Evoked Catecholamine Release Research Articles

Inhibition of Ca 2+-dependent catecholamine release by myosin light chain kinase inhibitor, wortmannin, in adrenal chromaffin cells

To elucidate the possible involvement of myosin light chain kinase (MLCK) in the mechanism of exocytosis, we studied effects of MLCK inhibitor, wortmannin, on the secretory function of bovine adrenal chromaffin cells. Preincubation of chromaffin cells with wortmannin inhibited both acetylcholine- and high K +-evoked catecholamine (CA) release. The IC 50 for high K +-evoked CA release was 1μM. When the cells were permeabilized with digitonin after wortmannin preincubation, Ca 2+-dependent exocytosis was inhibited in a dose-dependent manner (IC 50, 1μM). These findings suggest the implication of MLCK in the Ca 2+-triggered process in the machinery of exocytosis.

A pertussis-toxin-sensitive protein controls exocytosis in chromaffin cells at a step distal to the generation of second messengers

The role of GTP-binding proteins (G-proteins) in the secretory process in chromaffin cells was investigated by studying the effects of pertussis toxin (PTX) on catecholamine release and generation of various second messengers. PTX was found to stimulate the catecholamine secretion induced by nicotine, 59 mM-K+ or veratridine. PTX also potentiated Ca2(+)-evoked catecholamine release from permeabilized chromaffin cells, suggesting that PTX substrate(s) regulate the exocytotic machinery at a step distal to the rise in intracellular Ca2+. We have investigated the possible intracellular pathways involved in the stimulation of secretion by PTX. PTX did not modify the translocation of protein kinase C (PKC) to membranes in intact or permeabilized cells; in addition, neither inhibitors nor activators of PKC had any effect on catecholamine release induced by PTX. Thus it seems unlikely that the effect of PTX on secretion is mediated by activation of PKC. The effect of PTX is also cyclic AMP-independent, as PTX did not change cytoplasmic cyclic AMP levels. The relationship between PTX treatment and arachidonic acid release was also examined. We found that an increase in cytoplasmic arachidonic acid concentration enhanced Ca2(+)-evoked catecholamine release in permeabilized cells, but arachidonic acid did not mimic the effect of PTX on the Ca2(+)-dose-response curve for secretion. Furthermore, PTX did not significantly modify the release of arachidonic acid measured in resting or stimulated chromaffin cells, suggesting that the stimulatory effect of PTX on secretion is not mediated by an activation of phospholipase A2. Taken together, these results suggest that PTX may modulate the intracellular machinery of secretion at a step distal to the generation of second messengers. In alpha-toxin-permeabilized cells, full retention of the PTX-induced activation of secretion was observed even 30 min after permeabilization. In contrast, when chromaffin cells were permeabilized with streptolysin-O (SLO), there was a marked progressive loss of the PTX effect. We found that SLO caused the rapid leakage of three G-protein alpha-subunits which are specifically ADP-ribosylated by PTX. We propose that a PTX-sensitive G-protein may play an inhibitory role in the final stages of the Ca2(+)-evoked secretory process in chromaffin cells.

Modulation by β-adrenoceptors and angiotensin II receptors of splanchnic nerve evoked catecholamine release from the adrenal medulla

In the present study, we have evaluated the effect of both facilitatory beta 2-adrenoceptor and angiotensin II receptor on the release of adrenal catecholamines induced by electrical stimulation of the splanchnic nerve in anaesthetized and vagotomized dog. In these experiments, individual or combined treatments with the beta 2-adrenoceptor antagonist ICI 118551 (0.3 mg/kg i.v.), the converting enzyme inhibitor captopril (2 mg/kg i.v.), or the angiotensin II receptor antagonist saralasin (2 micrograms.kg-1.min-1 i.v.) were found to significantly decrease the release of adrenal catecholamines during splanchnic nerve stimulation (5-V pulses of 2 ms duration for 3 min at 1 Hz) whatever the order of administration of the drugs. On the other hand, the infusion of angiotensin II (20 ng.kg-1.min-1) was shown to potentiate the release of adrenal catecholamines in response to electrical stimulation, and this effect was totally blocked by treatment with saralasin (4 micrograms.kg-1.min-1 i.v.). This facilitating angiotensin mechanism differed from beta-adrenoceptor facilitating mechanism, since following beta-blockade with ICI 118551, angiotensin II infusion still significantly potentiated the release of catecholamines during splanchnic nerve stimulation. These observations thus suggest that both facilitating beta 2-adrenoceptors and angiotensin II receptors can independently modulate the release of adrenal catecholamines.

