Parathyroid hormone-activated calcium channels in an osteoblast-like clonal osteosarcoma cell line. cAMP-dependent and cAMP-independent calcium channels

Changes in free cytosolic calcium were measured in UMR-106 cells in response to parathyroid hormone (PTH) stimulation. Bovine PTH-(1-34) induced an increase in [Ca2+]i with the contour of the rise in [Ca2+]i occurring in three successive phases: a rapid increase in [Ca2+]i occurring within seconds, rapid decrement in [Ca2+]i to near-resting levels within 1 min, and slow increment in [Ca2+]i. Phase one and phase three increases in [Ca2+]i were dependent on medium calcium. The phase one rise in [Ca2+]i was inhibitable by the calcium channel blockers lanthanum and verapamil. Only the phase one rise in [Ca2+]i was blocked by preincubation of the cells with the phorbol ester, phorbol 12-myristate 13-acetate. This channel was also blocked when cellular cAMP levels were increased prior to PTH stimulation. The phase two decrement of [Ca2+]i was due to the rapid inactivation of the phase one calcium channel. The phase three rise in [Ca2+]i was mediated by cellular cAMP levels. This cAMP-dependent Ca2+ channel was insensitive to pretreatment of the cells with phorbol diesters and showed low sensitivity to Ca2+ channel blockers. It is concluded that UMR-106 cells respond to PTH stimulation by the activation of a cAMP-independent Ca2+ channel. This channel rapidly inactivates. The subsequent PTH-dependent increase in cellular cAMP is followed by activation of a cAMP-dependent Ca2+ channel resulting in a slow rise in [Ca2+]i.     

Stimulus-secretion coupling in bovine parathyroid cells. Dissociation between secretion and net changes in cytosolic Ca2+

The relationship between the concentration of cytosolic free Ca2+ ([Ca2+]i) and secretion of parathyroid hormone (PTH) was investigated in isolated bovine parathyroid cells using the fluorescent Ca2+ indicator, quin 2. Increasing the concentration of extracellular Ca2+ from 0.5 to 2.0 mM caused a 3-fold increase in [Ca2+]i (from 183 +/- 4 to 568 +/- 21 nM) which was associated with a 2-4-fold decrease in secretion of PTH. Decreasing extracellular Ca2+ to about 1 microM caused a corresponding fall in [Ca2+]i to 60-90 nM. Extracellular Ca2+-induced changes in [Ca2+]i were not affected by omission of extracellular Na+. Depolarizing concentrations of K+ (30 mM) depressed [Ca2+]i at all concentrations of extracellular Ca examined, and this was associated with increased secretion of PTH. Ionomycin (0.1 or 1 microM) increased [Ca2+]i at extracellular Ca2+ concentrations of 0.5, 1.0, and 2.0 mM, but inhibited secretion of PTH only at Ca concentrations near the "Ca2+ set point" (1.25 microM). In contrast, dopamine, norepinephrine (10 microM each), and Li+ (20 mM) potentiated secretion of PTH without causing any detectable change in [Ca2+]i. The results obtained with these latter secretagogues provide evidence for a mechanism of secretion which is independent of net changes in [Ca2+]i. The phorbol ester 12-O-tetradecanoyl phorbol 13-acetate (TPA) did not alter [Ca2+]i or secretion of PTH at low (0.5 mM) extracellular Ca2+ concentrations. At 2.0 mM extracellular Ca2+, however, TPA (20 nM or 1 microM) depressed [Ca2+]i and potentiated secretion of PTH. The addition of TPA prior to raising the extracellular Ca2+ concentration reduced the subsequent increase in [Ca2+]i. The results show that the effects of TPA on secretion in the parathyroid cell are not readily dissociated from changes in [Ca2+]i and suggest that some TPA-sensitive process, perhaps involving protein kinase C, may be involved in those mechanisms that regulate [Ca2+]i in response to changes in extracellular Ca2+.     

