Thyrotropin-releasing hormone and phorbol esters induce phosphatidylcholine synthesis in GH3 pituitary cells. Evidence for stimulation via protein kinase C

Phorbol esters have been shown to stimulate phosphatidylcholine synthesis via the CDP-choline pathway. The present study compares the effects of phorbol esters and thyrotropin-releasing hormone (TRH) on phosphatidylcholine metabolism in GH3 pituitary cells. In a previous study (Kolesnick, R.N., and Paley, A.E. (1987) J. Biol. Chem. 262, 9204-9210), the potent phorbol ester, 12-O-tetradecanoylphorbol 13-acetate (TPA) induced time- and concentration-dependent incorporation of 32Pi and [3H]choline into phosphatidylcholine in short-term labeling experiments. In this study, TPA is shown to activate choline-phosphate cytidylyltransferase (EC 2.7.7.15), the regulatory enzyme of the CDP-choline pathway, by stimulating redistribution of the inactive cytosolic form of the enzyme to the membrane. Redistribution was quantitative. TPA reduced cytosolic activity from 3.5 +/- 0.4 to 1.5 +/- 0.3 nmol . min-1 x 10(7) cells-1 and enhanced particulate activity from 2.5 +/- 0.4 to 4.9 +/- 0.6 nmol . min-1 x 10(7) cells-1. TRH also stimulated time- and concentration-dependent 32Pi and [3H]choline incorporation into phosphatidylcholine. An increase was detectable after 5 min; and after 30 min, the levels were 164 +/- 9 and 150 +/- 11% of control, respectively; EC50 congruent to 2 X 10(-10) M TRH. These events correlated directly with TRH-induced 32Pi incorporation into phosphatidylcholine. TRH also stimulated redistribution of cytidylyl-transferase specific activity. TRH reduced cytosolic activity 45% and enhanced particulate activity 51%. Neither TRH nor TPA stimulated phosphatidylcholine degradation. In cells down-modulated for protein kinase C (Ca2+/phospholipid-dependent protein kinase), the effects of TPA and TRH on 32Pi incorporation into phosphatidylcholine were abolished. However, TRH-induced incorporation into phosphatidylinositol still occurred. These studies provide evidence that hormones may regulate phosphatidylcholine metabolism via the protein kinase C pathway.     

1,2-Diacylglycerols overcome cyclic AMP-mediated inhibition of phosphatidylcholine synthesis in GH3 pituitary cells.

Previous studies showed that phorbol esters and thyrotropin-releasing hormone (TRH) stimulated phosphatidylcholine synthesis via protein kinase C in GH3 pituitary cells [Kolesnick (1987) J. Biol. Chem. 262, 14525-14530]. In contrast, 1,2-diacylglycerol-stimulated phosphatidylcholine synthesis appeared independent of protein kinase C. The present studies compare phosphatidylcholine synthesis stimulated by these agents with inhibition via the cyclic AMP system. The potent phorbol ester phorbol 12-myristate 13-acetate (PMA, 10 nM) increased [32P]Pi incorporation into phosphatidylcholine at 30 min to 159 +/- 6% of control. The adenylate cyclase activator cholera toxin (CT; 10 nM) and the cyclic AMP analogue dibutyryl cyclic AMP (1 mM) abolished this effect. CT similarly abolished TRH-induced phosphatidylcholine, but not phosphatidylinositol, synthesis. This is the first report of inhibiton of receptor-mediated phosphatidylcholine synthesis by the cyclic AMP system. The 1,2-diacylglycerol 1,2-dioctanoylglycerol (diC8) also stimulated concentration-dependent phosphatidylcholine synthesis. DiC8 (3 micrograms/ml) induced an effect quantitatively similar to that of maximal concentrations of PMA and TRH, whereas a maximal diC8 concentration (30 micrograms/ml) stimulated an effect 3-4-fold greater than these other agents. CT decreased the effect of diC8 (3 micrograms/ml) by 80%. Higher diC8 concentrations overcame the CT inhibition. Similar results were obtained with dibutyryl cyclic AMP. Additional differences were found between low and high concentrations of diC8. Low concentrations of diC8 failed to induce additive phosphatidylcholine synthesis with maximal concentrations of PMA, whereas high concentrations were additive. Hence, low concentrations of 1,2-diacylglycerols appear to be regulated similarly to phorbol esters, and higher concentrations appear to act via a pathway unavailable to phorbol esters.     

