Phorbol ester inhibits luteinizing hormone-, forskolin-, and dibutyryl cyclic AMP-induced progesterone production in chicken granulosa cells

The possible role of protein kinase C in avian granulosa cell steroidogenesis was studied in vitro by examining the effect of tumor-promoting phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) on progesterone synthesis in chicken granulosa cells in short-term (3h) incubations. TPA (1-100 nM) caused a marginal but nonsignificant increase in progesterone production in granulosa cells isolated from the largest preovulatory follicle. When incubated in combination with luteinizing hormone (5-100 ng/mL), TPA suppressed the stimulatory effects of submaximally and maximally effective doses of the gonadotropin in a concentration-related manner. Similarly, the phorbol ester inhibited the steroidogenic responses to forskolin and dibutyryl cyclic AMP. By comparison, TPA had no appreciable effect on the metabolism of exogenous pregnenolone substrate to progesterone. Our data indicate that the tumor-promoting phorbol ester influences steroidogenic steps distal to cyclic AMP generation but at or before pregnenolone formation, and that protein kinase C may be a negative regulator of steroid biosynthesis in chicken granulosa cells.     

Granulosa cells from pig follicles of different sizes demonstrate maturational differences in their steroidogenic responses to FSH, calcium ionophore A23187, and phorbol diester

Cultures of granulosa cells from small (less than 3 mm), medium (3-6 mm), or large (8-10 mm) pig follicles were treated as follows: (1) basal controls, (2) cyclic adenosine 3',5'-monophosphate (cAMP) pathway agonists (pig FSH: 100 ng/ml; forskolin: 10 microM; dibutyryl cAMP; 1 mM), (3) calcium ionophore A23187 (0.005-1 micrograms), or (4) phorbol 12-myristate 13-acetate (TPA; 0.05-4 ng/ml). The combination of A23187 or TPA together with cAMP agonists was also examined in cultures of granulosa cells from follicles of different sizes. All substances were added at the time of culture, and oestradiol and progesterone were measured in the culture media after 48 h. All cAMP agonists were most potent in their stimulation of steroidogenesis (as a % of control) in cells from small follicles (P less than 0.05) with the exception of forskolin, which increased oestradiol in cells from large follicles to a greater extent than in cells of small follicles (P less than 0.05) (cells from medium follicles demonstrated less stimulation than those from small follicles except in progesterone production, for which FSH was equipotent). With the exception of forskolin, however, granulosa from large follicles showed little (oestradiol) or no stimulation (progesterone) with cAMP agonists. Under basal conditions, A23187 inhibited progesterone in all groups (P less than 0.05), and oestradiol production was reduced in granulosa cells from small follicles (P less than 0.05), unchanged in cells from medium follicles, and significantly stimulated in cells from large follicles. A23187 inhibited the enhanced production of both hormones after administration of cAMP agonists from cells of small and medium follicles (P less than 0.05), with inhibition significantly greater in cells of small follicles compared with medium. In cells from large follicles challenged with cAMP agonists, A23187 inhibited progesterone but stimulated oestradiol production; substitution of TPA (a protein kinase C stimulator) for A23187 gave identical results under basal or FSH-treated cultures of granulosa cells from small-, medium- or large-sized follicles. Our results suggest that TPA, A23187 and cAMP agonists modulate steroidogenesis differently in pig granulosa cells, depending on the stage of maturation of the follicle. Oestradiol production in granulosa cells from large preovulatory follicles may come under the stimulatory control of regulators of protein kinase C as in follicles near ovulation.     

Tetradecanoyl phorbol acetate suppresses follicle-stimulating hormone-induced synthesis of the cholesterol side-chain cleavage enzyme complex in rat ovarian granulosa cells

