INCREASES OF INTRACELLULAR MAGNESIUM PROMOTE GLYCODEOXYCHOLATE-INDUCED APOPTOSIS IN RAT HEPATOCYTES

Retention of bile salts by the hepatocyte contributes to liver injury during cholestasis. Although cell injury can occur by one of two mechanisms, necrosis versus apoptosis, information is lacking regarding apoptosis as a mechanism of cell death by bile salts. Our aim was to determine if the bile salt glycodeoxycholate (GDC) induces apoptosis in rat hepatocytes. Morphologic assessment included electron microscopy and quantitation of nuclear fragmentation by fluorescent microscopy. Biochemical studies included measurements of DNA fragmentation, in vitro endonuclease activity, cytosolic free Ca2+ (Ca-i(2+)), and cytosolic free Mg2+ (Mg-i(2+)). Morphologic studies demonstrated typical features of apoptosis in GDC (50 mu M) treated cells. The ''ladder pattern'' of DNA fragmentation was also present in DNA obtained from GDC-treated cells. In vitro endonuclease activity was 2.5-fold greater with Mg2+ than Ca2+. Although basal Ca-i(2+) values did not change after addition of GDC, Mg-i(2+) increased twofold. Incubation of cells in an Mg2+-free medium prevented the rise in Mg-i(2+) and reduced nuclear and DNA fragmentation. In conclusion, GDC induces apoptosis in hepatocytes by a mechanism promoted by increases of Mg-i(2+) with stimulation of Mg2+-dependent endonucleases. These data suggest for the first time that changes of Mg-i(2+) may participate in the program of cellular events culminating in apoptosis.