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; caspase-3; cytochrome c; mitochondrion; thapsingargin; dimethyl-BAPTA; Ru360
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