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

We investigated whether vasoactive intestinal peptide (VIP) and pituit ary adenylate cyclase-activating polypeptide (PACAP), which stimulate melatonin biosynthesis in the mammalian pineal organ, cause phosphoryl ation of the cyclic AMP response element binding protein (CREB) in rat pinealocytes, Dispersed cells were treated with varying concentration s of VIP and PACAP for 10 to up to 240 min and then immunocytochemical ly analyzed with an antibody against phosphorylated CREB (pCREB). The experiments showed a dose- and time-dependent induction of pCREB immun oreactivity 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 s maller 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 contr ast, norepinephrine (NE) elicited pCREB immunoreaction in more than 95 % of the pinealocytes, and this response lasted as long as 300 min. Tr eatment of pinealocytes with forskolin or KCl induced pCREB immunoreac tion in the vast majority of pinealocytes, showing that in principle e levation of the intracellular concentrations of both cAMP and calcium can cause the response. Immunoblotting analyses confirmed that the imm unoreaction elicited by VIP, PACAP and NE is largely due to phosphoryl ation of a 42-kDa protein corresponding to CREB, but reflects to a min or extent also phosphorylation of two smaller proteins presumably rela ted to ATF-1. Immunocytochemical and immunochemical investigations wit h an antibody against total CREB showed that stimulation with VIP, PAC AP 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 a lso true for NE. However, VIP and PACAP affected only subpopulations o f pinealocytes and the reponses lasted for a shorter period of time th an those to NE. This conforms to previous results showing that both ne uropeptides are also less effective than NE in stimulating the melaton in biosynthesis in the rat pineal organ.