Phorbol ester-stimulated insulin secretion by RINm5F insulinoma cells is linked with membrane depolarization and an increase in cytosolic free Ca2+ concentration

In studying the regulation of insulin secretion by phorbol esters, we examined their effects on the cytosolic free Ca2+ concentration ([Ca2+]i), using the Ca2+ indicator fura-2 in the rat insulin-secreting beta-cell line RINm5F. [Ca2+]i was measured in parallel with the rate of insulin release. 50 nM 12-O-tetradecanoylphorbol-13-acetate (TPA), which may act via protein kinase C, stimulated insulin release and caused an increase in [Ca2+]i. Ca2+-free conditions eliminated the increase in [Ca2+]i and resulted in a reduced stimulation of insulin release by TPA. The Ca2+ channel blocker nitrendipine (300 nM) inhibited both the increase in [Ca2+]i and the increased rate of insulin secretion. Another phorbol ester, 4 beta-phorbol 12,13-didecanoate, which activates protein kinase C, also induced an increase in [Ca2+]i and in the rate of insulin release, while 4 alpha-phorbol 12,13-didecanoate, which fails to stimulate protein kinase C, was without effect. Further studies with bis-oxonol as an indicator of membrane potential showed that TPA depolarized the beta-cell plasma membrane. From these results, it is concluded that TPA depolarizes the plasma membrane, induces the opening of Ca2+ channels in the RINm5F beta-cell plasma membrane, increases [Ca2+]i, and results in insulin secretion. The action of TPA was next compared with that of a depolarizing concentration of KC1 (25 mM), which stimulates insulin secretion simply by opening Ca2+ channels. TPA consistently elicited less depolarization, a smaller rise of [Ca2+]i, but a greater release of insulin than KC1. Therefore an additional action of TPA is suggested, which potentiates the action of the elevated [Ca2+]i on insulin secretion.     

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

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

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.     

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

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

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)     

Muscarinic receptor-mediated increase in cytoplasmic free Ca2+ in isolated bovine adrenal medullary cells. Effects of TMB-8 and phorbol ester TPA

The change in cytoplasmic free calcium, [Ca2+]i in isolated bovine adrenal medullary cells during stimulation by acetylcholine (ACh) in Ca2+-free incubation medium was measured using the fluorescent Ca2+ indicator quin2. ACh (1-100 microM) caused an increase in [Ca2+]i by mobilization of Ca2+ from the intracellular pool. Nicotine (10 microM) did not increase [Ca2+]i in the absence of extracellular Ca2+. Pretreatment of the cells with atropine (10 microM) completely inhibited ACh-induced increase in [Ca2+]i, whereas pretreatment with hexamethonium (100 microM) did not. The intracellular Ca2+ antagonist 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8), inhibited ACh-induced increase in [Ca2+]i. The activator of protein kinase C 12-O-tetradecanoylphorbol-13-acetate (TPA), but not its 'inactive' analog 4 alpha-phorbol-12,13-didecanoate (PDD), also inhibited ACh-induced increase in [Ca2+]i. These findings suggest that in bovine adrenal medullary cells, stimulation of muscarinic ACh receptor causes an increase in [Ca2+]i by mobilizing Ca2+ from the intracellular pool and that protein kinase C is involved in 'termination' or 'down regulation' of this response.     

Phorbol ester-induced changes in cytoplasmic Ca2+ in human neutrophils. Involvement of a pertussis toxin-sensitive G protein