Bombesin and phorbol ester stimulate phosphatidylcholine hydrolysis by phospholipase C: evidence for a role of protein kinase C

Bombesin caused a marked stimulation of 32Pi into phosphatidylinositol (PI), with no apparent lag, and into phosphatidylcholine (PC), after a lag of about 20 min. Stimulation was blocked by the bombesin receptor antagonist, [D-Arg1, D-Pro2, D-Trp7,9, Leu11] substance P, indicating that the effects on both PI and PC were mediated through the same receptor. The tumor-promoting phorbol ester 12-0-tetradecanoylphorbol-13-acetate (TPA) and dioctanoylglycerol (diC8) both directly activate protein kinase C and in this report were shown to stimulate 32Pi incorporation into PC but not into Pl. In addition, TPA stimulated the release of [3H]choline and [3H]phosphocholine and the accumulation of [3H]diacyglycerol from prelabelled cells. These results strongly suggest that TPA activates a phospholipase C specific for PC. Pretreatment of cells with phorbol-12, 13-dibutyrate (PDBu) for 24 h depleted cellular protein kinase C activity and inhibited the ability of TPA to induce these effects suggesting a direct involvement of protein kinase C. Similarly the bombesin stimulation of 32Pi into PC and of [3H]choline and [3H]phosphocholine release was inhibited by PDBu pretreatment. DiC8 and, to a lesser extent, TPA stimulated the translocation of CTP:phosphocholine cytidylytransferase from the cytosolic to the particulate fraction. DiC8 also stimulated this translocation in cells depleted of protein kinase C. It was concluded that both bombesin and TPA activated protein kinase C leading to activation of a phospholipase C specific for PC.     

Phorbol ester tumor promoters specifically stimulate choline phospholipid metabolism in human leukemic cells

Human hairy cell leukemia (HCL) cells in culture showed a marked increase in both [1-14C]acetate and [14C]choline incorporation into phosphatidylcholine (PC) when treated with a 10 nM concentration of 12-O-tetradecanoylphorbol 13-acetate (TPA) for 3 h. Dramatic morphological changes occurred and synthesis of most phospholipids was stimulated. However, the most dramatic increase was seen in the [14C]acetate labeling of both long- and short-chain fatty acid-containing sphingomyelins (from 200-425% of control levels), sphingomyelin being especially enriched in HCL cells. Negligible incorporation of [14C]choline into sphingomyelin was observed and phospholipase inhibitor (U10029A) studies indicated that PC was the major source of sphingomyelin choline. These changes were most clearly seen by autoradiography of two-dimensional thin-layer chromatography plates. Chronic myelogenous leukemia (CML) blasts, which did not respond morphologically to TPA, showed no increased phospholipid synthesis under the same conditions and increases in sphingomyelin synthesis were modest. Other non-TPA-responding leukemic cells were similarly refractive. However, one out of four acute monomyelocytic leukemic (AMMoL) cells studied responded morphologically in a manner identical to HCL cells and exhibited the same dramatic increase in sphingomyelin synthesis. Data are presented which suggest that TPA may also stimulate PC phospholipase C activity in addition to activating the calcium-dependent protein kinase by mimicking diacylglycerol.     

Phorbol esters modulate the turnover of both ether- and ester-linked phospholipids in cultured mammalian cells

The effects of 12-O-tetradecanoylphorbol 13-acetate (TPA) on the metabolism of ester- and ether derivatives of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) were studied in HeLa and HEL-37 cells. TPA stimulated the incorporation of [3H]choline into diacyl-, alkylacyl- and alkenylacy/PC in HeLa cells, but inhibited the incorporation of [3H]ethanolamine into the corresponding derivatives of PE. TPA also stimulated the incorporation of [3H]ethanolamine into lysoPE and the release of labelled ethanolamine and phosphoethanolamine from HeLa cells prelabelled with [3H]ethanolamine. All responses to TPA were abolished in HeLa cells preincubated with the phorbol ester and which were deficient in protein kinase C. In HEL-37 cells TPA stimulated label incorporation into both ester- and ether-forms of PE. The marked effects of TPA on ether-lipid metabolism raises the possibility that hydrolysis products of this class of lipid are important in transmembrane signalling pathways.     

