Phosphorylation-dependent regulation of phospholipase A2 by G-proteins and Ca2+ in HL60 granulocytes.

We studied the regulation of arachidonic acid (AA) release by guanosine 5'-O-(3-thiotriphosphate (GTP gamma S) and Ca2+ in electropermeabilized HL60 granulocytes. Stimulation of AA release by GTP gamma S and Ca2+ was mediated by phospholipase A2 (PLA2) and required the presence of MgATP (EC50: 100-250 microM). The nucleotide effects were Ca(2+)-dependent (maximal effects detected at 1 microM free cation). UTP and ATP gamma S, which stimulate AA release in intact HL60 granulocytes with potencies and efficacies similar to those of ATP, were ineffective in supporting the effects of GTP gamma S in electropermeabilized cells. Pretreatment with pertussis toxin affected stimulation of AA release by ATP in intact cell, without altering the nucleotide effects in permeabilized cells. We observed the protein kinase C-dependent phosphorylation of PLA2 in permeabilized HL60 granulocytes, together with a correlation between the effects of phorbol esters and staurosporine on this reaction and on AA release. ATP-independent activation of PLA2 by GTP gamma S and/or Ca2+ was measured in subcellular fractions prepared from HL60 granulocytes. These data appear consistent with a model in which PLA2 activity in resting HL60 granulocytes is subjected to an inhibitory constraint that prevents its activation by Ca2+ and G-proteins. Removal of this constraint, either by the protein kinase C-dependent phosphorylation of the enzyme in vivo or physical disruption of the regulatory assembly (e.g. by N2 cavitation), allows its activation by Ca2+ and G-proteins.     

Regulation of phospholipase D in HL-60 granulocytes. Activation by phorbol esters, diglyceride, and calcium ionophore via protein kinase- independent mechanisms

It has recently been demonstrated that the chemotactic peptide N-formyl-Met-Leu-Phe activates phospholipase D (PLD) in dimethyl sulfoxide-differentiated HL-60 granulocytes to produce phosphatidic acid (PA) and, in the presence of ethanol, phosphatidylethanol (PEt) (Pai, J.-K., Siegel, M. I., Egan, R. W., and Billah, M. M. (1988) J. Biol. Chem. 263, 12472-12477). We now report that biologically active phorbol esters, a cell-permeable diacylglycerol, 1-oleoyl-2-acetylglycerol (OAG), and calcium ionophore A23187 are also potent inducers of PLD in these HL-60 granulocytes. HL-60 granulocytes have been selectively labeled in 1-O-alkyl-2-acyl-sn-glycero-3-phosphocholine (alkyl-PC) with 32P by incubating the cells with alkyl-[32P]lyso-phosphatidylcholine (PC). When these labeled cells are treated with phorbol 12-myristate 13-acetate (PMA), phorbol 12,13-dibutyrate, OAG, or A23187, alkyl-[32P]PA is formed. Because cellular ATP has not been labeled with 32P, the formation of alkyl-[32P]PA conclusively demonstrates PLD activation by these agents. In the presence of 0.5% ethanol, phorbol esters, OAG, and A23187 also induce formation of alkyl-[32P]PEt, demonstrating that the activated PLD catalyzes transphosphatidylation between the phosphatidyl moiety of the alkyl-[32P]PC and ethanol. Formation of alkyl-[32P]PA and alkyl-[32P]PEt in response to these various agents occurs in a time- and dose-dependent manner and exhibits differential Ca2+ requirements. Based on experiments with both [3H]alkyl-PC and alkyl-[32P]PC, it is concluded that alkyl-PA and alkyl-PEt formed in response to PMA, OAG, or A23187 are derived exclusively from PLD action on alkyl-PC. Furthermore, subthreshold concentrations of PMA (0.5-2.0 nM) or OAG (1.0-25 microM) combined with subthreshold levels of A23187 (15-60 nM) induce the formation of alkyl-[32P]PA and alkyl-[32P]PEt, suggesting that receptor-mediated activation of PLD might involve cooperative interactions between Ca2+ and diglyceride. Although PLD is activated by agents that also activate protein kinase C, the protein kinase C inhibitor, K252a, inhibits PMA-induced protein phosphorylation but causes only partial inhibition of PLD activation. We conclude that phorbol esters, OAG, and A23187 activate PLD in HL-60 granulocytes via protein kinase-independent as well as protein kinase-dependent mechanisms.     

