Transmembrane signals mediating neural peptide secretion: role of protein kinase C activators and arachidonic acid metabolites in luteinizing hormone-releasing hormone secretion

The present experiments were designed to determine the effects of different activators of protein kinase C on the secretion of LHRH from median eminence nerve terminals incubated in vitro. The release of prostaglandin E2 (PGE2), a metabolite of arachidonic acid intimately involved in the secretion of LHRH, was also evaluated. Synthetic diacylglycerol [1,2-didecanoylglycerol (DiC10)] significantly enhanced PGE2 release in a concentration-dependent manner. Blockade of phospholipase A2 (PLA2) activity nullified this effect. LHRH release, on the other hand, was not increased by DiC10. However, in the presence of a lipoxygenase inhibitor, DiC10 produced a concentration-related increase in LHRH release, which paralleled that in PGE2. Phospholipase C (PLC) increased both PGE2 and LHRH secretion. Again, blockade of the lipoxygenase pathway enhanced the release of LHRH by PLC without affecting the stimulated secretion of PGE2. A phorbol ester, phorbol 12,13-dibutyrate (PDBu), markedly increased LHRH secretion while inducing a modest increase in PGE2 release. Both effects of PDBu were unaffected by lipoxygenase inhibition. DiC10, PDBu, and PLC significantly augmented LHRH secretion from tissues in which metabolism of arachidonic acid had been prevented by inhibition of both cyclooxygenase and lipoxygenase pathways, suggesting that activation of protein kinase C, independent of PLA2 activation, can lead to the secretion of this neural peptide. Some lipoxygenase metabolites had either no effect on [5- and 15-hydroxyeicosatetraenoic (5- and 15-HETE)] or induced a marginal stimulation of (12-HETE) LHRH release. At certain concentrations, 12-HETE enhanced the stimulatory effect of the phorbol ester on LHRH release. Our results suggest that activation of protein kinase C can stimulate LHRH secretion from nerve terminals in vitro and, further, that diacylglycerol may represent an important intracellular messenger participating in the events leading to LHRH secretion. In addition, stimulation with DiC10 and PLC uncovered inhibitory [unknown arachidonic acid metabolite(s) via lipoxygenase] and stimulatory (PGE2 via cyclooxygenase) pathways through with arachidonic acid metabolites may participate in the intracellular transduction of signals modulating neural peptide secretion.     

Ethanol inhibits luteinizing hormone-releasing hormone (LHRH) secretion by blocking the response of LHRH neuronal terminals to nitric oxide.

It has previously been shown that alcohol can suppress reproduction in humans, monkeys, and small rodents by inhibiting release of luteinizing hormone (LH). The principal action is via suppression of the release of LH-releasing hormone (LHRH) both in vivo and in vitro. The present experiments were designed to determine the mechanism by which alcohol inhibits LHRH release. Previous research has indicated that the release of LHRH is controlled by nitric oxide (NO). The proposed pathway is via norepinephrine-induced release of NO from NOergic neurons, which then activates LHRH release. In the present experiments, we further evaluated the details of this mechanism in male rats by incubating medial basal hypothalamic (MBH) explants in vitro and examining the release of NO, prostaglandin E2 (PGE2), conversion of arachidonic acid to prostanoids, and production of cGMP. The results have provided further support for our theory of LHRH control. Norepinephrine increased the release of NO as measured by conversion of [14C]arginine to [14C]citrulline, and this increase was blocked by the alpha 1 receptor blocker prazosin. Furthermore, the release of LHRH induced by nitroprusside (NP), a donor of NO, is related to the activation of soluble guanylate cyclase by NO since NP increased cGMP release from MBHs and cGMP also released LHRH. Ethanol had no effect on the production of NO by MBH explants or the increased release of NO induced by norepinephrine. Therefore, it does not act at that step in the pathway. Ethanol also failed to affect the increase in cGMP induced by NP. On the other hand, as might be expected from previous experiments indicating that LHRH release was brought about by PGE2, NP increased the conversion of [14C]arachidonic acid to its metabolites, particularly PGE2. Ethanol completely blocked the release of LHRH induced by NP and the increase in PGE2 induced by NP. Therefore, the results support the theory that norepinephrine acts to stimulate NO release from NOergic neurons. This NO diffuses to the LHRH terminals where it activates guanylate cyclase, leading to an increase in cGMP. At the same time, it also activates cyclooxygenase. The increase in cGMP increases intracellular free calcium, activating phospholipase A2 to provide arachidonic acid, the substrate for conversion by the activated cyclooxygenase to PGE2, which then activates the release of LHRH. Since alcohol inhibits the conversion of labeled arachidonic acid to PGE2, it must act either directly to inhibit cyclooxygenase or perhaps it may act by blocking the increase in intracellular free calcium induced by cGMP, which is crucial for activation of of both phospholipase A2 and cyclooxygenase.     

