%) than the coding region. The cpa gene encodes a 398 amino acid polypeptide with a deduced molecular weight of 45,481 D. This is very similar to the estimated molecular weight (Mr) of the cpa primary gene product expressed in an in vitro transcription-translation system (Mr 46,000), but larger than the cpa gene product detected in E. coli minicells, E. coli whole cells or in C. perfringens cells (Mr 43,000), suggesting post-translational processing. The 28 N-terminal residues of the deduced alpha-toxin sequence possess the consensus features of a signal peptide and may be removed during secretion. The deduced alpha-toxin sequence shares significant structural homology with the phosphatidylcholine-preferring phospholipase C of Bacillus cereus.

Amino Acid Sequence↗

PubMed1994

Stimulation of phospholipase C-beta 2 by recombinant guanine-nucleotide-binding protein beta gamma dimers produced in a baculovirus/insect cell expression system. Requirement of gamma-subunit isoprenylation for stimulation of phospholipase C.

Recombinant wild-type beta 1 gamma 1 dimers of signal-transducing guanine nucleotide-binding proteins (G proteins) and beta 1 gamma 1 dimers carrying a mutation known to block gamma-subunit isoprenylation (beta 1 gamma 1 C71S) were expressed in baculovirus-infected insect cells. Both wild-type and mutant beta 1 gamma 1 dimers were found in soluble fractions of infected cells upon subcellular fractionation. Anion exchange chromatographic and metabolic-radiolabeling studies revealed that the soluble beta 1 gamma 1 preparation contained approximately equal amounts of non-isoprenylated and isoprenylated beta 1 gamma 1 dimers. Soluble wild-type and mutant beta 1 gamma 1 dimers and native beta 1 gamma 1 dimers purified from bovine retina were reconstituted with recombinant phospholipase C-beta 2. Only isoprenylated beta 1 gamma 1 dimers were capable of stimulating phospholipase C-beta 2. The results show that gamma-subunit isoprenylation and/or additional post-translational processing of the protein are required for beta gamma subunit stimulation of phospholipase C.

Animals↗

PubMed1987

Identification of a new phospholipase C activity by analysis of an insertional mutation in the hemolytic phospholipase C structural gene of Pseudomonas aeruginosa.

The phospholipase C (PLC) gene of Pseudomonas aeruginosa encodes a heat-labile secreted hemolysin which is part of a Pi-regulated operon. The structural gene for PLC, plcS, was mutated in vitro by insertion of a tetracycline resistance gene cartridge. Gene replacement techniques were used to introduce the mutated plcS gene into the P. aeruginosa chromosome in place of the wild-type gene. The precise replacement of wild-type sequences by mutant sequences was confirmed by Southern hybridization. The mutant strain, designated PLC S, is nonhemolytic and lacks a 78-kilodalton protein corresponding to the size of the wild-type PLC. However, there is an additional phospholipase activity present in PLC S capable of hydrolyzing p-nitrophenylphosphorylcholine, a synthetic PLC substrate, and phosphatidylcholine. This enzymatic activity is not a result of a truncated product produced from the mutated plcS gene. The phospholipase activity of PLC S was identified as a nonhemolytic PLC.

Chromatography, Thin Layer↗

PubMed1999

Activation of protein kinase A increases phospholipase D activity and inhibits phospholipase D activation by acetylcholine in tracheal smooth muscle.

Increased cAMP by stimulation of adenylyl cyclase with forskolin or by beta-adrenoceptor activation with isoproterenol increased phospholipase D (PLD) activity in tracheal smooth muscle strips. PLD activity was measured by the accumulation of phosphatidylethanol. A linear increase in the concentration of phosphatidylethanol was observed over 20 min in muscle strips treated with either forskolin or isoproterenol. Cholinergic stimulation with acetylcholine (ACh), by contrast, caused a rapid increase in phosphatidylethanol followed by a slow decline in the concentration of phosphatidylethanol from 5 to 20 min in the continued presence of ACh. Concomitant treatment with ACh and either forskolin or isoproterenol eliminated the rapid increases in phosphatidylethanol associated with ACh treatment. The response to forskolin or isoproterenol was not influenced by ACh. Inhibition of protein kinase C with calphostin C or bisindolylmaleimide I had no effect on isoproterenol- or forskolin-stimulated PLD activity but inhibited ACh-activated PLD activity. Protein kinase A (PKA) inhibitors H-89 and KT5720 significantly decreased forskolin- and isoproterenol-mediated activation of PLD activity. PKA inhibition also eliminated inhibition of ACh-stimulated PLD activity by forskolin or isoproterenol. Activation of adenylyl cyclase by forskolin or by isoproterenol caused increased phosphorylation of phospholipase C-beta(2) isoform and reduced the formation of inositol phosphates after ACh stimulation of muscarinic receptors. These results suggest that increasing the concentration of cAMP activates PLD via activation of PKA and that the increased activity of PKA also inhibits cholinergic stimulation of PLD, in part at least by inhibiting the activation of phospholipase C by ACh.

