Phosphatidylethanol Formation Research Articles

Phosphatidylcholine Hydrolysis and DNA Synthesis in Cultured Retinal Capillary Pericytes

In order to verify the role of activation of phosphatidylcholine (PC) hydrolysis by phospholipase D (PLD) in the initiation of mitogenic process of retinal capillary pericytes, platelet-derived growth factor (PDGF), a known PC hydrolysis stimulator, and exogenous PLD have been used to stimulate pericytes. Exogenous PLD ( Streptomyces chromofuscus PLD) or PDGF BB homodimer (PDGF) was added to a medium of quiescent pericytes prelabeled with [ 32P]orthophosphate. In the presence of ethanol (300 mm), phosphatidic acid (PA) and its stable transphosphatidylated product, phosphatidylethanol (PEt), were determined. In parallel, [ 3H]thymidine incorporation was measured. Downregulation of PKC was achieved by long-term treatment with a phorbol ester. The addition of exogenous PLD or PDGF stimulated both [ 3H]thymidine incorporation and [ 32P]PEt formation in a similar kinetic fashion, suggesting that PC hydrolysis is involved in PDGF-mitogenic signaling pathway. PDGF-stimulated [ 3H]PA formation was significantly higher in the presence than in the absence of PA phosphohydrolase (PAP) inhibitor, indicating the activation of PLD/PAP pathway. In the presence of ethanol, a substantial level of PA at the steady state can be abolished by an inhibitor of diacylglycerol (DAG) kinase. This phenomenon indicates the existence of PC-phospholipase C (PLC)/DAG kinase pathway in PC hydrolysis. Insulin potentiated both PLD- and PDGF-induced DNA synthesis. Though similarities occur in the induction of DNA synthesis and PC hydrolysis by exogenous PLD and PDGF, the maximum extent of DNA synthesis of exogenous PLD was only approximately 43% of that induced by PDGF. Moreover, exogenous PLD-induced DNA synthesis was not blunted, while PDGF-elicited DNA synthesis was markedly reduced, by PKC downregulation. In addition, PDGF-induced PC hydrolysis was attenuated by a tyrosine kinase inhibitor, whereas exogenous PLD-induced PC hydrolysis was unchanged. Taken together, exogenous PLD may mimic PDGF action and partially account for the efficacy on DNA synthesis elicited by PDGF. The signal transduction initiated by exogenous PLD is able to bypass the PKC- and PTK-dependent activation of endogenous PLD. These findings provide evidence for the importance of PLD-mediated PC hydrolysis in pericyte DNA synthesis stimulated by PDGF.

Protein kinase C and phospholipase D activation in rat parotid glands

We have previously demonstrated that muscarinic and α-adrenergic receptors regulated a phospholipase D (PLD) activity in parotid glands. Since phorbol 12-myristate, 13-acetate (PMA) induced production of phosphatidylethanol (PEt), a stable metabolite widely accepted as marker of PLD activation, we have investigated the role of protein kinase C (PKC) in PLD stimulation in parotid acini. We tested PKC inhibitors on PEt formation elicited by PMA, by muscarinic and adrenergic agents. Staurosporine and chelerythrine, which act on the catalytic domain of PKC, did not allow the attribution of a role for PKC in PLD activation. Indeed, staurosporine did not affect PMA-mediated PLD activity and chelerythrine showed an important non-specific effect, independent of PKC inhibition. On the other hand, calphostin C, which acts on the regulatory domain of PKC, affected PMA- and receptor-mediated PLD stimulation. We attributed this effect to PKC inhibition and we suggested PKC involvement in PLD regulation in parotid gland. Since only PKC inhibitor acting on the regulatory part of the enzyme affected PLD activity, we also suggested that PKC could be involved in PLD activation through a pathway independent of the phosphorylation mechanism.

