VALIDATION OF FURCHGOTT METHOD TO DETERMINE AGONIST-DEPENDENT A(1)-ADENOSINE RECEPTOR RESERVE IN GUINEA-PIG ATRIUM

1 The ubiquitous distribution of A(1)-adenosine receptors (A(1)AdoR) r epresents an impediment to achieve organ and/or response selectivity o f A(1)AdoR agonists. Differential receptor reserve may be exploited to overcome this problem. We hypothesize that A(1)AdoR reserve is agonis t-dependent and can be accurately estimated with Furchgott's method. 2 Concentration-response curves were constructed from measurement of th e atrial monophasic action potential duration in guinea-pig, isolated hearts treated with R(-) N-6-(2-phenylisopropyl)adenosine (R-PIA) or 2 -chloro-N-6-cyclopentyl-adenosine (CCPA) before and after treatment wi th the selective, irreversible A(1)AdoR antagonist uorosulphonyl)benzo yl]oxy]propyl]-1-propylxanthine (FSCPX). Using Furchgott's method, we determined the equilibrium dissociation constant (K-A) of R-PIA and CC PA, and the fraction of non-inactivated A(1)AdoRs remaining after FSCP X treatment (q(functional)) values of q(functional) were correlated to the fraction of specific binding sites after FSCPX treatment labelled by [H-3]-8-cyclopentyl-1,3-dipropylxanthine ([H-3]-CPX) derived from saturation binding normalized to control (q(binding)) 3 Both R-PIA and CCPA are full A(1)AdoR agonists, but have significantly different pot encies (pD(2) [EC50]=6.84+/-0.04 [145 nM] vs 7.36+/-0.04 [44 nM], resp ectively), 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 (r(2)=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 o btained binding data. 4 Radioligand binding studies validated the use of Furchgott's method to estimate A(1)AdoR reserve. A(1)AdoR reserve w as agonist-dependent. CCPA was shown to be a high intrinsic efficacy, low affinity agonist, whereas R-PIA was found to be a low intrinsic ef ficacy, high affinity agonist.