Stimulus-Secretion Coupling in Isolated Adrenal Chromaffin Cells: Calcium Channel Activation and Possible Role of Cytoskeletal Elements

Abstract: The catecholamine secretory function of a preparation of isolated bovine adrenal chromaffin cells has been further characterized under conditions designed to elucidate the mechanism of calcium channel activation and the possible role of cytoskeletal elements in stimulus-secretion coupling. Three related sets of data were obtained: (1) Differences in kinetics, Ca dependence, strength, and additivity of the secretory response to acetylcholine (ACh) versus excess K; (2) the effects on secretion of the Ca channel-blocking agents, Ni, Mg, and verapamil; and (3) the Ca dependence of vinblastine action on ACh- and K-evoked secretion. The results suggest that a major portion of the Ca influx required for catecholamine release enters the cell via voltage-dependent Ca channels with some additional Ca influx via the ACh receptor channel. Comparison of the present secretion data with corresponding known electrophysiological properties of isolated chromaffin cells provides added evidence for a role of chromaffin cell action potentials in regulation of Ca influx and the secretory response. Elevated Ca concentrations enhanced K-evoked secretion to levels comparable to that of ACh but did not induce a vinblastine block of K-evoked release. This provides further evidence against a role of microtubules in the common exocytosis event per se. However, a role of cytoskeletal elements in directing the movement of secretory granules, or an action of vinblastine at cholinergic receptors, remain distinct possibilities.     

M2 muscarinoceptor-associated ionophore at the cat adrenal medulla

Atropine and pirenzepine displaced 3H-quinuclydinyl-benzylate binding and inhibited methacholine-evoked catecholamine release with a similar order of potencies, atropine being 200 fold more potent than pirenzepine. In contrast to high-K, methacholine-evoked 45Ca uptake or catecholamine release were not blocked by (+)PN200-110. Bay-K-8644 did not modify the secretory response to methacholine either in the presence of Ca or Sr but potentiated K-evoked secretion. In depolarized glands, methacholine still evoked its usual secretory response. The results suggest that muscarinic stimulation of cat adrenal chromaffin cells stimulates Ca entry though an ionophore other than voltage-dependent Ca channels; such ionophore seems to be chemically operated through a M2 muscarinoceptor.     

Neurotransmitter release from bovine adrenal chromaffin cells is modulated by capacitative Ca(2+)entry driven by depleted internal Ca(2+)stores

Two potential mechanisms by which the intracellular Ca(2 stores might modulate catecholamine release from bovine adrenal chromaffin cells were investigated: (i) that the cytosolic Ca(2+)transient caused by Ca(2+)release from the intracellular stores recruits additional chromaffin granules to a readily releasable pool that results in augmented catecholamine release when this is subsequently evoked, and (ii) that the Ca(2+)influx that follows depletion of intracellular stores (i.e. store-operated Ca(2+)entry) triggers release per se thereby augmenting evoked catecholamine release. When histamine or caffeine were applied in Ca(2+)-free perfusion media, a transient elevation of intracellular free Ca(2+)occurred owing to mobilization of Ca(2+)from the stores. When Ca(2+)was later readmitted to the perfusing fluid there followed a prompt and maintained rise in intracellular Ca(2+)concentrations of magnitude related to the degree of store mobilization. In parallel experiments, increased catecholamine secretion was measured under the conditions when Ca(2+)influx following store-mobilization occurred. Furthermore, the size of the catecholamine release increment correlated with the degree of Ca(2+)influx. Store-operated Ca(2+)entry evoked by mobilization with histamine and/or caffeine did not augment nicotine-evoked secretion per se; that is, it augmented evoked catecholamine release only to the extent that it increased basal catecholamine release. The nicotine-evoked catecholamine release was sensitive to cytosolic BAPTA, which, at the concentration used (50 microM BAPTA-AM), reduced release by approximately 25%. However, the increment in basal catecholamine release which followed Ca(2+)influx triggered by Ca(2+)store mobilization was not reduced by intracellular BAPTA. This finding is inconsistent with the hypothesis that the elevated cytosolic Ca(2+)from store mobilization recruits additional vesicles of catecholamine to the sub-plasmalemmal release sites to augment subsequently evoked secretion. This position is supported by the observation that histamine (10 microM) in Ca(2+)-free medium caused a pronounced elevation of cytosolic free Ca(2+), but this caused no greater catecholamine release when Ca(2+)was re-introduced than did prior exposure to Ca(2+)-free medium alone, which caused no elevation of cytosolic free Ca(2+). It is concluded that intracellular Ca(2+)stores can modulate secretion of catecholamines from bovine chromaffin cells by permitting Ca(2+)influx through a store-operated entry pathway. The results do not support the notion that the Ca(2+)released from intracellular stores plays a significant role in the recruitment of vesicles into the ready-release pool under the experimental conditions reported here.     