Activation of phospholipase C and phospholipase D by stimulation of adenosine A1, bradykinin or P2U receptors does not correlate well with protein kinase C activation

Activation of adenosine A1-, bradykinin- or P2U-receptors on DDT1 MF-2 smooth muscle cells all increased the formation of inositol 1,4,5-trisphosphate and the mobilization of intracellular calcium. All three types of agents could increase [Ca2+]i in the same cell. Activation of the P2U receptor with ATP or UTP produced larger responses than activation of bradykinin- and adenosine A1-receptors, with bradykinin and N6-cyclopentyladenosine. When agonist-stimulated levels of diacylglycerol were determined, all agonists caused biphasic changes of similar magnitudes. If anything, ATP and UTP tended to give larger increases in the second phase of stimulation. Phospholipase D, measured as the formation of phosphatidylethanol in cells labeled with [3H]palmitic acid and activated in the presence of ethanol, was activated similarly as phospholipase C, i.e. ATP or UTP caused the largest increase in phosphatidylethanol formation, followed by N6-cyclopentyladenosine and bradykinin which caused weaker responses. Activation of PLD by P2U receptors was pertussis toxin insensitive. The activation of PLD by the agonists was only weakly affected by a PKC inhibitor, Ro 31-7549 (3-[1-(3-aminopropanyl)-3- indolyl]-4-(1-methyl-3-indolyl)-1H-pyrrole-2,5-dione). In contrast, ATP or UTP did not activate protein kinase C, determined in a permeabilized cell assay using two specific protein kinase C substrates, whereas N6-cyclopentyladenosine and bradykinin caused a substantial activation.(ABSTRACT TRUNCATED AT 250 WORDS)

CGMP antagonizes angiotensin-mediated phosphatidylcholine hydrolysis and C kinase activation in mesangial cells

Previous studies from this laboratory have shown that in cultured rat mesangial cells (MC), angiotensin II (ANG II) mediates its effects via activation of phosphatidylinositol-specific phospholipase C (PI-PLC) and phosphatidylcholine-specific phospholipase C (PC-PLC) and phospholipase D (PC-PLD). In addition, guanosine 3',5'-cyclic monophosphate (cGMP)-elevating maneuvers that stimulate particulate and soluble guanylate cyclase [atrial natriuretic factor (ANF) and sodium nitroprusside (SNP), respectively] antagonize ANG II-mediated PI-PLC activation. The current study explored whether cGMP impairs ANG II-mediated PC-PLC and PLD activity. The ANG II-stimulated release of the water-soluble metabolites of PC breakdown (phosphorylcholine and choline) was blocked by ANF and SNP. ANG II-stimulated phosphatidic acid and phosphatidylethanol formation were significantly reduced by ANF and SNP, confirming that cGMP blunted PLD activity. The inhibitory effect of cGMP on PLD could be reversed by N-(2-[methylamino]ethyl)-5-isoquinolinesulfonamide, a blocker of cGMP-dependent protein kinase. In parallel experiments, ANF and SNP abrogated sustained diacylglycerol (DAG) accumulation derived from ANG II stimulation of PC hydrolysis, confirming that cGMP diminished PC-PLC activity. Inhibition of PC-derived DAG accumulation by cGMP was associated with a concomitant decrement in ANG II-mediated translocation of protein kinase C (PKC) activity from the cytosol to the membrane. In summary, in MC, cGMP antagonizes ANG II-mediated PC hydrolysis, DAG formation, and PKC activation. We propose that cGMP-mediated inhibition of phospholipid metabolism and PKC translocation plays an important role in MC vasorelaxation.

