Phorbol ester and 1-oleoyl-2-acetylglycerol inhibit angiotensin activation of phospholipase C in cultured vascular smooth muscle cells

Angiotensin II acts on cultured rat aortic vascular smooth muscle cells (VSMC) to induce the rapid, phospholipase C-mediated generation of inositol trisphosphate from phosphatidylinositol 4,5-bisphosphate and mobilization of intracellular Ca2+. sn-1,2-Diacylglycerol, the other major product of inositol phospholipid breakdown, is known to activate protein kinase C, but its role in angiotensin II action on VSMC has not been defined. We report herein that, in cultured VSMC prelabeled with [3H]myoinositol, brief incubations (2-5 min) with 4 beta-phorbol 12-myristate 13-acetate (PMA) (1-100 nM) or 1-oleoyl-2-acetylglycerol (10-100 microM), two potent activators of protein kinase C, inhibit subsequent angiotensin II (100 nM)-induced increases in phosphatidylinositol 4,5-bisphosphate breakdown and inositol trisphosphate formation. In addition, pretreatment of VSMC with either PMA (IC50 approximately 1 nM) or 1-oleoyl-2-acetylglycerol (IC50 approximately 7.5 microM) also markedly inhibits angiotensin II (1 nM)-stimulated increases in cytosolic free Ca2+, as measured with the calcium-sensitive fluorescent indicator quin 2, or 45Ca2+ efflux. Neither PMA nor 1-oleoyl-2-acetylglycerol initiated phosphatidylinositol 4,5-bisphosphate breakdown or Ca2+ flux by itself. PMA treatment (10 nM, 5 min) did not influence the number or affinity of 125I-angiotensin II-binding sites in intact cells. These data suggest that one function of angiotensin II-generated sn-1,2-diacylglycerol in vascular smooth muscle may be to modulate, by protein kinase C-mediated mechanisms, angiotensin II receptor coupling to phospholipase C.     

Possible involvement of protein kinase C and calcium ion in growth factor-induced expression of c-myc oncogene in Swiss 3T3 fibroblasts

The addition of platelet-derived growth factor and fibroblast growth factor to quiescent cultures of Swiss 3T3 fibroblasts rapidly induced protein kinase C activation and Ca2+ mobilization and afterwards markedly increased c-myc mRNA levels. 1-Oleoyl-2-acetylglycerol, a membrane-permeable synthetic diacylglycerol, and 12-O-tetradecanoylphorbol 13-acetate, a tumor-promoting phorbol ester, stimulated protein kinase C activation without Ca2+ mobilization. Inversely, Ca2+ ionophores, A23187 and ionomycin, elicited Ca2+ mobilization without protein kinase C activation. Both protein kinase C-activating and Ca2+-mobilizing agents were able to increase c-myc mRNA levels in an additive manner. Prolonged treatment of the cells with phorbol 12,13-dibutyrate, another protein kinase C-activating phorbol ester, led to the down-regulation and complete disappearance of protein kinase C. In these cells, 1-oleoyl-2-acetylglycerol and 12-O-tetradecanoylphorbol 13-acetate did not increase c-myc mRNA levels, but platelet-derived growth factor, fibroblast growth factor, and the Ca2+ ionophores, all of which still induced Ca2+ mobilization, stimulated the increase of c-myc mRNA levels. These results strongly suggest that both protein kinase C and Ca2+ may be involved in platelet-derived growth factor- as well as fibroblast growth factor-induced expression of the c-myc oncogene in Swiss 3T3 cells.     

