Calpain Activity Increases in Hepatocytes Following Addition

Abstract

Our aim was to measure calpain protease activity during increases in cytosolic free calcium (Ca2+J after addition of extracellular ATP. The calpain protease substrate t-butoxycarbonyl-Leu-Met-7-amino-4-chloromethylcoumarin was synthesized. Nonfluorescent tbutoxycarbonyl-Leu-Met-7-amino-4-chloromethylcoumarin diffuses into the cell where it is conjugated to glutathione forming t-butoxycarbonyl-Leu-Met-7amino-4-methylcoumarin glutathione conjugate (BocLeu-Met-MAC-SG). The nonfluorescent, membrane impermeant Boc-Leu-Met-MAC-SG accumulates in the cell. Intracellular proteolytic hydrolysis of Boc-LeuMet-MAC-SG releases and unquenches the fluorescence of MAC-SG. Intracellular fluorescence of MACSG 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 166 2 13% of basal concurrent with a %fold rise of Ca2+i for 2-4 min. Thereafter, Ca2+i decreased to values of 1.6-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.

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