Plasma membrane preparations were purified as described (38). Briefly, membrane preparations from cardiac tissue were prepared by homogenization in ice-cold lysis buffer [25 mM tris (pH 7.4), 5 mM EDTA, aprotinin (1 μg/ml), and leupeptin (1 μg/ml)] and centrifuged at 1000g for 5 min at 4°C. The supernatant was centrifuged at 30,000g, and the crude membrane pellet was resuspended in lysis buffer containing 10% glycerol and stored at −80°C until use. The density of βAR on plasma membranes was determined by saturation binding experiments. Membrane preparations (25 μg of protein) were incubated with [125I]cyanopindolol (200 pM; PerkinElmer) in binding buffer [75 mM tris (pH 7.4), 2 mM EDTA, 12.5 mM MgCl2, aprotinin (1 μg/ml), and leupeptin (1 μg/ml)]. Incubations were performed in the presence or absence of propranolol (10 μM) to determine nonspecific binding. The reactions were performed in a 250-μl volume and allowed to equilibrate at 37°C for 1 hour before filtering the membranes 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) and 10 mM EDTA] to remove unbound ligand. The amount of total and nonspecific radiolabel bound to cell membranes was determined on a gamma counter. All assays were performed in triplicate. Receptor density was normalized to milligrams of membrane protein.

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