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.     

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.     

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.     

The role of Ca2+ and Ca2+-activated phospholipid-dependent protein kinase in degranulation of human neutrophils

The degranulation reactions of human neutrophils induced by 1-oleoyl-2-acetylglycerol (OAG), phorbol 12-myristate 13-acetate (PMA), and calcium ionophore A23187 or their combinations, were studied. OAG in the absence of the Ca2+-ionophore A23187 stimulated the releases of both lysozyme and lactoferrin, constituents of the specific granules, but did not stimulate the release of beta-glucuronidase, an enzyme of the azurophil granules. Electron microscopy revealed a selective decrease in the numbers of the specific granules in this case. The combined effects of A23187 at a concentration higher than 0.1 microM and OAG were essentially additive. W-7, known to be an inhibitor of both Ca2+-activated phospholipid-dependent protein kinase (C-kinase) and calmodulin, inhibited the degranulation induced by OAG or PMA, while it inhibited the reaction induced by A23187 less markedly. The release of lysozyme reached a plateau at about 0.1 microM A23187 and increased again at higher concentrations of A23187. The observations suggest that degranulation can be induced by the activation of the C-kinase, and the degranulation by A23187 at low concentrations may be due to the activation of the C-kinase; the effects of A23187 at high concentrations, however, could not be explained only in terms of the activation of the C-kinase.     

Chemotactic peptide, calcium and guanine nucleotide regulation of phospholipase C activity in membranes from DMSO-differentiated HL60 cells

Membranes prepared from DMSO-differentiated HL60 cells labeled with [3H]inositol hydrolyze polyphosphoinositides in a Ca2+-dependent manner, generating inositol 1,4-bisphosphate (IP2) and inositol 1,4,5-trisphosphate (IP3). Incubation of membranes with GTP or GTP gamma S reduces the concentration of Ca2+ required for activation. This nucleotide effect is potentiated by formyl-Met-Leu-Phe (FMLP). Pertussis toxin inhibits FMLP-induced augmentation, but not the induction of IP2/IP3 formation by GTP or GTP gamma S. These results suggest that differentiated HL60 cells contain a membrane-associated phospholipase C that degrades polyphosphoinositides and that activation of this enzyme is mediated by at least two guanine nucleotide binding proteins, one of which is linked to FMLP receptors and is pertussis toxin sensitive.     

fMLP-induced arachidonic acid release in db-cAMP-differentiated HL-60 cells is independent of phosphatidylinositol-4, 5-bisphosphate-specific phospholipase C activation and cytosolic phospholipase A(2) activation