Membrane Potential and Catecholamine Secretion by Bovine Adrenal Chromaffin Cells: Use of Tetraphenylphosphonium Distribution and Carbocyanine Dye Fluorescence

Changes in plasma membrane potential of isolated bovine adrenal chromaffin cells were measured independently by two chemical probe methods and related to corresponding effects on catecholamine secretion. The lipophilic cation tetraphenylphosphonium (TPP+) and the carbocyanine dye 3,3'-dipropylthiadicarbocyanine [DiS-C3-(5)] were used. The necessity of evaluating the subcellular distribution of TPP+ among cytoplasmic, mitochondrial, secretory granule, and bound compartments was demonstrated and the resting plasma membrane potential determined to be -55 mV. The relationship between membrane potential and catecholamine secretion was determined in response to variations in extracellular K+ and to the presence of several secretagogues including cholinergic receptor ligands, veratridine, and ionophores for Na+ and K+. The dependence of potential on K+ concentration fit the Goldman constant field equation with a Na/K permeability ratio of 0.1. The dependence of both K+- and veratridine-evoked catecholamine secretion on membrane potential exhibited a potential threshold of about -40 mV before a significant rise in secretion occurred. This is likely related to the threshold for opening of voltage-sensitive Ca2+ channels. Acetylcholine and nicotine evoked a large secretory response without a sufficiently sustained depolarization to be detectable by the relatively slow potential sensitive chemical probes. Decamethonium induced a detectable depolarization of the chromaffin cells. Veratridine and gramicidin evoked both membrane depolarization and catecholamine release. By contrast the K ionophore valinomycin evoked significant levels of secretion without any depolarization. This is consistent with its utilization of an intracellular source of Ca2+ and the independence of its measured secretory response on extracellular Ca2+.     

Mechanism of Na+ deprivation-induced catecholamine secretion from freshly isolated bovine adrenal chromaffin cells

We have studied the mechanism of Na+ deprivation-induced catecholamine secretion from freshly isolated bovine adrenal chromaffin cells. Na+ deprivation-induced catecholamine secretion depended on free extracellular Ca2+ concentrations and was almost parallel to 45Ca2+ influx into the cells under various experimental conditions. Furthermore, Na+ deprivation-induced 45Ca2+ influx and catecholamine secretion were actually induced by a relative Na+ concentration gradient across the plasma membrane, but not by simple omission of Na+ from the medium. These results indicate that the deprivation of Na+ from the medium changes the relative Na+ gradient across the plasma membrane and results in Ca2+ influx via a reverse mode of Na(+)-Ca(2+) exchange rather than by inducing Ca2+ entry through Ca2+ channels by eliminating the competition between extracellular Na+ and Ca2+.     

Possible Regulation of Caffeine-Induced Intracellular Ca2+ Mobilization by Intracellular Free Na+

To gain some understanding of the regulatory mechanism involved in caffeine-induced Ca2+ release in adrenal chromaffin cells, we took advantage of the paradoxical observation that removal of divalent cations potentiated the secretory response to caffeine. We measured the concentration of cytosolic free Ca2+ ([Ca]in) in isolated cat chromaffin cells, by fura-2 microfluorometry, to see whether there was any correlation between the secretory response and the rise in [Ca]in. The caffeine-induced [Ca]in rise and catecholamine secretion were increased by treatment of cells with a divalent cation-deficient solution. These potentiated responses were strongly inhibited either by pretreatment with ryanodine, by the reduction of the external Na+ concentration, or by the addition of Ca2+ channel blockers. Removal of divalent cations caused a large rise in the cytosolic free Na+ concentration ([Na]in), which was measured using SBFI microfluorometry. This rise in [Na]in was reduced either by adding Ca2+ channel blockers or by reducing the external Na+ concentration. These results show a good correlation between caffeine-induced Ca2+ release and [Na]in at the time of stimulation, suggesting that caffeine-induced Ca2+ release is regulated by [Na]in.     

