Calcium-activated neutral protease activity is decreased in PC12 cells after ethanol exposure

Calcium-activated neutral protease activity was determined in PC12 cells exposed to ethanol for 96 h using a fluorescence-based assay with N-succinyl-Leu-Tyr 7-amido-4-methylcoumarin as the substrate, Stimulated activity was measured at high (1,400 mu M) or low (140 mu M) Ca2+ concentrations in the presence of 20 mu M ionomycin. Kinetic parameters were derived by fitting a model relating fluorescence intensity to time: F-t = F-final* (1 - e(-kobst)). Cell extracts were subjected to nondenaturing gel electrophoresis and casein zymography with quantification of the activity of the two calpain isoforms. Exposure to ethanol significantly decreased whole cell calpain activity measured by k(obs) beginning at 20 mM, to 27.8% of control at 1,400 mu M Ca2+ and 29.2% of control at 140 mu M Ca2+ in the presence of 20 mu M ionomycin. No changes in mu-calpain or m-calpain activities were found in cell extracts from cells exposed to 20 mM ethanol, whereas at 40 and 80 mM ethanol, significant decreases in both mu-calpain and m-calpain activities were discovered.