Effects of ascorbic acid, dexamethasone, and insulin on the catecholamine and opioid peptide stores of cultured adrenal medullary chromaffin cells

Abstract

Bovine adrenal medullary chromaffin cells cultured in serum-free medium were examined for changes in their catecholamine and opioid peptide stores following exposure to dexamethasone, ascorbic acid, or insulin for 2 to 12 days. Dexamethasone failed to alter cellular catecholamine levels, measured by high performance liquid chromatography with electrochemical detection, or cellular opioid peptide content, measured by an enkephalin radioreceptor assay. Chromaffin cells cultured in medium supplemented with ascorbic acid retained high ascorbate contents for 2 to 3 days, despite the rapid loss of this vitamin from the culture medium (approximately 50% lost in 2 hr). The epinephrine and norepinephrine contents of chromaffin cell cultures supplemented with ascorbate for 7 days were increased approximately 10% compared to cultures without added ascorbic acid; ascorbate had no effect on chromaffin cell opioid peptide levels. Addition of insulin to chromaffin cell cultures produced a doubling of cellular protein and opioid peptide levels by 6 days and produced a concentration-dependent increase in the dopamine and norepinephrine contents of the cells with only a slight elevation in cell epinephrine. Chromaffin cells were also tested for the ability to resynthesize their catecholamine and opioid peptide stores following depletion as the result of secretion evoked by acetylcholine or nicotine. The cellular stores of norepinephrine and dopamine were resynthesized within 3 days following secretagogue-evoked depletion. Epinephrine stores were incompletely replenished with only 20% of the epinephrine lost via secretion recovered in 7 days. Opioid peptide levels were maximally recovered (50% of the amount secreted) within 1 day. Addition of ascorbic acid, dexamethasone, or insulin to the medium failed to enhance the recovery of catecholamine stores.