ADRENOCORTICOTROPIC HORMONE ACTIVATION OF ADENYLATE-CYCLASE IN RAPHE NEURONS - MULTIPLE REGULATORY PATHWAYS CONTROL SEROTONERGIC NEURONAL DIFFERENTIATION

The RN46A cell line was derived from embryonic day 13 rat medullary raphe cells by infection with a retrovirus encoding the temperature-sensitive mutant of SV40 large T antigen (tsT-ag). The RN46A cell line is neuronally restricted and constitutively differentiates following a shift to nonpermissive temperature, Differentiated RN46A cells express low levels of tryptophan hydroxylase (TPH) but no detectable levels of serotonin (5-HT). Treatment of cultures with the adrenocorticotropic hormone peptide ACTH(4-10) up-regulates the expression of TPH immunoreactivity in differentiated RN46A cells, but 5-HT synthesis requires initial treatment with ACTH(4-10), followed by partial membrane depolarizing conditions, Up-regulation of TPH by ACTH(4-10) is apparently due to activation of adenylate cyclase, whereas the increased 5-HT synthesis with membrane depolarization can be blocked with the voltage-sensitive Ca2+-channel blockers nifedipine and omega-conotoxin. ACTH(4-10) treatment also markedly up-regulates the expression of the 5-HT reuptake transporter, as do dibutyryl cyclic AMP and forskolin; chronic membrane depolarization has no effect on 5-HT reuptake. The expression of the high-affinity 5-HT1A receptor is increased threefold by ACTH(4-10) treatment during differentiation and fivefold by differentiation under partial membrane depolarizing conditions. Combining ACTH(4-10) treatment and membrane depolarization does not increase expression of the 5-HT1A receptor further. 5-HT release is constitutive in ACTH-treated RN46A cells and linked to spontaneous synaptic vesicle fusion in RN46A cells, Considered with previous results, these data indicate that multiple effecters, ACTH, brain-derived neurotrophic factor, and membrane depolarization, have both distinct and overlapping effects that regulate specific elements of the serotonergic neuronal phenotype during differentiation and maturation. (C) 1995 John Wiley & Sons, Inc.