Cellular signals regulating the release of ANF.

Atrial natriuretic factor (ANF), a peptide hormone that regulates salt and water balance and blood pressure, is synthesized, stored, and secreted from mammalian myocytes. Stretching of atrial myocytes stimulates ANF secretion, but the cellular processes involved in linking mechanical distension to ANF release are unknown. We reported that phorbol esters, which mimic the action of diacylglycerol by acting directly on protein kinase C and the Ca2+ ionophore A23187, which introduces free Ca2+ into the cell, both increase basal ANF secretion in the isolated perfused rat heart. Phorbol ester also increased responsiveness to Ca2+ channel agonists, such as Bay k8644, and to agents that increase cAMP, such as forskolin and membrane-permeable cAMP analogs. In neonatal cultured rat atrial myocytes, protein kinase C activation by 12-O-tetradecanoylphorbol 13-acetate stimulated ANF secretion, whereas the release was unresponsive to changes in intracellular Ca2+. Endothelin, which stimulates phospholipase C mediated hydrolysis of phosphoinositides and activates protein kinase C, increased both basal and atrial stretch-induced ANF secretion from isolated perfused rat hearts. Similarly, phorbol ester enhanced atrial stretch-stimulated ANF secretion, while the increase in intracellular Ca2+ appeared to be negatively coupled to the stretch-induced ANF release. Finally, phorbol ester stimulated ANF release from the severely hypertrophied ventricles of hypertensive animals but not from normal rat myocardium. These results suggest that the protein kinase C activity may play an important role in the regulation of basal ANF secretion both from atria and ventricular cells, and that stretch of atrial myocytes appears to be positively modulated by phorbol esters.     

Effects of protein kinase inhibitors on pig oocyte maturation in vitro.

Normal oocyte maturation depends on signal transmission between granulosa cells and the oocyte. We have analysed the effects of inhibiting (I) cyclic AMP-dependent protein kinase (protein kinase A, PK-A), (II) Ca2+/phospholipid-dependent protein kinase (protein kinase C, PK-C) and (III) calmodulin (CaM) on pig oocyte maturation in vitro, protein synthesis and phosphorylation. The inhibition of PK-A using a specific inhibitor H8, decreased the maturation rate (rate of germinal vesicle breakdown, GVBD) of cumulus-enclosed pig oocytes in a dose-dependent manner by approximately 12%, reaching a plateau at 100 microM. The inhibition of PK-C with H7, an inhibitor with some side-effects on PK-A, decreased the maturation rate of cumulus-enclosed oocytes in a dose-dependent manner to a maximum of 20% at a concentration of 100 microM. The calmodulin antagonist W7 up to a concentration of 200 microM had no effects on maturation of cumulus-enclosed pig oocytes. None of the inhibitors (H7, H8 and W7) altered the patterns of protein synthesis of either pig oocytes and cumulus cells after maturation in vitro. Oocyte phosphoprotein patterns were, however, clearly changed by W7. Cumulus cell protein phosphorylation patterns were changed by all 3 agents. Since inhibition of cyclic AMP and Ca2+ phospholipid pathways by PK-A and PK-C blocking chemicals affected only a limited proportion of oocytes (12 and 20%, respectively) and inhibition of Ca2+ binding to CaM was without effect on oocyte maturation, we conclude that these pathways modulate rather than regulate oocyte maturation in the pig.     

Protein kinase C mediated regulation of calcium channels in PC-12 pheochromocytoma cells

Depolarization of PC-12 pheochromocytoma cells with K+ produces an immediate increase in catecholamine release. The stimulation of release is blocked by Co2+, removal of extracellular Ca2+ or by dihydropyridine drugs such as nitrendipine. Release is enhanced by other dihydropyridines such as BAY K8644. Release is accompanied by a voltage dependent uptake of 45Ca2+ which is also blocked by Co2+ or nitrendipine and enhanced by BAY K8644. The phorbol ester phorbol 12-myristate-13-acetate (TPA) in the range 10(-9)-10(-6) M produced little effect by itself but augmented the K+ evoked release of catecholamine. An analog of TPA which does not activate protein kinase C was ineffective. In contrast, TPA in the same concentration range blocked influx of 45Ca2+ induced by 70 mM K+ or 70 mM K+/BAY K8644. 45Ca2+ influx produced by A23187 was not blocked by TPA. The results suggest a system by which protein kinase C may regulate the output of transmitters from secretory cells.     