Quantal size is dependent on stimulation frequency and calcium entry in calf chromaffin cells

To what extent the quantal hypothesis of transmitter release applies to dense-core vesicle (DCV) secretion is unknown. We determined the characteristics of individual secretory events in calf chromaffin cells using catecholamine amperometry combined with different patterns of stimulation. Raising the frequency of action potential trains from 0.25-10 Hz in 2 mM [Ca(2+)]o or [Ca(2+)]o from 0.25-7 mM at 7 Hz elevated the amount released per event (quantal size). With increased stimulation, quantal size rose continuously, not abruptly, suggesting that release efficiency from a single population of DCVs rather than recruitment of different-sized vesicles contributed to the effect. These results suggest that catecholamine secretion does not conform to the quantal model. Inhibition of rapid endocytosis damped secretion in successive episodes, implying an essential role for this process in the recycling of vesicles needed for continuous secretion.     

Enhancement by cytochalasin B of ouabain-stimulated catecholamine secretion from cultured bovine adrenal chromaffin cells: possible relation to alteration in Na+/K+-pump activity

1. Catecholamine secretion evoked by ouabain from cultured bovine adrenal chromaffin cells has previously been shown to be markedly enhanced by pretreatment of the cells with cytochalasin B (Morita et al., 1988). To elucidate a possible mechanism of this enhancement, the stimulatory action of ouabain on Ca2+ influx as well as catecholamine secretion was then examined in the cells pretreated with or without cytochalasin B. The effect of cytochalasin B pretreatment on the inhibitory action of ouabain on the Na+/K+ pump was also examined by measuring 86Rb+ uptake into the cells. 2. Pretreatment of the cells with cytochalasin B caused enhancement of ouabain-induced catecholamine secretion, and this enhancement was accompanied by the elevation of ouabain-stimulated 45Ca2+ uptake into the cells. The inhibitory action of ouabain on 86Rb+ uptake was significantly enhanced by pretreatment of the cells with cytochalasin B under the same conditions. 3. These results indicate that the enhancement of ouabain-induced catecholamine secretion caused by cytochalasin B pretreatment may be due to the increase in ouabain-stimulated Ca2+ influx into the cells and, furthermore, suggest the possibility that this increase in Ca2+ influx may be attributed to the potentiation of the inhibitory action of ouabain on the Na+/K+ pump in the adrenal chromaffin cell. Thus, the present study provides an evidence for a possible role of microfilaments as one of the intrinsic factors modulating the plasma membrane functions.     

Osmotic strength differentiates between two types of calcium transport pathways regulating catecholamine secretion from cultured bovine chromaffin cells

Calcium transport and catecholamine secretion was measured in cultured bovine chromaffin cells. Calcium ions which entered the cells following stimulation with either nicotine or 50 mM KCl (high potassium) triggered catecholamine release, but then inactivated the secretory process. The nicotine and the high potassium-induced calcium transport mechanisms were mechanistically distinct, but functionally dependent on each other. The specific evidence is that whereas the high potassium-induced Ca2+ influx was found to be inhibited by hyperosmotic medium, the nicotine-stimulated calcium influx was unaffected under these conditions. High potassium and nicotine-stimulated catecholamine release were also differently affected by hyperosmotic medium. While potassium-stimulated catecholamine release was profoundly inhibited by hyperosmolarity, nicotine-stimulated release was only moderately inhibited. Sequential treatments of cells with nicotine and high potassium, under isotonic physiological conditions, indicate that there is a functional, biochemical communication between the otherwise mechanistically distinct calcium channels. Calcium ions which were found to inactivate these channels may be the basis for such communication.     