Dissociation of protein kinase C activation from phorbol ester-induced maturation of HL-60 leukemia cells

The role of C-kinase in the induction of maturation of HL-60 promyelocytic leukemia cells was examined using two activators of this kinase, 12-O-tetradecanoyl phorbol 13-acetate (TPA) and 1-oleoyl-2-acetylglycerol (OAG). At 10(-8) M, a concentration that induced maturation, TPA effectively stimulated C-kinase activity in cell-free preparations by increasing the affinity of the enzyme for Ca2+. Similar activation was observed with 20 micrograms/ml of OAG. At these concentrations, addition of either compound to intact cells stimulated the phosphorylation of cellular proteins. Treatment with TPA resulted in an increased phosphorylation of 14 proteins, 9 of which also changed in response to OAG. In addition to the effects on protein phosphorylation, TPA and OAG both affected choline lipid metabolism. TPA at 10(-8) M stimulated the incorporation of [methyl-3H]choline into phosphatidylcholine, sphingomyelin, and lysophosphatidylcholine. OAG at 20 micrograms/ml had quantitatively similar effects on the labeling of the former two lipids, but did not affect incorporation of choline into lysophosphatidylcholine. Despite the similar biochemical effects of TPA and OAG, the diglyceride was unable to induce HL-60 cell maturation as measured by inhibition of cell growth, development of nonspecific esterase activity, phagocytosis, adherence of cells to plastic, and loss of transferrin receptor activity. The lack of effect is not due to metabolism of OAG; maturation could not be induced by treating cells with fresh OAG every 2 h for a period of 12 h. These results suggest a dissociation of the activation of C-kinase and the induction of HL-60 cell maturation by TPA.     

Effect of phorbol ester on stimulus-secretion coupling mechanisms in gonadotropin releasing hormone-stimulated pituitary gonadotrophs

The feedback regulatory control mechanism exerted by activated Ca2+/phospholipid-dependent protein C kinase upon gonadotropin releasing hormone (GnRH) binding, stimulation of phosphoinositide turnover and gonadotropin secretion was investigated in cultured pituitary cells. Addition of the tumor promoter phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), at concentrations which activate pituitary protein C kinase, to cultured pituitary cells resulted in up-regulation of GnRH receptors (155% at 4 h). The stimulatory effect of GnRH on [3H]inositol phosphates (Ins-P) production in myo-[2-3H]inositol prelabeled pituitary cells was not inhibited by prior treatment of the cells with TPA (10(-9)-10(-7) M). Higher concentrations of TPA (10(-6)-10(-5) M) inhibited the effect of GnRH on [3H]Ins-P production. Increasing concentrations of TPA or the permeable analog of diacylglycerol 1-oleoyl-2-acetylglycerol (OAG) stimulated luteinizing hormone (LH) release from cultured pituitary cells with ED50 values of 5 x 10(-9) M and 10 micrograms/ml, respectively. No consistent inhibition or additivity of LH release was observed when increasing doses of TPA or OAG were added with a submaximal dose of GnRH. These results suggest that protein C kinase might mediate the known homologous up-regulation of GnRH receptors during the reproductive cycle. Protein C kinase is positively involved in mediating the process of gonadotropin secretion. Unlike many other systems, activation of protein C kinase in pituitary gonadotrophs is not involved in negative feed-back regulation of stimulus-secretion-coupling mechanisms in GnRH-stimulated gonadotrophs.     

Differential regulation of phosphatidylcholine biosynthesis by 12-O-tetradecanoylphorbol-13-acetate and diacylglycerol in NG108-15 neuroblastoma x glioma hybrid cells

12-O-Tetradecanoylphorbol-13-acetate (TPA), a tumor promoter and potent activator of protein kinase C, stimulates [3H]choline incorporation into phosphatidylcholine (PtdCho) in NG108-15 cells (Liscovitch, M., Freese, A., Blusztajn, J. K. and Wurtman, R. J. (1986) J. Neurochem. 47, 1936-1941). In the present study we demonstrate that two cell-permeant diacylglycerols, sn-1-oleoyl-2-acetylglycerol and sn-1,2-dioctanoylglycerol, also stimulate [3H]choline incorporation into PtdCho. However, the effect of diacylglycerol is additional to that produced by a maximally effective concentration of TPA (0.5 microM), suggesting that the two agents may not act via the same mechanism. In addition, the protein kinase inhibitor 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (at 200 microM) inhibits the action of TPA by 59% while not affecting that of diacylglycerol. Finally, preincubation of the cells with TPA (0.1 microM) for 24 h reduces protein kinase C activity in the cells and completely abolishes the effect of additional TPA on choline incorporation. In contrast, diacylglycerol-induced stimulation of PtdCho biosynthesis was not inhibited in the cells that were desensitized to TPA. These results suggest that the effect of the two cell-permeant diacylglycerols on PtdCho biosynthesis either is not mediated by protein kinase C activation, or, is mediated by a TPA-insensitive isoenzyme of protein kinase C.     

