Adrenocorticotropic hormone activation of adenylate cyclase in raphe neurons: Multiple regulatory pathways control serotonergic neuronal differentiation

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Abstract
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 SV 40 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 adrenocorticotrophic hormone peptide ACTH4–10 up-regulates the expression of TPH immunoreactivity in differentiated RN46A cells, but 5-HT synthesis requires initial treatment with ACTH4–10, followed by partial membrane depolarizing conditions. Up-regulation of TPH by ACTH4–10 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 ω-conotoxin. ACTH4–10 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 ACTH4–10 treatment during differentiation and fivefold by differentiation under partial membrane depolarizing conditions. Combining ACTH4–10 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 effectors, 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. © 1995 John Wiley & Sons, Inc.