In inflammatory cells, agonist-stimulated arachidonic acid (AA) release is thought to be induced by activation of group IV Ca(2+)-dependent cytosolic phospholipase A(2) (cPLA(2)) through mitogen-activated protein kinase (MAP kinase)- and/or protein kinase C (PKC)-mediated phosphorylation and Ca(2+)-dependent translocation of the enzyme to the membrane. Here we investigated the role of phospholipases in N-formylmethionyl-l-leucyl-l-phenylalanine (fMLP; 1 nM-10 microM)-induced AA release from neutrophil-like db-cAMP-differentiated HL-60 cells. U 73122 (1 microM), an inhibitor of phosphatidyl-inositol-4,5-biphosphate-specific phospholipase C, or the membrane-permeant Ca(2+)-chelator 1, 2-bis?2-aminophenoxy?thane-N,N,N',N'-tetraacetic acid (10 microM) abolished fMLP-mediated Ca(2+) signaling, but had no effect on fMLP-induced AA release. The protein kinase C-inhibitor Ro 318220 (5 microM) or the inhibitor of cPLA(2) arachidonyl trifluoromethyl ketone (AACOCF(3); 10-30 microM) did not inhibit fMLP-induced AA release. In contrast, AA release was stimulated by the Ca(2+) ionophore A23187 (10 microM) plus the PKC activator phorbol myristate acetate (PMA) (0.2 microM). This effect was inhibited by either Ro 318220 or AACOCF(3). Accordingly, a translocation of cPLA(2) from the cytosol to the membrane fraction was observed with A23187 + PMA, but not with fMLP. fMLP-mediated AA release therefore appeared to be independent of Ca(2+) signaling and PKC and MAP kinase activation. However, fMLP-mediated AA release was reduced by approximately 45% by Clostridium difficile toxin B (10 ng/ml) or by 1-butanol; both block phospholipase D (PLD) activity. The inhibitor of phosphatidylcholine-specific phospholipase C (PC-PLC), D609 (100 microM), decreased fMLP-mediated AA release by approximately 35%. The effect of D609 + 1-butanol on fMLP-induced AA release was additive and of a magnitude similar to that of propranolol (0.2 mM), an inhibitor of phosphatidic acid phosphohydrolase. This suggests that the bulk of AA generated by fMLP stimulation of db-cAMP-differentiated HL-60 cells is independent of the cPLA(2) pathway, but may originate from activation of PC-PLC and PLD.     

Failure of 1-oleoyl-2-acetylglycerol to mimic the cell-differentiating action of 12-O-tetradecanoylphorbol 13-acetate in HL-60 cells

Treatment of human promyelocytic leukemia cells (HL-60 cells) with 12-O-tetradecanoylphorbol 13-acetate (TPA) results in terminal differentiation of the cells to macrophage-like cells. Treatment of the cells with TPA induced marked enhancement of the phosphorylation of 28- and 67-kDa proteins and a decrease in that of a 75-kDa protein. When the cells were treated with diacylglycerol, i.e. 50 micrograms/ml 1-oleoyl-2-acetylglycerol (OAG), similar changes in the phosphorylation of 28-, 67-, and 75-kDa proteins were likewise observed, indicating that OAG actually stimulates protein kinase C in intact HL-60 cells. OAG (1-100 micrograms/ml), which we used, activated partially purified mouse brain protein kinase C in a concentration-dependent manner. Treatment of HL-60 cells with 10 nM TPA for 48 h caused an increase by about 8-fold in cellular acid phosphatase activity. Although a significant increase in acid phosphatase activity was induced by OAG, the effect was scant compared to that of TPA (less than 7% that of TPA). After 48-h exposure to 10 nM TPA, about 95% of the HL-60 cells adhered to culture dishes. On the contrary, treatment of the cells either with OAG (2-100 micrograms/ml) or phospholipase C failed to induce HL-60 cell adhesion. Ca2+ ionophore A23187 failed to act synergistically with OAG. In addition, hourly or bi-hourly cumulative addition of OAG for 24 h also proved ineffective to induce HL-60 cell adhesion. Our present results do not imply that protein kinase C activation is nonessential for TPA-induced HL-60 cell differentiation, but do demonstrate that protein kinase C activation is not the sole event sufficient to induce HL-60 cell differentiation by means of this agent.     

Increase of [Ca(2+)]i and release of arachidonic acid via activation of M2 receptor coupled to Gi and rho proteins in oesophageal muscle