Protein kinase C activation enhances cAMP accumulation in Swiss 3T3 cells: inhibition by pertussis toxin.

Addition of phorbol 12,13-dibutyrate (PBt2) in the presence of forskolin or cholera toxin caused marked (6- to 8-fold) and rapid accumulation of cAMP in Swiss 3T3 cells. The effect of PBt2 is mediated by protein kinase C because the synthetic diacylglycerol 1-oleoyl-2 acetylglycerol substitutes for PBt2 in enhancing cAMP accumulation and because the enhancing effect of either PBt2 or 1-oleoyl-2-acetylglycerol was prevented by down-regulation of protein kinase C. Vasopressin, which activates protein kinase C but does not directly affect adenylate cyclase in 3T3 cells, also enhanced cAMP accumulation in cells treated with cholera toxin or forskolin. This effect was abolished by down-regulation of protein kinase C. Treatment with pertussis toxin blocked the enhancing effect of PBt2 in a concentration- and time-dependent manner. Pertussis toxin neither prevented protein kinase C activation by PBt2 nor other biologic responses elicited by PBt2. The results presented here suggest an unusual function for a pertussis toxin substrate--namely, coupling protein kinase C activation to cAMP production.     

Stimulation of cyclic adenosine 3',5'-monophosphate production enhances hypothalamic luteinizing hormone-releasing hormone release without increasing prostaglandin E2 synthesis: studies in prepubertal female rats

A role for cAMP in the process of LHRH release was suggested several years ago, but only recently has the validity of this notion come under close scrutiny. In the present experiments we have used three probes, which stimulate adenylate cyclase activity via different mechanisms, to determine whether an increase in endogenous cAMP results in LHRH release from the hypothalamus of prepubertal female rats. Median eminences from juvenile, 28-day-old animals were incubated in vitro with either forskolin (F), cholera toxin (CT), or pertussis toxin (PT). All three substances enhanced LHRH release. The estimated ED50 values were 28.7 microM and 20.0 ng/ml, for F and PT, respectively. The effect of CT appeared biphasic and thus no ED50 could be calculated. None of these agents increased the release of prostaglandin E2 (PGE2), an obligatory component in the process of norepinephrine-induced LHRH secretion. Doses of PGE2 and F, which were maximally effective in stimulating LHRH release when administered separately, did not produce any further response when administered concomitantly, thus suggesting that PGE2 and F act along a common pathway. Blockade of phosphodiesterase activity with 1-methyl-3-isobutylxanthine increased LHRH secretion without enhancing PGE2 release, implying that cAMP metabolism was elevated in the median eminence nerve terminals in vitro. Addition of 1-methyl-3-isobutylxanthine augmented the LHRH response to CT and PT, but it did not increase further the already marked LHRH response to PGE2 or F. The results indicate that both an increase in adenylate cyclase activity and a decrease in phosphodiesterase activity lead to LHRH release from the median eminence. They also suggest that, upon proper (neurotransmitter?) stimulation, cAMP production increases subsequent to the activation in PGE2 synthesis, which itself causes LHRH release. Furthermore, the capacity of PT to induce LHRH release suggests the involvement of an inhibitory guanine nucleotide-binding regulatory protein in transducing inhibitory inputs impinging on LHRH-secreting neurons.     

