Phorbol ester stimulates amylase secretion from rat parotid cells

Phorbol myristate acetate (PMA), a potent activator of Ca2+- and phospholipid-dependent protein kinase (protein kinase C), evoked amylase release from rat parotid cells. In dose-response studies, PMA stimulated amylase release independently of db-cAMP, but potentiated the effect of carbachol. PMA and A23187, a Ca2+ ionophore, synergistically increased amylase release. The maximum effect of carbachol was further enhanced by PMA but not by A23187, suggesting that protein kinase C is not fully activated by the muscarinic-cholinergic agonist under the condition where calcium is fully utilized for amylase secretion.     

Epidermal growth factor is synergistic with phorbol esters and vasopressin in stimulating arachidonate release and prostaglandin production in renal glomerular mesangial cells.

Epidermal growth factor (EGF) is produced in large quantities by the kidney. We identified EGF-binding sites on cultured rat renal glomerular mesangial cells. These cells serve as a model system for the investigation of renal prostaglandin biosynthesis. Since EGF has been shown to modulate phospholipase activity in other cell lines, we studied the ability of EGF to increase arachidonate release and prostaglandin E2 (PGE2) production in mesangial cells. We found that EGF stimulated arachidonate release and PGE2 production in the presence of the Ca2+ ionophore A23187. This stimulation was markedly potentiated by the addition of phorbol myristate acetate (PMA), which activates protein kinase C. However, down-regulation of protein kinase C by prolonged PMA treatment did not block the ability of EGF to stimulate PGE2 production in the presence of A23187. EGF also markedly potentiated the stimulation of PGE2 production by vasopressin, which increases intracellular Ca2+ and activates protein kinase C in these cells. The stimulatory effects of EGF were not the result of prolongation or enhancement of an increase in intracellular Ca2+ produced by ionophore or vasopressin. Furthermore, the synergistic interaction of EGF with PMA and vasopressin occurred despite the fact that these agents markedly decreased EGF binding in mesangial cells, presumably owing to protein-kinase-C-mediated phosphorylation of the EGF receptor. We conclude that there exists a distinct pathway for EGF-stimulated arachidonate release and PGE2 production in rat renal glomerular mesangial cells, which is synergistic with, but not dependent on, activation of protein kinase C. In contrast with long-term mitogenic responses to EGF, this rapid response may allow delineation of the membrane phospholipid changes and signalling steps involved in this aspect of EGF action.     

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.     

Phorbol esters reduce gonadotrope responsiveness to protein kinase C activators but not to Ca2+-mobilizing secretagogues. Does protein kinase C mediate gonadotropin-releasing hormone action?

The demonstration that activators of the Ca2+-activated, phospholipid-dependent protein kinase (protein kinase C), such as phorbol esters and diacylglycerols, can provoke luteinizing hormone (LH) release from pituitary gonadotropes, suggests a possible role for protein kinase C in stimulus-release coupling. We now report that administration of phorbol myristate acetate (PMA) to pituitary cell cultures causes a sustained reduction in Triton X-100-extracted protein kinase C activity. Further, phorbol ester- and diacylglycerol-stimulated LH release, as well as inhibition by PMA of gonadotropin-releasing hormone (GnRH)-stimulated inositol phosphate production, were reduced by pretreatment with PMA. The effects of phorbol ester pretreatment on PMA-stimulated LH release and protein kinase C activity were dose-dependent, sustained (greater than or equal to 24 h) and specific (no measurable effect with 4 alpha-phorbol didecanoate). The effect on PMA-stimulated LH release was apparently Ca2+-independent. In pituitary cell cultures with reduced protein kinase C activity, the gonadotropes have reduced responsiveness to PMA but release a similar proportion of cellular LH in response to Ca2+-mobilizing secretagogues (GnRH and A23187) as do control cells. The normal responsiveness to GnRH of cells with reduced responsiveness to protein kinase C activators calls into question the requirement for this enzyme for GnRH-stimulated LH release.     

Protein kinase C mediated regulation of calcium channels in PC-12 pheochromocytoma cells

Depolarization of PC-12 pheochromocytoma cells with K+ produces an immediate increase in catecholamine release. The stimulation of release is blocked by Co2+, removal of extracellular Ca2+ or by dihydropyridine drugs such as nitrendipine. Release is enhanced by other dihydropyridines such as BAY K8644. Release is accompanied by a voltage dependent uptake of 45Ca2+ which is also blocked by Co2+ or nitrendipine and enhanced by BAY K8644. The phorbol ester phorbol 12-myristate-13-acetate (TPA) in the range 10(-9)-10(-6) M produced little effect by itself but augmented the K+ evoked release of catecholamine. An analog of TPA which does not activate protein kinase C was ineffective. In contrast, TPA in the same concentration range blocked influx of 45Ca2+ induced by 70 mM K+ or 70 mM K+/BAY K8644. 45Ca2+ influx produced by A23187 was not blocked by TPA. The results suggest a system by which protein kinase C may regulate the output of transmitters from secretory cells.     

