NICOTINIC CHOLINERGIC REGULATION OF TYROSINE-HYDROXYLASE GENE-EXPRESSION AND CATECHOLAMINE SYNTHESIS IN ISOLATED BOVINE ADRENAL CHROMAFFIN CELLS

Isolated bovine adrenal chromaffin cells were used to study the nicotinic regulation of tyrosine hydroxylase (TH) gene expression. Continuous exposure of the cells to carbachol or the nicotinic receptor agonist 1,1-dimethyl-4-phenylpiperazinium (DMPP) produces a time- and concentration-dependent increase in TH enzyme activity, whereas muscarine has no effect. DMPP at 1 muM (EC50 = 0.3 muM) elicits a two- to threefold elevation of both TH activity and TH immunoreactive protein level after 3-5 days in the presence of 2.5 mM calcium; the increase in enzyme levels is significantly less at lower extracellular calcium levels. The rate of hydroxylation of tyrosine to dopamine (DA) in intact cells, an index of endogenous TH activity, increases in parallel with the rise in TH levels. The TH mRNA level is elevated before the increase in protein levels. As determined by nuclear run-on assays, TH gene transcription is stimulated two- to threefold within 30 min of addition of 1 muM DMPP to the cells; transcription returns to basal levels by 2 h. Nitrendipine (20 muM) blocks the stimulation of transcription by DMPP. Pretreatment of the cells with cycloheximide (5 muM) does not prevent the DMPP stimulation of transcription. Forskolin (10 muM) also increases TH transcription (fourfold in 15 min) by a mechanism that is not blocked by cycloheximide. These results show that nicotinic receptor stimulation increases TH mRNA synthesis, TH protein levels, and TH activity in a calcium-dependent manner. Furthermore, the nicotinic influence on TH gene expression does not appear to require the synthesis of a protein factor for its effects. That in situ DA synthesis rates are elevated consequent to the rise in TH levels demonstrates that TH induction serves as a mechanism for enhancing the catecholamine-synthesizing capacity of the chromaffin cell on a long-term basis.