The effect of tetradecanoylphorbol acetate (TPA) on follicle-stimulating hormone (FSH)-induced synthesis of the cholesterol side-chain cleavage (SCC) enzyme complex was studied in rat ovarian granulosa cells cultured for 48 h in serum-free medium. Cell proteins were radiolabeled with [35S]methionine, followed by immunoprecipitation of cholesterol side-chain cleavage cytochrome P-450 (P-450SCC) as well as the iron-sulfur protein adrenodoxin. Polyacrylamide gel electrophoresis and fluorography of the immunoprecipitates showed that TPA, when added in combination with FSH (50 ng/ml) or dibutyryl cAMP (Bt2cAMP; 1 mM), suppressed the stimulatory effects of these compounds on the synthesis of the SCC components in a concentration-dependent fashion. The effect of TPA was accompanied by decreased progesterone formation and decreased cAMP accumulation. The structural analog of TPA, phorbol-4 alpha-didecanoate, which does not activate protein kinase C (Ca2+/phospholipid-dependent enzyme), had no effect on the FSH- or Bt2cAMP-stimulated synthesis of SCC and progesterone or on cAMP formation. In addition to inhibiting the synthesis of these proteins, TPA greatly reduced the FSH- and Bt2cAMP-induced increase in levels of mRNA encoding the precursor form of P-450SCC. It is concluded that the effect of the phorbol ester TPA to inhibit FSH-stimulated progesterone formation in cultured ovarian granulosa cells of the rat involves decreased synthesis of the components of the SCC enzyme complex due to reduced levels of mRNA encoding the precursor forms of these proteins. The results are indicative that TPA not only inhibits FSH-mediated stimulation of cAMP formation but also may block cAMP-mediated induction of SCC synthesis. It is postulated that the effects of TPA may reflect the physiological role of protein kinase C in the regulation of ovarian steroidogenesis.     

Differential actions of phorbol ester and diacylglycerol on inhibition of granulosa cell maturation

Hormonal induction of granulosa cell maturation is inhibited by phorbol esters and permeant synthetic diacylglycerols, but these activators of protein kinase C differ in their effects on cAMP production and actions. Both agents prevented the induction of luteinizing hormone receptors and progesterone biosynthesis by follicle-stimulating hormone, choleragen, and forskolin, but only diacylglycerol abolished the cAMP responses to these stimuli. Granulosa cell aggregation and aromatase activity were inhibited by phorbol ester but not completely by diacylglycerol. In intact granulosa cells, cytosolic C kinase activity was rapidly decreased by phorbol ester but unaffected by diacylglycerol. Although diacylglycerol has a marked inhibitory action on cAMP production, the more prominent suppression of granulosa cell differentiation by phorbol ester may be related to its rapid and prolonged action on kinase C.     

Activation of protein kinase C potentiates cyclic AMP production and stimulates steroidogenesis in differentiated ovarian granulosa cells

Gonadotropin-stimulated steroidogenesis in the differentiating ovarian granulosa cell is mediated through the activation of cAMP-dependent protein kinase, and is also modulated by calcium-dependent mechanisms. Granulosa cells contain calcium-activated, phospholipid-dependent protein kinase (C kinase), and show an increase in phosphatidylinositol turnover in response to GnRH agonist analogs. To evaluate the role of C kinase in ovarian steroidogenesis, the potent phorbol ester, TPA, and the permeant diacylglycerol, OAG, were used to activate C kinase in granulosa cells from PMSG-treated immature rats. Both TPA and OAG caused dose-dependent stimulation of progesterone production without affecting intra- or extracellular cAMP levels. However, the maximum steroid responses to these compounds were less than those stimulated by cAMP. The ED50 for TPA-stimulated progesterone production was 3 nM, which is close to the known Km for activation of C kinase. Stimulation of steroidogenesis was only observed with biologically-active phorbol esters and permeant diacylglycerols such as OAG and DOG. Exposure of granulosa cells to phospholipase C also increased progesterone production in a dose-dependent manner without changing the cAMP content. Although TPA and OAG did not increase basal cAMP production, both agents enhanced the cAMP responses stimulated by hCG and forskolin; likewise, phospholipase C alone did not change cAMP production but caused a dose-dependent increase in the cAMP responses to hCG and forskolin. These results demonstrate that activation of C kinase promotes steroidogenesis in ovarian granulosa cells, and potentiates the activation of adenylate cyclase by hCG and forskolin. Such findings support the possibility that the calcium, phospholipid-dependent enzyme could be involved in the regulation of progesterone production by hormonal ligands such as gonadotropins and GnRH.     

