Direct and Continuous Detection of ATP Secretion from Primary Monolayer Cultures of Bovine Adrenal Chromaffin Cells

Abstract

A method was developed for direct and continuous detection of secretion of ATP from primary monolayer cultures of bovine adrenal chromaffin cells. ATP, which is costored with catecholamines within adrenal chromaffin cells, was released into the incubation medium, where it reacted with firefly luciferin-luciferase producing light detected by a photomultiplier located directly below the culture well. Acetylcholine, nicotine, the Ca2+ ionophore A23187, BaCl2, and KCl induced release of ATP. Induction of release of ATP by acetylcholine was dose dependent, with a threshold at 10-7 M and a maximum at 10-4 M. The dose-response curve for nicotine was bell shaped, with a threshold at 10-7 M, a maximum at 10-5 M, and diminished release at higher concentrations, an observation indicative of desensitization. Investigation of the initial rates of ATP secretion revealed that 10-4 M nicotine actually inhibited rlease of ATP at a faster rate than 10-5 M nicotine. However, the rate of ATP release evoked by 10-4 M nicotine began to decline by 6 s, a result indicating the onset of receptor desensitizations, whereas release induced by 10-5 N nicotine continued unabated. Induction of release of ATP by acetylcholine or nicotine was biphasic, with a rapid, initial phase of release followed by a plateau at 0.5-1.5 min and a second phase of release beginning at 1.5-2 min, reaching a maximum by 2-3 min. Comparison of the time course for ATP release evoked by 10-4 M nicotine with the time course for secretion of adrenaline and noradrenaline revealed that adrenaline was the predominant catecholamine released during the initial rapid phase of ATP release. A slight plateau for adrenaline release coincided with the plateau for ATP release. Finally, the second phase of ATP release coincided with the time at which release of noradrenaline exceeded the release of adrenaline. The initial phase of ATP release could originate primarily from adrenaline-containing cells, whereas the second phase could occur primarily from noradrenaline-containing cells in the monolayer culture.