Examination of the role of ADP-ribosylation factor and phospholipase D activation in regulated exocytosis in chromaffin and PC12 cells

The possible role of ADP-ribosylation factor (ARF)-activated and constitutive phospholipase D (PLD) activity in regulated exocytosis of preformed secretory granules in adrenal chromaffin and PC12 cells was examined. With use of digitonin-permeabilised cells, the effect of GTP analogues and exogenous ARF1 on PLD activity was determined. No evidence was seen for ARF-stimulated PLD activity in these cell types. Exocytosis from cytosol-depleted permeabilised chromaffin cells was not increased by adding recombinant nonmyristoylated or myristoylated ARF1, and exocytosis from both cell types was resistant to brefeldin A (BFA). Addition of bacterial PLD with demonstrably high activity in permeabilised chromaffin cells did not increase exocytosis in cytosol-depleted chromaffin cells. Diversion of PLD activity from production of phosphatidic acid (PA) due to the presence of 4% ethanol did not inhibit exocytosis triggered by Ca2+ or poorly hydrolysable GTP analogues in permeabilised chromaffin or PC12 cells. These results indicate that exocytosis in these cell types does not appear to require a BFA-sensitive ARF and the triggering of exocytosis does not require PLD activity and formation of PA. These findings rule out a general requirement for PLD activity during regulated exocytosis.     

Chromaffin granules release calcium on contact with annexin VI: implications for exocytosis

Chromaffin granules represent a substantial and exchangeable intracellular calcium pool which is thought to be regulated by a sodium/calcium exchange protein and also by a putative inositol trisphosphate-activated calcium channel. A family of calcium-binding proteins, called the annexins, has been shown to bind to chromaffin granules. We have therefore investigated the possible involvement of these proteins in the regulation of chromaffin granule sequestered calcium. Annexin VI (A-VI) produced a concentration-dependent release of 45Ca2+ from chromaffin granules; half-maximal release occurred at 1 microM A-VI, with near-maximum release being at 20 microM A-VI. The A-VI-induced release of 45Ca2+ was rapid, being essentially complete by our first time point of 7 s, and corresponded to 40% of the total sequestered 45Ca2+. A-VI-induced release occurred at extravesicular Ca2+ concentrations ranging from a pCa2+ of 4.12 to 6.86 and also appeared specific to this protein since neither annexin I nor annexin II (tetramer) could evoke any 45Ca2+ release. Given the predominant localization of A-VI to the apical plasmalemma, these results suggest that this protein could participate in the secretory event by mediating the localized release of Ca2+ at sites of contact between the chromaffin granule and plasma membrane.     

Activation of a calcium-calmodulin-dependent protein kinase I cascade in PC12 cells

It has been observed that the activity of Ca2+-calmodulin (CaM)-dependent protein kinase I is enhanced up to 50-fold by its phosphorylation in vitro by a distinct CaM kinase I kinase (Lee, J. C., and Edelman, A. M. (1994) J. Biol. Chem. 269, 2158-2164). It has, however, been unclear whether this event represents an acute form of cellular regulation. We demonstrate here the phosphorylation and activation of CaM kinase I in PC12 pheochromocytoma cells in response to elevation of intracellular Ca2+. Treatment of PC12 cells with the Ca2+-ionophore, ionomycin, or with a depolarizing concentration of KCl, led to rapid, biphasic phosphorylation of CaM kinase I and to increases in CaM kinase I activity of 5.1- and 7. 3-fold, respectively. Depolarization-induced activation of CaM kinase I was reduced by approximately 80% by blockade of Ca2+ influx through L-type voltage-dependent Ca2+ channels and completely abolished by removal of extracellular Ca2+. The ability of PC12 cell CaM kinase I to be phosphorylated and activated by purified CaM kinase I kinase in vitro was markedly reduced by prior depolarization of the cells, consistent with intracellular phosphorylation and activation of CaM kinase I by CaM kinase I kinase. These results demonstrate the existence in PC12 cells of a CaM kinase I cascade, the function of which may be to sensitize cells to signal-induced elevations of intracellular Ca2+.     

