Stimulatory effects of a short chain phosphatidate on superoxide anion production in guinea pig polymorphonuclear leukocytes

Treatment of guinea pig polymorphonuclear leukocytes (PMNL) with a phosphatidate containing short-chain fatty acids, 1,2-didecanoyl-3-sn-phosphatidate (PA10), induced substantial superoxide anion (O2-) production in a dose-dependent manner, whereas phosphatidates prepared from egg lecithin and 1,2-dioleoyl-3-sn-phosphatidate (PA18:1) had no such effect. Calcium was not involved in PA10-induced O2- production, since the production was also observed in the case of addition of EGTA prior to PA10 or pretreatment of PMNL with quin-2 and EGTA to eliminate contributions of both extracellular and intracellular calcium. We have reported in previous papers that the phosphorylation of 46K protein(s), which was commonly observed in parallel with an activation of NADPH oxidase in PMNL, was increased by treatment with 10 microM 1-oleoyl-2-acetylglycerol (OAG) with little change in the O2- production (Okamura et al. (1984) Arch. Biochem. Biophys. 228, 270-277; Ohtsuka et al. (1988) Arch. Biochem. Biophys. 260, 226-231). Treatment of PMNL with a combination of PA10, which slightly increased 46K protein phosphorylation, and such a low concentration of OAG induced a marked increase in the O2- production with the increase in 46K protein phosphorylation, which was probably due to OAG action. Thus, it is likely that this protein phosphorylation plays a significant role in the stimulation of the O2- production by phosphatidate in PMNL.     

Stimulation of superoxide anion production in guinea pig polymorphonuclear leukocytes by hypotonic conditions in combination with protein kinase C activators.

Conditions for superoxide anion (O2-) production were examined in guinea pig polymorphonuclear leukocytes (PMNL). When PMNL were suspended in the hypotonic medium, O2- production was significantly enhanced by concurrent treatment with low concentrations of 1-oleoyl-2-acetylglycerol (OAG), a cell-permeable protein kinase C activator. Such hypotonicity or OAG alone had little effect on the production. Other protein kinase C activators also markedly enhanced O2- production in combination with hypotonicity, but not in the isotonic medium. Protein kinase C inhibitors, H-7 and staurosporine, dose-dependently inhibited the production. These observations indicate that protein kinase C participates in such synergistic O2- production with hypotonicity. Phosphorylation of 46-kDa protein(s), which was commonly enhanced in paralleled with an activation of NADPH oxidase in guinea pig PMNL, was increased by treatment with 10 microM OAG, but the phosphorylation was little altered by hypotonic treatment. Intracellular calcium concentration, arachidonate release, and 1,2-diacylglycerol and phosphoinositide concentrations were slightly altered by hypotonic treatment. A change in phosphatidate (PA) production in PMNL was induced by hypotonic treatment either by itself or in combination with OAG treatment. These results suggest that the combination of cell membrane changes by hypotonic treatment accompanied by the increase in PA and 46-kDa protein phosphorylation by protein kinase C provides the conditions required for a marked increase in O2- production. Hypotonicity may be a good tool for studying the mechanism of priming in the activation of NADPH oxidase.     

Significance of phosphorylation/dephosphorylation of 46K protein(s) in regulation of superoxide anion production in intact guinea pig polymorphonuclear leukocytes

Superoxide anion (O2-) production stimulated by concanavalin A (Con A) in guinea pig polymorphonuclear leukocytes (PMNL) was suppressed by addition of methyl-alpha-mannoside, a Con A inhibitor, and resumed upon readdition of Con A. The reversible change in the O2- production was assumed to reflect the change in NADPH oxidase activity measured for the 30,000 X g particulate fraction. The stimulation by Con A of the phosphorylation of 46K protein(s), as observed previously with several membrane-perturbing agents in parallel with an activation of NADPH oxidase in intact guinea pig PMNL (Okamura, N., et al. (1984) Arch. Biochem. Biophys. 228, 270-277), was also suppressed by methyl-alpha-mannoside and resumed upon readdition of Con A. Similar parallelism between the phosphorylation and NADPH oxidase activity was also observed in the case of stimulation by N-formyl-methionyl-leucyl-phenylalanine (FMLP) and phorbol 12-myristate 13-acetate (PMA), though both processes were reversible after the stimulation by FMLP but not reversible after that by PMA. Thus, such a parallelism observed in both intact PMNL and 30,000 X g particulate fraction indicates possible involvement of the protein phosphorylation in the regulation of the production of active oxygen metabolites in PMNL.     

