Real‐Time Monitoring of the Secretory Function of Cultured Adrenal Chromaffin Cells

Abstract

A system to discriminate the real‐time dynamics of the secretory function in cultured adrenal chromaffin cells, using a cell bed perfusion technique and an amperometric detector, was established. Examination of basal conditions revealed that the electrode potential and flow rate are crucial factors for monitoring precise dynamics of the secretory process. Stimulation of the cells either with acetylcholine (ACh) or with high K* concentration caused a transient current response. The current responses showed concentration dependence for both stimuli, and also showed a high correlation with the amount of catecholamines (CA) in the respective peak fraction of perfusate. Either prolonged cholinergic stimulation or maintained depolarization produced a transient response, which is not attributable to a depletion of releasable storage of CA as indicated by double‐stimulation experiments. Stimulation with high K* concentration evoked an additional release of CA even after the cellular response to prolonged ACh was inactivated, whereas maintained depolarization with high K* produced both facilitatory and inhibitory effects on the cell responsiveness to ACh. Most probably the transient natures of the secretory responses to ACh and to high K* are mediated by different mechanisms. All the results suggest that the direct monitoring is profitable for studies on the regulatory mechanisms of the secretory function.

Characterization of Exocytotic Events From Single PC12 Cells: Amperometric Studies in Native PC12h, DA-Loaded PC12h and Bovine Adrenal Chromaffin Cells

“…The cells were cultured in DMEM media supplemented with 10% heat-inactivated horse serum, 5% fetal bovine serum and 50 units each of penicillin and streptomycin in 5% CO 2 atmosphere at 37 • C. The experiments were performed 4-6 days after plating. Bovine adrenal chromaffin cells were isolated from fresh adrenal glands by collagenase digestion as previously described (Kumakura et al, 1986;Ohara-Imaizumi et al, 1992) and cultured on a colagen-coated glassbase dishes (35 mm diameter). All the amperometric experiments were performed with the cells in culture for 5-7 days.…”