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.     

Ca2+-activated K+ channel blockers induce PKC modulated oscillatory contractions in guinea pig trachea

Mechanisms underlying the Ca2+-activated K+ channel (K(Ca)) blockers-induced oscillatory contractions were investigated in guinea pig tracheal smooth muscle. The mean oscillatory frequencies induced by charybdotoxin (ChTX; 100 nM) and iberiotoxin (IbTX; 100 nM) were 9.8+/-0.8 (counts/h) and 8.0+/-1.3 (counts/h), respectively. Apamin (1 microM ), a blocker of SK(Ca), induced no contraction in guinea pig trachea and did not affect ChTX-induced oscillatory contractions. In Ca2+ free solution, no ChTX-induced contraction was observed. Nifedipine (100 nM), a blocker of voltage-dependent Ca2+ channels, and SK&F 96365 (10 microM), a blocker of capacitative Ca2+ entry, completely abolished ChTX-induced oscillatory contractions. Ryanodine (1 microM) decreased the amplitude, but increased the frequency of the oscillatory contractions. Thapsigargin (1 microM) changed contractions from the oscillatory type to the sustained type. Moreover, the protein kinase C (PKC) inhibitor, bisindolylamaleimide I (1 microM), decreased the amplitude and frequency, but PKC activator, phorbol 12-myristate 13-acetate (1 microM), increased the frequency of oscillatory contractions. These results suggest that K(Ca) inhibitors-induced oscillatory contractions are initiated by Ca2+ influx through L-type voltage-dependent Ca2+ channels. The ryanodine-sensitive calcium release channels in the sarcoplasmic reticulum may play an important role in maintaining the oscillatory contractions. Moreover, PKC activity modulates these oscillatory contractions.     

12-O-Tetradecanoylphorbol 13-Acetate and Forskolin Modify Muscarinic Receptor-Linked Ca2+ Mobilization in SH-SY5Y Neuroblastoma Cells Through Different Mechanisms

The phorbol ester, 12-O-tetradecanoylphorbol 13-acetate (TPA), which causes differentiation of SH-SY5Y neuroblastoma cells, reduces carbachol binding and carbachol-stimulated Ca2+ mobilization in these cells. The decrease in responsiveness to carbachol is due partially to a reduction in the amount of Ca2+ released by the cells and partially to a decrease in the sensitivity of the cells to carbachol. These effects probably can be attributed to a reduction in muscarinic receptor number and a decrease in receptor affinity, respectively. Forskolin, an alkaloid known to cause an increase in cellular cyclic AMP, enhances Ca2+ influx into the cells without affecting the cytosolic free Ca2+ concentration. The alkaloid causes an apparent restoration of the reduced Ca2+ release, caused by TPA, but does not affect the sensitivity of the cells to carbachol. Forskolin increases the decay of carbachol-induced increase in cytosolic Ca2+. The effects of TPA appear to be linked directly to receptor function, whereas those of forskolin are due to the effect of cyclic AMP on cellular Ca2+ metabolism.     

Mutual Antagonism of κ-Opiate and α2-Adrenoceptor Agonist Effects on Intrasynaptosomal Free [Ca2+]i

Synaptosomes prepared from rat cerebral cortices on Percoll discontinuous density gradients were loaded with the fluorescent EGTA analogue Quin 2 to allow measurement of intracellular free [Ca2+]i. When either kappa-opiate or alpha 2-adrenoceptor agonists were incubated with the synaptosomes, there was a highly significant (p less than 0.004, p less than 2.7 X 10(-6), respectively) reduction in intrasynaptosomal free [Ca2+]i relative to controls. As these synaptosomes are not depolarised, the data suggest that both alpha 2-adrenoceptor agonists and kappa-opiate agonists inhibit neurotransmitter release, decreasing the availability of intraneuronal [Ca2+]i rather than altering Ca2+ entry. However, when these two agonists were coincubated, there was a complete abolition of the effects of either agonist; in fact, there was an apparent increase in the intrasynaptosomal free [Ca2+]i. Neither morphine nor [D-Ala2-D-Leu5]enkephalin, mu and delta opiate agonists respectively, had any significant effect on intrasynaptosomal free [Ca2+]i. These results show that the individual effects of clonidine and dynorphin A1-13 are in keeping with the role of these substances at autoreceptors controlling neurotransmitter release. The mutual antagonism of their effects on [Ca2+]i is more difficult to explain but it may be a mechanism that prevents the occurrence of excessive inhibition of neuronal systems.     

