Binding of the Adenosine A₂ Receptor Ligand [³H]CGS 21680 to Human and Rat Brain: Evidence for Multiple Affinity Sites

Abstract

A new radiolabeled adenosine receptor agonist, 2-[p-(2-carboxyethyl)phenethylamino]-5'-N-ethylcarboxamidoadeno sin e (CGS 21680), apparently specific for high-affinity binding sites of the A2 subtype in rat brain, was used to identify and pharmacologically characterize adenosine receptors in human brain. The binding of [3H]CGS 21680, as determined by standard radioligand binding technique in the presence of exogenously added adenosine deaminase, reached equilibrium after 40 min at 25 degrees C. In saturation studies, a single class of high-affinity binding sites with values for KD of 22 +/- 0.5 nM and Bmax of 444 +/- 63 fmol/mg of protein were observed. Similar binding characteristics were observed regardless of whether rapid filtration or centrifugation was used to separate bound versus free ligand. Of the 14 brain regions examined, [3H]CGS 21680 binding was highest in putamen, followed by globus pallidus and caudate nucleus. The level of [3H]CGS 21680 binding in these areas of basal ganglia was identical to 5'-N-[3H]ethylcarboxamidoadenosine ([3H]NECA) binding in the presence of 50 nM N6-cyclopentyladenosine (CPA). The rank order of agonist potencies as determined by a series of competition experiments was NECA greater than or equal to CGS 21680 greater than 2-chloroadenosine greater than N6-(R)-phenylisopropyladenosine greater than N6-cyclohexyladenosine greater than N6-(S)-phenylisopropyladenosine. This potency order was the same for the binding of [3H]CGS 21680 to rat, and of [3H]NECA in the presence of 50 nM CPA to rat and human, brain membranes.(ABSTRACT TRUNCATED AT 250 WORDS)

Further characterization of [3H]‐CGS 21680 binding sites in the rat striatum and cortex

“…Interestingly, when binding in the striatum was performed at pH 5.5, the same A2A profile was observed although there was a significant increase in affinity for this binding site for all the compounds tested with no apparent change in Bm.. This increase in affinity was probably due to the protonation of the histidine residues in the receptor binding site at low pH (Askalan & Richardson, 1994), since these residues have been shown to be involved in ligand binding to the A2A receptor (Jacobson et al, 1993) Wan et al, 1990). Such low levels of receptor may in part explain the failure of previous workers to detect mRNA in the cortex (Schiffman et al, 1991a,b;Fink et al, 1992 (Zhou et al, 1992;Y'akel et al, 1993).…”

“…In view of these reports and our reported A2A mediated inhibition of GABA release from the rat striatum, it was of interest to see whether two CGS 21680 binding sites could be identified. We therefore set out to determine whether the previously desBrkish Journal of Pharmacology (1995) 114,[537][538][539][540][541][542][543] cribed, but uncharacterized CGS 21680 binding site in the rat cerebral cortex Wan et al, 1990) was different from the well characterized striatal site.…”

Characterization of adenosine receptors in brush‐border membranes from pig kidney

“…Data from saturation isotherms and displacement curves were analyzed by non-linear regression with the ENZFIT-TER programme (Elsevier Biosoft) or other available programmes (Canela, 1984;Lopez-Cabrera et al, 1988) as described elsewhere (Casad6 et al, 1990a,b;1991 (Jarvis et al, 1989;Wan et al, 1990). In order to determine whether the kinetic characteristics reported in this paper correspond to species differences or to an adenosine receptor other than the A2a, the characterization of Figure 4b, the order of potency among adenosine receptor agonists was: CGS21680 (Ki = 12 ± 4 nM) = NECA (Ki = 12 3 nM)> CADO (Ki = 220 ± 60 nM)> R-PIA, (Ki = 2200 ± 400 nM), which is the same as that found for A2a receptors (Jarvis et al, 1989).…”

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

“…Until recently, the lack of highly selective A2 agents has generally meant that specific A2 binding has been determined by the use of a ligand such as [3H]-NECA, which binds to both Al and A2 receptors, with the binding study carried out in the presence of an Al agonist and rat striatum being used as a source of tissue (Bruns et al, 1986;Stone et al, 1988). More recently [3H]-CGS 21680 has been used to identify A2, binding sites in various regions of the brain of both human and rat (Wan et al, 1990), though no agent specific for the low affinity A2b site has yet been developed. (Bruns et al, 1987).…”