NOREPINEPHRINE STIMULATION OF PINEAL CYCLIC-AMP RESPONSE ELEMENT-BINDING PROTEIN-PHOSPHORYLATION - PRIMARY ROLE OF A BETA-ADRENERGIC-RECEPTOR CYCLIC-AMP MECHANISM

Norepinephrine (NE) regulates melatonin production and many other aspe cts of pineal function through actions involving cAMP. In the present study the effects of NE on the phosphorylation of the cAMP response el ement-binding protein (CREB) were studied to determine whether CREB ph osphorylation might be involved in cAMP signal transduction in this ti ssue. CREB was detected using gel mobility-shift analysis with the rad iolabeled Ca2+/cAMP response element of the c-fos promoter. CREB phosp horylation was estimated in the gel mobility-shift assay using an anti serum specific for phosphorylated CREB. This antiserum generates a sup ershifted CREB signal with protein extracts obtained from glands treat ed with NE (EC(50) congruent to 10 nM) in organ culture, demonstrating that NE stimulates CREB phosphorylation. CREB phosphorylation peaks 3 0-45 min after NE treatment is initiated and then gradually returns to base-line values. Pharmacological studies show that NE-stimulated CRE B phosphorylation is mediated primarily through beta(1)-adrenergic rec eptor-stimulated increases in cAMP. Activation of alpha(1)-adrenergic receptors, which is known to elevate the intracellular free Ca2+ conce ntration, does not cause CREB phosphorylation. However, it is possible to produce CREB phosphorylation with certain pharmacological agents t hat elevate the intracellular free Ca2+ concentration. In vivo studies show that CREB phosphorylation can be induced by treatment with isopr oterenol (1 mg/kg), demonstrating that phosphorylation of pineal CREB occurs in intact animals. These studies indicate that cAMP-dependent C REB phosphorylation could play a role in the adrenergic regulation of gene expression in pinealocytes.