The Adenosine Analogue N6-L-Phenylisopropyladenosine Inhibits Catecholamine Secretion from Bovine Adrenal Medulla Cells by Inhibiting Calcium Influx

We reported earlier that adenine nucleotides and adenosine inhibit acetylcholine-induced catecholamine secretion from bovine adrenal medulla chromaffin cells. In this article, we used an adenosine analogue, N6-L-phenylisopropyladenosine (PIA), to study the mechanism underlying inhibition of catecholamine secretion by adenosine. PIA inhibits secretion induced by a nicotinic agonist, 1,1-dimethyl-4-phenylpiperazinium, or by elevated external K+. The half-maximal effect on 1,1-dimethyl-4-phenylpiperazinium-induced secretion occurred at approximately 5 x 10(-5) M. The inhibition is immediate and reversible. Fura-2 measurements of cytosolic free Ca2+ indicate that PIA inhibits Ca2+ elevation caused by stimulation; measurements of 45Ca2+ influx show that PIA inhibits uptake of Ca2+. PIA does not inhibit calcium-evoked secretion from digitonin-permeabilized cells, nor does PIA cause any significant change in the dependence of catecholamine secretion on calcium concentration. These data suggest that inhibition by PIA occurs at the level of the voltage-sensitive calcium channel.     

Alpha-latrotoxin and its receptors CIRL (latrophilin) and neurexin 1 alpha mediate effects on secretion through multiple mechanisms

Alpha-Latrotoxin and its plasma membrane receptors cause a number of distinct effects in secretory cells. First, by tethering alpha-latrotoxin to the plasma membrane, CIRL/latrophilin and neurexin 1 alpha facilitate alpha-latrotoxin-induced channel formation. The stimulation of secretion by alpha-latrotoxin in neuroendocrine cells is a consequence of Ca(2+) influx through these alpha-latrotoxin-induced channels. In addition to channel formation, alpha-latrotoxin enhances secretion in permeabilized cells through interaction with the plasma membrane receptor CIRL/latrophilin. Finally, overexpression of CIRL/latrophilin inhibits Ca(2+)-dependent secretion in permeabilized chromaffin cells in the absence of alpha-latrotoxin. This effect represents a 'constitutive' action of the G-protein coupled receptor to specifically inhibit an ATP-dependent priming step in the secretory pathway. The effect suggests that the receptor may have an important modulatory role in synaptic transmission.     

Interleukin-1 inhibits voltage-dependent P/Q-type Ca2+ channel associated with the inhibition of the rise of intracellular free Ca2+ concentration and catecholamine release in adrenal chromaffin cells

Effects of interleukin (IL) on intracellular free Ca2+ concentration ([Ca2+]i) rise and catecholamine (CA) release were examined in isolated, cultured bovine adrenal chromaffin cells. IL-1alpha and IL-1beta inhibited the rise of [Ca2+]i and CA release induced by acetylcholine (ACh) and excess KCl both in normal and in Ca2+-sucrose medium. Pretreatment by IL-1 receptor antagonist (IL-1RA) blocked the inhibitory actions of IL-1alpha. IL-1alpha reduced CA release induced by veratridine in normal medium but not in the presence of diltiazem. Analysis using specific blockers for voltage-operated Ca2+ channels (VOCC) revealed that IL-1alpha and IL-1beta specifically inhibited the P/Q-type Ca2+ channel to reduce [Ca2+]i rise induced by excess KCl. IL-1 did not affect [Ca2+]i rise induced either by bradykinin or caffeine in Ca2+-deprived medium or via activation of store-operated Ca2+ channel (SOC). The inhibitory effects of IL-1alpha were blocked by pretreatments with herbimycin A, U0126 and PD 98054, but not with SB202190, SP 600125 or pertussis toxin (PTX). These results demonstrated that IL-1 inhibits stimulation-evoked [Ca2+]i rise and CA release in chromaffin cells by blocking voltage-operated P/O-type Ca2+ channels. The inhibitory action of IL-1 may be mediated through the tyrosine kinase and MEK/ERK pathways.     

