Catecholamine secretion from bovine adrenal chromaffin cells: the role of the Na+/Ca2+ exchanger and the intracellular Ca2+ pool

The role of the Na+/Ca2+ exchanger and intracellular nonmitochondrial Ca2+ pool in the regulation of cytosolic free calcium concentration ([Ca2+]i) during catecholamine secretion was investigated. Catecholamine secretion and [Ca2+]i were simultaneously monitored in a single chromaffin cell. After high-K+ stimulation, control cells and cells in which the Na+/Ca2+ exchange activity was inhibited showed similar rates of [Ca2+]i elevation. However, the recovery of [Ca2+]i to resting levels was slower in the inhibited cells. Inhibition of the exchanger increased the total catecholamine secretion by prolonging the secretion. Inhibition of the Ca2+ pump of the intracellular Ca2+ pool with thapsigargin caused a significant delay in the recovery of [Ca2+]i and greatly enhanced the secretory events. These data suggest that both the Na+/Ca2+ exchanger and the thapsigargin-sensitive Ca2+ pool are important in the regulation of [Ca2+]i and, by modulating the time course of secretion, are important in determining the extent of secretion.     

Modulations of early and late secretory processes by activation of protein kinases in the rat adrenal medulla

Modulatory effects of the activation of either protein kinase C (PKC) by phorbol 12,13-dibutyrate (PDBu) or protein kinase A (PKA) by forskolin on stimulant-evoked secretory processes in the perfused rat adrenal medulla were studied. PDBu or forskolin was applied during repetitive stimulation (30 s each at 10-min intervals) with nicotine, bradykinin, muscarine or histamine, and changes in [Ca2+]i (fura-2 microfluorometry) and catecholamine secretions (electrochemical detection) were simultaneously measured. PDBu markedly potentiated the nicotine-evoked secretion without altering the [Ca2+]i response. PDBu partially inhibited the muscarine-evoked secretion and almost completely blocked the histamine-evoked secretion, concomitantly with extensive suppressions of the [Ca2+]i responses to these stimulants. The bradykinin-evoked secretion was enhanced by PDBu despite a slight attenuation of the [Ca2+]i response. PDBu reduced bradykinin-induced intracellular Ca2+ release in a Ca2+-free medium but enhanced the secretion associated with the released Ca2+. These results suggest that PDBu-activated PKC modulates secretory processes at, at least, two different stages. An early-stage modulation may downregulate receptor/G protein systems, which accounts for the inhibitory effect of PDBu on the muscarine- and histamine-evoked responses. A late-stage modulation may generally promote Ca2+-triggered exocytosis after elevation of [Ca2+]i, which explains the potentiation of the nicotine-evoked secretion by PDBu. The late-stage modulation may counteract the early-stage modulation in bradykinin-stimulated cells. Forskolin potentiated the secretory responses to the four secretagogues without increasing the [Ca2+]i responses. PKA may modulate secretory process at a step(s) distal to the rise in [Ca2+]i as is the case with the late-stage modulation by PKC.     

Hypoxia enhances [3H]noradrenaline release evoked by nicotinic receptor activation from the human neuroblastoma SH-SY5Y

We have used the human sympathetic neuronal line SH-SY5Y to investigate the effects of hypoxia on noradrenaline (NA) release evoked by either raised [K+]o (100 mM) or the nicotinic acetylcholine receptor (nAChR) agonist dimethylphenylpiperazinium iodide (DMPP). NA release was monitored by loading cells with [3H]NA and collecting effluent fractions from perfused cells kept in a sealed perifusion chamber. Cells were challenged twice with either stimulus and release was expressed as that evoked by the second challenge as a fraction of that evoked by the first. K+-evoked release was unaffected by hypoxia (PO2 approximately 30-38 mm Hg), but release evoked by DMPP was significantly increased. For both stimuli, replacement of Ca2+o with 1 mM EGTA abolished NA release. K+-evoked release was also dramatically reduced in the presence of 200 microM Cd2+ to block voltage-gated Ca2+ channels, but DMPP-evoked release was less affected. In hypoxia, DMPP-evoked Cd2+-resistant NA release was dramatically increased. Our findings indicate that hypoxia increases NA release evoked from SH-SY5Y cells in response to nAChR activation by increasing Ca2+ influx through the nAChR pore, or by activating an unidentified Cd2+-resistant Ca2+-influx pathway. As acetylcholine is the endogenous transmitter at sympathetic ganglia, these findings may have important implications for sympathetic activity under hypoxic conditions.     

