J Appl Biomed 6:211-224, 2008 | DOI: 10.32725/jab.2008.025

Role of intracellular calcium on hydrogen peroxide-induced apoptosis in rat pancreatic acinar AR42J cells

Sara Morgado, María P. Granados, Ignacio Bejarano, José J. López, Ginés M. Salido, Antonio González, José A. Pariente^*

Department of Physiology, University of Extremadura, 06071-Badajoz, Spain

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 by hydrogen peroxide and thapsigargin is mediated by mitochondrial calcium uptake.

Keywords: programed cell death; caspase-3; cytochrome c; mitochondrion; thapsingargin; dimethyl-BAPTA; Ru360

Received: October 23, 2008; Revised: November 12, 2008; Published: July 31, 2008  Show citation