Pratt B L, Takahashi J S
Department of Neurobiology and Physiology, Northwestern University, Evanston, Illinois 60201.
J Neurosci. 1987 Nov;7(11):3665-74. doi: 10.1523/JNEUROSCI.07-11-03665.1987.
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.
雏鸡松果体表达褪黑素生物合成的昼夜节律，夜间水平升高，白天水平降低。褪黑素的节律受松果体自身的昼夜振荡器以及交感神经系统的肾上腺素能输入调节。先前的研究表明，通过血清素N-乙酰转移酶的活性测定，给予去甲肾上腺素会抑制褪黑素的生物合成。作为了解去甲肾上腺素调节雏鸡松果体褪黑素产生机制的第一步，我们确定了所涉及的肾上腺素能受体。制备了分离的雏鸡松果体细胞培养物，并在培养的第5天和第6天使用放射免疫测定法测量褪黑素释放。确定了肾上腺素能激动剂和拮抗剂对LD12:12光周期12小时黑暗部分期间褪黑素释放夜间增加的影响。去甲肾上腺素以剂量依赖性方式抑制褪黑素释放，平均EC50为19.7 nM±2.23（SEM）。根据去甲肾上腺素的剂量，褪黑素释放值范围为对照培养物中所见水平的100%至4%。对肾上腺素、去甲肾上腺素和异丙肾上腺素的生理反应具有立体特异性。（-）立体异构体的效力分别比（+）立体异构体高6、8和37倍。肾上腺素能激动剂的EC50值（以nM为单位）如下：α-甲基-（-）-去甲肾上腺素，2.46；曲马唑啉，3.06；胍那苄，3.31；可乐定，3.70；羟甲唑啉，4.29；（-）-肾上腺素，7.44；（-）-去甲肾上腺素，19.7；（-）-异丙肾上腺素，463；和（-）-去氧肾上腺素，659。使用Schild分析来确定肾上腺素能拮抗剂的相对效力。肾上腺素能拮抗剂的pA2值如下：萝芙辛，9.55；RX78 1094，8.32；育亨宾，8.14；酚妥拉明，7.11；哌唑嗪，5.93；和（-）-普萘洛尔，小于6。肾上腺素能激动剂和拮抗剂的相对效力均表明，α-2受体介导去甲肾上腺素诱导的雏鸡松果体细胞培养物中褪黑素释放的抑制。在雏鸡松果体细胞中鉴定α-2受体应有助于我们理解由受体激活引发的调节褪黑素合成的生化事件。
