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.     

Evidence for a catalytic role of phospholipase A in phorbol diester- and zymosan-induced mobilization of arachidonic acid in mouse peritoneal macrophages

Inositol phospholipid degradation and release of phospholipid-bound arachidonic acid was induced in intact peritoneal macrophages by exposure to phorbol myristate acetate (PMA) or zymosan particles. PMA, known to activate protein kinase C, selectively enhanced the deacylation of phosphatidylinositol (i.e., degradation by phospholipase A), while zymosan particles enhanced degradation via both phospholipase A and inositol lipid phosphodiesterase (phospholipase C). The release of arachidonic acid was found to correlate with the degradation of phosphatidylinositol by the phospholipase A pathway and could be dissociated from the phospholipase C-catalyzed cleavage of inositol phospholipids in several experimental situations: (i) when PMA was the stimulus, (ii) by the difference in Ca2+ dependence between the two enzymatic processes when zymosan was the stimulus and (iii) by the parallel inhibition by chlorpromazine of the phospholipase A pathway and arachidonic acid release, but not inositol phospholipid phosphodiesterase. In addition, phloretin, a reported inhibitor of protein kinase C, was found to inhibit arachidonic acid release and the deacylation of phosphatidylinositol. The results are consistent with a model in which arachidonic acid release is mediated by phospholipase(s) A and in which PMA or the phosphodiesterase-catalyzed degradation of phosphoinositides causes activation of the phospholipase A pathway via protein kinase C.     

Defining the role of protein kinase c in calcium-ionophore-(A23187)-mediated activation of phospholipase A2 in pulmonary endothelium.

We sought to investigate the mechanisms by which the calcium ionophore A23187 triggers arachidonic acid release in bovine pulmonary endothelial cells and to test the hypothesis that protein kinase C is involved in this process. Our results indicate that the mechanism by which A23187 increases phospholipase A2 activity and arachidonic acid release in bovine pulmonary arterial endothelial cells depends upon the concentration studied. At concentrations of 1 microM and 2.5 microM, A23187 increases phospholipase A2 activity and arachidonic acid release without stimulating protein kinase C. At concentrations of 5-12.5 microM, A23187 increases arachidonic acid release and phospholipase A2 activity in conjunction with a dose-dependent activation of membrane-bound protein kinase C. To test the hypothesis that these doses of A23187 increase phospholipase A2 activity by stimulating protein kinase C, we studied the effect of prior treatment with the protein kinase C inhibitor sphingosine. Sphingosine inhibits the increase in phospholipase A2 activity and arachidonic acid release caused by A23187 over the range 5-12.5 microM. To investigate further the potential role of protein kinase C, we studied the effects of the inactive phorbol ester 4 alpha-phorbol 12 beta-myristate 13 alpha-acetate (4 alpha-PMA) and an active phorbol ester 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (4 beta PMA). Neither 4 alpha-PMA nor 4 beta-PMA affected basal arachidonic acid release. 4 alpha-PMA also did not augment the effects of A23187. In contrast, 4 beta-PMA significantly augments the increase in phospholipase A2 activity and arachidonic acid release caused by lower doses of A23187. Under these conditions, sphingosine completely inhibits the stimulatory effects of 4 beta-PMA on protein kinase C translocation, phospholipase A2 and arachidonic acid release. Thus, at low doses (1 microM and 2.5 microM) A23187 increases phospholipase A2 activity and arachidonic acid release by a mechanism that does not involve protein kinase C. At these A23187 doses, activating membrane-bound protein kinase C with 4 beta-PMA causes a synergistic increase in phospholipase A2 activity and arachidonic acid release. At higher doses (5-12.5 microM), A23187 acts in large part by stimulating protein kinase C translocation. Overall, our results indicate that activating membrane-bound protein kinase C by itself is an insufficient stimulus to increase phospholipase A2 activity and arachidonic acid release in pulmonary endothelial cells, but activating protein kinase C can substantially augment the increase in phospholipase A2 activity and arachidonic acid caused by a small increase in intracellular calcium.     

Protein kinase C involved in zymosan-induced release of arachidonic acid and superoxide but not in calcium ionophore-elicited arachidonic acid release or formation of prostaglandin E2 from added arachidonate.

