Bombesin and platelet-derived growth factor stimulate formation of inositol phosphates and Ca2+ mobilization in Swiss 3T3 cells by different mechanisms.

Highly purified platelet-derived growth factor (PDGF) or recombinant PDGF stimulate DNA synthesis in quiescent Swiss 3T3 cells. The dose-response curves for the natural and recombinant factors were similar, with half-maximal responses at 2-3 ng/ml and maximal responses at approx. 10 ng/ml. Over this dose range, both natural and recombinant PDGF stimulated a pronounced accumulation of [3H]inositol phosphates in cells labelled for 72 h with [3H]inositol. In addition, mitogenic concentrations of PDGF stimulated the release of 45Ca2+ from cells prelabelled with the radioisotope. However, in comparison with the response to the peptide mitogens bombesin and vasopressin, a pronounced lag was evident in both the generation of inositol phosphates and the stimulation of 45Ca2+ efflux in response to PDGF. Furthermore, although the bombesin-stimulated efflux of 45Ca2+ was independent of extracellular Ca2+, the PDGF-stimulated efflux was markedly inhibited by chelation of external Ca2+ by using EGTA. Neither the stimulation of formation of inositol phosphates nor the stimulation of 45Ca2+ efflux in response to PDGF were affected by tumour-promoting phorbol esters such as 12-O-tetradecanoylphorbol 13-acetate (TPA). In contrast, TPA inhibited phosphoinositide hydrolysis and 45Ca2+ efflux stimulated by either bombesin or vasopressin. Furthermore, whereas formation of inositol phosphates in response to both vasopressin and bombesin was increased in cells in which protein kinase C had been down-modulated by prolonged exposure to phorbol esters, the response to PDGF was decreased in these cells. These results suggest that, in Swiss 3T3 cells, PDGF receptors are coupled to phosphoinositidase activation by a mechanism that does not exhibit protein kinase C-mediated negative-feedback control and which appears to be fundamentally different from the coupling mechanism utilized by the receptors for bombesin and vasopressin.     

Interleukin-3-stimulated haemopoietic stem cell proliferation. Evidence for activation of protein kinase C and Na+/H+ exchange without inositol lipid hydrolysis.

Interleukin 3 (IL-3) is an important regulator of haemopoietic stem cell proliferation both in vivo and in vitro. Little is known about the possible mechanisms whereby this growth factor acts on stem cells to stimulate cell survival and proliferation. Here we have investigated the role of intracellular pH and the Na+/H+ antiport in stem cell proliferation using the multipotential IL-3-dependent stem cell line, FDCP-Mix 1. Evidence is presented that IL-3 can stimulate the activation of an amiloride-sensitive Na+/H+ exchange via protein kinase C activation. IL-3-mediated activation of the Na+/H+ exchange is not observed in FDCP-Mix 1 cells where protein kinase C levels have been down-modulated by treatment with phorbol esters. Also the protein kinase C inhibitor H7 can inhibit IL-3-mediated increases in intracellular pH. This activation of Na+/H+ exchange via protein kinase C has been shown to occur with no measurable effects of IL-3 on inositol lipid hydrolysis or on cytosolic Ca2+ levels. Evidence is also presented that this IL-3-stimulated alkalinization acts as a signal for cellular proliferation in stem cells.     

Potentiation of thyrotropin releasing hormone-induced inositol phospholipid metabolism and Ca2+ mobilization by cyclic AMP-increasing agents

Thyrotropin releasing hormone (TRH) caused significant breakdown of phosphatidylinositol 4,5-bisphosphate (PIP2) in GH3 cells, but vasoactive intestinal peptide (VIP) did not. However, VIP enhanced the TRH-induced hydrolysis of PIP2, the conversion of phosphatidylinositol 4-phosphate (PIP) to PIP2 and the accumulation of phosphatidic acid (PA). On the other hand, the tumor promoter, tetradecanoyl phorbol acetate (TPA), suppressed the TRH-induced hydrolysis of PIP2. In the membrane fraction, the addition of cAMP inhibited the PI kinase activity in a dose-dependent manner, but stimulated the PIP kinase activity. TPA did not affect the PI and PIP kinase activities at all. VIP enhanced the first spike phase of the TRH-induced increase in the intracellular Ca2+ level, while TPA inhibited such Ca2+ mobilization. These results suggested that cAMP-increasing agents enhanced inositol phospholipid metabolism and Ca2+ mobilization induced by TRH in GH3 cells but that TPA inhibited them.     