We have previously shown that acetylcholine-induced contraction of oesophageal circular muscle depends on activation of phosphatidylcholine selective phospholipase C and D, which result in formation of diacylglycerol, and of phospholipase 2 which produces arachidonic acid. Diacylglycerol and arachidonic acid interact synergistically to activate protein kinase C. We have therefore investigated the relationship between cytosolic Ca(2+) and activation of phospholipase A(2) in response to acetylcholine-induced stimulation, by measuring the intracellular free Ca(2+) ([Ca(2+)]i), muscle tension, and [3H] arachidonic acid release. Acetylcholine-induced contraction was associated with increased [Ca(2+)]i and arachidonic acid release in a dose-dependent manner. In Ca(2+)-free medium, acetylcholine did not produce contraction, [Ca(2+)]i increase, and arachidonic acid release. In contrast, after depletion of Ca(2+) stores by thapsigargin (3 microM), acetylcholine caused a normal contraction, [Ca(2+)]i increase and arachidonic acid release. The increase in [Ca(2+)]i and arachidonic acid release were attenuated by the M2 receptor antagonist methoctramine, but not by the M3 receptor antagonist p-fluoro-hexahydro siladifenidol. Increase in [Ca(2+)]i and arachidonic acid release by acetylcholine were inhibited by pertussis toxin and C3 toxin. These findings indicate that contraction and arachidonic acid release are mediated through muscarinic M2 coupled to Gi or rho protein activation and Ca(2+) influx. Acetylcholine-induced contraction and the associated increase in [Ca(2+)]i and release of arachidonic acid were completely reduced by the combination treatment with a phospholipase A(2) inhibitor dimethyleicosadienoic acid and a phospholipase D inhibitor pCMB. They increased by the action of the inhibitor of diacylglycerol kinase R59949, whereas they decreased by a protein kinase C inhibitor chelerythrine. These data suggest that in oesophageal circular muscle acetylcholine-induced [Ca(2+)]i increase and arachidonic acid release are mediated through activation of M2 receptor coupled to Gi or rho protein, resulting in the activation of phospholipase A(2) and phospholipase D to activate protein kinase C.     

Evidence for the calcium-dependent activation of phospholipase D in thrombin-stimulated human erythroleukaemia cells.

Human erythroleukaemia (HEL) cells were exposed to thrombin and other platelet-activating stimuli, and changes in radiolabelled phospholipid metabolism were measured. Thrombin caused a transient fall in PtdInsP and PtdInsP2 levels, accompanied by a rise in diacylglycerol and phosphatidic acid, indicative of a classical phospholipase C/diacylglycerol kinase pathway. However, the rise in phosphatidic acid preceded that of diacylglycerol, which is inconsistent with phospholipase C/diacylglycerol kinase being the sole source of phosphatidic acid. In the presence of ethanol, thrombin and other agonists (platelet-activating factor, adrenaline and ADP, as well as fetal-calf serum) stimulated the appearance of phosphatidylethanol, an indicator of phospholipase D activity. The Ca2+ ionophore A23187 and the protein kinase C activator phorbol myristate acetate (PMA) also elicited phosphatidylethanol formation, although A23187 was at least 5-fold more effective than PMA. Phosphatidylethanol production stimulated by agonists or A23187 was Ca2(+)-dependent, whereas that with PMA was not. These result suggest that phosphatidic acid is generated in agonist-stimulated HEL cells by two routes: phospholipase C/diacylglycerol kinase and phospholipase D. Activation of the HEL-cell phospholipase D in response to agonists may be mediated by a rise in intracellular Ca2+.     

Phospholipase A2 activation in human neutrophils. Differential actions of diacylglycerols and alkylacylglycerols in priming cells for stimulation by N-formyl-Met-Leu-Phe

