CONTROL OF CREB PHOSPHORYLATION AND ITS ROLE FOR INDUCTION OF MELATONIN SYNTHESIS IN RAT PINEALOCYTES

Phosphorylation of the transcription factor CREB appears as an importa nt step in the signal transduction cascade that activates melatonin bi osynthesis in the mammalian pineal organ. We have studied the mechanis ms causing CREB phosphorylation by immunocytochemical and immunochemic al demonstration of phosphorylated CREB (pCREB) in isolated, immunocyt ochemically identified rat pinealocytes kept in vitro and in the rat p ineal organ in situ. Norepinephrine (NE), the most potent stimulator o f the melatonin biosynthesis was shown to induce pCREB immunoreaction (ir) in the vast majority of pinealocytes in a time- and dose-dependen t manner. This response was elicited by stimulation of beta-adrenergic receptors resulting in an increase in the intracellular cAMP concentr ation. Activation of alpha(1)-adrenergic receptors that causes a rise in intracellular calcium via stimulation of intracellular stores and s ubsequent increase in calcium influx did not evoke pCREB ir on its own and did not potentiate the beta-adrenergic response. VIP and PACAP th at activate the melatonin biosynthesis to a lesser extent than NE indu ced pCREB ir in only 50-60% of the pinealocytes. Immunoblotting showed that a protein of 43 kDa corresponding to CREB accounts for the pCREB ir and confirmed that VIP and PACAP are less effective in inducing CR EB phosphorylation than NE. The amount of total (phosphorylated and un phosphorylated) CREB was not changed upon stimulation of the cells wit h NE, VIP or PAC:AP. In an attempt to identify the protein kinase cata lyzing CREB phosphorylation in rat pinealocytes, the cAMP-dependent pr otein kinases (cAK) present in the rat pineal were identified with the use of antibodies recognizing different catalytic and regulatory subu nits. Application of cAK agonists and antagonists showed that the cAK type II is responsible for CREB phosphorylation. Correlations between the melatonin concentration in the medium and the CREB phosphorylation in pinealocytes revealed a tight connection between these two paramet ers. Phosphorylation of CREB appears important for the stimulation of melatonin biosynthesis also under natural conditions because our inves tigations of whole pineal organs taken from rats during different time points of the 24 h light-dark cycle revealed a strong induction of pC REB ir in the first part of the night. ((C) Elsevier, Paris).