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.     

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

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

Phorbol esters and dioctanoylglycerol block anti-IgM-stimulated phosphoinositide hydrolysis in the murine B cell lymphoma WEHI-231

Cross-linking of membrane IgM (mIgM) on both normal resting B cells and on the murine B cell lymphoma WEHI-231 activates the phosphoinositide signal transduction pathway. The initial event in this pathway is the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PtdInsP2), which results in the generation of two second-messengers: inositol trisphosphate (InsP3), which can cause the release of Ca2+ from intracellular stores, and diacylglycerol (DG), which activates protein kinase C. In examining the effects of exogenous activation of protein kinase C on WEHI-231 cells, we found that phorbol esters blocked some of the biologic effects of anti-IgM on WEHI-231 cells. The mechanism of this effect was investigated. Phorbol ester treatment of WEHI-231 cells blocked the ability of anti-IgM to stimulate production of inositol phosphates and accumulation of phosphatidic acid, the phosphorylated product of DG. Phorbol esters also blocked the ability of anti-IgM to cause an increase in intracellular Ca2+. Thus, it is clear that phorbol esters block anti-IgM-stimulated PtdInsP2 hydrolysis in WEHI-231 cells. In addition, a synthetic DG, dioctanoylglycerol (diC8), also blocked anti-IgM-stimulated inositol phosphate production and the anti-IgM-stimulated rise in cytoplasmic Ca2+. The ability of phorbol esters and diC8 to block mIgM-mediated signaling may reflect a feedback inhibition mechanism by which activated protein kinase C limits the magnitude and duration of receptor signaling.     

Effects of phorbol esters, diglyceride, and cholinergic agonists on the subcellular distribution of protein kinase C in intact or digitonin-permeabilized adrenal chromaffin cells

Phorbol esters which activate protein kinase C increased the percentage of membrane-bound protein kinase C activity in bovine adrenal chromaffin cells from less than 10 to 20-50% within 30 min. Permeabilization of chromaffin cells with digitonin in the absence of Ca2+ and phorbol esters caused virtually 100% of the protein kinase C activity to leave the cells within 1 h, which is consistent with protein kinase C being soluble and cytosolic. However, if cells were incubated for 15-30 min with 12-O-tetradecanoylphorbol-13-acetate (TPA) prior to permeabilization, 50-60% of the protein kinase C activity exited from the cells within 1 h of permeabilization. In cells not incubated with phorbol ester, permeabilization in the presence of 1-10 microM Ca2+ also decreased the rate at which protein kinase C exited from the cells. The slower release of protein kinase C caused by prior incubation of the cells with TPA or because of the presence of micromolar Ca2+ in permeabilized cells was associated with increased membrane-bound protein kinase C. The effects of TPA and permeabilization in the presence of micromolar Ca2+ were approximately additive. Active phorbol esters had different abilities to cause retention of protein kinase C in digitonin-treated cells. Dioctanoylglycerol, which activates protein kinase C in vitro and enhanced Ca2+-dependent secretion from permeabilized chromaffin cells similarly to TPA, also increased membrane-bound protein kinase C in intact cells, but had no effect on the retention of protein kinase C in permeabilized cells in the presence or absence of Ca2+. The different abilities of protein kinase C activators to cause retention of protein kinase C in subsequently permeabilized cells suggest differences in the reversibility of the binding. The mixed nicotinic-muscarinic agonist carbachol and the nicotinic agonist 1,1-dimethyl-4-phenylpiperazinium, but not the muscarinic agonist muscarine, caused 3-10% of the total protein kinase C activity to become membrane-bound within 3 min in intact chromaffin cells. Thus, nicotinic stimulation of chromaffin cells may rapidly activate protein kinase C.     

