Wilcox R A, Challiss R A, Liu C, Potter B V, Nahorski S R
Department of Pharmacology and Therapeitucs, University of Leicester, UK.
Mol Pharmacol. 1993 Oct;44(4):810-7.
myo-Inositol-1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4]-induced Ca2+ mobilization was examined in saponin-permeabilized SH-SY5Y cells using myo-inositol hexakisphosphate-supplemented buffer to prevent Ins(1,3,4,5)P4-3-phosphatase-catalyzed back-conversion of exogenous Ins(1,3,4,5)P4 to myo-inositol-1,4,5-trisphosphate [Ins(1,4,5)P3]. The Ins(1,3,4,5)P4 concentration-response curve for Ca2+ release in SH-SY5Y cells exhibited an EC50 of 2.5 microM, compared with 52 nM for Ins(1,4,5)P3, with the maximally effective concentration of Ins(1,3,4,5)P4 (100 microM) mobilizing the entire Ins(1,4,5)P3-sensitive pool. Both Ins(1,3,4,5)P4- and Ins(1,4,5)P3-induced Ca2+ mobilizations were heparin sensitive. Further, L-chiro-inositol-2,3,5-trisphosphorothioate, a recently identified low intrinsic activity Ins(1,4,5)P3 receptor partial agonist, shifted both the Ins(1,4,5)P3 and Ins(1,3,4,5)P4 concentration-response curves significantly rightward, with similar potencies. However, binding studies demonstrate that L-chiro-inositol-2,3,5-trisphosphorothioate interacts very poorly (IC50 > 30 microM) with specific Ins(1,3,4,5)P4 binding sites that have been previously characterized in pig cerebellum. Carbachol-pretreated SH-SY5Y cells (1 mM, > or 6 hr) exhibit a decrease in Ins(1,4,5)P3 receptor number, accompanied by both a rightward shift and a reduced maximal Ca2+ release in their Ins(1,4,5)P3 concentration-response curve. Here both Ins(1,4,5)P3 and Ins(1,3,4,5)P4 concentration-response curves were found to exhibit identically reduced maximal Ca2+ release responses and about 4-fold rightward shifts in EC50 values. Together, these observations provide compelling evidence for our hypothesis that Ins(1,3,4,5)P4 exhibits weak but full agonist status at Ins(1,4,5)P3 receptor-operated Ca2+ channels in SH-SY5Y cells.
在皂素通透的SH-SY5Y细胞中，使用补充了肌醇六磷酸的缓冲液来防止外源性肌醇-1,3,4,5-四磷酸[Ins(1,3,4,5)P4]被Ins(1,3,4,5)P4-3-磷酸酶催化逆转为肌醇-1,4,5-三磷酸[Ins(1,4,5)P3]，进而检测了Ins(1,3,4,5)P4诱导的Ca2+动员情况。SH-SY5Y细胞中Ca2+释放的Ins(1,3,4,5)P4浓度-反应曲线的半数有效浓度（EC50）为2.5微摩尔，而Ins(1,4,5)P3的EC50为52纳摩尔，Ins(1,3,4,5)P4的最大有效浓度（100微摩尔）可动员整个Ins(1,4,5)P3敏感池。Ins(1,3,4,5)P4和Ins(1,4,5)P3诱导的Ca2+动员均对肝素敏感。此外，L-手性肌醇-2,3,5-三硫代磷酸酯是最近鉴定出的一种内在活性较低的Ins(1,4,5)P3受体部分激动剂，它使Ins(1,4,5)P3和Ins(1,3,4,5)P4的浓度-反应曲线均显著右移，且效力相似。然而，结合研究表明，L-手性肌醇-2,3,5-三硫代磷酸酯与先前在猪小脑中鉴定出的特定Ins(1,3,4,5)P4结合位点的相互作用非常弱（IC50>30微摩尔）。用卡巴胆碱预处理的SH-SY5Y细胞（1毫摩尔，≥6小时）显示Ins(1,4,5)P3受体数量减少，同时其Ins(1,4,5)P3浓度-反应曲线出现右移且最大Ca2+释放减少。在此，发现Ins(1,4,5)P3和Ins(1,3,4,5)P4的浓度-反应曲线均表现出最大Ca2+释放反应同等程度的降低以及EC50值右移约4倍。这些观察结果共同为我们的假设提供了有力证据，即Ins(1,3,4,5)P4在SH-SY5Y细胞中Ins(1,4,5)P3受体介导的Ca2+通道上表现出弱但完全的激动剂状态。
