Oxidized ATP decreases beta-actin expression and intracellular superoxide concentrations in RBA-2 type-2 astrocytes independently of P2X7 receptor.

Periodate-oxidized 2',3'-dialdehyde ATP (oxidized ATP) has been used extensively as a selective antagonist at P2X(7) receptors, although P2X(7)-independent actions on pro-inflammatory cytokine release have also been reported. Because P2X(7) receptors in astrocytes have been suggested as potential targets of anti-inflammatory drug therapy, we examined the effect of oxidized ATP on beta-actin expression and superoxide production of RBA-2 type-2 astrocytes known to possess P2X(7) receptors. Oxidized ATP per se decreased beta-actin expression time and dose dependently. Treatment with oxidized ATP for 8 h caused an approximately 50% decrease in beta-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 P2X(7) 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-1beta, converting enzyme (ICE)-like protease activity in these astrocytes. To further elucidate the mechanism, the effects of oxidized ATP on intracellular superoxide concentration and beta-actin expression were examined in a P2X(7) 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 beta-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 beta-actin expression in RBA-2 type-2 astrocytes. In addition, oxidized ATP decreased intracellular superoxide concentrations and altered f-actin cytoskeleton arrangement in both P2X(7) 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 P2X(7) receptors.

Elucidation of ATP-stimulated stress protein expression of RBA-2 type-2 astrocytes: ATP potentiate HSP60 and Cu/Zn SOD expression and stimulates pI shift of peroxiredoxin II.

ATP has been shown to mediate stress responses in the brain. The present study examined the ATP-stimulated stress protein expression of RBA-2 type-2 astrocytes. Our results revealed that ATP stimulated HSP60 expression in a dose- and time-dependent manner. The stimulation requires a minimal ATP concentration of 500 microM and high concentration of extracellular ATP (1 mM) stimulated a significant increase of HSP60 expression from 2 to 24 h. In addition, the ATP-stimulated HSP60 expressions were inhibited by inhibitors for protein kinase C (PKC) and phospholipase D (PLD), and by antioxidants, resveratrol, and catalase. Furthermore, ATP stimulated the expression of Cu/Zn superoxide dismutase (SOD). In addition, ATP and P2X7 receptor selective agonist BzATP also decreased mitochondria membrane potential measured by flow cytometry. To further examine the proteins involving in ATP-mediated stress responses, we conducted proteomic analysis. We found that RBA-2 astrocytes possess abundant peroxiredoxin II (Prx II), an antioxidant enzyme. ATP and exogenous H2O2 stimulated Prx II shifting from oxidized form to reduced form. Thus, we concluded that ATP potentiated the expression of HSP60 and Cu/Zn SOD, and decreased mitochondria membrane potential. In addition, RBA-2 astrocytes expressed Prx II that might also serve as a protective mechanism to control the concentration of reactive oxygen species.

Roles of protein kinase C in regulation of P2X7 receptor-mediated calcium signalling of cultured type-2 astrocyte cell line, RBA-2.

The role of protein kinase C (PKC) on regulation of P2X(7) receptor-mediated Ca(2+) signalling was examined on RBA-2 astrocytes. Activation of PKC decreased the receptor-mediated Ca(2+) signalling and the decrease was restored by PKC inhibitors. Down regulation of PKC also caused a decrease in the Ca(2+) signalling. Thus PKC might play a dual role on the P2X(7) receptor signalling. Successive stimulation of the P2X(7) receptor induced a gradual decline of Ca(2+) signalling but PKC inhibitors failed to restore the decline. Nevertheless, PMA stimulated translocation of PKC-alpha, -betaI, -betaII, and -gamma, but only anti-PKC-gamma co-immunoprecipitated the receptors. To examine the role of PKC-gamma, Ca(2+) signalling was measured by Ca(2+) imaging. Our results revealed that the agonist-stimulated Ca(2+) signalling were reduced in the cells that the transfection of either P2X(7) receptor or PKC-gamma morpholino antisense oligo was identified. Thus, we concluded that PKC-gamma interacted with P2X(7) receptor complex and positively regulated the receptor-mediated Ca(2+) signalling.