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+.     

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.     

The phorbol ester TPA enhances A23187--but not carbachol- and high K+-induced catecholamine secretion from cultured bovine adrenal chromaffin cells

The effect of the phorbol ester TPA on catecholamine secretion was studied in cultured bovine adrenal chromaffin cells. The pretreatment of chromaffin cells with TPA caused the enhancement of catecholamine secretion induced by the calcium ionophore, A23187. By contrast, neither carbachol- nor high K+-induced secretion was changed by TPA pretreatment. These results support the concept that protein kinase C plays an important role as a factor transducing the Ca2+ signal to the exocytotic process of catecholamine secretion in bovine adrenal chromaffin cells.     

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.     

22Na+ Uptake and Catecholamine Secretion by Primary Cultures of Adrenal Medulla Cells

The uptake of 22Na+ and secretion of catecholamines by primary cultures of adrenal medulla cells under the influence of a variety of agonists and antagonists were determined. Veratridine, batrachotoxin, scorpion venom, and nicotine caused a parallel increase in 22Na+ uptake and Ca2+-dependent catecholamine secretion. Ba2+, depolarizing concentrations of K+, and the Ca2+ ionophore Ionomycin stimulated secretion of catecholamines but did not increase the uptake of 22Na+. Tetrodotoxin inhibited both 22Na+ uptake and catecholamine secretion evoked by veratridine, batrachotoxin, and scorpion venom, but had no effect on 22Na+ uptake and catecholamine secretion caused by nicotine. On the other hand, histrionicotoxin, which blocks the acetylcholine receptor-linked ion conductance channel, blocked nicotine-stimulated 22Na+ uptake and catecholamine secretion, but only partially inhibited veratridine-stimulated catecholamine secretion and had no effect on veratridine-stimulated 22Na+ uptake. The combination of veratridine plus tetrodotoxin, which has been shown to prevent nicotine-stimulated secretion of catecholamines by adrenal medulla cells, also prevented nicotine-stimulated 22Na+ uptake by the primary cultures. These studies demonstrate the presence of tetrodotoxin-sensitive Na+ channels in adrenal medulla cells which are functionally linked to Ca2+-dependent catecholamine secretion. However, These channels are not utilized for Na+ entry upon activation of nicotinic receptors; in this case Na+ entry occurs through the receptor-associated ion conductance channel.     

Differential control of adrenal and sympathetic catecholamine release by alpha 2-adrenoceptor subtypes

In the adrenergic system, release of the neurotransmitter norepinephrine from sympathetic nerves is regulated by presynaptic inhibitory alpha2-adrenoceptors, but it is unknown whether release of epinephrine from the adrenal gland is controlled by a similar short feedback loop. Using gene-targeted mice we demonstrate that two distinct subtypes of alpha2-adrenoceptors control release of catecholamines from sympathetic nerves (alpha 2A) and from the adrenal medulla (alpha 2C). In isolated mouse chromaffin cells, alpha2-receptor activation inhibited the electrically stimulated increase in cell capacitance (a correlate of exocytosis), voltage-activated Ca2+ current, as well as secretion of epinephrine and norepinephrine. The inhibitory effects of alpha2-agonists on cell capacitance, voltage-activated Ca2+ currents, and on catecholamine secretion were completely abolished in chromaffin cells isolated from alpha 2C-receptor-deficient mice. In vivo, deletion of sympathetic or adrenal feedback control led to increased plasma and urine norepinephrine (alpha 2A-knockout) and epinephrine levels (alpha 2C-knockout), respectively. Loss of feedback inhibition was compensated by increased tyrosine hydroxylase activity, as detected by elevated tissue dihydroxyphenylalanine levels. Thus, receptor subtype diversity in the adrenergic system has emerged to selectively control sympathetic and adrenal catecholamine secretion via distinct alpha2-adrenoceptor subtypes. Short-loop feedback inhibition of epinephrine release from the adrenal gland may represent a novel therapeutic target for diseases that arise from enhanced adrenergic stimulation.     

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.     

Role of protein kinase C in catecholamine secretion from digitonin-permeabilized bovine adrenal medullary cells