A diacylglycerol kinase inhibitor, R 59 022, potentiates superoxide anion production and 46-kDa protein phosphorylation in guinea pig polymorphonuclear leukocytes

A diacylglycerol (DG) kinase inhibitor, R 59 022, potentiated superoxide anion (O2-) production in guinea pig polymorphonuclear leukocytes (PMNL) induced by N-formyl-methionyl-leucyl-phenylalanine (FMLP). R 59 022 also potentiated O2- production induced by 1-oleoyl-2-acetylglycerol, a permeable DG. However, the production induced by phorbol 12-myristate 13-acetate (PMA), a direct activator for protein kinase C, was not potentiated by R 59 022. R 59 022 by itself had no significant effects on unstimulated O2- production. The potentiation of FMLP-induced O2- production by R 59 022 was correlated closely with increased formation of DG and decreased formation of phosphatidic acid, a product of DG kinase. R 59 022 had no effect on the breakdown of phosphoinositides. Phosphorylation of 46-kDa protein(s) by protein kinase C was also examined in relation to O2- production in PMNL. In coincidence with the increase in O2- production, the phosphorylation was potentiated by R 59 022 in the response to FMLP, but not in the response to PMA. In addition, staurosporine, a protein kinase C inhibitor, inhibited increases in both O2- production and phosphorylation of the 46-kDa protein(s) after PMA stimulation. Similar inhibitory effects of staurosporine were also observed upon stimulation with FMLP, irrespective of the presence of R 59 022. These results indicate that retention of DG as a result of the inhibition of further metabolism induces marked stimulation of O2- production via protein kinase C activation in PMNL. These results also provide further evidence for the close relationship between 46-kDa protein phosphorylation by protein kinase C and stimulation of O2- production in PMNL.     

Phospholipase A2 activation in chemotactic peptide-stimulated HL60 granulocytes: synergism between diacylglycerol and Ca2+ in a protein kinase C-independent mechanism

In dimethylsulfoxide-differentiated HL60 granulocytes, the chemotactic peptide N-formyl-Met-Leu-Phe (FMLP) augments arachidonic acid (AA) release via phospholipase A2 activity induced by the Ca2+-ionophore, A23187. Evidence indicates that this augmentation is mediated by diacylglycerols formed endogenously during FMLP receptor activation: The augmentation is mimicked by the synthetic diglyceride 1-oleoyl-2-acetyl-glycerol (OAG) and the tumor promoting phorbol ester 12-O-tetradecanoylphorbol-13-acetate; Pertussis toxin inhibits FMLP-induced augmentation but not OAG-induced augmentation: At suboptimal concentrations FMLP and OAG act cooperatively to augment ionophore A23187-induced AA release but not at optimal concentrations. These data indicate that phospholipase A2 activation in FMLP-stimulated HL60 granulocytes involves cooperative interactions between diacylglycerol formed endogenously and Ca2+. Interestingly, this effect of diacylglycerol appears not to be mediated by protein kinase C, since a specific protein kinase C inhibitor, 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H7) does not inhibit receptor-mediated release of AA by stimulated HL60 granulocytes.     

Synergism between protein kinase C activator and fatty acids in stimulating superoxide anion production in guinea pig polymorphonuclear leukocytes

Treatment of guinea pig polymorphonuclear leukocytes (PMNL) with various fatty acids elicited superoxide anion (O2-) production and an increase in intracellular Ca2+ [( Ca2+]i). Both responses, however, were seldom observed when PMNL were treated at lower concentrations. But, simultaneous addition of 1-oleoyl-2-acetylglycerol (OAG), a protein kinase C activator, caused an increase in O2- production even at the lower concentrations of fatty acids. In contrast to the synergism in O2- production, [Ca2+]i remained at almost the basal level irrespective of the presence of OAG. Among saturated fatty acids, those with carbon numbers of 14 to 18 were most effective in stimulating O2- production in combination with OAG. Unsaturated fatty acids with a carbon number of 18 were almost equally effective irrespective of the number of double bonds.     

