Relation between alpha adrenergic receptor occupation and contractile response: radioligand and physiologic studies...

The relation between occupation of alpha-1 adrenergic receptors by an agonist and the resulting force was examined in the canine aorta in vitro. Methods of preparing membrane preparations for radioligand studies were improved to maximize the yield of receptors from strips of aortic wall. [3H]Prazosin was a reliable ligand, binding to a single class of receptors with a dissociation constant of 97 (+/- 10) pM. Agonists and antagonists displaced [3H]prazosin in a manner consistent with binding to a single class of sites, with Hill coefficients near unity. The rank order of drug potencies determined by competition with [3H]prazosin was characteristic of alpha adrenergic receptors of the alpha-1 subtype. Conventional measurements of maximum isometric force and dose-response relations for l-phenylephrine in strips of ascending aorta in vitro were followed by radioligand studies on homogenates made from the same strips. The number of receptors was 38.8 (+/- 2.9) fmol/mg of protein in the homogenate, corresponding to 580 (+/- 41) fmol/g of aortic wall, a value at least twice as high as any reported previously. Maximum active stress was 440 (+/- 23) g/cm2. Concentration-response curves to l-phenylephrine were relatively shallow, with a Hill coefficient of 0.63 and an ED50 of 1.1 (+/- 0.24) microM. The curve relating receptor occupation to response (force development) in the ascending aorta was consequently nonlinear; 50% of the maximum response occurred when only 10% of the receptors were occupied. Occupation of virtually all receptors (greater than or equal to 97%) was required to produce maximum response, suggesting an absence of "spare" receptors.