The effects of staurosporine and K-252a, potent inhibitors of protein kinases, and 12-O-tetradecanoylphorbol-13-acetate (TPA) on catecholamine secretion and protein phosphorylation in digitonin-permeabilized bovine adrenal medullary cells were investigated. Staurosporine and K-252a (0.01-10 microM) did not cause large changes in catecholamine secretion evoked by Ca2+ in digitonin-permeabilized cells whereas these compounds strongly prevented TPA-induced enhancement of catecholamine secretion in a concentration-dependent manner. Incubation of digitonin-permeabilized cells with [gamma-32P]ATP resulted in 32Pi incorporation into a large number of proteins, detected as several major bands and darkened background in autoradiograms. Ca2+ and TPA increased phosphorylation of these proteins. Staurosporine and K-252a markedly inhibited Ca(2+)-induced and TPA-induced increases in protein phosphorylation as well as basal (0 Ca2+) protein phosphorylation in digitonin-permeabilized cells. Long term treatment (24 h) of adrenal medullary cells with 1 microM TPA markedly decreased total cellular protein kinase C activity to about 5.3% of control. Pretreatment of the cells with 1 microM TPA strongly inhibited the TPA-induced enhancement of catecholamine secretion whereas it did not cause large changes in total cellular catecholamine amounts, Ca(2+)-induced catecholamine secretion, and cAMP-induced enhancement of catecholamine secretion from digitonin-permeabilized cells. From these results we conclude that protein kinase C plays a modulatory role in catecholamine secretion rather than being essential for initiating catecholamine secretion.     

Arachidonic Acid Release and Catecholamine Secretion from Digitonin-Treated Chromaffin Cells: Effects of Micromolar Calcium, Phorbol Ester, and Protein Alkylating Agents

The relationship between catecholamine secretion and arachidonic acid release from digitonin-treated chromaffin cells was investigated. Digitonin renders permeable the plasma membranes of bovine adrenal chromaffin cells to Ca2+, ATP, and proteins. Digitonin-treated cells undergo exocytosis of catecholamine in response to micromolar Ca2+ in the medium. The addition of micromolar Ca2+ to digitonin-treated chromaffin cells that had been prelabeled with [3H]arachidonic acid caused a marked increase in the release of [3H]arachidonic acid. The time course of [3H]arachidonic acid release paralleled catecholamine secretion. Although [3H]arachidonic acid release and exocytosis were both activated by free Ca2+ in the micromolar range, the activation of [3H]arachidonic acid release occurred at Ca2+ concentrations slightly lower than those required to activate exocytosis. Pretreatment of the chromaffin cells with N-ethylmaleimide (NEM) or p-bromophenacyl bromide (BPB) resulted in dose-dependent inhibition of 10 microM Ca2+-stimulated [3H]arachidonic acid release and exocytosis. The IC50 of NEM for both [3H]arachidonic acid release and exocytosis was 40 microM. The IC50 of BPB for both events was 25 microM. High concentrations (5-20 mM) of Mg2+ caused inhibition of catecholamine secretion without altering [3H]arachidonic acid release. A phorbol ester that activates protein kinase C, 12-O-tetradecanoylphorbol-13-acetate (TPA), caused enhancement of both [3H]arachidonic acid release and exocytosis. The findings demonstrate that [3H]arachidonic acid release is stimulated during catecholamine secretion from digitonin-treated chromaffin cells and they are consistent with a role for phospholipase A2 in exocytosis from chromaffin cells. Furthermore the data suggest that protein kinase C can modulate both arachidonic acid release and exocytosis.     

Inhibition of voltage-gated Ca(2+) current by PACAP in rat adrenal chromaffin cells.

It is well established that pituitary adenylate cyclase-activating polypeptide (PACAP) can stimulate catecholamine biosynthesis and secretion in adrenal chromaffin cells. Recent studies from this laboratory demonstrated that PACAP pretreatment inhibits nicotine (NIC)-induced intracellular Ca(2+) transients and catecholamine secretion in porcine adrenal chromaffin cells. Mechanistically, this effect is mediated by protein kinase C (PKC), and based on indirect evidence, is thought to primarily target voltage-gated Ca(2+) channels. The present study used whole-cell patch-clamp analysis to test this possibility more directly in rat chromaffin cells. Consistent with the porcine data, pretreatment with PACAP or with phorbol ester [phorbol myristate acetate (PMA)] significantly suppressed NIC-induced intracellular Ca(2+) transients and catecholamine secretion in rat chromaffin cells. Exposure to PACAP and PMA significantly reduced peak Ca(2+) current in rat cells. The effects of both PACAP and PMA on Ca(2+) current could be blocked by treating cells with the PKC inhibitor staurosporine. Exposure to selective channel blockers demonstrated that rat chromaffin cells contain L-, N- and P/Q-type Ca(2+) channels. PACAP pretreatment significantly reduced Ca(2+) current gated through all three channel subtypes. These data suggest that PACAP can negatively modulate NIC-induced catecholamine secretion in both porcine and rat adrenal chromaffin cells.     

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+.     

