INTERACTION OF ETHYL ALCOHOL WITH CELLULAR CYSTEINE PROTEASES

某些类型的细胞半胱氨酸蛋白酶可能在调节G蛋白偶联受体的活性中发挥重要作用。这类酶的活性受到细胞质和核抑制物的密切调控。在PC12细胞系的细胞培养中，研究了酒精暴露对半胱氨酸蛋白酶活性的影响。我们先前已经证实，PC12细胞在酒精中暴露96小时会导致钙刺激的蛋白酶活性下降。我们开发了测定GSH、GSSG和NO的方法，并确定GSH和GSSG都是钙蛋白酶活性的重要调节因子。GSH是一种非竞争性的抑制物，而GSSG是钙蛋白酶的竞争性抑制物，其抑制常数在细胞中发现的GSH和GSSG的浓度范围内。我们还确定，一氧化氮也强烈地抑制了蛋白酶的活性。这些结果可能与先前的报道有关，即缺血和氧化应激都会增加几种哺乳动物细胞中钙蛋白酶的活性，而这两个过程都已知会降低GSH和GSSG。我们现在正在研究酒精暴露如何改变GSH、GSSG和NO，作为酒精诱导的蛋白酶活性改变的可能机制。

Certain types of cellular cysteine proteases may play an important role in modulating the activity of G-protein coupled receptors. The activity of this class of enzymes is closely regulated by cytoplasmic and nuclear inhibitors. The effects of ethyl alcohol exposure on cysteine protease activity has been studied in cell culture utilizing a PC12 cell line. We have previously established that exposure of PC12 cells to ethyl alcohol for 96 hours results in a decrease in calcium-stimulated protease activity. We developed assays to measure GSH, GSSG, and NO and we have determined that both GSH and GSSG are important modulators of calpain activity. GSH is an uncompetitive inhibitor while GSSG is a competitive inhibitor of calpains, with inhibition constants well within range of concentrations of GSH and GSSG found in cells. We have also determined that nitric oxide also strongly inhibits protease activity. These results may relate to the previous reports that both ischemia and oxidative stress increase calpain activity in several mammalian cell types, and both processes are known to decrease both GSH and GSSG. We are now examining how alcohol exposure alters GSH, GSSG and NO as possible mechanisms for alcohol induced alterations in protease activity.