Ca2+-triggered peptide secretion in single cells imaged with green fluorescent protein and evanescent-wave microscopy

Green fluorescent protein fused to human chromogranin B or neuropeptide Y was expressed in PC12 cells and caused bright, punctate fluorescence. The fluorescent points colocalized with the endogenous secretory granule marker dopamine beta-hydroxylase. Stimulation of live PC12 cells with elevated [K+], or of permeabilized PC12 cells with Ca2+, led to Ca2+-dependent loss of fluorescence from neurites. Ca2+ stimulated secretion of both fusion proteins equally well. In living cells, single fluorescent granules were imaged by evanescent-wave fluorescence microscopy. Granules were seen to migrate; to stop, as if trapped by plasmalemmal docking sites; and then to disappear abruptly, as if through exocytosis. Evidently, GFP fused to secreted peptides is a fluorescent marker for dense-core secretory granules and may be used for time-resolved microscopy of single granules.     

Primary central nervous system lymphomas in immunocompetent individuals: histology, Epstein-Barr virus genome, Ki-67 proliferation index, p53 and bcl-2 gene expression

We investigated the effects of potassium channel inhibitors on electrical activity, membrane ionic currents, intracellular calcium concentration ([Ca2+]i) and hormone release in GH3/B6 cells (a line of pituitary origin). Patch-clamp recordings show a two-component after hyperpolarization (AHP) following each action potential (current clamp) or a two-component tail current (voltage-clamp). Both components can be blocked by inhibiting Ca2+ influx. Application of D-tubocurarine (dTc) (20-500 microM) reversibly suppressed the slowly decaying Ca2+-activated K+ tail current (I AHPs) in a concentration-dependent manner. On the other hand, low doses of tetraethylammonium ions (TEA+) only blocked the rapidly decaying voltage- and Ca2+-activated K+ tail current (I AHPf). Therefore, GH3/B6 cells exhibit at least two quite distinct Ca2+-dependent K+ currents, which differ in size, voltage- and Ca2+-sensitivity, kinetics and pharmacology. These two currents also play quite separate roles in shaping the action potential. d-tubocurarine increased spontaneous Ca2+ action potential firing, whereas TEA increased action potential duration. Thus, both agents stimulated Ca2+ entry. I AHPs is activated by a transient increase in [Ca2+]i such as a thyrotrophin releasing hormone-induced Ca2+ mobilization. All the K+ channel inhibitors we tested: TEA, apamin, dTC and charybdotoxin, stimulated prolactin and growth hormone release in GH3/B6 cells. Our results show that I AHPs is a good sensor for subplasmalemmal Ca2+ and that dTc is a good pharmacological tool for studying this current.     

Progress in research towards a world without leprosy. Report of a WHO meeting in Ethiopia, February 1998

Rat cortex synaptosomes have been successfully perforated with high concentrations (> or = 400 U/ml) of Staphylococcus aureus alpha-toxin. The free Ca2+-concentration dependence of [3H]-noradrenaline release was similar to that observed for PC 12 and chromaffin cells. Release from the alpha-toxin perforated synaptosomes was not significantly inhibited by omega-conotoxin GVIA. Initially, Ca2+-dependent release was independent of MgATP (for 0.5 min), but became increasingly dependent on MgATP with time. Lactate dehydrogenase efflux from alpha-toxin-perforated synaptosomes was not different than efflux from control synaptosomes, and an antibody to N-ethylmaleimide-sensitive fusion protein did not enter the synaptosomes. [3H]-noradrenaline release was temperature and alpha-toxin-concentration dependent. Ca2-dependent release was more resistant to rundown from alpha-toxin- than from streptolysin-O-perforated synaptosomes. This preparation of perforated synaptosomes should be useful for studies of regulated exocytosis from nerve endings.     