Enhancement of adenylate cyclase activity in S49 lymphoma cells by phorbol esters. Putative effect of C kinase on alpha s-GTP-catalytic subunit interaction

Addition of 12-O-tetradecanoylphorbol-13-acetate (TPA) to S49 lymphoma cells (wild type and a cyclic AMP-dependent protein kinase-lacking clone) has little effect alone but doubles accumulation of cyclic AMP in response to isoproterenol. The effect is immediate and has an apparent affinity and order of potency characteristic of the activation of protein kinase C by phorbol esters. Enhancement does not reflect an altered time course of the beta-adrenergic response, enhanced affinity of the cellular beta-receptor for agonist, or decreased degradation and export of cellular cyclic AMP. Reduction of the beta-adrenergic response by somatostatin does not remove the effect of TPA nor does TPA abolish the effect of somatostatin. Phorbol ester enhances cyclic AMP accumulation in response to cholera toxin in wild type and UNC clones but not in H21a or cyc-. TPA also enhances cAMP accumulation in response to forskolin in wild type cells. The effect of TPA is stable to rapid preparation of membranes. In adenylate cyclase assays on membranes from cells treated with TPA, the activation by guanosine 5'-(beta, gamma-imino)triphosphate is enhanced by 40% with no change in lag time; the effect of beta-agonist plus Gpp(NH)p is similarly enhanced; activation by Mn2+ is unchanged. We conclude that phorbol ester facilitates the productive interaction of the alpha subunit of the transducer protein Gs with the catalytic unit of adenylate cyclase, hypothetically via an action of protein kinase C.     

Activation of protein kinase C sensitizes the cyclic AMP signalling system of T51B rat liver cells

Activation of protein kinase C (PKC) bu phorbol esters (TPA) results in a modification of the cyclic AMP system leading to either attenuation or amplification of the cyclic AMP signal. In the non-neoplastic T51B rat live cell line, TPA, when added to intact cells, had no effect on the basal level of cyclic AMP synthesis but caused a 1.5 fold amplification of the stimulation induced by β-adrenergic agents, cholera toxin and forskolin. The effect appeared to be mediated by PKC since diacylglycerols caused the same amplification as did TPA while inactive phorbol esters were without effect. Phosphorylation of Gs or the catalytic subunit of adenylate cyclase by PKC is likely to be responsible for the enhancement of cyclic AMP synthesis. TPA also caused translocation of PKC; however, the time course of the translocation was loner than the time course of the enhancement of adenylate cyclase activity. Thus, the ability of TPA to amplify cyclic AMP synthesis is probably mediated by activation of PKC that is already present in the membrane.     

Evidence for a functionally active inhibitory guanine nucleotide-binding regulatory protein in the swine ovary

Incubation of particulate fractions of swine granulosa cells or luteal minces with purified pertussis toxin (islet-activating protein) and [32P]-NAD catalyzed the (32P)-ADP ribosylation of a 41,000 dalton membrane protein. ADP-ribosylation was markedly reduced by prior incubation of intact cells with toxin. The functional relevance of this presumptive inhibitory guanine nucleotide-binding protein in pig granulosa cells was indicated by the ability of prior treatment with pertussis toxin to increase cyclic AMP generation and progesterone production significantly in response to follicle stimulating hormone. Prior cellular intoxication also enhanced cyclic AMP production stimulated by luteinizing hormone and choleratoxin, but not basally or after forskolin. These results demonstrate the presence of an inhibitory guanine nucleotide-binding protein in both the follicular (granulosa cell) and luteal compartments of the mammalian ovary, and indicate its functional relevance in cyclic AMP generation and progesterone secretion.     

Phorbol esters enhance basal and stimulated adenylate cyclase activity in a pituitary cell line

We reported in anterior pituitary cells that hormone stimulation of cyclic AMP levels is amplified by agents that activate protein kinase C (e.g., phorbol esters). We utilized the 235-1 pituitary cell line to explore the mechanism of this response. PGE1- and forskolin-stimulated cyclic AMP accumulation and adenylate cyclase activity are enhanced by exposing viable cells to phorbol esters. Adenylate cyclase activity in the presence of PGE1 demonstrated a biphasic stimulatory, then inhibitory response to increasing GTP concentrations; phorbol esters attenuated this inhibition. These data support the hypothesis that protein kinase C can covalently change the functional state of the adenylate cyclase holoenzyme, amplifying its response to certain hormones.     

