Asermely K E, O'Neill J J
Department of Pharmacology, Temple University School of Medicine, Philadelphia, Pennsylvania 19140, USA.
Life Sci. 1996;59(25-26):2113-28. doi: 10.1016/s0024-3205(96)00568-1.
Vesamicol (AH5183) is an inhibitor (IC50, 50 nM) of acetylcholine (ACh) vesicle packaging. Vesamicol increases the phosphorylation pattern of synaptophysin (p38), identified as a vesicle-specific phosphoprotein involved in vesicle-mediated neurotransmitter release. Percoll fractionation of the rat cortex yielded a cholinergic-enriched synaptosomal Fraction 4. Fraction 4 contained the highest enrichment of cholineacetyl-transferase activity (86 +/- 4.6 mumole AcCh/g protein/hr.) in the Percoll gradient. Fraction 4 demonstrated oxygen consumption (108 +/- 23.4 nmole/mg protein), levels of adenosine triphosphate, ATP, (10.29 +/- 0.45 nmole/mg protein) and adenosine diphosphate, ADP, (10.54 +/- 2.72 nmole/mg protein), energy potential (ATP/[ADP] [Pi], (0.49) phosphate uptake (65-80 nmoles phosphate/mg tissue), 32Pi labelling (130 +/- 12 x 10(5) DPM/mg tissue; 74 +/- 9.8 x 10(2) nmoles phosphate/mg tissue). Synaptophysin was identified by Western blotting and confirmed by qualitative immunoprecipitation. Synaptophysin phosphorylation was confirmed by autoradiograph. Synaptophysin phosphorylation increased (225%) in the presence of vesamicol (ED50, 1 nM) in Fraction 4. Vesamicol (50 nM) and vanadate (54 microM) were compared for their effects on synaptophysin. This study suggests that during the inhibition of acetylcholine packaging by vesamicol that synaptophysin is phosphorylated. Therefore, the phosphorylation and dephosphorylation of synaptophysin may be involved in the transport of acetylcholine in or out of the synaptic vesicle.
维萨米柯（AH5183）是一种乙酰胆碱（ACh）囊泡包装抑制剂（IC50为50 nM）。维萨米柯可增加突触素（p38）的磷酸化模式，突触素是一种参与囊泡介导的神经递质释放的囊泡特异性磷蛋白。对大鼠皮层进行 Percoll 分级分离得到富含胆碱能的突触体组分4。组分4在 Percoll 梯度中胆碱乙酰转移酶活性的富集程度最高（86±4.6微摩尔乙酰胆碱/克蛋白/小时）。组分4显示出氧消耗（108±23.4纳摩尔/毫克蛋白）、三磷酸腺苷（ATP）水平（10.29±0.45纳摩尔/毫克蛋白）和二磷酸腺苷（ADP）水平（10.54±2.72纳摩尔/毫克蛋白）、能量势（ATP/[ADP][Pi]，0.49）、磷酸盐摄取（65 - 80纳摩尔磷酸盐/毫克组织）、32Pi标记（130±12×10⁵ DPM/毫克组织；74±9.8×10²纳摩尔磷酸盐/毫克组织）。通过蛋白质印迹法鉴定突触素，并通过定性免疫沉淀法进行确认。通过放射自显影片确认突触素磷酸化。在组分4中，维萨米柯（ED50为1 nM）存在时突触素磷酸化增加（225%）。比较了维萨米柯（50 nM）和钒酸盐（54 microM）对突触素的影响。本研究表明，在维萨米柯抑制乙酰胆碱包装过程中，突触素会发生磷酸化。因此，突触素的磷酸化和去磷酸化可能参与乙酰胆碱进出突触囊泡的运输。