Modification of luteinizing hormone secretion by activators of Ca2+/phospholipid-dependent protein kinase

We investigated the role of Ca2+/phospholipid-dependent protein kinase (protein kinase C) in LH secretion using rat anterior pituitary pieces obtained at known stages of the estrous cycle and superfused in vitro. Secretagogues were administered as 10-min (LHRH) or 30-min (all others) pulses. Activation of protein kinase C with phorbol 12-myristate 13-acetate (PMA) results 2 h later in an amplification of LHRH-induced LH secretion in a concentration (1 nM to 1 microM)-and protein synthesis-dependent manner in proestrous, but not estrous, pituitaries; the diacylglycerol analog 1-oleoyl-2-acetylglycerol (OAG) also augments subsequent LHRH-induced secretion. At 1 microM, PMA alone increases the LH secretory rate, but with a pattern different from that induced by LHRH; the characteristics of the PMA response are affected by prior exposure to LHRH, estrous cycle stage, and cycloheximide. Pretreatment with either 8-bromo-cAMP or forskolin results in augmentation of subsequent LHRH-induced secretion without affecting baseline secretion. If the cells are exposed simultaneously to forskolin and OAG, but not 8-bromo-cAMP and OAG, the augmentation is dampened. This preliminary result suggests a possible interaction between protein kinase C and cAMP-dependent protein kinase in LH secretion regulation. We conclude that, regarding initiation of LH release, protein kinase C appears to be but one of a complex of mediators required for the secretory response to LHRH. Regarding the amplification of LHRH-induced release, activation of protein kinase C may be a component of the LHRH self-priming response.     

Nitric oxide mediates norepinephrine-induced prostaglandin E2 release from the hypothalamus.

Nitric oxide (NO), formed by conversion of arginine to citrulline and NO by NO synthase, mediates relaxation of vascular smooth muscle. NO synthase has been demonstrated by immunocytochemical methods in neurons in various parts of the central nervous system including the hypothalamus. The latter finding suggested to us that NO might play a role in controlling the release of hypothalamic peptides. We have previously shown that norepinephrine mediates the release of luteinizing hormone-releasing hormone (LHRH) from LHRH terminals in the median eminence into the hypophyseal portal veins, which transport LHRH to the anterior pituitary gland to trigger release of luteinizing hormone from gonadotrophs. LHRH release from these terminals requires increased release of prostaglandin E2 (PGE2). PGE2 activates adenylate cyclase to produce cAMP, and then cAMP induces the exocytosis of LHRH secretory granules. In view of the evidence above and because of the developing evidence for the importance of NO in the central nervous system, it occurred to us that NO might be involved in this process. Consequently, we evaluated the role of NO in the release of PGE2 from medial basal hypothalamic fragments. As previously reported, norepinephrine (10 microM) increased PGE2 release from the hypothalamic fragments. The inhibitor of NO synthase NG-monomethyl-L-arginine (NMMA, 300 microM) blocked the stimulation of PGE2 release induced by norepinephrine but had no effect on the basal release of PGE2. Sodium nitroprusside (100 microM), which liberates NO, also elevated PGE2 release from the hypothalamic fragments. This elevation was not affected by NMMA, presumably because NMMA blocks enzymatic generation of NO but does not alter NO liberated by nitroprusside. When the NO liberated by nitroprusside was inactivated by hemoglobin (2 micrograms/ml), the effect of nitroprusside on PGE2 release was completely inhibited. Neither NMMA nor hemoglobin altered the basal release of PGE2, which indicates that NO is not responsible for basal PGE2 release. Addition of L-arginine (10 microM to 1 mM), the substrate for NO synthase, had no effect on basal PGE2 production. These results indicate that NO synthase is not activated in unstimulated hypothalamic fragments in vitro. The results suggest that norepinephrine activates NO synthase leading to the production of NO, which subsequently activates cyclooxygenase and results in the production of PGE2. PGE2 then activates adenylate cyclase leading to generation of increased cAMP, which induces exocytosis of secretory granules of LHRH and other neuropeptides released by PGE2. The indication that NO is essential to norepinephrine-induced release of PGE2 from hypothalamic fragments provides insight into the mechanism of LHRH release and the results open the possibility that the importance of NO to neuronal functions may be widespread in the nervous system.     

Protein kinase C activators and calcium-mobilizing agents synergistically increase GH, LH, and TSH secretion from anterior pituitary cells

A series of studies was designed to determine the effects of protein kinase C activators on TSH, LH, and GH release from anterior pituitary cells. A 15-min incubation of cultured pituitary cells with synthetic diacylglycerol or phorbol myristate acetate, stimulators of protein kinase C, increased GH, LH, and TSH release. Similarly phospholipase C, which liberates endogenous diacylglycerol, stimulated GH, LH, and TSH secretion. The potentiation of the effects of protein kinase C activators is achieved by calcium mobilization in various cell types. The results of the present studies show that calcium ionophore A23187 or calcium channel activator maitotoxin potentiate diacylglycerol-, phorbol ester-, or phospholipase C-induced GH, LH, or TSH release. These findings suggest that activation of protein kinase C by diacylglycerol and mobilization of calcium may be synergistically involved in the regulation of GH, LH, and TSH release.     

