Schomerus C, Maronde E, Laedtke E, Korf H W
Center of Morphology, Section on Neurobiology, Johann Wolfgang Goethe University, Theodor Stern-Kai 7, D-60590 Frankfurt/Main, Germany.
Cell Tissue Res. 1996 Dec;286(3):305-13. doi: 10.1007/s004410050700.
We investigated whether vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP), which stimulate melatonin biosynthesis in the mammalian pineal organ, cause phosphorylation of the cyclic AMP response element binding protein (CREB) in rat pinealocytes. Dispersed cells were treated with varying concentrations of VIP and PACAP for 10 to up to 240 min and then immunocytochemically analyzed with an antibody against phosphorylated CREB (pCREB). The experiments showed a dose- and time-dependent induction of pCREB immunoreactivity in the nuclei of subpopulations of pinealocytes identified by the S-antigen immunoreaction. Stimulation with VIP elicited pCREB immunoreaction in approximately 50-65% of the S-antigen immunoreactive pinealocytes. The number of PACAP-responsive pinealocytes was often smaller and more variable. Maximal responses to both neuropeptides were seen after 30 min. pCREB immunoreaction gradually declined within 2 h and could not be induced again by an additional stimulation. In contrast, norepinephrine (NE) elicited pCREB immunoreaction in more than 95% of the pinealocytes, and this response lasted as long as 300 min. Treatment of pinealocytes with forskolin or KCl induced pCREB immunoreaction in the vast majority of pinealocytes, showing that in principle elevation of the intracellular concentrations of both cAMP and calcium can cause the response. Immunoblotting analyses confirmed that the immunoreaction elicited by VIP, PACAP and NE is largely due to phosphorylation of a 42-kDa protein corresponding to CREB, but reflects to a minor extent also phosphorylation of two smaller proteins presumably related to ATF-1. Immunocytochemical and immunochemical investigations with an antibody against total CREB showed that stimulation with VIP, PACAP and NE did not affect the level of CREB. All findings indicate that the stimulatory effects of VIP and PACAP on rat pinealocytes involve phosphorylation of transcription factors of the CREB family as holds also true for NE. However, VIP and PACAP affected only subpopulations of pinealocytes and the reponses lasted for a shorter period of time than those to NE. This conforms to previous results showing that both neuropeptides are also less effective than NE in stimulating the melatonin biosynthesis in the rat pineal organ.
我们研究了血管活性肠肽（VIP）和垂体腺苷酸环化酶激活多肽（PACAP）这两种在哺乳动物松果体器官中刺激褪黑素生物合成的物质，是否会导致大鼠松果体细胞中环状AMP反应元件结合蛋白（CREB）的磷酸化。将分散的细胞用不同浓度的VIP和PACAP处理10至240分钟，然后用抗磷酸化CREB（pCREB）抗体进行免疫细胞化学分析。实验表明，在通过S抗原免疫反应鉴定的松果体细胞亚群的细胞核中，pCREB免疫反应性呈剂量和时间依赖性诱导。用VIP刺激可在约50 - 65%的S抗原免疫反应性松果体细胞中引发pCREB免疫反应。对PACAP有反应的松果体细胞数量通常较少且更具变异性。对两种神经肽的最大反应在30分钟后出现。pCREB免疫反应在2小时内逐渐下降，再次刺激不能再次诱导。相比之下，去甲肾上腺素（NE）可在超过95%的松果体细胞中引发pCREB免疫反应，且该反应可持续长达300分钟。用福斯可林或氯化钾处理松果体细胞可在绝大多数松果体细胞中诱导pCREB免疫反应，表明原则上细胞内cAMP和钙浓度的升高均可引起该反应。免疫印迹分析证实，VIP、PACAP和NE引发的免疫反应主要是由于与CREB相对应的42 kDa蛋白的磷酸化，但在较小程度上也反映了可能与ATF - 1相关的两种较小蛋白的磷酸化。用抗总CREB抗体进行的免疫细胞化学和免疫化学研究表明，VIP、PACAP和NE刺激不影响CREB水平。所有发现表明，VIP和PACAP对大鼠松果体细胞的刺激作用涉及CREB家族转录因子的磷酸化，去甲肾上腺素也是如此。然而，VIP和PACAP仅影响松果体细胞亚群，且反应持续时间比去甲肾上腺素短。这与先前的结果一致，即这两种神经肽在刺激大鼠松果体器官中的褪黑素生物合成方面也不如去甲肾上腺素有效。
