Patel S, Taylor C W
Department of Pharmacology, University of Cambridge, U.K.
Biochem J. 1995 Dec 15;312 ( Pt 3)(Pt 3):789-94. doi: 10.1042/bj3120789.
Submaximal concentrations of inositol 1,4,5-trisphosphate (InsP3) rapidly release only a fraction of the InsP3-sensitive intracellular Ca2+ stores, despite the ability of further increases in InsP3 concentration to evoke further Ca2+ release. The mechanisms underlying such quantal Ca2+ mobilization are not understood, but have been proposed to involve regulatory effects of cytosolic Ca2+ on InsP3 receptors. By examining complete concentration-effect relationships for InsP3-stimulated 45Ca2+ efflux from the intracellular stores of permeabilized hepatocytes, we demonstrate that, at 37 degrees C, responses to InsP3 are quantal in Ca(2+)-free media heavily buffered with either EGTA or BAPTA [1,2-bis-(2-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid]. Lower concentrations of InsP3 were used to examine the kinetics of Ca2+ mobilization at 2 degrees C, because at the lower temperature the stores were more sensitive to InsP3: the concentration of InsP3 causing half-maximal Ca2+ release (EC50) after a 30 s incubation decreased from 281 +/- 37 nM at 37 degrees C to 68 +/- 3 nM at 2 degrees C. At 2 degrees C, the EC50 for InsP3-stimulated Ca2+ mobilization decreased as the duration of exposure to InsP3 was increased: the EC50 was 68 +/- 3 nM after 30 s, and 29 +/- 2 nM after 420 s. InsP3-stimulated Ca2+ mobilization is therefore non-quantal at 2 degrees C: InsP3 concentration determines the rate, but not the extent, of Ca2+ release. By initiating quantal responses to InsP3 at 37 degrees C and then simultaneously diluting and chilling cells to 2 degrees C, we demonstrated that the changes that underlie quantal responses do not rapidly reverse at 2 degrees C. At both 37 degrees C and 2 degrees C, modest increases in cytosolic Ca2+ increased the sensitivity of the stores to InsP3, whereas further increases were inhibitory; both Ca2+ effects persisted after prior removal of ATP. We conclude that the effects of Ca2+ on InsP3 receptors are unlikely either to be enzyme-mediated or to underlie the quantal pattern of Ca2+ release evoked by InsP3.
次最大浓度的肌醇1,4,5 - 三磷酸（InsP3）仅能迅速释放一小部分对InsP3敏感的细胞内钙库中的钙，尽管InsP3浓度进一步升高能够引发更多的钙释放。这种量子化钙动员的潜在机制尚不清楚，但有人提出这涉及胞质钙对InsP3受体的调节作用。通过研究InsP3刺激通透化肝细胞内钙库中45Ca2+流出的完整浓度 - 效应关系，我们证明，在37℃时，在无钙培养基中用EGTA或1,2 - 双（2 - 氨基苯氧基）乙烷 - N,N,N',N' - 四乙酸（BAPTA）进行重缓冲时，对InsP3的反应是量子化的。使用较低浓度的InsP3来研究2℃时钙动员的动力学，因为在较低温度下钙库对InsP3更敏感：孵育30秒后引起半数最大钙释放（EC50）的InsP3浓度从37℃时的281±37 nM降至2℃时的68±3 nM。在2℃时，InsP3刺激的钙动员的EC50随着暴露于InsP3的持续时间增加而降低：30秒后EC50为68±3 nM，420秒后为29±2 nM。因此，InsP3刺激的钙动员在2℃时是非量子化的：InsP3浓度决定钙释放速率，但不决定释放程度。通过在37℃时引发对InsP3的量子反应，然后同时将细胞稀释并冷却至2℃，我们证明量子反应背后的变化在2℃时不会迅速逆转。在37℃和2℃时，胞质钙适度增加会增加钙库对InsP3的敏感性，而进一步增加则具有抑制作用；在预先去除ATP后，两种钙效应仍然存在。我们得出结论，钙对InsP3受体的作用不太可能是酶介导的，也不太可能是InsP3引发的钙释放量子模式的基础。
