Identification and characterization of an endogenous P2X7 (P2Z) receptor in CHO-K1 cells.
CHO-K1 cells were examined for their cellular responses to the P2 receptor agonist, 2'- and 3'-O-(4-benzoylbenzoyl)-ATP (DbATP), and for the presence of mRNA for P2X receptors. Reverse transcriptase-polymerase chain reactions, using primers directed against the rat P2X subunits, detected the presence of P2X7 but not P2X1-P2X6 subunits. DbATP (EC50 approximately equal to 100 microM) evoked non-desensitizing inward currents which reversed at approximately equal to 0 mV, suggesting activation of a non-selective cation channel. ATP also evoked inward currents but was less potent than DbATP. DbATP also stimulated the accumulation of 45calcium (45Ca2+) and the DNA binding dye, YO-PRO-1, in CHO-KI cells. Both responses were inhibited by NaCl and MgCl2. In 280 mM sucrose buffer, 45Ca2+ accumulation was measurable within 10-20 s of agonist addition, whereas YO-PRO-1 accumulation was only detectable after 8 min. ATP and ATPgammaS were also agonists but were less potent than DbATP, while UTP, 2-methylthio ATP, ADP and (alphabeta)methylene ATP were inactive at concentrations up to 100 microM. DbATP increased lactate dehydrogenase release from CHO-K1 cells, suggesting cell lysis, although this effect was only pronounced after 60-90 min. These data suggest that CHO-K1 cells express an endogenous P2X7 receptor which can be activated by DbATP to cause a rapid inward current and accumulation of 45Ca2+. Prolonged receptor activation results in a delayed, increased permeability to larger molecules such as YO-PRO-1 and ultimately leads to cell lysis. Importantly, the presence of an endogenous P2X7 receptor should be considered when these cells are used to study recombinant P2X receptors.
Michel
A D
AD
Glaxo Institute of Applied Pharmacology, Department of Pharmacology, University of Cambridge.
Chessell
I P
IP
Hibell
A D
AD
Simon
J
J
Humphrey
P P
PP
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D000345:Affinity Labels / Q000494:pharmacology; D000818:Animals; D001583:Benzoxazoles; D016466:CHO Cells / Q000378:metabolism / Q000648:ultrastructure*; D002118:Calcium / Q000493:pharmacokinetics; D002132:Calcium Radioisotopes; D006224:Cricetinae; D018076:DNA, Complementary / Q000235:genetics / Q000378:metabolism; D004594:Electrophysiology; D005456:Fluorescent Dyes / Q000493:pharmacokinetics; D005784:Gene Amplification; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D058910:Purinergic P2 Receptor Agonists; D011806:Quinolinium Compounds; D012333:RNA, Messenger / Q000378:metabolism; D018048:Receptors, Purinergic P2 / Q000235:genetics / Q000502:physiology*; D058486:Receptors, Purinergic P2X7; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013268:Stimulation, Chemical
D000345:Affinity Labels; D001583:Benzoxazoles; D002132:Calcium Radioisotopes; D018076:DNA, Complementary; D005456:Fluorescent Dyes; C546470:P2rx7 protein, rat; D058910:Purinergic P2 Receptor Agonists; D011806:Quinolinium Compounds; D012333:RNA, Messenger; D018048:Receptors, Purinergic P2; D058486:Receptors, Purinergic P2X7; C089813:YO-PRO 1; C033901:3'-O-(4-benzoyl)benzoyladenosine 5'-triphosphate; D000255:Adenosine Triphosphate; D002118:Calcium