Functional coupling of secretion and capacitative calcium entry in PC12 cells

The caffeine-evoked effects on the intracellular Ca2+ concentration ([Ca2+]i) and on the release of dopamine by PC12 cells were investigated. Stimulation by caffeine resulted in a transient Ca2+ release which was followed by a sustained phase of Ca2+ entry through a non-voltage dependent pathway. Treatment with cyclopiazonic acid (CPA) or thapsigargin, inhibitors of the Ca2+ ATPase pump of the endoplasmic reticulum, resulted in only a sustained rise in [Ca2+]i in the presence of extracellular Ca2+. Pretreatment of cells with CPA or thapsigargin abolished the subsequent Ca2+ responses to caffeine. Caffeine also evoked the release of dopamine from the cells only in the presence of extracellular Ca2+, which was mimicked by CPA. These results suggest that store-dependent Ca2+ entry evoked by caffeine has an indispensable role in the secretory response in an excitable cell line, PC12 cells.     

Cysteine string protein functions directly in regulated exocytosis

Cysteine string protein (Csp) is essential for neurotransmitter release in Drosophila. It has been suggested that Csp functions by regulating the activity of presynaptic Ca2+ channels, thus controlling exocytosis. We have examined the effect of overexpressing Csp1 in PC12 cells, a neuroendocrine cell line. PC12 cell clones overexpressing Csp1 did not show any changes in morphology, granule number or distribution, or in the levels of other key exocytotic proteins. This overexpression did not affect intracellular Ca2+ signals after depolarization, suggesting that Csp1 has no gross effect on Ca2+ channel activity in PC12 cells. In contrast, we show that Csp1 overexpression enhances the extent of exocytosis from permeabilized cells in response to Ca2+ or GTPgammaS in the absence of Ca2+. Because secretion from permeabilized cells is not influenced by Ca2+ channel activity, this represents the first demonstration that Csp has a direct role in regulated exocytosis.     

Localization of the Rab3 small G protein regulators in nerve terminals and their involvement in Ca2+-dependent exocytosis

The Rab3 small G protein subfamily (Rab3) consists of four members, Rab3A, -B, -C, and -D. We have recently isolated and characterized the Rab3 regulators, GDP/GTP exchange protein (GEP) and GTPase activating protein (GAP), both of which are specific for the Rab3 subfamily. Rab3 GEP stimulates the conversion of the GDP-bound inactive form to the GTP-bound active form, whereas Rab3 GAP stimulates the reverse reaction. Of the four members of the Rab3 subfamily, evidence is accumulating that Rab3A is involved in Ca2+-dependent exocytosis, particularly in neurotransmitter release. We first analyzed the subcellular localization of Rab3 GEP and GAP in rat brain. Subcellular fractionation analysis showed that both Rab3 GEP and GAP were enriched in the synaptic soluble fraction. Immunocytochemical analysis in primary cultured rat hippocampal neurons showed that both Rab3 GEP and GAP were concentrated at the presynaptic nerve terminals. We then examined whether Rab3 GEP and GAP were involved in Ca2+-dependent exocytosis by use of human growth hormone (GH) co-expression assay system of cultured PC12 cells. Overexpression of the deletion mutant of Rab3 GEP possessing the catalytic activity reduced the high K+-induced GH release without affecting the basal GH release, whereas that of the deletion mutant lacking the catalytic activity showed no effect on the high K+-induced GH release. In contrast, overexpression of Rab3 GAP or its deletion mutant possessing the catalytic activity did not affect the high K+-induced GH release or the basal GH release. These results indicate that Rab3 GEP and GAP are colocalized with Rab3A at the synaptic release sites and suggest that they regulate the activity of Rab3A and are involved in Ca2+-dependent exocytosis.     