Comparative study on the stimulation of superoxide production in guinea-pig eosinophils by the calcium ionophore A23187

The effect of calcium and/or magnesium on O2- production by guinea-pig eosinophils stimulated by the calcium ionophore A23187 was studied in comparison to neutrophils. In the absence of calcium, A23187 did not stimulate O2- production in resting eosinophils and neutrophils, regardless of the presence of extracellular magnesium. The A23187-induced O2- production by both cells increased linearly with extracellular Ca2+ concentrations. However, the concentration of Ca2+ required for maximum O2- production in eosinophils was about 10-times lower than that required of neutrophils. The addition of Mg2+ strongly inhibited O2- production, especially in eosinophils at low Ca2+ concentrations. The intracellular Ca2+ concentration was lower in eosinophils than in neutrophils in the resting state, and the enhancement of the intracellular Ca2+ concentration in response to A23187 was much lower in eosinophils than in neutrophils. The activation of the NADPH-dependent O2(-)-forming enzyme (NADPH oxidase) in eosinophils depended on extracellular calcium, as observed in O2- production. However, the NADPH oxidase activity in the particulate fraction from A23187-stimulated eosinophils was only slightly affected by the addition of divalent cations or EDTA. The compound W-7 (N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride), a calmodulin antagonist, significantly inhibited O2- production by both cells. On the other hand, the compound H-7 (1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride), a protein kinase C antagonist, was less effective on O2- production than was W-7. H-7 had little effect on O2- production of eosinophils. These findings suggest that NADPH oxidase might be activated by a smaller Ca2+ concentration through the calmodulin-dependent reaction. This was not observed with protein kinase C, at least in eosinophils.     

Pretreatment with phorbol esters abrogates mast cell adenosine responsiveness

Adenosine potentiates preformed mediator release from mouse bone marrow-derived mast cells stimulated with specific Ag or the calcium ionophore A23187. When these mast cells were cultured for 30 to 120 min with the phorbol ester PMA (10(-8) or 10(-7) M), protein kinase C activity was increased and Ag-stimulated beta-hexosaminidase release was modestly inhibited, whereas A23187-stimulated release was synergistically enhanced. However, in both cases, exogenous adenosine failed to augment beta-hexosaminidase release. Overnight PMA exposure produced a decrease in protein kinase C activity and a decrease in both Ag- and A23187-stimulated preformed mediator release, as well as a lack of responsiveness to adenosine. This hyporesponsiveness could be reversed by 24 h after washing the cells free of PMA. The generation of the arachidonic acid metabolite leukotriene C4 was not altered by mast cell PMA exposure. The ability of adenosine to increase intracellular cAMP concentrations was modestly blunted by high doses of PMA, and PMA abrogated the increase in intracellular free calcium levels usually observed in cells stimulated with Ag in the presence of 10(-5) M adenosine. PMA exposure induces a hyporesponsiveness to adenosine in mast cells, either by a direct effect on protein kinase C activity and/or by an effect on adenosine receptor expression or recycling.     

Synergistic signals in the mechanism of antigen-induced exocytosis in 2H3 cells: evidence for an unidentified signal required for histamine release

The aim of this study was to determine whether the increase in cytosolic free Ca2+ concentration ([Ca2+]i) in response to antigen (aggregated ovalbumin) on IgE-primed 2H3 cells was sufficient to account for exocytosis. When the [Ca2+]i responses to antigen and the Ca2+ ionophore A23187 were compared, A23187 was much less effective at releasing histamine at equivalent [Ca2+]i increases, and little or no stimulated histamine release occurred with A23187 concentrations that matched the [Ca2+]i response to antigen concentrations that stimulated maximal histamine release. The [Ca2+]i response to antigen is not, therefore, sufficient to account for exocytosis, although extracellular Ca2+ is necessary to initiate both the [Ca2+]i response and histamine release: the antigen must generate an additional, unidentified, signal that is required for exocytosis. To determine whether this signal was the activation of protein kinase C, the effects of the phorbol ester 12-0-tetradecanoyl phorbol 13-acetate (TPA) on the responses to antigen were examined. TPA blocked the antigen-induced [Ca2+]i response and the release of inositol phosphates but had little effect on histamine release and did not stimulate exocytosis by itself. The unidentified signal from the antigen is therefore distinct from the activation of protein kinase C and is generated independently of the [Ca2+]i response or the release of inositol phosphates. Taken together with other data that imply that there is very little activation of protein kinase C by antigen when the rate of histamine release is maximal, it is concluded that the normal exocytotic response to antigen requires the synergistic action of the [Ca2+]i signal together with an unidentified signal that is not mediated by protein kinase C.     