Regulation of inositol phospholipid and inositol phosphate metabolism in chemoattractant-activated human polymorphonuclear leukocytes

Binding of chemoattractants to specific cell surface receptors on polymorphonuclear leukocytes (PMNs) initiates a series of biochemical responses leading to cellular activation. A critical early biochemical event in chemoattractant (CTX) receptor-mediated signal transduction is the phosphodiesteric cleavage of plasma membrane phosphatidylinositol 4,5-bisphosphate (PIP2), with concomitant production of the calcium mobilizing inositol-1,4,5-trisphosphate (IP3) isomer, and the protein kinase C activator, 1,2-diacylglycerol (DAG). The following lines of experimental evidence collectively suggest that CTX receptors are coupled to phospholipase C via a guanine nucleotide binding (G) protein. Receptor-mediated hydrolysis of PIP2 in PMN plasma membrane preparations requires both fMet-Leu-Phe and GTP, and incubation of intact PMNs with pertussis toxin (which ADP ribosylates and inactivates some G proteins) eliminates the ability of fMet-Leu-Phe plus GTP to promote PIP2 breakdown in isolated plasma membranes. Studies with both PMN particulate fractions and with partially purified fMet-Leu-Phe receptor preparations indicate that guanine nucleotides regulate CTX receptor affinity. Finally, fMet-Leu-Phe stimulates high-affinity binding of GTP gamma S to PMN membranes as well as GTPase activity. A G alpha subunit has been identified in phagocyte membranes which is different from other G alpha subunits on the basis of molecular weight and differential sensitivity to ribosylation by bacterial toxins. Thus, a novel G protein may be involved in coupling CTX receptors to phospholipase C. Studies in intact and sonicated PMNs demonstrate that metabolism of 1,4,5-IP3 proceeds via two distinct pathways: 1) sequential dephosphorylation to 1,4-IP2, 4-IP1 and inositol, or 2) ATP-dependent conversion to inositol 1,3,4,5-tetrakisphosphate (IP4) followed by sequential dephosphorylation to 1,3,4-IP3, 3,4-IP2, 3-IP1 and inositol. Receptor-mediated hydrolysis of PIP2 occurs at ambient intracellular Ca2+ levels; but metabolism of 1,4,5-IP3 via the IP4 pathway requires elevated cytosolic Ca2+ levels associated with cellular activation. Thus, the two pathways for 1,4,5-IP3 metabolism may serve different metabolic functions. Additionally, inositol phosphate production appears to be controlled by protein kinase C, as phorbol myristate acetate (PMA) abrogates PIP2 hydrolysis by interfering with the ability of the activated G protein to stimulate phospholipase C. This implies a physiologic mechanism for terminating biologic responses via protein kinase C mediated feedback inhibition of PIP2 hydrolysis.     

Stimulation of insulin release by the phorbol ester 12-O-tetradecanoylphorbol 13-acetate in the clonal cell line RINm5F despite a lowering of the free cytoplasmic Ca2+ concentration

The effects of 12-O-tetradecanoylphorbol 13-acetate (TPA) on the handling of Ca2+ and insulin release were investigated in the clonal insulin-producing cell line RINm5F. The presence of the phorbol ester lowered the free cytoplasmic Ca2+ and suppressed the increase obtained by depolarization with high concentrations of K+. Despite the lowering in cytoplasmic Ca2+ by TPA, there was a concomitant stimulation of insulin release indicating that one feature of protein kinase C activation is to make the secretory system more sensitive to Ca2+. Furthermore, there was no interaction of TPA with the mechanisms responsible for inositol 1,4,5-tris(phosphate) induced Ca2+ release or Ca2+ uptake in permeabilized cells. Although TPA slightly depolarized the RINm5F cells there was no interference with K+-induced depolarization. It is suggested that an additional effect of protein kinase C activation in these cells, is to stimulate the extrusion of Ca2+ over the plasma membrane.     

