Vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) induce phosphorylation of the transcription factor CREB in subpopulations of rat pinealocytes: immunocytochemical and immunochemical evidence

Abstract.

We investigated whether vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP), which stimulate melatonin biosynthesis in the mammalian pineal organ, cause phosphorylation of the cyclic AMP response element binding protein (CREB) in rat pinealocytes. Dispersed cells were treated with varying concentrations of VIP and PACAP for 10 to up to 240 min and then immunocytochemically analyzed with an antibody against phosphorylated CREB (pCREB). The experiments showed a dose- and time-dependent induction of pCREB immunoreactivity in the nuclei of subpopulations of pinealocytes identified by the S-antigen immunoreaction. Stimulation with VIP elicited pCREB immunoreaction in approximately 50–65% of the S-antigen immunoreactive pinealocytes. The number of PACAP-responsive pinealocytes was often smaller and more variable. Maximal responses to both neuropeptides were seen after 30 min. pCREB immunoreaction gradually declined within 2 h and could not be induced again by an additional stimulation. In contrast, norepinephrine (NE) elicited pCREB immunoreaction in more than 95% of the pinealocytes, and this response lasted as long as 300 min. Treatment of pinealocytes with forskolin or KCl induced pCREB immunoreaction in the vast majority of pinealocytes, showing that in principle elevation of the intracellular concentrations of both cAMP and calcium can cause the response. Immunoblotting analyses confirmed that the immunoreaction elicited by VIP, PACAP and NE is largely due to phosphorylation of a 42-kDa protein corresponding to CREB, but reflects to a minor extent also phosphorylation of two smaller proteins presumably related to ATF-1. Immunocytochemical and immunochemical investigations with an antibody against total CREB showed that stimulation with VIP, PACAP and NE did not affect the level of CREB. All findings indicate that the stimulatory effects of VIP and PACAP on rat pinealocytes involve phosphorylation of transcription factors of the CREB family as holds also true for NE. However, VIP and PACAP affected only subpopulations of pinealocytes and the reponses lasted for a shorter period of time than those to NE. This conforms to previous results showing that both neuropeptides are also less effective than NE in stimulating the melatonin biosynthesis in the rat pineal organ.