Alpha-2 adrenergic regulation of melatonin release in chick pineal cell cultures

The chick pineal gland expresses a circadian rhythm of melatonin biosynthesis, with elevated levels at night and low levels during the day. The rhythm of melatonin is regulated both by circadian oscillators located within the gland itself and by adrenergic input from the sympathetic nervous system. Previous work has shown that norepinephrine administration inhibits melatonin biosynthesis, as measured by the activity of the enzyme serotonin N-acetyltransferase. As a first step toward understanding the mechanisms by which norepinephrine regulates melatonin production in the chick pineal, we have identified the adrenergic receptor involved. Dissociated chick pineal cell cultures were prepared and melatonin release was measured on days 5 and 6 of culture using radioimmunoassay. The effects of adrenergic agonists and antagonists on the nocturnal increase of melatonin release during the 12 hr dark portion of a LD12:12 light cycle were determined. Norepinephrine inhibited melatonin release in a dose-dependent manner, with an average EC50 of 19.7 nM +/- 2.23 (SEM). Melatonin release values ranged from 100 to 4% of the level seen in control cultures, depending on the dose of norepinephrine. The physiological response to epinephrine, norepinephrine, and isoproterenol was stereospecific. The (-) stereoisomer was 6, 8, and 37 times more potent than the (+) stereoisomer, respectively. EC50 values (in nM) for adrenergic agonists were as follows: alpha-methyl-(-)-norepinephrine, 2.46; tramazoline, 3.06; guanabenz, 3.31; clonidine, 3.70; oxymetazoline, 4.29; (-)-epinephrine, 7.44; (-)-norepinephrine, 19.7; (-)-isoproterenol, 463; and (-)-phenylephrine, 659. Schild analysis was used to determine the relative potency of adrenergic antagonists. pA2 values for adrenergic antagonists were as follows: rauwolscine, 9.55; RX78 1094, 8.32; yohimbine, 8.14; phentolamine, 7.11; prazosin, 5.93; and (-)-propranolol, less than 6. The relative potencies of both adrenergic agonists and antagonists demonstrate that alpha-2 receptors mediate norepinephrine-induced inhibition of melatonin release in chick pineal cell cultures. The identification of alpha-2 receptors in chick pineal cells should aid in our understanding of the biochemical events initiated by receptor activation that regulate melatonin synthesis.