Activation of the C3b receptor: effect of diacylglycerols and calcium mobilization

The plasma membrane expression and the phagocytic function of the C3b receptor (CR1) on human neutrophils (PMN) are under the control of cellular regulatory mechanisms, and phorbol esters are one class of agents that modulate both membrane expression and function. Phorbol esters also activate protein kinase C; however, the physiologic activation of protein kinase C is thought to be mediated by diacylglycerol. Diacylglycerols are generated during phosphatidyl inositol turnover, which is associated with a rise in intracellular calcium due to another product of polyphosphoinositide metabolism, inositol trisphosphate. We therefore studied the effects of synthetic diacylglycerols and calcium mobilization on CR1 function. In our experiments, treatment of neutrophils with two synthetic diacylglycerols, 1-oleoyl-2-acetoyl-sn-3-glycerol (OAG) and sn-1,2-dioctanoylglycerol, like phorbol esters, induced ligand-independent internalization of CR1. In contrast, the addition of exogenous phospholipase C had no effect on receptor internalization over the time course studied. OAG treatment also enabled neutrophils to specifically phagocytose via CR1. Calcium mobilization with the calcium ionophore A23187 (1 microM) had a synergistic effect on phorbol ester-induced internalization of CR1, but abrogated the phorbol ester enhancement of CR1-dependent phagocytosis. Both trimethoxybenzoate, the intracellular calcium antagonist, and chlorpromazine inhibited phorbol ester-induced internalization of CR1, whereas chelation of extracellular calcium did not. We conclude that activation of protein kinase C modulates the expression and function of CR1, and that calcium mobilization also influences these processes. We speculate that polyphosphoinositide turnover may be involved in the physiologic regulation of CR1.     

Down-regulation of protein kinase C potentiates angiotensin II-stimulated polyphosphoinositide hydrolysis in vascular smooth-muscle cells.

In smooth-muscle cells (SMC) isolated from rat aorta, angiotensin II stimulates a phospholipase C with subsequent formation of inositol trisphosphate (InsP3). Short-term (10 min) pretreatment of SMC with 12-O-tetradecanoylphorbol 13-acetate (TPA; 100 nM) decreases the angiotensin II-induced InsP3 formation. However, this inhibition is not observed after incubating the cells for 2 h with TPA. Longer-term pretreatments even lead to an enhanced generation of InsP3. This increased response to angiotensin II occurs without a significant change in the receptor number or Kd value of angiotensin II binding to the cells. The biologically inactive phorbol ester 4 alpha-phorbol 12,13-didecanoate was without effect on angiotensin II-stimulated InsP3 generation, irrespective of the time of preincubation. In parallel with this potentiation of angiotensin II-induced generation of InsP3 by TPA, a down-regulation of protein kinase C activity is observed. A 24 h pretreatment of SMC with TPA decreases protein kinase C activity to less than 10% of that of control cells. Longer-term pretreatment also increases the angiotensin II-induced release of Ca2+ and delays the decay of the transient Ca2+ increase. All these data suggest that protein kinase C exerts a negative feedback control on angiotensin II-stimulated polyphosphoinositide turnover, and that protein kinase C is an important factor in limiting the production of InsP3 in stimulated cells.     

Regulation of calcium channels in aortic muscle cells by protein kinase C activators (diacylglycerol and phorbol esters) and by peptides (vasopressin and bombesin) that stimulate phosphoinositide breakdown

Voltage-dependent Ca2+ channels of the aortic cell line A7r5 were studied using 45Ca2+ flux experiments. Ca2+ channels which have been studied belong to the L-type and are very sensitive to inhibitors and activators in the 1,4-dihydropyridine series as well as to (-)desmethoxyverapamil and d-cis-diltiazem. L-type Ca2+ channels in these smooth muscle cells are not affected by cyclic 8-bromo-AMP and dibutyryl cyclic AMP. However, the activity of these channels is strongly depressed after treatment with diacylglycerols (1-oleyl 2-acetylglycerol and 1,2-dioctanoylglycerol). Phorbol esters, which like diacylglycerols are well-known activators of protein kinase C (the Ca2+- and phospholipid-dependent enzyme), inhibit 70% of Ca2+ channel activity (K0.5 = 25 nM for phorbol 12-myristate 13-acetate and K0.5 = 200 nM for phorbol 12,13-dibutyrate). Phorbol esters that are inactive on kinase C are without effect on Ca2+ channel activity. [Arg8]Vasopressin and bombesin, two peptides that are well known for their action on polyphosphoinositide metabolism, inhibit Ca2+ channel activity to the same extent as active phorbol esters (65-70%). Oxytocin has the same type of effect presumably by acting at the V1-receptor. Both effects of [Arg8]vasopressin and oxytocin are suppressed by [1-(beta-mercapto-beta,beta-diethylpropionic acid)4-valine]arginine vasopressin, a specific vasopressin antagonist at the V1-receptor.     

