Characterization of P2X3, P2Y1 and P2Y4 receptors in cultured HEK293-hP2X3 cells and their inhibition by ethanol and trichloroethanol

Membrane currents and changes in the intracellular Ca2+ concentration ([Ca2+](i)) were measured in HEK293 cells transfected with the human P2X(3) receptor (HEK293-hP2X(3)). RT-PCR and immunocytochemistry indicated the additional presence of endogenous P2Y(1) and to some extent P2Y(4) receptors. P2 receptor agonists induced inward currents in HEK293-hP2X(3) cells with the rank order of potency alpha,beta-meATP approximate to ATP > ADP-beta-S > UTP. A comparable rise in [Ca2+](i) was observed after the slow superfusion of ATP, ADP-beta-S and UTP; alpha,beta-meATP was ineffective. These data, in conjunction with results obtained by using the P2 receptor antagonists TNP-ATP, PPADS and MRS2179 indicate that the current response to alpha,beta-meATP is due to P2X(3) receptor activation, while the ATP-induced rise in [Ca2+](i) is evoked by P2Y(1) and P2Y(4) receptor activation. TCE depressed the alpha,beta-meATP current in a manner compatible with a non-competitive antagonism. The ATP-induced increase of [Ca2+](i) was much less sensitive to the inhibitory effect of TCE than the current response to alpha,beta-meATP. The present study indicates that in HEK293-hP2X(3) cells, TCE, but not ethanol, potently inhibits ligand-gated P2X(3) receptors and, in addition, moderately interferes with G protein-coupled P2Y(1) and P2Y(4) receptors. Such an effect may be relevant for the interruption of pain transmission in dorsal root ganglion neurons following ingestion of chloral hydrate or trichloroethylene.