Both 1,2-diacyl- and 1-O-alkyl-2-acylglycerols are formed during stimulation of human neutrophils (PMN), and both can prime respiratory burst responses for stimulation by the chemotactic peptide, N-formyl-Met-Leu-Phe (fMLP); however, mechanisms of priming are unknown. Arachidonic acid (AA) release through phospholipase A2 activation and metabolism by 5-lipoxygenase are important activities of PMN during inflammation and could be involved in the process of primed stimulation. Therefore, we have examined the ability of diacyl- and alkylacylglycerols to act as priming agents for AA release and metabolism in human neutrophils. After prelabeling PMN phospholipids with [3H]AA, priming was tested by incubating human PMN with the diacylglycerol, 1-oleoyl-2-acetylglycerol (OAG), or its alkylacyl analog, 1-O-delta 9-octadecenyl-2-acetylglycerol (EAG) before stimulating with fMLP. fMLP (1 microM), OAG (20 microM), or EAG (20 microM) individually caused little or no release of labeled AA. However, after priming PMN with the same concentrations of either OAG or EAG, stimulation with 1 microM fMLP caused rapid (peak after 1 min) release of 6-8% of [3H]AA from cellular phospholipids; total release was similar with either diglyceride. Priming cells with OAG also enhanced conversion of released AA to leukotriene B4 (LTB4) and 5-hydroxyeicosatetraenoic acid (5-HETE) upon subsequent fMLP stimulation, but AA metabolites were not increased in EAG-primed PMN. If fMLP was replaced with the calcium ionophore A23187 (which directly causes release of AA and production of LTB4 and 5-HETE), priming by both diglycerides again enhanced release of [3H]AA, but only OAG priming increased lipoxygenase activity. Indeed, EAG pretreatment markedly reduced LTB4 and 5-HETE production. Thus, both diglycerides prime release of AA from membrane phospholipids but have opposite actions on the subsequent metabolism of AA.     

Phorbol esters and oleoyl acetoyl glycerol enhance release of arachidonic acid in platelets stimulated by Ca2+ ionophore A23187

Washed human platelets prelabeled with [14C]arachidonic acid and then exposed to the Ca2+ ionophore A23187 mobilized [14C]arachidonic acid from phospholipids and formed 14C-labeled thromboxane B2, 12-hydroxy-5-8,10-heptadecatrienoic acid, and 12-hydroxy-5,8,10,14-eicosatetraenoic acid. Addition of phorbol myristate acetate (PMA) by itself at concentrations from 10 to 1000 ng/ml did not release arachidonic acid or cause the formation of any of its metabolites, nor did it affect the metabolism of exogenously added arachidonic acid. When 1 microM A23187 was added to platelets pretreated with 100 ng of PMA/ml for 10 min, the release of arachidonic acid, and the amount of all arachidonic acid metabolites formed, were greatly increased (average 4.1 +/- 0.5-fold in eight experiments). This effect of PMA was mimicked by other stimulators of protein kinase C, such as phorbol dibutyrate and oleoyl acetoyl glycerol, but not by 4-alpha-phorbol 12,13-didecanoate, which does not stimulate protein kinase C. However, phosphorylation of the cytosolic 47-kDa protein, the major substrate for protein kinase C in platelets, was produced at lower concentrations of PMA and at a much higher rate than enhancement of arachidonic acid release by PMA, suggesting that 47-kDa protein phosphorylation is not directly involved in mobilization of the fatty acid. PMA also potentiated arachidonic acid release when stimulation of phospholipase C by the ionophore (which is due to thromboxane A2 and/or secreted ADP) was blocked by aspirin plus ADP scavengers, i.e. apyrase or creatine phosphate/creatine phosphokinase. Increased release of arachidonic acid was attributable to loss of [14C]arachidonic acid primarily from phosphatidylcholine (79%) with lesser amounts derived from phosphatidylinositol (12%) and phosphatidylethanolamine (8%). Phosphatidic acid, whose production is a sensitive indicator of phospholipase C activation, was not formed. Thus, the potentiation of arachidonic acid release by PMA appeared to be due to phospholipase A2 activity. These results suggest that diacylglycerol formed in response to stimulation of platelet receptors by agonists may cooperatively promote release of arachidonic acid via a Ca2+/phospholipase A2-dependent pathway.     

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.     

Cholecystokinin-evoked Ca(2+) waves in isolated mouse pancreatic acinar cells are modulated by activation of cytosolic phospholipase A(2), phospholipase D, and protein kinase C.