Protein phosphorylation and secretion in digitonin-permeabilized adrenal chromaffin cells. Effects of micromolar Ca2+, phorbol esters, and diacylglycerol

The effects of phorbol esters, dioctanoylglycerol (DiC8), and micromolar Ca2+ on protein phosphorylation and catecholamine secretion in digitonin-treated chromaffin cells were investigated. [gamma-32P]ATP was used as a substrate for phosphorylation in the permeabilized cells. 12-O-Tetradecanoylphorbol-13-acetate (TPA) enhanced Ca2+-dependent catecholamine secretion from digitonin-permeabilized cells. The enhancement required MgATP. Only those phorbol esters which activate protein kinase C in vitro enhanced both catecholamine secretion and protein phosphorylation. DiC8, which activates protein kinase C in vitro and mimics phorbol ester effects in situ, also enhanced both catecholamine secretion and protein phosphorylation. Preincubation of intact cells with TPA or DiC8 was necessary for maximal effects on both catecholamine secretion and protein phosphorylation in subsequently digitonin-treated chromaffin cells. The TPA-induced enhancement of protein phosphorylation was almost entirely Ca2+-independent, whereas DiC8-induced enhancement of protein phosphorylation was mainly Ca2+-dependent. Micromolar Ca2+ alone also enhanced the phosphorylation of a large number of proteins. Most of the proteins phosphorylated in response to TPA or potentiated by DiC8 in combination with Ca2+ were also phosphorylated by micromolar Ca2+ in the absence of exogenous protein kinase C activators. In intact cells, 1,1-dimethyl-4-phenylpiperazinium (DMPP) induced Ca2+-dependent phosphorylation of at least 17 proteins which were detected by two-dimensional gel electrophoresis. All of the proteins phosphorylated upon incubation with 1,1-dimethyl-4-phenylpiperazinium were phosphorylated upon incubation with micromolar Ca2+ in digitonin-treated cells. These results demonstrate that TPA- or DiC8-enhanced Ca2+-dependent catecholamine secretion is associated with enhanced protein phosphorylation which is probably mediated by protein kinase C and that activation of protein kinase C modulates catecholamine secretion from digitonin-treated chromaffin cells.     

Control of exocytosis from adrenal chromaffin cells

1. Calcium-dependent exocytosis of catecholamines from intact and digitonin-permeabilized bovine adrenal chromaffin cells was investigated. 2. 45Ca2+ uptake and secretion induced by nicotinic stimulation or depolarization in intact cells were closely correlated. The results provide strong support for Ca2+ entry being the trigger for exocytosis. 3. Experiments in which the H+ electrochemical gradient across the intracellular secretory granule (chromaffin granule) membrane was altered indicated that the gradient does not play an important role in exocytosis. 4. Ca2+ entry into the cells is associated with activation of phospholiphase C and a rapid translocation of protein kinase C to membranes. 5. The plasma membrane of chromaffin cells was rendered permeable to Ca2+, ATP, and proteins by the detergent digitonin without disruption of the intracellular secretory granules. In this system in which the intracellular milieu can be controlled, micromolar Ca2+ directly stimulated catecholamine secretion. 6. Treatment of the cells with phorbol esters and diglyceride, which activate protein kinase C, enhanced phosphorylation and subsequent Ca2+-dependent secretion in digitonin-treated cells. 7. Phorbol ester-induced secretion could be specifically inhibited by trypsin. The experiments indicate that protein kinase C modulates but is not necessary for Ca2+-dependent secretion.     