Inhibition of nicotinic receptor-mediated responses in bovine chromaffin cells by diltiazem

1. The effects of diltiazem on various functional parameters were studied in bovine cultured adrenal chromaffin cells stimulated with the nicotinic receptor agonist dimethylphenylpiperazinium (DMPP) or with depolarizing Krebs-HEPES solutions containing high K+ concentrations. 2. The release of [3H]-noradrenaline induced by DMPP (100 microM for 5 min) was gradually and fully inhibited by increasing concentrations of diltiazem (IC50 = 1.3 microM). In contrast, the highest concentration of diltiazem used (10 microM) inhibited the response to high K+ (59 mM for 5 min) by only 25%. 3. 45Ca2+ uptake into cells stimulated with DMPP (100 microM for 1 min) was also blocked by diltiazem in a concentration-dependent manner (IC50 = 0.4 microM). Again, diltiazem blocked the K(+)-evoked 45Ca2+ uptake (70 mM K+ for 1 min) only by 20%. In contrast, the N-P-Q-type Ca2+ channel blocker omega-conotoxin MVIIC depressed the K+ signal by 70%. In the presence of this toxin, diltiazem exhibited an additional small inhibitory effect, indicating that the compound was acting on L-type Ca2+ channels. 4. Whole-cell Ba2+ currents through Ca2+ channels in voltage-clamped chromaffin cells were inhibited by 3-10 microM diltiazem by 20-25%. The inhibition was readily reversed upon washout of the drug. 5. The whole-cell currents elicited by 100 microM DMPP (IDMPP) were inhibited in a concentration-dependent and reversible manner by diltiazem. Maximal effects were found at 10 microM, which reduced the peak IDMPP by 70%. The area of each curve represented by total current (QDMPP) was reduced more than the peak current. At 10 microM, the inhibition amounted to 80%; the IC50 for QDMPP inhibition was 0.73 microM, a figure close to the IC50 for 45Ca2+ uptake (0.4 microM) and [3H]-noradrenaline release (1.3 microM). The blocking effects of diltiazem developed very quickly and did not exhibit use-dependence; thus the drug blocked the channel in its closed state. The blocking effects of 1 microM diltiazem on IDMPP were similar at different holding potentials (inhibition by around 30% at -100, -80 or -50 mV). Diltiazem did not affect the current flow through voltage-dependent Na+ channels. 6. These data are compatible with the idea that diltiazem has little effect on Ca2+ entry through voltage-dependent Ca2+ channels in bovine chromaffin cells. Neither, does diltiazem affect INa. Rather, diltiazem acts directly on the neuronal nicotinic receptor ion channel and blocks ion fluxes, cell depolarization and the subsequent Ca2+ entry and catecholamine release. This novel effect of diltiazem might have clinical relevance since it might reduce the sympathoadrenal drive to the heart and blood vessels, thus contributing to the well established antihypertensive and cardioprotective effects of the drug.     

Potentiation of histamine-induced catecholamine secretion by ouabain in cultured bovine adrenal chromaffin cells is dependent on calcium and sodium influx

The effects of histamine on catecholamine secretion from cultured bovine adrenal chromaffin cells were studied in the presence of ouabain, an inhibitor of Na+-K+ ATPase. The purpose of this study was to determine whether Na+, as well as Ca2+, was involved in histamine receptor-mediated catecholamine secretion. Histamine (10(-8)-10(-5) M)-induced catecholamine secretion was markedly potentiated by addition of ouabain (10(-5) M) and was inhibited by a histamine-H1 receptor antagonist or incubation in a Ca2+-free medium. Histamine-induced 45Ca2+ influx was also potentiated by addition of ouabain. Ouabain alone or in the presence of histamine increased 22Na+ influx into the cells. In an additional set of experiments, cells were preincubated in the presence or absence of Na+ for 30 min (+/- histamine and ouabain), washed and then catecholamine secretion was measured following exposure to 2.2 mM Ca2+ for 15 min. Preincubation with histamine alone with or without Na+ had no effect of Ca2+-induced secretion of catecholamine. Preincubation with ouabain alone or with ouabain plus histamine produced a slight stimulation of catecholamine secretion in Na+-free medium and a large stimulation in Na+-containing medium. These results suggested that stimulation of the histamine-H1 receptor and inhibition of the Na+ pump both increase intracellular Na+ levels, resulting in increases in Ca2+ influx and catecholamine secretion.     

