Identification of Adenosine Receptor in Rat Pineal Gland: Evidence for A-2 Selectivity

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Abstract
We have examined the binding of the adenosine agonist radioligands [3H]cyclohexyladenosine ([3H]CHA), R-N6-[3H]phenylisopropyladenosine ([3H]R-PIA), and 5''-N-ethylcarboxamido[3H]adenosine ([3H]NECA) to membranes prepared from rat pineal gland. The results showed that the A-1-selective ligands (CHA and R-PIA) had .ltoreq. 10% specific binding. By contrast, [3H]NECA, a nonselective A-1/A-2 ligand, gave 72% specific binding of the total binding. This specific binding was insensitive to cyclopentyladenosine (50 nM) or R-PIA (50 .mu.M). To characterize this binding, we used the N-ethylmaleimide pretreatment method. Under these conditions, this binding was of high affinity with a KD of 51 .+-. 10 nM and an apparent Bmax of 1,060 .+-. 239 fmol/mg of protein. Specific binding was unaffected by the presence of MgCl2 (10 mM) but was sensitive to guanylylimidodiphosphate (100 .mu.M) (-25%), a result suggesting the involvement of an N-protein mechanism in the coupling of the adenosine receptor labeled by [3H]NECA to other components of the receptor complex. The rank of activity of adenosine analogues in displacing specific [3H]NECA binding was NECA > 2-chloroadenosine > S-adenosyl-L-homocysteine > CHA. Binding was also displaced by 3-isobutyl-1-methylxanthine (IC50 = 23.6 .mu.M). These findings are consistent with the selective labaling by [3H]NECA of an A-2-type adenosine receptor in rat pineal membranes.