Possible involvement of adenylyl cyclase-cAMP-protein kinase a pathway in somatostatin inhibition of growth hormone release from chicken pituitary cells.

Somatostatin (SRIF) reduces growth hormone releasing hormone (GRF)-stimulated growth hormone (GH) release from avian and mammalian adenohypophyseal cells. The present studies examined the intracellular mechanisms mediating SRIF inhibition of GRF-stimulated GH release from chicken pituitary cells. Increases (P less than 0.05) in GH release were observed in the presence of (1) GRF; (2) the adenylyl cyclase stimulator, forskolin; (3) a cAMP analog, 8-bromo-cAMP; (4) the phosphodiesterase inhibitor 3-isobutyl-l-methyl-xanthine (IBMX) combined with GRF; (5) a tumor-promoting phorbol ester and protein kinase C activator, phorbol 12-myristate, 13-acetate (PMA); (6) a diacylglycerol analog, 1,2-dioctanoyl-glycerol (DiC8); and (7) a calcium ionophore, A23187, alone and in combination with PMA. Somatostatin (10 ng/ml) reduced the release of GH stimulated by GRF, forskolin, and 8-bromo cAMP and the GRF-provoked release of GH in the presence of IBMX (P less than 0.05). Somatostatin, however, did not influence GH release in the presence of the protein kinase C activators, PMA or DiC8, or the calcium ionophore A23187. These data suggest that SRIF inhibits GRF-provoked GH release by reducing the ability of the cAMP-protein kinase A but not of the calcium or protein kinase C intracellular message pathways to stimulate GH release.     

Regulation of protein kinase C by extracellular calcium in bovine parathyroid cells.

Regulation of protein kinase C in the parathyroid gland was investigated by testing the effects of phorbol ester, exogenous phospholipase C, and low and high calcium concentrations on enzyme activity. Treatment of bovine parathyroid cells with phorbol ester, which activates protein kinase C directly, and with phospholipase C, which produces diacylglycerol, an activator of protein kinase C, significantly stimulated protein kinase C activity. Both agents also enhanced the release of parathyroid hormone. Acute exposure of bovine parathyroid cells to low extracellular calcium (0.5 mM) caused a 5- to 6-fold increase in protein kinase C activity associated with the particulate fraction. In contrast, high extracellular calcium (1.75 mM and 2.5 mM) markedly decreased membrane protein kinase C activity. These data suggest that the effects of extracellular calcium on parathyroid hormone secretion are due, at least in part, to regulation of protein kinase C activity in the parathyroid-cell membrane.     

Inhibition of gonadotropin-induced granulosa cell differentiation by activation of protein kinase C.

The induction of granulosa cell differentiation by follicle-stimulating hormone (FSH) is characterized by cellular aggregation, expression of luteinizing hormone (LH) receptors, and biosynthesis of steroidogenic enzymes. These actions of FSH are mediated by activation of adenylate cyclase and cAMP-dependent protein kinase and can be mimicked by choleragen, forskolin, and cAMP analogs. Gonadotropin releasing hormone (GnRH) agonists inhibit these maturation responses in a calcium-dependent manner and promote phosphoinositide turnover. The phorbol ester phorbol 12-myristate 13-acetate (PMA) also prevented FSH-induced cell aggregation and suppressed cAMP formation, LH receptor expression, and progesterone production, with an ID50 of 0.2 nM. In FSH-treated cells, PMA did not reduce the initial increase in cAMP formation during the first 24 hr of culture but prevented its secondary increase from 24 to 48 hr. PMA also inhibited LH receptor induction by cholera toxin, forskolin, and 8-bromo-cAMP, but it did not impair cAMP responses to the former two agents, indicating that the site of action of the phorbol ester is distal to adenylate cyclase. The early stimulation of cAMP-dependent protein kinase activity by FSH was also unaffected by PMA, consistent with its lack of effect on the initial cAMP response to FSH. However, PMA caused a marked decrease in cytosolic protein kinase C activity within 1 min of its addition to the cells. The permeant diacylglycerols, 1-oleoyl-2-acetoyl-sn-glycerol and sn-1,2-dioctanoyl glycerol, also inhibited LH receptor formation, while the nonpermeant diacylglycerol, diolein, was inactive. These results indicate that in situ activation of protein kinase C by PMA or permeant diacylglycerols inhibits cAMP-dependent granulosa cell differentiation, and suggest that the inhibitory actions of GnRH agonists on granulosa cell maturation are also mediated by protein kinase C.     