Difference in the effectiveness of Ca2+ to evoke catecholamine secretion between adrenaline- and noradrenaline-containing cells of bovine adrenal medulla

Differential adrenaline (Ad) and noradrenaline (NA) secretions evoked by secretagogues were investigated using digitonin-permeabilized adrenal chromaffin cells, cultured adrenal chromaffin cells, and perfused adrenal glands of the ox. In digitonin-permeabilized cells, Ca2+ (0.8-160 microM) caused a concentration-dependent increase in catecholamine secretion, which was characterized by a predominance of NA over Ad secretion. Acetylcholine (10-1,000 microM), high K+ (14-56 mM), and bradykinin (0.1-1,000 nM) all were confirmed to induce the release of more NA than Ad at all concentrations used. There was no apparent difference in the ratios of NA/Ad between Ca(2+)-induced catecholamine secretion from digitonin-permeabilized cells and those induced by secretagogues from cultured cells. Qualitatively the same result was obtained in the secretory responses to acetylcholine and high K+ in perfused adrenal glands. These results indicate that the effectiveness of Ca2+ for catecholamine secretion is higher in the secretory apparatus of NA cells than in that of Ad cells of the bovine adrenal medulla. This may be one of the reasons why the secretagogues cause a predominance of NA secretion over Ad secretion in the bovine adrenal medulla.     

Pituitary adenylate cyclase-activating polypeptide causes rapid Ca2+ release from intracellular stores and long lasting Ca2+ influx mediated by Na+ influx-dependent membrane depolarization in bovine adrenal chromaffin cells

Pituitary adenylate cyclase-activating polypeptide (PACAP) has been reported to increase intracellular Ca2+ concentrations ([Ca2+]i) and catecholamine release in adrenal chromaffin cells. We measured [Ca2+]i with fura-2 and recorded ion currents and membrane potentials with the whole cell configuration of the patch-clamp technique to elucidate the mechanism of PACAP-induced [Ca2+]i increase in bovine adrenal chromaffin cells. PACAP caused [Ca2+]i to increase due to Ca2+ release and Ca2+ influx, and this was accompanied by membrane depolarization and inward currents. The Ca2+ release was suppressed by ryanodine, an inhibitor of caffeine-sensitive Ca2+ stores, but was unaffected by cinnarizine, an inhibitor of inositol trisphosphate-induced Ca2+ release. Ca2+ influx and inward currents were both inhibited by replacement of extracellular Na+, and Ca2+ influx was inhibited by nicardipine, an L-type Ca2+ channel blocker, or by staurosporine, a protein kinase C (PKC) inhibitor, but was unaffected by a combination of omega- conotoxin-GVIA, omega-agatoxin-IVA, and omega-conotoxin- MVIIC, blockers of N-, P-, and Q-type Ca2+ channels. Moreover, 1-oleoyl-2-acetyl-sn-glycerol, a PKC activator, induced inward currents and Ca2+ influx. These results indicate that PACAP causes both Ca2+ release, mainly from caffeine-sensitive Ca2+ stores, and Ca2+ influx via L-type Ca2+ channels activated by membrane depolarization that depends on PKC-mediated Na+ influx.     

Potentiation by ouabain of catecholamine secretion from bovine adrenal chromaffin cells in culture induced by pituitary adenylate cyclase-activating polypeptide: evidence for involvements of Na+ and Ca2+ movements

The effect of pituitary adenylate cyclase-activating polypeptide (PACAP) on catecholamine secretion with ouabain, an inhibitor of Na(+)-K+ ATPase, in cultured bovine adrenal chromaffin cells was examined, to determine whether movement of Na+, as well as Ca2+, is involved in the secretory process. PACAP (10(-10)-10(-6)M)-induced catecholamine secretion was markedly potentiated by addition of ouabain (10(-5)M). When cultured cells were preincubated with PACAP for 30 min in Ca(2+)-free medium in the presence of ouabain and then stimulated for 15 min with Ca(2+)-containing medium without PACAP or ouabain, their catecholamine secretion was dependent on the external Ca2+ concentration, and 45Ca2+ influx into the cells was increased. When the cells had been preincubated with PACAP and ouabain in Na(+)-free sucrose medium, their Ca(2+)-induced catecholamine secretion was greatly reduced. PACAP increased 22Na+ influx into cells treated with ouabain. These results suggest that stimulation by PACAP and inhibition of the Na(+)-pump both increase the intracellular Na+ level, resulting in increase in Ca2+ influx and catecholamine secretion.     