Stimulation of cyclic ADP-ribose synthesis by acetylcholine and its role in catecholamine release in bovine adrenal chromaffin cells

Cyclic ADP-ribose (cADPR) is suggested to be a novel messenger of ryanodine receptors in various cellular systems. However, the regulation of its synthesis in response to cell stimulation and its functional roles are still unclear. We examined the physiological relevance of cADPR to the messenger role in stimulation-secretion coupling in cultured bovine adrenal chromaffin cells. Sensitization of Ca2+-induced Ca2+ release (CICR) and stimulation of catecholamine release by cADPR in permeabilized cells were demonstrated along with the contribution of CICR to intracellular Ca2+ dynamics and secretory response during stimulation of intact chromaffin cells. ADP-ribosyl cyclase was activated in the membrane preparation from chromaffin cells stimulated with acetylcholine (ACh), excess KCl depolarization, and 8-bromo-cyclic-AMP. ACh-induced activation of ADP-ribosyl cyclase was dependent on the influx of Ca2+ into cells and on the activation of cyclic AMP-dependent protein kinase. These and previous findings that ACh activates adenylate cyclase by Ca2+ influx in chromaffin cells suggested that ACh induces activation of ADP-ribosyl cyclase through Ca2+ influx and cyclic AMP-mediated pathways. These results provide evidence that the synthesis of cADPR is regulated by cell stimulation, and the cADPR/CICR pathway forms a significant signal transduction for secretion.     

Reduced expression of E-cadherin in oral squamous cell carcinoma: relationship with DNA methylation of 5'' CpG island

The caffeine-evoked effects on the intracellular Ca2+ concentration ([Ca2+]i) and on the release of dopamine by PC12 cells were investigated. Stimulation by caffeine resulted in a transient Ca2+ release which was followed by a sustained phase of Ca2+ entry through a non-voltage dependent pathway. Treatment with cyclopiazonic acid (CPA) or thapsigargin, inhibitors of the Ca2+ATPase pump of the endoplasmic reticulum, resulted in only a sustained rise in [Ca2+]i in the presence of extracellular Ca2+. Pretreatment of cells with CPA or thapsigargin abolished the subsequent Ca2+ responses to caffeine. Caffeine also evoked the release of dopamine from the cells only in the presence of extracellular Ca2+, which was mimicked by CPA. These results suggest that store-dependent Ca2+ entry evoked by caffeine has an indispensable role in the secretory response in an excitable cell line, PC12 cells.     

Gonadotropin response to naloxone in the mare: effect of time of year and reproductive status

Effects of a novel dihydropyridine type of antihypertensive drug, cilnidipine, on the regulation of the catecholamine secretion closely linked to the intracellular Ca2+ were examined using nerve growth factor (NGF)-differentiated rat pheochromocytoma PC12 cells. By measuring catecholamine secretion with high-performance liquid chromatography coupled with an electrochemical detector, we showed that high K+ stimulation evoked dopamine release from PC12 cells both before and after NGF treatments. Cilnidipine depressed dopamine release both from NGF-treated and untreated PC12 cells in a concentration-dependent manner. In contrast, inhibition by nifedipine was markedly decreased in the differentiated PC12 cells. With intracellular Ca2+ concentration ([Ca2+]i) measurements using fura 2, the elevation of high K+-evoked [Ca2+]i was separated into nifedipine-sensitive and -resistant components. The nifedipine-resistant [Ca2+]i increase was also blocked by cilnidipine, as well as omega-conotoxin-GVIA. By the use of the conventional whole-cell patch-clamp technique, the compositions of the high-voltage-activated Ca2+ channel currents in the NGF-treated PC12 cells were divided into types: L-type, N-type, and residual current components. It was also estimated that cilnidipine at 1 and 3 micromol/L strongly blocked the N-type current without affecting the residual current. These results suggest that cilnidipine inhibits catecholamine secretion from differentiated PC12 cells by blocking Ca2+ influx through the N-type Ca2+ channel, in addition to its well-known action on the L-type Ca2+ channel.     