Regulation of 3-O-methyl-D-glucose uptake in isolated bovine adrenal chromaffin cells

We showed earlier that insulin stimulated sugar transport in adrenal chromaffin cells (Bigornia, L. and Bihler, I. Biochim. Biophys. Acta 885, 335-344). Transport regulation and its Ca2+ -dependence was further investigated in isolated bovine adrenal chromaffin cells, serving as a model of a homogeneous neuronal cell population. Uptake of the nonmetabolizable glucose analogue, 3-O-methyl-D-glucose was stimulated by hyperosmolar medium, and this effect was abolished in the absence of external Ca2+, or depressed in the presence of La3+ or the slow Ca2+ channel blocker methoxyverapamil. Basal transport was also stimulated by factors (acetylcholine, carbamylcholine, low-Na+ medium), which cause Ca2+ -dependent catecholamine release, and these effects were abolished in Ca2+ -free medium. In addition insulin, acetylcholine, hyperosmolar and low-Na+ medium significantly increased 45Ca uptake. Thus, glucose transport in adrenal chromaffin cells was stimulated by insulin and hyperosmolarity in a Ca2+ -dependent manner, as in muscle. Sensitivity to secretory stimuli, a regulatory feature perhaps characteristic of this cell type, was also demonstrated. In contrast to muscle, sugar transport was not affected by Na+ -pump inhibition, metabolic inhibitors or the Na+ ionophore monensin, suggesting that Ca2+ influx by Na+/Ca2+ exchange does not play a significant role in the activation of sugar transport in chromaffin cells.     

Ryanodine Inhibits Caffeine-Evoked Ca2+ Mobilization and Catecholamine Secretion from Cultured Bovine Adrenal Chromaffin Cells

The effects of ryanodine, a selective inhibitor of the Ca(2+)-induced Ca2+ release mechanism, on caffeine-evoked changes in cytosolic Ca2+ concentration ([Ca2+]i) and catecholamine secretion were investigated using cultured bovine adrenal chromaffin cells. Caffeine (5-40 mM) caused a concentration-dependent transient rise in [Ca2+]i and catecholamine secretion in Ca2+/Mg(2+)-free medium containing 0.2 mM EGTA. Ryanodine (5 x 10(-5) M) alone had no effect on either [Ca2+]i or catecholamine secretion. Although the application of ryanodine plus caffeine caused the same increase in both [Ca2+]i and catecholamine secretion as those induced by caffeine alone, ryanodine (4 x 10(-7) - 5 x 10(-5) M) irreversibly prevented the increase in both [Ca2+]i and catecholamine secretion resulting from subsequent caffeine application over a range of concentrations. The secretory response to caffeine was markedly enhanced by replacement of Na+ with sucrose in Ca2+/Mg(2+)-free medium, and this enhanced response was also blocked by ryanodine. Caffeine was found to decrease the susceptibility of the secretory apparatus to Ca2+ in digitonin-permeabilized cells. These results indicate that caffeine mobilizes Ca2+ from intracellular stores, the function of which is irreversibly blocked by ryanodine, resulting in the increase in catecholamine secretion in the bovine adrenal chromaffin cell.     

Barium ions enter chromaffin cells via voltage-dependent calcium channels and induce secretion by a mechanism independent of calcium

Barium ions enter chromaffin cells via voltage-sensitive calcium channels, although the intracellular site of barium action is distinct from that of calcium. The entry of barium primarily through voltage-sensitive channels was indicated by experiments showing inhibition of 133Ba2+ uptake by blockers of voltage-dependent calcium channels. In addition, 133Ba2+ uptake was stimulated by 50 mM KCl but not by nicotine. Furthermore, 133Ba2+ uptake was inhibited by hyperosmolarity, which specifically blocks the voltage-sensitive calcium channel but not the receptor-associated calcium channel. These conclusions from studies on barium uptake were also borne out by experiments measuring catecholamine secretion. Thus, blockers of voltage-dependent calcium channels which inhibited barium uptake also inhibited barium-induced catecholamine secretion. In other experiments, simultaneous stimulation with nicotine and barium in the presence of calcium evoked coincident and additive catecholamine secretion. By contrast, when 50 mM KCl was substituted for nicotine in the same experimental design, barium ions inhibited potassium-induced catecholamine secretion at low calcium concentrations. Only at high calcium concentrations were barium-induced and potassium-induced secretion additive. These data also indicate that barium and calcium compete at the voltage-sensitive pathway. Furthermore, these additivity data suggest that once inside the cell, barium and calcium have two distinct mechanisms of action. As predicted by this hypothesis, in digitonin-permeabilized chromaffin cells either calcium or barium stimulated catecholamine release, and in the presence of both cations catecholamine secretion was equivalent to the sum of secretion with either cation alone. Additional support of this concept comes from experiments showing that while calcium-mediated catecholamine secretion is sensitive to trifluoperazine and imipramine, barium-mediated secretion is not. Taken together, all these data indicate that there are two distinct intracellular sites of action for barium and calcium. In contrast to catecholamine secretion, non-exocytotic ascorbic acid secretion was induced by nicotine and potassium in the presence of calcium, but not by barium alone. These data provide additional evidence that barium acts by a different mechanism than calcium, in still another secretory system in chromaffin cells.     

