Norepinephrine-dependent phosphorylation of the transcription factor cyclic adenosine monophosphate responsive element-binding protein in bovine pinealocytes.

Norepinephrine (NE)-dependent activation of transcription factors is of central importance for the rhythmic production of melatonin in the rodent pineal gland. At variance with rodents, NE regulates melatonin biosynthesis through post-translational mechanisms in ungulates, and it is not yet known whether transcription factors play any role in ungulate pineal functions. Here, we investigated in isolated bovine pinealocytes the NE-dependent phosphorylation of the transcription factor cyclic adenosine monophosphate (cAMP) responsive element-binding protein (CREB) and compared the effects of NE with those of vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP). Treatment with 10(-7) m NE for 30 min induced a strong nuclear phosphorylated CREB (pCREB) immunoreaction in cells that were identified as pinealocytes by immunocytochemical demonstration of serotonin, a pinealocyte-specific marker. Immunoblots showed that the NE-induced immunoreaction was due to phosphorylation of the transcription factor CREB and another protein, presumably the activating transcription factor 1 (ATF-1). 10(-7) m isoproterenol (ISO) or 10(-5) m forskolin mimicked the response to NE indicating that NE acts through the beta-adrenergic/cAMP pathway. Also 10(-7) m PACAP, but not 10(-7) m VIP-enhanced CREB phosphorylation; however, only a subpopulation of cells was responsive to PACAP. Our results suggest that, irrespective of whether or not melatonin production is controlled via transcriptional mechanisms, NE-induced CREB phosphorylation represents a very conserved element in pineal physiology of mammals because NE increases pCREB levels in all mammalian species investigated so far. However, the genes targeted by pCREB may vary from one mammalian species to the other. Our results also suggest that transcription factors other than pCREB, like ATF-1, may play a role in pineal functions of mammals.