Characterization of Adenosine Receptors in the PC 12 Pheochromocytoma Cell Line Using Radioligand Binding: Evidence for A‐2 Selectivity

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Abstract
Examination of the binding characteristics of the adenosine agonist radioligands [3H]N6-cyclohexyladenosine ([3H]CHA), [3H]cyclopentyladenosine ([3H]CPA), and [3H]5''-N-ethylcarboxamido adenosine ([3H]NECA) to membranes prepared from PC12 cells showed that the A-1-selective ligands (CHA and CPA) had minimal binding, which was not amenable to analysis using curve-fitting programs. However, [3H]NECA, a nonselective A-1/A-2 agonist, gave reproducible binding, which was enhanced by removal of endogenous adenosine, using the catabolic enzyme adenosine deaminase. This binding was of high affinity (KD = 4.7 nM) with limited capacity (263 fmol/mg of protein). Specific binding of [3H]NECA was unaffected by the presence of either CPA (50 nM) or MgCl2 (10 mM) but was sensitive to guanylylimidodiphosphate (100 .mu.M), a finding suggesting involvement of an N-protein mechanism in the coupling of the adenosine receptor labeled by [3H]NECA to other components of the receptor complex. Binding of [3H]NECA to PC12 cell membranes was stereoselective, with the R isomer of N6-phenylisopropyladenosine (PIA) being .apprx. 12 times more active than S-PIA. The A-I-selective agonist CPA was a weak inhibitor of [3H]NECA binding (Ki = 251 nM). The rank order of activity of adenosine agonists in displacing specific [3H]NECA binding was NECA .ltoreq. 2-chloroadenosine > CHA .gtoreq. 5''-N-methylcarboxamido adenosine .gtoreq. R-PIA > CPA > S-PIA. Binding was also displaced by the marine adenosine agonist 1-methylisoguanosine and by a series of xanthine antagonists with the activity order being 1,3-dipropyl-8-(2-amino-4-chloro)phenylxanthine > 8-phenyltheophylline > 8-sulfophenyltheophylline. These findings are consistent with the selective labeling by [3H]NECA of an A-2 type adenosine receptor in the PC12 phenochromocytoma cell line.