125i cyanopindolol 125i cyp

The density of βAR on cardiac membranes, prepared as previously described 13 , or NRVM were determined by saturation binding experiments. Cardiac membranes (25 μg of protein) or single cell suspensions (1×10⁵ cells) of NRVM were incubated with [¹²⁵I]-cyanopindolol ([¹²⁵I]-CYP; 200 pM; PerkinElmer) in binding buffer (75 mM Tris, pH 7.4, 2 mM EDTA, 12.5 mM MgCl₂, 1 μg/mL aprotinin, 1 μg/mL leupeptin). Incubations were performed in the presence or absence of propranolol (10 μM) to determine non-specific binding of CGP20712A and ICI 118,551 (100 nM) to determine βAR subtype expression. Reactions were performed in a 250 μL volume and allowed to equilibrate at 37 °C for 1 h before filtering through a glass fiber filter (Whatman GF/C; Brandel). Each filter was washed five times with 5 mL of ice-cold wash buffer (10 mM Tris, pH 7.4, 10 mM EDTA) to remove unbound drug. The amount of total and nonspecific radiolabel bound to cells was determined on a gamma counter and receptor density was normalized to cell number (NRVM) or protein amount (cardiac membranes). All assays were performed in duplicate.