Beta-Adrenoceptor Quantification by Radioligand Binding

To assess beta-adrenoceptor density, a radioligand binding assay was performed on left ventricles snap frozen in liquid nitrogen. Crude cell membrane fractions were prepared as described by Krobert et al. (23). Radioligand binding was performed as described by Ramberg et al. (24), where membranes were incubated with [¹²⁵I]-(–)iodocyanopincolol (0.066 nM) and the indicated concentration of either CGP20712A or ICI118551 for 2 h at 23°C. Data were fitted to a two-site binding model, and high and low binding affinities (pK_i) were calculated in GraphPad Prism 8.0.1 using a K_d of 0.04 nM (affinity of [¹²⁵I]-(–)iodocyanopincolol was determined in the left ventricular membranes). Beta₁-adrenoceptor density was determined as an average of high-affinity CGP20712A and low-affinity ICI118551 binding in the same heart. Similarly, beta₂-adrenoceptor density was determined as an average of high-affinity ICI118551 and low-affinity CGP20712A binding in the same heart.