Acetylcholine↗

PubMed1973

Phospholipase B activity of a purified phospholipase A from Vipera palestinae venom.

Phospholipase was isolated (in two fractions) from Vipera palestinae venom and it was shown to possess phospholipase A activity (hydrolyzing diacyl-sn-glycerophosphorylcholines, e.g., lecithin, in the 2-position) as well as lysophospholipase (phospholipase B) activity (hydrolyzing 1-monoacyl-sn-glycerophosphorylcholines, e.g., lysolecithin, yielding free fatty acid and glycerophosphorylcholine). Each of the two purified enzyme fractions was homogeneous as judged by electrophoresis on acrylamide gel and by immunodiffusion and immunoelectrophoresis, and both had essentially equal activities. The ratio of the specific activity, at various purification stages, to the specific activity of the whole venom was the same for A activity (substrate lecithin) as for B activity (substrate lysolecithin). The enzyme has a molecular weight of 16,000, six S-S bridges, and no free thiol groups. At pH 7, dimerization was observed in the ultracentrifuge. A dissociation constant of about 10(-5) m was estimated. The amino acid composition for both fractions (140 amino acid residues) was found to be essentially the same. The A activity had a pH optimum at 9; B activity was low at this pH but increased steadily beyond pH 10.5. For the hydrolysis of lysolecithin the Lineweaver-Burk plot was found to be linear, giving K(m) = 1.1 mm and k(cat) = 0.55 sec(-1) at 37 degrees C and pH 10. 2-Deoxylysolecithin was also hydrolyzed by the enzyme at pH 10, with k(cat) = 0.01 sec(-1) (zero-order kinetics in the range 0.5-2.5 mm). For lecithin these constants could not be determined, but at 0.25 mm substrate the hydrolysis rate (at pH 9) of lecithin was about 1000 times the hydrolysis rate of lysolecithin (at pH 10).

Animals↗

PubMed2004

Expression of inflammatory secretory phospholipase A2 and cytosolic phospholipase A2 in premalignant and malignant Barrett's oesophagus.

AIMS: To establish the prevalence of inflammatory secretory phospholipase A2 (sPLA2) and cytoplasmic phospholipase A2 (cPLA2) expression in a surgical series of Barrett's adenocarcinoma and associated preneoplastic lesions and to correlate this expression with clinicopathological data and prognosis. METHODS: sPLA2 and cPLA2 were analysed by means of immunohistochemistry in surgical specimens of 67 and 73 cases of Barrett's adenocarcinomas, respectively. Barrett's mucosa was analysed in 31 cases. RESULTS: Expression of sPLA2 was detected in 48% of Barrett's mucosa negative for intraepithelial neoplasia and 63% of Barrett's adenocarcinoma. Semi-quantitative analysis revealed a significant increase in sPLA2 expression between Barrett's mucosa negative for intraepithelial neoplasia and adenocarcinoma. cPLA2 expression was detected in 18% of Barrett's adenocarcinoma. An inverse correlation was found between cPLA2 expression and depth of tumour infiltration, neoplastic vascular invasion and neoplastic perineural invasion. Survival analysis showed no significant prognostic value for sPLA2 and cPLA2. CONCLUSION: sPLA2 is frequently expressed in Barrett's oesophagus. The increasing expression of sPLA2 that we observed from Barrett's mucosa to adenocarcinoma suggests that sPLA2 could be involved in Barrett's carcinogenesis. In contrast, cPLA2 expression is less frequently observed in Barrett's oesophagus and is inversely associated with aggressive pathological features of the tumours.

