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

Naunyn Schmiedebergs Arch Pharmacol. 2005 Jun;371(6):466-72. doi: 10.1007/s00210-005-1070-6. Epub 2005 Jul 16.


ATP, ADPbetaS 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 ADPbetaS 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 ADPbetaS, 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 ADPbetaS, 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 ADPbetaS; 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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenosine Triphosphate / analogs & derivatives
  • Adenosine Triphosphate / pharmacology
  • Calcium Signaling / drug effects
  • Cell Line
  • Chelating Agents
  • Fluorescent Dyes
  • Fura-2
  • Gene Expression
  • Humans
  • Purinergic P2 Receptor Agonists
  • RNA, Messenger / metabolism
  • Receptors, Purinergic P2 / biosynthesis*
  • Receptors, Purinergic P2 / genetics
  • Receptors, Purinergic P2Y1
  • Receptors, Purinergic P2Y2
  • Thionucleotides / pharmacology
  • Uridine Triphosphate / pharmacology


  • Chelating Agents
  • Fluorescent Dyes
  • P2RY1 protein, human
  • P2RY2 protein, human
  • Purinergic P2 Receptor Agonists
  • RNA, Messenger
  • Receptors, Purinergic P2
  • Receptors, Purinergic P2Y1
  • Receptors, Purinergic P2Y2
  • Thionucleotides
  • purinoceptor P2Y4
  • adenosine 5'-O-(2-thiotriphosphate)
  • Adenosine Triphosphate
  • Fura-2
  • Uridine Triphosphate