Protein Kinase C Activation Enhances K+-Stimulated Endogenous Dopamine Release from Rat Striatal Synaptosomes in the Absence of an Increase in Cytosolic Ca2+

The possibility that protein kinase C modulates neurotransmitter release in brain was investigated by examining the effects of 12-O-tetradecanoylphorbol 13-acetate (TPA) on Ca2+ transport and endogenous dopamine release from rat striatal synaptosomes. TPA (0.16 and 1.6 microM) significantly increased dopamine release by 24 and 33%, respectively, after a 20-min preincubation with TPA followed by 60 s of depolarization with 30 mM KCl. Depolarization-induced 45Ca2+ uptake, measured simultaneously with dopamine release, was not significantly increased by TPA. Neither 45Ca2+ uptake nor dopamine release was altered under resting conditions. When the time course of K+-stimulated 45Ca2+ uptake and dopamine release was examined, TPA (1.6 microM) enhanced dopamine release after 15, 30, and 60 s, but not 1, 3, or 5 s, of depolarization. A slight increase in 45Ca2+ uptake after 60 s of depolarization was also seen. The addition of 30 mM KCl to synaptosomes which had been preloaded with the Ca2+-sensitive fluorophore fura-2 increased the cytosolic free Ca2+ concentration ([Ca2+]i) from 445 nM to 506 nM after 10 s of depolarization and remained elevated after 60 s. TPA had no effect on [Ca2+]i under depolarizing or resting conditions. Replacing extracellular Ca2+ with 100 microM EGTA reduced K+-stimulated (60 s) endogenous dopamine release by 53% and decreased [Ca2+]i to 120 nM. In Ca2+-free medium, 30 mM KCl did not produce an increase in the [Ca2+]i. TPA (1.6 microM) did not alter the [Ca2+]i under resting or depolarizing conditions, but did increase K+-stimulated dopamine release in Ca2+-free medium.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evidence that phorbol ester interferes with stimulated Ca2+ redistribution by activating Ca2+ efflux in neutrophil leucocytes.

The free calcium ion concentration, [Ca2+]i, in the cytoplasmic matrix of quin2-loaded neutrophil leucocytes increases rapidly after addition of concanavalin A. This increase is effectively abolished by a short (3 min) preincubation with 10 nM-TPA (12-O-tetradecanoylphorbol 13-acetate). TPA also inhibits a [Ca2+]i rise of similar magnitude induced by low concentrations (10 nM) of calcium ionophore A23187, suggesting that phorbol ester does not interfere with a physiological influx mechanism. To investigate the effects of TPA further, cells were depleted of Ca2+ during quin2 loading and then re-equilibrated with normal extracellular [Ca2+]. The return to a stable [Ca2+]i value was preceded by a transient overshoot in [Ca2+]i, implying delayed activation of an efflux mechanism by rising [Ca2+]i. TPA abolished the transient, suggesting preactivation by TPA of the efflux mechanism before Ca2+ influx. TPA also stimulates net Ca2+ efflux from neutrophils and neutrophil cytoplasts. These observations are consistent with the thesis that TPA stimulates a Ca2+-efflux mechanism in these cells.     

Autoregulation of prostaglandin E2-induced Ca2+ influx in osteoblast-like cells: inhibition by self-induced activation of protein kinase C.

In cloned osteoblast-like MC3T3-E1 cells, prostaglandin E2 (PGE2) stimulated 45Ca2+ influx even in the presence of nifedipine, a Ca2+ antagonist that inhibits voltage-dependent Ca2+ channel, in a dose-dependent manner, attaining a maximum at 0.5 microM. Dose of PGE2 above 0.5 microM caused less than maximal stimulation. While PGE2 stimulated the formation of inositol trisphosphate dose dependently in the range between 1 nM and 10 microM. 12-O-Tetradecanoylphorbol-13-acetate (TPA), a protein kinase C (PKC)-activating phorbol ester, which by itself had little effect on 45Ca2+ influx, significantly suppressed the 45Ca2+ influx induced by PGE2 in a dose-dependent manner between 1 nM and 1 microM. 4 alpha-Phorbol 12,13-didecanoate, a phorbol ester which is inactive for PKC, showed little effect in this capacity. Staurosporine, a PKC inhibitor, enhanced the PGE2-induced 45Ca2+ influx. On the other hand, dibutyryl cAMP had little effect on the 45Ca2+ influx induced by PGE2. Our data suggest that PGE2 regulates Ca2+ influx through self-induced activation of PKC. These results indicate that there is an autoregulatory mechanism in signal transduction by PGE2, and PGE2 modulates osteoblast functions through the interaction between Ca2+ influx and phosphoinositide hydrolysis in osteoblast-like cells.     

