Abbreviations used: CRE, cyclic AMP-response element; DBH, dopamine β-hydroxylase; DMEM/F12, Dulbecco's modified Eagle medium/nutrient mixture F12; DPBS, Dulbecco's phosphate-buffered saline; H-89, N-[2-(p-bromocinnamylamine)ethyl]-5-isoquinolinesulfonamide; PKA, protein kinase A; PNMT, phenylethanolamine N-methyltransferase; SSC, saline-sodium citrate; TH, tyrosine hydroxylase.
Abstract: Studies have shown that the cyclic AMP-regulated pathway is involved in the activation of tyrosine hydroxylase (TH) and in the induction of gene expression of the three catecholamine-synthesizing enzymes, TH, dopamine β-hydroxylase (DBH), and phenylethanolamine N-methyltransferase (PNMT). In the present study we investigated further the role of protein kinase A (PKA) in the regulation of both basal and cyclic AMP-inducible transcription of the three catecholamine-synthesizing enzymes in primary cultured bovine chromaffin cells by using the PKA-specific inhibitor N-[2-(p-bromocinnamylamine)ethyl]-5-isoquinolinesulfonamide (H-89). In the presence of 40 µM H-89, mRNA levels of TH, DBH, and PNMT were reduced to 17 ± 8, 19 ± 8, and 14 ± 2% of the untreated control, respectively, in 24 h, and intracellular norepinephrine and epinephrine levels were decreased to 20 and 34%, respectively, in 72 h. At 20 µM, although the basal enzyme gene expression levels were little affected, their induction by forskolin was abolished and norepinephrine and epinephrine levels fell to 55 and 74%. This reduction in catecholamines at 20 µM was probably due to changes in the phosphorylation state of TH, as its enzymatic activity was found to be decreased to 66 and 69% in 48 and 72 h, respectively. Thus, PKA activity in bovine adrenal medullary cells coordinately regulates both basal and cyclic AMP-inducible gene expression of specific catecholamine-synthesizing enzymes, resulting in changes in intracellular catecholamine levels available for consequent neurohormonal activities.