Evidence for the existence of P2Y1,2,4 receptor subtypes in HEK-293 cells: reactivation of P2Y1 receptors after repetitive agonist application.
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.
Fischer
Wolfgang
W
Rudolf-Boehm-Institut für Pharmakologie und Toxikologie, Universität Leipzig, Haertelstrasse 16-18, 04107 Leipzig, Germany. Wolfgang.Fischer@medizin.uni-leipzig.de
Franke
Heike
H
Gröger-Arndt
Helke
H
Illes
Peter
P
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D020013:Calcium Signaling / Q000187:drug effects; D002460:Cell Line; D002614:Chelating Agents; D005456:Fluorescent Dyes; D016257:Fura-2; D015870:Gene Expression; D006801:Humans; D058910:Purinergic P2 Receptor Agonists; D012333:RNA, Messenger / Q000378:metabolism; D018048:Receptors, Purinergic P2 / Q000096:biosynthesis* / Q000235:genetics; D058472:Receptors, Purinergic P2Y1; D058473:Receptors, Purinergic P2Y2; D013873:Thionucleotides / Q000494:pharmacology; D014544:Uridine Triphosphate / Q000494:pharmacology
D002614:Chelating Agents; D005456:Fluorescent Dyes; C546382:P2RY1 protein, human; C546385:P2RY2 protein, human; D058910:Purinergic P2 Receptor Agonists; D012333:RNA, Messenger; D018048:Receptors, Purinergic P2; D058472:Receptors, Purinergic P2Y1; D058473:Receptors, Purinergic P2Y2; D013873:Thionucleotides; C104749:purinoceptor P2Y4; C022570:adenosine 5'-O-(2-thiotriphosphate); D000255:Adenosine Triphosphate; D016257:Fura-2; D014544:Uridine Triphosphate