The inhibition by pertussis and tetanus toxins of evoked catecholamine release from intact and permeabilized bovine adrenal chromaffin cells

Pertussis toxin stimulates both basal and nicotine-evoked catecholamine secretion from intact bovine adrenal chromaffin cells, as well as Ca 2+-evoked release from permeabilized cells. Tetanus toxin inhibits all these effects; it reduces the secretion of intact cells treated with pertussis toxin to the basal level, and decreases by about 50% Ca 2+-evoked release from permeabilized cells whether or not previously stimulated by pertussis toxin.

Involvement of phosphoinositide metabolism in GABA-induced catecholamine release from cultured bovine adrenal chromaffin cells

The effects of GABA on catecholamine release and phosphoinositide metabolism were studied in cultured bovine adrenal chromaffin cells. GABA and muscimol, a specific agonist for the GABA A receptor, each evoked a gradual secretion of catecholamines from the cells in the presence of ouabain, an inhibitor of Na +,K +-ATPase. This release was inhibited by bicuculline, a specific antagonist for the GABA A receptor, or by picrotoxin, a blocker of GABA-gated Cl − channels, and was potentiated by diazepam or pentobarbital. GABA or muscimol induced a concentration-dependent formation of inositol phosphates. This accumulation of inositol phosphates was also inhibited by bicuculline, picrotoxin or removal of extracellular Ca 2+, and also potentiated by diazepam and pentobarbital. Nicardipine suppressed GABA-induced catecholamine release in the presence of ouabain and accumulation of inositol phosphates, while verapamil, diltiazem, and ω-conotoxin failed to inhibit these responses to GABA. The phosphoinositide-specific phospholipase C inhibitor neomycin also inhibited both GABA-induced accumulation of inositol phosphates and stimulation of catecholamine release in the presence of ouabain. These results taken together indicate that GABA evoked catecholamine release from the chromaffin cells in the presence of ouabain by stimulation of phosphoinositide metabolism in a Ca 2+-sensitive manner via activation of GABA A receptor-coupled Cl − channels.

Enhancement by GABA of the stimulation-evoked catecholamine release from cultured bovine adrenal chromaffin cells

The possible involvement of GABAergic mechanisms in the catecholamine (CA) release from adrenal medulla was investigated in a primary culture of bovine adrenal chromaffin cells. GABA elicited CA release and enhanced acetylcholine (ACh)-, excess K(+)- and veratridine-evoked CA release. Muscimol, a selective GABAA receptor agonist, mimicked the action of GABA on CA release. On the other hand, baclofen, a GABAB receptor agonist, failed to affect basal or evoked CA release. Furthermore, bicuculline and picrotoxin blocked the enhancement by GABA of veratridine-evoked CA release without affecting basal CA release and CA release evoked by veratridine. In Ca2(+)-free medium, GABA failed to affect basal and caffeine-evoked CA release. ACh-evoked CA release was slightly reduced by bicuculline, whereas excess K(+)-evoked CA release was not, suggesting the involvement of endogenous GABA in CA release evoked by ACh. These results suggest a facilitatory modulation by GABA of basal and evoked release of CA from bovine adrenal medulla through GABAA receptor-mediated mechanisms.

Tetanus antitoxin binds to intracellular tetanus toxin in permeabilized chromaffin cells without restoring Ca 2+-induced exocytosis

Tetanus toxin blocks Ca 2+-evoked catecholamine release from permeabilized bovine adrenal chromaffin cells preloaded with gangliosides. Tetanus toxin preincubated with its specific antibodies F(ab′) 2 is without any effect on exocytosis. Specific antitetanus F(ab′) 2 presented to chromaffin cells which are pretreated with tetanus toxin and permeabilized by digitonin cannot restore exocytosis. Under the same conditions, however, 125I-labeled F(ab′) 2 accumulates in chromaffin cells. The accumulation depends on the presence and concentration of tetanus toxin and can be prevented by an excess of unlabeled F(ab′) 2. Once tetanus toxin has initiated block of exocytosis, it cannot be neutralized by binding to its specific antibody.