Role for protein kinase C in A23187 induced glomerular arachidonate release and PGE2 production

A high concentration of the calcium ionophore A23187 (10 uM) stimulated release of labeled arachidonate and immunoreactive PGE2 from isolated superfused glomeruli. A lower concentration of A23187 (1 uM) or 12-0-tetradecanoyl phorbol-13-acetate (TPA, 0.1 uM), a direct activator of protein kinase C, when added alone was without effect on these parameters. Combined addition of A23187 (1 uM) and TPA (0.1 uM) synergistically stimulated arachidonate release and PGE2 production. 1-(5-isoquinolinyl)-2-methylpiperazine (H-7) a known inhibitor of protein kinase C in other tissues, suppressed increases in arachidonate release and PGE2 production mediated by A23187 (10 uM) or TPA plus A23187 (1 uM). H-7 inhibited while TPA stimulated protein kinase C activity that had been partially purified from soluble fractions of glomerular homogenates. These results support a role for protein kinase C in A23187 mediated arachidonate release.     

Phorbol myristate acetate and the calcium ionophore A23187 synergistically induce release of LTB4 by human neutrophils: involvement of protein kinase C activation in regulation of the 5-lipoxygenase pathway

Phorbol myristate acetate (PMA), a tumor-promoting phorbol ester, and the calcium ionophore A23187 synergistically induced the noncytotoxic release of leukotriene B4 (LTB4) and other 5-lipoxygenase products of arachidonic acid metabolism from human neutrophils. Whereas neutrophils incubated with either A23187 (0.4 microM) or PMA (1.6 microM) alone failed to release any 5-lipoxygenase arachidonate products, neutrophils incubated with both stimuli together for 5 min at 37 degrees C released LTB4 as well as 20-COOH-LTB4, 20-OH-LTB4, 5-(S),12-(R)-6-trans-LTB4, 5-(S),12-(S)-6-trans-LTB4, and 5-hydroxyeicosatetraenoic acid, as determined by high pressure liquid chromatography. This synergistic response exhibited concentration dependence on both PMA and A23187. PMA induced 5-lipoxygenase product release at a concentration causing a half-maximal effect of approximately 5 nM in the presence of A23187 (0.4 microM). Competition binding experiments showed that PMA inhibited the specific binding of [3H]phorbol dibutyrate ([3H]PDBu) to intact neutrophils with a 50% inhibitory concentration (IC50) of approximately 8 nM. 1-oleoyl-2-acetyl-glycerol (OAG) also acted synergistically with A23187 to induce the release of 5-lipoxygenase products. 4 alpha-phorbol didecanoate (PDD), an inactive phorbol ester, did not affect the amount of lipoxygenase products released in response to A23187 or compete for specific [3H]PDBu binding. PMA and A23187 acted synergistically to increase arachidonate release from neutrophils prelabeled with [3H]arachidonic acid but did not affect the release of the cyclooxygenase product prostaglandin E2. Both PMA and OAG, but not PDD, induced the redistribution of protein kinase C activity from the cytosol to the membrane fraction of neutrophils, a characteristic of protein kinase C activation. Thus, activation of protein kinase C may play a physiologic role in releasing free arachidonate substrate from membrane phospholipids and/or in modulating 5-lipoxygenase activity in stimulated human neutrophils.     

Synergistic stimulation of luteinizing hormone (LH) release by protein kinase C activators and Ca2+-ionophore

When cultured pituitary cells were stimulated with synthetic diacylglycerol such as 1-oleoyl-2-acetylglycerol (OAG), or with a potent tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA), which are known stimulators of Ca2+-activated, phospholipid-dependent protein kinase (protein kinase C), enhanced release of luteinizing hormone (LH) was observed. Similarly, LH release was also stimulated by the Ca2+-ionophore, A23187. Simultaneous presence of A23187 and OAG or TPA resulted in a synergistic response that mimicked the full physiological response to gonadotropin releasing hormone (GnRH). Removal of extracellular Ca2+ only slightly affected the stimulatory action of TPA and OAG on LH release, but completely blocked the effect of GnRH. The results suggest that the stimulatory effect of GnRH on LH release may be mediated by two intracellular pathways involving Ca2+ and diacylglycerol as second messengers.     

