Recently a series of 2-amino-3 benzoylthiophenes were shown to be allosteric enhancers (AEs) of the actions of adenosine (Ado) on the A1- Ado receptor (A1-AdoR) in the rat brain and in the FRTL-5 thyroid cell line. Positive allosterism of the cardiac A1-AdoR system has not been previously investigated. This research proposal is designed to 1) characterize and establish the specificity of the enhancement caused by the prototype AE, PD 81,723 (PD), of the cardiac actions of exogenous and endogenous Ado, 2) define the selectivity of PD for the A1-AdoR vs other AdoR subtypes, and mathematically model the allosteric enhancement, 3) investigate species and receptor-subtype differences in response to PD, and determine the PD binding domain(s) using recombinant AdoRs, and 4) synthesize and screen new Aes with improved potency an selectivity for AdoR subtypes. To accomplish these broad objectives, a multidisciplinary (pharmacology, molecular biology, chemistry) approach is proposed. Studies will be carried out with guinea pig isolated hearts and freshly isolated cardiomyocytes, cultured cell lines, and cells stably transfected with native and mutated recombinant AdoR subtypes. The effect of PD on AdoR-mediated slowing of AV nodal conduction in isolated heats and on membrane currents (e.g., IKAdo) in single atrial and ventricular myocytes will be quantitated using electrophysiological techniques. The effects of PD on membrane binding of, and cardiac responses to, drugs that have effects similar to those of Ado (e.g., carbachol) will be determined to assess the specificity of PD as enhancer of the actions of Ado. To investigate potentiation by Aes of cardiac responses to endogenous Ado, hypoxia, ischemia, and inhibitors of Ado metabolism will be used to elevate the myocardial interstitial concentration of Ado in the presence and absence of PD. To determine the selectivity of PD for a given AdoR subtype, PD's effects on A1-mediated slowing of AV nodal conduction and on A2-mediated coronary vasodilation will be compared, and radioligand binding of AdoR agonists to recombinant A1,A2a,A2b and A3-AdoRs permanently expressed in CHO cells and to endogenous A1- and A2-AdoRs in DDT1MF-2 and PC-12 cell lines will be quantitated. The nature of the allosteric interaction between PD and the AdoR will be assessed by studying the effect of Aes on a) AdoR agonist, and antagonist radioligand binding parameters, b) affinity states of the A1-AdoR, and c) coupling of AdoRs to G proteins. The effects of PD on binding of AdoR agonists to cloned and expressed AdoR subtypes from various species (including human) and to chimeric receptor constructs will be used to guide site-directed mutagenesis efforts to identify the region(s) of the AdoR important for AE activity. The ultimate goal of this proposal is to demonstrate that allosteric enhancers of the actions of Ado are useful agents to increase the response to locally released Ado in the heart and thereby act as site=specific drugs. These compounds may prove to be an ideal therapeutic approach to selectively and specifically amplify the actions of endogenous adenosine.