Activation of neutrophils by most soluble stimuli is associated with a marked increase in intracellular free Ca2+ ([Ca2+]i). However, under physiological conditions (Na+-rich media), the potent activator 12-O-tetradecanoylphorbol-13-acetate (TPA) causes no change or a decrease in [Ca2+]i. We report here that the [Ca2+]i response to phorbol esters varies depending on the ionic composition of the medium. A marked increase in [Ca2+]i was detected in Na+-free solutions. Maximal effects were observed when N-methyl-D-glucammonium+ or choline+ were substituted for Na+, whereas an intermediate response was recorded in K+ medium. The increase in [Ca2+]i was substantially (approximately 65%) inhibited by removal of external Ca2+. A [Ca2+]i increase was also elicited by other beta-phorbol diesters and by diacylglycerol, but not by unesterified phorbol or by alpha-phorbol diesters, indicating involvement of protein kinase C. The increase in [Ca2+]i observed in Na+-free media is not due to inhibition of Na+/Ca2+ exchange, since no change in [Ca2+]i in response to TPA was observed in: 1) cells suspended in Li+, which is not countertransported for Ca2+; 2) cells preloaded with Na+ to eliminate the driving force for Na+/Ca2+ exchange; and 3) cells treated with 3',4'-dichlorobenzamyl, an inhibitor of Na+/Ca2+ exchange. Similarly, the [Ca2+]i increase in Na+-free media is not linked to the absence of Na+/H+ exchange and the associated cytoplasmic acidification since: 1) it was not observed in Na+ media in the presence of inhibitors of the Na+/H+ antiport and 2) it was not mimicked by inducing acidification with nigericin. Pretreatment with pertussis toxin largely inhibited the phorbol ester-induced change in [Ca2+]i, while activation of protein kinase C under these conditions was unaffected. It is concluded that in the absence of extracellular Na+ (or Li+), activation of protein kinase C leads to a net Ca2+ influx into the cytoplasm through a process mediated by a GTP-binding or G protein. Opening of a Na+-sensitive Ca2+ channel could partially explain these observations. Alternatively, the nature of the monovalent cation could conceivably affect the conformation of a G protein or of an associated receptor, inducing the appearance of a site susceptible to an activating phosphorylation by protein kinase C.     

Relationship between cytosolic Ca2+ concentration and amylase release in rat parotid acinar cells following muscarinic stimulation.

Carbachol (CCh), a muscarinic-cholinergic agonist, increased both cytosolic free calcium concentration ([Ca2+]i) and amylase release in rat parotid acinar cells or acini in a dose-dependent manner. Treatment of acinar cells with the intracellular Ca2+ antagonist, 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8), or the intracellular Ca2+ chelator, 1,2-bis(O-aminophenoxy)ethane-N,N,N'N'-tetraacetic acid (BAPTA), strongly attenuated the increases in [Ca2+]i evoked by CCh, but amylase release from acini was not significantly suppressed by the treatment with TMB-8 or BAPTA. Low concentrations (0.02-0.5 microM) of ionomycin, a Ca2+ ionophore, caused increases in [Ca2+]i comparable to those induced by CCh, but the same concentrations had only a little effect on amylase release. The protein kinase C activator, 12-O-tetradecanoylphorbol-13-acetate (TPA), stimulated amylase release in quantities similar to those induced by CCh, although TPA alone did not cause any change in [Ca2+]i. Combined addition of TPA and ionomycin potentiated only modestly amylase release stimulated by TPA alone. Staurosporine, a protein kinase C-inhibitor, similarly inhibited both the CCh- and TPA-induced amylase release. These results suggest that an increase in [Ca2+]i elicited by CCh does not play an essential role for inducing amylase release in rat parotid acini. Amylase release by muscarinic stimulation may be mediated mainly by activation of protein kinase C.     

Synergistic signals in the mechanism of antigen-induced exocytosis in 2H3 cells: evidence for an unidentified signal required for histamine release