Biochemical studies on the release of catecholamines from the rat carotid body in vitro

The effects of hypoxia and carbachol on the release of newly synthesized catecholamines from superfused rat carotid bodies have been examined. Hypoxic superfusion medium was found to evoke catecholamine release which was dependent on the extracellular calcium concentration and was reduced by nitrendipine and atropine. Superfusion with the muscarinic agonist, carbachol, stimulated catecholamine release independently of the oxygen tension of the medium. The effect of carbachol on catecholamine release was abolished by atropine, suggesting that it was mediated by activation of cholinergic receptors of the muscarinic type. Both hypoxia and carbachol stimulated the release of 45Ca from carotid bodies prelabelled with 45Ca. The release of 45Ca with either stimulus was reduced by atropine and nitrendipine. These results suggest that although extracellular calcium plays an important role in the exocytotic secretory process of the carotid body, the mobilization of intracellular calcium pools may also contribute to the secretory response.

The effect of etorphine on the secretion of endogenous catecholamines and total tritium evoked by nerve- and acetylcholine-stimulation in perfused rat adrenal glands

Isolated perfused rat adrenal glands were prelabeled with 3H-norepinephrine and catecholamine secretion was evoked by nerve stimulation (10 Hz, supramaximal voltage for 30 seconds) or acetylcholine (ACh)(5.4 μg) injection. Nerve stimulation evoked significant increases in tritium (16371±2109 cpm) and catecholamine (11.5 ± 1.0 ng norepinephrine [NE], 123 ± 13.0 ng epinephrine [EP]) release from the adrenal medulla. ACh injection evoked catecholamine release, but failed to increase tritium release. In the presence of etorphine, the nerve stimulation-mediated release of tritium, NE and EP was inhibited. In contrast, the ACh-mediated release of NE but not EP was inhibited by etorphine. In a previous publication (1), we have shown that 3H-NE is taken up by sympathetic nerve endings contained in extra adrenal tissue removed along with the adrenal gland during the surgery, but not by chromaffin cells. Therefore, the inhibitory effect of etorphine on NE, EP and tritium release evoked by nerve stimulation suggests a functional role for opiate receptors on transmitter release from sympathetic and splanchnic nerve endings. However, the differential effect of etorphine on NE and EP release evoked by ACh injection indicates that opiate receptors on chromaffin cells modulates NE but not EP release.

Sodium-dependent inhibition by PN200-110 enantiomers of nicotinic adrenal catecholamine release.

1. Dimethylphenylpiperazinium (DMPP) or high K concentrations evoke catecholamine release from perfused cat adrenal glands; in both cases the secretory response was significantly enhanced in the absence of Na. Tetrodotoxin did not modify the nicotinic secretory response. 2. The (+)- and (-)-enantiomers of the dihydropyridine Ca channel blocker PN200-110 show a high degree of stereoselectivity in the inhibition of catecholamine secretion evoked by high K or by DMPP in the presence of Na, the (+)-enantiomer being 57 and 80 times more potent, respectively, than the (-)-enantiomer. Both, noradrenaline and adrenaline release were equally depressed by PN200-110. 3. The IC50 values for (+)- and (-)-PN200-110 for blockade of the secretory response induced by K or DMPP in the presence of Na are in the same range. In the absence of Na, (-)-PN200-110 did not affect DMPP-evoked secretion; however, the (+)-enantiomer partially inhibited it. 4. The results suggest that the physiological catecholamine release from chromaffin cells is preceded by Na entry through the nicotinic receptor-associated ionophore; this causes cell depolarization, opening of voltage-dependent, dihydropyridine-sensitive Ca channels and Ca entry into the cell. In the absence of Na, additional Ca influx through an alternative pathway (the nicotinic cholinoceptor ionophore?) might also activate secretion.

Potentiation of evoked adrenal catecholamine release by cyanide: Possible role of calcium

The effect of cyanide on release of catecholamines was evaluated in isolated bovine adrenal glands stimulated with 4 different agonists. Cyanide (0.1 – mM) increased catecholamine release induced by barium or cadmium 2−3-fold. Acetylcholine or potassium induced secretion of adrenal catecholamines was also enhanced by cyanide, but only to the extent of 30–50%. These data suggest that cyanide acts by multiple mechanisms to enhance evoked catecholamine release. The above results may be partly explained by the fact that cyanide inhibited 45Ca efflux from stimulated bovine adrenals. Changes in plasma membrane permeability may be crucial in the alterations of ion flux and evoked catecholamine release caused by cyanide.

