Synergistic induction of ornithine decarboxylase by diacylglycerol, A23187, and cholera toxin in guinea pig lymphocytes

When guinea pig lymphocytes were cultured with 1-oleoyl-2-acetyl-glycerol (OAG), A23187, and cholera toxin, ornithine decarboxylase activity was induced synergistically, peaking at 6 h. Addition of 12-O-tetradecanoyl-phorbol 13-acetate (TPA), A23187, and dibutyryl cAMP caused the same kind of induction. Cholera toxin potentiated the ability of A23187 to induce ornithine decarboxylase, but not that of OAG. Dibutyryl cAMP augmented the induction caused by A23187 but not by TPA. These results suggest that both the activation of Ca++-sensitive, phospholipid-dependent protein kinase (protein kinase C) and the increase in intracellular levels of Ca++ and cAMP are necessary for this induction. cAMP may potentiate the induction by modulating a Ca++ messenger system other than that for protein kinase C activation.     

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

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

Induction of ornithine decarboxylase activity by 1-oleoyl-2-acetyl-glycerol in isolated mouse epidermal cells

1-Oleoyl-2-acetyl-glycerol induced a rise in ornithine decarboxylase activity in isolated epidermal cells in a concentration-dependent manner. The time course of the induction of ornithine decarboxylase by 1-oleoyl-2-acetyl-glycerol was similar to that by 12-O-tetradecanoylphorbol-13-acetate. A23187 did not enhance the enzyme induction caused by 1-oleoyl-2-acetyl-glycerol. Palmitoyl-DL-carnitine prevented the induction of the enzyme either by 1-oleoyl-2-acetyl-glycerol or 12-O-tetradecanoyl-phorbol-13-acetate. These results suggest that the activation of protein kinase C is an initial and essential event in the process of ornithine decarboxylase induction caused by 12-O-tetradecanoyl-phorbol-13-acetate.     

Mesocestoides lineatus: trypsin induced development to adult mediated by Ca2+ and protein kinase C

A mode of action of the inducible treatment with trypsin for the development of Mesocestoides lineatus tetrathyridium to adult was analyzed by administering various agents effective on Ca2+-dependent metabolic pathways in the cells: protein kinase C activators such as a synthetic diacylglycerol, 1-oleoyl-2-acetylglycerol, and a tumor promoting phorbol, 12-O-tetra-decanoyl-phorbol-13-acetate, enhanced the trypsin induced developmental processes. On the contrary, a calmodulin inhibitor, N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide, cyclic adenosine 3',5'-monophosphate, and adenylate cyclase activators such as forskolin and cholera toxin, inhibited the triggering action of trypsin. Furthermore, a combined administration of Ca2+ ionophore (A23187) and the phorbol showed a similar effect with trypsin treatment, and sodium taurocholate acted as a potent enhancer like the activators of protein kinase C. These results strongly suggest that the initiation of development to adult in this cestode may be regulated synergistically by Ca2+ and protein kinase C, and that a bile acid may be involved in an activation mechanism of protein kinase C.     

Synergistic stimulation of luteinizing hormone (LH) release by protein kinase C activators and Ca2+-ionophore

When cultured pituitary cells were stimulated with synthetic diacylglycerol such as 1-oleoyl-2-acetylglycerol (OAG), or with a potent tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA), which are known stimulators of Ca2+-activated, phospholipid-dependent protein kinase (protein kinase C), enhanced release of luteinizing hormone (LH) was observed. Similarly, LH release was also stimulated by the Ca2+-ionophore, A23187. Simultaneous presence of A23187 and OAG or TPA resulted in a synergistic response that mimicked the full physiological response to gonadotropin releasing hormone (GnRH). Removal of extracellular Ca2+ only slightly affected the stimulatory action of TPA and OAG on LH release, but completely blocked the effect of GnRH. The results suggest that the stimulatory effect of GnRH on LH release may be mediated by two intracellular pathways involving Ca2+ and diacylglycerol as second messengers.     