Pathway for Ca2+ influx into cells by trichosporin-B-VIa, an alpha-aminoisobutyric acid-containing peptide, from the fungus Trichoderma polysporum

Trichosporin (TS) -B-VIa, a fungal alpha-aminoisobutyric acid (Aib) -containing peptide consisting of 19 amino acid residues and a phenylalaninol, produced both 45Ca2+ influx into bovine adrenal chromaffin cells and catecholamine secretion from the cells. The secretion induced by TS-B-VIa at lower concentrations (2-5 microM) was completely dependent on the external Ca2+, while that induced by TS-B-VIa at higher concentrations (10-30 microM) was partly independent of the Ca2+. The concentration-response curves (2-5 microM) for the TS-B-VIa-induced Ca2+ influx and secretion correlated well. The TS-B-VIa (at 5 microM) -induced secretion was not antagonized by diltiazem, a blocker of L-type voltage-sensitive Ca2+ channels. The treatment of fura-2-loaded C6 glioma cells with TS-B-VIa (2-5 microM) led to an increase in the intracellular free Ca2+ concentration ([Ca2+]i) in a concentration-dependent manner but the stimulatory effects of TS-B-VIa on [Ca2+]i were only slightly observed in Ca(2+)-free medium, indicating that TS-B-VIa causes Ca2+ influx from the external medium into the C6 cells. The TS-B-VIa-induced increase in [Ca2+]i in the C6 cells was not antagonized by diltiazem and by SK&F 96365, a novel blocker of receptor-mediated Ca2+ entry. High K+ increased neither [Ca2+]1 in the C6 cells nor Mn2+ influx into the cells, while TS-B-VIa increased Mn2+ influx. Also in other non-excitable cells, bovine platelets, similar results were obtained. These results strongly suggest that the mechanism of Ca2+ influx by TS-B-VIa at the lower concentrations is distinct from the event of Ca2+ influx through receptor-operated or L-type voltage-sensitive Ca2+ channels in both excitable cells (the chrornaffin cells) and non-excitable cells (the C6 cells and the platelets) and that TS-B-VIa per se may form Ca(2+)-permeable ion channels in biological membranes. On the other hand, the peptide at the higher concentrations seems to damage cell membranes.     

Ca(2+)/calmodulin-dependent protein kinase II inhibitor KN-62 inhibits adrenal medullary chromaffin cell functions independent of its action on the kinase

KN-62, an inhibitor of Ca2+/calmodulin-dependent protein kinase II (CaM kinase II), inhibited significantly catecholamine secretion and tyrosine hydroxylase activity stimulated by acetylcholine in cultured bovine adrenal medullary cells. KN-62, however, showed an additional inhibitory effect on acetylcholine-induced 45Ca2+ influx, which is essential for functional responses. Carbachol-stimulated 22Na+ influx, veratridine-induced 22Na+ influx, and 56 mM K(+)-evoked 45Ca2+ influx were also attenuated by KN-62. Inhibitions by KN-62 of these ion influxes were correlated closely with those of catecholamine secretion. KN-04, which is a structural analogue of KN-62 but does not inhibit CaM kinase II activity, elicited inhibitory effects on the three kinds of stimulant-evoked ion influxes with an inhibitory potency similar to KN-62. These results suggest that KN-62 inhibits catecholamine secretion and tyrosine hydroxylase activation due to mainly its ion channel blockade on the plasma membrane rather than the inhibition of CaM kinase II activity in the cells.     

Solvent polarity-dependent structural refolding: a CD and NMR study of a 15 residue peptide

We have studied osmotically induced catecholamine secretion from bovine adrenal chromaffin cells by combining patch-clamp measurements, electrochemical detection of secretion, and Fura-2 measurements of intracellular free calcium concentration ([Ca2+]i). We find that osmotically induced catecholamine release is exocytotic and calcium dependent. Furthermore, we demonstrate that cell swelling is coupled to such secretion via a volume-activated current, carrying predominantly chloride, which causes a plateau depolarization of the cell membrane potential and thus promotes voltage-activated calcium influx. Therefore, cell volume changes may modulate the secretory activity.     