Benzodiazepine inhibition of the stimulation-evoked catecholamine release from bovine adrenomedullary cells in culture

Effect of benzodiazepines on evoked catecholamine (CA) release from a primary culture of bovine adrenal medullary cells was investigated. Midazolam at high doses (> 10 ? M) inhibited CA release evoked by acetylcholine (ACh), excess K(+) and veratridine but not by A23187 or caffeine in Ca(2+) -free media. Other benzodiazepines, diazepam, clonazepam, nitrazepam and R05-4864, as well as 1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinolinecarboxamide (PK11195) and ethyl-?-carboline-3-carboxylate (?CCE) also inhibited ACh-evoked CA release but only at high concentrations. The inhibitory effect of midazolam on ACh-evoked CA release was not affected by R015-1788, a central-type benzodiazepine receptor antagonist which itself had no effect on basal and ACh-evoked CA release. Facilitatory action of Bay K 8644 on CA release evoked by 20 mM K(+) was reduced by midazolam, PK11195 and R05-4864. Further, ACh-evoked (45)Ca uptake was markedly reduced by midazolam and R05-4864 in association with the inhibition of CA release. These results suggest that benzodiazepines at high doses, inhibit the evoked CA release from adrenal chromaffin cells possibly through the blockade of Ca(2+) influx. Possible involvement of receptor subtypes of benzodiazepines in regulating CA secretion is discussed.

Down regulation of protein kinase C in neuronal cells: effects on neurotransmitter release

We investigated the effects of phorbol esters on protein kinase C (PKC) activity and on neurotransmitter release from cultured neuronal cells. Both differentiated and undifferentiated PC12 pheochromocytoma cells contained high levels of protein PKC. Under normal conditions all the enzyme activity was found in the cytoplasm. Addition of the phorbol esters phorbol 12-myristate-13-acetate (TPA) or phorbol 12,13-dibutyrate (PDBu) caused a rapid translocation of PKC from the cytoplasm to the particulate fraction. Continued culture of cells with these phorbol esters resulted in the decline of total PKC activity. After 10-20 hr of culture, both membrane and cytoplasmic PKC activity had declined to background levels. cAMP-dependent and Ca2+/calmodulin-dependent protein kinase activities were only slightly affected by chronic phorbol ester treatment. Addition of active phorbol esters to PC12 cells produced an enhancement of the depolarization-induced release of 3H-norepinephrine. Following chronic phorbol ester treatment, the ability of these substances to enhance evoked catecholamine release was lost. Furthermore, depolarizing stimuli released considerably less 3H-norepinephrine than in control untreated cells. Phorbol esters also enhanced depolarization-induced 3H-norepinephrine release from primary cultures of rat sympathetic neurons. Chronic treatment of these neurons with phorbol esters also resulted in the loss of their ability to enhance transmitter release and in a large reduction in the extent of depolarization-evoked transmitter release. Chronic phorbol ester treatment also resulted in the disappearance of PKC from sympathetic neurons, but had little effect on cAMP-dependent or Ca2+/calmodulin-dependent kinase activities. These results demonstrate that PKC-deficient neurons can be prepared. The data also demonstrate that depolarization-induced neurotransmitter release is mediated by both protein kinase C-dependent and independent pathways.

Potentiation of hemorrhage-evoked catecholamine release by prior blood loss in cats.