Activation of adenosine A1 and bradykinin receptors increases protein kinase C and phospholipase D activity in smooth muscle cells

Since adenosine A1 receptors activate phospholipase C (PLC) in DDT1 MF-2 smooth muscle cells we have examined whether phospholipase D (PLD) and protein kinase C (PKC) activities are also increased. The formation of diacylglycerol was also measured. PKC activity was determined by measuring the phosphorylation of two peptide substrates after rapidly permeabilizing the cells. PLD activity was determined by measuring the formation of phosphatidylethanol. N6-cyclopentyladenosine, a selective adenosine A1 receptor agonist (100 nM) and bradykinin (1 microM) both stimulated the formation of diacylglycerol. The activation was biphasic with a rapid, transient increase (within 1 min) followed by a second increase. N6-cyclopentyladenosine increased the activity of PKC (EC50 5.6 nM) and PLD (EC50 18.7 nM). This was blocked by treatment of cells with pertussis toxin or the adenosine A1 receptor selective antagonist, 8-cyclopentyl-1,3-dipropylxanthine. Ki values (3 nM for PKC; 0.1 nM for PLD) were consistent with responses mediated via adenosine A1 receptors. Bradykinin (1 microM) also increased PKC and PLD activity, but these responses were insensitive to pertussis toxin treatment. The activation of PKC by N6-cyclopentyladenosine or bradykinin was transient, reaching a maximum at 1-2 min, and was preceded by increases in the formation of diacylglycerol. When adenosine A1 and bradykinin receptors were activated simultaneously, a synergistic activation of PKC was seen. There was no synergistic effect on PLD activity. In summary, the present study shows that activation of adenosine receptors of the A1 subtype increases PKC and PLD activity. Simultaneous activation of adenosine A1 and bradykinin receptors causes a synergistic increase in PKC.

Diverging signal transduction pathways activated by interleukin-8 and related chemokines in human neutrophils: interleukin-8, but not NAP-2 or GRO alpha, stimulates phospholipase D activity

Interleukin-8 (IL-8) and the structurally related cytokines neutrophil- activating peptide-2 (NAP-2) and GRO alpha are powerful chemotactic agents for human neutrophils. Although these three chemokines act by binding to overlapping but not identical receptor subsets, the data available to date have stressed the similarities in their mechanisms of action. The present studies were undertaken to further our understanding of the signal transduction mechanisms associated with these neutrophil agonists. IL-8, NAP-2, and GRO alpha stimulated similar increases in the level of cytoplasmic free calcium. They were also shown to stimulate qualitatively similar increases in the levels of protein tyrosine phosphorylation. In contrast, only IL-8 enhanced the formation of phosphatidylethanol (PEt), the product catalyzed by phospholipase D (PLD) in the presence of ethanol. The formation of PEt stimulated by IL-8 was inhibited by pertussis toxin and the tyrosine kinase inhibitors erbstatin and herbimycin A. The ability of IL-8 to stimulate the activity of PLD was additively enhanced, or primed, by cytochalasin B and by tumor necrosis factor alpha. Although all three chemokines increased the level of free cytoplasmic calcium to the same extent, IL-8 was significantly more potent than either NAP-2 or GRO alpha with respect to its ability to enhance CD11b expression and to stimulate chemotactic and oxidative responses. The differences between IL-8, NAP-2, and GRO alpha in their ability to stimulate PLD is likely to be related to their respective binding affinities for the two IL-8 receptors (IL-8R-A and IL-8R-B). These results suggest that the signalling pathways activated by IL-8R-A and IL-8R-B diverge at a step preceding the activation of PLD.

Diverging Signal Transduction Pathways Activated by Interleukin-8 and Related Chemokines in Human Neutrophils: Interleukin-8, But Not NAP-2 or GROα, Stimulates Phospholipase D Activity