Platelet activation by simultaneous actions of diacylglycerol and unsaturated fatty acids.

Several cis-unsaturated fatty acids such as oleic, linoleic, linolenic, eicosapentaenoic, and docosahexaenoic acids added directly to intact human platelets greatly enhance protein kinase C activation as judged by the phosphorylation of its specific endogenous substrate, a 47-kDa protein. This enhancement absolutely requires the presence of a membrane-permeant diacylglycerol, 1,2-dioctanoylglycerol, or a tumor-promoting phorbol ester, phorbol 12-myristate 13-acetate. In the presence of ionomycin and either 1,2-dioctanoylglycerol or phorbol 12-myristate 13-acetate, the release of serotonin from the platelets is also remarkably increased by cis-unsaturated fatty acids. The effect of these fatty acids is observed at concentrations less than 50 microM. Saturated fatty acids and trans-unsaturated fatty acids are inactive. Titration of ionomycin to induce a release reaction and measurement of the intracellular Ca2+ level by the fura-2 procedure indicate that cis-unsaturated fatty acids increase an apparent sensitivity of the platelet response to Ca2+. The results suggest that cis-unsaturated fatty acids, which are presumably produced from phosphatidylcholine by signal-dependent activation of phospholipase A2, may take part directly in cell signaling through the protein kinase C pathway.     

12-O-tetradecanoyl phorbol-13-acetate stimulates rat growth hormone (GH) release through different pathways from that of human pancreatic GH-releasing factor

The mechanism(s) of action of 12-O-tetradecanoyl phorbol-13-acetate (TPA) on rat (r) GH release was studied in primary rat pituitary cell cultures. TPA stimulated rGH release (3.2- to 4.1-fold above control value) and rTSH and rLH release (1.4- and 1.7-fold above control values, respectively), but not rPRL release. The ED50 of TPA on rGH secretion was 1.3 X 10(-9) M compared to 4.5 X 10(-11) M for human pancreatic GH-releasing factor [hpGRF-(1-44)]. If maximally effective doses of TPA or hpGRF-(1-44) were added to the cells, the magnitudes of the increase in rGH release were quite similar for both agents when the incubation period was less than 12 h. When (Bu)2cAMP was added simultaneously with various doses of TPA, (Bu)2cAMP increased rGH release beyond the maximal effect of TPA. There was an additive effect when hpGRF-(1-44) and TPA were used to stimulate rGH release. These results indicate that TPA enhances rGH release through a different pathway than hpGRF-(1-44). TPA failed to increase the formation of intra- and extracellular cAMP, whereas hpGRF-(1-44) increased both, suggesting that TPA stimulates rGH release through an cAMP-independent pathway(s). Protein kinase C has been postulated to be a receptor for TPA in human platelets. When phospholipase C, which activates protein kinase C via the formation of diacylglycerol, was added to the cells, rGH release was stimulated in a dose-dependent manner. This effect was not blocked by indomethacin. These results may suggest that activation of protein kinase C leads to rGH release. The observations are consistent with the hypothesis that TPA activates protein kinase C and causes the release of rGH in normal pituitary cells in culture. These findings indicate that the mechanism(s) of action of TPA on rGH release is different from that of hpGRF-(1-44).     

