Vol. 58, Issue 6, 1502-1510, December 2000

Competitive Antagonism of Recombinant P2X_2/3 Receptors by 2',3'-O-(2,4,6-Trinitrophenyl) Adenosine 5'-Triphosphate (TNP-ATP)

Edward C. Burgard, Wende Niforatos, Tim van Biesen, Kevin J. Lynch, Karen L. Kage, Edward Touma, Elizabeth A. Kowaluk, and Michael F. Jarvis

Neurological and Urological Diseases Research, Pharmaceutical Products Division, Abbott Laboratories, Abbott Park, Illinois

TNP-ATP has become widely recognized as a potent and selective P2X receptor antagonist, and is currently being used to discriminate between subtypes of P2X receptors in a variety of tissues. We have investigated the ability of TNP-ATP to inhibit ,-methylene ATP (,-meATP)-evoked responses in 1321N1 human astrocytoma cells expressing recombinant rat or human P2X_2/3 receptors. Pharmacological responses were measured using electrophysiological and calcium imaging techniques. TNP-ATP was a potent inhibitor of P2X_2/3 receptors, blocking both rat and human receptors with IC₅₀ values of 3 to 6 nM. In competition studies, 10 to 1000 µM ,-meATP was able to overcome TNP-ATP inhibition. Schild analysis revealed that TNP-ATP was a competitive antagonist with pA₂ values of 8.7 and 8.2. Inhibition of P2X_2/3 receptors by TNP-ATP was rapid in onset, reversible, and did not display use dependence. Although the onset kinetics of inhibition were concentration-dependent, the TNP-ATP off-kinetics were concentration-independent and relatively slow. Full recovery from TNP-ATP inhibition did not occur until 5 s after removal of the antagonist. Because of the slow off-kinetics of TNP-ATP, full competition with ,-meATP for receptor occupancy could be seen only after both ligands had reached a steady-state condition. It is proposed that the slowly desensitizing P2X_2/3 receptor allowed this competitive interaction to be observed over time, whereas the rapid desensitization of other P2X receptors (P2X₃) may mask the detection of competitive inhibition by TNP-ATP.