氨的慢性作用对星形胶质细胞内Ca2+浓度和谷氨酸释放的影响

Brain ammonia levels reach low millimolar concentrations in acute liver failure, and astrocyes are selectively sensitive to increased concentrations of ammonia. Brain extracellular concentration of glutamate is increased in animal models of liver failure. Previously, we have reported that ammonia induced release of ouabain-like compounds which mediated EGFR transactivation and regulation of gene expression in astrocytes. Astrocytes are non-excitable cells. In astrocytes, Ca2+ signaling is important for their excitability and release of gliotransmitters, such as glutamate and ATP. In preliminary experiments, we have found that chronic treatment with ammonia up-regulated gene expression of TRPC1 and Cav1.2, and increased extracellular high K+-induced increase of intracellular Ca2+ concentration ([Ca2+]i). In addition, the effect of ammonia could be inhibited by ouabain inhibiotr, canrenone. Therefore, we hypothesize that chronic treatment with ammonia will stimulate Ca2+-dependent glutamate release by either increase of Ca2+ influx mediated by Cav1.2 or increase of Ca2+- induced Ca2+ release mediated by RyR and IP3R from intracellular Ca2+ store. This study will not only contribute to the understanding of brain pathophysiology of acute liver failure, but may also eventually lead to a new therapeutic strategy of the disease.

体内高血氨在星形胶质细胞引起的改变是肝性脑病病理变化的基础。我们曾报道氨可刺激内源性哇巴因释放，后者引起EGF受体间接激活，此信号通路可调节细胞基因表达。[Ca2+]i升高是维持星形胶质细胞兴奋性的重要途径，参与胶质递质谷氨酸的释放。与神经元不同，星形胶质细胞[Ca2+]i升高来源于外钙内流和钙库释放，且以后者为主。预实验发现氨慢性处理星形胶质细胞，增强高钾引起的[Ca2+]i升高，并上调TRPC1和Cav1.2基因表达。同时哇巴因拮抗剂坎利酮可抑制氨引起的TRPC1上调。TRPC1是钙库操纵性钙通道的主要组件，参与钙库再充，其中和性抗体可降低谷氨酸释放。我们推测氨引起的TRPC1和Cav1.2功能异常是肝性脑病细胞外谷氨酸浓度升高的病理基础。本项目中，我们将研究氨慢性处理对IP3R和RyR引起的钙库释放、L-型钙通道激活所致钙内流和钙依赖性谷氨酸释放的影响，为治疗肝性脑病提供新的途径。

在肝功能衰竭时，脑内氨浓度可高达5 mM。脑内氨水平增高是肝衰竭时肝性脑病发生的主要因素。目前研究认为氨的中枢神经系统损伤机制与谷氨酸、ATP等脑内神经递质释放有关，还可直接或间接损伤线粒体功能进而影响脑内的代谢过程。我们在前期研究中发现氨慢性处理星形胶质细胞可增强细胞外高钾引起的[Ca2+]i升高。这一作用依赖于氨诱导的TRPC1和Cav1.2 mRNA表达增强，进而导致SOC通道介导的Ca2+内流及Cav1.2/RyR系统的钙库内Ca2+释放增加。在此基础上，本项目对氨慢性应用后Ca2+依赖性的胶质递质谷氨酸和ATP释放，以及Ca2+依赖性的线粒体损伤进行了研究。. 本项目研究发现氨慢性作用引起的星形胶质细胞Ca2+依赖性谷氨酸和ATP释放增加，此作用依赖于[Ca2+]i水平升高。而且参与星形胶质细胞胞吐作用的Ca2+主要来源于细胞外Ca2+内流及细胞内钙库的Ca2+释放，与氨诱导的TRPC1和Cav1.2 mRNA表达增强密切相关。应用氨早期信号传导通路抑制剂可抑制TRPC1和Cav1.2 mRNA表达，进而降低Ca2+依赖性的谷氨酸和ATP释放。本项目在原定研究内容基础上，进一步研究了氨慢性作用对星形胶质细胞能量代谢的影响。我们发现氨慢性作用可引起Ca2+依赖性的线粒体功能损伤，体现为：ROS生成增加，ATP生成相关氧消耗量降低。本项目的研究阐明了星形胶质细胞[Ca2+]i异常与肝性脑病病理生理机制间的关系，并为寻找肝性脑病防治措施提供了理论依据。