Dual pathways for carbamylcholine-stimulated arachidonic acid release in rat pancreatic acini

Recent studies suggested the involvement of arachidonic acid in the mediation of pancreatic amylase release. However, an effect of carbamylcholine on arachidonic acid release has not yet been reported in the exocrine pancreas. This study was performed to evaluate the effect of carbamylcholine on arachidonic acid release and determine the underlying intracellular mechanisms. From enzymatic assays, phospholipase A₂ and diacylglycerol lipase were activated by carbamylcholine and these activations were inhibited by the phospholipase A₂ inhibitors, mepacrine and aristolochic acid, and by the diacylglycerol lipase inhibitor RHC 80267. Carbamylcholine also increased arachidonic acid release in a concentration-dependent manner. Both phospholipase A₂ and diacylglycerol inhibitors partially inhibited carbamylcholine-stimulated arachidonic acid release. The phospholipase C inhibitor U73122 and the protein kinase C inhibitor staurosporine also caused partial inhibition. Arachidonic acid release by carbamylcholine was suppressed by the simultaneous addition of RHC 80267 with either phospholipase A₂ inhibitors. Our data demonstrate that phospholipase A₂ and diacylglycerol lipase are activated and arachidonic acid is released in pancreatic acini by carbamylcholine. Dual pathways are responsible for carbamylcholine-induced arachidonic acid release. One such pathway involves the sequential action of phospholipase C, protein kinase C and diacylglycerol lipase, whereas the other involves phospholipase A₂ activation.     

Potential role of phospholipase A2 in HL-60 cell differentiation to macrophages induced by protein kinase C activation.

2-Lysophosphatidylcholine and cis-unsaturated fatty acids such as linoleic and linolenic acids, which are the products of the hydrolysis of phosphatidylcholine catalyzed by phospholipase A2 (EC 3.1.1.4), significantly potentiate the differentiation of HL-60 cells to macrophages that is induced by either a membrane-permeant diacylglycerol or a phorbol ester. The cell differentiation was assayed by measuring the expression of CD11b, one of the cell surface markers of macrophages, and also by the appearance of phagocytic activity. Snake venom phospholipase A2 added directly to the cells is also active for this potentiation. Neither lysophosphatidylcholine, fatty acid, nor phospholipase A2 is active unless a membrane-permeant diacylglycerol or a phorbol ester is present. The results presented provide further evidence that activation of phospholipase A2 may be intimately related to the signal transduction pathway through protein kinase C.     

Ghrelin and growth hormone (GH) secretagogues potentiate GH-releasing hormone (GHRH)-induced cyclic adenosine 3',5'-monophosphate production in cells expressing transfected GHRH and GH secretagogue receptors

GHRH stimulates GH secretion from somatotroph cells of the anterior pituitary via a pathway that involves GHRH receptor activation of adenylyl cyclase and increased cAMP production. The actions of GHRH to release GH can be augmented by the synthetic GH secretagogues (GHS), which bind to a distinct G protein-coupled receptor to activate phospholipase C and increase production of the second messengers calcium and diacylglycerol. The stomach peptide ghrelin represents an endogenous ligand for the GHS receptor, which does not activate the cAMP signaling pathway. This study investigates the effects of GHS and ghrelin on GHRH-induced cAMP production in a homogenous population of cells expressing the cloned GHRH and GHS receptors. Each epitope-tagged receptor was shown to be appropriately expressed and to functionally couple to its respective second messenger pathway in this heterologous cell system. Although activation of the GHS receptor alone had no effect on cAMP production, coactivation of the GHS and GHRH receptors produced a cAMP response approximately twice that observed after activation of the GHRH receptor alone. This potentiated response is dose dependent with respect to both GHRH and GHS, is dependent on the expression of both receptors, and was observed with a variety of peptide and nonpeptide GHS compounds as well as with ghrelin-(1-5). Pharmacological inhibition of signaling molecules associated with GHS receptor activation, including G protein betagamma-subunits, phospholipase C, and protein kinase C, had no effect on GHS potentiation of GHRH-induced cAMP production. Importantly, the potentiation appears to be selective for the GHRH receptor. Treatment of cells with the pharmacological agent forskolin elevated cAMP levels, but these levels were not further increased by GHS receptor activation. Similarly, activation of two receptors homologous to the GHRH receptor, the vasoactive intestinal peptide and secretin receptors, increased cAMP levels, but these levels were not further increased by GHS receptor activation. Based on these findings, we speculate that direct interactions between the GHRH and GHS receptors may explain the observed effects on signal transduction.     

Protein kinase C, an endogenous regulator of hormone-induced cyclic AMP induction in porcine luteal cells.