Differential effects of phorbol ester on phenylephrine and vasopressin-induced Ca2+ mobilization in isolated hepatocytes

Receptor-mediated breakdown of PtdIns(4,5)P2 produces two cellular signals, Ins(1,4,5)P3, which can release intracellular Ca2+, and diacylglycerol, which activates a Ca2+- and phospholipid-dependent protein kinase (protein kinase C). This study assesses the significance of protein kinase C in relation to phenylephrine- and vasopressin-induced Ca2+ mobilization in hepatocytes. Phorbol ester (4 beta-phorbol-12-myristate-13-acetate), which can directly activate protein kinase C, had no effect either on Ca2+ efflux from the cell (measured with arsenazo III) or on Ca2+ influx (measured with Quin-2), processes which are inhibited and stimulated, respectively, by both phenylephrine and vasopressin. No evidence of synergism between phorbol ester pretreatment of hepatocytes and the Ca2+ ionophore (ionomycin)-mediated effects on the increase of cytosolic free Ca2+ and phosphorylase activation could be obtained. These findings suggest that protein kinase C is not obligatorily involved in the regulation of hepatocyte Ca2+ fluxes. Pretreatment of hepatocytes with phorbol ester (PMA) or 1-oleoyl-2-acetylglycerol totally inhibited the effects of phenylephrine in elevating the cytosolic free Ca2+; half-maximal inhibitory effects occurred at PMA and 1-oleoyl-2-acetylglycerol concentrations of 1 ng/ml and 12 micrograms/ml, respectively. In contrast, pretreatment with PMA had a much smaller effect on Ca2+ mobilization induced by vasopressin. These observations suggest that protein kinase C may be involved in "down-regulation" of the alpha 1-receptor in hepatocytes and may thus exert a negative influence on the Ca2+-signalling pathway.     

The role of Ca2+ and Ca2+-activated phospholipid-dependent protein kinase in degranulation of human neutrophils

The degranulation reactions of human neutrophils induced by 1-oleoyl-2-acetylglycerol (OAG), phorbol 12-myristate 13-acetate (PMA), and calcium ionophore A23187 or their combinations, were studied. OAG in the absence of the Ca2+-ionophore A23187 stimulated the releases of both lysozyme and lactoferrin, constituents of the specific granules, but did not stimulate the release of beta-glucuronidase, an enzyme of the azurophil granules. Electron microscopy revealed a selective decrease in the numbers of the specific granules in this case. The combined effects of A23187 at a concentration higher than 0.1 microM and OAG were essentially additive. W-7, known to be an inhibitor of both Ca2+-activated phospholipid-dependent protein kinase (C-kinase) and calmodulin, inhibited the degranulation induced by OAG or PMA, while it inhibited the reaction induced by A23187 less markedly. The release of lysozyme reached a plateau at about 0.1 microM A23187 and increased again at higher concentrations of A23187. The observations suggest that degranulation can be induced by the activation of the C-kinase, and the degranulation by A23187 at low concentrations may be due to the activation of the C-kinase; the effects of A23187 at high concentrations, however, could not be explained only in terms of the activation of the C-kinase.     

Protein kinase C of human erythrocytes phosphorylates bands 4.1 and 4.9

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

Effects of Ca2+ ionophore A23187, 1,2-dioctanoylglycerol, and dibutyryl cAMP on the activity and expression of ornithine decarboxylase in guinea pig lymphocytes

The Ca2+ ionophore A23187 induced small increases in ornithine decarboxylase activity and ornithine decarboxylase mRNA in guinea pig lymphocytes. 1,2-Dioctanoylglycerol potentiated the A23187-induced ornithine decarboxylase activity and the accumulation of mRNA for this enzyme. Dibutyryl cAMP also potentiated the enzyme activity, but had little effect on the accumulation of mRNA. 1,2-Dioctanoylglycerol and 12-O-tetradecanoylphorbol-13-acetate potentiated ornithine decarboxylase activity that had been increased by treatment with both A23187 and dibutyryl cAMP with a consistent increase in the ornithine decarboxylase mRNA. However, dibutyryl cAMP augmented ornithine decarboxylase activity that had been increased by the combination of A23187 and 1,2-dioctanoylglycerol without affecting the ornithine decarboxylase mRNA level. These results suggest that the protein kinase C and cyclic AMP pathways are involved in the enhancement of ornithine decarboxylase activity in guinea pig lymphocytes, but that the mechanisms of the enhancement differ for each pathway, the former increasing the ornithine decarboxylase mRNA level, but not the latter.     

