[Single cell analysis of H(2)O(2) mediated Fas mRNA expression and Ca(2+) influx in LO(2) cells]

To investigate whether apoptotic injury is induced by Fas mRNA expression mediated by H(2)O(2) and concerned with intracellular Ca(2+) unbalance at a single cell level.

Fas mRNA expression in human hepatocyte (LO(2) cell) was analyzed by whole-cell patch-clamp combined with single cell reverse transcription polymerase chain reaction (RT-PCR). Cytosolic Ca(2+) concentration was measured with another whole-cell patch-clamp combined with single-cell microfluorescence [Ca(2+)] i measurement at the same time. The index of early phase apoptotic cells (Annexin-V(+) cells) measured with flow cytometry (FCM) and the hepatocytic morphological changes were observed by scanning electron microscopy.

Fas mRNA of LO(2) cells cultured with H(2)O(2) (10 micromol/L) was expressed at 2 h. The single cell [Ca(2+)] i analyzed by another whole-cell patchclamp increased to 1 115 nmol/L +/- 227 nmol/L (P < 0.01, t = 13.37); at the same time, an elevated Ca(2+) wave was recorded. The index of Annexin-V(+) cells was as high as 16.18 +/- 0.65 (P < 0.01, t = 48.41), and the membrane surface protuberances were observed.

The activation of Fas mRNA, as a major apoptotic factor mediated by H(2)O(2) in LO(2) cells, is probably associated with the imbalance of intracellular Ca(2+).