Multiple regulation of proenkephalin gene expression by protein kinase C

In the present study we investigated the role of protein kinase C (Ca2+/phospholipid-dependent enzyme)-mediated processes in the regulation of proenkephalin gene expression in primary cultures of bovine adrenal chromaffin cells. Activators of protein kinase C such as 1-oleoyl-2-acetylglycerol, mezerein, and the phorbol esters phorbol 12-myristate 13-acetate (PMA) and phorbol 12,13-didecanoate induced a time-dependent increase in proenkephalin mRNA levels, whereas the inactive phorbol ester 4 alpha-phorbol 12,13-didecanoate had no effect. The increase in phorbol ester-induced proenkephalin mRNA was potentiated by low concentrations of the Ca2+ ionophore A23187, suggesting an interaction between protein kinase- and Ca2+-mediated processes in the regulation of proenkephalin mRNA. The phorbol ester-induced stimulation does not appear to be mediated by an interaction with the cAMP-generating system or increases in Ca2+ uptake. However, when proenkephalin mRNA levels were stimulated by KCl (10 mM) and the dihydropyridine BayK8644, PMA exhibited an inhibitory effect on proenkephalin mRNA, which was detectable at a 10-fold lower concentration of PMA than the stimulatory effect. This inhibitory effect appears to be mediated by an inhibition of Ca2+ entry through voltage-dependent Ca2+ channels, as suggested by 45Ca2+ uptake experiments. Thus, the net effect of PMA depends on and varies with the state of voltage-dependent Ca2+ channel activity. A third mode of action by protein kinase C to modulate proenkephalin gene expression is by interaction with the phosphatidylinositol second messenger system. Stimulation of phosphoinositide hydrolysis and proenkephalin mRNA by histaminic H1-receptor activation was inhibited by low concentrations of PMA. We suggest that protein kinase C may act as a positive and negative regulator of proenkephalin gene expression by interacting with at least three receptor-coupled second messenger systems.     

Ethanol causes desensitization of receptor-mediated phospholipase C activation in isolated hepatocytes.

The effect of ethanol on receptor-mediated phospholipase C-linked signal transduction processes was investigated in isolated rat hepatocytes. Pretreatment of the cells with ethanol (6-300 mM) markedly inhibited a subsequent stimulation of phospholipase C by vasopressin, angiotensin II, or epidermal growth factor. By contrast, the effects of the alpha 1-adrenergic agonist phenylephrine and of glucagon were not affected by ethanol pretreatment. Ethanol inhibited the agonist-induced decrease in polyphosphoinositides, the formation of inositol phosphates, and the increase in cytosolic free Ca2+ levels, as detected with the intracellular Ca2+ indicator indo-1. The effects of ethanol were concentration dependent and were pronounced at low concentrations of agonists but were not significant at saturating levels. Pretreatment of the cells with the protein kinase C inhibitor H7 partly prevented the inhibition by ethanol of vasopressin-induced phospholipase C activation. By contrast, pretreatment of the cells with (Rp)-adenosine cyclic 3':5'-phosphorothioate [Rp)-cAMP-S), a competitive inhibitor of protein kinase A, potentiated the inhibitory effect of ethanol on the Ca2+ mobilization by vasopressin. (Rp)-cAMP-S similarly potentiated the inhibition of phospholipase C by the protein kinase C-activating phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). The kinase A inhibitor also made the Ca2+ mobilization by phenylephrine sensitive to ethanol, indicating that the formation of cAMP in the cells played a role in suppressing the sensitivity to ethanol. Pretreatment of the cells with ethanol enhanced the inhibitory effects of TPA on the vasopressin-induced phospholipase C activation at all concentrations of the hormone; however, these synergistic effects were prevented when TPA was added prior to ethanol, a condition that prevents the activation of phospholipase C by ethanol. The data indicate that ethanol causes desensitization of the receptor-mediated phospholipase C secondary to the ethanol-induced activation of phospholipase C and activation of protein kinase C. Ethanol treatment also affects the sensitivity of the phospholipase C system to control by protein kinases A and C. The data indicate that ethanol can affect the control of intracellular signal transduction processes in liver cells under physiologically relevant conditions.     

Potentiation of stimulus-induced insulin secretion in protein kinase C-deficient RINm5F cells.