Phorbol ester stimulates catecholamine synthesis in isolated bovine adrenal medullary cells

In isolated bovine adrenal medullary cells, the phorbol ester 12-O-tetradecanoyl phorbol 13-acetate (TPA), an activator of protein kinase C, stimulated [14C]catecholamine synthesis from [14C]tyrosine, but not from [14C]DOPA. This stimulatory effect of TPA on [14C]catecholamine synthesis was not dependent upon extracellular Ca2+, and TPA did not affect the uptake of 45Ca2+ or the release of catecholamine by the cells. TPA also did not affect the intracellular cyclic AMP (cAMP) level. 4 alpha-Phorbol 12, 13-didecanoate, which is not an activator of protein kinase C, did not stimulate the synthesis of [14C]catecholamine from [14C]tyrosine. The stimulatory effect of TPA on [14C]catecholamine synthesis was additive with that of carbamylcholine, but not with that of dibutyryl cAMP (DB-cAMP). From these results, it was suggested that protein kinase C is involved in the regulation of tyrosine hydroxylase activity and that this regulatory mechanism might be similar to that involving cAMP.     

Second-messenger control of catecholamine release from PC12 cells. Role of muscarinic receptors and nerve-growth-factor-induced cell differentiation.

The role of various intracellular signals and of their possible interactions in the control of neurotransmitter release was investigated in PC12 cells. To this purpose, agents that affect primarily the cytosolic concentration of Ca2+, [Ca2+]i (ionomycin, high K+), agents that affect cyclic AMP concentrations (forskolin; the adenosine analogue phenylisopropyladenosine; clonidine) and activators of protein kinase C (phorbol esters) were applied alone or in combination to either growing chromaffin-like PC12-cells, or to neuron-like PC12+ cells differentiated by treatment with NGF (nerve growth factor). In addition, the release effects of muscarinic-receptor stimulation (which causes increase in [Ca2+]i, activation of protein kinase C and decrease in cyclic AMP) were investigated. Two techniques were employed to measure catecholamine release: static incubation of [3H]dopamine-loaded cells, and perfusion incubation of unlabelled cells coupled to highly sensitive electrochemical detection of released catecholamines. The results obtained demonstrate that: (1) release from PC12 cells can be elicited by both raising [Ca2+]i and activating protein kinases (protein kinase C and, although to a much smaller extent, cyclic AMP-dependent protein kinase); and (2) these various control pathways interact extensively. Activation of muscarinic receptors by carbachol induced appreciable release responses, which appeared to be due to a synergistic interplay between [Ca2+]i and protein kinase C activation. The muscarinic-induced release responses tended to become inactivated rapidly, possibly by feedback desensitization of the receptor mediated by protein kinase C. Muscarinic inactivation was prevented (or reversed) by agents that increase, and accelerated by agents that decrease, cyclic AMP. Agents that stimulate release primarily through the Ca2+ pathway (ionomycin and high K+) were found to be equipotent in both PC12- and PC12+ cells, whereas the protein kinase C activator 12-O-tetradecanoyl-phorbol 13-acetate was approx. 10-fold less potent in PC12+ cells, when administered either alone or in combination with ionomycin. In contrast, the cell binding of phorbol esters was not greatly modified by NGF treatment. Thus control of neurotransmitter release from PC12 cells is changed by differentiation, with a diminished role of the mechanism mediated by protein kinase C.     

Catecholamine secretion from digitonin-treated PC12 cells. Effects of Ca2+, ATP, and protein kinase C activators

PC12 cells, a cloned rat pheochromocytoma cell line, were treated with digitonin to render the plasma membrane permeable to ions and proteins. At a cell density of 2-6 X 10(5) cells/cm2, incubation with 7.5 microM digitonin permitted a Ca2+-dependent release of 25-40% of the catecholamine within 18 min in the presence of 10 microM Ca2+. Half-maximal secretion occurred at 0.5-1 microM Ca2+. PC12 cultures at lower cell densities were more sensitive to digitonin and gave more variable results. Secretion in the presence of digitonin and Ca2+ began after a 2-min lag and continued for up to 30 min. When cells were treated for 3 min in digitonin and then stimulated with Ca2+ in the absence of digitonin, secretion occurred in the same manner but without the initial lag. Optimal secretion from PC12 cells was also dependent upon the presence of Mg2+ and ATP. Permeabilized PC12 cells exhibited a slow time-dependent loss of secretory responsiveness which was correlated with the release of a cytosolic marker, lactate dehydrogenase (134 kDa). This suggests that digitonin permeabilization allows soluble constituents necessary for secretion to leave the cell in addition to allowing Ca2+ and ATP access into the cell interior. Ca2+-dependent secretion was completely inhibited by exposure of digitonin-permeabilized cells to 100 micrograms/ml trypsin (27 kDa), whereas secretion was only slightly inhibited by trypsin exposure prior to digitonin treatment. Thus, an intracellular, trypsin-sensitive protein is probably involved in secretion. The data also indicate that the same population of digitonin-treated cells which responded to Ca2+ was permeable to a 27-kDa protein. 1,2-Dioctanoylglycerol and phorbol esters which activate protein kinase C enhanced the Ca2+-dependent and Ca2+-independent secretion in digitonin-permeabilized PC12 cells. Thus, protein kinase C appears to be involved in the regulation of catecholamine secretion from permeabilized PC12 cells.     

