Evidence for the existence of P2Y1,2,4 receptor subtypes in HEK-293 cells: reactivation of P2Y1 receptors after repetitive agonist application

ATP, ADPβS 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 ADPβS has no marked effect on UTP-induced [Ca2+]i transients and vice versa. Whereas the P2Y1 receptor-selective antagonist 2′-deoxy-N6-methyladenosine 3′,5′-diphosphate (MRS 2179) abolished the responses to ADPβS, it decreased and did not alter the responses to ATP and UTP respectively. Although the P2Y1/P2Y4 receptor-preferential antagonist pyridoxalphosphate-6-azophenyl-2′,4′-disulphonic acid (PPADS) abolished the responses to ADPβS, and decreased those to ATP, it also depressed the UTP-induced [Ca2+]i transients. Suramin, an antagonist with preference for P2Y2 receptors decreased both the ATP- and UTP-induced [Ca2+]i reactions. After numerous splittings, HEK-293 cells failed to react to ADPβS; however, repeated superfusion with this P2Y1 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 P2Y1, P2Y2 and P2Y4 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.