The aim of this study was to determine whether the increase in cytosolic free Ca2+ concentration ([Ca2+]i) in response to antigen (aggregated ovalbumin) on IgE-primed 2H3 cells was sufficient to account for exocytosis. When the [Ca2+]i responses to antigen and the Ca2+ ionophore A23187 were compared, A23187 was much less effective at releasing histamine at equivalent [Ca2+]i increases, and little or no stimulated histamine release occurred with A23187 concentrations that matched the [Ca2+]i response to antigen concentrations that stimulated maximal histamine release. The [Ca2+]i response to antigen is not, therefore, sufficient to account for exocytosis, although extracellular Ca2+ is necessary to initiate both the [Ca2+]i response and histamine release: the antigen must generate an additional, unidentified, signal that is required for exocytosis. To determine whether this signal was the activation of protein kinase C, the effects of the phorbol ester 12-0-tetradecanoyl phorbol 13-acetate (TPA) on the responses to antigen were examined. TPA blocked the antigen-induced [Ca2+]i response and the release of inositol phosphates but had little effect on histamine release and did not stimulate exocytosis by itself. The unidentified signal from the antigen is therefore distinct from the activation of protein kinase C and is generated independently of the [Ca2+]i response or the release of inositol phosphates. Taken together with other data that imply that there is very little activation of protein kinase C by antigen when the rate of histamine release is maximal, it is concluded that the normal exocytotic response to antigen requires the synergistic action of the [Ca2+]i signal together with an unidentified signal that is not mediated by protein kinase C.     

The biphasic stimulation of insulin secretion by bombesin involves both cytosolic free calcium and protein kinase C.

Members of the bombesin family of peptides potently stimulate insulin release by HIT-T15 cells, a clonal pancreatic cell line. The response to bombesin consists of a large burst in secretion during the first 30 s, followed by a smaller elevation of the secretory rate, which persists for 90 min. The aim of this study was to identify the intracellular messengers involved in this biphasic secretory response. Addition of 100 nM-bombesin to cells for 20 s increased the cellular accumulation of [3H]diacylglycerol (DAG) by 40% and that of [3H]inositol monophosphate (InsP), bisphosphate (InsP2) and trisphosphate (InsP3) by 40%, 300%, and 800%, respectively. In contrast, cyclic AMP concentrations were unaffected. Bombesin stimulation of [3H]InsP3 formation was detected at 2 s, before the secretory response, which was not measurable until 5 s. Furthermore, the potency of bombesin to stimulate [3H]InsP3 generation (ED50 = 14 +/- 9 nM) agreed with its potency to stimulate insulin release (ED50 = 6 +/- 2 nM). Consistent with its effects on [3H]InsP3 formation, bombesin raised the intracellular free Ca2+ concentration [( Ca2+]i) from a basal value of 0.28 +/- 0.01 microM to a peak of 1.3 +/- 0.1 microM by 20 s. Chelation of extracellular Ca2+ did not abolish either the secretory response to bombesin or the rise in [Ca2+]i, showing that Ca2+ influx was not required. Although the Ca2+ ionophore ionomycin (100 nM) mimicked the [Ca2+]i response to bombesin, it did not stimulate secretion. However, pretreating cells with ionomycin decreased the effects of bombesin on both [Ca2+]i and insulin release, suggesting that elevation of [Ca2+]i was instrumental in the secretory response to this peptide. To determine the role of the DAG produced upon bombesin stimulation, we examined the effects of another activator of protein kinase C, the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA). TPA did not affect [Ca2+]i, but it increased insulin secretion after a 2 min lag. However, an immediate increase in secretion was observed when ionomycin was added simultaneously with TPA. These data indicate that the initial secretory burst induced by bombesin results from the synergistic action of the high [Ca2+]i produced by InsP3 and DAG-activated protein kinase C. However, activation of protein kinase C alone appears to be sufficient for a sustained secretory response.     

Effect of thapsigargin on cytosolic Ca2+ level and amylase release in rat parotid acinar cells.

At concentrations greater than 0.01 microM, thapsigargin (ThG) dose-dependently caused an increase in cytosolic free Ca2+ concentration ([Ca2+]i) in rat parotid acinar cells, as measured by the fluorescent Ca(2+)-indicator fura-2. In the absence of extracellular Ca2+, a transient increase in [Ca2+]i by ThG was observed, and subsequent addition of carbachol (CCh) did not produce a further [Ca2+]i response, suggesting that ThG released Ca2+ from the CCh-sensitive intracellular Ca2+ pool. Since ThG did not stimulate formation of inositol phosphates, the ThG-induced Ca2+ mobilization is independent of phosphoinositide breakdown. High concentrations (greater than 0.1 microM) of ThG induced amylase release from rat parotide acini, but the effect was very poor as compared with that of CCh or the protein kinase C activator, PMA (phorbol 12-myristate 13-acetate). Combined addition of ThG and PMA modestly potentiated amylase release induced by PMA alone. These results support the view that amylase release by muscarinic stimulation is mediated mainly by activation of protein kinase C rather than a rise in [Ca2+]i, although Ca2+ may modulate the secretory response.     

