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

ATP, ADPS 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 ADPS 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 ADPS, 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 ADPS, 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 ADPS; 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.