The effect of prior blood loss on the plasma catecholamine response to acute hemorrhage (H) was assessed in alpha-chloralose-urethane anesthetized cats. Animals sustained an initial H period of 0 (samples only), 10, or 20% H total blood volume. Ninety minutes after reinfusion of the shed blood, all animals sustained a rapid 20% H. The catecholamine and arterial pressure responses to this second 20% H were assessed every 2 min for 20-min duration. Plasma norepinephrine increased modestly in the 0/20% group (+0.63 +/- 0.13 ng/ml) and 10/20% group (+0.66 +/- 0.07 ng/ml), whereas the 20/20% group showed a much larger (P less than 0.01) mean increase of 3.58 +/- 1.16 ng/ml. Plasma epinephrine did not increase after 0/20% (+0.05 +/- 0.02 ng/ml), increased slightly after 10/20% H (+0.10 +/- 0.05 ng/ml), and demonstrated a large significant increase after 20/20% H (+0.48 +/- 0.18 ng/ml). The differential effect of 20% H on catecholamine release, depending on the magnitude of prior blood loss, was not the result of altered mean arterial or pulse pressure responses to H. Correlation analyses revealed that the mean increases in epinephrine and norepinephrine during the post-H sampling period were well correlated in each animal (r = 0.864, P less than 0.001). The data indicate that the priming effect of prior blood loss on H evoked catecholamine release: requires an initial blood loss of 20% of total blood volume; occurs rapidly, as it is seen by 90 min after the initial H period; and affects epinephrine and norepinephrine similarly. We conclude that the immediate past secretory history of the sympathoadrenal system affects its responsiveness to subsequent blood loss.

Potassium ion is indispensable to the catecholamine releasing response of dog adrenals to gamma-aminobutyric acid.

The effect of potassium ion on the GABA-evoked catecholamine (CA) release from isolated perfused adrenal glands of the dog was investigated. When omitting the external potassium ion, the basal release of CA was increased. During this period GABA no longer caused the increase in CA release and moreover the increased basal release was diminished reversibly by GABA. 3-Amino-1-propane-sulfonic acid, a GABAA agonist, mimicked the action of GABA in K+-free solution, while baclofen, a GABAB agonist, did not cause CA release in normal solution and did not alter the basal release in K+-free solution. The inhibition by GABA of the basal CA release in K+-free solution was blocked by bicuculline. The potency of the CA releasing action of GABA was dependent on the concentration of external K+ between 1-10 mM. Reintroduction of K+ to glands which had been perfused with K+-free solution immediately reduced the basal release of CA whereas it recovered the CA releasing action of GABA. These results suggest that GABA-evoked CA release is dependent on potassium ion. The possible mechanisms by which GABA evoked CA release are discussed.

Inhibition of calcium uptake and catecholamine release by 8-( n, n-diethylamino)- octyl-3,4,5-trimethoxybenzoate hydrochloride (TMB-8) In cultured bovine adrenal chromaffin cells

Effects of intracellular calcium antagonists, 8-( f, N-diethylamino)-octyl-3,4,5-trimethoxybenzoate hydrochloride (TMB-8) and 1-(5-( p-nitrophenyl)-furfurylidene-amino) hydantoin sodium hydrate (dantrolene sodium), on catecholamine release and 45Ca 2+ uptake were studied using cultured bovine adrenal chromaffin cells. TMB-8 inhibited carbamylcholine-evoked catecholamine release and 45Ca 2+ uptake in a concentration-dependent manner with a similar potency. On the contrary, dantrolene sodium did not show obvious inhibitory effects of catecholamine release and 45Ca 2+ uptake. Although TMB-8 inhibited the high K +-evoked catecholamine release and 45Ca 2+ uptake, the potency of the drug was approximately 100-fold less than when used to inhibit the carbamylcholine-evoked catecholamine release and 45Ca 2+ uptake. The inhibitory effect of TMB-8 on the carbamylcholine-evoked catecholamine release was not overcome by an increase in an extracellular calcium concentration, and was not due to competitive antagonism at the nicotinic receptor site. Moreover, TMB-8 inhibited the carbamylcholine-stimulated 45Ca 2+ efflux, but dantrolene sodium failed to affect it. These results suggest that TMB-8, a well-known intracellular calcium antagonist, prevents the cellular calcium uptake in cultured adrenal chromaffin cells, and thus prevents catecholamine release.