Induction of 5-lipoxygenase activation in polymorphonuclear leukocytes by 1-oleoyl-2-acetylglycerol

1,2-Diacylglycerols (DAGs) can prime polymorphonuclear leukocytes (PMNL) for enhanced release of arachidonic acid (AA) and generation of 5-lipoxygenase (5-LO) products upon subsequent agonist stimulation. Here, we demonstrate that in isolated human PMNL, 1-oleoyl-2-acetylglycerol (OAG) functions as a direct agonist stimulating 5-LO product formation (up to 42-fold). OAG caused no release of endogenous AA, but in the presence of exogenous AA, the magnitude of 5-LO product synthesis induced by OAG was comparable to that obtained with the Ca(2+)-ionophore A23187. Interestingly, OAG-induced 5-LO product synthesis was not connected with increased 5-LO nuclear membrane association. Examination of diverse glycerides revealed that the sn-2-acetyl-group is important, thus, also 1-O-hexadecyl-2-acetylglycerol (EAG) stimulated 5-LO product formation (up to 8-fold).Treatment of PMNL with OAG did not alter the mobilization of Ca(2+) but removal of intracellular Ca(2+) abolished the upregulatory OAG effects. Notably, the PKC activator phorbol-myristate-acetate hardly increased 5-LO product synthesis and PKC inhibitors failed to suppress the effects of OAG. Although OAG rapidly activated p38 MAPK and p42/44(MAPK), which can stimulate 5-LO for product synthesis, specific inhibitors of these kinases could not prevent 5-LO activation by OAG. Together, OAG acts as a direct agonist for 5-LO product synthesis in PMNL stimulating 5-LO by novel undefined mechanisms.     

Leukocytic functions in burn-injured patients

Anergy associated with an increase in suppressor helper T cell (Tc) ratio and a decrease in natural killer (NK) is one main cause of death following thermal injury (Tl). Recently, in vitro studies have shown that LTB4 can induce human Tc to exert suppressor cell activity, and incubation of lymphocytes with LTB4 for 24 hours significantly suppressed NK cell activity. Thus, we undertook an investigation of both AA metabolism and immunologic response in 20 patients who suffered 40-90% total body surface area (TBSA) burns. Cyclooxygenase (CO:RIA) and lipoxygenase (LO;HPLC det.) metabolites and superoxide (O2 X-) production were measured in stimulated polymorphonuclear cells (PMNL) (A 23187 +/- AA for icosanoid release; phorbol myristate acetate for O2 X-production). Lyso-paf-acether (P-LPA) was measured in plasma samples. Ca2+-dependent K+permeability in PMNL was measured by the cell K+ release induced by A 23187. Tc and Tc subsets were determined using monoclonal antibodies (OKT3+, OKT4+ and OKT8+). A biphasic sequential release of the different substances (leukocytic icosanoids and O2 X-) was monitored: increase (approximately 36-48 h after Tl) and decrease (greater than or equal to 72 h after Tl). The increase in AA stimulation was more transient than that of O2 X -. The decline in the release of AA metabolites and O2 X-production was associated with the anergic phase (decrease OKT4+/OKT8+ ratio) and with the clinical outcome of the patients. The decrease in LTB4 and other LO metabolites could explain the impairment of neutrophil chemotaxis. Ca2+-dependent K+ permeability increased early up to 2 or 3 times normal. In order to go further with the mechanism of inhibition of LTB4 and O X-release, the effect of Tl plasma was assayed on normal leukocytes: a 10 min incubation with such plasma was sufficient to abolish LTB4 secretion. A less important inhibition was observed with O2 X-release (-32%) and Ca2+-dependent K+ permeability (-30%). Plasma inhibition seems to be due to a thermolabile factor(s) [protein(s): "suppressive factor(s) of membrane activation "SFMA] which is (are) under active investigation using gel-filtration chromatography and fast protein liquid chromatography (FPLC). Among the SFMAs, certain acute phase proteins could play a key role: i.e., incubation (10 min) of normal PMNL with ceruloplasmin (1 mg/ml) abolished LO products and O X 2-release.     

Diverse involvements of Ni protein in superoxide anion production in polymorphonuclear leukocytes depending on the type of membrane stimulants

Effects of islet-activating protein (IAP) were examined to assess the involvement of the guanine nucleotide-binding regulatory protein responsible for inhibition of adenylate cyclase system (Ni protein) in the superoxide anion (O-2) production in polymorphonuclear leukocytes (PMNL) stimulated with various agents. N-Formyl-methionyl-leucyl-phenylalanine (FMLP)-stimulated O-2 production was inhibited by the pretreatment with IAP. O-2 production induced by each of phorbol myristate acetate, concanavalin A, and A23187, however, was rather resistant to the pretreatment with IAP. This observation indicates that the Ni protein does not involve in the common pathway for the O-2 production. in PMNL, and the involvement is rather specific for the FMLP-induced production. O-2 production in PMNL stimulated with various membrane perturbing agents was also diverse in the requirement of extracellular Ca2+.     

