The binding of 1,3‐[3H]‐dipropyl‐8‐cyclopentylxanthine to adenosine Ai receptors in rat smooth muscle preparations

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Abstract
1 The binding of 1,3-[3H]-dipropyl-8-cyclopentylxanthine ([3H]-DPCPX), an antagonist radioligand selective for adenosine A1 receptors, was studied in rat duodenum, colon muscularis mucosae and longitudinal muscle, urinary bladder and vasa deferentia. 2 [3H]-DPCPX bound with high affinity to a single site in all membrane preparations studied with the exception of the rat urinary bladder in which no specific binding was detected. The affinity (Kd) of the binding site for [3H]-DPCPX was similar in all membrane preparations, the colon longitudinal muscle (1.18 ± 0.47 nm), colon muscularis mucosae (0.84 ± 0.15 nm), duodenum (1.59 ± 0.18 nm) and vasa deferentia (0.93 ± 0.17 nm). The density of [3H]-DPCPX binding sites was similar in the duodenum (38.8 ± 4fmol mg−1 protein), muscularis mucosae (43 ± 3.5 fmol mg−1 protein) and vasa deferentia (43.3 ± 12.2 fmol mg−1 protein), but in the longitudinal muscle 6–7 fold more binding sites (295 ± 70 fmol mg−1 protein) were identified. 3 Inhibition studies using DPCPX (0.1–100 nm), N6-cyclopentyladenosine (CPA) (0.1–100 nm), 5′-N-ethylcarboxamidoadenosine (NECA) (2nM-10 μm) and (R)-N6-phenylisopropyladenosine (R-PIA) (1 nm-1 μm) to displace the binding of [3H]-DPCPX at a concentration around the Kd value (1 nm), demonstrated an order of potency of displacement in all tissues of DPCPX ≥ CPA > R- PIA > NECA. This potency order is characteristic of an A1 receptor, indicating that [3H]-DPCPX binds to adenosine A1 receptors in the rat duodenum, colon and vasa deferentia. Two site analysis revealed that the agonists bind to both a high and low affinity state of the receptor. 4 The existence of A1 binding sites in the rat vasa deferentia, colon muscularis mucosae and duodenum, and their absence in the urinary bladder, is consistent with previous functional studies. However, in contrast to the findings of the [3H]-DPCPX binding assay, no functional response mediated by adenosine A1 receptors could be detected by measuring contractile or relaxant responses to CPA in the colon longitudinal muscle. The functional significance of the binding sites in this tissue has therefore yet to be determined.