Arachidonic Acid Release and Catecholamine Secretion from Digitonin-Treated Chromaffin Cells: Effects of Micromolar Calcium, Phorbol Ester, and Protein Alkylating Agents

The relationship between catecholamine secretion and arachidonic acid release from digitonin-treated chromaffin cells was investigated. Digitonin renders permeable the plasma membranes of bovine adrenal chromaffin cells to Ca2+, ATP, and proteins. Digitonin-treated cells undergo exocytosis of catecholamine in response to micromolar Ca2+ in the medium. The addition of micromolar Ca2+ to digitonin-treated chromaffin cells that had been prelabeled with [3H]arachidonic acid caused a marked increase in the release of [3H]arachidonic acid. The time course of [3H]arachidonic acid release paralleled catecholamine secretion. Although [3H]arachidonic acid release and exocytosis were both activated by free Ca2+ in the micromolar range, the activation of [3H]arachidonic acid release occurred at Ca2+ concentrations slightly lower than those required to activate exocytosis. Pretreatment of the chromaffin cells with N-ethylmaleimide (NEM) or p-bromophenacyl bromide (BPB) resulted in dose-dependent inhibition of 10 microM Ca2+-stimulated [3H]arachidonic acid release and exocytosis. The IC50 of NEM for both [3H]arachidonic acid release and exocytosis was 40 microM. The IC50 of BPB for both events was 25 microM. High concentrations (5-20 mM) of Mg2+ caused inhibition of catecholamine secretion without altering [3H]arachidonic acid release. A phorbol ester that activates protein kinase C, 12-O-tetradecanoylphorbol-13-acetate (TPA), caused enhancement of both [3H]arachidonic acid release and exocytosis. The findings demonstrate that [3H]arachidonic acid release is stimulated during catecholamine secretion from digitonin-treated chromaffin cells and they are consistent with a role for phospholipase A2 in exocytosis from chromaffin cells. Furthermore the data suggest that protein kinase C can modulate both arachidonic acid release and exocytosis.     

Effect of the phorbol ester TPA on PTH secretion. Evidence for a role for protein kinase C in the control of PTH release

Parathyroid hormone (PTH) secretion is stimulated by low extracellular calcium (Ca2+) in association with a reduction in cyosolic Ca2+, indicating that this cell type does not conform to classical models of stimulus-secretion coupling. We used the phorbol ester TPA (12-O-tetradecanoyl phorbol 13-acetate), which directly activates protein kinase C, to investigate the possible role of this enzyme in the unusual secretory properties of the parathyroid cell. TPA causes a dose-dependent stimulation of PTH release inhibited by high extracellular Ca2+ (EC50 = 10 nM) but has relatively little effect on secretion stimulated by low Ca2+. This effect was mimicked by the beta 4-isomer of phorbol 12,13-didecanoate which also activates kinase C, but not by the alpha 4-isomer, which has no effect on this enzyme. TPA does not modify cellular cAMP or cytosolic Ca2+ in the parathyroid cell indicating that its effects on PTH secretion are not mediated indirectly via changes in these second messengers. These results suggest that inhibition of PTH release at high Ca2+ might be related to a reduction in protein kinase C activity which can be overcome when the enzyme is directly activated by TPA.     