Involvement of Protein Kinase C in Ca2+-Signaling Pathways to Activation of AP-1 DNA-Binding Activity Evoked via NMDA- and Voltage-Gated Ca2+ Channels

Abstract: Stimulation of cultured cerebellar granule cells with N -methyl- d -aspartate (NMDA) or kainic acid (KA) leads to activation of activator protein-1 (AP-1) DNA-binding activity, which can be monitored by an increase in 12- O -tetradecanoylphorbol 13-acetate (TPA)-responsive element (TRE)-binding activity, in concert with c- fos induction. For this increase in TRE-binding activity, Ca²⁺ influx across the plasma membrane is essential. Treatment of cells with an intracellular Ca²⁺ chelator, BAPTA-AM, abolished this increase. Close correspondence between the dose-response curves of ⁴⁵Ca²⁺ uptake and TRE-binding activity by NMDA or KA suggested that Ca²⁺ influx not only triggered sequential activation of Ca²⁺-signaling processes leading to the increase in TRE-binding activity, but also controlled its increased level. Stimulation of non-NMDA receptors by KA mainly caused Ca²⁺ influx through voltage-gated Ca²⁺ channels, whereas stimulation of NMDA receptors caused Ca²⁺ influx through NMDA-gated ion channels. The protein kinase C (PKC) inhibitors staurosporine and calphostin C inhibited the increase in TRE-binding activity caused by NMDA and KA at the same concentration at which they inhibited that caused by TPA. Furthermore, down-regulation of PKC inhibited the increase in TRE-binding activity by NMDA and KA. Thus, a common pathway that includes PKC could, at least in part, be involved in the Ca²⁺-signaling pathways for the increase in TRE-binding activity coupled with the activation of NMDA- and non-NMDA receptors.     

Differential Sensitivity of the Short and Long Human Dopamine D2 Receptor Subtypes to Protein Kinase C

The human dopamine D2L (long form) and D2S (short form) receptors were expressed separately in mouse Ltk- fibroblast cells to investigate whether there is a difference in transmembrane signaling of these D2 receptors. Both receptors induced two signals, a phosphatidylinositol-linked mobilization of intracellular calcium and an inhibition of cyclic adenosine 3'-5' monophosphate (cAMP) accumulation, each with similar response magnitudes and identical pharmacology. Both calcium and cAMP signals were sensitive to pretreatment with pertussis toxin (PTX), indicating mediation by coupling to Gi/Go proteins. However, the two forms of D2 receptor were distinguished by acute prior activation of protein kinase C (PKC) with 12-O-tetradecanoyl 4 beta-phorbol 13-acetate (TPA): TPA blocked the D2S-mediated increase in cytosolic free calcium concentration ([Ca2+]i) in a concentration-dependent manner (between 10 nM and 1 microM), whereas the D2L receptor-induced increase in [Ca2+]i was resistant to TPA and was only partially (60%) inhibited by 100 microM TPA. By contrast, TPA did not alter the inhibition of cAMP accumulation induced by activation of either D2S or D2L receptors. We conclude that, in the L cell system, prior activation of PKC differentially modulates the transmembrane signaling of the D2L and D2S receptors, preferentially inhibiting the D2S receptor-mediated calcium signal but not altering the dopamine-induced inhibitory cAMP signal of either receptor subtype.     

Effects of Ca2+ Channel Blockers on Ca2+ Translocation Across Synaptosomal Membranes