Adenocarcinoma↗

PubMed2004

Labour is associated with increased expression of type-IIA secretory phospholipase A2 but not type-IV cytosolic phospholipase A2 in human myometrium.

Human labour is associated with increased prostaglandin synthesis within the uterus. The aim of this study was to examine the expression of the type-IV cytosolic phospholipase A2 (cPLA2-IV) and the type IIA secretory phospholipase A2 (sPLA2-IIA) in myometrium in association with labour onset at term and preterm deliveries. These enzymes are important for the release of the prostaglandin precursor, arachidonic acid, from phospholipid membrane stores. RT-PCR was used to determine differences in gene expression between non-labour and labour groups. Expression of sPLA2-IIA in human myometrium was significantly increased with pregnancy, and with labour, both at term and preterm. Expression of cPLA2-IV in myometrium was not significantly altered with respect to pregnancy or labour. Immunohistochemical analysis demonstrated differences in the spatial localization of cPLA2-IV and sPLA2-IIA protein in upper and lower segment myometrium. cPLA2-IV was predominantly in vascular endothelial cells, while sPLA2-IIA was observed in vascular, endothelial and smooth muscle cells. In addition, sPLA2-IIA showed a distinct nuclear or perinuclear localization in myometrial smooth muscle cells of the lower segment. We postulate that the increased expression of sPLA2-IIA rather than cPLA2-IV in the myometrium may play a role in the onset and/or maintenance of human parturition.

Adult↗

PubMed1990

CD2 triggering stimulates a phospholipase A2 activity beside the phospholipase C pathway in human T lymphocytes.

In previous studies we demonstrated the triggering of the phospholipase C (PLC) pathway during the activation of an Ag-specific human CD4+ T lymphocyte clone by a mitogenic pair of CD2 (X11,D66) mAb. Similar conditions were applied to investigate a possible involvement of a phospholipase A2 (PLA2) acting as an additional alternative pathway during human T cell activation. Our results show that arachidonic acid or its derivatives are released after CD2 triggering. This release is largely independent of PLC activation and is mediated by a PLA2 because: 1) phosphatidylcholine is the preferential source of [3H]arachidonate release; 2) [3H]arachidonic acid release and phosphatidylcholine hydrolysis are blocked by two inhibitors of solubilized PLA2, mepacrine, and 4-p-bromophenacylbromide; and 3) we evidenced a PLA2 activity in cell homogenates. Extracellular calcium appears to play a critical role because the effects of CD2 mAb were inhibited in a Ca2(+)-depleted medium. In contrast, protein kinase C is not implicated since PMA, a protein kinase C activator, neither stimulated arachidonic acid release nor modulated CD2-induced arachidonic acid release. Cyclic AMP which has been proved to regulate the activity of the PLC in T lymphocytes does not appear to play an important role in the regulation of PLA2 activity since PGE2 has only a minimal effect on [3H]-arachidonate release. Altogether, these findings suggest that CD2 triggering stimulates a PLA2 activity in T lymphocytes via an extracellular Ca2(+)-dependent PLC protein kinase C independent mechanism.

Antigens, Differentiation, T-Lymphocyte↗

PubMed1997

Effects of prenatal ethanol exposure on phospholipase C-beta 1 and phospholipase A2 in hippocampus and medial frontal cortex of adult rat offspring.

Previous studies in our laboratory using a rat model of fetal alcohol exposure (FAE) suggest that FAE-induced behavioral deficits are, in part, linked to neurochemical and electrophysiological deficits in long-term potentiation (LTP) in the entorhinal cortical perforant path projection to the hippocampal formation. Several findings suggest that signal-activated phospholipase C (PLC) and phospholipase A2 (PLA2) are critical to the induction and maintenance of LTP. Thus, alterations in phospholipid metabolism may play a significant role in the LTP deficits observed in FAE offspring. To test this hypothesis, we measured PLC-beta 1 and PLA2 activities in the hippocampus and medial frontal cortex of adult rats prenatally exposed to ethanol. PLC-beta 1 activities were significantly decreased by 20 to 30% in both the hippocampus and medial frontal cortex of FAE rats, compared with ad libitum and pair-fed controls. Total Ca(2+)-dependent PLA2 activity was 25% lower in the medial frontal cortex of FAE rats, but did not significantly differ from controls in the hippocampal formation. Approximately 30% of the measured activity in both the medial frontal cortex and hippocampal formation of ad libitum and pair-fed animals was associated with an 85 kDa cytosolic PLA2 form. Cytosolic PLA2 activities were significantly reduced in both the medial frontal cortex and hippocampal formation of FAE rats, compared with controls. These changes in Ca(2+)-dependent PLA 2 and PLC-beta 1 activities, coupled with reports of FAE-induced deficits in protein kinase C activity, indicate that prenatal exposure to moderate quantities of ethanol causes profound and long-lasting deficits in the cellular signaling mechanisms associated with activity-dependent synaptic plasticity and memory formation.