The release of acetylcholine and of catecholamine from the cat's adrenal gland

The cat's adrenal gland was perfused in situ with Krebs solution containing eserine; the amount of acetylcholine and of catecholamine released was measured. Splanchnic nerve stimulation (5 Hz for 2 min) increased the release of acetylcholine and catecholamine; the molar ratio of evoked release of catecholamine to acetylcholine was122 ± 8. It is suggested that this amplification is achieved because a chromaffin cell granule contains more mediator than does the acetylcholine quantum that releases it. The release per impulse of catecholamine during splanchnic nerve stimulation at 30 Hz was less than that released by stimulation at 1 or 5 Hz. This depression is attributed to a presynaptic failure, because the release of acetylcholine was similarly frequency dependent. The release of catecholamine was linearly related to the release of acetylcholine over the range tested, indicating that the input-output relationship at the splanchnic-adrenal medullary junction is linear. During continuous stimulation of the splanchnic nerve (5 Hz), catecholamine release declined to a level that was32 ±2% of the initial output. This fatigue is attributed primarily to a postsynaptic depression, because the release of acetylcholine was maintained at71±6% of its initial level. The presence of eserine in the perfusate was necessary for the release of acetylcholine to be detected, but in the presence of eserine catecholamine release was90±10% that in the drug's absence. It is concluded that released acetylcholine is hydrolysed at some distance from its site of release and action. Glands perfused with raised K + released acetylcholine and catecholamine. During a 10 min exposure to K +, catecholamine release was less well maintained than was acetylcholine release. It is suggested that calcium channels on adrenal cells desensitize more readily than those on cholinergic nerve terminals. Muscarine or nicotine released catecholamine but not acetylcholine; acetylcholine antagonists depressed evoked catecholamine release but did not alter acetylcholine release. These findings are interpreted to indicate that compounds secreted from the adrenal medulla have no physiologically important feed-back effects on cholinergic nerve terminals.

Effects of hypertonic solutions on catecholamine release from cat adrenal glands

The effects of hypertonic solutions on the spontaneous and evoked catecholamine release from isolated, perfused cat adrenal glands were studied. Hypertonic solutions enhanced three times the spontaneous release of the amines, but markedly inhibited the release evoked by nicotine 15μM for 2 min or high K +(17.7 mM for 2 min). Using sucrose as osmoticant, the secretory response to high K + was decreased to 53 and 15% of controls at 344 and 416 mosM, respectively At 512 mosM sucrose inhibited poorly the release of catecholamines evoked by nicotine, but reduced it by 90% at 1000 mosM; sodium chloride behaved similarly to sucrose. A rise in the osmolarity of only 7.5% with choline chloride produed a complete inhibition of the K +-evoked response. These effects were not seen using isotonic choline chloride; on the contrary, isotonic choline chloride enhanced the K + response, probably by stimulating nicotinic receptors. Since atropine did not modify this effect, it seems that secretion of catecholamines evoked by choline is mediated by nicotinic receptors. While the inhibitory effects of sucrose and NaCl were completely reversed when the tonicity of the perfusion medium was restored to its normal value (320 mosM), the effects of choline seemed to be long lasting and were reversed only partially. It is worth noting that the inhibitory effects of hyperosmotic solutions developed very fast. It is concluded that an increase in the tonicity of the medium bathing the chromaffin cell depresses profoundly the exocytotic catecholamine release response to nicotine and high K + these experiments suggest that the existence of an osmotic gradient across the plasma and or vesicular membranes might be a prerequisite for fusion of both membranes and or the subsequent extracellular discharge of chromaffin granule contents to the cell exterior.

O-111) - The stimulatory effect of forskolin on evoked catecholamine release from adrenal chromaffin cells

O-111) - The stimulatory effect of forskolin on evoked catecholamine release from adrenal chromaffin cells

The nature of the stimulatory action of gamma-aminobutyric acid in the isolated perfused dog adrenals.

The effect of gamma-aminobutyric acid (GABA) on catecholamine (CA) release from adrenal medulla was investigated. GABA and GABA agonists, 3-amino-1-propane-sulfonic acid and imidazole-4-acetic acid caused CA release from isolated perfused dog adrenals in a dose-dependent manner, and no tachyphylaxis to GABA was observed. CA release elicited by GABA was antagonized by bicuculline and picrotoxin. This antagonism was specific for GABA- and GABA agonist-induced responses, response to acetylcholine being unaffected. Pretreatment with atropine plus hexamethonium did not affect the response to GABA. GABA-induced CA release was abolished by the removal of Ca2+ from perfusion medium, but not by the removal of Na+ or Cl-. Verapamil, CoCl2 and dibucaine blocked the effect of GABA. A Na+ channel blocker, tetrodotoxin did not reduce GABA-evoked CA release. These results suggest that GABA may interact with its receptor to evoke CA release from adrenal medulla in a fashion of Ca2+-dependence and independence on external Na+ or Cl-.