LH, in addition to increasing cyclic AMP (cAMP) in ovarian cells, stimulates phosphoinositide hydrolysis producing inositol trisphosphate and diacylglycerol (DG). DG activates phospholipid- and calcium-dependent protein kinase (PKC). In the present study, we have used both PKC activators and inhibitors to examine the interactions of the PKC pathway on hormone-induced cAMP production in porcine luteal cells. Phorbol 12-myristate 13-acetate (PMA) enhanced LH- and forskolin-induced cAMP production. A time-course study indicated that the facilitatory effect of PMA was greater when added to incubation tubes following addition LH or forskolin. The non-tumour-promoting phorbol ester 4 alpha-phorbol 12,13-didecanoate, which does not stimulate PKC activation, did not facilitate hormone-induced cAMP induction. PKC inhibitors polymyxin B, sphingosine and 1-(5-isoquinolinesulphonyl)-2-methylpiperazine (H7) antagonized the facilitatory effect of PMA on LH-induced cAMP production. The cAMP induction by both LH and forskolin was inhibited in the presence of PKC inhibitors. Polymyxin E, which differs from polymyxin B by a single amino acid and does not inhibit PKC activation, did not inhibit LH- or forskolin-induced cAMP induction. The results of this study provide evidence for a facilitative action of the PKC effector system on hormonally stimulated cAMP production. Furthermore, PKC may be an important endogenous regulator of adenylate cyclase activity in porcine luteal cells.     

Calcium mobilization potentiates prolactin release induced by protein kinase C activators

The in vitro effect of synthetic diacylglycerol (DG) and phorbol myristate acetate (PMA), potent stimulators of protein kinase C, was studied on prolactin release. These substances increased, in a concentration-dependent manner, prolactin release from primary cultures of anterior pituitary cells. Similarly, exposure of pituitary cells to phospholipase C, which liberates endogenous DG from various substrates, also enhanced prolactin release. The effect of Ca²⁺ mobilization on PMA-, synthetic DG- or phospholipase C-induced prolactin release was examined. A23187 at 400 nM or 2 ng/ml maitotoxin, a Ca²⁺ channel activator, did not affect prolactin release by themselves, but enhanced the release of prolactin induced by DG, PMA or phospholipase C. The stimulatory effects of DG, PMA and phospholipase C on prolactin release were reduced by co-incubation with dopamine. These results suggest that the presumed activation of protein kinase C by DG and mobilization of Ca²⁺ may be synergistically involved in the regulation of prolactin release. Dopamine appears to inhibit prolactin release at a point distal to the DG-enhanced stimulation of the process.     

Pituitary adenylate cyclase polypeptide (PACAP) stimulates cyclic AMP formation in pituitary fibroblasts and 3T3 tumor fibroblasts: lack of enhancement by protein kinase C activation.

A number of neuropeptides were shown to produce potent mitogenic effects on Swiss 3T3 fibroblasts by activating the phospholipase C pathway. Here we provide evidence for the activation by PACAP of the adenylate cyclase pathway in 3T3, as well as in non-tumoral pituitary fibroblasts, similarly to what was seen in pituitary endocrine cells. In these cells, PACAP triggered elevation of both intracellular and extracellular contents of cAMP and the effect was time- and dose-dependent, with half-maximal stimulations being induced with about 0.1 nM. Following activation of protein kinase C (PKC) by the phorbol ester phorbol 12-myristate 13-acetate (PMA), PACAP-induced cAMP production was amplified in pituitary endocrine cells, but was either unchanged or dampened in 3T3 and pituitary fibroblasts, respectively. Pretreatment of cells with pertussis toxin (PT) failed to change the effect of PMA on PACAP-stimulated adenylate cyclase activity, irrespective of the cell type being used. However, PT dramatically reduced the potentiation by PMA of cAMP production enhanced by forskolin in 3T3 cells. These results provide new evidence pointing to the presence in fibroblasts of receptors for PACAP, coupled to cAMP production, which may play a role in the modulation of the mitogenic signal. They also indicate that, compared with pituitary endocrine cells, PKC activation in fibroblasts differentially affected PACAP-induced cAMP formation and that these effects were unaltered upon inhibition by PT of Gi-like proteins.     

Rapid formation of diacylglycerol from phosphatidylcholine: a pathway for generation of a second messenger.