Pituitary thyrotropin-releasing hormone (TRH) receptors: effects of TRH, drugs mimicking TRH action, and chlordiazepoxide

Binding of TRH to specific cell surface receptors on clonal GH4C1 cells is followed within 10 min by receptor sequestration and over 24 h by receptor down-regulation. These experiments were designed to determine if TRH-activated second messenger systems are responsible for changes in receptor localization or number. BAY K8644 and A23187, which increase intracellular calcium, alone or together with 12-O-tetradecanoyl phorbol acetate (TPA), which activates protein kinase C, did not appear to internalize TRH receptors. Drug treatment did not alter the rate of [3H]MeTRH association or internalization, determined by resistance to an acid/salt wash, or the amount of [3H]MeTRH able to bind at 0 C, where only surface receptors are accessible. TPA (0-100 nM) alone or in combination with BAY K8644 or A23187, also failed to change receptor number or affinity after 48 h when TRH caused a 75% decrease in the density of specific binding sites. Chlordiazepoxide has been reported antagonize TRH binding and TRH-induced phospholipid breakdown. Chlordiazepoxide shifted the dose-response curves for TRH stimulation of PRL release and synthesis to the right, and did not change PRL release alone. The affinity of receptors for chlordiazepoxide was not affected by a nonhydrolyzable analog of GTP whereas affinity for TRH was decreased; these properties are consistent with the classification of chlordiazepoxide as a competitive antagonist. Several experiments tested whether chlordiazepoxide would cause receptor internalization and down-regulation. Chlordiazepoxide did not appear to internalize TRH receptors, because TRH-binding sites became available rapidly and at the same rate after they had been saturated with chlordiazepoxide at 0 or 37 C.(ABSTRACT TRUNCATED AT 250 WORDS)     

A novel mechanism of diglyceride formation. 12-O-tetradecanoylphorbol-13-acetate stimulates the cyclic breakdown and resynthesis of phosphatidylcholine

12-O-Tetradecanoylphorbol-13-acetate (TPA) treatment of Madin-Darby canine kidney cells resulted in an increased incorporation of 32Pi and [methyl-3H]choline into choline-containing phosphoglycerides (PC). In pulse-chase experiments, TPA treatment caused an increased release of [methyl-3H]choline from the PC fraction of prelabeled cells. When cells were prelabeled with [3H]arachidonic acid and [14C]palmitic acid, TPA treatment resulted in an increased synthesis of 14C, 3H-diglycerides. Further studies were done to determine the relationship between PC breakdown and diglyceride synthesis. Cells were preincubated with ether-linked 1-O-[3H]hexadecyl-2-lyso-sn-glycero-3-phosphocholine which was acylated to form 1-O-[3H]hexadecyl-2-acyl-sn-glycero-3-phosphocholine. Subsequent treatment of these cells with TPA resulted in an increased synthesis of 1-O-[3H]hexadecyl-2-acyl-sn-glycerol compared to cells not stimulated with TPA. These findings demonstrate that TPA stimulates PC turnover in Madin-Darby canine kidney cells and provide evidence for a novel mechanism of diglyceride formation.     

Stimulation of Phospholipase D Activity by Phorbol Esters in Cultured Astrocytes

The phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) was found to stimulate phospholipase D activity in cultured primary astrocytes. Both the hydrolysis and the transphosphatidylation reaction catalyzed by phospholipase D were studied in cells labeled with [3H]glycerol. Phosphatidic acid (PA) synthesis was increased after addition of 100 nM TPA. When ethanol was present in the cell culture medium, phosphatidylethanol (Peth), a product of phospholipase D-catalyzed transphosphatidylation, was formed. The half-maximum effective concentrations (EC50) of TPA were 25 nM for PA increase as well as for Peth formation. The formation of Peth in ethanol-treated cells was accompanied by an inhibition of the TPA-induced increase in labeled PA. Increasing ethanol concentrations led to an increase in [3H]Peth and a decrease in [3H]PA. A protein kinase C inhibitor, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H7), inhibited both the synthesis of PA and the formation of Peth observed after TPA addition to the astrocytes. Dioctanoyl-glycerol (100 microM) stimulated the formation of Peth in the presence of ethanol. In addition to the induction of Peth formation in astrocytes, TPA induced Peth formation in ethanol-treated neurons. The present results indicate that phospholipase D activity is stimulated by TPA in cultured primary brain cells. Modulation of phospholipase D activity by protein kinase C is a mechanism that may be important in signal transduction cascades.     