Activation of protein kinase C is not required for exocytosis from bovine adrenal chromaffin cells. The effects of protein kinase C(19-31), Ca/CaM kinase II(291-317), and staurosporine

We examined whether protein kinase C activation plays a modulatory or an obligatory role in exocytosis of catecholamines from chromaffin cells by using PKC(19-31) (a protein kinase C pseudosubstrate inhibitory peptide), Ca/CaM kinase II(291-317) (a calmodulin-binding peptide), and staurosporine. In permeabilized cells, PKC (19-31) inhibited the phorbol ester-mediated enhancement of Ca2(+)-dependent secretion as much as 90% but had no effect on Ca2(+)-dependent secretion in the absence of phorbol ester. The inhibition of the phorbol ester-induced enhancement of secretion by PKC (19-31) was correlated closely with the ability of the peptide to inhibit in situ phorbol ester-stimulated protein kinase C activity. PKC(19-31) also blocked 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced phosphorylation of numerous endogenous proteins in permeabilized cells but had no effect on Ca2(+)-stimulated phosphorylation of tyrosine hydroxylase. Ca/CaM kinase II(291-317), derived from the calmodulin binding region of Ca/calmodulin kinase II, had no effect on Ca2(+)-dependent secretion in the presence or absence of phorbol ester. The peptide completely blocked the Ca2(+)-dependent increase in tyrosine hydroxylase phosphorylation but had no effect on TPA-induced phosphorylation of endogenous proteins in permeabilized cells. To determine whether a long-lived protein kinase C substrate might be required for secretion, the lipophilic protein kinase inhibitor, staurosporine, was added to intact cells for 30 min before permeabilizing and measuring secretion. Staurosporine strongly inhibited the phorbol ester-mediated enhancement of Ca2(+)-dependent secretion. It caused a small inhibition of Ca2(+)-dependent secretion in the absence of phorbol ester which could not be readily attributed to inhibition of protein kinase C. Staurosporine also inhibited the phorbol ester-mediated enhancement of elevated K(+)-induced secretion from intact cells while it enhanced 45Ca2+ uptake. Staurosporine inhibited to a small extent secretion stimulated by elevated K+ in the absence of TPA. The data indicate that activation of protein kinase C is modulatory but not obligatory in the exocytotoxic pathway.     

Mechanisms of secretory responses to gonadotropin-releasing hormone and phorbol esters in cultured pituitary cells. Participation of protein kinase C and extracellular calcium mobilization

The role of protein kinase C in luteinizing hormone (LH) release was analyzed in studies on the actions of gonadotropin releasing hormone (GnRH) and phorbol esters in cultured pituitary cells. During incubation in normal medium, GnRH stimulated LH release with an ED50 of 0.35 nM. Incubation in Ca2+-deficient medium (Ca2+-free, 10 microM) substantially decreased but did not abolish the LH responses to GnRH. The extracellular Ca2+-dependent component of GnRH action could be mimicked by high K+ concentrations, consistent with the presence of voltage-sensitive calcium channels (VSCC) in pituitary gonadotrophs. Ca2+ channel agonist (Bay K 8644) and antagonist (nifedipine) analogs, respectively, enhanced or partially inhibited LH responses to GnRH and also to K+, the latter confirming the participation of two types of VSCC (dihydropyridine-sensitive and -insensitive) in K+-induced secretion. Phorbol esters, including 12-O-tetradecanoylphorbol-13-acetate (TPA), 4 beta-phorbol-12,13-dibenzoate, and 4 beta-phorbol-12,13-diacetate, stimulated LH release with ED50s of 5, 10, and 1000 nM, respectively, and with about 70% of the efficacy of GnRH. Phorbol ester-stimulated LH secretion was decreased but not abolished by progressive reduction of [Ca2+]e in the incubation medium, and the residual LH response was identical with that elicited by GnRH in Ca2+-deficient medium. TPA increased [Ca2+]i to a peak after 20 s in normal medium but not in the absence of extracellular Ca2+, indicating that protein kinase C (Ca2+/phospholipid-dependent enzyme) promotes calcium entry but can also mediate secretory responses without changes in calcium influx and [Ca2+]i. The extracellular Ca2+-dependent action of TPA on LH release was blocked by Co2+. However, nifedipine did not alter TPA action on [Ca2+]i and LH release. These observations indicate that protein kinase C can participate in GnRH-induced LH release that is independent of Ca2+ entry, but also promotes the influx of extracellular Ca2+ through dihydropyridine-insensitive Ca2+-channels.     

