Characterization of A‐2 Receptors in Postmortem Human Pineal Gland

We have examined the binding of the adenosine agonist radioligands [ 3 H] N 6 ‐cyclohexyladenosine ([ 3 H]CHA) and [ 3 H]5′‐ N ‐ethylcarboxamidoadenosine ([ 3 H]NECA) to membranes prepared from postmortem human pineal glands. The results showed that the A‐1‐specific ligand CHA did not bind to membranes. By contrast, [ 3 H]NECA, a nonselective A‐ 1/A‐2 ligand, gave 68% specific binding of the total binding. This specific binding was nearly insensitive to the TV‐ethylmaleimide pretreatment method. To characterize this binding, we used cyclopentyladenosine (50 n M ). Under those conditions [ 3 H]NECA binding at 30°C was rapid and reversible; the K D determined from the kinetic studies was 141 n M . In postmortem human pineal gland, the rank order of potency of adenosine analogues and drugs competing with [ 3 H]NECA showed the specificity for an A‐2 receptor: NECA > 2‐chloroadenosine > l‐ N 6 (2‐phenylisopropyl)adenosine > 8‐phenyltheophylline > 3‐isobutyl‐1‐methylxanthine > caffeine. Guanylylimidodiphosphate (100 μ M ) induced a decrease in the affinity of [ 3 H]NECA, a result suggesting the involvement of a G protein mechanism in the coupling of the adenosine receptor to other components of the receptor complex. Scatchard analysis revealed one class of binding sites for [ 3 H]NECA with K D and B max ranging from 175 to 268 n M and 11.0 to 14.1 pmol/mg protein, respectively. The binding of [ 3 H]NECA was not affected by age, sex, or postmortem delay. [ 3 H]NECA should be a useful tool to assess brain A‐2 receptor density in a variety of neuropsychiatric disorders.