The binding of [3H]nimodipine to purified synaptic plasma membranes (SPM) isolated from sheep brain cortex was characterized, and the effects of nimodipine, nifedipine, and (+)-verapamil on the [3H]nimodipine binding were compared to the effects on 45Ca2+ translocation under conditions that separate 45Ca2+ fluxes through Ca2+ channels from 45Ca2+ uptake via Na+/Ca2+ exchange. [3H]Nimodipine labels a single class of sites in SPM, with a KD of 0.64 +/- 0.1 nM, a Bmax of 161 +/- 27 fmol X mg-1 protein, and a Hill slope of 1.07, at 25 degrees C. Competition of [3H]nimodipine binding to purified SPM with unlabelled Ca2+ channel blockers shows that: nifedipine and nimodipine are potent competitors, with IC50 values of 4.7 nM and 5.9 nM, respectively; verapamil and (-)-D 600 are partial competitors, with biphasic competition behavior. Thus, (+)-verapamil shows an IC50 of 708 nM for the higher affinity component and the maximal inhibition is 50% of the specific binding, whereas for (-)-verapamil the IC50 is 120 nM, and the maximal inhibition is 30%; (-)-D 600 is even less potent than verapamil in inhibiting [3H]nimodipine binding (IC50 = 430 nM). However, (+)-verapamil, nifedipine, and nimodipine are less potent in inhibiting depolarization-induced 45Ca2+ influx into synaptosomes in the absence of Na+/Ca2+ exchange than in competing for [3H]nimodipine binding. Thus, (+)-verapamil inhibits Ca2+ influx by 50% at about 500 microM, whereas it inhibits 50% of the binding at concentrations 200-fold lower, and the discrepancy is even larger for the dihydropyridines. The Na+/Ca2+ exchange and the ATP-dependent Ca2+ uptake by SPM vesicles are also inhibited by the Ca2+ channel blockers verapamil, nifedipine, and d-cis-diltiazem, with similar IC50 values and in the same concentration range (10(-5)-10(-3) M) at which they inhibit Ca2+ influx through Ca2+ channels. We conclude that high-affinity binding of the Ca2+ blockers by SPM is not correlated with inhibition of the Ca2+ fluxes through channels in synaptosomes under conditions of minimal Na+/Ca2+ exchange. Furthermore, the relatively high concentrations of blockers required to block the channels also inhibit Ca2+ translocation through the Ca2+-ATPase and the Na+/Ca2+ exchanger. In this study, clear differentiation is made of the effects of the Ca2+ channel blockers on these three mechanisms of moving Ca2+ across the synaptosomal membrane, and particular care is taken to separate the contribution of the Na+/Ca2+ exchange from that of the Ca2+ channels under conditions of K+ depolarization.     

Distinct mechanisms for two amplification systems of insulin release.

The mechanisms whereby activation of the cyclic AMP-dependent protein kinase A or the Ca2+-phospholipid-dependent protein kinase C amplifies insulin release were studied with mouse islets. Forskolin and the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) were used to stimulate adenylate cyclase and protein kinase C respectively. The sulphonylurea tolbutamide was used to initiate insulin release in the presence of 3 mM-glucose. Tolbutamide alone inhibited 86Rb+ efflux, depolarized beta-cell membrane, triggered electrical activity, accelerated 45Ca2+ influx and efflux and stimulated insulin release. Forskolin alone only slightly inhibited 86Rb+ efflux, but markedly increased the effects of tolbutamide on electrical activity, 45Ca2+ influx and efflux, and insulin release. In the absence of Ca2+, only the inhibition of 86Rb+ efflux persisted. TPA (100 nM) alone slightly accelerated 45Ca2+ efflux and insulin release without affecting 45Ca2+ influx or beta-cell membrane potential. It increased the effects of tolbutamide on 45Ca2+ efflux and insulin release without changing 86Rb+ efflux, 45Ca2+ influx or electrical activity. Omission of extracellular Ca2+ suppressed all effects due to the combination of TPA and tolbutamide, but not those of TPA alone. Though ineffective alone, 10 nM-TPA amplified the releasing action of tolbutamide without affecting its ionic and electrical effects. In conclusion, the two amplification systems of insulin release involve at least partially distinct mechanisms. The cyclic AMP but not the protein kinase C system initiating signal (Ca2+ influx) triggered by the primary secretagogue.     

