DETERMINATION OF BETA-ADRENOCEPTOR SUBTYPE ON RAT ISOLATED VENTRICULAR MYOCYTES BY USE OF HIGHLY SELECTIVE BETA-ANTAGONISTS

1 The relative proportions of beta(1)- and beta(2)-adrenoceptors were determined by radioligand binding studies in three different rat myocardial preparations: membranes prepared from rat ventricle (ventricular membranes), membranes prepared from rat isolated ventricular myocytes (myocyte membranes), and myocytes isolated from rat ventricle (myocytes). 2 Competition experiments using CCP 20712A or ICI 118,551 with [I-125]-iodocyanopindolol ([I-125]-ICYP) revealed high- and low-affinity binding sites in ventricular membranes. The concentration at which each beta-antagonist occupied 100% of its high-affinity binding sites was 300 nM for CGP 20712A (beta(1)-adrenoceptor) and 50 nM for ICI 118,551 (beta(2)-adrenoceptor). 3 The density of high-affinity (beta(1)-adrenoceptor) and low-affinity (beta(2)-adrenoceptor) binding sites for CGP 20712A was measured by a saturation experiment using [I-125]-ICYP in the presence and absence of 300 nM CGP 20712A. In ventricular membranes, the proportions of high-affinity and low-affinity binding sites for CGP 20712A were 73% and 27%, respectively, whereas in myocyte membranes, the corresponding figures were 90% and 10%, respectively. The density of low-affinity binding sites for CGP 20712A in ventricular membranes, defined as [I-125]-ICYP-specific binding in the presence of 300 nM CGP 20712A, was decreased by addition of 50 nM ICI 118,551, whereas that in myocyte membranes was not affected. 4 In myocytes, specific binding of [I-125]-ICYP and [H-3]-CGP 12177 was not detected by saturation experiments performed in the presence of 300 nM CGP 20712A. 5 In myocytes, the activation of adenylate cyclase caused by beta(2)-adrenaceptors was not detected in the presence of 10 nM, 100 nM or 1000 nM CGP 20712A, which selectively antagonized beta(1)-adrenoceptors. Furthermore, the concentration-response curve for isoprenaline-stimulated cyclic AMP accumulation was not shifted by 10 nM or 100 nM ICI 118,551, which selectively antagonized beta(2)-adrenoceptors, but was shifted to the right by 1000 nM ICI 118,551. 6 These results indicate that beta(2)-adrenoceptors are not present on rat ventricular myocytes and that beta(2)-adrenoceptor stimulation does not cause any detectable production of cyclic AMP. We conclude that only beta(1)-adrenoceptors exist on rat ventricular myocytes.