Effects of dietary vitamin E on the biosynthesis of 5-lipoxygenase products by rat polymorphonuclear leukocytes (PMNL)

Activation of polymorphonuclear neutrophils (PMNL) leads to the release of arachidonate from cellular phospholipids via a phospholipase A2, and conversion of products of the 5-lipoxygenase pathway. Evidence to date indicates the dietary vitamin E ((R,R,R)-alpha-tocopherol) can influence both cyclooxygenase and phospholipase A2 activities and that the effect of this vitamin is cell/tissue specific. The present study was undertaken in order to examine the effects of varying dietary tocopherol on PMNL tocopherol content and 5-lipoxygenase product profile using the ionophore A23187 as stimulant in the presence and absence of exogenous arachidonate. Feeding semi-purified diets containing 0, 30 or 3000 ppm of (R,R,R)-alpha-tocopherol acetate to weanling rats for 17 weeks resulted in a dose-related enrichment of PMNL tocopherol. Stimulation of PMNL elicited a significant and rapid loss of tocopherol. When PMNL were stimulated with A23187 alone, the synthesis of 5-HETE, LTB4 and 19-hydroxy-LTB4 was decreased in proportion to increasing dietary tocopherol concentrations. However, when exogenous arachidonate was provided with A23187, intermediate amounts of dietary tocopherol (30 ppm) still suppressed the formation of 5-lipoxygenase products, but high doses (3000 ppm) did not have any additional inhibitory effect. This differential response to high concentrations of vitamin E in the presence and absence of exogenous arachidonate highly suggest that at these concentrations, tocopherol may act principally at the level of substrate release whereas at lower concentrations, 5-lipoxygenase is inhibited. Data from this study demonstrated that attenuation of the formation of 5-lipoxygenase products in PMNL can be achieved by dietary vitamin E enrichment.     

The effects of the phospholipase A2 inhibitors aristolochic acid and PGBx on A23187-stimulated mobilization of arachidonate in human neutrophils are overcome by diacylglycerol or phorbol ester.

Aristolochic acid and PGBx, two structurally unrelated, protein-targeted inhibitors of isolated phospholipases A2, are effective antagonists of calcium ionophore A23187-stimulated mobilization of [3H]arachidonate from human neutrophils. We now report that preincubation of neutrophils with oleoylacetylglycerol (OAG, 15 microM) substantially reverses the inhibitory effect of 200 microM aristolochic acid (from 70 to 24% inhibition). Similarly, OAG increases the IC50 for PGBx from 2.5 to greater than 20 microM. The effects of OAG on inhibition by either aristolochic acid or PGBx are dose-dependent, with an ED50 of 2.5 microM. Protection against inhibition by either aristolochic acid or PGBx is also observed with phorbol myristate acetate (PMA, ED50 3 nM), but not 4-alpha-phorbol didecanoate. Aristolochic acid and PGBx do not inhibit PMA-stimulated superoxide generation, and are thus not protein kinase C inhibitors. Furthermore, neither aristolochic acid nor PGBx inhibit diglyceride generation through the phospholipase D/phosphatidate phosphohydrolase pathway. A23187-stimulated [3H]arachidonate mobilization is increased by 20-50% when neutrophils are preincubated with OAG or PMA. The present results indicate that OAG and PMA also modulate the A23187-stimulated [3H]arachidonate mobilization so as to render it less sensitive to inhibitors of phospholipase A2.     

Arachidonic acid release by basophilic leukemia cells and macrophages stimulated by Ca2+ ionophores, antigen and diacylglycerol: essential role for protein kinase C and prevention by glucocorticosteroids