Interleukin-8 (IL-8) and the structurally related cytokines neutrophil-activating peptide-2 (NAP-2) and GRO alpha are powerful chemotactic agents for human neutrophils. Although these three chemokines act by binding to overlapping but not identical receptor subsets, the data available to date have stressed the similarities in their mechanisms of action. The present studies were undertaken to further our understanding of the signal transduction mechanisms associated with these neutrophil agonists. IL-8, NAP-2, and GRO alpha stimulated similar increases in the level of cytoplasmic free calcium. They were also shown to stimulate qualitatively similar increases in the levels of protein tyrosine phosphorylation. In contrast, only IL-8 enhanced the formation of phosphatidylethanol (PEt), the product catalyzed by phospholipase D (PLD) in the presence of ethanol. The formation of PEt stimulated by IL-8 was inhibited by pertussis toxin and the tyrosine kinase inhibitors erbstatin and herbimycin A. The ability of IL-8 to stimulate the activity of PLD was additively enhanced, or primed, by cytochalasin B and by tumor necrosis factor alpha. Although all three chemokines increased the level of free cytoplasmic calcium to the same extent, IL-8 was significantly more potent than either NAP-2 or GRO alpha with respect to its ability to enhance CD11b expression and to stimulate chemotactic and oxidative responses. The differences between IL-8, NAP-2, and GRO alpha in their ability to stimulate PLD is likely to be related to their respective binding affinities for the two IL-8 receptors (IL-8R-A and IL-8R-B). These results suggest that the signalling pathways activated by IL-8R-A and IL-8R-B diverge at a step preceding the activation of PLD.

Impaired activation of phospholipase D in polycythaemia vera-implications for the pathogenesis of the disease?

A series of studies have demonstrated a stimulus-specific defect in PMN oxidative metabolism after stimulation with surface receptor dependent stimuli such as fMLP, leukotriene B4 and platelet activating factor (PAF), whereas the response to phorbol myristate acetate was normal. Having discovered this defect, studies of the stimulus response coupling for oxidative responses were performed showing a normal interaction of fMLP with it's receptor, as well as an intact activation of phospholipase C, as measured by the generation of 1,4,5-inositoltrisphosphate, and the subsequent rise of intracellular calcium. In contrast, the formation of diacylglycerol and phosphatidylethanol was decreased in PV PMN, denoting an impaired activation of phospholipase D (PLD). It was shown by flow cytometry analyses that the hampered oxidative response was present both in single PMN and monocytes. Moreover, platelets from PV patients, whose PMN exhibit a lower oxidative response to PAF, also have a diminished aggregatory response to PAF. Thus three different cell lineages in PV have been revealed to respond abnormally to surface receptor dependent stimuli, indicating that the proposed impairment of PLD might be relevant for changes in the malignant stem cell clone. Since phosphatidic acid, produced as a result of PLD activation, may be implicated in the regulation of several oncogenes, perturbations of the PLD system could theoretically be important for the development of PV.

Activation mechanism of phospholipase D involved in the generation of lipid mediators in cultured Madin-Darby canine kidney cells.

Addition of 100 nM phorbol 12-myristate 13-acetate (PMA), an active phorbol diester, to quiescent cultured Madin-Darby canine kidney (MDCK) cells caused a maximal stimulation of phosphatidylethanol formation within 1-2 h in the presence of 1% ethanol, indicating the activation of phospholipase D (PLD). The specificity of phorbol diesters for the activation of PLD activation was confirmed by the fact that phorbol 12,13-dibutyrate (PDBu) was effective, whereas 4 alpha-phorbol 12,13-didecanoate (4 alpha-PDD) was without effect. Down-regulation caused by the long-term pretreatment of the cells with active phorbol diesters significantly decreased the production of phosphatidylethanol. Staurosporine, a well known protein kinase (PK)C inhibitor at 1 microM, decreased the activation of PLD. Taken together, these observations suggested the involvement of PKC in the activation of PLD. The cellular PLD activity was found to be selectively localized in the particulate fraction by centrifugation at 12,000 x g. The particulate PLD showed the selective substrate specificity for phosphatidylcholine rather than phosphatidylethanolamine. In response to the addition of 100 nM PMA, 1,2-diacylglycerol (DG) increased in a biphasic fashion. In view of the time course of the activation of PLD, the second increase in the 1,2-DG around 20 min was contributed by the activation of PLD. In response to the simultaneous addition of 100 nM PMA and 100 nM A23187, the cultured MDCK cells activated the arachidonate cascades to form prostaglandin (PG)E2 and PGF2 alpha as major products, requiring slower 24 h to reach maximal levels.(ABSTRACT TRUNCATED AT 250 WORDS)