Cyclic AMP Analogues Potentiate k-Opiate and Attenuate α2-Adrenoceptor Agonist Effects on Intrasynaptosomal Free Calcium

The intrasynaptosomal free calcium concentration ([Ca2+]i) was measured in quin2-loaded synaptosomes prepared from rat cerebral cortex. Membrane-permeant cyclic adenosine-3',5'-monophosphate (cAMP) analogues [8-bromo-cyclic adenosine-3',5'-monophosphate (8-Br-cAMP) and dibutyryl-cyclic adenosine-3',5'-monophosphate (db-cAMP)] increased [Ca2+]i in a dose-dependent manner; The maximal increases were approximately 50% for 8-Br-cAMP and 35% for db-cAMP and occurred at approximately 10 microM with both analogues. Clonidine (1 microM) alone reduced [Ca2+]i by 26.5%; db-cAMP and 8-Br-cAMP attenuated this reduction to 14.2 and 8.2%, respectively. In contrast, the reduction (19.9%) in [Ca2+]i induced by the preferential kappa-opiate agonist dynorphin A(1-13) was not attenuated by the cAMP analogues; in fact, db-cAMP and 8-Br-cAMP potentiated the effect of dynorphin A(1-13) (1 microM), producing decreases in [Ca2+]i of 33.6 and 29.6%, respectively. We conclude that although alpha 2-adrenergic and kappa-opiate receptors both reduce [Ca2+]i, the alpha 2-adrenoceptor-mediated response and the kappa-opiate receptor-mediated response involve different effector mechanisms. It appears that presynaptic alpha 2-adrenoceptor agonist effects are linked to reductions in adenylate cyclase activity and cAMP production and a resultant increase in Ca2+ sequestration, Ca2+-channel blockade, or both. On the other hand, the kappa-opiate-mediated effects possibly involve an increase in cAMP production and a blockade of Ca2+ entry.     

Temporal integration of alpha 1-adrenergic responses in BC3H-1 muscle cells. Regulation of glycogen phosphorylase activity

Regulation of Ca2+-dependent glycogen phosphorylase activity by alpha 1-adrenergic and H1-histamine receptors has been examined in BC3H-1 muscle cells. Stimulation by either norepinephrine or histamine elevates the phosphorylase activity ratio within 5 s from a resting value of 0.37 +/- 0.03 to maximal values of 0.8-0.9. Phosphorylase activation by alpha-adrenergic agonists is sustained over 20-30 min of agonist exposure, whereas histamine exposure only transiently activates phosphorylase during the initial 5 min of stimulation. The initial activation of phosphorylase by either receptor is not attenuated by treated cells with Ca2+-deficient and [ethylenebis(oxyethylenenitrilo)]tetraacetic acid-supplemented buffer, whereas the response to sustained adrenergic stimulation depends largely, but not totally, upon extracellular Ca2+. The involvement of protein kinase C in agonist responses was tested by treating cells with phorbol 12-myristate 13-acetate. Phorbol 12-myristate 13-acetate inhibits receptor-mediated mobilization of intracellular Ca2+ (IC50 = 3.6 nM) yet activates phosphorylase independently of agonist. Phorbol 12-myristate 13-acetate has no effect on cellular 45Ca2+ fluxes in the absence of agonist. Thus, the two receptors coordinately regulate intracellular signaling through Ca2+- and protein kinase C-mediated pathways. alpha 1-Adrenergic receptors elicit sustained phosphorylase activation whereas H1-histaminergic receptors desensitize.     

Effects of Hypoxia on the Catecholamine Release, Ca2+ Uptake, and Cytosolic Free Ca2+ Concentration in Cultured Bovine Adrenal Chromaffin Cells

The purpose of the present study is to clarify the effects of hypoxia on catecholamine release and its mechanism of action. For this purpose, using cultured bovine adrenal chromaffin cells, we examined the effects of hypoxia on high (55 mM) K(+)-induced increases in catecholamine release, in cytosolic free Ca2+ concentration ([Ca2+]i), and in 45Ca2+ uptake. Experiments were carried out in media preequilibrated with a gas mixture of either 21% O2/79% N2 (control) or 100% N2 (hypoxia). High K(+)-induced catecholamine release was inhibited by hypoxia to approximately 40% of the control value, but on reoxygenation the release returned to control levels. Hypoxia had little effect on ATP concentrations in the cells. In the hypoxic medium, [Ca2+]i (measured using fura-2) gradually increased and reached a plateau of approximately 1.0 microM at 30 min, whereas the level was constant in the control medium (approximately 200 nM). High K(+)-induced increases in [Ca2+]i were inhibited by hypoxia to approximately 30% of the control value. In the cells permeabilized by digitonin, catecholamine release induced by Ca2+ was unaffected by hypoxia. Hypoxia had little effect on basal 45Ca2+ uptake into the cells, but high K(+)-induced 45Ca2+ uptake was inhibited by hypoxia. These results suggest that hypoxia inhibits high K(+)-induced catecholamine release and that this inhibition is mainly the result of the inhibition of high K(+)-induced increases in [Ca2+]i subsequent to the inhibition of Ca2+ influx through voltage-dependent Ca2+ channels.     

