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- chloromethyl-coumarin 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+i decreased to values of 1.5-fold above basal and protease activity returned to normal. Incubation of cells in Ca(2+)-free buffer abolished the rise in Ca2+i and calpain protease activity. Calpain protease activity increases concomitantly with increases of Ca2+i supporting the hypothesis that calpain proteases participate in Ca(2+)-mediated signal transduction.
Rosser
B G
BG
Department of Internal Medicine, Mayo Medical School, Rochester, Minnesota 55905.
Powers
S P
SP
Gores
G J
GJ
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D002154:Calpain / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism; D005978:Glutathione / Q000378:metabolism; D008099:Liver / Q000201:enzymology*; D008297:Male; D008856:Microscopy, Fluorescence; D051381:Rats; D017207:Rats, Sprague-Dawley
D005456:Fluorescent Dyes; D000255:Adenosine Triphosphate; D002154:Calpain; D005978:Glutathione