Potentiation by ouabain of bradykinin-induced catecholamine secretion and calcium influx into cultured bovine adrenal chromaffin cells: evidence for involvement of Na+ influx associated with the bradykinin B2-receptor

The effect of bradykinin (BK), in the presence of ouabain, an inhibitor of Na(+)-K+ ATPase, on catecholamine (CA) secretion was studied in cultured bovine adrenal chromaffin cells, to determine whether Na+, as well as Ca2+, is involved in BK-receptor mediated CA secretion. BK (10(-8)-10(-5) M)-induced CA secretion was markedly potentiated by addition of ouabain (10(-5) M), was blocked by a BK-B2 receptor antagonist, and was decreased in Ca(2+)-free medium. BK-induced increase in 45Ca2+ influx was also potentiated by addition of ouabain. The cultured cells were first incubated with BK for 30 min in Ca(2+)-free medium in the presence or absence of ouabain and then stimulated for 15 min with Ca(2+)-medium without BK or ouabain. Prior stimulation of the cells, BK induced 22Na+ influx and increased Ca(2+)-induced CA secretion and these stimulatory effects of BK were potentiated by added ouabain. When the cells were stimulated with BK and ouabain in Na(+)-free sucrose medium, the Ca(2+)-induced CA secretion was greatly reduced. These results indicated that activation of the BK-B2 receptor and inhibition of the Na+ pump both increase the intracellular Na+ level, resulting in increase in Ca2+ influx and CA secretion.     

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In Fura-2 loaded-single guinea pig adrenal chromaffin cells, muscarine, nicotine and KCl all caused an early peak rise in intracellular Ca concentration ([Ca2+]i) followed by a sustained rise. In Ca(2+)-free solution, muscarine, but neither nicotine nor KCl, caused a transient increase in [Ca2+]i, which was partially reduced by preceding application of caffeine or by treatment with ryanodine plus caffeine. In voltage-clamped cells at a holding potential of -60 mV, the muscarine-induced [Ca2+]i rise, especially its sustained phase, decreased in magnitude. Intracellular application of inositol 1,4,5-trisphosphate caused a transient increase in [Ca2+]i and inhibited the following [Ca2+]i response to muscarine without affecting responses to nicotine and a depolarizing pulse. Muscarine evoked membrane depolarization following brief hyperpolarization in most cells tested. There was a significant positive correlation between the amplitude of the depolarization and the magnitude of the sustained rise in [Ca2+]i. Muscarine-induced sustained [Ca2+]i rise was much greater in the current-clamp mode than that in the voltage-clamp mode. The sustained phase of [Ca2+]i rise and Mn2+ influx in response to muscarine were suppressed by a voltage-dependent Ca2+ channel blocker, methoxyverapamil. These results suggest that stimulation of muscarinic receptors causes not only extracellular Ca2+ entry, but also Ca2+ mobilization from inositol 1,4,5-trisphosphate-sensitive intracellular stores. Voltage-dependent Ca(2+)-channels may function as one of the Ca2+ entry pathways activated by muscarinic receptor in guinea pig adrenal chromaffin cells.     

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.     

Cytosolic Ca2+ acts by two separate pathways to modulate the supply of release-competent vesicles in chromaffin cells

Recovery from depletion of the readily releasable pool of vesicles (RRP) in adrenal chromaffin cells was studied at differing basal [Ca2+]i or following protein kinase C (PKC) activation by phorbol esters. Following depletion, the pool size was estimated at varied times from cell capacitance jumps in response to paired depolarizations. The experimentally observed RRP recovery time course and steady-state size could be predicted from the measured [Ca2+]i signal assuming a Michaelis-Menten-type regulation of the vesicle supply by Ca2+. An elevated recruitment activity was observed at increased [Ca2+]i even when protein kinase C was blocked, but maximum effects could be obtained only after stimulation of PKC by phorbol esters or by prolonged elevations in [Ca2+]i. We suggest that, in chromaffin cells, elevated cytosolic Ca2+ modulates exocytotic plasticity via PKC-dependent and -independent pathways.     

Roles for protein kinase C and mitogen-activated protein kinase in nicotine-induced secretion from bovine adrenal chromaffin cells