The effects of cAMP,Ca2+, and phorbol esters on ouabain-induced depression of acetylcholine responses inHelix neurons

1. Using internal perfusion and concentration-clamp procedures applied to Helix neurons, the effects of cAMP, Ca2+, and phorbol esters on ouabain-induced depression of acetylcholine Cl-dependent responses were determined. 2. Intracellular cAMP (10(-4) M) depressed those acetylcholine responses which were blocked by ouabain but had no effect on ouabain-insensitive acetylcholine responses. In the presence of elevated intracellular cAMP, ouabain had no further depressant effect on these acetylcholine responses. Both elevated cAMP and ouabain reduced the acetylcholine response without altering the current-voltage curves. 3. An increase in intracellular Ca2+ concentration depressed the amplitude of current induced by application of acetylcholine in neurons with ouabain-sensitive responses and shifted the dose-response relationship to the right. However, elevated Ca2+ did not reduce the maximal response induced by acetylcholine, nor did it prevent the reduction of that response by ouabain. 4. 12-O-Tetradecanoylphorbol-13-acetate (TPA), a potent stimulator of protein kinase C activity, caused depression of both the ouabain-sensitive and the ouabain-insensitive acetylcholine responses. The inhibitory effect of TPA was markedly enhanced after addition of ATP to the intracellular medium and was greatly reduced by cooling to 5 degrees C. The blocking effect of ouabain, however, reexamined in the presence of TPA. 5. These observations are consistent with the hypothesis that the depression of acetylcholine induced Cl--responses in Helix neurons is a result of an increase in intracellular cAMP concentration but is unrelated to activation of protein kinase C or increases in intracellular Ca2+.     

Role of cyclic adenosine 3',5'-monophosphate-dependent protein kinase in hormone-stimulated beta-endorphin secretion in AtT20 cells.

Secretion of beta-endorphin from mouse pituitary AtT20 cells is stimulated by a variety of compounds that raise intracellular cAMP and Ca2+. To investigate the role of cAMP-dependent protein kinases in secretion, AtT20 cells were transfected with an expression vector coding for a regulatory (R) subunit of cAMP-dependent protein kinase containing mutations in both cAMP-binding sites. Expression of the mutant regulatory subunit in stable transformants (RAB cells) results in a dominant inhibition of cAMP-dependent protein kinase activity. Isoproterenol (1 microM) or analogs of cAMP stimulated beta-endorphin secretion from AtT20 cells, but failed to stimulate secretion in RAB cells expressing the mutant R subunit. Secretion in response to CRF (100 nM) was inhibited by 80% in these mutant clones, whereas the secretory response to vasoactive intestinal peptide (VIP; 100 nM) or phorbol ester (100 nM phorbol myristate acetate) was not inhibited by the R subunit mutation. Intracellular cAMP was elevated in response to CRF (11- to 15-fold), isoproterenol (5- to 10-fold), and VIP (4- to 8-fold) in RAB cells. Similar concentrations of VIP were required to evoke beta-endorphin secretion in either RAB cells or AtT20 cells. As with most secretagogues, VIP-induced secretion was inhibited in the presence of either EGTA or a voltage-sensitive Ca2+ channel antagonist, PN200-110. The secretory response to VIP was unaffected by down-regulation of protein kinase-C. These results suggest that CRF and isoproterenol work via cAMP-dependent protein kinase to activate beta-endorphin secretion, whereas VIP can act by a different mechanism that does not involve cAMP-dependent protein kinase or protein kinase-C.     

Regulation of calcium-dependent [3H]noradrenaline release from rat cerebrocortical synaptosomes by protein kinase C and modulation of the actin cytoskeleton.