Alpha 2-adrenergic receptor stimulation mobilizes intracellular Ca2+ in human erythroleukemia cells

Human erythroleukemia cells are a model system for studies of alpha 2-adrenergic receptors and their coupling to inhibition of adenylate cyclase (McKernan, R. M., Howard, M. J., Motulsky, H. J., and Insel, P. A. (1987) Mol. Pharmacol. 32, 258-265). Using Fura-2, we show that alpha 2-adrenergic receptor stimulation also increases intracellular Ca2+ in these cells by 80-250 nM. Although epinephrine only inhibited forskolin-stimulated cAMP generation when beta-adrenergic receptors were blocked, the Ca2+ increase was not affected by beta-adrenergic receptor blockade. The Ca2+ increase was not affected by forskolin or 8-bromo-cAMP. Thus, alpha 2-adrenergic receptors independently couple to elevation of intracellular Ca2+ and adenylate cyclase inhibition. Chelating all extracellular Ca2+ did not reduce the response, demonstrating mobilization of intracellular, rather than influx of extracellular Ca2+. The epinephrine-stimulated Ca2+ mobilization occurred prior to any detectable increase in inositol-(1,4,5)-trisphosphate. It was abolished by pretreatment with pertussis toxin (which blocks some G protein-mediated processes), but not by aspirin and indomethacin (which inhibit cyclooxygenase), nordihydroguaiaretic acid (which inhibits lipoxygenase), or Na+-free buffer (to block any Na+H+ exchange). We conclude, therefore, that alpha 2-adrenergic receptors on human erythroleukemia cells couple to mobilization of intracellular Ca2+ via a (pertussis toxin-sensitive) G protein-mediated mechanism that is independent of inhibition of adenylate cyclase.     

Phosphorylation of the inhibitory guanine-nucleotide-binding protein as a possible mechanism of inhibition by protein kinase C of agonist-induced Ca2+ mobilization in human platelet.

Increases in the intracellular Ca2+ concentration of human platelets caused by receptor agonists, such as thrombin, 9,11-epithio-11,12-methanothromboxane A2 (STA2), platelet-activating factor (PAF) and arginine-vasopressin, were inhibited by prior addition of 12-O-tetradecanoylphorbol 13-acetate (TPA) in time-dependent and concentration-dependent manners. The inhibitions were mostly reversed by staurosporine, and inhibitor of protein kinase C, added 1 min before TPA. Prior treatment of platelets with thrombin or STA2, the efficacious Ca2+ mobilizer, suppressed the increase in the intracellular Ca2+ concentration of the cells to other agonists, but treatment with less efficacious PAF or vasopressin did not. The heterologous receptor desensitizations were also reversed by staurosporine. The antibody, directed against the carboxy-terminal region of the alpha subunits 1 and 2 of the inhibitory guanine-nucleotide-binding proteins (Gi1 alpha and Gi2 alpha), was raised in rabbit and was used to immunoprecipitate Gi alpha in 32P-labeled platelets. The radioactivity was detected in Gi alpha after incubation of 32P-labeled platelets with TPA, thrombin or STA2, but not in the cells incubated with PAF or vasopressin. The time-dependency or concentration-dependency of TPA-induced phosphorylation of Gi alpha was similar to the dependency of its inhibitory action on agonist-induced Ca2+ mobilization. Thus, strong activation of Ca2+/phospholipid-dependent protein kinase C by phorbol ester or agonists of certain Ca(2+)-mobilizing receptors leads to phosphorylation of the alpha subunit of guanine-nucleotide-binding protein, thereby impairing the coupling of the G protein to receptors as a feedback regulatory component of the receptor-triggered intracellular Ca(2+)-mobilizing system.     

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.     