Opposing effects of calcium entry and phorbol esters on fusion of chick muscle cells

Studies utilizing cultured muscle cells have shown that myoblast fusion requires extracellular Ca2+ and involves transient coordinated changes in cell membrane topography and cytoskeletal organization. However, neither the mechanisms by which Ca2+ influences these changes nor its cellular sites of action are known. We have investigated the effects of Ca2+ channel modulators and phorbol esters on fusion of embryonic chick myoblasts in culture. Myoblast fusion was inhibited by the Ca2+ channel blockers D600 and nitrendipine and stimulated by the Ca2+ channel activator Bay K 8644. We have obtained evidence that the tumor promoting phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) inhibits fusion through activation of protein kinase C. Myoblasts prevented from fusing by Ca2+ channel blockers or TPA display a distinctive elongated morphology that is characteristic of cells prevented from fusion by Ca2+ deprivation. The inhibition of fusion by D600 and TPA is significantly diminished in the presence of the Ca2+ ionophore A23187. TPA arrest of myoblast fusion was found to be accompanied by an increase in phosphorylation of the 20-kDa light chain of cytoplasmic myosin in a dose- and time-dependent manner. The effects of TPA on myoblast fusion and phosphorylation of myosin light chain were mimicked by the cell permeant diacylglycerol sn-1,2-dioctanoylglycerol, a potent activator of protein kinase C. The present results suggest that activators of protein kinase C block fusion by interfering with a Ca2+ signal transduction pathway and that this interference may be associated with a protein kinase C catalyzed inhibitory phosphorylation of myosin light chain.     

Intracellular protein phosphorylation in oat (Avena sativa L.) protoplasts by phytochrome action: involvement of protein kinase C

Phosphorylations of two proteins (27 KDa, 32 KDa) in oat cells were dependent on phytochrome action. To determine which kinase system(s) for the phosphorylation of these two proteins are controlled by the phytochrome, involvement of the Ca2+/DG dependent protein kinase (protein kinase C) was first investigated. When a protein kinase C inhibitor (1-(5-isoquinoline sulfonyl)-2-methylpiperazine:H-7) or the inositol phospholipid metabolic blocker Li+ was added into the cell suspension, respectively, the phosphorylations of these two proteins were substantially reduced. On the other hand, an addition of 1-oleoyl-2-acetyl-sn-glycerol (OAG:activator of protein kinase C) or phorbol 12-myristate 13-acetate (TPA: tumor promoting phorbol ester) enhanced the phosphorylations of these proteins. These results suggest that phytochrome action is certainly connected with the protein phosphorylation via the activation of protein kinase C or a similar molecule with protein kinase C.     

Diacylglycerol, but not inositol 1,4,5-trisphosphate, accounts for platelet-derived growth factor-stimulated proliferation of BALB 3T3 cells

Recently we found that an intracellular event related to phosphatidylinositol 4,5-bisphosphate (PIP2) is crucial for platelet-derived growth factor (PDGF)-induced mitogenesis in fibroblastic cells (Matuoka, K., et al.: Science 239:640-643, 1988). In the present study we examined the mitogenic effects of PIP2 and its hydrolysis products introduced into the cytoplasm of BALB 3T3 cells by micro-injection to confirm the role of PIP2 hydrolysis in PDGF stimulation of cell proliferation. Injection of 1,2-dioleylglycerol (diolein) into serum-deprived quiescent cells induced DNA synthesis with the same time course as that induced by exposure of the cells to PDGF and, in the presence of PDGF, caused no additional increase in the cell population entering S phase. The injection of PIP2, inositol 1,4,5-trisphosphate, or 1,2-dioleylphosphatidic acid into the cells did not induce mitogenesis. Consistent results were obtained in experiments in which the cells were exposed to 1-oleyl-2-acetylglycerol (OAG) and ionomycin; namely, OAG stimulated proliferation of BALB 3T3 cells, but ionomycin did not induce any mitogenesis. Desensitization of the protein kinase C pathway by prolonged exposure of the cells to phorbol ester abolished the induction of cell proliferation by subsequent injection of diolein or exposure to phorbol ester or OAG as well as by PDGF challenge. These findings strongly suggest that activation of the protein kinase C system following formation of diacylglycerol by PIP2 hydrolysis is mainly responsible for the mitogenic action of PDGF on BALB 3T3 cells.     