Survey of antimicrobial resistance in bacterial isolates from diseased cattle in France

Adrenaline and noradrenaline are released from adrenal medullary chromaffin cells by regulated exocytosis from stored secretory granules. Many aspects of the mechanisms by which exocytosis is activated in chromaffin cells are now understood in detail and these cells have provided an important model for the study of neuroendocrine secretion in general. Exocytosis is triggered by Ca2+ influx which activates a multistep process involving at least two Ca(2+)-binding proteins with distinct Ca2+ affinities. Several cytosolic and membrane proteins have been implicated by functional studies as components of the exocytotic machinery. The likely roles of these proteins in exocytosis are discussed in this review and the questions that remain for the understanding of the molecular basis of catecholamine release are highlighted.     

Two distinct, calcium-mediated, signal transduction pathways can trigger deflagellation in Chlamydomonas reinhardtii

The molecular machinery of deflagellation can be activated in detergent permeabilized Chlamydomonas reinhardtii by the addition of Ca2+ (Sanders, M. A., and J. L. Salisbury, 1989. J. Cell Biol. 108:1751-1760). This suggests that stimuli which induce deflagellation in living cells cause an increase in the intracellular concentration of Ca2+, but this has never been demonstrated. In this paper we report that the wasp venom peptide, mastoparan, and the permeant organic acid, benzoate, activate two different signalling pathways to trigger deflagellation. We have characterized each pathway with respect to: (a) the requirement for extracellular Ca2+; (b) sensitivity to Ca2+ channel blockers; and (c) 45Ca influx. We also report that a new mutant strain of C. reinhardtii, adf-1, is specifically defective in the acid-activated signalling pathway. Both signalling pathways appear normal in another mutant, fa-1, that is defective in the machinery of deflagellation (Lewin, R. and C. Burrascano. 1983. Experientia. 39:1397-1398; Sanders, M. A., and J. L. Salisbury. 1989. J. Cell Biol. 108:1751-1760). We conclude that mastoparan induces the release of an intracellular pool of Ca2+ whereas acid induces an influx of extracellular Ca2+ to activate the machinery of deflagellation.     

A specific cyclic ADP-ribose antagonist inhibits cardiac excitation-contraction coupling

BACKGROUND: Cyclic ADP-ribose (cADPR) has been shown to act as a potent cytosolic mediator in a variety of tissues, regulating the release of Ca2+ from intracellular stores by a mechanism that involves ryanodine receptors. There is controversy over the effects of cADPR in cardiac muscle, although one possibility is that endogenous cADPR increases the Ca2+ sensitivity of Ca2+-induced Ca2+ release (CICR) from the sarcoplasmic reticulum. We investigated this possibility using 8-amino-cADPR, which has been found to antagonize the Ca2+-releasing effects of cADPR on sea urchin egg microsomes and in mammalian cells (Purkinje neurons, Jurkat T cells, smooth muscle and PC12 cells). RESULTS: In intact cardiac myocytes isolated from guinea-pig ventricle, cytosolic injection of 8-amino-cADPR substantially reduced contractions and Ca2+ transients accompanying action potentials (stimulated at 1Hertz). These reductions were not seen with injection of HEPES buffer, with heat-inactivated 8-amino-cADPR, or in cells pretreated with ryanodine (2 microM) to suppress sarcoplasmic reticulum function before injection of the 8-amino-cADPR. L-type Ca2+ currents and the extent of Ca2+ loading of the sarcoplasmic reticulum were not reduced by 8-amino-cADPR. CONCLUSIONS: These observations are consistent with the hypothesis that endogenous cADPR plays an important role during normal contraction of cardiac myocytes. One possibility is that cADPR sensitizes the CICR mechanism to Ca2+, an action antagonized by 8-amino-cADPR (leading to reduced Ca2+ transients and contractions). A direct effect of 8-amino-cADPR on CICR cannot be excluded, but observations with caffeine are not consistent with a non-selective block of release channels.     