The role of protein kinase C (PKC) in stimulus recognition and insulin secretion was investigated after long-term (24 h) treatment of RINm5F cells with phorbol 12-myristate 13-acetate (PMA). Three methods revealed that PKC was no longer detectable, and PMA-induced insulin secretion was abolished. Such PKC-deficient cells displayed enhanced insulin secretion (2-6-fold) in response to vasopressin and carbachol (activating phospholipase C) as well as to D-glyceraldehyde and alanine (promoting membrane depolarization and voltage-gated Ca2+ influx). Insulin release stimulated by 1-oleoyl-2-acetylglycerol (OAG) was also greater in PKC-deficient cells. OAG caused membrane depolarization and raised the cytosolic Ca2+ concentration ([Ca2+]i), both of which were unaffected by PKC down-regulation. Except for that caused by vasopressin, the secretagogue-induced [Ca2+]i elevations were similar in control and PKC-depleted cells. The [Ca2+]i rise evoked by vasopressin was enhanced during the early phase (observed both in cell suspensions and at the single cell level) and the stimulation of diacylglycerol production was also augmented. These findings suggest more efficient activation of phospholipase C by vasopressin after PKC depletion. Electrically permeabilized cells were used to test whether the release process is facilitated after long-term PMA treatment. PKC deficiency was associated with only slightly increased responsiveness to half-maximally (2 microM) but not to maximally stimulatory Ca2+ concentrations. At 2 microM-Ca2+ vasopressin caused secretion, which was also augmented by PMA pretreatment. The difference between intact and permeabilized cells could indicate the loss in the latter of soluble factors which mediate the enhanced secretory responses. However, changes in cyclic AMP production could not explain the difference. These results demonstrate that PKC not only exerts inhibitory influences on the coupling of receptors to phospholipase C but also interferes with more distal steps implicated in insulin secretion.     

Protein kinase C activation stimulates plasma membrane Ca2+ pump in cultured vascular smooth muscle cells

We examined the effect of phorbol myristate acetate (PMA), a potent activator of protein kinase C, on Ca2+ extrusion from cultured vascular smooth muscle cells (VSMCs) incubated in the absence of added extracellular Na+ (Na+o). Previously, strong experimental evidence was presented that the Na+o-independent Ca2+ extrusion from VSMCs is effected by the plasma membrane Ca2+ pump (Furukawa, K.-I., Tawada, Y., and Shigekawa, M. (1988) J. Biol. Chem. 263, 8058-8065). Brief (2 min) pretreatment of VSMCs with 30-300 nM PMA suppressed the intracellular Ca2+ transient induced with 1 microM ionomycin to about 60% of the control, whereas it accelerated the concomitant Na+o-independent 45Ca2+ extrusion by up to 20%. When the Ca2+ transient was induced with 0.1 microM angiotensin II, the PMA pretreatment markedly suppressed it and reduced also the rate of 45Ca2+ efflux from cells slightly. These effects of PMA were mimicked by 1-oleoyl-2-acetylglycerol, another protein kinase C activator, but were abolished by prior treatment of cells with staurosporine, an inhibitor of protein kinase C, or prior long incubation of cells with PMA. Analysis of the effect of PMA on [Ca2+]i dependence of the rate of Na+o-independent 45Ca2+ efflux revealed that PMA increased the maximum Ca2+ efflux rate without a significant change in the affinity for Ca2+. These results strongly suggest that the plasma membrane Ca2+ pump in VSMCs can be stimulated by PMA and that protein kinase C is involved in regulation of [Ca2+]i in intact VSMCs.     

Protein kinase C enhances Ca2+ mobilization in human platelets by activating Na+/H+ exchange

Control of cytoplasmic pH (pHi) by a Na+/H+ antiport appears a general property of most eukaryotic cells. In human platelets activation of the Na+/H+ exchanger enhances Ca2+ mobilization and aggregation induced by low concentrations of thrombin (Siffert, W., and Akkerman, J. W. N. (1987) Nature 325, 456-458). Several observations indicate that the exchanger is regulated by protein kinase C. (i) Inhibitors of protein kinase C (trifluoperazine, sphingosine) inhibit the increase in pHi seen during thrombin stimulation as well as Ca2+ mobilization; artificially increasing pHi by monensin or NH4Cl then restores Ca2+ mobilization. (ii) Direct activation of protein kinase C by 1-oleoyl-2-acetylglycerol initiates an increase in pHi that depends on the presence of extracellular Na+ and is sensitive to inhibition by ethylisopropylamiloride. The pHi sensitivity of thrombin-induced Ca2+ mobilization is particularly evident in the range between pH 6.8 and 7.4 and at low thrombin concentrations, whereas thrombin concentrations of more than 0.2 unit/ml bypass the pH sensitivity. In the absence of thrombin an increase in pHi, either induced artificially (by addition of the ionophores nigericin or monensin) or via activation of protein kinase C (by addition of 1-oleoyl-2-acetylglycerol), does not induce Ca2+ mobilization. We conclude that activation of protein kinase C is essential for Ca2+ mobilization in platelets stimulated by low concentrations of thrombin and that protein kinase C exerts this effect via activation of the Na+/H+ exchanger.     

