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SUMMARY The authors investigated whether cytosolic free calcium concentration ([Ca2+]c) plays a role in hydrogen peroxide-induced pancreatic acinar AR42J cells apoptosis. We analysed mitochondrial depolarization, [Ca2+]c determination and caspase-3 activity by fluorimetric methods, and cytochrome c release by subcellular fractionation and western blotting. The data shown that hydrogen peroxide, which causes a sustained [Ca2+]c increase, induces mitochondrial depolarization and cytochrome c release, and activation of caspase-3. Dimethyl-BAPTA loading did not affect hydrogen peroxide-evoked mitochondrial apoptosis, suggesting that these responses are independent of increases in [Ca2+]c. Treatment with thapsigargin, to induce extensive calcium store depletion and subsequent increases in [Ca2+]c, also stimulates mitochondrial depolarization , cytochrome c release, and caspase-3 activation. Similar results were observed in AR42J cells loaded with dimethyl-BAPTA, suggesting that activation of apoptosis by thapsigargin does not require rises in [Ca2+]c. However, the blockade of mitochondrial calcium entry by pretreating with Ru360 showed protection against hydrogen peroxide- and thapsigargin-induced mitochondrial apoptosis. These results indicate that the apoptosis evoked y hydrogen peroxide and thapsigargin is mediated by mitochondrial calcium uptake. KEY WORDS programed cell death; casase-3; cytochrome c;mitochondrion; thapsingargin; dimethyl-BAPTA; Ru360; REFERENCES Ashkenazi A, Dixit VM: Death receptors: signaling and modulation. Science 281:1305-1308, 1998. Bejarano I, Lozano GM, Ortiz, Garcia JF, Paredes SD, Rodriguez AB, Pariente JA: Caspase 3 activation in human spermatozoa in response to hydrogen peroxide and progesterone. Fertility and Sterility 90:1340-1347, 2008. 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