Stimulatory effect of enkephalins on calcium efflux from bovine adrenal chromaffin cells in culture

The effects of leucine- and methionine-enkephalin, opiate peptides, on Ca2+ efflux from cultured bovine adrenal chromaffin cells were examined. These enkephalins stimulated the efflux of 45Ca2+ from cells in a concentration-dependent manner (10(-8) M-10(-6) M). Leucine-enkephalin did not increase the intracellular free Ca2+ level, 45Ca2+ uptake, catecholamine secretion, cAMP level or cGMP level. The peptide-stimulated 45Ca2+ efflux was not inhibited by incubation in Ca2+-free medium, but was inhibited by incubation in Na+-free medium. These results indicate that enkephalins stimulate extracellular Na+-dependent 45Ca2+ efflux from cultured bovine adrenal chromaffin cells, probably by stimulating membrane Na+/Ca2+ exchange.     

Stimulation of catecholamine secretion from cultured chromaffin cells by an ionophore-mediated rise in intracellular sodium

The significance of intracellular Na+ concentration in catecholamine secretion of cultured bovine adrenal chromaffin cells was investigated using the monovalent carboxylic ionophore monensin. This ionophore, which is known to mediate a one-for-one exchange of intracellular K+ for extracellular Na+, induces a slow, prolonged release of catecholamines which, at 6 h, amounts of 75-90% of the total catecholamines; carbachol induces a rapid pulse of catecholamine secretion of 25-35%. Although secretory granule numbers appear to be qualitatively reduced after carbachol, multiple carbachol, or Ba2+ stimulation, overall granule distribution remains similar to that in untreated cells. Monensin-stimulated catecholamine release requires extracellular Na+ but not Ca2+ whereas carbachol-stimulated catecholamine release requires extracellular Ca2+ and is partially dependent on extracellular Na+. Despite its high selectivity for monovalent ions, monensin is considerably more effective in promoting catecholamine secretion than the divalent ionophores, {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187 and ionomycin, which mediate a more direct entry of extracellular Ca2+ into the cell. We propose that the monensin-stimulated increase in intracellular Na+ levels causes an increase in the availability of intracellular Ca2+ which, in turn, stimulates exocytosis. This hypothesis is supported by the comparable stimulation of catecholamine release by ouabain which inhibits the outwardly directed Na+ pump and thus permits intracellular Na+ to accumulate. The relative magnitudes of the secretion elicited by monensin, carbachol, and the calcium ionophores, are most consistent with the hypothesis that, under normal physiological conditions, Na+ acts by decreasing the propensity of Ca2+- sequestering sites to bind the Ca2+ that enters the cell as a result of acetylcholine stimulation.     

Internal Ca2+ Mobilization by Muscarinic Stimulation Increases Secretion from Adrenal Chromaffin Cells Only in the Presence of Ca2+ Influx

The cytosolic free Ca2+ concentration ([Ca2+]in) in single cat and bovine adrenal chromaffin cells was measured to determine whether or not there was any correlation between the [Ca2+]in and the catecholamine (CA) secretion caused by muscarinic receptor stimulation. In cat chromaffin cells, methacholine (MCh), a muscarinic agonist, raised [Ca2+]in by activating both Ca2+ influx and intracellular Ca2+ mobilization with an accompanying CA secretion. In bovine cells, MCh elevated [Ca2+]in by mobilizing intracellular Ca2+ but did not cause CA secretion. The MCh-induced rise in [Ca2+]in in cat cells was much higher than that in bovine cells, but when Ca2+ influx was blocked, the rise was reduced, with a concomitant loss of secretion, to a level comparable to that in bovine cells. Intracellular Ca2+ mobilization due to muscarinic stimulation substantially increased secretion from depolarized bovine and cat cells, where a [Ca2+]in elevated above basal values was maintained by a continuous Ca2+ influx. These results show that Ca2+ released from internal stores is not effective in triggering secretion unless Ca2+ continues to enter across the plasma membrane, a conclusion suggesting that secretion depends on [Ca2+]in in a particular region of the cell.     

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.     

Catecholamine secretion by hamster adrenal cells.

Abstract— Suspensions of isolated adrenal cells were prepared by digesting hamster adrenal glands with collagenase, and the secretion of catecholamine from these cells was studied. Acetylcholine (ACh) produces a dose-dependent increase in catecholamine secretion; half-maximal secretion is produced by 3 μm -ACh, and maximal secretion by 100 μm -ACh. The cholinergic receptor in these cells appears to be nicotinic, since catecholamine secretion is stimulated by the nicotinic agonists nicotine and dimeth-ylphenylpiperaziniurn, but not by the muscarinic agonists pilocarpine or oxotremorine. ACh-induced catecholamine secretion is inhibited by hexamethonium, tubocurarine, and atropine, but is not inhibited by α-bungarotoxin. ACh-induced catecholamine secretion is dependent upon the presence of extracellular Ca²⁺, and appears to occur by exocytosis, since the release of catecholamine is accompanied by the release of dopamine β-monooxygenase, but not of lactate dehydrogenase. These biochemical studies complement the morphological evidence for exocytosis in hamster adrenal glands, and indicate that catecholamine secretion from hamster chromaffin cells is similar to that from chromaffin cells of other species.     