Effects of pH and free Mg2+ on the Keq of the creatine kinase reaction and other phosphate hydrolyses and phosphate transfer reactions.

The observed equilibrium constants (Kobs) of the creatine kinase (EC 2.7.3.2), myokinase (EC 2.7.4.3), glucose-6-phosphatase (EC 3.1.3.9), and fructose-1,6-diphosphatase (EC 3.1.3.11) reactions have been determined at 38 degrees C, pH 7.0, ionic strength 0.25, and varying free magnesium concentrations. The equilibrium constant (KCK) for the creatine kinase reaction defined as: KCK = [sigma ATP] [sigma creatine] divided by ([sigma ADP] [sigma creatine-P] [H+]) was measured at 0.25 ionic strength and 38 degrees C and was shown to vary with free [Mg2+]. The value was found to be 3.78 x 10(8) M-1 at free [Mg2+] = 0 and 1.66 x 10(9) M-1 at free [Mg2+] = 10(-3) M. Therefore, at pH 7.0, the value of Kobs, defined as Kobs = KCK[H+] = [sigma ATP] [sigma creatine] divided by ([sigma ADP] [sigma creatine-P] was 37.8 at free [Mg2+] = 0 and 166 at free [Mg2+] = 10(-3) M. The Kobs value for the myokinase reaction, 2 sigma ADP equilibrium sigma AMP + sigma ATP, was found to vary with free [Mg2+], being 0.391 at free [Mg2+] = 0 and 1.05 at free [Mg2+] = 10(-3) M. Taking the standard state of water to have activity equal to 1, the Kobs of glucose-6-P hydrolysis, sigma glucose-6-P + H2O equilibrium sigma glucose + sigma Pi, was found not to vary with free [Mg2+], being 110 M at both free [Mg2+] = 0 and free [Mg2+] = 10(-3) M. The Kobs of fructose-1,6-P2 hydrolysis, sigma fructose-1,6-P2 equilibrium sigma fructose-6-P + sigma Pi, was found to vary with free [Mg2+], being 272 M at free [Mg2+] = 0 and 174 M at free [Mg2+] = 0.89 x 10(-3) M.     

Cyclic 3',5'-adenosine monophosphate-dependent kinase and phosphoproteins in human amnion

3',5'-Cyclic adenosine monophosphate (cAMP) modulates prostaglandin production in human amnion membranes. The major effects of cAMP are presumably mediated through the phosphorylation of specific regulatory phosphoproteins following cAMP activation of cAMP-dependent protein kinase. Cyclic AMP-dependent protein kinase and phosphoproteins have not previously been characterized in human amnion. Total homogenates, cytosol, and membrane fractions from human amnion were examined for [3H]cAMP binding activity and cAMP-dependent kinase activity. cAMP-dependent kinase activity was barely detectable in crude amnion fractions. Cytosol was therefore partially purified by DEAE column chromatography for further examination. Two peaks of coincident [3H]cAMP binding and cAMP-dependent kinase activity were demonstrated at 70 and 140 mM NaCl, characteristic of the Type I and Type II cAMP-dependent protein kinase isozymes. [3H]cAMP binding to the material from both peak fractions was saturable and reversible. Scatchard analysis of [3H]cAMP binding to the peak fractions was linear for peak I and curvilinear for peak II. Assuming a one-site model, [3H]cAMP binding to the Type I isozyme showed a KD = 4.17 x 10(-8) M and Bmax = 73 pmole/mg protein; using a two-site model, [3H]cAMP binding to the high-affinity site for the Type II isozyme had a KD = 3.94 x 10(-8) M and Bmax = 6.3 pmole/mg protein. Other cyclic nucleotides competed for these [3H]cAMP binding sites with a potency order of cAMP much greater than cGMP greater than (BU)2cAMP.cAMP caused a dose-dependent increase in cAMP-dependent kinase activity in the peak fractions; half-maximal activation was observed with 5.0 x 10(-8) M cAMP. The ability of cAMP to increase phosphorylation of endogenous proteins in both crude amnion cytosol and cytosol from cultures of amnion epithelial cells was assessed using [32P]ATP, SDS-polyacrylamide gel electrophoresis and autoradiography. cAMP stimulated 32P incorporation into three proteins having Mr = 80,000, 54,000, and 43,000 (P less than .01). Half-maximal 32P incorporation into these proteins occurred at 1.0 x 10(-7) M cAMP. cAMP-dependent kinase is present in human amnion; specific cAMP-enhanced phosphoproteins are also present. Hormones elevating cAMP levels in amnion may exert their effects by activating cAMP-dependent kinase and phosphorylating these phosphoproteins.     