Protein kinase C and calcium ion in mitogenic response of macrophage-depleted human peripheral lymphocytes

For mitogenic response of macrophage-depleted human peripheral lymphocytes, 12-O-tetradecanoylphorbol-13-acetate (TPA) or 1-oleoyl-2-acetylglycerol (OAG) and Ca2+ ionophore are both needed in addition to a small quantity of plant lectin, phytohemagglutinin (PHA). PHA alone is not sufficient to produce the cellular response. The addition of TPA or OAG to these cells induces the activation of protein kinase C as assayed by the phosphorylation of its endogenous substrates. Apparently, TPA or OAG and A23187 together substitute for macrophages and act synergistically to potentiate the DNA synthesis of this lymphocyte preparation. The results suggest that protein kinase C activation and Ca2+ mobilization are essential and that additional receptor occupation by PHA is necessary for producing cell proliferation.     

Phospholipase A2 activation in chemotactic peptide-stimulated HL60 granulocytes: synergism between diacylglycerol and Ca2+ in a protein kinase C-independent mechanism

In dimethylsulfoxide-differentiated HL60 granulocytes, the chemotactic peptide N-formyl-Met-Leu-Phe (FMLP) augments arachidonic acid (AA) release via phospholipase A2 activity induced by the Ca2+-ionophore, A23187. Evidence indicates that this augmentation is mediated by diacylglycerols formed endogenously during FMLP receptor activation: The augmentation is mimicked by the synthetic diglyceride 1-oleoyl-2-acetyl-glycerol (OAG) and the tumor promoting phorbol ester 12-O-tetradecanoylphorbol-13-acetate; Pertussis toxin inhibits FMLP-induced augmentation but not OAG-induced augmentation: At suboptimal concentrations FMLP and OAG act cooperatively to augment ionophore A23187-induced AA release but not at optimal concentrations. These data indicate that phospholipase A2 activation in FMLP-stimulated HL60 granulocytes involves cooperative interactions between diacylglycerol formed endogenously and Ca2+. Interestingly, this effect of diacylglycerol appears not to be mediated by protein kinase C, since a specific protein kinase C inhibitor, 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H7) does not inhibit receptor-mediated release of AA by stimulated HL60 granulocytes.     

Involvement of calcium-sensitive phospholipid-dependent protein kinase in control of acid secretion by isolated rat parietal cells.

The relative potency with which phorbol esters inhibited histamine-stimulated aminopyrine accumulation (an index of acid secretion) paralleled that which has been established for the activation of purified protein kinase C. The inhibitory effect of 1-oleoyl-2-acetylglycerol on aminopyrine accumulation stimulated by various secretagogues was similar to that of 12-O-tetradecanoylphorbol 13-acetate. Protein kinase C activity was present in a parietal-cell-enriched fraction. In conclusion, protein kinase C could be involved in mechanisms regulating gastric acid secretion.     

Failure of 1-oleoyl-2-acetylglycerol to mimic the cell-differentiating action of 12-O-tetradecanoylphorbol 13-acetate in HL-60 cells