Vascular smooth-muscle cells contain AT1 angiotensin receptors coupled to phospholipase D activation

We previously showed that angiotensin II (Ang II) and angiotensin-(2-8)-peptide [Ang-(2-8)] activate a phosphoinositide-specific phospholipase C (PLC) and cause calcium mobilization in rat aortic vascular smooth-muscle cells (VSMC), while Ang II and Ang-(1-7) produce prostaglandins. To define further the signal-transduction mechanisms activated by angiotensin peptides in smooth-muscle cells, we measured diacylglycerol (DAG) accumulation in response to different angiotensin peptides and its inhibition by subtype-selective receptor antagonists. Both an initial (10 s) and secondary (10 min) phase of DAG production in response to 100 nM Ang II were inhibited by 1 microM losartan (DuP 753), an AT1 antagonist, while 1 microM PD 123177, an AT2 antagonist, was ineffective. In contrast, the heptapeptide Ang-(1-7) did not produce DAG in VSMC. Ang II also caused the hydrolysis of phosphatidylinositol and phosphatidylcholine, the formation of phosphatidic acid and the formation of phosphatidylethanol (PEt) in the presence of ethanol, through activation of a PLD and a PLD-induced transphosphatidylation reaction. A similar concentration of Ang-(2-8) also activated PLD; in contrast, Ang-(1-7) was ineffective. PEt production by 100 nM Ang II was significantly attenuated by the AT1 antagonists losartan, its metabolite EXP 3174 or L-158,809 (all at 1 microM), whereas a similar concentration of the AT2 antagonists CGP 42112A or PD 123177 was ineffective. The production of PEt by Ang II was also partially attenuated by the removal of extracellular calcium and potentiated by increasing calcium concentrations, indicating that PLD activity is partially dependent on extracellular calcium. Thus VSMC PLD is coupled to an AT1 receptor and occurs in response to Ang II or Ang-(2-8), but not Ang-(1-7). Since AT1 receptors in VSMC are also coupled to activation of PLC, both PLC and PLD may be coupled to the same or a different AT1 receptor. Alternatively, PLD may be sequentially activated in response to Ang II activation of PLC and a subsequent increase in calcium concentration.

Selective Activation by Atrial Natriuretic Factor of Phosphatidylcholine-specific Phospholipase Activities in Purified Heart Muscle Plasma Membranes

We have characterized a membrane-bound phosphatidylcholine (PC) specific phospholipase C (PC-PLC) in plasma membranes from rat cardiac muscle, and have investigated the role of PC-PLC and PC-specific phospholipase D (PC-PLD) activities in the mechanism of action of atrial natriuretic factor (ANF). In purified sarcolemma, ANF stimulated over a wide range of concentrations with a maximum at 10-11 M the hydrolysis of phosphatidylcholine through PC-PLD giving phosphatidate and choline, whereas higher concentrations of ANF (10-10 M) preferentially stimulated PC breakdown through PC-PLC to form diacylglycerol and phosphocholine. To confirm the involvement of the PC-PLD in the mechanism of ANF action, we measured the transphosphatidylation reaction, a specific assay for this phospholipase which in the presence of ethanol catalyses the phosphatidylethanol formation from PC. ANF stimulated phosphatidylethanol formation with the same dose-response behavior as phosphatidate formation. The significant diacylglycerol increase at 10-10 M ANF, in the presence of propranolol, a potent inhibitor of phosphatidate phosphatase which can hydrolyse phosphatidate to give diacylglycerol, suggested a direct involvement of PC-PLC. The use of GTPγ-S, a non hydrolysable analog of GTP, and of pertussis toxin showed the involvement of a pertussis toxin insensitive G protein in PC-PLC mediated ANF signal transduction. We suggest a differential effect of ANF on PC breakdown by phospholipases C and D depending on the concentration of the peptide.