Differential effects of protein kinase C activators on carbamylcholine- and high K+-induced rises in intracellular free calcium concentration in cultured adrenal chromaffin cells

Pretreatment of adrenal chromaffin cells with protein kinase C activators, i.e. 12-O-tetradecanoyl phorbol-13-acetate (TPA) and 1-oleoyl 2-acetyl glycerol (OAG), partially inhibited carbamylcholine (CCh)-induced rise in intracellular free Ca2+ concentration ([Ca2+]i). The apparent IC50 values of TPA and OAG were 3 nM and 25 microM, respectively. The effect of TPA on the CCh-induced rise in [Ca2+]i was overcome by pretreatment of the cells with a protein kinase C inhibitor, 1-(5-isoquinidinesulfonyl)-2-methylpiperazine hydrochloride (H-7). In contrast, KCl-induced rise in [Ca2+]i was not affected by pretreating the cells with TPA or OAG. An inactive phorbol ester, 4 alpha-phorbol 12,13-didecanoate failed to affect the CCh-induced rise in [Ca2+]i. CCh-induced 45Ca2+ uptake was also partially inhibited by pretreatment of the cells with TPA or OAG, but KCl-induced 45Ca2+ uptake was not affected by these pretreatments. These results indicate that protein kinase C activation causes an uncoupling of signal transduction between the nicotinic receptors and Ca2+ channels.     

Sphingosine stimulates calcium mobilization in rat parotid acinar cells

In fura-2-loaded parotid acinar cells, 50-200 microM sphingosine induced an increase in cytosolic Ca2+ ([Ca2+]i). When extracellular Ca2+ was chelated by EGTA, 50 microM sphingosine failed to increase [Ca2+]i, but 100 or 200 microM sphingosine induced a slight and transient increase in [Ca2+]i. The addition of LaCl3 to the medium resulted in the same effect as chelation of extracellular Ca2+. When cells were incubated in low Ca2+ medium containing sphingosine, and extracellular Ca2+ was subsequently added, a rapid increase in [Ca2+]i depending on the concentration of sphingosine was shown. In low Ca2+ medium, a slight increase in [Ca2+]i induced by high concentrations of sphingosine was not shown after the transient increase in [Ca2+]i elicited by methacholine. Inhibitors of protein kinase C, H-7 and K252a, did not mimic the effect of sphingosine on [Ca2+]i. These results suggest that sphingosine stimulates Ca(2+)-influx and further stimulates the release of Ca2+ from agonist-sensitive intracellular pools by a mechanism that is independent of protein kinase C.     

Parathyroid hormone secretion does not respond to changes of free calcium in electropermeabilized bovine parathyroid cells, but is stimulated with phorbol ester and cyclic AMP

The secretion of parathyroid hormone (PTH) is suppressed in bovine parathyroid cells by raised extracellular [Ca2+], and 12-0-tetradecanoyl-phorbol-13-acetate (TPA) stimulates the release of PTH from cells suppressed by high extracellular [Ca2+]. Extracellular and cytosolic free [Ca2+] are proportionally related in intact cells. To assess the role of cytosolic free [Ca2+] on PTH secretion, bovine parathyroid cells were rendered permeable by brief exposure to an intense electric field. PTH secretion was comparable at 40 nM, 500 nM, 5 microM, 28 microM, 0.5 mM and 2 mM [Ca2+] (release of total cellular PTH 3.7 +/- 0.5%, 3.9 +/- 0.4%, 3.4% +/- 0.3%, 3.9 +/- 0.4%, 3.1 +/- 0.3%, 3.5 +/- 0.7%, respectively), but the secretion was stimulated twofold (P less than 0.05 vs. control) in a dose and ATP dependent manner with TPA (100 nM) and cyclic AMP (1 mM). As a result, free [Ca2+] in the range of those observed in intact cells during regulation of PTH secretion by changes of extracellular [Ca2+] did not affect the release of PTH in permeabilized cells. The [Ca2+] independent stimulation of PTH release by TPA and cyclic AMP indicates that changes of cytosolic free [Ca2+] may represent a secondary event not related to the regulation of PTH secretion.     