Lipopolysaccharide and phorbol esters induce differentiation but have opposite effects on phosphatidylinositol turnover and Ca2+ mobilization in 70Z/3 pre-B lymphocytes

We have recently shown that both lipopolysaccharide (LPS) and the phorbol ester, 12-O-tetradecanoyl phorbol 13-acetate (TPA) induce differentiation in the transformed murine pre-B lymphocyte cell line 70Z/3 by enhancing Na+-H+ exchange across the plasma membrane through an amiloride-sensitive transport system (Rosoff, P.M., Stein, L.F., and Cantley, L.C. (1984) J. Biol. Chem. 259, 7056-7060). These data suggested that the activation of protein kinase C indirectly by LPS and directly by TPA was the critical step in the initiation of differentiation in these cells. We extend these observations to show that LPS rapidly stimulates an increase in phosphatidylinositol turnover, leading to a rise in the levels of diacylglycerol and inositol 1,4,5-trisphosphate and a concomitant decrease in the amount of phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate. There is also a rapid elevation of intracellular free [Ca2+] which is independent of the presence of extracellular Ca2+ or Na+. These results suggest that the increase in cytosolic [Ca2+] is due to release of cation from internal stores. TPA, which also causes differentiation in these cells, and the synthetic diacylglycerol, 1-oleoyl-2-acetylglycerol, have opposite effects from LPS on both phosphatidylinositol turnover and cellular Ca+ mobilization. These data suggest that protein kinase C inhibits the activity of phospholipase C. Thus protein kinase C plays a pivotal role in the regulation of mitogen-induced differentiation in these cells by both transducing a positive stimulus to the Na+-H+ exchange system as well as feedback regulating its own stimulatory pathway.     

The hormone-sensitive hepatic Na+-pump. Evidence for regulation by diacylglycerol and tumor promoters

Ouabain-sensitive 86Rb+ uptake by isolated rat hepatocytes was studied to elucidate how Ca2+-mobilizing hormones stimulate the Na+-pump. Stimulation of this uptake was observed with concentrations of vasopressin ([8-arginine]vasopressin, AVP), angiotensin II, and norepinephrine which elicited Ca2+ mobilization and phosphorylase activation. These results suggested that changes in cytosolic Ca2+, mediated by inositol trisphosphate, might trigger sodium pump stimulation by AVP. However, in hepatocytes incubated in Ca2+-free Krebs-Henseleit buffer, Na+-pump activity was not altered over 15 min by either 1.5 mM EGTA or 1.5 mM Ca2+. Furthermore, incubation of cells in 5 mM EGTA for 15-30 min drastically impaired the ability of AVP to increase cytosolic Ca2+, but only modestly attenuated AVP-stimulated Na+-pump activity. Two tumor promoters, phorbol myristate acetate (PMA) and mezerein, stimulated Na+/K+-ATPase-mediated transport activity. Similarly, addition of synthetic diacylglycerols or of exogenous phospholipase C from Clostridium perfringens to increase endogenous diacylglycerol levels also resulted in a stimulation of the Na+-pump in the absence of changes in cytosolic or total cellular Ca2+ levels. Stimulation of the Na+-pump by the combination of maximal concentrations of PMA and AVP did not produce an additive response, and both agents displayed a transient time course, suggesting that the two agents share a common mechanism. Stimulation of the Na+-pump by AVP and PMA was not blocked by amiloride analogs which inhibit Na+/H+ exchange, but these compounds blocked the action of insulin. These data suggest that the elevated Na+/K+-ATPase-mediated transport activity observed in hepatocytes following exposure to Ca2+-mobilizing hormones is a consequence of stimulated diacylglycerol formation and may involve protein kinase C.     

