A selective binding site for 3H-NECA that is not an adenosine A 2 receptor

Abstract

In homogenates of NG108-15 cells, adenosine analogues activate adenylate cyclase with the following order of potency: N- ethylcarboxamidoadenosine ( NECA) >2- chloroadenosine >N 6-( L- phenylisopropyl)- adenosine (PIA) = cyclohexyladenosine = 2-phenylaminoadenosine. Adenosine receptor antagonists inhibit NECA-stimulated adenylate cyclase activity with the order of potency 3-isobutyl-1-methyl-xanthine (IBMX)>theophyllin > caffeine. These data suggest that these ligands act at an adenosine A 2 receptor. There is an apparently homogenous population of saturable 3H-NECA binding sites in homogenates of NG108-15 cells. These sites have an affinity for 3H-NECA of t~1 ωM, and are present at a density of ≈10 pmol/mg protein. Unlabelled NECA, 2-chloroadenosine, IBMX and theophylline displace 3H-NECA binding, with an order of potency that suggests that the 3H-NECA binding site may represent an adenosine A 2 receptor. However, PIA, cyclohexyladenosine and 2-phenylaminoadenosine produce no detectable displacement of 3H-NECA binding at concentrations that produce a maximal stimulation of adenylate cyclase activity. Pretreatment of NG108-15 cells with either NECA or PIA produces a homologous desensitization of subsequent responses to all the adenosine analogues, with no effect on subsequent responses to a prostacyclin receptor agonist or NaF. This suggests that all the adenosine analogues examined activate an adenosine A 2 receptor. Therefore, the 3H-NECA site at which PIA is inactive cannot represent this receptor.