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 IC(50)) at human and rat P2X(7) receptors. However, there were species differences in antagonist potency with PPADS, pyridoxal 5'-phosphate (P5P), and periodate-oxidized ATP (OxATP) exhibiting 20- to 500-fold higher potency for human than for mouse P2X(7) receptors. HMA (5-(N,N-hexamethylene)amiloride) was also selective for human over rat P2X(7) receptors but potentiated responses at mouse P2X(7) receptors. Coomassie Brilliant Blue G (CBB) was a nonselective antagonist with high potency at mouse P2X(7) receptors (IC(50) approximately 100 nM). All compounds were noncompetitive antagonists, and potency could only be quantified by measuring IC(50) values. These values were similar when determined against EC(50) concentrations of ATP or 2'- and 3'-O-4(-benzoylbenzoyl)-ATP and, for most compounds, only slightly (3- to 5-fold) affected by agonist concentration. However, IC(50) values for KN62 (1-[N,O-bis(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazine) and suramin, varied up to 25-fold depending upon agonist concentration. Furthermore, IC(50) values for KN62 and OxATP were 10-fold lower at 22 degrees C than at 37 degrees C, whereas IC(50) values for PPADS, P5P, suramin, and OxATP were up to 20-fold lower in NaCl than in sucrose buffer. Potency estimates for CBB and PPADS decreased 5-fold in the presence of bovine serum albumin, possibly due to protein binding. 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.