Rats were treated i.v. with 0.1 mg/kg of (R)-Timolol Rats were treated p.o. with 1-10mg/kg of (R)-Timolol

Rabbit eyes and heart ventricle were from New Zealand albino males, 3 to 6 months old. Chary process villae were separated carefully from any contaminating iris or ciliary body, washed once in 150 mM NaCl and homogenized (10 mg/ml) by hand in an all-glass homogenizer in 6 mM Tris maleate buffer, pH 7.4. Heart tissue was first minced and then homogenized (20-50 mg/ml). Aparticulate (P1) preparation was prepared by diluting the homogenate in 20 volumes of 6 mM Tris maleate buffer, centrifuging at 100,000 x g for 30 min, and resuspending the pellet in the initial volume of buffer used for homogenization. Kinetic constants obtained using the P1 fraction as a source of adenylate cyclase were similar to those obtained using homogenates. Inhibition (by various concentrations of an antagonist) of beta adrenergic receptor-mediated activation of adenylate cyclase activity was used to determine the inhibitory constants. IC50 values were determined utilizing 12 to 16 data points per dose-response curve. For the inhibition curves, various concentrations of antagonist were tested against a fixed concentration of (-)-isoproterenol. Stimulation in the presence of isoproterenol plus antagonist was calculated as the increase over that seen in the presence of antagonist alone.