Complexities of measuring antagonist potency at P2X(7) receptor orthologs

The ability of P2 antagonists to affect agonist-stimulated fluorescent dye accumulation in cells expressing human, rat, or mouse P2X(7) receptors was examined. Several compounds, including pyridoxalphosphate-6-azophenyl-2',4' -disulfonic acid (PPADS), which was previously thought to be a weak P2X(7) receptor antagonist, possessed high potency (nanomolar IC50) at human and r at P2X(7) receptors. However, there were species differences in antagonist potency with PPADS, pyridoxal 5'-phosphate (P5P), and periodate-oxidized AT P (OxATP) exhibiting 20- to 500-fold higher potency for human than for mous e P2X(7) receptors. HMA (5-(N,N-hexamethylene) amiloride) was also selectiv e for human over rat P2X(7) receptors but potentiated responses at mouse P2 X(7) receptors. Coomassie Brilliant Blue G (CBB) was a nonselective antagon ist with high potency at mouse P2X(7) receptors (IC50 similar to 100 nM). A ll compounds were noncompetitive antagonists, and potency could only be qua ntified by measuring IC50 values. These values were similar when determined against EC50 concentrations of ATP or 2'- and 3'-O-4(-benzoylbenzoyl)-ATP and, for most compounds, only slightly (3- to 5-fold) affected by agonist c oncentration. However, IC50 values for KN62 (1-[N,O-bis(5-isoquinolinesulfo nyl)- N-methyl-L-tyrosyl]-4-phenylpiperazine) and suramin, varied up to 25- fold depending upon agonist concentration. Furthermore, IC50 values for KN6 2 and OxATP were 10-fold lower at 22 degrees C than at 37 degrees C, wherea s IC50 values for PPADS, P5P, suramin, and OxATP were up to 20-fold lower i n NaCl than in sucrose buffer. Potency estimates for CBB and PPADS decrease d 5-fold in the presence of bovine serum albumin, possibly due to protein b inding. Given the species differences, and the effects of assay conditions on antagonist potency, caution must be exercised when interpreting results obtained with the available antagonists.