Role of store-operated Ca entry mechanism in signal transduction in gastrointestinal intrinsic neurons

本项目的目的是确定胃肠内神经元的容性钙进入的调控机制。为此，我们分析了容性钙离子进入的特性、细胞内信使和通道分子的调控机制。1)建立单培养胃肠道内禀神经元Ca信号可视化实验系统。2) ATP通过细胞内钙离子的释放，激活大鼠胃肠道内神经元的钙离子进入通道和P2X2通道，诱导钙离子的增加。3)缓激肽通过肠胶质细胞分泌PGE2，激活胃肠道内禀神经元的B2受体，引起Ca升高。4)克隆的TRPC5通道在PC12细胞中表达，作为Ca进入通道与gtp结合蛋白受体偶联。5)克隆了TRPV1的互补DNA，并在HEK293细胞中异种表达。我们发现TRPV1通道具有高钙通透性的多模受体通道功能。这些结果表明，在胃肠内神经元中，电容性钙通道在功能性钙通道中起着重要的作用，这些通道受ATP和缓激素等多种内在因子的调节。克隆的TRPC5和TRPV1构成钙离子进入通道，调控细胞内钙离子信号。

The aim of this project was to identify the regulatory mechanisms of capacitative Ca entry in gastrointestinal intrinsic neurons. For these purposes, we analyzed the properties of capacitative Ca entry, its regulatory mechanisms with intracellular messengers and putative channel molecules. 1) We established experimental system for visualization of Ca signaling in single cultured gastrointestinal intrinsic neurons. 2) ATP elicited Ca increases through release of Ca from intracellular stores, the activation of capacitative Ca entry and P2X2 channels in rat cultured gastrointestinal intrinsic neurons. 3) Bradykinin evoked the elevation of Ca through B2 receptor activation on gastrointestinal intrinsic neurons via PGE2 secreted from enteric glial cells. 4) Cloned TRPC5 channels expressed in PC12 cells functioned as Ca entry channels coupled with GTP-binding protein receptors. 5) Complimentary DNA for TRPV1 was cloned and was hetelogeously expressed in HEK293 cells. We revealed that TRPV1 channels functioned as polymodal receptor channels with high Ca permeability. These results indicate that capacitative Ca entry channels play a role for functional Ca entry pathways in gastrointestinal intrinsic neurons, which are regulated by various intrinsic factors such as ATP and bradykinin. Cloned TRPC5 and TRPV1 constitute Ca entry channels for regulation of intracellular Ca signallings.