Evidence for the existence of P2Y12,4 receptor subtypes in HEK-293 cells:: reactivation of P2Y1 receptors after repetitive agonist application

ATP, ADP beta S and UTP induced a comparable rise in the intracellular Ca2+ concentration ([Ca2+](i)) in HEK-293 cells using fura-2 microfluorimetry. The responses persisted in Ca2+-free medium, but were abolished following depletion of intracellular Ca2+ stores by cyclopiazonic acid. Cross-desensitisation experiments demonstrated that exposure to ADPOS has no marked effect on UTP-induced [Ca2+]i transients and vice versa. 6 Whereas the P2Y(1) receptor-selective antagonist 2'-deoxy-N-6-methyladenosine 3',5'-diphosphate (MRS 2179) abolished the responses to ADP S, it decreased and did not alter the responses to ATP and UTP respectively. Although the P2Y(1)/ P2Y(4) receptor-preferential antagonist pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS) abolished the responses to ADPOS, and decreased those to ATP, it also depressed the UTP-induced [Ca2+](i) transients. Suramin, an antagonist with preference for P2Y(2) receptors decreased both the ATP- and UTP-induced [Ca2+](i) reactions. After numerous splittings, HEK-293 cells failed to react to ADPOS; however, repeated superfusion with this P2Y(1) receptor agonist restored the [Ca2+](i) signals. In agreement with the functional data, real-time polymerase chain reaction and immunocytochemical studies indicated the presence of P2Y(1), P2Y(2) and P2Y(4) receptors. Our findings raise doubt with respect to the reliability of HEK-293 cells as expression systems for recombinant P2X receptors, because of a possible functional interaction with endogenous P2Y receptors.