Studies of protein kinase C in the rat basophilic leukemia (RBL-2H3) cell reveal that antigen-induced signals are not mimicked by the actions of phorbol myristate acetate and Ca2+ ionophore

Exogenous activators of protein kinase C such as PMA in combination with a Ca2+ ionophore (A23187), cause secretion in rat basophilic (RBL-2H3) cells,but they do so through stimulatory signals that are not the same as those generated by Ag or oligomers of IgE. On the one hand, the synergy between PMA and A23187 and the suppression of Ag-mediated signals (hydrolysis of inositol phospholipids and rise in concentration of cytosolic Ca2+) by PMA were totally dependent on protein kinase C. The loss of synergistic and inhibitory actions of PMA, for example, correlated with the loss of protein kinase C (as determined by immunoblotting techniques) when cells were continuously exposed to PMA. Furthermore, the permeabilization of RBL-2H3 cells resulted in the loss of both protein kinase C and the inhibitory action of PMA, but both were retained if cells were exposed to PMA before permeabilization Ag-induced secretion, on the other hand, was not as dependent on the presence of protein kinase C. The potent inhibitor of this enzyme, staurosporine, which blocked completely the secretory response to the combination of PMA and A23187, did not inhibit Ag-induced secretion except at concentrations (greater than 10 nM) that inhibited Ag-stimulated hydrolysis of inositol phospholipids as well. Also RBL-2H3 cells still showed some secretory-response (approximately 25% of normal) to Ag when cells were depleted (greater than 98%) of protein kinase C by prolonged treatment with PMA. Previous studies have indicated that the secretory response to PMA and A23187 is much lower than that elicited by Ag when the concentrations of stimulants were matched to give the same increase in concentrations of cytosolic Ca2+.     

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.     

Protein kinase C: subcellular redistribution by increased Ca2+ influx. Evidence that Ca2+-dependent subcellular redistribution of protein kinase C is involved in potentiation of beta-adrenergic stimulation of pineal cAMP and cGMP by K+ and A23187

Phenylephrine is known to stimulate translocation of protein kinase C in rat pinealocytes (Sugden, D., Vanecek, J., Klein, D.C., Thomas, T.P., and Anderson, W. B. (1985) Nature 314, 359-361). In the present study, the receptor mediating this effect was found to belong to the alpha 1-adrenoceptor subclass. Activation of this receptor is also known to produce a sustained increase in [Ca2+]i by increasing net influx (Sugden, A. L., Sugden, D., and Klein, D. C. (1985) J. Biol. Chem. 261, 11608-11612), which points to the possible importance of Ca2+ influx in the subcellular redistribution (activation) of protein kinase C in intact cells. This possibility was investigated by reducing extracellular Ca2+ ((Ca2+]o) with EGTA or by inhibiting Ca2+ influx with inorganic Ca2+ blockers. These treatments reduced alpha 1-adrenoceptor-mediated translocation of protein kinase C. This suggested that elevation of Ca2+ influx alone triggers activation of protein kinase C. In support of this, it was found that treatments which elevate Ca2+ influx, including increased extracellular K+ and addition of the Ca2+ ionophore A23187, cause redistribution of protein kinase C. The effect of K+ was blocked by nifedipine and that of A23187 by EGTA, indicating that effects of these agents are Ca2+-dependent. The possible role of phospholipase C activation in these effects was examined by measuring the formation of [3H]diacylglycerol by cells labeled with [3H]arachidonic acid. Although [3H]diacylglycerol formation was easily detected in the presence or absence of an effective concentration of an inhibitor of diacylglycerol kinase, none of the agents which cause rapid translocation of protein kinase C were found to cause a rapid increase in the generation of [3H]diacylglycerol. These findings establish that an increase in Ca2+ influx is sufficient to trigger translocation of protein kinase C. In addition, we found that a very close correlation exists between translocation of protein kinase C by phenylephrine, K+, and A23187 and their ability to potentiate beta-adrenergic stimulation of cAMP and cGMP accumulation. This provides strong support to the proposal that translocation of protein kinase C is required for potentiation of beta-adrenergic stimulation of pinealocyte cAMP and cGMP accumulation.     

