Analysis of functional role of capacitative Ca entry in enteric neurons

本项目的主要目的是阐明肠神经元容积性钙内流机制的调节作用。为此，以嗜铬细胞和PC12细胞为神经模型，以培养的肠神经细胞为模型，研究了钙离子内流途径的特性及其调控。1)内源性阿片类药物通过G蛋白抑制嗜铬细胞电压依赖性钙通道。2)细胞内钙释放调节膜离子通道的活性和分泌反应。3)抗Pgp 9.5免疫反应阳性的肠神经细胞内[Ca~(2+)]i明显升高。细胞内储存的钙被ATP和thapsigargin耗竭后，产生持续的[Ca^&lt；2+&gt；]i，而SK&F96365、Ni^&lt；2+&gt；和La^&lt；3+&gt；可抑制[Ca^&lt；2+]i，表明肠神经细胞存在电容钙内流。5)逆转录-聚合酶链式反应(RT-PCR)结果显示，在大鼠脑内和肠神经细胞中有多种被认为是钙进入途径的色氨酸蛋白的表达。5)在TRP5转基因的PC12细胞中，缓激肽和UTP等G蛋白偶联受体刺激引起的[Ca~(2+)]i升高明显大于模型细胞。这些结果表明，神经细胞内钙内流途径受到多种机制的调节，其中电容性钙内流机制可能是由神经递质刺激的肠神经细胞内钙内流途径。进一步的研究有必要阐明该通道蛋白的分子基础和肠神经细胞电容性钙内流的细胞内调控机制。

The main aim of this project was to clarify the regulatory role of capacitative Ca entry mechanisms in enteric neurons. For this purpose the properties of Ca influx pathways and their regulation were examined using chromaffin and PC12 cells as neural models, and cultured enteric neurons. 1) Voltage-dependent Ca channels in chromaffin cells were received an inhibitory control through G-proteins by endogenous opioids. 2) Ca release from the intracellular stores regulated the activity of membrane ionic channels as well as secretory responses. 3) ATP evoked [Ca^<2+>]I increase in enteric neural cells with anti-PGP 9.5-immunoreactivity. After intracellular stored Ca was depleted with ATP and thapsigargin, a sustained [Ca^<2+>]I was produced, which was suppressed by the treatment with SK&F96365, Ni^<2+> and La^<3+>, indicating that the presence of capacitative Ca entry in enteric neuronal cells. 5) RT-PCR revealed that several TRP-proteins, being considered as Ca entry pathways, were expressed in rat brain and enteric neurons. 5) In TRP5-transfected PC12 cells, [Ca^<2+>]I increases induced by stimulation of G-protein coupled receptors such as bradykinin and UTP were significantly larger than those in mock-cells. These results indicate that Ca entry pathways in neural cells were regulated under various mechanisms, and capacitive Ca entry mechanisms play a role as Ca influx pathway stimulated by the neurotransmitters in enteric neurons. Further research is necessarily to elucidate the molecular basis of the channels protein and intracellular regulatory mechanism on capacitative Ca entry in enteric neural cells.