Zymosan and phorbol ester induced in liver macrophages the release of arachidonic acid, prostaglandin E2, and superoxide; the calcium ionophore A 23187 elicited a release of arachidonic acid and prostaglandin E2 but not of superoxide, and exogenously added arachidonic acid led to the formation of prostaglandin E2 only. The zymosan- and phorbol-ester-induced release of arachidonic acid, prostaglandin E2, and superoxide was dose-dependently inhibited by staurosporine and K252a, two inhibitors of protein kinase C, and by pretreatment of the cells with phorbol ester which desensitized protein kinase C. The release of arachidonic acid or prostaglandin E2 following the addition of A 23187 or arachidonic acid was not affected by these treatments. Zymosan and phorbol ester but not A 23187 or arachidonic acid induced a translocation of protein kinase C from the cytosol to membranes in intact cells. These results demonstrate an involvement of protein kinase C in the zymosan- and phorbol-ester-induced release of arachidonic acid, prostaglandin E2, and superoxide; the release of arachidonic acid and prostaglandin E2 elicited by A 23187 and the formation of prostaglandin E2 from exogenously added arachidonic acid, however, is independent of an activation of protein kinase C.     

Protein kinase C inhibition by sphingoid long-chain bases: effects on secretion in human neutrophils

Sphingoid long-chain bases (sphinganine and sphingosine) have recently been shown to inhibit protein kinase C both in vitro [Y. Hannun et al. (1986) J. Biol. Chem. 261, 12604-12609] and in intact human neutrophils, in which they block activation of the superoxide-generating respiratory burst [E. Wilson et al. (1986) J. Biol. Chem. 261, 12616-12623]. In the present study we have used sphingosine to investigate the pathways for agonist-induced secretion of neutrophil granule contents. Induction of secretion of the specific granule component lactoferrin by a variety of agonists [phorbol 12-myristate-13-acetate (PMA), formyl-methionyl-leucyl-phenylalanine (fMLP), and calcium ionophore A23187] was completely inhibited by sphingosine with an ED50 of 6 to 10 microM. PMA-induced secretion of lysozyme (present in both the azurophilic and specific granules) was completely blocked with an ED50 of 10 microM, whereas fMLP-induced secretion was only about 50% inhibited. Secretion of the azurophilic granule proteins beta-glucuronidase and myeloperoxidase was activated by fMLP and A23187, but not by PMA, and was not affected by sphingosine. The use of A23187 in the presence of sphingosine allowed differentiation between calcium activation of protein kinase C-dependent versus-independent pathways. The effect of sphingosine was not mediated by neutralizing intracellular acidic compartments, since treatment of neutrophils with inhibitory concentrations of sphingosine did not significantly alter the uptake of labeled methylamine. We conclude that at least two mechanisms participate in the regulation of specific and azurophilic granule secretion, respectively: a protein kinase C-dependent pathway and a calcium-dependent pathway which does not involve protein kinase C.     

Intracellular Ca2+ mobilization and not calcium influx promotes phorbol ester-stimulated thromboxane A2 synthesis in human platelets.

Phorbol esters, potent activators of protein kinase C (PKC), greatly enhance the release of arachidonic acid and its metabolites (TXA2, HETES, HHT) by Ca2+ ionophores in human platelets. In this paper, we report the relationship between intracellular Ca2+ mobilization and external calcium influx into platelets and the ability of PMA plus A23187 to promote thromboxane A2 (TXA2) synthesis. The enhanced levels of TXA2 due to the synergistic stimulation of the platelets with A23187 and phorbol esters are not affected significantly by the presence of external Ca2+ or the calcium-chelator EGTA. PKC inhibitors, staurosporine and sphingosine, abolished phorbol myristate acetate (PMA) potentiation of TXA2 production which strongly supports the role of PKC in the synergism. Platelet aggregation is more sensitive to PMA and external calcium than TXA2 formation. PMA increased TXA2 production as much as 4-fold at low ionophore concentrations. The A23187-induced rise in [Ca2+]i was reduced by pretreatment of human platelets with phorbol esters, both in the presence and absence of EGTA, and staurosporine reversed this inhibitory effect. These results indicate that the synergistic stimulation of TXA2 production by A23187 and phorbol esters is promoted by intracellular Ca2+ mobilization and not by external calcium influx. Our data also suggest that PKC is involved in the regulation of Ca2+ mobilization from some specific intracellular stores and that PKC may also stimulate the Ca(2+)-dependent phospholipase A2 at suboptimal Ca2+i concentrations.     