Stimulation of dopamine synthesis and activation of tyrosine hydroxylase by phorbol diesters in rat striatum

In rat striatal synaptosomes, 4 beta-phorbol 12-myristate 13-acetate (PMA) and 4 beta-phorbol 12,13-dibutyrate (PDBu), two activators of Ca2+-phospholipid-dependent protein kinase (protein kinase C) increased dopamine (DA) synthesis measured by following the release of 14CO2 from L-[1-14C] tyrosine. Maximal stimulation (21-28% increase of basal rate) was produced by 0.5 microM PMA and 1 microM PDBu. 4 beta-Phorbol and 4 beta-phorbol 13-acetate, which are not activators of protein kinase C, were ineffective at 1 microM. PMA did not change the release of 14CO2 from L-[1-14C]DOPA. Addition of 1 mM EGTA to a Ca2+-free incubation medium failed to affect PMA stimulation. KC1 (60 mM) enhanced DA synthesis by 25%. Exposure of synaptosomes to either PMA or PDBu prior to KC1 addition resulted in a more than additive increase (80-100%) of DA synthesis. A similar synergistic effect was observed when the phorbol diesters were combined with either veratridine or d-amphetamine but not with forskolin and dibutyryl cyclic AMP. Pretreatment of striatal synaptosomes with phorbol diesters produced an activation on of tyrosine hydroxylase (TH) associated with a 60% increase of the Vmax and a decrease of the Km for the pterine cofactor 6-methyl-5,6,7,8-tetrahydropterin. These results indicate that protein kinase C participates in the regulation of striatal TH in situ and that its activation may act synergistically with DA releasing agents in stimulating DA synthesis.     

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.     

Biochemical responses in activated human neutrophils mediated by protein kinase C and a Ca2+-requiring proteinase

Low concentrations of phorbol 12-myristate 13-acetate (PMA) elicit a specific response in human neutrophils, characterized by the production of oxygen radicals and the release into the medium of a membrane-bound serine proteinase (Pontremoli, S., Melloni, E., Michetti, M., Sacco, O., Sparatore, B., Salamino, F., Damiani, G. and Horecker, B. L. (1986) Proc. Natl. Acad. Sci. U. S. A., 83, 1685-1689). The following evidence indicates that this response is mediated by membrane-bound protein kinase C: 1) it is blocked by inhibitors of protein kinase C; and 2) it is enhanced in cells preloaded with leupeptin which prevents proteolysis of protein kinase C and its subsequent dissociation from the cell membrane. This response is not accompanied by significant exocytosis of granule enzymes. With higher concentrations of PMA, and more particularly on stimulation with formylmethionyl-leucyl-phenylalanine (fMLP) plus cytochalasin B, a substantial exocytosis of constituents of both specific and azurophil granules is observed. With fMLP, exocytosis of granule enzymes is the predominant event, with little production of H2O2 and negligible release of membrane-bound serine proteinase. Exocytosis promoted either by a high concentration of PMA or by fMLP is inhibited by leupeptin, indicating that it is due to the action of an intracellular Ca2+-dependent thiol proteinase (calpain), either directly or by conversion by calpain of membrane-bound protein kinase C to the soluble Ca2+/phospholipid-independent form. Intracellular mobilization of Ca2+ is also observed following stimulation with either PMA or fMLP, but only the latter results in a net increase in the intracellular concentration of free Ca2+; under these conditions maximum exocytosis of granule contents is observed.     

PMA-sensitive protein kinase C is not necessary in TRH-stimulated prolactin release from female rat primary pituitary cells.

In GH3 cells and other clonal rat pituitary tumor cells, TRH has been shown to mediate its effects on prolactin release via a rise of cytosolic Ca2+ and activation of protein kinase C. In this study, we examined the role of protein kinase C in TRH-stimulated prolactin release from female rat primary pituitary cell culture. Both TRH and PMA stimulated prolactin release in a dose-dependent manner. When present together at maximal concentrations, TRH and PMA produced an effect which was slightly less than additive. Pretreatment of rat pituitary cells with 10(-6) M PMA for 24 hrs completely down-regulated protein kinase C, since such PMA-pretreated cells did not release prolactin in response to a second dose of PMA. Interestingly, protein kinase C down-regulation had no effect on TRH-induced prolactin release from rat pituitary cells. In contrast, PMA-pretreated GH3 cells did not respond to a subsequent stimulation by either PMA or TRH. Pretreatment of rat pituitary cells with TRH (10(-7) M, 24 hrs) inhibited the subsequent response to TRH, but not PMA. Forskolin, an adenylate cyclase activator, stimulated prolactin release by itself and in a synergistic manner when incubated together with TRH or PMA. The synergistic effects of forskolin on prolactin release was greater in the presence of PMA than TRH. Down-regulation of protein kinase C by PMA pretreatment abolished the synergistic effect produced by PMA and forskolin but had no effect on those generated by TRH and forskolin. sn-1,2-Dioctanylglycerol (DOG) pretreatment attenuated the subsequent response to DOG and PMA but not TRH. The effect of TRH, but not PMA, on prolactin release required the presence of extracellular Ca2+. In conclusion, the mechanism by which TRH causes prolactin release from rat primary pituitary cells is different from that of GH3 cells; the former is a protein kinase C-independent process whereas the latter is at least partially dependent upon the activation of protein kinase C.     