Both the Ca2+/phospholipid-dependent protein kinases (protein kinases C, PKCs) and mitogen-activated protein kinases (MAPKs) have been implicated as participants in the secretory response of bovine adrenomedullary chromaffin cells. To investigate a possible role for these kinases in exocytosis and the relationship of these kinases to one another, intact chromaffin cells were treated with agents that inhibited each of the kinases and analyzed for catecholamine release and MAPK/extracellular signal-regulated kinase (ERK) kinase (MEK)/MAPK activation after stimulation with secretagogues of differential efficacy. Of the three secretagogues tested, inactivation of PKCs by long-term phorbol 12-myristate 13-acetate (PMA) treatment or incubation with GF109203X had the greatest inhibitory effect on nicotine-induced catecholamine release and MEK/MAPK activation, a moderate effect on KCl-induced events, and little, if any, effect on Ca2+ ionophore-elicited exocytosis and MEK/MAPK activation. These results indicate that PKC plays a significant role in events induced by the optimal secretagogue nicotine and a lesser role in exocytosis elicited by the suboptimal secretagogues KCl and Ca2+ ionophore. Treatment of cells with the MEK-activation inhibitor PD098059 completely inhibited MEK/MAPK activation (IC50 1-5 microM) and partially inhibited catecholamine release induced by all secretagogues. However, PD098059 was more effective at inhibiting exocytosis induced by suboptimal secretagogues (IC50 approximately 10 microM) than that induced by nicotine (IC50 approximately 30 microM). These results suggest a more prominent role for MEK/MAPK in basic secretory events activated by suboptimal secretagogues than in those activated by the optimal secretagogue nicotine. However, PD098059 also partially blocked secretion potentiated by short-term PMA treatment, suggesting that PKC can function in part by signaling through MEK/MAPK to enhance secretion. Taken together, these results provide evidence for the preferential involvement of MEK/MAPK in basic secretory events activated by the suboptimal secretagogues KCl and Ca2+ ionophore and the participation of both PKC and MEK/MAPK in optimal, secretion induced by nicotine.     

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.     

Novel autocrine feedback control of catecholamine release. A discrete chromogranin a fragment is a noncompetitive nicotinic cholinergic antagonist

Catecholamine secretory vesicle core proteins (chromogranins) contain an activity that inhibits catecholamine release, but the identity of the responsible peptide has been elusive. Size-fractionated chromogranins antagonized nicotinic cholinergic-stimulated catecholamine secretion; the inhibitor was enriched in processed chromogranin fragments, and was liberated from purified chromogranin A. Of 15 synthetic peptides spanning approximately 80% of chromogranin A, one (bovine chromogranin A344-364 [RSMRLSFRARGYGFRGPGLQL], or catestatin) was a potent, dose-dependent (IC50 approximately 200 nM), reversible secretory inhibitor on pheochromocytoma and adrenal chromaffin cells, as well as noradrenergic neurites. An antibody directed against this peptide blocked the inhibitory effect of chromogranin A proteolytic fragments on nicotinic-stimulated catecholamine secretion. This region of chromogranin A is extensively processed within chromaffin vesicles in vivo. The inhibitory effect was specific for nicotinic cholinergic stimulation of catecholamine release, and was shared by this chromogranin A region from several species. Nicotinic cationic (Na+, Ca2+) signal transduction was specifically disrupted by catestatin. Even high-dose nicotine failed to overcome the inhibition, suggesting noncompetitive nicotinic antagonism. This small domain within chromogranin A may contribute to a novel, autocrine, homeostatic (negative-feedback) mechanism controlling catecholamine release from chromaffin cells and neurons.     

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.     

Puerarin inhibits tetrodotoxin-resistant sodium current in rat dorsal root ganglion neurons

The effect of 7-nitroindazole (7-NI), an inhibitor of neuronal nitric oxide synthase (nNOS) on the dimethylphenylpiperazinium(DMPP)-evoked release of [3H]noradrenaline ([3H]NA) from rat hippocampal slices was studied. The effect of DMPP (20 microM) to increase the basal release of [3H]NA was significantly potentiated by 7-NI (40 microM). In our previous study we showed that the response to DMPP has two components, a nicotinic receptor-mediated, [Ca2+]-dependent exocytosis followed by a [Ca2+]-independent, uptake blocker-sensitive carrier-mediated release. To clarify which part of the response was affected by the inhibition of nNOS, we investigated the effect of 7-NI on the nicotine-evoked NA release (nicotine has only receptor-mediated effect) and on the DMPP-evoked NA release in Ca(2+)-free medium where the receptor-mediated component is abolished. Nicotine (100 microM) significantly increased the basal release of [3H]NA but this release was not affected, whereas in Ca(2+)-free medium the response to DMPP (20 microM) was still potentiated by 7-NI (40 microM). In the presence of the NA uptake blocker desipramine (10 microM) DMPP (20 microM) was unable to provoke NA release independently from the presence or absence of 7-NI (40 microM). Our data show that 7-NI influences the carrier-mediated component of DMPP-evoked [3H]NA release, which indicates that nitric oxide produced by nNOS may play a role in the regulation of the NA uptake carrier.