Adenosine Receptor Subtypes: Binding Studies

Summary

Adenosine receptors are divided into two main subtypes, designated A₁ and A₂. Although the two subtypes are presently defined by structure-activity relationships, the original definition based on adenylate cyclase is still useful: A₁ receptors inhibit adenylate cyclase, whereas A₂ receptors stimulate the enzyme. The two receptors also contribute in different ways to adenosine’s role in maintaining oxygen supply/demand balance: A₁ responses generally bring about a reduction in oxygen demand (e. g., reductions in heart rate and contractility, inhibition of locomotor activity, hypothermia, inhibition of lipolysis), whereas A₂ responses generally increase oxygen supply (e. g., vasodilation, inhibition of platelet aggregation).

A₁ receptor binding assays are now well-characterized, and we routinely use ³H-N⁶γ-cyclohexyl-adenosine for this purpose. To determine the relative A₁/A₂ affinities of compounds, we developed an A₂ receptor binding assay, based on the ³H-NECA binding assay of Yeung and Green. ³H-NECA binding was performed in rat striatal membranes, with 50 nM N⁶-cyclopenty-ladenosine (CPA) included in the assay to eliminate A₁ binding of ³H-NECA. Under these conditions, ³H-NECA binding showed the characteristics expected of an A₂ receptor. Affinities of six adenosine agonists in ³H-NECA binding were closely correlated with literature values for affinities in the dog coronary artery system (r = 0.986, P < 0.001), whereas no such correlation was seen for ³H-CHA binding (r = 0.530, n. s.). CPA was the most highly A₁-selective agonist (780-fold), and 2-(phenylamino)adenosine (CV-1808) was the most highly A₂-selective (5-fold). The antagonist PD 116,948 (8-cyclopentyl-1,3-dipropylxanthine) showed 740-fold A₁-selectivity. PD 115,199, N-[2-(dimethylamino)ethyl]-N-methyl-4-(2,3,6,7-tetrahydro-2,6-dioxo-1,3-dipropyl-1H-purin-8-yl)benzenesulfonamide, a moderately soluble xanthine derivative, had a high affinity in A₂ binding but was equally potent in A₁ binding. ³H-PD 116,948 and ³H-PD 115,199 have been used to label A₁ and A₂ receptors, respectively.

Several groups have observed that A₂ receptors can differ markedly in their affinity for adenosine, and it has been proposed that A₂ receptors can be divided into high- and low-affinity subclasses. The high-affinity A₂ receptors, which we propose to call A_2a, stimulate broken-cell adenylate cyclase and are localized to the striatum, whereas the low-affinity A_2b receptors stimulate cyclic AMP accumulation in brain slices and are widely distributed in the brain. The ³H-NECA binding site has a high affinity for adenosine agonists and is highly localized to the striatum, indicating that it belongs to the A_2a subclass. 2-(4-Methoxyphenyl) adenosine (CV-1674) appears to be quite selective for the A_2a receptor: it has 600 nM affinity in ³H-NECA binding but is inactive at 1 mM in human fibroblasts (A_2b response). The antagonist ligand ³H-PD 115,199 does not show any detectable binding to the A_2b receptor, confirming that this receptor is not simply an antagonist-preferring coupling state of the A_2a receptor.

Some new structure-activity criteria to distinguish A₁ and A₂ receptors are proposed.