NEURAL CONTROL OF DOPAMINE-BETA-HYDROXYLASE IN-VIVO - ACUTE AND CHRONIC EFFECTS

Reserpine treatment was used to examine whether short- and long-term neural stimulation regulates rat adrenal medullary dopamine beta-hydroxylase (DBH, EC 1.14.17.1) through transcriptional activation and to examine the extent of coordinate control of DBH and phenylethanolamine N-methyltransferase (PNMT, EC 2.1.1.28). A single dose of reserpine (10 mg/kg i.p.) elevates DBH mRNA 1.5-fold by 6 h post-injection. Chronic treatment (10 mg/kg i.p., 4 alternate day injections) continues the rise in DBH mRNA, with a peak of 3.4-fold control values after 2 doses of reserpine and a plateau at this level thereafter. Even though DBH mRNA is elevated 6 h after a single injection of reserpine, DBH activity does not change in parallel. A 1.3-fold rise in activity occurs at 24 h post-injection. In contrast, chronic reserpine treatment concommitantly increases DBH activity and mRNA. As observed for DBH mRNA, enzyme activity peaks and plateaus after 2 injections on alternate days. However, the rise in enzymatic activity is less than the rise in mRNA (2,4-fold versus 3.4-fold). Ribosomal loading experiments demonstrate that the DBH mRNA pool is fully utilized for protein synthesis with an apparent decrease in the number of ribosomes loaded per molecule of mRNA. Western analysis and thermal denaturation studies indicate that an altered form of DBH may be expressed. With a single dose of reserpine, the enzyme shows a decline in specific activity while repeated treatment leads to an enzyme with higher specific activity. In both cases, the protein appears to be more stable. Reserpine treatment also markedly elevates adrenal glucocorticoids. A 1.5-fold increment in glucocorticoid receptor mRNA accompanies the corticosteroid rise, with the receptor mRNA peaking at 6 h and remaining at this level thereafter. The up-regulation of glucocorticoid receptor mRNA expression, together with the presence of a putative glucocorticoid response element in the 5' flanking region of the DBH gene, suggests that neural and hormonal regulatory mechanisms may work in concert to control DBH gene transcription. Finally, by comparison to PNMT, activation of DBH appears to require sustained stimulation of the neural axis, since acute changes in mRNA lead to only minor changes in enzyme expression. Similar to PNMT, continuous neural stimulation increases both DBH mRNA and enzymatic activity. However, the discordance in the magnitude of these indices suggests that other regulatory controls may be important in setting the ultimate limits on DBH expression, glucocorticoids perhaps being one such influence.