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

肌醇多磷酸盐（InsPPs），如肌醇11、3、4、5、6-五磷酸肌醇（InsP5）和肌醇六磷酸肌醇（InsP6）结合突触tagmins I和II （Syt）的C2B结构域，抑制递质释放。在肾上腺染色质细胞中，我们已经证明Ca2+与C2A结构域的结合逆转了InsPPs对胞外分泌的抑制，可能是将InsPPs与Syt分离。据报道，内源性InsP5和InsP6在去极化刺激后在肾上腺染色质细胞、NIE-1 - 15细胞和大鼠小脑颗粒细胞中迅速积累。如此快速的积累可能是由于它们与Syt的C2B结构域分离的结果。为了验证这一假设，我们检测了C2A和C2B结构域抗体（抗C2A Ab，抗C2B Ab）对Ca2+诱导的肾上腺色素细胞中InsP5和InsP6积累的影响。Ca2+刺激诱导胞质成分中inspp的快速和短暂增加。抗c2b抗体本身引起胞质成分中inspp的显著积累，并增加儿茶酚胺（CA）的自发释放。抗c2a Ab可消除Ca2+诱导的细胞基质中InsP5和InsP6的升高。与抗c2b Ab相比，抗c2a Ab抑制Ca2+诱导的CA释放，对CA的自发释放影响不大。此外，在完整的染色质细胞中微量注射InsP6而非InsS6可显著抑制自发和尼古丁诱发的胞外事件。这些结果表明，Ca2+结合到C2A结构域诱导内源性InsPPs从Syt的C2B结构域分离。

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.