Puerarin protects rat pancreatic islets from damage by hydrogen peroxide

Periodate-oxidized 2′,3′-dialdehyde ATP (oxidized ATP) has been used extensively as a selective antagonist at P2X₇ receptors, although P2X₇-independent actions on pro-inflammatory cytokine release have also been reported. Because P2X₇ receptors in astrocytes have been suggested as potential targets of anti-inflammatory drug therapy, we examined the effect of oxidized ATP on β-actin expression and superoxide production of RBA-2 type-2 astrocytes known to possess P2X₇ receptors. Oxidized ATP per se decreased β-actin expression time and dose dependently. Treatment with oxidized ATP for 8 h caused an approximately 50% decrease in β-actin expression whereas other P2 receptor antagonists, brilliant blue G (BBG), suramin and pyridoxal phosphate-6-azophenyl-2′,4′-disulfonic acid (PPADS), were not effective. In addition, oxidized ATP per se decreased the intracellular superoxide concentration, whereas ATP and the P2X₇ receptor-selective agonist 3′-O-(4-benzoylbenzoyl)adenosine 5′-triphosphate (BzATP) stimulated intracellular superoxide production, an effect inhibited by oxidized ATP. In addition, oxidized ATP neither affected cellular viability nor affected interleukin-1β, converting enzyme (ICE)-like protease activity in these astrocytes. To further elucidate the mechanism, the effects of oxidized ATP on intracellular superoxide concentration and β-actin expression were examined in a P2X₇ receptor-negative astrocyte cell line, IA-1g1. Oxidized ATP-induced a time-dependent decrease in intracellular superoxide concentration whereas oxidized ATP had no effect on β-actin expression. Nevertheless, oxidized ATP altered f-actin cytoskeleton arrangement in IA-1g1 astrocytes. Taken together, these results indicate that oxidized ATP per se caused a cell specific decrease in β-actin expression in RBA-2 type-2 astrocytes. In addition, oxidized ATP decreased intracellular superoxide concentrations and altered f-actin cytoskeleton arrangement in both P2X₇ receptor-positive and -negative astrocytes. Thus, we conclude from these results that the effects of oxidized ATP on actin and superoxide are mediated through mechanisms that are at least in part, independent of P2X₇ receptors.