Treatment of human promyelocytic leukemia cells (HL-60 cells) with 12-O-tetradecanoylphorbol 13-acetate (TPA) results in terminal differentiation of the cells to macrophage-like cells. Treatment of the cells with TPA induced marked enhancement of the phosphorylation of 28- and 67-kDa proteins and a decrease in that of a 75-kDa protein. When the cells were treated with diacylglycerol, i.e. 50 micrograms/ml 1-oleoyl-2-acetylglycerol (OAG), similar changes in the phosphorylation of 28-, 67-, and 75-kDa proteins were likewise observed, indicating that OAG actually stimulates protein kinase C in intact HL-60 cells. OAG (1-100 micrograms/ml), which we used, activated partially purified mouse brain protein kinase C in a concentration-dependent manner. Treatment of HL-60 cells with 10 nM TPA for 48 h caused an increase by about 8-fold in cellular acid phosphatase activity. Although a significant increase in acid phosphatase activity was induced by OAG, the effect was scant compared to that of TPA (less than 7% that of TPA). After 48-h exposure to 10 nM TPA, about 95% of the HL-60 cells adhered to culture dishes. On the contrary, treatment of the cells either with OAG (2-100 micrograms/ml) or phospholipase C failed to induce HL-60 cell adhesion. Ca2+ ionophore A23187 failed to act synergistically with OAG. In addition, hourly or bi-hourly cumulative addition of OAG for 24 h also proved ineffective to induce HL-60 cell adhesion. Our present results do not imply that protein kinase C activation is nonessential for TPA-induced HL-60 cell differentiation, but do demonstrate that protein kinase C activation is not the sole event sufficient to induce HL-60 cell differentiation by means of this agent.     

Multiple regulation of proenkephalin gene expression by protein kinase C

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

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

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

The multifunctional Ca2+/calmodulin-dependent protein kinase mediates Ca2+-dependent phosphorylation of tyrosine hydroxylase

Stimulation of rat pheochromocytoma PC12 cells with ionophore A23187, carbachol, or high K+ medium, agents which increase intracellular Ca2+, results in the phosphorylation and activation of tyrosine hydroxylase (Nose, P., Griffith, L. C., and Schulman, H. (1985) J. Cell Biol. 101, 1182-1190). We have identified three major protein kinases in PC12 cells and investigated their roles in the Ca2+-dependent phosphorylation of tyrosine hydroxylase and other cytosolic proteins. A set of PC12 proteins were phosphorylated in response to both elevation of intracellular Ca2+ and to protein kinase C (Ca2+/phospholipid-dependent protein kinase) activators. In addition, distinct sets of proteins responded to either one or the other stimulus. The three major regulatory kinases, the multifunctional Ca2+/calmodulin-dependent protein kinase, the cAMP-dependent protein kinase, and protein kinase C all phosphorylate tyrosine hydroxylase in vitro. Neither the agents which increase Ca2+ nor the agents which directly activate kinase C (12-O-tetradecanoylphorbol-13-acetate or 1-oleyl-2-acetylglycerol) increase cAMP or activate the cAMP-dependent protein kinase, thereby excluding this pathway as a mediator of these stimuli. The role of protein kinase C was assessed by long term treatment of PC12 cells with 12-O-tetradecanoylphorbol-13-acetate, which causes its "desensitization." In cells pretreated in this manner, agents which increase Ca2+ influx continue to stimulate tyrosine hydroxylase phosphorylation maximally, while protein kinase C activators are completely ineffective. Comparison of tryptic peptide maps of tyrosine hydroxylase phosphorylated by the three protein kinases in vitro with phosphopeptide maps generated from tyrosine hydroxylase phosphorylated in vivo indicates that phosphorylation by the Ca2+/calmodulin-dependent kinase most closely mirrors the in vivo phosphorylation pattern. These results indicate that the multifunctional Ca2+/calmodulin-dependent protein kinase mediates phosphorylation of tyrosine hydroxylase by hormonal and electrical stimuli which elevate intracellular Ca2+ in PC12 cells.     