Ethanol-induced phospholipase C activation is inhibited by phorbol esters in isolated hepatocytes.

Ethanol causes a transient activation of the phosphoinositide-specific phospholipase C in intact hepatocytes and mimics the action of receptor-mediated agonists [Hoek, Thomas, Rubin & Rubin (1987) J. Biol. Chem. 262, 682-691]. Preincubation of the hepatocytes with phorbol esters which activate protein kinase C prevented this effect of ethanol: phorbol ester treatment inhibited the ethanol-induced phosphorylase activation, the increase in intracellular free Ca2+ concentrations measured in quin 2-loaded hepatocytes, and the changes in concentrations of inositol phosphates, phosphoinositides and phosphatidic acid. Several lines of evidence indicate that these effects were mediated by protein kinase C. Phorbol esters acted in a concentration range where they activate protein kinase C; phorbol esters that do not activate protein kinase C were not effective in inhibiting the effects of ethanol. The permeant diacylglycerol oleoyl-acetylglycerol also inhibited the effects of ethanol, but other diacylglycerols were not effective in the intact cells. The inhibition of ethanol-induced Ca2+ mobilization by phorbol esters was prevented by preincubating the cells with the protein kinase C inhibitors 1-(5-isoquinolinesulphonyl)-2-methylpiperazine (H7) and sphingosine. H7 also enhanced the Ca2+ mobilization induced by ethanol in cells that were not pretreated with phorbol esters, indicating that the transient nature of the ethanol-induced Ca2+ mobilization may be due to an activation of protein kinase C caused by the accumulation of diacylglycerol. These data support a model whereby ethanol activates the phosphoinositide-specific phospholipase C, possibly by affecting receptor-G-protein-phospholipase C interactions in the membrane.     

Phorbol esters reduce gonadotrope responsiveness to protein kinase C activators but not to Ca2+-mobilizing secretagogues. Does protein kinase C mediate gonadotropin-releasing hormone action?

The demonstration that activators of the Ca2+-activated, phospholipid-dependent protein kinase (protein kinase C), such as phorbol esters and diacylglycerols, can provoke luteinizing hormone (LH) release from pituitary gonadotropes, suggests a possible role for protein kinase C in stimulus-release coupling. We now report that administration of phorbol myristate acetate (PMA) to pituitary cell cultures causes a sustained reduction in Triton X-100-extracted protein kinase C activity. Further, phorbol ester- and diacylglycerol-stimulated LH release, as well as inhibition by PMA of gonadotropin-releasing hormone (GnRH)-stimulated inositol phosphate production, were reduced by pretreatment with PMA. The effects of phorbol ester pretreatment on PMA-stimulated LH release and protein kinase C activity were dose-dependent, sustained (greater than or equal to 24 h) and specific (no measurable effect with 4 alpha-phorbol didecanoate). The effect on PMA-stimulated LH release was apparently Ca2+-independent. In pituitary cell cultures with reduced protein kinase C activity, the gonadotropes have reduced responsiveness to PMA but release a similar proportion of cellular LH in response to Ca2+-mobilizing secretagogues (GnRH and A23187) as do control cells. The normal responsiveness to GnRH of cells with reduced responsiveness to protein kinase C activators calls into question the requirement for this enzyme for GnRH-stimulated LH release.     