A dual role for calcium in regulation of superoxide generation by stimulated rat alveolar macrophages

Superoxide production in alveolar macrophages is stimulated by agonists which act through Ca2+-mediated (concanavalin A) and/or protein kinase C (phorbol ester or diacylglycerol analogues) -mediated events. Simultaneous addition of saturating concentrations of concanavalin A and a protein kinase C activator (either phorbol 12-myristate-13-acetate or 1-oleoyl-2-acetyl-sn-glycerol) caused a supra-additive enhancement of the initial rate of O2-. production. This synergism closely correlated with the known time-course of Ca2+ mobilization induced by concanavalin A; however, it occurred under conditions in which protein kinase C activation is reportedly not Ca2+ dependent. Phorbol ester-induced O2-. production was partially inhibited by the Ca2+ ionophore, A23187. Although phorbol ester-stimulated O2-. production initially was enhanced by concanavalin A, the duration of this O2-. production was reduced in comparison to that induced by phorbol ester alone. These results suggest a dual role for intracellular Ca2+ in both stimulatory and inhibitory regulation of O2-. production.     

Phorbol esters and diacylglycerols amplify bradykinin-stimulated prostaglandin synthesis in Swiss 3T3 fibroblasts. Possible independence from protein kinase C

When Swiss 3T3 fibroblasts were incubated with bradykinin, prostaglandin E2 (PGE2) synthesis was stimulated. Phorbol esters or the diacylglycerol analog 1-oleoyl-2-acetylglycerol (OAG), by themselves, did not acutely stimulate PGE2 synthesis. However, when cells were preincubated with phorbol esters or OAG, bradykinin-stimulated PGE2 synthesis was potentiated markedly. When phorbol esters and OAG were added together, bradykinin-stimulated PGE2 synthesis was potentiated in an additive manner. When cells were preincubated for 48 h with phorbol esters, then bradykinin added, amplification of bradykinin-stimulated PGE2 synthesis by phorbol ester or OAG was still apparent, even though prolonged pretreatment with phorbol esters abolished protein kinase C (Ca2+/phospholipid-dependent enzyme) activity in cell-free preparations. Further, the protein kinase C antagonist, H-7, only slightly inhibited phorbol ester or OAG amplification of bradykinin-stimulated PGE2 synthesis. The possibility is raised that diacylglycerol, formed in response to many receptors, may serve as a transducer of receptor-receptor interactions. Since desensitization or inhibition of protein kinase C only partially reduced the amplification of bradykinin-stimulated PGE2 synthesis by phorbol esters or OAG, the possibility is raised that diacylglycerol mimetics may have actions in addition to activation of protein kinase C.     

Studies on the hepatic calcium-mobilizing activity of aluminum fluoride and glucagon. Modulation by cAMP and phorbol myristate acetate

The effects of submaximal doses of AlF4- to mobilize hepatocyte Ca2+ were potentiated by glucagon (0.1-1 nM) and 8-p-chlorophenylthio-cAMP. A similar potentiation by glucagon of submaximal doses of vasopressin, angiotensin II, and alpha 1-adrenergic agonists has been previously shown (Morgan, N. G., Charest, R., Blackmore, P. F., and Exton, J. H. (1984) Proc. Natl. Acad. Sci. U. S. A. 81, 4208-4212). When hepatocytes were pretreated with the protein kinase C activator 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (PMA), the effects of AlF4- to mobilize Ca2+, increase myo-inositol 1,4,5-trisphosphate (IP3), and activate phosphorylase were attenuated. Treatment of hepatocytes with PMA likewise inhibits the ability of vasopressin, angiotensin II, and alpha 1-adrenergic agonists to increase IP3 and mobilize Ca2+ (Lynch, C. J., Charest, R., Bocckino, S. B., Exton, J. H., and Blackmore, P. F. (1985) J. Biol. Chem. 260, 2844-2851). In contrast, the ability of AlF4- or angiotensin II to lower cAMP or inhibit glucagon-mediated increases in cAMP was unaffected by PMA. The ability of AlF4- to lower cAMP was attenuated in hepatocytes from animals treated with islet-activating protein, whereas Ca2+ mobilization was not modified. These results suggest that the lowering of cAMP induced by AlF4- and angiotensin II was mediated by the inhibitory guanine nucleotide-binding regulatory protein of adenylate cyclase, whereas Ca2+ mobilization was not. Addition of glucagon, forskolin, or 8CPT-cAMP to hepatocytes raised IP3 and mobilized Ca2+. Both effects were blocked by PMA pretreatment, whereas cAMP and phosphorylase a levels were only minimally affected by PMA. The mobilization of Ca2+ induced by cAMP in hepatocytes incubated in low Ca2+ media was not additive with that induced by maximally effective doses of vasopressin, angiotensin II, or alpha 1-adrenergic agonists, indicating that the Ca2+ pool(s) affected by agents which increase cAMP is the same as that affected by Ca2+-mobilizing hormones which do not increase cAMP. These findings support the proposal that AlF4- mimics the effects of the Ca2+-mobilizing hormones in hepatocytes by activating a guanine nucleotide-binding regulatory protein (Np) which couples the hormone receptors to a phosphatidylinositol 4,5-bisphosphate (PIP2)-specific phosphodiesterase. They also suggest that Np, PIP2 phosphodiesterase, or a factor involved in their interaction is activated following phosphorylation by cAMP-dependent protein kinase and inhibited after phosphorylation by protein kinase C.(ABSTRACT TRUNCATED AT 400 WORDS)     