ATP-Stimulated Ca2+ Influx and Phospholipase D Activities of a Rat Brain-Derived Type-2 Astrocyte Cell Line, RBA-2, Are Mediated Through P2X7 Receptors

This study characterizes and examines the P2 receptor-mediated signal transduction pathway of a rat brain-derived type 2 astrocyte cell line, RBA-2. ATP induced Ca2+ influx and activated phospholipase D (PLD). The ATP-stimulated Ca2+ influx was inhibited by pretreating cells with P2 receptor antagonist, pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS), in a concentration-dependent manner. The agonist 2'- and 3'-O-(4-benzoylbenzoyl)adenosine 5'-triphosphate (BzATP) stimulated the largest increases in intracellular Ca2+ concentrations ([Ca2+]i); ATP, 2-methylthioadenosine triphosphate tetrasodium, and ATPgammaS were much less effective, whereas UTP, ADP, alpha,beta-methylene-ATP, and beta,gamma-methylene-ATP were ineffective. Furthermore, removal of extracellular Mg2+ enhanced the ATP- and BzATP-stimulated increases in [Ca2+]i. BzATP stimulated PLD in a concentration- and time-dependent manner that could be abolished by removal of extracellular Ca2+ and was inhibited by suramin, PPADS, and oxidized ATP. In addition, PLD activities were activated by the Ca2+ mobilization agent, ionomycin, in an extracellular Ca2+ concentration-dependent manner. Both staurosporine and prolonged phorbol ester treatment inhibited BzATP-stimulated PLD activity. Taken together, these data indicate that activation of the P2X7 receptors induces Ca2+ influx and stimulates a Ca2+-dependent PLD in RBA-2 astrocytes. Furthermore, protein kinase C regulates this PLD.     

Capacitative Ca2+ entry is involved in regulating soluble amyloid precursor protein (sAPPalpha) release mediated by muscarinic acetylcholine receptor activation in neuroblastoma SH-SY5Y cells

Previous studies have demonstrated that stimulation of phospholipase C-linked G-protein-coupled receptors, including muscarinic M1 and M3 receptors, increases the release of the soluble form of amyloid precursor protein (sAPPalpha) by alpha-secretase cleavage. In this study, we examined the involvement of capacitative Ca2+ entry (CCE) in the regulation of muscarinic acetylcholine receptor (mAChR)-dependent sAPPalpha release in neuroblastoma SH-SY5Y cells expressing abundant M3 mAChRs. The sAPPalpha release stimulated by mAChR activation was abolished by EGTA, an extracellular Ca2+ chelator, which abolished mAChR-mediated Ca2+ influx without affecting Ca2+ mobilization from intracellular stores. However, mAChR-mediated sAPPalpha release was not inhibited by thapsigargin, which increases basal [Ca2+]i by depletion of Ca2+ from intracellular stores. While these results indicate that the mAChR-mediated increase in sAPPalpha release is regulated largely by Ca2+ influx rather than by Ca2+ mobilization from intracellular stores, we further investigated the Ca2+ entry mechanisms regulating this phenomenon. CCE inhibitors such as Gd3+, SKF96365, and 2-aminoethoxydiphenyl borane (2-APB), dose dependently reduced both Ca2+ influx and sAPPalpha release stimulated by mAChR activation, whereas inhibition of voltage-dependent Ca2+ channels, Na+/Ca2+ exchangers, or Na+-pumps was without effect. These results indicate that CCE plays an important role in the mAChR-mediated release of sAPPalpha.     

α2-Adrenergic,k-Opiaie,and PrPurinergic Autoreceptors Have Mutually Antagonistic Effects: A New Regulatory Mechanism?

Rat cortical synaptosomes prepared on four-step discontinuous Percoll density gradients were loaded with the fluorescent Ca2+-indicator fura-2 to allow measurement of the intrasynaptosomal free calcium concentration ([Ca2+]i). When P1-purinergic, alpha 2-adrenergic, or kappa-opiate agonists were incubated with these synaptosomes for 1 min, there was a highly significant, dose-dependent reduction in [Ca2+]i. The effects of these agonists were blocked by inclusion of appropriate specific antagonists. When alpha 2-adrenergic and P1-purinergic agonists were coincubated, a mutual antagonism of their effects was observed, and, in fact, an increase rather than a decrease in [Ca2+]i was apparent. This mutual antagonism was reversed by addition of either a P1-purinergic or a alpha 2-adrenergic antagonist. Parallel studies in which kappa-opiate and P1-purinergic agonists were coincubated also demonstrated a mutual antagonism between the individual effects that was reversed by prior inclusion of either a kappa-opiate or P1-purinergic antagonist. As these mutually antagonistic effects have been observed between alpha 2-adrenergic, kappa-opiate, and P1-purinergic receptor-mediated events, we suggest that this may be a general phenomenon and may be a regulatory mechanism at nerve endings.