Validation of Furchgott's method to determine agonist-dependent A₁-adenosine receptor reserve in guinea-pig atrium

Abstract

1. The ubiquitous distribution of A₁-adenosine receptors (A₁AdoR) represents an impediment to achieve organ and/or response selectivity of A₁AdoR agonists. Differential receptor reserve may be exploited to overcome this problem. We hypothesize that A₁AdoR reserve is agonist-dependent and can be accurately estimated with Furchgott's method.

2. Concentration-response curves were constructed from measurement of the atrial monophasic action potential duration in guinea-pig, isolated hearts treated with R(−) N⁶-(2-phenylisopropyl)adenosine (R-PIA) or 2-chloro-N⁶-cyclopentyl-adenosine (CCPA) before and after treatment with the selective, irreversible A₁AdoR antagonist 8-cyclopentyl-3-[3-[[4-(fluorosulphonyl)benzoyl]oxy]propyl]-1-propyl-xanthine (FSCPX). Using Furchgott's method, we determined the equilibrium dissociation constant (K_A) of R-PIA and CCPA, and the fraction of non-inactivated A₁AdoRs remaining after FSCPX treatment (q_functional). Values of q_functional were correlated to the fraction of specific binding sites after FSCPX treatment labelled by [³H]-8-cyclopentyl-1,3-dipropylxanthine ([³H]-CPX) derived from saturation binding normalized to control (q_binding).

3. Both R-PIA and CCPA are full A₁AdoR agonists, but have significantly different potencies (pD₂ [EC₅₀]=6.84±0.04 [145 nM] vs 7.36±0.04 [44 nM], respectively), receptor affinities (pK_A [K_A]= 6.54±0.10 [288 nM] vs 6.13±0.03 [734 nM]), and pharmacological shift ratios defined as K_A/EC₅₀ (2.2±0.6 vs 15.9±1.5). Values for q_functional and q_binding were highly correlated (r²=0.96). The ratio between the intrinsic efficacies of CCPA and R-PIA derived from Furchgott's analysis was 5.9, a value similar to the ratio of 6.2–6.6 calculated from previously obtained binding data.

4. Radioligand binding studies validated the use of Furchgott's method to estimate A₁AdoR reserve. A₁AdoR reserve was agonist-dependent. CCPA was shown to be a high intrinsic efficacy, low affinity agonist, whereas R-PIA was found to be a low intrinsic efficacy, high affinity agonist.

Full Text

The Full Text of this article is available as a PDF (517.3 KB).