Poitras M, Bernier S, Servant M, Richard D E, Boulay G, Guillemette G
Department of Pharmacology, Faculty of Medicine, University of Sherbrooke, Québec, Canada.
J Biol Chem. 1993 Nov 15;268(32):24078-82.
Inositol 1,4,5-trisphosphate (InsP3) is a second messenger responsible for the rapid and discontinuous release of Ca2+ from intracellular stores. In this study, the effects of the sulfhydryl reagent thimerosal were investigated on Ca2+ mobilization and on InsP3 binding. Thimerosal was shown to release Ca2+, in a dose-dependent manner, with an EC50 of 135.8 +/- 5.2 microM, from bovine adrenal cortex microsomes. Thimerosal-induced Ca2+ release was not prevented by heparin (250 micrograms/ml), ruling out a participation of InsP3 receptor in that effect. The slow rate of thimerosal-induced Ca2+ release rather suggested an inhibition of microsomal Ca2+ ATPase. At submaximal concentration, thimerosal (100 microM) was also shown to potentiate the release of Ca2+ induced by InsP3. Dose-response experiments revealed that thimerosal enhanced the apparent affinity of InsP3 by a factor 2.21 +/- 0.28, without modifying the maximal amount of Ca2+ released by InsP3. Thimerosal also enhanced, in a dose-dependent manner, [3H]InsP3 binding to adrenal cortex microsomes (EC50 = 43.3 +/- 7.6 microM). A similar effect was also observed on [3H]InsP3 binding to solubilized receptors, suggesting a direct modification of the receptor protein by thimerosal. The effects of thimerosal on Ca2+ release and [3H]InsP3 binding were abolished in the presence of the reducing agent dithiothreitol (1 mM), suggesting a modification by thimerosal of specific thiol groups on these microsomal proteins. Scatchard analysis revealed that thimerosal (100 microM) increased InsP3 receptor affinity by 1.87 +/- 0.26-fold. Kinetic analysis indicated that this increased affinity was due to an enhancement of InsP3 association rate constant. The concomitant increases of binding affinity and Ca2+ releasing potency suggest that the high affinity state of InsP3 receptor is a functional state.
肌醇1,4,5 - 三磷酸（InsP3）是一种第二信使，负责从细胞内储存库中快速且不连续地释放Ca2+。在本研究中，研究了巯基试剂硫柳汞对Ca2+动员和InsP3结合的影响。结果表明，硫柳汞能以剂量依赖的方式从牛肾上腺皮质微粒体中释放Ca2+，其半数有效浓度（EC50）为135.8±5.2微摩尔。肝素（250微克/毫升）不能阻止硫柳汞诱导的Ca2+释放，排除了InsP3受体参与该效应的可能性。硫柳汞诱导Ca2+释放的速率较慢，这表明其对微粒体Ca2+ ATP酶有抑制作用。在亚最大浓度下，硫柳汞（100微摩尔）还能增强InsP3诱导的Ca2+释放。剂量反应实验表明，硫柳汞使InsP3的表观亲和力提高了2.21±0.28倍，而不改变InsP3释放Ca2+的最大量。硫柳汞还以剂量依赖的方式增强了[3H]InsP3与肾上腺皮质微粒体的结合（EC50 = 43.3±7.6微摩尔）。在[3H]InsP3与可溶性受体的结合中也观察到了类似的效应，这表明硫柳汞对受体蛋白有直接修饰作用。在存在还原剂二硫苏糖醇（1毫摩尔）的情况下，硫柳汞对Ca2+释放和[3H]InsP3结合的影响被消除，这表明硫柳汞对这些微粒体蛋白上的特定巯基进行了修饰。Scatchard分析表明，硫柳汞（100微摩尔）使InsP3受体亲和力提高了1.87±0.26倍。动力学分析表明，这种亲和力的增加是由于InsP3结合速率常数的提高。结合亲和力和Ca2+释放能力的同时增加表明，InsP3受体的高亲和力状态是一种功能状态。
