NF449: a subnanomolar potency antagonist at recombinant rat P2X(1) receptors

Antagonistic effects of the novel suramin analogue 4,4',4",4"'-(carbonylbis (imino-5,1,3-benzenetriyl-bis(carbonylimino)))tetrakis-benzene- 1,3-disulfo nic acid (NF449) were studied on contractions of the rat vas deferens elici ted by alpha,beta -methylene ATP ((alpha beta meATP; mediated by P2X(1) rec eptors), contractions of the guinea-pig ileal longitudinal smooth muscle el icited by (alpha beta meATP (mediated by P2X(3) receptors) or adenosine 5'- O-(2-thiodiphosphate) (ADP betaS; mediated by P2Y(1) receptors), ATP-induce d increases of [Ca2+](i), in human embryonic kidney (HEK) 293 cells (mediat ed by P2Y(2) receptors), inward currents evoked by ATP in follicle cell-fre e Xenopus laevis oocytes expressing rP2X(1) or rP2X(3) receptors and degrad ation of ATP by ecto-nucleotidases in folliculated Xenopus laevis oocytes. In addition, NF449 was examined for its P2 receptor specificity in rat vas deferens (alpha (1A)-adrenoceptors) and guinea-pig ileum (histamine H-1 and muscarinic M-3 receptors). At native (pIC(50)=7.15) and recombinant (pIC(50)=9.54) P2X(1) receptors, N F449 was a highly potent antagonist. The P2X3 receptors present in guinea-p ig ileum (pIC(50)= 5.04) or expressed in oocytes (pIC(50)approximate to5.6) were much less sensitive for NF449. It also was a very weak antagonist at P2Y(1) receptors in guinea-pig ileum (pIC(50)=4.85) and P2Y(2) receptors in HEK 293 cells (pIC(50)=3.86), and showed very low inhibitory potency on ec to-nucleotidases (pIC(50)<3.5). NF449 (100 muM) did not interact with alpha (1A)-adrenoceptors or histamine H-1 and muscarinic M-3 receptors. Thus, th e antagonism by NF449 is highly specific for P2 receptors. In conclusion, the subnanomolar potency at rP2X(1) receptors and the rank o rder of potency, P2X(1) > > P2X(3) > P2Y(1) > P2Y(2) > ecto-nucleotidases, make NF449 unique among the P2 receptor antagonists reported to date. NF449 may fill the long-standing need for a P2X(1)-selective radioligand.