Protein kinase C-mediated phospholipase D activity is increased by linolenic acid supplementation in NG 108-15 cells

Phospholipase D activation was studied in NG 108-15 cells after manipulation of the phospholipid fatty acid composition. Cultivation of cells in media containing different polyunsaturated fatty acids induced extensive and specific changes in the phospholipid fatty acid composition. General for all phospholipids was an increase in polyunsaturated fatty acids at the expense of monounsaturated fatty acids. To examine phospholipase D activation, cells were stimulated with phorbol esters in the presence of ethanol and the formation of phosphatidylethanol was analyzed. In cells cultured with linolenic acid, a significantly higher amount of phosphatidylethanol was formed compared to control cells. On the other hand, supplementation with linoleic, arachidonic or docosahexaenoic acids did not induce any changes in phospholipase D activity. The effect was not due to free fatty acids in the cell culture medium and thus probably induced by fatty acids incorporated into membrane phospholipids or fatty acid metabolites. The results indicate a specific effect of linolenic acid and/or its metabolites on protein kinase C-mediated phospholipase D activity.

Diacylglycerol formation induced by human plasmin in cultured endothelial cells

Treatment of cultured bovine carotid artery endothelial cells with 10 −7M plasmin increased the cellular diacylglycerol which was determined by the formation of [ 3H]palmitate-labeled diacylglycerol and diacylglycerol mass. Upon the stimulation with plasmin, a gradual increase in diacylglycerol formation was observed within 20 min then slightly declined. The maximal effect during the 1-h time course study was 45 and 55% increases in [ 3H]palmitate-labeled diacylgycerol and diacylglycerol mass, respectively, at 20 min after plasmin treatment. Formation of phosphatidylethanol was also studied in [ 3H]palmitate-prelabeled cells in the presence of ethanol. Treatment with plasmin for 20 min induced a significant 45% increase in phosphatidylethanol formation. The present results indicate that the plasmin-induced diacylglycerol formation in endothelial cells was at least in part mediated through the phospholipase D activation.

Vitamin K 3 Preferentially Inhibits Stimulation of Phospholipase D-Mediated Hydrolysis of Phosphatidylethanolamine by Protein Kinase C Activators in NIH 3T3 Fibroblasts

Vitamin K 3 (menadione), a synthetic vitamin K congener, inhibits the growth of tumor cells. Here, we examined possible effects of vitamin K 3 On phospholipase D (PLD) activity, an enzyme which produces growth regulatory substances. In NIH 3T3 fibroblasts, vitamin K 3 (50-100 μM) alone had no effect on PLD-catalyzed formation of phosphatidylethanol, a marker of PLD activity, but it slightly (10-21%) inhibited the stimulatory effect of phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C (PKC). Of the two major substrates of PLD, phosphatidylcholine (PtdCho) and phosphatidylethanolamine (PtdEtn), vitamin K 3 (10-100 μM) preferentially inhibited PtdEtn hydrolysis when stimulated by PMA or platelet-derived growth factor, the latter being a hormonal activator of PKC. Vitamin K 3 had no inhibitory effect on sphingosine- or staurosporine-induced hydrolysis of PtdEtn or PtdCho. Inhibition of PMA-induced PtdEtn hydrolysis by vitamin K 3 was effectively reduced by both cysteine (1 mM) and reduced glutathione (1 mM) and was mimicked by the superoxide-generating xanthine/xanthine oxidase system. The results show that vitamin K 3 preferentially inhibits the effects of PKC activators on PLD-mediated hydrolysis of PtdEtn by a mechanism which may involve oxidation of thiols in a critically important regulatory component.