We employed confocal laser-scanning microscopy to monitor cholecystokinin (CCK)-evoked Ca(2+) signals in fluo-3-loaded mouse pancreatic acinar cells. CCK-8-induced Ca(2+) signals start at the luminal cell pole and subsequently spread toward the basolateral membrane. Ca(2+) waves elicited by stimulation of high-affinity CCK receptors (h.a.CCK-R) with 20 pM CCK-8 spread with a slower rate than those induced by activation of low-affinity CCK receptors (l.a. CCK-R) with 10 nM CCK-8. However, the magnitude of the initial Ca(2+) release was the same at both CCK-8 concentrations, suggesting that the secondary Ca(2+) release from intracellular stores is modulated by activation of different intracellular pathways in response to low and high CCK-8 concentrations. Our experiments suggest that the propagation of Ca(2+) waves is modulated by protein kinase C (PKC) and arachidonic acid (AA). The data indicate that h.a. CCK-R are linked to phospholipase C (PLC) and phospholipase A(2) (PLA(2)) cascades, whereas l.a.CCK-R are coupled to PLC and phospholipase D (PLD) cascades. The products of PLA(2) and PLD activation, AA and diacylglycerol (DAG), cause inhibition of Ca(2+) wave propagation by yet unknown mechanisms.     

Mechanisms of bombesin-induced arachidonate mobilization in Swiss 3T3 fibroblasts

A peptide mitogen bombesin, which activates the phospholipase C-protein kinase C signaling pathway, induces a mepacrine-sensitive, dose-dependent increase in the release of [3H]arachidonic acid and its metabolites ([3H]AA) from prelabeled Swiss 3T3 fibroblasts. The effect is temporally composed of two phases, i.e. an initial transient burst that is essentially independent of extracellular Ca2+, and a following sustained phase that is absolutely dependent on the extracellular Ca2+. The initial transient [3H]AA liberation occurs concomitantly with bombesin-induced 45Ca efflux from prelabeled cells: both responses being substantially attenuated by loading cells with a Ca2+ chelator quin2. However, bombesin-induced intracellular Ca2+ mobilization by itself is not sufficient as a signal for the initial transient [3H]AA liberation, since A23187 potently stimulates 45Ca efflux to an extent comparable to bombesin but fails to induce [3H]AA release in the absence of extracellular Ca2+. The second sustained phase of the bombesin-induced [3H]AA release is abolished by reducing extracellular Ca2+ to 0.03 mM, although bombesin effects on phospholipase C and protein kinase C activation are barely affected by the same procedure. A protein kinase C activator phorbol 12,13-dibutyrate induces an extracellular Ca(2+)-dependent, slowly developing sustained increase in [3H]AA release, and markedly potentiates both phases of bombesin-induced [3H]AA release. Down-regulation of cellular protein kinase C completely abolishes all of the effects of phorbol dibutyrate, and partially inhibits the second but not the first phase of bombesin-induced [3H]AA release. These results indicate that bombesin-induced receptor-mediated activation of phospholipase A2 involves multiple mechanisms, including intracellular Ca2+ mobilization for the first phase, protein kinase C activation plus Ca2+ influx for the second phase, and as yet unknown mechanism(s) independent of intracellular Ca2+ mobilization or protein kinase C for both of the phases.     

Inhibitory effect of the intrauterine infusion of phorbol 12-myristate 13-acetate and 1-oleoyl-2-acetylglycerol on the decidual cell reaction in rats

In rats, prostaglandins (PGs) have an essential role in the decidual cell reaction (DCR), but their mechanism of action at the cellular level within the endometrium is at present uncertain. To test the hypothesis that both protein kinase C activation and calcium mobilization mediate the action of PGs within the endometrium during decidualization, the phorbol ester phorbol 12-myristate 13-acetate (PMA) or the synthetic diacylglycerol 1-oleoyl-2-acetyl-glycerol (OAG), activators of protein kinase C in vitro, and the calcium ionophore A23187, which causes calcium mobilization, were infused, alone or combined, into the uterine lumen of rats sensitized for the DCR. The results obtained indicate that both PMA and OAG have an inhibitory effect on the DCR in rats. The calcium ionophore A23187, although having no apparent effect by itself, had a synergistic effect with PMA, but not with OAG, in inhibiting the DCR. The intrauterine infusion of PMA and/or A23187 had no effect on the increase in endometrial vascular permeability (EVP), which precedes the DCR. The inhibitory effect of PMA or PMA plus A23187 on decidualization is probably not mediated by a decrease in uterine PG synthesis, as assessed by the measurement of uterine prostaglandin E concentrations at various times during the intraluminal infusion. These data suggest that activation of protein kinase C can modulate the DCR.     