Calcium Is Released by Exocytosis Together with Catecholamines from Bovine Adrenal Medullary Cells

We have tested the hypothesis that exocytosis is a possible export route for calcium from bovine adrenal medullary cells. After prelabelling cells in primary tissue culture with 45Ca, evoked 45Ca export and catecholamine secretion show the same time course, a similar fraction of the total pool of 45Ca and catecholamine is released, and the same concentrations of carbamylcholine or KCl are required for half-maximal triggered release. Increasing the osmolarity of the extracellular medium or treating the cells with botulinum toxin type D inhibits both evoked catecholamine secretion and 45Ca export to the same extent without inhibiting 45Ca influx. Incorporation of 45Ca into chromaffin granules is very slow, however, and incorporated 45Ca is not immediately releasable. 45Ca entering the cell during short-term stimulation is not found in the releasable pool during a second period of triggered secretion. Our data suggest that chromaffin granules are the largest pool of intracellular calcium in bovine adrenal medullary cells and that most of the calcium in chromaffin granules does not rapidly exchange with cytoplasmic Ca, but can be released directly by exocytosis. Exocytosis does not appear to play a major role in exporting Ca that enters the cell during short-term stimulation.     

Lysophosphatidic acid-LPA1 receptor-Rho-Rho kinase-induced up-regulation of Nav1.7 sodium channel mRNA and protein in adrenal chromaffin cells: enhancement of 22Na+ influx, 45Ca2+ influx and catecholamine secretion

In cultured bovine adrenal chromaffin cells, chronic (> or = 24 h) treatment with lysophosphatidic acid (LPA) augmented veratridine-induced 22Na+ influx via Na(v)1.7 by approximately 22% (EC(50) = 1 nmol/L), without changing nicotine-induced 22Na+ influx via nicotinic receptor-associated channel. LPA enhanced veratridine (but not nicotine)-induced 45Ca2+ influx via voltage-dependent calcium channel and catecholamine secretion. LPA shifted concentration-response curve of veratridine for 22Na+ influx upward, without altering the EC(50) of veratridine. Ptychodiscus brevis toxin-3 allosterically enhanced veratridine-induced 22Na+ influx by twofold in non-treated and LPA-treated cells. Whole-cell patch-clamp analysis showed that peak Na+ current amplitude was greater by 39% in LPA (100 nmol/L for 36 h)-treated cells; however, I-V curve and steady-state inactivation/activation curves were comparable between non-treated and LPA-treated cells. LPA treatment (> or = 24 h) increased cell surface [3H]saxitoxin binding by approximately 28%, without altering the K(d) value; the increase was prevented by cycloheximide, actinomycin D, or Ki16425, dioctylglycerol pyrophosphate 8:0 (two inhibitors of LPA(1) and LPA3 receptors), or botulinum toxin C3 (Rho inhibitor), Y27632 (Rho kinase inhibitor), consistent with LPA(1) receptor expression in adrenal chromaffin cells. LPA raised Nav1.7 mRNA level by approximately 37%. Thus, LPA-LPA(1) receptor-Rho/Rho kinase pathway up-regulated cell surface Nav1.7 and Nav1.7 mRNA levels, enhancing veratridine-induced Ca2+ influx and catecholamine secretion.     