Interaction of SK(Ca) channels and L-type Ca(2+) channels in catecholamine secretion in the rat adrenal gland

We elucidated the interaction of small-conductance Ca(2+)-activated K(+) (SK(Ca)) channels and L-type Ca(2+) channels in muscarinic receptor-mediated control of catecholamine secretion in the isolated perfused rat adrenal gland. The muscarinic agonist methacholine (10-300 microM) produced concentration-dependent increases in adrenal output of epinephrine and norepinephrine. The SK(Ca) channel blocker apamin (1 microM) enhanced the methacholine-induced catecholamine responses. The facilitatory effect of apamin on the methacholine-induced catecholamine responses was not observed during treatment with the L-type Ca(2+) channel blocker nifedipine (3 microM) or Ca(2+)-free solution. Nifedipine did not affect the methacholine-induced catecholamine responses, but it inhibited the responses during treatment with apamin. The L-type Ca(2+) channel activator Bay k 8644 (1 microM) enhanced the methacholine-induced catecholamine responses, whereas the enhancement of the methacholine-induced epinephrine and norepinephrine responses were prevented and attenuated by apamin, respectively. These results suggest that SK(Ca) channels are activated by muscarinic receptor stimulation, which inhibits the opening of L-type Ca(2+) channels and thereby attenuates adrenal catecholamine secretion.     

Na(+)-dependent Ca2+ influx in bovine adrenal chromaffin cells

Bovine adrenal chromaffin cells were loaded with Na+ via either acetylcholine receptor-associated ion channels or voltage-sensitive Na+ channels. There were increases in [Ca2+]i, 45Ca2+ uptake and catecholamine secretion in both types of Na(+)-loaded cells relative to control cells in which Na+ loading had been prevented by hexamethonium and tetrodotoxin, respectively. These results show the presence of Na(+)-dependent Ca2+ influx activity in chromaffin cells which is probably mediated by the reverse mode of a Na+/Ca2+ exchanger.     

Studies on the Effect of Insulin-Like Growth Factor-I on Catecholamine Secretion from Chromaffin Cells

Chromaffin cells cultured in serum-free medium secreted a smaller percentage of their catecholamine stores in response to stimulation by high K+ (55 mM) than did cells cultured in serum-containing medium. Addition of insulin-like growth factor-I (IGF-I) to serum-free medium restored high K(+)-stimulated catecholamine secretion to the levels seen in serum-treated cultures. In contrast, addition of IGF-I to serum-containing medium had little effect on catecholamine secretion. These results suggest that serum contains IGF-I or another factor that maintains the secretory responsiveness of chromaffin cells. IGF-I not only enhanced high K(+)-stimulated catecholamine secretion, but also augmented secretion elicited by the nicotinic agonist dimethyl-phenylpiperazinium, the dihydropyridine agonist Bay K 8644, and Ba2+. IGF-I did not affect the dependence of catecholamine secretion on extracellular Ca2+ concentration nor did it affect the time course of secretion. Experiments using 45Ca2+ demonstrated that IGF-I treatment enhanced Ca2+ uptake into the cells. When cells were permeabilized by treatment with digitonin, Ca2(+)-dependent catecholamine secretion was slightly, but consistently, greater from IGF-I-treated cells than from untreated cells. Our results suggest that IGF-I may enhance catecholamine secretion partly by increasing Ca2+ entry into the cells and partly by affecting a step distal to Ca2+ entry.     

Inhibition of 45Ca2+ uptake and catecholamine secretion by phenothiazines and pimozide in adrenal medulla cells cultures

The inhibition by several phenothiazine drugs and pimozide of the uptake of 45Ca2+ and secretion of catecholamines by cultured adrenal medulla cells stimulated with nicotine, veratridine, 50 mM K+, ionomycin and Ba2+ was studied. The inhibition of 45Ca2+ uptake, except for ionomycin, closely parallelled the inhibition of catecholamine secretion. The nicotine-and veratridine-stimulated effects were several fold more sensitive to inhibition by the drugs than were those stimulated by 50mM K+, ionomycin and Ba2+; the ionomycin-stimulated effects were least sensitive to inhibition. These studies indicate that the drugs have multiple effects on stimulus-secretion coupling in adrenal medulla cells. It is suggested that inhibition of the veratridine- and nicotine-stimulated events is due to membrane perturbations caused by the drugs, that inhibition of the 50mM K+- and Ba2+-stimulated events is due to alterations in the voltage sensitive membrane Ca2+ channel, and that inhibition of secretion elicited by ionomycin may be due to inhibition of Ca2+-calmodulin reactions or to more profound non specific membrane effects.