Time course of release of catecholamine and other granular contents from perifused adrenal chromaffin cells of guinea-pig.

Experiments were carried out to investigate the time course of the release of catecholamine, dopamine-beta-hydroxylase (DBH) and adenine nucleotides from isolated chromaffin cells of guinea-pig adrenal gland. 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 micrograms/ml.), catecholamine was released into the medium. Catecholamine secretion induced by veratridine or scorpion venom was inhibited by tetrodotoxin (1 microM) but not by atropine (0.1 mM) plus hexamethonium (0.1 mM). On the other hand, the secretory response to ACh was abolished by the cholinergic blocking drugs but not by tetrodotoxin. 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 +/- 0.55, 6.60 +/- 0.27 and 8.91 +/- 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). 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. 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. The secretion of adenine nucleotides induced by both secretagogues ceased much faster than the catecholamine secretion, so that molar ratio of catecholamine to adenine nucleotides was gradually increased during and after stimulation. 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 secretory granules of isolated chromaffin cells are suggested to be heterogeneous with respect to the ratio of catecholamine to ATP.