The role of protein kinase C in phospholipase A2 (PLA2) activation in rat basophilic leukemia cells (RBL-2H3) and macrophages was investigated. 12-O-Tetradecanoyl phorbol 13-acetate (TPA) doubled ionomycin-induced PLA2 activity, assessed by [3H]arachidonate release. Protein kinase C inhibitors, staurosporine and K252a (100 nM) or H-7 (15 micrograms/ml) inhibited ionomycin-stimulation of PLA2 activity by 62, 75 and 80%, respectively. Down-regulation of protein kinase C by prolonged treatment with TPA inhibited Ca2(+)-ionophore A23187 or antigen-stimulation of [3H]arachidonate release by 80%. We examined whether the inhibitory effect of dexamethasone (DEX) on PLA2 activity is related to modulation of protein kinase C activity. The 50% inhibition by DEX of ionomycin elevation of [3H]arachidonate release was almost overcome by addition of TPA. The Ca2+ ionophore and antigen-induced increase in [3H]TPA binding to intact RBL cells was not impaired by DEX. However, DEX markedly reduced phosphorylation of several proteins. 1-Oleoyl-2-acetyl-glycerol (OAG) had a sustained stimulatory effect on PLA2 activity in isolated plasma membranes derived from treated bone-marrow intact mouse macrophages, while both DEX and staurosporine reduced elevated PLA2 activity by 68 and 84%, respectively. The results support an essential role for protein kinase C in regulation of PLA2 activity.     

Phorbol ester, 1,2-diacylglycerol, and collagen induce inhibition of arachidonic acid incorporation into phospholipids in human platelets

We have shown that phorbol myristate acetate (PMA) enhanced A-23187-induced arachidonate release and thromboxane synthesis in human platelets (Mobley, A., and Tai, H. H. (1985) Biochem. Biophys. Res. Commun. 130, 717-723). The mechanism of enhancement by PMA was not elucidated. In the present study, we have shown that PMA-treated platelets exhibited significantly less [1-14C]arachidonate incorporation than did control platelets. However, no significant change in uptake of labeled linoleate or oleate was observed by PMA treatment. Examination of the two enzyme activities involved in arachidonate incorporation into phospholipids indicated that both arachidonoyl-coenzyme A (CoA) synthase and arachidonoyl-CoA lysophosphatide acyltransferase were inactivated following treatment with PMA or 1-oleoyl-2-acetyl glycerol. When platelets were stimulated with A-23187 plus PMA which produced a significant synergism in thromboxane synthesis, both enzyme activities were substantially less than those in platelets treated with A-23187 alone. In addition to PMA and 1-oleoyl-2-acetyl glycerol induced decreases in both enzyme activities, collagen, a platelet agonist which can activate protein kinase C (Ca2+/phospholipid-dependent enzyme), was also found to cause a concentration-dependent attenuation of both enzyme activities. These results suggest that protein kinase C activation induced by PMA or collagen may cause inactivation of both arachidonoyl-CoA synthase and arachidonoyl-CoA lysophosphatide acyltransferase resulting in inhibition of the reincorporation of arachidonate released by A-23187 and, consequently, greater availability of arachidonate for thromboxane synthesis.     

Ca2+-independent synergistic augmentation of O2- production by FMLP and PMA in HL-60 cells

N-Formyl-Met-Leu-Phe (FMLP) and phorbol 12-myristate 13-acetate (PMA) caused a synergistic augmentation of superoxide anion (O2-) production in neutrophil-like HL-60 cells differentiated with dibutyryl cAMP. The present study was undertaken to investigate the mechanism of the synergistic augmentation of O2- production. FMLP increased intracellular free Ca2+ concentration ([Ca2+]i), which was slightly suppressed by PMA and completely inhibited by an intracellular Ca2+ chelating agent, O,O'-bis(2-aminophenyl)ethyleneglycol-N,N,N',N'-tetraacetic acid tetraacetoxymethyl ester (BAPTA-AM). Although FMLP-induced O2- production was inhibited by BAPTA-AM, a major part of the synergistic augmentation of O2- production by FMLP and PMA remained after BAPTA-AM treatment, suggesting that a Ca2+-independent mechanism might be involved in the augmentation. FMLP and PMA caused an activation of phospholipase D (PLD) almost additively in a Ca2+-sensitive manner. The synergistic activation of mitogen-activated protein kinase (MAPK) was evoked by combined addition of PMA and FMLP in a BAPTA-AM resistant manner. However, PD98059, a MAPK kinase inhibitor, did not affect the synergistic augmentation of O2- production, although it potently inhibited the synergistic augmentation of tyrosine phosphorylation of MAPK. Wortmannin, a phosphatidylinositol 3-kinase inhibitor, inhibited FMLP-induced O2- production, but it did not inhibit the synergistic augmentation of O2- production by PMA and FMLP. In contrast, staurosporine and GF109203X, protein kinase C inhibitors, reduced the synergistic augmentation induced by PMA and FMLP. In addition, pertussis toxin (PT) abolished the synergistic augmentation of O2- production. It is concluded that the synergistic augmentation of O2- production induced by PMA and FMLP is mediated through a PT-sensitive G protein and a protein kinase C in a Ca2+-independent manner.     