Effects of protein kinase C activation on the regulation of the stimulus-secretion coupling in pancreatic beta-cells.

Effects of protein kinase C (PKC) activation on the insulin-secretory process were investigated, by using beta-cell-rich suspensions obtained from pancreatic islets of obese-hyperglycaemic mice. The phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA), which is known to activate PKC directly, the muscarinic-receptor agonist carbamoylcholine and high glucose concentration enhanced the phosphorylation of a specific 80 kDa PKC substrate in the beta-cells. At a non-stimulatory glucose concentration, 10 nM-TPA increased insulin release, although there were no changes in either the cytoplasmic free Ca2+ concentration ([Ca2+]i) or membrane potential, as measured with the fluorescent indicators quin-2 and bisoxonol respectively. At a stimulatory glucose concentration TPA caused a lowering in [Ca2+]i, whereas membrane potential was unaffected. Despite the decrease in [Ca2+]i, there was a large stimulation of insulin release. Addition of TPA lowered [Ca2+]i also in beta-cells stimulated by tolbutamide or high K+, although to a lesser extent than in those stimulated by glucose. There was no effect of TPA on either Ca2+ buffering or the ability of Ins(1,4,5)P3 to release Ca2+ in permeabilized beta-cells. However, the phorbol ester inhibited the rise in [Ca2+]i in response to carbamoylcholine, which stimulates the formation of InsP3, in intact beta-cells. Down-regulation of PKC influenced neither glucose-induced insulin release nor the increase in [Ca2+]i. Hence, although PKC activation is of no major importance in glucose-stimulated insulin release, this enzyme can serve as a modulator of the glucose-induced insulin-secretory response. Such a modulation involves mechanisms promoting both amplification of the secretory response and lowering of [Ca2+]i.     

Glucose-induced increase in cytoplasmic pH in pancreatic beta-cells is mediated by Na+/H+ exchange, an effect not dependent on protein kinase C.

Glucose-induced changes in cytoplasmic pH (pHi) were investigated using pancreatic beta-cells isolated from obese hyperglycemic mice. Glucose, at concentrations above 3-5 mM, depolarized the beta-cell and increased pHi, cytoplasmic free Ca2+ ([Ca2+]i), and insulin release. This increase in pHi was dependent on the presence of extracellular Na+ and was inhibited by 5-(N-ethyl-N-isopropyl) amiloride, a blocker of Na+/H+ exchange. Stimulation of protein kinase C with phorbol ester also induced an alkalinization. However, when protein kinase C activity was down-regulated, glucose stimulation still induced alkalinization. At 20 mM glucose, 10 mM NH4Cl induced a marked rise in pHi, paralleled by repolarization, inhibition of electrical activity, and decreases in both [Ca2+]i and insulin release. Reduction in [Ca2+]i was prevented by 200 microM tolbutamide, but not by 10 mM tetraethylammonium. At 4 mM glucose, NH4Cl induced a transient increase in insulin release, without changing [Ca2+]i. Exposure of beta-cells to 10 mM sodium acetate caused a persistent decrease in pHi, an effect paralleled by a small transient increase in [Ca2+]i. Acidification per se did not change the beta-cell sensitivity to glucose, not excluding that the activity of the ATP-regulated K+ channels may be modulated by changes in pHi.     

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.