Pharmacological characterization of heterologously expressed ATP-gated cation channels (P2x purinoceptors).
cDNAs encoding P2x purinoceptors from human bladder smooth muscle and from rat PC-12 cells were expressed in oocytes and human embryonic kidney 293 cells. Agonist potencies of 2-methylthio-ATP = 2-chloro-ATP = ATP > = 2'- and 3'-O-(4-benzoylbenzoyl)-ATP > or = adenosine-5'-O-(3-thio)-triphosphate > or = P1,P5-di(adenosine-5') pentaphosphate >> ADP prevailed for both P2x purinoceptors. There were two main differences in agonist sensitivity between the two receptors. First, ATP was 10 times more potent at the receptor from bladder (EC50, 0.8 microM) than at the receptor from PC-12 cells (EC50, 8.2 microM). Second, alpha,beta-methylene-ATP and L- and D-beta,gamma-methylene-ATP were agonists in cells expressing the bladder smooth muscle receptor (EC50, 1-3 microM) but were ineffective in cells expressing the PC-12 receptor. The P2 purinoceptor antagonists suramin, pyridoxal phosphate 6-azophenyl-2',4'-disulfonic acid, and pyridoxal-5-phosphate acted similarly at both receptor forms, producing noncompetitive inhibition, with IC50 values of 1-5 microM for suramin and pyridoxal phosphate 6-azophenyl-2',4'-disulfonic acid and 10-20 microM for pyridoxal-5-phosphate. 4,4'-Diisothiocyanatostilbene-2,2'-disulfonic acid distinguished receptor subtypes, producing potent inhibition of the bladder smooth muscle P2x-mediated response, with an IC50 value of 3 microM; it inhibited the PC-12 form by < 40% at 100 or 300 microM. This study thus defines the pharmacological properties of homo-oligomeric forms of these two types of cloned P2x receptor channels.
Evans
R J
RJ
Glaxo Institute for Molecular Biology, Geneva, Switzerland.
Lewis
C
C
Buell
G
G
Valera
S
S
North
R A
RA
Surprenant
A
A
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000494:pharmacology; D000227:Adenine Nucleotides / Q000737:chemistry / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000502:physiology*; D000818:Animals; D002460:Cell Line; D003001:Cloning, Molecular; D018076:DNA, Complementary; D006801:Humans; D015640:Ion Channel Gating; D007473:Ion Channels / Q000187:drug effects* / Q000235:genetics / Q000502:physiology; D016716:PC12 Cells; D011732:Pyridoxal Phosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D051381:Rats; D018048:Receptors, Purinergic P2 / Q000187:drug effects* / Q000235:genetics / Q000502:physiology; D013498:Suramin / Q000494:pharmacology
D000227:Adenine Nucleotides; D018076:DNA, Complementary; D007473:Ion Channels; D018048:Receptors, Purinergic P2; C077792:pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid; D011732:Pyridoxal Phosphate; D013498:Suramin; D000255:Adenosine Triphosphate; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid