Calpain activity increases in hepatocytes following addition of ATP: Demonstration by a novel fluorescent approach

Our aim was to measure calpain protease activity during increases in cytosolic free calcium (Ca²⁺_i) after addition of extracellular ATP. The calpain protease substrate t-butoxycarbonyl-Leu-Met-7-amino-4-chloromethylcoumarin was synthesized. Nonfluorescent t-butoxycarbonyl-Leu-Met-7-amino-4-chloromethylcoumarin diffuses into the cell where it is conjugated to glutathione forming t-butoxycarbonyl-Leu-Met-7-amino-4-methylcoumarin glutathione conjugate (Boc-Leu-Met-MAC-SG). The nonfluorescent, membrane impermeant Boc-Leu-Met-MAC-SG accumulates in the cell. Intracellular proteolytic hydrolysis of Boc-Leu-Met-MAC-SG releases and unquenches the fluorescence of MAC-SG. Intracellular fluorescence of MAC-SG was quantitated in single, cultured rat hepatocytes using digitized video fluorescent microscopy. Enhancement of intracellular fluorescence generation by increases in Ca²⁺_i- and inhibition by a calpain inhibitor indicated the probe was a calpain substrate. After addition of ATP, calpain protease activity increased to 156 ± 13% of basal concurrent with a 3-fold rise of Ca²⁺_i for 2-4 min. Thereafter, Ca²⁺, decreased to values of 1.5-fold above basal and protease activity returned to normal. Incubation of cells in Ca²⁺-free buffer abolished the rise in Ca²⁺, and calpain protease activity. Calpain protease activity increases concomitantly with increases of Ca²⁺_i supporting the hypothesis that calpain proteases participate in Ca²⁺-mediated signal transduction.