Control of CREB phosphorylation and its role for induction of melatonin synthesis in rat pinealocytes

Phosphorylation of the transcription factor CREB appears as an important step in the signal transduction cascade that activates melatonin biosynthesis in the mammalian pineal organ. We have studied the mechanisms causing CREB phosphorylation by immunocytochemical and immunochemical demonstration of phosphorylated CREB (pCREB) in isolated, immunocytochemically identified rat pinealocytes kept in vitro and in the rat pineal organ in situ. Norepinephrine (NE), the most potent stimulator of the melatonin biosynthesis was shown to induce pCREB immunoreaction (i.r.) in the vast majority of pinealocytes in a time- and dose-dependent manner. This response was elicited by stimulation of beta-adrenergic receptors resulting in an increase in the intracellular cAMP concentration. Activation of alpha 1-adrenergic receptors that causes a rise in intracellular calcium via stimulation of intracellular stores and subsequent increase in calcium influx did not evoke pCREB ir on its own and did not potentiate the beta-adrenergic response. VIP and PACAP that activate the melatonin biosynthesis to a lesser extent than NE induced 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 CREB phosphorylation than NE. The amount of total (phosphorylated and unphosphorylated) CREB was not changed upon stimulation of the cells with NE, VIP or PACAP. In an attempt to identify the protein kinase catalyzing CREB phosphorylation in rat pinealocytes, the cAMP-dependent protein kinases (cAK) present in the rat pineal were identified with the use of antibodies recognizing different catalytic and regulatory subunits. 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 parameters. Phosphorylation of CREB appears important for the stimulation of melatonin biosynthesis also under natural conditions because our investigations of whole pineal organs taken from rats during different time points of the 24 h light-dark cycle revealed a strong induction of pCREB ir in the first part of the night.