The effects that active phorbol esters, staurosporine, and changes in actin dynamics, might have on Ca2+ -dependent exocytosis of [3H]-labelled noradrenaline, induced by either membrane-depolarizing agents or a Ca2+ ionophore, have been examined in isolated nerve terminals in vitro. Depolarization-induced openings of voltage-dependent Ca2+ channels with 30 mM KCl or 1 mM 4-aminopyridine induced limited exocytosis of [3H]noradrenaline, presumably from a readily releasable vesicle pool. Application of the Ca2+ ionophore calcimycin (10 microM) induced more extensive [3H]noradrenaline release, presumably from intracellular reserve vesicles. Stimulation of protein kinase C with phorbol 12-myristate,13-acetate increased release evoked by all secretagogues. Staurosporine (1 microM) had no effect on depolarization-induced release, but decreased ionophore-induced release and reversed all effects of the phorbol ester. When release was induced by depolarization, internalization of the actin-destabilizing agent DNAase I into the synaptosomes gave a slight increase in [3H]NA release and strongly increased the potentiating effect of the phorbol ester. In contrast, when release was induced by the Ca2+ ionophore, DNAase I had no effect, either in the absence or presence of phorbol ester. The results indicate that depolarization of noradrenergic rat synaptosomes induces Ca2+ -dependent release from a releasable pool of staurosporine-insensitive vesicles. Activation of protein kinase C increases this release by staurosporine-sensitive mechanisms, and destabilization of the actin cytoskeleton further increases this effect of protein kinase C. In contrast, ionophore-induced noradrenaline release originates from a pool of staurosporine-sensitive vesicles, and although activation of protein kinase C increases release from this pool, DNAase I has no effect and also does not change the effect of protein kinase C. The results support the existence of two functionally distinct pools of secretory vesicles in noradrenergic CNS nerve terminals, which are regulated in distinct ways by protein kinase C and the actin cytoskeleton.     

Prostaglandin E receptor enhancement of catecholamine release may be mediated by phosphoinositide metabolism in bovine adrenal chromaffin cells

In the presence of ouabain, prostaglandin (PG) E2 stimulated a gradual secretion of catecholamines from cultured bovine adrenal chromaffin cells. PGE2 or ouabain alone evoked a marginal secretory response. The synergism of ouabain was also observed with muscarine. PGE2, like muscarine, induced a concentration-dependent formation of inositol phosphates: rapid rises in inositol trisphosphate and inositol bisphosphate followed by a slower accumulation of inositol monophosphate. This effect on phosphoinositide metabolism was accompanied by an increase in cytosolic free Ca2+. The potency of PGs (PGE2 greater than PGF2 alpha greater than PGD2) to stimulate catecholamine release was well correlated with that to affect phosphoinositide metabolism and that to increase the level of intracellular Ca2+. PGE2 did not stimulate cAMP generation significantly in bovine chromaffin cells. The effect of PGE2 on catecholamine release was mimicked by 12-O-tetradecanoylphorbol 13-acetate and A23187, but not by the cAMP analogue dibutyryl cAMP nor by forskolin. These results indicate that PGE2 may enhance catecholamine release from chromaffin cells by activating protein kinase C in concert with the increment of intracellular Ca2+.     

Mechanism of activation of lymphocyte Na+/H+ exchange by concanavalin A. A calcium- and protein kinase C-independent pathway

Treatment of thymic lymphocytes with the mitogenic lectin concanavalin A (ConA) increases the intracellular free Ca2+ concentration and stimulates phosphoinositide turnover. ConA also induced a rapid, amiloride-sensitive, Na+-dependent increase in cytosolic pH of 0.13 +/- 0.01, indicative of stimulation of the Na+/H+ antiport. To investigate the mechanism underlying activation of Na+/H+ exchange by ConA, the intracellular free Ca2+ concentration changes induced by this lectin were precluded by loading the cells with Ca2+-buffering agents and suspension in Ca2+-free media. Under these conditions, the ConA-induced cytoplasmic alkalinization proceeded normally. Two approaches were used to assess the role of protein kinase C. First, this enzyme was inhibited by the addition of 1-(5-isoquinolinysulfonyl)-2-methylpiperazine. In the presence of this potent antagonist, stimulation of the antiport by 12-O-tetradecanoylphorbol-13-acetate was greatly inhibited. In contrast, stimulation by ConA was unaffected. Second, protein kinase C was depleted by overnight incubation with phorbol esters. Following this treatment, Na+/H+ exchange was no longer activated by 12-O-tetradecanoyl-13-acetate, but was still stimulated by ConA. These data suggest that a Ca2+- and protein kinase C-independent mechanisms mediates the activation of Na+/H+ exchange by ConA. The possible role of GTP-binding proteins in the activation was also studied. The antiport was not stimulated by either fluoroaluminate or vanadate. Moreover, pretreatment with pertussis toxin failed to inhibit the ConA-induced cytoplasmic alkalinization. In contrast, preincubation with cholera toxin partially inhibited activation. Under these conditions, cholera toxin significantly elevated intracellular cAMP levels. Inhibition was also observed in cells treated with forskolin at concentrations that increased [cAMP]. The data suggest that a novel cAMP-sensitive signaling mechanism not involving Ca2+ and protein kinase C is involved in the stimulation of Na+/H+ exchange by mitogens in T lymphocytes.     