Hyperoxia alters effect of calcium on rat alveolar macrophage superoxide production

The effect of hyperoxia on the Ca2+ dependence of stimulated superoxide anion radical (O2-.) production (the respiratory burst) of rat alveolar macrophages was investigated. Enhancement of the concanavalin A (con A)-stimulated respiratory burst by extracellular Ca2+ was suppressed by O2 exposure. Similarly, the inhibitory effect of verapamil on the con A-stimulated respiratory burst was reduced by O2 exposure. O2 exposure also inhibited con A stimulation that was independent of Ca2+ entry. Exposure to O2 also caused a decline in O2-. production stimulated by either A23187 or phorbol myristate acetate (PMA). With A23187 stimulation, extracellular Ca2+ was essential for either air-exposed (control) or O2-exposed cells. With PMA, stimulation was independent of extracellular Ca2+ for either air or O2-exposed macrophages and verapamil did not inhibit. Free intracellular Ca2+ concentration ([Ca2+]i) was measured in control and O2-exposed alveolar macrophages. Hyperoxic exposure did not alter [Ca2+]i in unstimulated cells. In controls, con A stimulated an immediate increase in [Ca2+]i followed by a rapid decrease and a second rise and fall. The second elevation was suppressed by verapamil or ethyleneglycol-bis (beta-aminoethylether)-N,N'-tetraacetic acid or O2 exposure. The results of both the respiratory burst assays and measurement of con A-stimulated changes in [Ca2+]i suggest that Ca2+ entry involved in stimulus-response coupling is suppressed in cellular O2 toxicity.     

Protein kinase C activity appears to be required for the synthesis of platelet-activating factor and leukotriene B4 by human neutrophils

Human neutrophils synthesize platelet-activating factor (PAF) and leukotriene B4 (LTB4) when stimulated with the Ca2+ ionophore A23187. These processes are enhanced to a variable extent by phorbol 12-myristate 13-acetate (PMA), a direct activator of protein kinase C. The long chain amines sphingosine, stearylamine (Hannun, Y.A., Loomis, C.R., Merrill, A.H., Jr., and Bell, R.M. (1986) J. Biol. Chem. 261, 12604-12609), and palmitoylcarnitine competitively inhibit activation of purified protein kinase C in vitro and inhibit protein kinase C-mediated activation of the respiratory burst in human neutrophils (Wilson, E., Olcott, M.C., Bell, R.M., Merrill, A.H., Jr., and Lambeth, J.D. (1986) J. Biol. Chem. 261, 12616-12623). These amines were found to inhibit A23187-induced PAF and LTB4 synthesis. Inhibition of PAF and LTB4 synthesis occurred in parallel; half-maximal inhibition by sphingosine occurred at 7 microM, with complete inhibition at 15 microM. PMA by itself did not induce the synthesis of PAF or LTB4, although it did enhance PAF and LTB4 synthesis at suboptimal concentrations of A23187. PMA reversed long chain amine inhibition of PAF and LTB4 accumulation. Reversal of the inhibition of PAF and LTB4 accumulation occurred in parallel, was concentration-dependent, and was complete by approximately 3 x 10(-8) M PMA. The inactive 4 alpha-phorbol didecanoate ester did not reverse inhibition at these concentrations. Sphingosine completely prevented the A23187-induced release of [3H]arachidonate and its various metabolites from [3H]arachidonate-labeled cells. PMA, but not 4 alpha-phorbol didecanoate, restored arachidonate release and its metabolism. Therefore, while activation of protein kinase C is not sufficient to induce PAF and LTB4 synthesis, its action appears to be required to couple a rise in intracellular Ca2+ to their synthesis. This coupling occurs at the level of the initial reaction in the production of lipid mediators, a phospholipase A2-like activity that mobilizes the two substrates 1-O-alkyl-sn-glycero-3-phosphocholine and arachidonic acid from complex lipids.     

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.