A two-step model for acetylcholine control of exocytosis via nicotinic receptors

The view that Ca²⁺ entry through voltage-dependent Ca²⁺ channels (VDCC) and through nicotinic receptors for acetylcholine (nAChRs) causes equal catecholamine release responses in chromaffin cells, was reinvestigated here using new protocols. We have made two-step experiments consisting in an ACh prepulse followed by a depolarizing pulse (DP). In voltage-clamped bovine chromaffin cells an ACh prepulse caused a slow-rate release but augmented 4.5-fold the much faster exocytotic response triggered by a subsequent depolarizing pulse (measured with capacitance and amperometry). If the ACh prepulse was given with mecamylamine or in low external Ca²⁺, the secretion increase disappeared. This suggests a two-step model for the effects of ACh: (1) meager Ca²⁺ entry through nAChRs mostly serves to keep loaded with vesicles the secretory machine; and (2) in this manner, the cell is prepared to respond with an explosive secretion of catecholamine upon depolarization and fast high Ca²⁺ entry through VDCC.     

Fast exocytosis mediated by T- and L-type channels in chromaffin cells: distinct voltage-dependence but similar Ca<Superscript>2+</Superscript>-dependence

Expression, spatial distribution and specific roles of different Ca(2+) channels in stimulus-secretion coupling of chromaffin cells are intriguing issues still open to discussion. Most of the evidence supports a role of high-voltage activated (HVA) Ca(2+) channels (L-, N-, P/Q- and R-types) in the control of exocytosis: some suggesting a preferential coupling of specific Ca(2+) channel subunits with the secretory apparatus, others favoring the idea of a contribution to secretion proportional to the expression density and gating properties of Ca(2+) channels. In this work we review recent findings and bring new evidence in favor of the hypothesis that also the LVA (low-voltage-activated, T-type) Ca(2+) channels effectively control fast exocytosis near resting potential in adrenal chromaffin cells of adult rats. T-type channels recruited after long-term treatments with pCPT-cAMP (or chronic hypoxia) are shown to control exocytosis with the same efficacy of L-type channels, which are the dominant Ca(2+) channel types expressed in rodent chromaffin cells. A rigorous comparison of T- and L-type channel properties shows that, although operating at different potentials and with different voltage-sensitivity, the two channels possess otherwise similar Ca(2+)-dependence of exocytosis, size and kinetics of depletion of the immediately releasable pool and mobilize vesicles of the same quantal size. Thus, T- and L-type channels are coupled with the same Ca(2+)-efficiency to the secretory apparatus and deplete the same number of vesicles ready for release. The major difference of the secretory signals controlled by the two channels appear to be the voltage range of operation, suggesting the idea that stressful conditions (hypoxia and persistent beta-adrenergic stimulation) can lower the threshold of cell excitability by recruiting new Ca(2+) channels and activate an additional source of catecholamine secretion.     

p-Chloromercuribenzoate Causes Ca2+-Dependent Exocytotic Catecholamine Secretion from Cultured Bovine Adrenal Medullary Cells

Incubation of cultured bovine adrenal medullary cells with p-chloromercuribenzoate (50-500 microM), a sulfhydryl-reacting agent, caused an increase in the secretion of catecholamines, p-Chloromercuriphenyl sulfonate, a p-chloromercuribenzoate analogue that poorly penetrates the cell membrane, caused a similar increase in catecholamine secretion. In both cases, catecholamine secretion was dependent on extracellular Ca2+. Furthermore, p-chloromercuribenzoate caused both 45Ca2+ influx into the cells and an increase in the intracellular free Ca2+ concentration. The increases in catecholamine secretion and 45Ca2+ influx behaved similarly in relation to p-chloromercuribenzoate concentration. The time courses of the increased secretion, 45Ca2+ influx, and intracellular free Ca2+ concentration by p-chloromercuribenzoate were also quite similar. The stimulation of catecholamine secretion by p-chloromercuribenzoate was reversed by washing the cells with dithiothreitol-containing medium, but not by dithiothreitol-free medium. When the cells were treated with p-chloromercuribenzoate, dopamine-beta-hydroxylase, an enzyme present in the chromaffin granules along with catecholamines, was also released. However, p-chloromercuribenzoate did not cause release of phenylethanolamine-N-methyltransferase, an enzyme present in the cytoplasm. These results indicate that catecholamine secretion due to p-chloromercuribenzoate occurs by Ca2+-dependent exocytosis.