组胺H3受体信号转导中G蛋白和离子通道作用机制的研究

In the present study , we cultured the AtT-20 cells line from a murine anterior pituitary gland tumor that were reported containing high affinity histamine H3 receptors and observed the effects of histamine agonists and antagonists on the release of adrenocorticotropic hormone (ACTH). Therefore we investigated the signal transduction mechanisms of histamine H3 receptors. The contents of development and innovation presented in our study involves the follwing aspects as:1. Histamine H3 receptor mediates release of ACTH from AtT-20 cells : Detecting ACTH levels in the supernatants of AtT-20 cells by radio immunoassay at the given time points following administrating reagents, we observed that histamine (HA) 10-6mol/L increased the release of ACTH in time-dependent form and the H3 receptor-specific agonist R-(α)-methylhistamine (R-(a)-MeHA) 10-7mol/L mimicked the effects of HA, while the H1 receptor agonist 2-methylhistamine 10-6mol/L and the H2 agonist impromidine 10-5mol/L had no significant influence. Furthermore, above responses could be blocked by thioperamide 10-5mol/L, an H3 receptor-specific antagonist, but not be done by the H1 antagonist chloropheniramine 10-5mol/L or H2 antagonist cimetidine 10-5 mol/L. R-(α)-MeHA 10-5mol/L～10-9mol/L had no significant effects on the cells proliferation compared with the control group 8 h and 24 h after the adminitration. The results indicated that R-(α)-MeHA increased not the cells number but the secret function significantly. Specific activation of H3 receptor could evoke the excitation-secretion coupling process. The effective research system made the foundation of study for the signal transduction mechanism of H3 receptor.2. Effect of G protein and Ca2+ level to histamine H3 receptor induced consequence: By this research system, pretreatment with G protein inactivator, N-ethylmaleimide 10-5mol/L 30min, the effects of R-(α)-MeHA on the release of ACTH could be abolished. This result suggested that in H3 receptor's signal pathway, G protein involved in the excitation-secretion coupling process evoked by specific activation of H3 receptor. In other way, we observed the influences of different extracellular culture solutions on the H3 receptor's effects. R-(α)-MeHA could increase ACTH release more significantly in the presence of extracellular Ca2+ than in the absence of extracellular Ca2+. It indicated that the effects of R-(α)-MeHA acted through the intracellular Ca2+, not dependent on the extracelluler Ca2+, but might be elevated by the later. That is to say, both the intracellular Ca2+ and the extracelluler Ca2+ involved in the effects of R-(α)-MeHA on ACTH release on AtT-20 cells.3.The intracellular signal pathway and effectors activated by G protein :. After R-(α)-MeHA 10-7 mol/L administrated 10min, 30min, 2h and 8h, there is no significant changes of the intracellular cAMP detected. But the IP3 was increased after R-(α)-MeHA administrated 30min and the translocated PKC on the membrane (activated PKC) showed increased by immunohistorical essay. Also, R-(α)-MeHA could induce NO synthase (iNOS) and make levels of cGMP increased. The results suggested that the IP3/Ca2+, DG/PKC and NO/cGMP involved in the process of histamine H3 receptor signal transduction pathway. It seemed less relation between cAMP and H3 receptor.4.The possible pathway of the changes of intracellular calcium followed H3 receptor activation: Making use of laser confocal microscope observation and analysis system, we studied the effects of R-(α)-MeHA on the changes of intracellular calcium fluorescence intensity which could reflex the [Ca2+]i changes. ( 1 ) Histamine H3 receptor agonist R-(α)-MeHA 10-7mol/L could elevate [Ca2+]i slowly both in presence and in absence of extracellular Ca2+. [Ca2+]i elevated to peak earlier but lower in absence of extracellular Ca2+ than in presence of extracellular Ca2+, in which [Ca2+]i was elevated to peak later but reached much more higher peak values and lasted longer. It was showed that the way of R-(α)-MeHA to elevated the

目前对组胺H3受体信号系统方面的认识尚为空白。利用膜片钳单通道记录和生化分析技术，将直接反映神经细胞的离子活动与细胞内信使物质变化相结合，同步观察激动或阻断H3受体细胞内的信号传递和细胞膜离子通道开放与关闭的变化规律，从分子水平揭示G蛋白和离子ǖ涝贖3受体信号通路中的作用。目前在H3受体研究领域，国内外未见类似的文献报道。