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.     

Leupeptin selectively inhibits human platelet responses induced by thrombin and trypsin; a role for proteolytic activation of phospholipase C

Thrombin and trypsin induce serotonin release and aggregation in human platelets. Both proteases induce activation of phospholipase C as reflected by formation of inositol phosphates and phosphorylation of the resultant 1,2-diacylglycerol to phosphatidic acid. Also, thrombin and trypsin activate protein kinase C and myosin light chain kinase as indicated, respectively, by phosphorylation of the 40,000 and 20,000 dalton proteins. Leupeptin, a known inhibitor of serine proteases, blocks all the observed responses of human platelets to trypsin and thrombin. Leupeptin does not inhibit serotonin release and aggregation induced by other platelet stimuli such as collagen, platelet-activating factor, ionophore A23187, and arachidonic acid. The implication of a proteolytic-mediated pathway in the transmembrane signalling involved in platelet activation is discussed.     

Protein kinase C-dependent and -independent pathways of proto-oncogene induction in human astrocytoma cells

We compared the abilities of the muscarinic agonist carbachol, epidermal growth factor (EGF), and phorbol 12-myristate 13-acetate (PMA) to induce proto-oncogene mRNA accumulation and other cellular responses in normal and protein kinase C-deficient 1321-N1 human astrocytoma cells. PMA, carbachol, and EGF all stimulated rapid accumulation of mRNA for the proto-oncogenes c-fos and c-myc in the normal cells; in the protein kinase C-deficient cells, carbachol and EGF, but not PMA, retained this effect, which was not mimicked by the calcium ionophore A23187. Both carbachol and PMA activated protein kinase C in these cells, as evidenced by the stimulated phosphorylation of an acidic Mr 80,000 protein kinase C substrate protein with phosphoamino acid and peptide map identity. This response was mimicked by several other neurotransmitters in these cells, including epinephrine, histamine, oxotremorine, and serotonin, and was abolished in cells made protein kinase C-deficient by preincubation with high concentrations of PMA. Both PMA and carbachol promoted the phosphorylation of the ribosomal protein S6 and activated an S6 protein kinase in the normal but not in the protein kinase C-deficient cells. EGF, in contrast, did not appear to activate protein kinase C, but promoted the phosphorylation of S6 and activation of the S6 kinase in both normal and protein kinase C-deficient cells. We conclude that, in 1321-N1 cells, induction of c-fos and c-myc mRNA can occur through a protein kinase C-dependent pathway and one or more independent pathways, exemplified by the responses to carbachol and EGF in the protein kinase C-deficient cells.     

Modes of inhibitory action of 4 beta-phorbol 12-myristate 13-acetate in thrombin-stimulated arachidonic acid release in intact and permeabilized platelets

The tumor-promoting phorbol ester 4 beta-phorbol 12-myristate 13-acetate (PMA) inhibited thrombin-stimulated arachidonic acid (AA) release in rabbit and human platelets. PMA was effective over the same concentration range that activates protein kinase C in intact rabbit platelets: IC50 vs thrombin = 0.5 nM, greater than 90% inhibition at 10 nM. Suppression of thrombin-stimulated AA release was evident within 5 min of pretreatment with 1 nM PMA. A non-tumor-promoting phorbol ester, 4-O-methyl PMA, showed a very weak ability to inhibit AA release. Thrombin-stimulated serotonin secretion was progressively inhibited by PMA pretreatment in platelets, while PMA was a stimulus for secretion at higher concentrations. 1-(5-Isoquinolinylsulfonyl)-2-methyl-piperazine (H-7), a selective inhibitor of protein kinase C, blocked PMA-induced inhibition of AA release. Furthermore, H-7 enhanced the effect of thrombin on AA release. PMA pretreatment reduced the inhibitory effect of thrombin on forskolin-stimulated cAMP accumulation, but had no effect on nonstimulated cAMP metabolism in the presence of thrombin. PMA did not inhibit AA release caused by A23187 or melittin. In digitonin-permeabilized platelets, thrombin plus guanosine 5'-(3-O-thio)triphosphate (GTP gamma S)-stimulated AA release, but not GTP gamma S- and AIF4(-)-stimulated AA release, was abolished by PMA pretreatment. These results suggest that activation of protein kinase C may exert negative feedback on the receptor-mediated activation of phospholipase A2. A possible uncoupling of thrombin receptor to GTP-binding protein leading to activation of phospholipase A2 by PMA pretreatment is discussed.     

