McNulty S, Ross A W, Shiu K Y, Morgan P J, Hastings M H
Department of Anatomy, University of Cambridge, UK.
J Neuroendocrinol. 1996 Aug;8(8):635-45.
This study used a combination of Western blotting and immunocytochemistry to test whether signalling pathways independent of cyclic AMP have the potential to induce phospho-CREB (pCREB)-like immunoreactivity (-ir) in the oPT. Western blot analysis of extracts of primary cultures of oPT using an antiserum against CREB, revealed a major band of CREB-ir at 44 KDa. The intensity of this band did not vary systematically with treatment. In extracts from untreated cells, Western blot analysis revealed a major band of pCREB-ir at 42 KDa which was not sensitive to agonist treatment. Treatment of cells with forskolin (10(-6) M) increased the intensity of a number of other pCREB-ir bands at between ca. 38 and 44 KDa. The band at 44 KDa probably represented native pCREB whilst the other bands induced by forskolin probably represented pCREB-like proteins. Melatonin (10(-6) M) alone had no effect on pCREB-ir, but it did inhibit the effect of forskolin on the ca. 38 and 44 KDa pCREB-ir bands. Treatment with lamb serum (1%) consistently increased the intensity of the ca. 38 and 44 KDa pCREB-ir bands relative to control cells, as assessed by Western blot. However, Western blot analysis did not reveal a consistent effect of melatonin on the pCREB-ir response to serum. The effect of serum on pCREB-ir in oPT cells was characterized further by immunocytochemical analysis. In contrast to experiments utilizing Western blotting, untreated cells did not possess detectable pCREB-ir. In serum-starved oPT and oPD cultures, treatment with serum induced exclusively nuclear pCREB-ir. A large majority of oPT cells (> or = 90%) were sensitive to serum (1%), and serum caused a time- and dose-dependent increase of nuclear pCREB-ir. Melatonin attenuated the response to serum in the oPT. This inhibition of the response to serum was not apparent in the oPD, demonstrating that the effect of melatonin was specific for a tissue known to express melatonin receptors. In oPT cultures, physiological concentrations of melatonin (10(-9) M) partially reversed (ca. 70%) the inductive effect of 0.1% serum on nuclear pCREB-ir. However, in contrast to studies applying forskolin, the induction of pCREB-ir by serum occurred in the absence of measurable changes in the concentration of cyclic AMP, indicating that components of serum are able to stimulate the phosphorylation of CREB in the oPT through mechanisms independent of cyclic AMP. Both adenosine and prostaglandin E2 (PGE2) also induced nuclear pCREB-ir in the absence of increased levels of cyclic AMP. These results demonstrate that transcriptional activities in the oPT which are under the control of CREB may be modulated by convergent cyclic AMP-dependent and cyclic AMP-independent pathways. Regulation of these pathways by melatonin and other factors present in serum may be an important control-point in the generation of seasonal neuroendocrine cycles.
本研究采用蛋白质免疫印迹法（Western blotting）和免疫细胞化学法相结合的方法，来检测不依赖环磷酸腺苷（cAMP）的信号通路是否有可能在视前区（oPT）诱导出磷酸化环磷腺苷反应元件结合蛋白（pCREB）样免疫反应性（-ir）。使用抗CREB抗血清对oPT原代培养物提取物进行蛋白质免疫印迹分析，结果显示在44 kDa处有一条主要的CREB-ir条带。该条带的强度不会随处理而发生系统性变化。在未处理细胞的提取物中，蛋白质免疫印迹分析显示在42 kDa处有一条主要的pCREB-ir条带，其对激动剂处理不敏感。用福斯可林（10⁻⁶ M）处理细胞会增加一些其他位于约38至44 kDa之间的pCREB-ir条带的强度。44 kDa处的条带可能代表天然pCREB，而福斯可林诱导产生的其他条带可能代表pCREB样蛋白。褪黑素（10⁻⁶ M）单独作用时对pCREB-ir没有影响，但它确实抑制了福斯可林对约38和44 kDa pCREB-ir条带的作用。通过蛋白质免疫印迹评估，用羊血清（1%）处理相对于对照细胞持续增加了约38和44 kDa pCREB-ir条带的强度。然而，蛋白质免疫印迹分析未显示褪黑素对血清诱导的pCREB-ir反应有一致的影响。通过免疫细胞化学分析进一步表征了血清对oPT细胞中pCREB-ir的影响。与使用蛋白质免疫印迹的实验不同，未处理的细胞未检测到可检测到的pCREB-ir。在血清饥饿的oPT和oPD培养物中，血清处理仅诱导细胞核pCREB-ir。绝大多数oPT细胞（≥90%）对血清（1%）敏感，血清导致细胞核pCREB-ir呈时间和剂量依赖性增加。褪黑素减弱了oPT中对血清的反应。在oPD中未观察到这种对血清反应的抑制，这表明褪黑素的作用对已知表达褪黑素受体的组织具有特异性。在oPT培养物中，生理浓度的褪黑素（10⁻⁹ M）部分逆转（约70%）了0.1%血清对细胞核pCREB-ir的诱导作用。然而，与应用福斯可林的研究不同，血清诱导pCREB-ir时环磷酸腺苷浓度没有可测量的变化，这表明血清成分能够通过不依赖环磷酸腺苷的机制刺激oPT中CREB的磷酸化。腺苷和前列腺素E2（PGE₂）在环磷酸腺苷水平未升高的情况下也诱导了细胞核pCREB-ir。这些结果表明，受CREB控制的oPT中的转录活性可能受到cAMP依赖性和cAMP非依赖性途径的共同调节。褪黑素和血清中存在的其他因素对这些途径的调节可能是季节性神经内分泌周期产生过程中的一个重要控制点。