Protein kinase A and/or C inhibitors potentiate isoproterenol-induced cyclic AMP accumulation in intact human lymphocytes

The objective of the present study was to investigate the roles of protein kinase A and/or C in agonist-induced beta adrenoceptor activation in intact human lymphocytes. LYmphocytes from healthy subjects were incubated with isoproterenol and phosphodiesterase inhibitor (IBMX, 1.0 mM) after 20 minutes of preincubation with (or without) various compounds possessing protein kinase A and/or C inhibitory activities. These compounds included the relatively selective protein kinase C (PK-C) inhibitors (W-7, calmidazolium, polymyxin B, neomycin, tamoxifen and clomiphene), purified protein inhibitors of protein kinase A (PK-A) (obtained synthetically, or purified from bovine hearts and porcine hearts) and the two compounds (H-7, H-9), which have been found to inhibit both PK-A and PK-C. The results showed that all PK-C inhibitors alone decreased cellular basal cAMP levels while inhibitors of PK-A as well as both H-7 and H-9 increased basal cAMP levels in a dose dependent manner at certain concentrations. All inhibitors studied potentiated isoproterenol-induced cAMP accumulation. The protein kinase A and C inhibitor, H-7, also potentiated PGE1 (but not forskolin)-induced cAMP accumulation. In contrast, the protein kinase C activator, PMA, inhibited isoproterenol- and PGE1- (but not forskolin) induced cAMP accumulation. These data suggest that the potentiating effects of PK-A and/or C inhibitors may be related to the inhibition of PK-A and/or PK-C, both of which have been shown to be involved in beta 2 adrenoceptor desensitization and phosphorylation.     

Cross-talk between protein kinase C and multifunctional Ca2+/calmodulin-dependent protein kinase.

Protein kinase C (PKC) exhibits both negative and positive cross-talk with multifunctional Ca2+/calmodulin-dependent protein kinase (CaM kinase) in PC12 cells. PKC effects negative cross-talk by inhibiting the mobilization of intracellular Ca2+ stores and by inhibiting Ca2+ influx through voltage-sensitive Ca2+ channels. In the absence of cross-talk, Ca2+ influx induced by depolarization with 56 mM K+ stimulates CaM kinase and its autophosphorylation and converts up to 50% of the enzyme to a Ca(2+)-independent or autonomous species. Acute treatment with phorbol myristate acetate (PMA) elicits a parallel reduction in depolarization-induced Ca2+ influx and in generation of autonomous CaM kinase. Negative cross-talk also occurs during stimulation of the phosphatidylinositol signaling system with bradykinin, which activates both PKC and CaM kinase. The extent of CaM kinase activation is attenuated by the simultaneous activation of PKC; it is enhanced by prior down-regulation of PKC. PKC also exhibits positive cross-talk with CaM kinase. Submaximal activation of CaM kinase by ionomycin is potentiated by concurrent activation of PKC with PMA. Such PMA treatment is found to increase the level of cytosolic calmodulin. Enhanced activation of CaM kinase by PKC may result from PKC-mediated phosphorylation of calmodulin-binding proteins, such as neuromodulin and MARCKS, and the subsequent increase in the availability of previously bound calmodulin for activation of CaM kinase.