Phorbol ester-stimulated hydrolysis of phosphatidylcholine and phosphatidylethanolamine by phospholipase D in HeLa cells. Evidence that the basal turnover of phosphoglycerides does not involve phospholipase D

12-O-Tetradecanoylphorbol-13-acetate (TPA) stimulated the release of [3H]ethanolamine from HeLa cells prelabeled with [3H]ethanolamine within 2 min, and of [3H]choline from cells prelabeled with [3H]choline after a lag of 10-20 min. This result suggests that TPA activates phospholipase D. Propranolol alone or propranolol plus TPA stimulated phosphatidic acid (PA) labeling in cells prelabeled with [3H]hexadecanol. In the presence of ethanol, TPA stimulated the accumulation of labeled phosphatidylethanol (PEth); no PEth was formed in the absence of TPA. TPA-dependent PEth accumulation was not observed in cells pretreated with TPA to down-regulate protein kinase C, whereas propranolol-induced accumulation of PA was unaffected by TPA pretreatment. Incubation of prelabeled cells with propranolol alone caused a rapid loss of label and phospholipid mass from both phosphatidylethanolamine and phosphatidylcholine (PC) together with an accumulation of PA and phosphatidylinositol plus phosphatidylserine. When [3H]hexadecanol-prelabeled cells were pulse labeled with 32P to label nucleotide pools, propranolol induced the accumulation of both 3H- and 32P-labeled PA. When cells were prelabeled with lyso-PC double labeled with 3H and 32P, and incubated with propranolol, only 3H-labeled PA accumulated, indicating that the pathways involved in the basal turnover of PC resulted in the loss of 32P from the lipid. These results suggest that the basal turnover of phosphatidylethanolamine and PC involves the sequential actions of phospholipase C, diglyceride kinase, and PA phosphohydrolase.     

Endothelin-1 activates phospholipase D and thymidine incorporation in fibroblasts overexpressing protein kinase C beta 1.

Endothelins (ETs) are a family of extremely potent vasoconstrictor peptides. In addition, ET-1 acts as a potent mitogen and activates phospholipase C in smooth muscle cells and fibroblasts. We examined the effects of ET-1 on phosphatidylcholine (PC) metabolism and thymidine incorporation in control Rat-6 fibroblasts and in cells that overexpress protein kinase C beta 1 (PKC). PC pools were labeled with [3H]myristic acid, and formation of phosphatidylethanol (PEt), an unambiguous marker of phospholipase D (PLD) activation, was monitored. ET-1 stimulated much greater PEt formation in the PKC overexpressing cells. ET-1 action was dose-dependent with a half-maximal effect at 1.0 x 10(-9) M. With increasing ethanol concentrations, [3H]PEt formation increased at the expense of [3H]phosphatidic acid (PA). Propranolol, an inhibitor of PA phosphohydrolase, increased [3H]PA accumulation and decreased [3H]diacylglycerol (DAG) formation. These data are consistent with the formation of [3H]DAG from PC by the sequential action of PLD and PA phosphohydrolase. Phorbol esters are known to stimulate thymidine incorporation and PLD activity to a greater extent in PKC overexpressing cells than in control cells. ET-1 also stimulates thymidine incorporation to a greater extent in the PKC overexpressing cells. The effect of ET-1 on thymidine incorporation into DNA in the overexpressing cells was also dose-dependent with a half-maximal effect at 0.3 x 10(-9) M. Enhanced PLD activity induced by ET-1 in the overexpressing cells may contribute to the mitogenic response, especially in light of a possible role of the PLD product, PA, in regulation of cell growth.     

1,2-Diacylglycerols but not phorbol esters stimulate sphingomyelin hydrolysis in GH3 pituitary cells

