Analysis of functional role of capacitative Ca entry in enteric neurons

本项目的主要目的是阐明在肠神经元中，能性钙进入机制的调控作用。为此，我们以嗜铬蛋白和PC12细胞为神经模型，以及培养的肠神经元，研究了钙内流通路的特性及其调控。1)内源性阿片通过g蛋白抑制染色质细胞电压依赖性Ca通道。2)细胞内钙离子的释放调节了细胞膜离子通道的活性和分泌反应。3) ATP诱导肠神经细胞[Ca^<2+>]I升高，具有抗pgp 9.5免疫反应性。在细胞内储存的Ca被ATP和thapsigarin耗尽后，产生持续的[Ca^<2+>]I, SK&F96365、Ni^<2+>和La^<3+>处理抑制了这一过程，表明在肠神经元细胞中存在capacitative Ca entry。5) RT-PCR结果显示，在大鼠脑和肠神经元中表达了几种被认为是钙进入途径的trp蛋白。5)在trp5转染的PC12细胞中，缓激肽、UTP等g蛋白偶联受体刺激引起的[Ca^<2+>]I升高明显大于模拟细胞。这些结果表明，钙在神经细胞内的进入途径受到多种机制的调控，其中电容性钙进入机制是受肠内神经元神经递质刺激的钙内流途径。通道蛋白的分子基础和钙离子在肠神经细胞内的调节机制有待进一步研究。

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.