Superoxide anion release by human endothelial cells: synergism between a phorbol ester and a calcium ionophore

In order to study the signal transduction mechanism of human endothelial cells (EC), the regulation of superoxide anion (O2-)release in EC has been investigated using the calcium ionophore A23187 and phorbol myristate acetate (PMA), a potential activator of the Ca2+ activated, phospholipid-dependent protein kinase, designated "protein kinase C." PMA enhanced O2- release from EC, and this enhancement occurred regardless of the presence or absence of extracellular Ca2+. A similar increase was produced by A23187; omission of extracellular Ca2+ prevented this increase. Simultaneous stimulation with PMA and A23187 produced a large increase in O2- release at submaximal concentrations of these agents, which, when added separately, caused minimal effects. These findings indicate that the activation of protein kinase C and mobilization of Ca2+ evoked by PMA and A23187 respectively are synergistically effective for eliciting a full physiological response of EC in the generation and release of O2-.     

The effects of phorbol ester, diacylglycerol, phospholipase C and Ca2+ ionophore on protein phosphorylation in human and sheep erythrocytes.

Treatment of human or sheep erythrocytes with PMA (phorbol myristate acetate) enhanced [32P]phosphate labelling of membrane polypeptides of approx. 100, 80 and 46 kDa. The 80 kDa and 46 kDa polypeptides coincided with bands 4.1 and 4.9 respectively on Coomassie-Blue-stained gels. Similar but smaller effects were obtained by treating human cells with 1-oleoyl-2-acetyl-rac-glycerol (OAG), exogenous bacterial phospholipase C or ionophore A23187 + Ca2+, each of which treatments would be expected to raise the concentration of membrane diacylglycerol. In contrast, sheep cells, which do not increase their content of diacylglycerol when treated with phospholipase C or A23187 + Ca2+, only showed enhanced phosphorylation with OAG. Neither human nor sheep cells showed any enhanced [32P]phosphate labelling of phosphoproteins when treated with 1-mono-oleoyl-rac-glycerol. It is concluded that diacylglycerol from a variety of sources can activate erythrocyte protein kinase C, but that the most effective diacylglycerol is that derived from endogenous polyphosphoinositides. In contrast with bacterial phospholipase C and A23187, which stimulate synthesis of phosphatidate by increasing the cell-membrane content of diacylglycerol in human erythrocytes, PMA, OAG or 1-mono-oleoyl-rac-glycerol caused no change in phospholipid metabolism.     

Synergistic release of arachidonic acid from platelets by activators of protein kinase C and Ca2+ ionophores. Evidence for the role of protein phosphorylation in the activation of phospholipase A2 and independence from the Na+/H+ exchanger

The protein kinase C activators phorbol myristate acetate (PMA), mezerein, oleoylacetylglycerol, and (-)-indolactam V, although without direct effect on arachidonic acid release, greatly enhance the release of platelet arachidonic acid caused by the Ca2+ ionophores A23187 and ionomycin. In contrast, 4 alpha-phorbol 12,13-didecanoate and (+)-indolactam V, which lack the ability to activate kinase C, do not potentiate arachidonate release. Release of arachidonic acid occurs without activation of phospholipase C and is therefore mediated by phospholipase A2. Synergism between PMA and A23187 is not affected by inactivation of the Na+/H+ exchanger with dimethylamiloride. The time course and dose-response for the effect of PMA at 23 degrees C closely correlate with the phosphorylation of a set of relatively "slowly" phosphorylated proteins (P20, P35, P41, P60), but not the rapidly phosphorylated P47 protein. P20 is myosin light chain, and P41 is probably Gi alpha, but the other proteins have not been positively identified. Depletion of metabolic ATP stores by antimycin A plus 2-deoxyglucose abolishes both protein phorphorylation and the potentiation of arachidonate release by PMA, but does not prevent fatty acid release by the ionophores. Similarly, the kinase C inhibitors H-7 and staurosporine produce, respectively, partial and complete inhibition of PMA-potentiated arachidonic acid release and protein phosphorylation, without affecting the direct response to ionophores. These results indicate that protein phosphorylation, mediated by kinase C, promotes the phospholipase A2 dependent release of arachidonic acid in platelets when intracellular Ca2+ is elevated by Ca2+ ionophores.