Suppression of cyclic AMP-dependent protein kinase activity in murine melanoma cells by 12-0-tetradecanoylphorbol-13-acetate

The effect of the potent tumor promoter 12-0-tetradeconylphorbol-13-acetate (TPA) on the cyclic AMP metabolism of B16 mouse melanoma cells was examined. TPA (10^?7M) slightly increased the growth rate and inhibited melanin production by these cells. Although TPA had little effect on basal or hormone stimulated cyclic AMP levels, it did significantly suppress cyclic AMP-dependent protein kinase activity from treated cells in a dose-dependent fashion. Other phorbol ester and non-phorbol ester tumor promoters also suppressed cyclic AMP-dependent protein kinase activity while the non-promoter, phorbol, did not alter cyclic AMP-dependent protein kinase activity.     

Activation of protein kinase C is coupled to prostaglandin E2 synthesis in swine granulosa cells

We have examined the role of phospholipid-sensitive calcium-dependent protein kinase (protein kinase C) in prostaglandin E2 synthesis by monolayer cultures of swine granulosa cells. Specific phorbol ester derivatives known to activate protein kinase C significantly augmented the production of prostaglandin E2. These stimulatory actions were dose and time-dependent, and could be abolished by the cyclooxygenase inhibitor, indomethacin, or the protein synthesis inhibitor, cycloheximide. Moreover, the rank order of potency of phorbol esters in enhancing prostaglandin E2 production was concordant with that demonstrated for activation of protein kinase C. Phorbol ester in conjunction with the divalent cation ionophore, A23187, increased prostaglandin E2 production synergistically. In addition, a non-phorbol stimulator of protein kinase C, 1-octanoyl-2-acetylglycerol, also significantly enhanced prostaglandin E2 biosynthesis. The stimulated synthesis of prostaglandin E2 was confirmed by high-pressure liquid chromatographic purification of this radiolabeled metabolite of 3H-arachidonic acid, and by capillary gas chromatography high-resolution mass spectrometry. Thus, the present studies indicate that the protein kinase C effector pathway is functionally coupled to prostaglandin E2 production in the swine granulosa cell.     

Regulation of the corpus luteum by protein kinase C. I. Phosphorylation activity and steroidogenic action in large and small ovine luteal cells

The activity and steroidogenic action of protein kinase C were evaluated in small and large steroidogenic ovine luteal cells. Protein kinase C activity (per mg protein) was threefold greater in large than in small luteal cells, whereas protein kinase A activity was similar in the two cell types. Phorbol 12-myristate 13-acetate (PMA) activated protein kinase C in luteal cells as demonstrated by membrane association of 91% of available protein kinase C within 15 min of PMA treatment. Longer treatments with PMA produced cells with low protein kinase C activity (protein kinase C-deficient cells) but did not affect cellular viability or protein kinase A activity. Activation of protein kinase C caused an acute, dose-dependent inhibition of progesterone production in unstimulated large and luteinizing hormone (LH)-stimulated small luteal cells. This inhibition by PMA appeared to be specific for protein kinase C since it was greatly attenuated in protein kinase C-deficient cells and since an inactive phorbol ester, 4 alpha-phorbol, had no effect on luteal progesterone production. The inhibitory locus of protein kinase C action in small luteal cells appeared to be distal to the adenylate cyclase enzyme because progesterone production was inhibited similarly in cells stimulated with LH, forskolin, or dibutyryl cyclic adenosine 3',5'-monophosphate. Cholesterol side-chain cleavage activity, as measured by metabolism of 25-hydroxycholesterol, was inhibited by PMA in large, but not in small, luteal cells. These data indicate that activation of protein kinase C specifically inhibits progesterone production in both large and small ovine luteal cells, although the intracellular mechanisms invoked appear to differ in the two cell types.     

Stimulation by glucose of cyclic AMP accumulation in mouse pancreatic islets is mediated by protein kinase C.