Protein kinase C of human erythrocytes phosphorylates bands 4.1 and 4.9

Addition of 10 nM 12-O-tetradecanoylphorbol 13-acetate (TPA) to intact human erythrocytes results in rapid phosphorylation of two cytoskeletal components, bands 4.1 and 4.9. The synthetic diacylglycerol, 1-oleoyl-2-acetylglycerol, shows a similar effect, while the biologically inactive phorbol ester, 4 alpha-phorbol didecanoate, fails to enhance phosphorylation. That TPA and 1-oleoyl-2-acetylglycerol stimulate this phosphorylation suggests that protein kinase C is being activated. In the presence of TPA, bands 4.1 and 4.9 incorporate 1.5 mol Pi/mol protein and 1.2 mol Pi/mol protein, respectively. The pattern and extent of phosphorylation shows that it is not due to cAMP-dependent protein kinases, which also phosphorylate bands 4.1 and 4.9. Ca2+-phospholipid-dependent protein kinase activity is demonstrable in the soluble fraction of erythrocytes, and has been partially purified (2200-fold) from the hemolysate by affinity chromatography (Uchida and Filburn, 1984. J. Biol. Chem. 259, 12311-12314). The affinity purified erythrocyte kinase has a 42 A Stokes' radius and phosphorylates purified bands 4.1 and 4.9 in vitro in a Ca2+- and phospholipid-dependent manner. These results show that human erythrocytes contain protein kinase C, and that band 4.1 and 4.9 are the major endogenous substrates for this kinase.     

Phorbol esters and calcium-mobilizing hormones increase membrane-associated protein kinase C activity in rat hepatocytes

Vasopressin, angiotensin II, epinephrine (alpha 1-adrenergic action) and phorbol 12-myristate 13-acetate (PMA) induce increases in membrane-associated protein kinase C activity concomitant with decreases in the cytosolic activity. The data indicate that the calcium-mobilizing hormones and the active phorbol ester induce translocation from the cytosol to the plasma membrane of this protein kinase. The protein kinase C inhibitor, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine, blocked the translocation to the membrane of this protein kinase induced by PMA and vasopressin.     

Prostaglandins E1 and E2 enhance the stimulation of superoxide release by 1-oleoyl-2-acetylglycerol from human neutrophils

Superoxide release from human neutrophils was stimulated either by receptor activation (using fMet-Leu-Phe) or by activating, independently, each of the two pathways considered to be involved in signal transduction--calcium mobilization (using the ionophore, A23187) and protein kinase C activation (using phorbol myristate acetate or 1-oleoyl-2-acetylglycerol). Prostaglandin E1 (3 X 10(-5) M) decreased fMet-Leu-Phe-stimulated superoxide release, had no effect on superoxide release stimulated by A23187, or by phorbol myristate acetate, and markedly enhanced the superoxide release stimulated by 1-oleoyl-2-acetylglycerol. Similar enhancement was obtained with prostaglandin E2.     

