Witt-Enderby P A, Masana M I, Dubocovich M L
Department of Molecular Pharmacology and Biological Chemistry, Northwestern University Institute for Neuroscience, Northwestern University Medical School, Chicago, Illinois 60611, USA.
Endocrinology. 1998 Jul;139(7):3064-71. doi: 10.1210/endo.139.7.6102.
Here, we report the effects of short exposure to melatonin on the human mt1 (h mt1) melatonin receptor-mediated signaling in Chinese hamster ovary (CHO) cells, and the consequences of an exposure that resembles the physiological pattern of melatonin release on cAMP-mediated signal transduction. Short exposure (10 min) of h mt1 melatonin receptors to melatonin (400 pM) inhibited forskolin-stimulated cAMP formation, cAMP-dependent protein kinase activity, and phosphorylation of the cAMP response element-binding protein. However, treatment of mt1-CHO cells with melatonin in a manner that closely mimics the in vivo activation of melatonin receptors (i.e. 400 pM melatonin for 8 h to mimic darkness) resulted in a supersensitization of the cAMP-dependent signal transduction cascade during the period of withdrawal (i.e. 16 h without melatonin to mimic the light cycle of a diurnal photoperiod). During the period of withdrawal, forskolin induced a time-dependent (1-16 h) increase in cAMP formation (approximately 200% of control cells). This effect of melatonin was dependent on the presence of the h mt1 melatonin receptor, as no potentiation of forskolin-induced cAMP formation was observed in CHO cells transfected only with the neomycin resistance plasmid. The time-dependent increase in forskolin-stimulated cAMP levels resulted in a potentiation of cAMP-dependent protein kinase activity 1 h after withdrawal (approximately 130% of control cells; P < 0.05) and in the number of cells containing the phosphorylated form of cAMP response element-binding protein (approximately 75% of cells at 1 and 16 h compared with 30% in control cells; P < 0.05). An increase in the undissociated state (G alphabetagamma) of Gi proteins may underlie this phenomenon as demonstrated by the increase in pertussis toxin-catalyzed ADP-ribosylation of G proteins (217 +/- 48% of control; P < 0.05) after melatonin withdrawal. This increase in the ribosylation was not due to an up-regulation of Galpha(i) protein, as no significant change in Galpha(i) protein levels occurred at this time. We demonstrated that activation of the h mt1 melatonin receptor in a manner that resembles the physiological pattern of melatonin exposure alters signaling, as potentiation of cAMP-mediated signal transduction events is observed after hormone withdrawal. The CHO cells expressing the human melatonin receptor may provide an in vitro cellular model in which to investigate the putative signaling mechanisms leading to gene regulation by melatonin.
在此，我们报告了短时间暴露于褪黑素对中国仓鼠卵巢（CHO）细胞中人类褪黑素1型（h mt1）褪黑素受体介导的信号传导的影响，以及类似于褪黑素释放生理模式的暴露对cAMP介导的信号转导的影响。h mt1褪黑素受体短时间（10分钟）暴露于褪黑素（400 pM）可抑制福斯可林刺激的cAMP形成、cAMP依赖性蛋白激酶活性以及cAMP反应元件结合蛋白的磷酸化。然而，以紧密模拟褪黑素受体体内激活的方式（即400 pM褪黑素处理8小时以模拟黑暗）用褪黑素处理mt1-CHO细胞，在撤药期间（即16小时无褪黑素以模拟昼夜光周期的光循环）导致cAMP依赖性信号转导级联超敏化。在撤药期间，福斯可林诱导cAMP形成呈时间依赖性（1 - 16小时）增加（约为对照细胞的200%）。褪黑素的这种作用依赖于h mt1褪黑素受体的存在，因为在仅转染了新霉素抗性质粒的CHO细胞中未观察到福斯可林诱导的cAMP形成增强。福斯可林刺激的cAMP水平的时间依赖性增加导致撤药1小时后cAMP依赖性蛋白激酶活性增强（约为对照细胞的130%；P < 0.05）以及含有磷酸化形式的cAMP反应元件结合蛋白的细胞数量增加（1小时和16小时时约为细胞总数的75%，而对照细胞中为30%；P < 0.05）。Gi蛋白未解离状态（Gαβγ）的增加可能是这一现象的基础，如褪黑素撤药后百日咳毒素催化的G蛋白ADP核糖基化增加所示（为对照的217 ± 48%；P < 0.05）。这种核糖基化的增加并非由于Gα(i)蛋白上调，因为此时Gα(i)蛋白水平无显著变化。我们证明，以类似于褪黑素暴露生理模式的方式激活h mt1褪黑素受体可改变信号传导，因为在激素撤药后观察到cAMP介导的信号转导事件增强。表达人类褪黑素受体的CHO细胞可能提供了一个体外细胞模型，用于研究导致褪黑素对基因调控的假定信号传导机制。
