Stimuli which entrain the circadian clock of the neonatal Syrian hamster in vivo regulate the phosphorylation of the transcription factor CREB in the suprachiasmatic nucleus in vitro
Stimuli which entrain the circadian clock of the neonatal Syrian hamster in vivo regulate the phosphorylation of the transcription factor CREB in the suprachiasmatic nucleus in vitro
复制标题
体内诱导新生叙利亚仓鼠生物钟的刺激在体外调节视交叉上核转录因子 CREB 的磷酸化
DOI：
10.1046/j.1460-9568.1998.00114.x
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发表时间：
1998
影响因子：
3.4
通讯作者：
M. Hastings
中科院分区：
文献类型：
--
作者：
S. Mcnulty;I. Schurov;P. Sloper;M. Hastings
体内成年哺乳动物昼夜节律钟的光重置与视交叉上核（SCN）视网膜受体区钙/环AMP反应元件结合蛋白（CREB）的Ser 133残基磷酸化相关。蛋白质印迹和免疫细胞化学被用来调查是否激动剂已知重置的新生仓鼠在体内的时钟也能够影响CREB的磷酸化在视交叉上的下丘脑在体外。针对合成CREB肽序列产生的抗血清用于区分总CREB和CREB的Ser 133磷酸化形式（pCREB）。从1日龄叙利亚仓鼠的视交叉上组织中分离的蛋白质的Western印迹分析显示了与总CREB和pCREB对应的约45 kDa的条带。用谷氨酸能激动剂[N-甲基-d-天冬氨酸（NMDA）、氨基甲基丙酸（AMPA）和红藻氨酸盐，均为1 μm]或天然谷氨酸盐（1 μm）的混合物处理组织对总CREB信号没有影响，但增加了pCREB信号，表明激动剂刺激的CREB在Ser 133上的磷酸化。用多巴胺（1 μm）或毛喉素（1 μm）治疗视交叉上阻滞后观察到类似的效果。同时用褪黑激素（1 μm）处理显著减弱了毛喉素的刺激。在含有体内视交叉上核特征性细胞类型混合物的原代培养物中研究了激动剂对核pCREB免疫反应性（-ir）的影响。核总CREB‐ir的基础表达高，而pCREB‐ir的表达低。用谷氨酸（1 μm）或多巴胺（1 μm）处理对总CREB‐ir没有影响，但分别增加了约50%和30%的细胞中的pCREB‐ir，而毛喉素（1 μm）增加了几乎所有细胞中的pCREB‐ir（> 90%）。所有三种激动剂的作用都是快速的（< 15分钟），并且具有剂量和时间依赖性。褪黑激素逆转了毛喉素在混合培养物中的作用，但在纯星形胶质细胞培养物中没有。双重免疫细胞化学（ICC）显示，谷氨酸（1 μm）增加了微管相关蛋白II（MAP II-ir）免疫反应性细胞的核pCREB-ir，但不增加其他细胞的pCREB-ir，表明主要对神经元产生影响。这同样发生在γ-氨基丁酸（GABA）-ir和非GABA-ir神经元中。多巴胺（1 μm）更具选择性，仅增加GABA-ir神经元中的pCREB-ir，而毛喉素增加所有细胞中的pCREB-ir。多巴胺对GABA-ir神经元中pCREB-ir的特异性刺激被褪黑素逆转，但褪黑素对谷氨酸诱导的GABA-ir神经元中pCREB-ir的增加没有影响。这些结果表明，已知在体内携带生物钟的激动剂调节来自新生儿视交叉上核的GABA-ir神经元中CREB的磷酸化。
Photic resetting of the adult mammalian circadian clock in vivo is associated with phosphorylation of the Ser133 residue of the calcium/cyclic AMP response‐element binding‐protein (CREB) in the retinorecipient region of the suprachiasmatic nucleus (SCN). Western blotting and immunocytochemistry were used to investigate whether agonists known to reset the clock of neonatal hamsters in vivo are also able to influence the phosphorylation of CREB in the suprachiasmatic hypothalamus in vitro. Antisera raised against synthetic CREB peptide sequences were used to differentiate between total CREB and the Ser133 phosphorylated form of CREB (pCREB). Western blot analysis of proteins isolated from suprachiasmatic tissue of 1‐day‐old Syrian hamsters revealed bands at ≈ 45 kDa corresponding to total CREB and pCREB. Treatment of the tissue with a mixture of glutamatergic agonists [N‐methyl‐d‐aspartate (NMDA), amino‐methyl proprionic acid (AMPA) and kainate, all at 1 μm], or native glutamate (1 μm) had no effect on the total CREB signal, but increased the pCREB signal, indicative of agonist‐stimulated phosphorylation of CREB on Ser133. A similar effect was seen following treatment of the suprachiasmatic blocks with either dopamine (1 μm) or forskolin (1 μm). Simultaneous treatment with melatonin (1 μm) significantly attenuated stimulation by forskolin. The effect of the agonists on nuclear pCREB‐immunoreactivity (‐ir) was investigated in primary cultures which contained a mixture of cell types characteristic of the suprachiasmatic nuclei in vivo. Basal expression of nuclear total CREB‐ir was high, whereas expression of pCREB‐ir was low. Treatment with glutamate (1 μm) or dopamine (1 μm) had no effect on total CREB‐ir, but increased pCREB‐ir in ≈ 50 and 30% of cells, respectively, whereas forskolin (1 μm) increased pCREB‐ir in almost all cells (> 90%). The effects of all three agonists were rapid (< 15 min), and dose and time dependent. Melatonin reversed the effects of forskolin in mixed cultures, but not in pure astrocyte cultures. Dual‐immunocytochemistry (ICC) revealed that glutamate (1 μm) increased nuclear pCREB‐ir in cells immunoreactive for microtubule‐associated protein II (MAP II‐ir), but not other cells, indicating an effect predominantly on neurons. This occurred equally in γ‐amino butyric acid (GABA)‐ir and non‐GABA‐ir neurons. Dopamine (1 μm) was more selective, increasing pCREB‐ir only in GABA‐ir neurons, whereas forskolin increased pCREB‐ir in all cells. The specific stimulation of pCREB‐ir in GABA‐ir neurons by dopamine was reversed by melatonin, but melatonin had no effect on the increase in pCREB‐ir induced in GABA‐ir neurons by glutamate. These results demonstrate that agonists known to entrain the circadian clock in vivo modulate phosphorylation of CREB in GABA‐ir neurons derived from the neonatal suprachiasmatic nuclei.
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影响因子：
56.9
作者：
Crosthwaite, SK;Dunlap, JC;Loros, JJ
通讯作者：
Loros, JJ
影响因子：
56.9
作者：
GINTY, DD;KORNHAUSER, JM;GREENBERG, ME
通讯作者：
GREENBERG, ME
影响因子：
56.9
作者：
DING, JM;CHEN, D;GILLETTE, MU
通讯作者：
GILLETTE, MU
影响因子：
56.9
作者：
BADING, H;GINTY, DD;GREENBERG, ME
通讯作者：
GREENBERG, ME
新生儿褪黑激素注射诱导叙利亚仓鼠昼夜节律活动。
DOI：
10.1152/ajpregu.1996.270.3.r533
发表时间：
1996
期刊：
The American journal of physiology.
影响因子：
--
作者：
Grosse,J;Velickovic,A;Davis,FC
通讯作者：
Davis,FC