It has recently been proposed that degradation products of sphingolipids may serve as physiologic inhibitors of protein kinase C. The present study was performed to determine the effect of 1,2-diacylglycerols and phorbol esters, known activators of protein kinase C, on sphingomyelin metabolism. 1,2-Dioctanoylglycerol (diC8) caused time- and concentration-dependent reduction in the level of sphingomyelin labeled to equilibrium with [3H]choline. diC8 (200 micrograms/ml) reduced [3H]sphingomyelin to 81 +/- 3% of control (p less than 0.005) by 15 min, and the level was 58 +/- 5% of control after 1 h; an EC50 for this event was 56 micrograms/ml. To evaluate the mechanisms of stimulated hydrolysis, the sphingoid base backbone of sphingomyelin was labeled with [14C] serine, and the effects of diC8 were quantitated. diC8 (100 micrograms/ml) reduced the level of sphingomyelin to 66 +/- 7% of control by 1 h from 375 +/- 12 pmol/10(6) cells to 245 +/- 26 pmol/10(6) cells. There was a concomitant increase in ceramide from 89 +/- 4 pmol/10(6) cells to 252 +/- 27 pmol/10(6) cells consistent with activation of the enzyme, sphingomyelinase (EC 3.1.4.12). In support of this contention, 1,2-diacylglycerols appeared to enhance the activity of an acid, but not a neutral, sphingomyelinase in homogenates of GH3 cells. The 1,2-diacylglycerol, 1-oleyl-2-acetylglycerol, produced similar effects. In contrast, the phorbol esters, 12-O-tetradecanoylphorbol 13-acetate and phorbol 12,13-dibutyrate, failed to stimulate sphingomyelin hydrolysis. Further, these effects of the 1,2-diacylglycerols occurred in cells down-modulated for protein kinase C. These studies demonstrate that 1,2-diacylglycerols stimulate sphingomyelin hydrolysis by a mechanism independent of the protein kinase C which mediates phorbol ester action. This is the first report of stimulated sphingomyelin hydrolysis by a physiologic effector molecule.     

Phosphatidylcholine Metabolism in Nuclei of Phorbol Ester-Activated LA-N-1 Neuroblastoma Cells

The agonist stimulation of a variety of cells results in the induction of specific lipid metabolism in nuclear membranes, supporting the hypothesis of an important role of the lipids in nuclear signal transduction. While the existence of a phosphatidylinositol cycle has been reported in cellular nuclei, little attention has been given to the metabolism of phosphatidylcholine in nuclear signaling. In the present study the metabolism of phosphatidylcholine in the nuclei of neuro-blastoma cells LA-N-1 was investigated. The incubation of LA-N-1 nuclei with radioactive choline, phosphocholine or CDP-choline led to the production of labelled phosphatidylcholine. The incorporation of choline and phosphocholine but not CDP-choline was enhanced in nuclei of TPA treated cells. Moreover the presence of choline kinase, phosphocholine cytidylyltransferase and phosphocholine transferase activities were detected in the nuclei and the TPA treatment of the cells stimulated the activity of the phosphocholine cytidylyltransferase. When cells prelabelled with [³H]palmitic acid were stimulated with TPA in the presence of ethanol, an increase of labelled diacylglycerol and phosphatidylethanol in the nuclei was observed. Similarly, an increase of labelled diacylglycerol and phosphatidic acid but not of phosphatidylethanol occurred in [³H]palmitic acid prelabelled nuclei stimulated with TPA in the presence of ethanol. However the production of phosphatidylethanol was observed when the nuclei were treated with TPA in the presence of ATP and GTPS. The stimulation of [³H]choline prelabelled nuclei with TPA also generated the release of free choline and phosphocholine. The results indicate the presence of PLD and probably PLC activities in LA-N-1 nuclei and the involvement of phosphatidylcholine in the production of nuclear lipid second messengers upon TPA stimulation of LA-N-1 cells. The correlation of the disappearance of phosphatidylcholine, the production of diacylglycerol and phosphatidic acid with the stimulation of phosphatidylcholine synthesis in nuclei of TPA treated LA-N-1 suggests the existence of a phosphatidylcholine cycle in these nuclei.     

Phorbol diester enhances calcium ionophore A23187-induced [3H]acetate incorporation into platelet-activating factor in murine macrophages: predominant incorporation into 1-O-acyl-2-acetyl-sn-glycero-3-phosphocholine