Inhibition of prostaglandin E1-induced elevation of cytoplasmic free calcium ion by protein kinase C-activating phorbol esters and diacylglycerol in Swiss 3T3 fibroblasts

In quiescent cultures of Swiss 3T3 cells, prostaglandin E1 (PGE1) known to elevate cAMP increased rapidly cytoplasmic free Ca2+ concentration ([Ca2+]i) as measured with the fluorescent Ca2+ indicator quin2. The primary source of the PGE1-induced elevation of [Ca2+]i was extracellular. Pretreatment of the cells with various doses of 12-O-tetradecanoylphorbol-13-acetate (TPA), a potent protein kinase C-activating phorbol ester, inhibited the PGE1-induced elevation of [Ca2+]i in a dose-dependent manner. Inversely, TPA enhanced slightly the PGE1-induced increase of cAMP. TPA alone did not affect the basal level of [Ca2+]i or cAMP in the absence of PGE1. The inhibitory action of TPA on the PGE1-induced elevation of [Ca2+]i was mimicked by other protein kinase C-activating agents such as phorbol 12,13-dibutyrate and 1-oleoyl-2-acetylglycerol. 4 alpha-Phorbol 12,13-didecanoate known to be inactive for protein kinase C was ineffective in this capacity. Prolonged treatment of the cells with phorbol 12,13-dibutyrate resulted in the down-regulation and disappearance of protein kinase C. In these protein kinase C-deficient cells, PGE1 still elevated [Ca2+]i to the same extent as that in the control cells, but TPA did not inhibit the PGE1-induced elevation of [Ca2+]i. These results strongly suggest that protein kinase C serves as an inhibitor for PGE1-induced Ca2+ influx in Swiss 3T3 cells.     

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.     

Attenuation of hen granulosa cell steroidogenesis by a phorbol ester and 1-oleoyl-2-acetylglycerol

A series of studies was conducted to evaluate the effects of phorbol esters and a diacylglycerol analog on basal and hormone-stimulated steroidogenesis in granulosa cells from the largest preovulatory follicle of the domestic hen. Agents that previously have been shown to activate protein kinase C, such as the tumor-promoting phorbol ester, phorbol 12-myristate 13-acetate (PMA), and the synthetic diacylglycerol analog, 1-oleoyl-2-acetylglycerol (OAG), suppressed luteinizing hormone (LH)-induced progesterone (PMA at levels of 10 and 100 ng/tube; OAG at levels of 10 and 25 micrograms/tube), and androgen (10 and 100 ng PMA; 25 micrograms OAG) production, but had no effect on basal levels of either steroid. Furthermore, PMA decreased the ability of vasoactive intestinal peptide to induce steroidogenesis, suggesting that protein kinase C activation may generally modulate the activity of hormones that act via the adenylyl cyclase/cyclic 3',5'-adenosine monophosphate (cAMP) second messenger system. In further support of this proposal was the finding that PMA and OAG decreased the production of cAMP in response to LH, and attenuated the steroidogenic response in granulosa cells exposed to 10 mM 8-bromo-cAMP. By contrast, the induction of calcium mobilization using a calcium ionophore (A23187; 0.5-2.0 microM) stimulated progesterone and androgen production without increasing intracellular levels of cAMP, and this stimulatory effect on steroidogenesis was not inhibited by the presence of 100 ng PMA/tube. From these data, we suggest that the activation of protein kinase C in granulosa cells of the hen may provide a physiological mechanism by which receptor-mediated steroidogenesis, involving the adenylyl cyclase second messenger system, is modulated.     

Dissimilar effects of 1-oleoyl-2-acetylglycerol and phorbol 12-myristate 13-acetate on fatty acid synthesis in isolated rat-liver cells

Exogenous 1-oleoyl-2-acetylglycerol (OAG) is known to mimic the action of tumour-promoting phorbol esters in various cell types. However, in isolated rat hepatocytes OAG depressed the rate of de novo fatty acid synthesis and the activity of the key enzyme acetyl-CoA carboxylase (EC 6.4.1.2), in contrast to the pronounced stimulation of both parameters by phorbol 12-myristate 13-acetate (PMA). The inhibition by OAG appeared to be dose- and time-dependent. On the other hand, medium-chain 1,2-diacylglycerols like 1,2-dioctanoyl-sn-glycerol did mimic the stimulatory action of PMA. The anomalous effect of OAG may well be explained by its metabolic breakdown leading to liberation of oleate and subsequent inhibition of acetyl-CoA carboxylase activity by endogenously formed oleoyl-CoA. The stimulatory effects of both PMA and medium-chain diacylglycerols are likely to be mediated by protein kinase C.