Animals↗

PubMed1989

Phospholipase A2 engineering: design, synthesis, and expression of a gene for bovine (pro)phospholipase A2.

A gene coding for the (pro)phospholipase A2 (PLA2) from bovine pancreas has been designed, synthesized, and expressed in Escherichia coli. The gene was designed with a variety of restriction sites that will facilitate future mutagenesis studies. Codons occurring frequently in prokaryotic systems were chosen whenever possible. The total gene spans 404 base pairs and was divided into 33 oligonucleotides. The gene was constructed in two halves of 224 and 180 base pairs from the oligonucleotides by the shotgun ligation technique using pBSM13- as the cloning vehicle. The two fragments were then ligated and cloned into pBSM13- to complete the gene. The (pro)PLA2 gene was then verified by restriction site mapping and dideoxy sequencing. The gene was expressed to high levels from a high copy number vector, designated as pJPN, derived from the E. coli secretion vector pIN-III-ompA3. Although the protein failed to be excreted and was in the form of insoluble inclusion body, active PLA2 could be obtained by renaturation of the inclusion body pellet followed by tryptic activation, which removes the signal sequence and the pro-peptide of proPLA2. The PLA2 thus obtained reacted with the antisera raised against the natural PLA2 purified from bovine pancreas, and the specific activity of the expressed PLA2 was identical to that of the natural PLA2. The shotgun ligation and synthetic gene approaches are simple and inexpensive and can be adapted to express most of the enzymes in the phospholipase A2 family.

Amino Acid Sequence↗

PubMed1993

Inducers of the heat shock response stimulate phospholipase C and phospholipase A2 activity in mammalian cells.

Although the mammalian heat shock response has been well characterized, the processes that mediate the induction of the response and the regulation of heat shock protein function are not completely understood. We have investigated the potential role in heat-shocked cells of phosphoinositide-specific phospholipase C (PLC), a membrane enzyme activity involved in transmembrane signal transduction. Our studies indicate that heat shock activates PLC in each of seven cell lines, including cells of human, rat, mouse, and hamster origin. Heat shock produced increases in inositol phosphate concentrations comparable in magnitude to those achieved after simulation with growth factors, indicating that heat shock might initiate transmembrane signaling cascades of potential importance in cellular regulation. Common cellular responses to heat and growth factors also included feedback modulation of PLC by its products and the parallel stimulation of phospholipase A2 activity. In addition to heat shock, other agents that induce the stress response stimulated PLC activity. The data indicate a close correlation between expression of the mammalian heat shock response and stimulation of PLC activity and indicate a possible role for this enzyme activity in the regulation of some aspects of the stress response.

Animals↗

PubMed1993

Differential involvement of phospholipase A2/arachidonic acid and phospholipase C/phosphoinositol pathways during cholecystokinin receptor activated Ca2+ oscillations in pancreatic acini.

In pancreatic acini, administration of the phospholipase C inhibitor, U-73122, abolished Ca2+ oscillations and amylase secretion induced by CCK but had much less effect on the action of CCK analog JMV-180. In contrast, the phospholipase A2 inhibitor, ONO-RS-082, inhibited both Ca2+ spikes and amylase secretion induced by JMV-180, but it had little effect on the action of CCK-8. Both arachidonic acid (AA) and a cytochrome P-450 inhibitor, SKF-96365, generated Ca2+ spikes from the agonist-sensitive pool. AA was capable of releasing Ca2+ from the endoplasmic reticulum (ER), suggesting the direct Ca2+ releasing pathway. There is no evidence of Ca(2+)-induced Ca2+ release (CICR) since neither caffeine, a CICR potentiator, nor ryanodine, a CICR inhibitor, modulated agonist-induced Ca2+ oscillations and Ca2+ release from the ER. On the contrary, increasing concentrations of caffeine abolished agonist-induced Ca2+ spikes. Therefore we have demonstrated that depending on the agonists used, CCK receptor activation may result in the differential involvement of the phosphoinositol and arachidonic acid pathways to mediate calcium oscillation and amylase secretion.

