School of Life Sciences, Tokyo University of Pharmacy and Life Science, Horinouchi, Hachioji, Tokyo 192-0392, Japan.
Brain Res. 2013 Oct 16;1535:1-13. doi: 10.1016/j.brainres.2013.08.018. Epub 2013 Aug 23.
The purpose of this study was to determine whether calmodulin (CaM) plays a role in neurotransmitter release by examining the effect that ophiobolin A (OBA), a CaM antagonist, on neurotransmitter release from clonal rat pheochromocytoma PC12 cells, primary cortical neurons, and primary cerebellar granule cells. OBA inhibited Ca²⁺/CaM-dependent phosphorylation of cAMP response element binding protein in all cell types tested. Moreover, Ca²⁺-dependent release of dopamine and acetylcholine from PC12 cells were remarkably reduced by OBA in a dose-dependent and temporal manner, but neurotransmitter release partially recovered with the addition of CaM in membrane permeabilized PC12 cells. OBA and several synthetic CaM antagonists suppressed Ca²⁺-dependent glutamate release from cerebral cortical neurons, but not from cerebellar granule cells. Myosin Va, a CaM binding protein, localized to synaptic vesicles of PC12 cells and cerebral cortical neurons, but not in cerebellar granule cells. OBA suppressed Ca²⁺-induced myosin Va dissociation from secretory vesicles, and inhibited secretory vesicle motility in PC12 cells. These results suggest that CaM, although not essential, regulates neurotransmitter release in a subset of neurons and secretory cells, and myosin Va is a possible target of OBA in this process.
本研究旨在通过考察钙调蛋白（CaM）拮抗剂蛇菰菌素 A（OBA）对克隆大鼠嗜铬细胞瘤 PC12 细胞、原代皮质神经元和原代小脑颗粒细胞神经递质释放的影响，来确定 CaM 是否在神经递质释放中发挥作用。OBA 抑制了所有测试细胞类型中 Ca²⁺/CaM 依赖性 cAMP 反应元件结合蛋白的磷酸化。此外，OBA 以剂量和时间依赖性方式显著减少 PC12 细胞中 Ca²⁺依赖性多巴胺和乙酰胆碱的释放，但在透膜 PC12 细胞中添加 CaM 后，神经递质释放部分恢复。OBA 和几种合成 CaM 拮抗剂抑制了大脑皮质神经元中 Ca²⁺依赖性谷氨酸的释放，但不抑制小脑颗粒细胞中的谷氨酸释放。肌球蛋白 Va，一种 CaM 结合蛋白，定位于 PC12 细胞和大脑皮质神经元的突触小泡中，但不在小脑颗粒细胞中。OBA 抑制了 Ca²⁺诱导的肌球蛋白 Va 从小泡中的解离，并抑制了 PC12 细胞中分泌小泡的运动。这些结果表明，CaM 虽然不是必需的，但在一部分神经元和分泌细胞中调节神经递质的释放，肌球蛋白 Va 可能是 OBA 在该过程中的作用靶点。