The classic pathway for agonist-induced generation of diacylglycerol is via activation of a phospholipase C-mediated hydrolysis of the "phosphoinositides." We now report findings from a variety of cell types, which indicate that tumor-promoting phorbol diesters, serum, and platelet-derived growth factor activate within seconds the hydrolysis of phosphatidylcholine, as detected by the formation of diacylglycerol and phosphocholine. It is known that phorbol diesters do not stimulate hydrolysis of the phosphoinositides. Yet, in cells prelabeled with either [14C]oleate or [32P]orthophosphate, addition of the tumor promoter phorbol dibutyrate (PBt2) resulted in the rapid generation of both diacylglycerol and phosphatidate in a time- and dose-dependent manner. The fatty acid composition of the phosphatidate most resembled the fatty acid profile of phosphatidylcholine from the same cell type. Taken together, these findings suggested a role for protein kinase C in the generation of diacylglycerol (and phosphatidate) from phosphatidylcholine. To define further the pathways involved, the metabolism of cellular phosphatidylcholine was studied. In cells prelabeled with [3H]choline, addition of PBt2, but not 4 alpha-phorbol, stimulated the formation of intracellular phosphocholine within 45 sec. Furthermore, addition of platelet-derived growth factor (PDGF) or serum to "serum-starved" cells prelabeled with [3H]choline resulted in increased levels of intracellular phosphocholine within 15-30 sec. Thus, the data suggest that agonists that stimulate protein kinase C either directly (e.g., PBt2) or indirectly via activation of phosphoinositide hydrolysis (e.g., PDGF and serum) may stimulate degradation of phosphatidylcholine by phospholipase C in intact cells. However, prior down-regulation of protein kinase C by prolonged pretreatment of cells with PBt2 almost totally abolished subsequent stimulation of phosphatidylcholine degradation by PBt2 but only partially attenuated subsequent stimulation by PDGF and serum. These observations suggest that PDGF and serum act, at least partially, through a protein kinase C-independent mechanism. Lastly, the size of the cellular choline and CDP-choline pools were shown to be small and relatively insensitive to agonist addition, as compared to the size and behavior of the phosphocholine pool. Thus, the rapidly increased levels of phosphocholine (and diacylglycerol) arising in response to agonist addition appear to be derived directly from phosphatidylcholine by a phospholipase C-mediated mechanism.     

Activation of phospholipase D by glyceraldehyde in isolated islet cells follows protein kinase C activation.

Our recent studies have demonstrated the presence in neonatal islet cells and intact adult islets of a phosphatidylcholine-directed phospholipase D (PLD) which is activated after phorbol ester stimulation. The present study describes PLD activation in the presence of a carbohydrate insulin secretagogue. At the highest concentration tested (20 mM) the triose, glyceraldehyde, induced formation of phosphatidic acid in cells prelabeled with [14C]arachidonic acid or [3H]myristic acid (164 +/- 7 and 210 +/- 9% of basal phosphatidic acid values, respectively). Experimental confirmation of a concentration-dependent specific activation of PLD was provided by the formation of a transphosphatidylation product, phosphatidylethanol, after stimulation with glyceraldehyde in the presence of added ethanol (1.5%). Additionally, there was an early (within 5 min) rise in [14C]arachidonate-labeled diacylglycerol (139 +/- 7% of basal) accompanied by an increase in intracellular diacylglycerol mass (51 +/- 2 pmol/mg protein) and an increase in membrane-associated protein kinase C activity (183 +/- 5% of basal) which preceded the activation of PLD, as indicated by the time course of glyceraldehyde-stimulated phosphatidylethanol formation in the presence of ethanol. Pretreatment of islet cells with 2 microM 12-O-tetradecanoylphorbol-13-acetate for 18 h, to down-regulate protein kinase C, was without effect on diacylglycerol and phosphatidic acid production after 5 min but inhibited completely the production of phosphatidylethanol at 30 min. The phosphohydrolase inhibitor propranolol (100 microM) potentiated the accumulation of phosphatidic acid and phosphatidylethanol incubation following incubation with glyceraldehyde. These findings demonstrate for the first time that a physiological nutrient activates a phospholipase directed against endogenous phosphatidylcholine in intact islet cells; furthermore, they indicate a role for PLD in a delayed formation of phosphatidic acid in the islet cell. The finding of an early rise in glyceraldehyde-stimulated diacylglycerol (which may be formed de novo or by the action of phospholipase C), suggests that PLD is recruited by the activation of protein kinase C by this nutrient.