Induction of ornithine decarboxylase in guinea-pig lymphocytes. Synergistic effect of diacylglycerol and calcium

Calmodulin antagonists, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), N-(6-aminohexyl)-1-naphthalenesulfonamide (W-5) and trifluoperazine inhibited ornithine decarboxylase induction in lymphocytes activated with phytohemagglutinin or inophore A23187. W-7, a more potent calmodulin antagonist than W-5, suppressed ornithine decarboxylase induction in a higher extent than did W-5. These results suggest that calmodulin may play an important role in ornithine decarboxylase induction in the activated lymphocytes. However, the extent of ornithine decarboxylase induction was greater in cells pretreated with Clostridium phospholipase C and then incubated with ionophore A23187 than in cells incubated with ionophore A23187 without the pretreatment. Moreover, combined treatment of cells with ionophore A23187 and tumor promotor, phorbol 12-myristate 13-acetate, caused synergistic induction of ornithine decarboxylase activity. These results, taken together, suggest that both activations of Ca2+-activated phospholipid-dependent protein kinase by diacylglycerol and of calmodulin-dependent function resulted from an elevation of cytosolic Ca2+ concentration may operate in the induction of ornithine decarboxylase in the activated lymphocytes.     

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

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

Protein kinase C-activated calcium channel in the osteoblast-like clonal osteosarcoma cell line UMR-106

The effects of protein kinase C stimulation on free cytosolic Ca2+ [( Ca2+]i) were studied in Fura 2-loaded UMR-106 cells. Stimulation of the protein kinase C with the tumor-promoting phorbol esters 12-O-tetradecanoylphorbol 13-acetate (TPA) and phorbol 12,13-diacetate or 1-oleoyl-2-acetylglycerol was followed by an increase in [Ca2+]i. The protein kinase C-induced increase in [Ca2+]i has a lag period, the duration of which was dependent on the stimulant and medium Ca2+ concentrations. With 2 microM TPA, the rise in [Ca2+]i peaked within 1.5 min, after which [Ca2+]i returned partially toward base line. The increase in [Ca2+]i was absolutely dependent on the presence of medium Ca2+ and was inhibited by the Ca2+ channel blockers nicardipine and verapamil. Cell stimulation also results in Ca2+ release from intracellular pool(s) which appears to be mediated by a Ca2+-dependent Ca2+ release mechanism. The reduction in [Ca2+]i was due to channel inactivation. Pretreatment of the cells with 1 nM TPA, 2 units/ml parathyroid hormone (PTH), or 15 microM forskolin blocked the effect of 2 microM TPA on [Ca2+]i. TPA and PTH were more potent inhibitors than was forskolin. The properties of this channel are compared to the cAMP-independent PTH-stimulated Ca2+ channel present in these cells.     

Ca2+-stimulated catecholamine release from alpha-toxin-permeabilized PC12 cells: biochemical evidence for exocytosis and its modulation by protein kinase C and G proteins

Two possible cellular pathways of catecholamines from the chromaffin vesicles of PC12 cells to the surrounding medium are explored in this study. The direct one circumventing the cytoplasm can be activated in alpha-toxin-permeabilized cells with micromolar levels of free Ca2+. Catecholamine metabolites formed in the cytoplasm (i.e., 3,4-dihydroxyphenylacetic acid and 3,4-dihydroxyphenylethanol) are neither formed nor released from the cells under these conditions. However, when vesicular catecholamines were discharged into the cytoplasm by addition of the ionophore nigericin, such metabolites are formed and released into the medium independent of Ca2+. Both types of experiments provide direct evidence for the operation of Ca2+-induced exocytosis of dopamine and noradrenaline in permeabilized PC12 cells. The Ca2+ dependence of dopamine or noradrenaline release, as measured by the determination of the endogenous catecholamines using the high-performance liquid chromatography technique, exhibits two different phases. One is already activated below 1 microM free Ca2+ and plateaus at 1-5 microM free Ca2+, while a second occurs in the presence of larger amounts of free Ca2+ (10-100 microM). Ca2+-induced catecholamine release from the permeabilized cells can be modulated in different ways: It is enhanced by the phorbol ester 12-O-tetradecanoylphorbol 13-acetate and the diacylglycerol 1-oleyl-2-acetylglycerol provided Mg2+/ATP is present, and it is inhibited by guanosine 5'-O-(3-thiotriphosphate). The latter effect is abolished by pretreatment of the cells with pertussis toxin but not by cholera toxin. Thus, it appears that Ca2+-induced exocytosis can be modulated via the protein kinase C system, as well as via GTP binding proteins.