Pretreatment of macrophages with 12-O-tetradecanoylphorbol-13-acetate (TPA) has been shown to enhance the release of arachidonic acid from cell phospholipids in response to agonist stimulation. This study describes the ability of TPA to also alter calcium ionophore A23187-induced incorporation of [3H]acetate into platelet activating factor (PAF). Cultured murine peritoneal macrophages were preincubated with [3H]acetate (25 muCi) and TPA (10 ng/ml) for 10 min, and subsequently incubated with 0.1 microM A23187 for 0.5-10 min. Buffer and cells were then extracted and PAF resolved by normal-phase HPLC. Sequential exposure to TPA and A23187 resulted in a greatly enhanced incorporation (11,861 dpm/10(6) cells) of [3H]acetate into PAF compared to TPA alone, which did not significantly influence [3H]acetate incorporation into PAF, and 0.1 microM A23187, which induced minimal incorporation (688 dpm/10(6) cells). Macrophage-produced [3H]PAF was resolved by HPLC, extracted, treated with phospholipase-C, and acetylated to facilitate quantitation of 1-O-alkyl-2-acetyl-GPC (PAF) from 1-O-acyl-2-acetyl-GPC (acylPAF). A23187 alone (1 microM) produced 72% 1-O-acyl-2-[3H]acetyl-GPC, and A23187 (0.1 microM) following TPA pretreatment produced 81% 1-O-acyl-2-[3H]acetyl-GPC. Less than 2% of the radioactivity of acylPAF was in the acyl moiety. These data support a role for protein kinase C in modulating agonist-induced PAF synthesis. The results also suggest that acetyltransferase of murine macrophages does not possess specificity for 1-O-alkyl-2-lyso-GPC, and that availability of specific species of lyso-phospholipid may determine the type of PAF produced.     

Dual actions of phorbol esters on cytosolic free Ca2+ concentrations and reconstitution with ionomycin of acute thyrotropin-releasing hormone responses

We have used phorbol esters, such as 12-O-tetradecanoyl phorbol 13-acetate (TPA), to study the actions of protein kinase C (a TPA receptor) on cytosolic free Ca2+ concentrations [( Ca2+]i) and hormone secretion in rat pituitary cells (GH cells), and to elucidate the role of diacylglycerol (a protein kinase C activator) in thyrotropin-releasing hormone (TRH) action. TPA had a dual action on [Ca2+]i, inducing a stimulatory phase from 300 (basal) to 420 nM, which was interrupted in 30-60 s by an inhibitory phase which transiently lowered [Ca2+]i to 240 nM and rose in 3-10 min to yield the stimulatory phase. TPA-mediated changes in [Ca2+]i were induced by other phorbol esters and mezerein but not by phorbol or activators of kinases different from protein kinase C. Both phases of TPA action on [Ca2+]i were abolished by 5-min pretreatment with ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) (1.33 mM) or Ca2+ channel antagonists (verapamil or nifedipine). TPA also enhanced the rate of sustained hormone secretion without inducing a burst of hormone release (unlike TRH). Also, stimulation of secretion by TPA was not inhibited by Ca2+ channel antagonists and was resistant (10%) to EGTA. Simultaneous addition of TPA with the ionophore ionomycin (100 nM) reconstituted a TRH-like spike, nadir and plateau of [Ca2+]i. Ionomycin generated the spike in [Ca2+]i by releasing TRH-sensitive Ca2+ stores, while TPA induced the nadir (inhibitory phase), and a nifedipine/verapamil-sensitive plateau of [Ca2+]i (stimulatory phase). Concurrent (but not separate) addition of ionomycin and TPA also reconstituted a TRH-like burst of hormone secretion. These and previous results indicate that activation of protein kinase C by TPA or diacylglycerol (which is elevated by TRH) and a simultaneous spike in [Ca2+]i are required for burst secretion. Diacylglycerol may also mediate the TRH-induced nadir and plateau of [Ca2+]i; the latter process contributes to Ca2+-dependent stimulation of steady secretion by TRH.     

Stimulation of choline release from NG108-15 cells by 12-O-tetradecanoylphorbol 13-acetate.

The effects of the potent tumour-promoting phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) on phosphatidylcholine (PtdCho) metabolism were investigated in the neuroblastoma X glioma hybrid cell line NG108-15. TPA (100 nM) stimulated by 150-200% the release into the medium of 3H radioactivity from cells that had been pre-labelled with [3H]choline. H.p.l.c. analysis of the medium revealed that TPA stimulated the release of only free [3H]choline (212 +/- 11% of control), without affecting such other labelled metabolites as [3H]phosphocholine and [3H]glycerophosphocholine. This effect was concentration-dependent, with a half-maximal effect obtained at 27.5 +/- 6.8 nM, and was observable as early as 5-10 min after exposure to TPA. The TPA-induced release of [3H]choline into the medium was accompanied by a small and variable decrease in cellular [3H]PtdCho (to 93 +/- 4% of control). However, the radioactivity associated with water-soluble cellular choline metabolites (mainly [3H]phosphocholine and [3H]glycerophosphocholine) remained unchanged. TPA also stimulated the release of [3H]choline derived from [3H]PtdCho that had been produced via the methylation pathway from [3H]methionine. These data suggest that phosphatidylcholine may serve as the source of free choline released from the cells in response to TPA. The possible enzymic mechanisms underlying this response are discussed.