Activators of protein kinase C trigger cortical granule exocytosis, cortical contraction, and cleavage furrow formation in Xenopus laevis oocytes and eggs

Prophase I oocytes, free of follicle cells, and metaphase II eggs of the amphibian Xenopus laevis were subjected to transient treatments with the protein kinase C activators, phorbol 12-myristate 13-acetate (PMA), phorbol 12,13-didecanoate, and 1-olyeoyl-2-acetyl-sn-glycerol. In both oocytes and eggs, these treatments triggered early events of amphibian development: cortical granule exocytosis, cortical contraction, and cleavage furrow formation. Surprisingly, activation of oocytes occurred in the absence of meiotic resumption, resulting in cells with an oocytelike nucleus and interior cytoplasm, but with a zygotelike cortex. PMA-induced activation of oocytes and eggs did not require external calcium, a prerequisite for normal activation of eggs. PMA-induced activation of eggs was inhibited by retinoic acid, a known inhibitor of protein kinase C. In addition, pretreatment of eggs with retinoic acid prevented activation by mechanical stimulation and inhibited activation by calcium ionophore A23187. The results suggest that protein kinase C activation is an integral component of the Xenopus fertilization pathway.     

Phospholipid metabolism, calcium flux, and the receptor-mediated induction of chemotaxis in rabbit neutrophils

Rabbit neutrophils were stimulated with the chemotactic peptide fMet-Leu-Phe in the presence of the methyltransferase inhibitors homocysteine (HCYS) and 3-deazaadenosine (3-DZA). HCYS and 3-DZA inhibited chemotaxis, phospholipid methylation, and protein carboxymethylation in a dose-dependent manner. The chemotactic peptide-stimulated release of [14C]arachidonic acid previously incorporated into phospholipid was also partially blocked by the methyltransferase inhibitors. Stimulation by fMet-Leu-Phe or the calcium ionophore A23187 caused release of arachidonic acid but not of previously incorporated [14C]-labeled linoleic, oleic, or stearic acids. Unlike the arachidonic acid release caused by fMet-Leu-Phe, release stimulated by the ionophore could not be inhibited by HCYS and 3-DZA, suggesting that the release was caused by a different mechanism or by stimulating a step after methylation in the pathway from receptor activation to arachidonic acid release. Extracellular calcium was required for arachidonic acid release, and methyltransferase inhibitors were found to partially inhibit chemotactic peptide-stimulated calcium influx. These results suggest that methylation pathways may be associated with the chemotactic peptide receptor stimulation of calcium influx and activation of a phospholipase A2 specific for cleaving arachidonic acid from phospholipids.     

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.     

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.     

Epidermal growth factor enhances glomerular mesangial cell soluble phospholipase A2 activity

We have previously characterized a hormonally regulated soluble form of phospholipase A2 (PLA2) in the cultured renal mesangial cell which is similar and possibly identical to the major form in rat kidney. In an attempt to further characterize the mechanisms of regulation of this enzyme we have used epidermal growth factor (EGF), which does not activate polyphosphoinositide-specific phospholipase C in these cells. EGF-enhanced PLA2 activity as assayed by the ability of the soluble extracts of cells to cleave arachidonic acid from the sn-2 position of phosphatidylcholine and phosphatidylethanolamine. This represents a direct demonstration of EGF-induced PLA2 activation which is preserved in a cell-free extract. Phorbol myristate acetate (PMA), as well as 1-oleoyl-2-acetylglycerol, also enhanced PLA2 activity. By contrast, the calcium ionophore A23187 had no effect on extract PLA2 activity. The EGF- and PMA-induced enhanced activity was recovered following fractionation by Mono-Q anion exchange chromatography. The peak of activity comigrated for both agonists, suggesting that both EGF and PMA stimulated the same form of the enzyme. Down-regulation of protein kinase C by pretreatment with PMA resulted in loss of the PMA-induced, but not the EGF-induced, enhancement in PLA2 activity. 8-Bromo-cAMP had no effect upon the PLA2 activity, and did not modulate the EGF effect. Pertussis toxin induced G protein ADP-ribosylation but had no effect upon PLA2 activity, and did not alter the EGF effect. In summary, EGF results in a stable modification of PLA2 activity in glomerular mesangial cells. This enhanced activity is independent of polyphosphoinositide hydrolysis, insensitive to protein kinase C down-regulation, and is not affected by cAMP or pertussis toxin pretreatment of the cells.     

