Antagonist effects on human P2X(7) receptor-mediated cellular accumulationof YO-PRO-1

1 We have examined the interaction of P2 antagonists with the human P2X(7) receptor by studying their effect on 2' and 3'-O-benzoyl-benzoyl-ATP (DbATP ) stimulated cellular accumulation of the fluorescent, DNA binding dye, YO- PRO-1 (MW = 375Da). 2 In suspensions of HEK293 cells expressing human recombinant P2X(7) recept ors, DbATP produced time and concentration-dependent increases in YO-PRO-1 fluorescence. This response presumably reflects YO-PRO-1 entry through P2X( 7) receptor channels and binding to nucleic acids. When studies were perfor med in a NaCl-free, sucrose-containing buffer, full concentration-effect cu rves to DbATP could be constructed. 3 The P2 antagonists, pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS) and periodate oxidized ATP (oATP), reduced the potency of DbATP an d decreased its maximum response. 1-[N,O-bis(1,5-isoquinolinesulphonyl)-N-m ethyl-L-tyrosyl]-4-phenylpiperazine (KN62) and its analogue, KN04, reduced the potency of DbATP. Schild slopes for KN62 and KN04 were shallow and exhi bited a plateau at concentrations of compound greater than 1 mu M, indicati ng that these compounds were not competitive antagonists. 4 Callmidazolium and a monoclonal antibody to human P2X(7) receptors attenu ated DbATP-stimulated YO-PRO-1 accumulation but they were not competitive a ntagonists and only produced 2-3 fold decreases in the potency of DbATP. 5 The effects of PPADS and KN62 were partially reversible whereas those of oATP were not. PPADS protected cells against the irreversible antagonist ef fects of oATP suggesting a common site of action. In contrast KN62 was not effective suggesting that it may bind at a different site to oATP and PPADS . 6 This study has demonstrated that P2X(7) receptor function can be quantifi ed by measuring DbATP stimulated YO-PRO-1 accumulation and has provided add itional information about the interaction of P2 receptor antagonists with t he human P2X(7) receptor.