Champeil P, Combettes L, Berthon B, Doucet E, Orlowski S, Claret M
Unité de Recherche Institut National de la Santé et de la Recherche Médicale, Université Paris Sud, Orsay, France.
J Biol Chem. 1989 Oct 25;264(30):17665-73.
We used a stopped-flow method for determining the kinetic properties (between 10 ms and 10 s) of the Ca2+ release induced by inositol 1,4,5-trisphosphate (InsP3) in saponin-treated rat hepatocytes. Preliminary experiments ensured that the indicator was able to monitor rapid changes in free Ca2+ reliably. At 20 degrees C, a maximally efficient concentration of 10 microM InsP3 released Ca2+ with a half-time of 150-300 ms, the initial rate being about 1-2 nmol of Ca2+/mg of cell protein/s. The delay between the addition of 10 microM InsP3 and the onset of Ca2+ release was shorter than 20 ms, suggesting that the opening process of Ca2+ channels after binding of InsP3 to receptors is completed within a few milliseconds. Half-maximal initial rates for Ca2+ release occurred at about 1 microM InsP3 (Hill index was 1.6). The resulting Ca2+ efflux had a moderate temperature dependence. It could not be fitted to a single exponential. After low speed centrifugation of saponin-treated cells (1000 x g for 1 min), part of the InsP3-sensitive Ca2+ pool was recovered in the cell-free supernatant fraction, suggesting that the response to InsP3 arises from a vesicular fraction which may diffuse from the saponin-treated cells into the medium.
我们采用停流法来测定皂角苷处理的大鼠肝细胞中由肌醇1,4,5-三磷酸（InsP3）诱导的Ca2+释放的动力学特性（10毫秒至10秒之间）。初步实验确保了该指示剂能够可靠地监测游离Ca2+的快速变化。在20摄氏度下，10微摩尔/升的InsP3最大有效浓度释放Ca2+的半衰期为150 - 300毫秒，初始速率约为1 - 2纳摩尔Ca2+/毫克细胞蛋白/秒。添加10微摩尔/升InsP3与Ca2+释放开始之间的延迟短于20毫秒，这表明InsP3与受体结合后Ca2+通道的开放过程在几毫秒内完成。Ca2+释放的初始速率达到半数最大值时InsP3浓度约为1微摩尔/升（希尔系数为1.6）。所产生的Ca2+外流对温度有适度的依赖性。它不能用单一指数拟合。对皂角苷处理的细胞进行低速离心（1000×g，1分钟）后，部分InsP3敏感的Ca2+池在无细胞上清液部分中被回收，这表明对InsP3的反应源自可能从皂角苷处理的细胞扩散到培养基中的囊泡部分。