Inhibition of antigen and calcium ionophore induced secretion from RBL-2H3 cells by phosphatase inhibitors

The role of serine/threonine protein phosphatases PP1 and PP2A in mast cell secretion was investigated using the phosphatase inhibitors okadaic acid and calyculin A. Calyculin A (5-25 nm) inhibited antigen-induced secretion from a rat mucosal mast cell line (RBL-2H3) when added in conjunction with the activator. Okadaic acid (250-1000 nm) inhibited secretion only when added before activation and did so in a time- and concentration-dependent manner. Both inhibitors caused the cells to become rounder, but only calyculin A induced membrane blebbing and a loss of adherence. Okadaic acid also inhibited secretion induced by the calcium ionophore A23187, in the presence or absence of PMA, indicating that the phosphatase inhibitors act on a component of the secretory pathway downstream of calcium mobilization. Okadaic acid increased the phosphorylation of a number of proteins, as did an analogue methyl okadaate, which also inhibited secretion, but less effectively. Okadaic acid induced the phosphorylation of triton-insoluble proteins of 55, 18 and 16 kDa. The 55 kDa protein was identified as vimentin and okadaic acid induced its partial translocation to the triton-soluble fraction. Our data indicate that full secretory function in mucosal mast cells requires phosphatase activity.     

Ca2+ requirement of prostanoid but not of superoxide production by rat Kupffer cells

The release of the prostanoids prostaglandin D2 (PGD2), prostaglandin E2 (PGE2) and thromboxane induced by zymosan and phorbol ester in cultured rat Kupffer cells was found to depend on the extracellular concentration of Ca2+ to some extent. Prostanoid formation following the addition of the calcium ionophore A 23187 was totally inhibited when calcium ions were withdrawn from the medium whereas the prostanoid synthesis from added arachidonic acid was independent of Ca2+. A half-maximal rate of PGE2 release by cells treated with zymosan, phorbol ester or A23187 was obtained at 0.6-0.7 microM free extracellular Ca2+ and greater than or equal to 100 microM free Ca2+ was required to stimulate PGE2 formation maximally. The calmodulin antagonist R24571 partially inhibited the release of PGE2 elicited by zymosan and A23187 but not by phorbol ester or arachidonic acid. Verapamil and nifedipine, two calcium channel blockers, had no effect on the formation of PGE2 irrespective of the stimulus. TMB 8 [3,4,5-trimethoxybenzoic acid 8-(diethylamino)-octyl ester] an intracellular calcium antagonist, inhibited the synthesis of PGE2 induced by zymosan and phorbol ester. The superoxide formation following the addition of zymosan and phorbol ester was not influenced by removal of calcium ions from the medium or by addition of the various calcium antagonists. The data presented here suggest that Ca2+-dependent reactions are involved in the synthesis of prostanoids induced by zymosan and phorbol ester and that both extracellular Ca2+ and mobilization of Ca2+ from intracellular stores are needed to induce maximally the production of prostanoids in cultured rat Kupffer cells.     

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.     

Reactive oxygen production associated with arachidonic acid metabolism by peritoneal macrophages

The nature of the calcium-dependent chemiluminescence observed in peritoneal macrophages after exposure to the calcium ionophore A23187 or during the phagocytosis of zymosan has been investigated. Eicosatetraynoic acid, an inhibitor of the lipoxygenase and cyclooxygenase pathways of arachidonic acid metabolism, inhibited the calcium-dependent chemiluminescence whereas indomethacin, a selective inhibitor of the cyclooxygenase pathway, did not. Arachidonic acid induced chemiluminescence only in phagocytosing cells, whilst 15-HPETE, an intermediate of the lipoxygenase pathway, generated a similar, transient chemiluminescent response in either unstimulated or phagocytosing cells. The results suggest that the lipoxygenase pathway may be a significant source of the reactive species of oxygen that give rise to chemiluminescence. Prostaglandin E₁ inhibited the chemiluminescence induced by zymosan and A23187, but did not affect that generated in response to 15-HPETE or arachidonic acid, suggesting that the inhibition is directed at a step either connected with or occurring prior to the release of free arachidonic acid by the cells.