Aminobenzoates↗

PubMed1999

Differentiation of U937 cells enables a phospholipase D-dependent pathway of cytosolic phospholipase A2 activation.

Treatment with dibutyryl cyclic AMP (dBcAMP) of the human, premonocytic U937 cell line results in differentiation toward a monocyte/granulocyte-like cell. This differentiation enables the cell to activate cytosolic phospholipase A2 (cPLA2) to release arachidonate upon stimulation. In contrast, undifferentiated cells are unable to release arachidonate even when stimulated with calcium ionophores. In the present research, a role for phospholipase D (PLD) in the regulation of cPLA2 was shown based on a number of observations. First, the ionomycin- and fMLP-stimulated production of arachidonate in differentiated cells was sensitive to ethanol (2% (v/v)). Ethanol acts as an alternate substrate in place of water for PLD producing phosphatidylethanol (PEt) instead of phosphatidic acid. Indeed, ionomycin stimulation of differentiated cells produced a 14-fold increase in PEt levels. Further evidence for the involvement of PLD in the regulation of cPLA2 came from the observation that the stimulated production of diacylglycerol (for which phosphatidic acid is a major source) was greatly diminished in undifferentiated cells as compared to differentiated cells. Moreover, the normally deficient activation of cPLA2 in undifferentiated cells could be stimulated to release arachidonate if the cells were electroporated in the presence of GTP[gamma]S and MgATP. This treatment stimulates phosphatidylinositol-4,5-bisphosphate (PIP2) production which appears to activate PLD and cPLA2 in subsequent steps. The phosphatidic acid (and diacylglycerol derived from phosphatidic acid) appears to greatly regulate the action of cPLA2 by an unknown mechanism, and undifferentiated cells lack the ability to stimulate PLD activity due to a dysfunction of PIP2 production.

Adenosine Triphosphate↗

PubMed1998

Phospholipase A2-mediated activation of phospholipase D in rat heart sarcolemma.

The effect of phospholipase A2 (PLA2)-dependent release of unsaturated fatty acids (FA) on phospholipase D (PLD) function was examined in purified sarcolemmal (SL) membranes isolated from rat heart. PLD hydrolytic activity was determined by measuring either [14C] phosphatidic acid formation from exogenous [14C] phosphatidylcholine (PtdCho) or [3H] choline release from prelabelled SL Ptd[3H]choline. SL membranes with endogenous [3H] PtdCho that were prelabelled with [3H] myristic acid were used for testing PLD transphosphatidylation activity. Exogenous cis-unsaturated FA, arachidonate and oleate, significantly enhanced the [3H] choline formation at 50 and 100 microM, respectively; their effect was maximal at 250 microM and declined at higher concentrations. Use of melittin (which stimulates membrane-bound PLA2, thus releasing FA) or exogenous PLA2 reproduced the stimulatory effect of added arachidonate and oleate. Under melittin, PLA2-dependent FA release was strongly correlated (r = 0.99) to the PLD-dependent phosphatidic acid formation. Arachidonate- or melittin-enhanced PLD transphosphatidylation activity confirmed the augmented catalytic rate of PLD by these agents. Melittin-evoked PLD activation was completely blocked by 1 microM E-6-(bromomethylene) tetrahydro-3-(1-naphthalenyl)-2H-pyran-2-one, a selective inhibitor of Ca(2+)-independent v Ca(2-)-dependent PLA2, thus indicating that PLD stimulation under melittin occurred via PLA2. Activity measurement and Western blotting studies revealed the presence of a Ca(2+)-independent, high molecular weight (110 kDa) PLA2 in the SL membrane, and its immunoprecipitation by monoclonal antibodies significantly reduced the melittin-related PLD stimulation. These results suggest that Ca(2+)-independent PLA2 and subsequent endogenous mobilization of sn-2 unsaturated FA modulate PLD activity in heart SL membranes. This event may occur in physiological conditions via hormonal stimulation of membranal PLA2 as well as in heart diseases characterized by PLA2 pathological dysfunction.

Animals↗