Phosphatidylethanol formation and degradation in brains of acutely and repeatedly ethanol-treated rats

The formation of the abnormal phospholipid phosphatidylethanol (PEth) was studied in hippocampus, cerebellum and cerebrum of rat brain after intraperitoneal ethanol administration. Prior to analysis by high performance thin layer chromatography PEth was purified. After one injection, PEth levels reached a maximum after 2 h and remained detectable for 14–24 h in all three regions. Repeated injections led to additional accumulation. Maximum in vivo levels of 30–50 nmol/g wet wt. were reached.

Leupeptin inhibits phospholipases D and C activation in rat hepatocytes

The relationship between phospholipase D and C activation was studied in intact rat hepatocytes and rat liver plasma membranes. In intact hepatocytes, in the presence of ethanol, vasopressin, phorbol ester, and calcium independently stimulated phosphatidylethanol (PETH) formation, a specific marker of phospholipase D activity. Leupeptin (10–1500 μM) inhibited PETH formation induced by vasopressin, but was ineffective in response to phorbol ester or calcium. Leupeptin also inhibited the formation of inositol phosphates in intact cells in response to vasopressin. In liver plasma membranes, GTP[S] induced the production of phosphatidic acid and, in the presence of ethanol, PETH. Plasma membrane-associated phospholipase D did not require calcium and was insensitive to protein kinase C inhibitors. Leupeptin inhibited PETH formation in response to GTP[S]. The inhibition by leupeptin could be overcome by increasing the concentration of GTP[S]. In plasma membranes, the inhibitory effects of leupeptin on phospholipase D occurred at doses that far exceed those required to maximally inhibit proteolysis. These data highlight a central role for phospholipase C in the activation of phospholipase D, and a minor role for a direct G-protein activation. The findings also demonstrate a novel use of leupeptin as an inhibitor of phospholipases D and C perhaps at the level of a G protein.

Role of diradylglycerol formation in H2O2 and lactoferrin release in adherent human polymorphonuclear leukocytes

Polymorphonuclear leukocytes (PMNs) adherent to fibrinogen exhibit a delay in the release of H2O2 in response to fMLP. Previously, we demonstrated that H2O2 release in adherent PMNs coincides with the exocytosis of lactoferrin-containing specific granules and activation of phospholipase D (PLD). We also found that chelation of intracellular calcium blocked both lactoferrin and H2O2 release in stimulated PMNs in spite of the fact that adhesion and spreading remained normal. Since diradylglycerol (DRG) formation has been implicated in PMN secretion and oxidant release, we determined the effect of intracellular calcium chelation on PLD activation and DRG formation to ascertain whether DRG formation was coupled to lactoferrin and H2O2 release. We observed that chelation of intracellular calcium with bis-(O-aminophenoxy)-ethanol-N,N;N'-tetraacetic acid (BAPTA) prevented PLD activation as monitored by inhibition of phosphatidylethanol formation. Formation of DRG derived from phosphatidic acid (PA) was also inhibited in the presence of BAPTA. Following the addition of the calcium ionophore ionomycin to the BAPTA-treated PMNs, lactoferrin and H2O2 release was coincident with the onset of DRG formation. Also the addition of sn-1,2-didecanoylglycerol to the BAPTA-treated PMNs stimulated them to release H2O2. Our studies support the hypothesis that DRG derived from PLD activation is required for degranulation of specific granules and associated H2O2 release from adherent PMNs.

The cloned vasopressin V1a receptor stimulates phospholipase A 2, phospholipase C, and phospholipase D through activation of receptor-operated calcium channels