Protein Kinase C of Sympathetic Neuronal Membrane Is Activated by Phorbol Ester-Correlation Between Transmitter Release, 45Ca2+ Uptake, and the Enzyme Activity

The effects of phorbol esters [phorbol 12,13-dibutyrate (PDB), 12-O-tetradecanoylphorbol 13-acetate (TPA), and phorbol 13-acetate] were investigated on the release of [3H]norepinephrine, 45Ca2+ accumulation, and protein kinase C activity in cultured sympathetic neurons of the chick embryo. Sympathetic neurons derived from 10-day-old chick embryo were cultured in serum-free medium supplemented with insulin, transferrin, and nerve growth factor. After 3 days, neurons were loaded with [3H]-norepinephrine and the release of [3H]norepinephrine was determined before and after electrical stimulation. Stimulation at 1 Hz for 15 s increased the release of [3H]-norepinephrine over the nonstimulation period. Stimulation-evoked release gradually declined with time during subsequent stimulation periods. Incubation of neurons in Ca2+-free Krebs solution containing 1 mM EGTA completely blocked stimulation-evoked release of [3H]-norepinephrine. Stimulation-evoked release of [3H]-norepinephrine was markedly facilitated by 3 and 10 nM PDB or TPA. The spontaneous release was also enhanced by PDB and TPA. The net accumulation of 45Ca2+ during stimulation of sympathetic neurons was increased by two- to fourfold in the presence of PDB or TPA. PDB at 1-100 nM produced a concentration-dependent increase in the activation of protein kinase C. PDB at 30 nM increased the activity of protein kinase C of the particulate fraction from 0.09 to 0.58 pmol/min/mg protein. There was no significant change in protein kinase C activity of the cytosolic fraction (0.14 pmol/min/mg versus 0.13 pmol/min/mg protein). The ratio of the particulate to cytosolic protein kinase C increased from a control value of 0.62 to 4.39 after treatment with 30 nM PDB. TPA (10 and 30 nM) also increased protein kinase C activity of the particulate fraction by six- to eightfold. Phorbol 13-acetate had no effect on protein kinase C activity, [3H]norepinephrine release, and 45Ca2+ accumulation. These results provide direct evidence that activation of protein kinase C enhances Ca2+ accumulation, which in turn leads to the facilitation of transmitter release in sympathetic neurons.     

Phorbol ester modulates serotonin-stimulated phosphoinositide breakdown in cultured vascular smooth muscle cells

Stimulation of cultured rabbit aortic vascular smooth muscle cells (VSMC) with serotonin (5HT) induced a rapid generation of inositol phosphates from receptor-mediated hydrolysis of inositol phospholipids. Pretreatment of these cells with 500ng/ml of pertussis toxin for 24h prior to addition of 5HT reduced 5HT-induced formation of inositol phosphates. Phorbol esters, such as 12-O-tetradecanoylphorbol-13-acetate (TPA) or phorbol-12,13-dibutyrate (PDBu), are known to activate protein kinase C (PKC), but their role on cultured VSMC stimulated by 5HT has not been defined. TPA exhibited a rapid inhibition of 5HT-stimulated phosphoinositide breakdown, although 4 alpha-phorbol-12,13-didecanoate (4 alpha PDD), an inactive phorbol ester, did not inhibit it. These data suggest that a guanine nucleotide inhibitory (Gi) protein couples 5HT receptor to phospholipase C and TPA modulates 5HT-stimulated hydrolysis of inositol phospholipids in cultured VSMC through activation of PKC.     

Dual role for guanine nucleotides in stimulus-secretion coupling

We have made mast cells and neutrophils permeable to gain access to the cytosol and thus to manipulate the composition of the cytosol. Secretion from both cell types can be triggered by elevation of cytosol Ca2+ to concentrations approaching 10-6 M; alternatively, secretion from mast cells, and of beta-glucuronidase (but not lysozyme) from neutrophils, can be triggered in the absence of Ca2+ by introducing stable analogs of GTP. We propose that GTP acts at two intracellular guanine nucleotide regulatory proteins (N proteins) in the stimulus-secretion sequence. By interaction with Np located on the inner face of the plasma membrane, it activates polyphosphoinositide phosphodiesterase to yield inositol phosphates and diacylglycerol. By interaction with Ne, situated distal to the site of action of Ca2+ and protein kinase C, it directly activates the exocytotic process without intervention of the products of polyphosphoinositide hydrolysis.