Glucocorticoid receptors and regulation of phenylethanolamine-N-methyltransferase activity in cultured chromaffin cells

Abstract

Glucocorticoids are known to regulate the enzyme phenylethanolamine-N-methyltransferase (PNMT) in the adrenal medulla of the rat and are thereby thought to control the synthesis of epinephrine. We have examined the details of this relationship in a simplified system, chromaffin cell primary cultures derived from bovine adrenal medulla. Cultured chromaffin cells were found to have a cytosolic, high affinity, saturable glucocorticoid-binding protein with the steroid specificity of a classical glucocorticoid receptor and a K[d] of ~ 1 nM. Treatment of cultured cells with dexamethasone or hydrocortisone at any time up to 21 days in culture increased PNMT activity in the soluble fraction of the cell. The concentration of hormone required to produce a half-maximal response was 10 nM dexamethasone when cells were cultured in the presence of 5% fetal calf serum, or 1 nM in a defined serum-free medium. These dose-response relationships are consistent with mediation of this effect by the glucocorticoid receptor. Unexpectedly, however, the glucocorticoid-induced increment in PNMT activity was not inhibited by cycloheximide at concentrations up to 50 μM, and an acceleration of protein synthesis by insulin treatment did not augment the glucocorticoid effect on PNMT. Treatment of the cells with dexamethasone (100 μM) prevented the decline in the epinephrine-to-norepinephrine ratio seen over time in culture, an effect consistent with increased PNMT activity. However, there was no effect of dexamethasone on the ability of the cells to secrete catecholamines in response to stimulation with high KCl or 30 μM nicotine. We interpret these data to suggest that PNMT activity and, thus, epinephrine biosynthesis can be controlled in cultured chromaffin cells by a mechanism which is probably tied to the glucocorticoid receptor detected in these cultures. Furthermore, these primary cultures provide a model for detailed biochemical analysis of steroid regulation of catecholamine metabolism.