Interactions of amiloride with alpha- and beta-adrenergic receptors: amiloride reveals an allosteric site on alpha 2-adrenergic receptors.

Interaction of amiloride with adrenergic receptors was studied using radioligand binding techniques. Amiloride competed for [3H]prazosin binding to alpha 1-adrenergic receptors on rat renal cortical membranes and BC3H-1 muscle cell membranes. Non-linear regression analysis of radioligand binding isotherms showed that amiloride increased Kd without a change in Bmax, suggesting the drug binds competitively in a mutually exclusive manner with the radioligand at the receptor-binding site. Similarly, amiloride competitively blocked [125I]iodocyanopindolol binding to beta-adrenergic receptors on both tissues. The addition of guanylyl 5'-imidodiphosphate or sodium chloride did not alter the interaction of amiloride with alpha 1- or beta-adrenergic receptors. The interaction of amiloride with alpha 2-adrenergic receptors was more complex and revealed an allosteric site. In both rat renal cortical membranes and intact human platelets, amiloride increased the Kd for [3H]rauwolscine binding, as well as decreasing the apparent Bmax. In binding experiments where amiloride competed for [3H]rauwolscine-binding sites, pseudo-Hill slopes of less than 1.0 were obtained for both platelet and renal alpha 2 receptors. In addition, amiloride increased the rate of [3H]rauwolscine dissociation from renal alpha 2 receptors. In the presence of 100-120 mM sodium chloride, the Ki for amiloride competition was decreased an average of 54% in renal membranes; in contrast, sodium increased the Kd of the agonist epinephrine. Taken together, these data support the hypothesis that alpha 2-adrenergic receptors, but not alpha 1- or beta-adrenergic receptors, have an allosteric site to which amiloride binds and which we propose to be a cation-binding site.