Specific binding of [ 3H]prazosin to myocardial cells isolated from adult rats

Abstract

The characteristics of α-adrenoceptors in rat myocardium were investigated by specific binding of [ 3H]prazosin to cells isolated from adult rat heart by perfusion with collagenase and hyaluronidase. The cells were incubated in Krebs-Ringer bicarbonate buffer gassed with 95% O 2 and 5% CO 2 at 31° with the appropriate concentrations of the different ligands. Non-specific binding was defined by the addition of 10 −5 mole/1. phentolamine. The binding of [ 3H]prazosin was saturable and reached equilibrium within 15 min. Scatchard analysis showed a straight line giving an apparent dissociation constant, K d , equal to 155.9±8.0 pmole/1. and a maximal number of binding sites equal to 76.7±11.1 fmole/mg protein. Inhibition of specific [ 3H]prazosin binding by different adrenergic blockers showed the order of potency characteristics of α 1-adrenoceptors: prazosin ⪢ phentolamine > yohimbine ⪢ propranolol. Inhibition by adrenergic agonists showed the order of potency: adrenaline > noradrenaline = phenylephrine > isoprenaline. The same orders of potency were observed in the presence of propranolol. However, propranolol slightly decreased the affinity for noradrenaline and phenylephrine. Hofstee analyses of the inhibition curves showed two binding components for all ordinary α-adrenoceptor blockers and agonists including unlabelled prazosin. In contrast, [ 3H]prazosin showed only one binding component. Both binding components were of the α 1-adrenoceptor subtype according to the order of potency of blockers. The different ligands had different affinity ratios for the two binding components giving them different profiles. Trifluoperazine, a phenothiazine compound, also had high affinity for the [ 3H]prazosin binding sites. This drug, however, apparently detected one class of binding sites only, as interpreted from the Hofstee analysis. Hill analyses of the inhibition data consistently yielded Hill constant, n H , in the range 0.75–0.85 except for [ 3H]prazosin, where n H = 1.02 and for trifluoperazine, where n H = 1.07. Although the two binding components may serve different functions, it seems impossible at present to relate the negative and the positive inotropic components, respectively, of the α-adrenergic inotropic response observed in functional studies only to one or the other binding component.