Protein kinase C activation amplifies prostaglandin F2 alpha-induced prostaglandin E2 synthesis in osteoblast-like cells.

In cloned osteoblast-like cells, MC3T3-E1, prostaglandin F2 alpha (PGF2 alpha) stimulated arachidonic acid (AA) release in a dose-dependent manner in the range between 1 nM and 10 microM. 12-O-tetradecanoylphorbol-13-acetate (TPA), a protein kinase C (PKC) activator, which by itself had little effect on AA release, markedly amplified the release of AA stimulated by PGF2 alpha in a dose-dependent manner. 4 alpha-phorbol 12,13-didecanoate, a phorbol ester which is inactive for PKC, showed little effect on the PGF2 alpha-induced AA release. 1-oleoyl-2-acetylglycerol (OAG), a specific activator for PKC, mimicked TPA by enhancement of the AA release induced by PGF2 alpha. H-7, a PKC inhibitor, markedly suppressed the effect of OAG on PGF2 alpha-induced AA release. Quinacrine, a phospholipase A2 inhibitor, showed partial inhibitory effect on PGF2 alpha-induced AA release, while it suppressed the amplification by OAG of PGF2 alpha-induced AA release almost to the control level. Furthermore, TPA enhanced the AA release induced by melittin, known as a phospholipase A2 activator. On the other hand, TPA inhibited the formation of inositol trisphosphate stimulated by PGF2 alpha. Under the same condition, PGF2 alpha indeed stimulated prostaglandin E2 (PGE2) synthesis and TPA markedly amplified the PGF2 alpha-induced PGE2 synthesis as well as AA release. These results indicate that the activation of PKC amplifies PGF2 alpha-induced both AA release and PGE2 synthesis through the potentiation of phospholipase A2 activity in osteoblast-like cells.     

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.     

Pertussis toxin-sensitive GTP-binding proteins may regulate phospholipase A2 in response to thrombin in rabbit platelets

Incubation of rabbit platelets with thrombin resulted in rapid accumulations of inositol trisphosphate (IP3) in [3H]inositol-labeled platelets, increases of [3H]arachidonic acid [( 3H]AA) release, and [3H]serotonin secretion from the platelets prelabeled with these labeled compounds. The experiments using phospholipase A2 or C inhibitor suggested that not only phospholipase C but also phospholipase A2 activity plays an important role in serotonin secretion. We then studied the regulatory mechanisms of phospholipase A2 activity. Guanosine 5'-(3-O-thio)triphosphate (GTP gamma S), guanyl-5'-(beta,gamma-iminio)triphosphate), or AlF4- caused a significant liberation of AA in digitonin-permeabilized platelets but not in intact platelets. Thrombin-stimulated AA release was not observed in permeabilized platelets, whereas thrombin acted synergistically with GTP or GTP analogs to stimulate AA release. GTP analog-stimulated AA release was inhibited by guanosine 5'-(2-O-thio)diphosphate) and was also inhibited by decreased Mg2+ concentrations. Thrombin-induced, GTP-dependent AA release, but not IP3 formation, was diminished by 100 ng/ml of pertussis toxin, associated with ADP-ribosylation of membrane 41-kDa protein(s). Thrombin-stimulated AA release from intact platelets and GTP gamma S-stimulated release from permeabilized platelets were both markedly dependent on Ca2+. However, Ca2+ addition could not enhance AA release without GTP gamma S even when Ca2+ was increased up to 10(-4) M in permeabilized platelets. The results show that thrombin-stimulated AA release from rabbit platelets is mainly mediated by phospholipase A2 activity, not by phospholipase C activity, and that Ca2+ is an important factor to the activation of phospholipase A2 but is not the sole factor to the regulation. GTP-binding protein(s) is involved in receptor-mediated activation of phospholipase A2.