Arginine vasopressin mediates its effects through vasopressin receptor activation and second messenger production. Recent cloning of the V1a receptor provided the opportunity to investigate the possible signal transduction pathways associated with this single vasopressin receptor subtype. When stably expressed in CHO cells, vasopressin stimulated several signal transduction pathways simultaneously including calcium influx, phospholipase A 2, phospholipase C, and phospholipase D. Vasopressin-stimulated release of arachidonic acid, IP 3 formation, and phosphatidylethanol formation (in the presence of 1% ethanol) were used as indexes of phospholipase A 2, phospholipase C, and phospholipase D activation, respectively. V1a receptor-activation stimulated a peak followed by a sustained plateau phase of intracellular calcium. The plateau phase was dependent on extracellular calcium, insensitive to blockers of voltage sensitive calcium channels, blocked by heavy metals, and quenched when MnCl 2 was present in the extracellular media. Removal of extracellular calcium blunted the release of IP 3, and blocked the release of arachidonic acid and phosphatidylethanol indicating that these responses were at least in part regulated by receptor-operated calcium influx. Vasopressin-stimulated release of arachidonic acid and phosphatidylethanol were augmented with the phorbol ester PMA, and this augmentation was blocked by inhibitors of protein kinase C and absent with long-term PMA treatment. Vasopressin-stimulated IP 3 release was inhibited with PMA and the inhibition reversed with protein kinase C inhibitors.

Insulin-stimulated hydrolysis of phosphatidylcholine by phospholipase C and phospholipase D in cultured rat hepatocytes

We have investigated the mechanism of action by which insulin increases phosphatidate (PA) and diacylglycerol (DAG) levels in cultured rat hepatocytes. Insulin initially stimulated phosphatidylcholine-dependent phospholipase D (PC-PLD) with a significant increase in both PA and intracellular as well as extracellular choline. The involvement of phospholipase D was confirmed by the formation of PC-derived phosphatidylethanol in the presence of ethanol. The DAG increase appeared to be biphasic. Only the early phase of DAG production was inhibited by propranolol, an inhibitor of the phosphatidate phosphatase (PAP) responsible for the conversion of PA into DAG, suggesting that initially the DAG increase is due to the PLD-PAP pathway. The delayed DAG increase was in parallel with increased intracellular and extracellular phosphocholine and probably derived directly from PC-PLC activity. Experiments performed in the presence of 1 μM phorbol 12-myristate 13-acetate (PMA) indicated that protein kinase C (PKC) mediated the insulin effect on PC-PLC, but not on PC-PLD. These findings were confirmed using the PKC inhibitors calphostin, H7 and staurosporine. The dual activation of these phospholipases with a biphasic elevation of DAG levels and activation of specific PKC isoenzymes could be necessary to elicit both early and delayed effects of insulin.

Agonist-stimulated and basal phosphatidylethanol formation in neutrophils from alcoholics.

Phospholipase D has been shown to be a key enzyme in the signal transduction systems involved in neutrophil activation. In the presence of ethanol, the enzyme catalyzes a transphosphatidylation reaction through which phosphatidylethanol is formed instead of the normal product phosphatidic acid. The effects of ethanol on the formation of phosphatidylethanol and phosphatidic acid was studied in neutrophils from human alcoholics in vitro. Neutrophils were isolated and cellular lipids were labeled with [3H]oleate, whereafter the cells were preincubated with cytochalasin B. Subsequently, cells were stimulated with the chemotactic peptide formyl-Met-Leu-Phe in the presence of ethanol concentration ranging from 0 to 200 mM. In the presence of ethanol, both neutrophils from alcoholics and controls produced phosphatidylethanol, with a concomitant reduction of the production of phosphatidic acid. The amounts of phosphatidyl-ethanol and phosphatidic acid formed were dependent on the concentration of ethanol. In neutrophils from alcoholics, a higher apparent Km for the phospholipase D-mediated transphosphatidylation reaction was noted (58 mM ethanol compared with 28 mM in controls). The in vivo mass of phosphatidylethanol in recently drinking alcoholics was also analyzed in neutrophils. Measurable phosphatidyl-ethanol levels (average 5.6 pmol/10(8) neutrophils) were found in alcoholics up to 23 hr after the last intake of ethanol. Thus, in addition to the ethanol-induced changes in the normal production of phosphatidic acid, phosphatidylethanol accumulated in vivo in alcoholics may be expected to influence neutrophil function.