Ligand recognition sites on P2X receptors studied by quantitative autoradiography of [3H]alpha,beta-methylene-ATP binding in rat brain.
The specificity of alpha, beta-methylene-ATP for P2X receptor binding sites in the CNS has been examined by testing the effects of several ATP analogues and other ATP-related substances on the binding of 10 nM [3H]alpha,beta-methylene-ATP to 20 microns thick sections of fresh-frozen rat brain. The labelling of the putative P2X receptor binding sites by [3H]alpha,beta-methylene-ATP was evaluated by quantitative densitometry. [3H]alpha,beta-methylene-ATP binding was strongly inhibited by two close ATP analogues, 3'-O-(trinitrophenyl)-adenosine-5'-triphosphate and beta,gamma-imido-ATP (IC50 2.5 microM). beta,gamma-Methylene-ATP was, however, less potent (< 50% inhibition at 25 microM). Inosine-5'-triphosphate, guanosine-5'-triphosphate, uridine-5'-triphosphate, and cytidine-5'-triphosphate were practically inactive up to concentrations of 100 microM. Periodate oxidised ATP and 1, N6-etheno-ATP produced < 50% inhibition at 100 and 500 microM concentrations, respectively. Cations (K+, Rb+, Cs+, and Mg2+ at 5 mM and Na+ at 150 mM) reduced [3H]alpha,beta-methylene-ATP binding by no more than 50%. Several agents known to interact with Ca2+- and/or ATP-related cationic channels (Cd2+, glibenclamide, dantrolene, nifedipine, and thapsigargin) had no effect. We conclude that [3H]alpha,beta-methylene-ATP at low nanomolar concentrations binds to a site that has very strict structural requirements and is pharmacologically similar to ATP P2X receptors.
Department of Anatomy and Histology, University of Sydney, NSW, Australia.
R A
RA
Worthington
M A
MA
Hansen
M R
MR
Bennett
J A
JA
Barden
V J
VJ
Balcar
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D000818:Animals; D001345:Autoradiography; D001665:Binding Sites; D001921:Brain / Q000378:metabolism*; D008024:Ligands; D051381:Rats; D017207:Rats, Sprague-Dawley; D018048:Receptors, Purinergic P2 / Q000378:metabolism*
D008024:Ligands; D018048:Receptors, Purinergic P2; D000255:Adenosine Triphosphate; C002630:alpha,beta-methyleneadenosine 5'-triphosphate