The mechanism of glucose-stimulated cyclic AMP accumulation in mouse pancreatic islets was studied. In the presence of 3-isobutyl-1-methylxanthine, both glucose and the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA), an activator of protein kinase C, enhanced cyclic AMP formation 2.5-fold during 60 min of incubation. Both TPA-stimulated and glucose-stimulated cyclic AMP accumulations were abolished by the omission of extracellular Ca2+. The Ca2+ ionophore A23187 did not affect cyclic AMP accumulation itself, but affected the time course of TPA-induced cyclic AMP accumulation, the effect of A23187 + TPA mimicking the time course for glucose-induced cyclic AMP accumulation. A 24 h exposure to TPA, which depletes islets of protein kinase C, abolished the effects of both TPA and glucose on cyclic AMP production. Both TPA-induced and glucose-induced cyclic AMP productions were inhibited by anti-glucagon antibody, and after pretreatment with this antibody glucose stimulation was dependent on addition of glucagon. Pretreatment of islets with TPA for 10 min potentiated glucagon stimulation and impaired somatostatin inhibition of adenylate cyclase activity in a particulate fraction of islets. Carbamoylcholine, which is supposed to activate protein kinase C in islets, likewise stimulated cyclic AMP accumulation in islets. These observations suggest that glucose stimulates islet adenylate cyclase by activation of protein kinase C, and thereby potentiates the effect of endogenous glucagon on adenylate cyclase.     

Effects of pretreatment with tetradecanoyl phorbol acetate on regulation of growth hormone and prolactin secretion from ovine anterior pituitary cells

Tetradecanoyl phorbol acetate (TPA) stimulates growth hormone (GH) and prolactin secretion from ovine anterior pituitary cells. Pretreatment of the cells with TPA abolishes this effect, presumably due to down-regulation of protein kinase C. Such pretreatment did not alter effects of thyrotropin-releasing hormone or dopamine on prolactin secretion, suggesting no involvement of protein kinase C. Pretreatment with TPA attenuated actions of GH-releasing hormone on GH release (but not actions on cyclic AMP levels), possibly due to depletion of cellular stores of GH. Such pretreatment also attenuated inhibition of GH release by somatostatin, possibly due to phosphorylation of receptors or associated proteins by protein kinase C.     

Evidence that the mitochondrial activator of phosphorylated branched-chain 2-oxoacid dehydrogenase complex is the dissociated E1 component of the complex

The ability of glucagon (10 nM) to increase hepatocyte intracellular cyclic AMP concentrations was reduced markedly by the tumour-promoting phorbol ester TPA (12-O-tetradecanoyl phorbol-13-acetate). The half-maximal inhibitory effect occurred at 0.14 ng/ml TPA. This action occurred in the presence of the cyclic AMP phosphodiesterase inhibitor isobutylmethylxanthine (1 mM) indicating that TPA inhibited glucagon-stimulated adenylate cyclase activity. TPA did not affect either the binding of glucagon to its receptor or ATP concentrations within the cell. TPA did inhibit the increase in intracellular cyclic AMP initiated by the action of cholera toxin (1 microgram/ml) under conditions where phosphodiesterase activity was blocked. TPA did not inhibit glucagon-stimulated adenylate cyclase activity in a broken plasma membrane preparation unless Ca2+, phosphatidylserine and ATP were also present. It is suggested that TPA exerts its inhibitory effect on adenylate cyclase through the action of protein kinase C. This action is presumed to be exerted at the point of regulation of adenylate cyclase by guanine nucleotides.     

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.     

Glucocorticoid stimulation of amnion cell prostaglandin synthesis: suppression by protein kinase C inhibitors and independence of phorbol ester-sensitive protein kinase C.

