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Biological activities of InsP5 and InsP6 in neuronal cells : Analysis by single cell amperometory.

InsP5 和 InsP6 在神经元细胞中的生物活性：单细胞安培分析。

基本信息

批准号：
11670101
负责人：
SASAKAWA Nobuyuki
金额：
$ 2.3万
依托单位：
Sophia University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2000
项目状态：
已结题

项目摘要

Inositol polyphosphates (InsPPs) such as Inosito 11,3,4,5,6-pentakisphosphate (InsP5) and inositol hexakisphosphate (InsP6) bind to C2B domain of synaptotagmins I and II (Syt), and inhibit transmitter release. In adrenal chromaffln cells, we have shown that binding of Ca2+ to the C2A domain reverses the inhibition of exocytosis by InsPPs probably dissociating InsPPs ftom Syt. A rapid accumulation of endogenous InsP5 and InsP6 after depolarizing stimuli have been reported in adrenal chromaffin cells, NIE-1 15 cells and rat cerebellar granule cells. Such a rapid accumulation of InsPPs might be a result from their dissociation from C2B domain of Syt. To test this hypothesis, we examined the effects of antibodies against C2A and C2B domains (anti-C2A Ab, anti-C2B Ab) on the accumulation of InsP5 and InsP6 induced by Ca2+ in digitoninpermeabilized adrenal chromafrln Cells. Stimulation with Ca2+ induced a rapid and transient increase of InsPPs in the cytosolic components. Anti-C2B Ab by itself caused a significnt accumulation of InsPPs in the cytosolic components, and increased spontaneous release of catecholamines (CA). Anti-C2A Ab abolished Ca2+-induced increase of InsP5 and InsP6 in cytosohc component. In contrast to anti-C2B Ab, anti-C2A Ab inhibited Ca2+ -evoked release of CA with little effect on the spontaneous release. Moreover, microinjection of InsP6 but not inositol hexakissulfate (InsS6) into the intact chromaffin cells remarkably inhibited both spontaneous and nicotine- evoked exocytotic events. These results suggest that binding of Ca2+ to the C2A domain induces dissociation of endogenous InsPPs from the C2B domain of Syt.

肌醇多磷酸（InsPPs）如肌醇11，3，4，5，6-五磷酸（InsP 5）和肌醇六磷酸（InsP 6）与突触结合蛋白I和II（Syt）的C2B结构域结合，并抑制递质释放。在肾上腺嗜铬细胞中，我们已经表明，结合的Ca 2+的C2 A结构域逆转抑制胞吐的InsPP可能解离InsPP从Syt。在肾上腺嗜铬细胞、NIE-115细胞和大鼠小脑颗粒细胞中，去极化刺激后内源性InsP 5和InsP 6迅速积累。InsPPs的这种快速积累可能是它们与Syt的C2B结构域解离的结果。为了验证这一假设，我们检测了抗C2 A和C2B结构域的抗体（抗C2 A Ab、抗C2B Ab）对Ca ~（2+）诱导的InsP 5和InsP 6在毛地黄酮不透化的肾上腺嗜铬细胞中蓄积的影响。用Ca 2+刺激诱导胞质组分中InsPPs的快速和瞬时增加。抗C2B抗体本身可引起InsPPs在胞浆中的大量积聚，并增加儿茶酚胺（CA）的自发释放。抗C2 A抗体可阻断Ca ~（2+）诱导的InsP 5和InsP 6在胞浆中的表达。与抗C2B抗体相反，抗C2 A抗体抑制Ca ~（2+）诱发的CA释放，对CA的自发释放影响不大。此外，在完整的嗜铬细胞中微量注射InsP 6而不是肌醇六硫酸酯（InsS 6）显著抑制自发和尼古丁诱发的胞吐事件。这些结果表明，Ca 2+的C2 A结构域的结合诱导的内源性InsPPs从Syt的C2B结构域的解离。