Genetic risk of neuropsychological impairment in schizophrenia: a study of monozygotic twins discordant and concordant for the disorder

We investigated the effect of the adenosine receptor agonist 5'-(N-ethylcarboxamido)adenosine (NECA) in catecholamine secretion from adrenal chromaffin cells that exhibit only the A2b subtype adenosine receptor. NECA reduced catecholamine release evoked by the nicotinic agonist 1,1-dimethyl-4-phenylpiperazinium (DMPP) in a time-dependent manner. Inhibition reached 25% after 30-40-min exposure to NECA. This effect on DMPP-evoked catecholamine secretion was mirrored by a similar (27.7 +/- 3.3%), slowly developing inhibition of [Ca2+]i transients induced by DMPP that peaked at 30-min preincubation with NECA. The capacity of the chromaffin cells to buffer Ca2+ load was not affected by the treatment with NECA. Short-term treatment with NECA failed both to modify [Ca2+]i levels and to increase endogenous diacylglycerol production, showing that NECA does not activate the intracellular Ca2+/protein kinase C signaling pathway. The inhibitory effects of NECA were accompanied by a 30% increase of protein phosphatase activity in chromaffin cell cytosol. We suggest that dephosphorylation of a protein involved in DMPP-evoked Ca2+ influx pathway (e.g., L-type Ca2+ channels) could be the mechanism of the inhibitory action of adenosine receptor stimulation on catecholamine secretion from adrenal chromaffin cells.     

Physical and functional interactions of Doc2 and Munc13 in Ca2+-dependent exocytotic machinery

Doc2 has two C2 domains that interact with Ca2+ and phospholipid. Munc13 has two C2 domains and one C1 domain that interacts with phorbol ester or diacylglycerol (DAG) and phospholipid. Both Doc2 and Munc13 are implicated in Ca2+-dependent neurotransmitter release, but their modes of action still remain unclear. We show here that Doc2 interacts with Munc13 both in a cell-free system and in intact PC12 cells during the high K+-induced Ca2+-dependent exocytosis. The Doc2-Munc13 interactions are stimulated by phorbol ester through the C1 domain of Munc13. Overexpression of the Doc2-interacting domain of Munc13 reduces the Ca2+-dependent exocytosis from PC12 cells, and co-expression with Doc2 suppresses this reduction. These results, together with the earlier findings that secretagogues produce DAG and elevate cytoplasmic Ca2+, suggest that the DAG-induced Doc2-Munc13 interactions play an important role in Ca2+-dependent exocytotic machinery.     

Ca2+-induced Ca2+ release in chromaffin cells seen from inside the ER with targeted aequorin

The presence and physiological role of Ca2+-induced Ca2+ release (CICR) in nonmuscle excitable cells has been investigated only indirectly through measurements of cytosolic [Ca2+] ([Ca2+]c). Using targeted aequorin, we have directly monitored [Ca2+] changes inside the ER ([Ca2+]ER) in bovine adrenal chromaffin cells. Ca2+ entry induced by cell depolarization triggered a transient Ca2+ release from the ER that was highly dependent on [Ca2+]ER and sensitized by low concentrations of caffeine. Caffeine-induced Ca2+ release was quantal in nature due to modulation by [Ca2+]ER. Whereas caffeine released essentially all the Ca2+ from the ER, inositol 1,4, 5-trisphosphate (InsP3)- producing agonists released only 60-80%. Both InsP3 and caffeine emptied completely the ER in digitonin-permeabilized cells whereas cyclic ADP-ribose had no effect. Ryanodine induced permanent emptying of the Ca2+ stores in a use-dependent manner after activation by caffeine. Fast confocal [Ca2+]c measurements showed that the wave of [Ca2+]c induced by 100-ms depolarizing pulses in voltage-clamped cells was delayed and reduced in intensity in ryanodine-treated cells. Our results indicate that the ER of chromaffin cells behaves mostly as a single homogeneous thapsigargin-sensitive Ca2+ pool that can release Ca2+ both via InsP3 receptors or CICR.