Glucocorticoids stimulate the prostaglandin E2 production of confluent amnion cell cultures, but have no stimulatory effect on the PGE2 output of freshly isolated human amnion cells. Since protein phosphorylation may modify the responsiveness of target cells to steroids, and activators of protein kinase C (PKC), as well as corticosteroids, promote amnion cell PGE2 output by stimulating the synthesis of prostaglandin endoperoxide H synthase (PGHS), we investigated the possibility that PKC is involved in the glucocorticoid-induction of PGE2 synthesis in cultured amnion cells. The dexamethasone-induced PGE2 output of arachidonate-stimulated cells was blocked by the protein kinase inhibitors staurosporine, K-252a, H7, HA1004, and sphinganine, in a manner consistent with their effect on PKC. However, dexamethasone increased the PGE2 production of cultures treated with maximally effective concentrations of the PKC-activator compound TPA. Moreover, dexamethasone stimulated PGE2 synthesis in cultures which were desensitized to TPA-stimulation by prolonged phorbol ester treatment. Concentration-dependence studies showed that staurosporine completely (greater than 95%) blocked glucocorticoid-provoked PGE2 synthesis at concentrations which did not inhibit TPA-stimulated prostaglandin output, and that K-252a inhibited the effect of TPA by more than 95% at concentrations which decreased the effect of dexamethasone only moderately (approximately 40%). Dibutyryl cyclic AMP had no influence on the basal- or dexamethasone-stimulated PGE2 production, and on the staurosporine inhibition of the steroid effect. These results show that glucocorticoids and phorbol esters control amnion PGE2 production by separate regulatory mechanisms. It is suggested that the response of human amnion cells to glucocorticoids is modulated by protein kinase(s) other than phorbol ester-sensitive PKC and cyclic AMP-dependent protein kinase.     

Evidence that arachidonic acid influences hen granulosa cell steroidogenesis and plasminogen activator activity by a protein kinase C-independent mechanism

We recently proposed that arachidonic acid serves as a second messenger within granulosa cells from the largest preovulatory follicle of the hen. The present studies were conducted to determine whether the inhibitory effects of arachidonic acid on LH-induced cAMP accumulation and on the ability of cells to convert 25-hydroxycholesterol to progesterone are mediated via the protein kinase C pathway. Furthermore, we determined the effects of arachidonic acid on plasminogen activator activity in granulosa cells. In the first experiment, the putative protein kinase C inhibitor, staurosporine, completely reversed the inhibitory effects of phorbol 12-myristate 13-acetate (PMA) on LH-promoted cAMP formation, but failed to overcome the inhibitory effects of arachidonic acid. Prolonged pretreatment (18 h) with 1.6 microM PMA depleted granulosa cells of both cytosolic and membrane-associated protein kinase C, and subsequently attenuated the inhibitory effects of PMA on LH-induced progesterone production; however, such depletion did not alter the inhibitory effects of phospholipase A2 (PLA2; an agent that increases intracellular levels of arachidonic acid). PMA, but not arachidonic acid, caused a rapid (within 2 min) translocation of protein kinase C from the cytosol to the membrane (a characteristic of agents that activate protein kinase C). Finally, both arachidonic acid and PLA2 inhibit plasminogen activator (PA) activity in a dose-dependent fashion, whereas activation of protein kinase C with PMA stimulates PA activity. Taken together, the data suggest that the effects of arachidonic acid in granulosa cells can occur independently of protein kinase C activation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Activation of protein kinase C is coupled to prostaglandin E2 synthesis in swine granulosa cells

We have examined the role of phospholipid-sensitive calcium-dependent protein kinase (protein kinase C) in prostaglandin E₂ synthesis by monolayer cultures of swine granulosa cells. Specific phorbol ester derivatives known to activate protein kinase C significantly augmented the production of prostaglandin E₂. These stimulatory actions were dose and time-dependent, and could be abolished by the cyclooxygenase inhibitor, indomethacin, or the protein synthesis inhibitor, cycloheximide. Moreover, the rank order of potency of phorbol esters in enhancing prostaglandin E₂ production was concordant with that demonstrated for activation of protein kinase C. Phorbol ester in conjunction with the divalent cation ionophore, A23187, increased prostaglandin E₂ production synergistically. In addition, a non-phorbol stimulator of protein kinase C, 1-octanoyl-2-acetylglycerol, also significantly enhanced prostaglandin E₂ biosynthesis. The stimulated synthesis of prostaglandin E₂ was confirmed by high-pressure liquid chromatographic purification of this radiolabeled metabolite of ³H-arachidonic acid, and by capillary gas chromatography high-resolution mass spectrometry. Thus, the present studies indicate that the protein kinase C effector pathway is functionally coupled to prostaglandin E₂ production in the swine granulosa cell.