Binding of [3H]Dihydroergocryptine to an a-Adrenergic Site in the Stalk Median Eminence of the Steer*

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Abstract
Dihydroergocryptine (DHE), a potent dopamine agonist and α-adrenergic antagonist, has been used as a radioligand to characterize both dopamine and α-adrenergic receptors. In the present study, the binding of [3H]DHE to participate fractions of the steer stalk median eminence was characterized using a filtration assay. Specific binding was defined by the presence of 10 μM phentolamine or by an iterative nonlinear hyperbolic curve-fitting program. Scatchard analysis of equilibrium isotherms of specific binding defined a single high affinity (Kd = 1.78 ± 0.22 nM), saturable (maximum binding, 481 ± 39 fmol/mg protein), stereoselective binding site, the Kd, calculated from the ratio of the rate constants k2 and k1, was 2.8 ± 0.14 nM. The rank order of potency of agonists to compete for [3H]DHE binding (l-epinephrine > l-norepinephrine > dopamine > l-isoproterenol) was consisten with interactions at an αadrenergic site. The rank order of potency of α-antagonists (phentolamine > yohimbine > prazosin) suggested that this was an α2-adrenergic receptor. The affinity of dopamine agonists for the [3H]DHEbinding site was 10-fold lower relative to their potency at known dopamine receptors, while the affinity of dopaminergic antagonists was 100-fold lower. Furthermore, Scatchard Analysis of specific [3H]DHE binding in the presence of a concentration of spiperone which should saturate dopamine receptorp, only decreased the number of binding sites by 9%. These data demonstrate the presence of large numbers of a-adrenergic receptors in the stalk median eminence of the steer. Only a small number of dopaminergic binding sites for [3H]DHE appeared to be present.