Experiments on perfused adrenal glands of guinea‐pigs were carried out to study the catecholamine output induced by veratridine in the presence of hexamethonium and atropine. Veratridine (10 μm to 200 μm) caused a dose‐dependent increase in catecholamine output. The addition of veratridine to the perfusion medium for a period of 3 min caused an increase in catecholamine output which reached a maximum 5 min to 10 min after withdrawal of the drug. The catecholamine output then gradually declined and reached near resting values within 30 minutes. It was never sustained for a longer period, even when veratridine was infused for 1 hour. Veratridine failed to increase the catecholamine output in the absence of extracellular Ca2+. However, the addition of Ca2+ after an infusion of veratridine (100 μm) in the absence of Ca2+ caused an increase in the catecholamine output which was proportional to the concentration of Ca2+ (0.55 mm to 8.8 him) used. Veratridine did not increase the catecholamine output in the absence of extracellular Na+ ions, NaCl being replaced by equimolar choline chloride or LiCl. Veratridine also failed to evoke catecholamine output in a Na+‐ffee solution in which Na+ was replaced by sucrose; this was the case even in the presence of a high concentration of Ca2+ (8.8 mm). Tetrodotoxin (0.1 μm) and excess Mg2+ (20 mm) reversibly inhibited the catecholamine output induced by veratridine. Ouabain (10 μm) significantly potentiated the veratridine‐induced catecholamine output. It is suggested that Na+‐dependent Ca2+ influx as well as voltage‐dependent Ca2+ influx mechanisms may be involved in the catecholamine output induced by veratridine.

Veratridine (0.1 mM) was found to be effective in producing an increase in the catecholamine output from perfused guinea‐pig adrenal glands in the presence of high concentrations of hexamethonium (1.83 mM) and atropine (28.8 μM). The response to veratridine was abolished by removal of either Na+ or Ca2+ from perfusion media and by the addition of tetrodotoxin (0.1 μM). It is suggested that the response to veratridine may be due to an increase in the tetrodotoxin‐sensitive Na+ permeability of chromaffin cell membranes.

SUMMARY1. Experiments were carried out to investigate the time course of the release of catecholamine, dopamine-,8-hydroxylase (DBH) and adenine nucleotides from isolated chromaffin cells of guinea-pig adrenal gland.2. When the isolated chromaffin cells were incubated with medium containing acetylcholine (ACh) (0-1 mM), veratridine (0-1 mM) or scorpion (Leiurus quinquestriatus) venom, (10 ,ug/ml.), catecholamine was released into the medium. Catecholamine secretion induced by veratridine or scorpion venom was inhibited by tetrodotoxin (1 gM) but not by atropine (0-1 mM) plus hexamethonium (041 mM). On the other hand, the secretary response to ACh was abolished by the cholinergic blocking drugs but not by tetrodotoxin.3. DBH was released together with catecholamine into the medium in which cells were suspended with these drugs. The ratio of catecholamine (n-mole) to DBH activity (n-mole/hr) appearing in the supernatant was 7-08 + 055, 6-60 + 0X27 and 8X91 + 0 47 for ACh, veratridine and scorpion venom, respectively. These values were close to that found in the lysate of chromaffin granules obtained from guinea-pig adrenal glands (7-37 + 0-39).4. The application of ACh or veratridine to perifused chromaffin cells was found to cause a parallel increase in catecholamine and DBH secretion in the perifusion medium without corresponding amounts of phenylethanolamine-N-methyltransferase leakage. However, DBH secretion tended to last for a longer period than catecholamine secretion.5. Adenine nucleotides were released from perifused chromaffin cells together with catecholamine, by ACh and veratridine. ATP added to the perifusion medium was metabolized to ADP and AMP, of which the ratio (ATP, 21-6 %; ADP, 34 %; AMP, 17-9 %) was close to those of adenine nucleotides released from the cells.6. The secretion ofadenine nucleotides induced by both secretagogues ceased much faster than the catecholamine secretion, so that the molar ratio of catecholamine to adenine nucleotides was gradually increased during and after stimulation. 7. The results indicate that catecholamine secretion is accompanied with a simultaneous release of DBH and ATP from adrenal chromaffin cells. Therefore, it is suggested that the delayed output of DBH, unlike catecholamine secretion, in perfused adrenal glands results from the presence of a diffusion barrier for this protein. The releasable secretary granules of isolated chromaffin cells are suggested to be heterogeneous with respect to the ratio of catecholamine to ATP.

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