Abstract

Two novel amiloride analogs have been synthesized during the course of efforts to develop a photoaffinity label for the amiloride allosteric domain on alpha 2-adrenergic receptors. One of these, 5-[N-2'-aminoethyl-N'-isopropyl]amiloride-N-[4"-azidosalicylamide] (A-EIA-AS), markedly accelerates the rate of dissociation of [3H]yohimbine from affinity-purified alpha 2-adrenergic receptors, an assay for allosteric modulation of receptor-adrenergic ligand interactions. In contrast, this agent does not appreciably inhibit Na+/H+ exchange, measured as 5-(N-ethyl-N-isopropyl)amiloride (EIA)-inhibitable 22Na+ uptake into cultured renal epithelial cells. A second analog, 5-[N-2'-(4"-azidosalicylamidino)ethyl-N'- isopropyl]amiloride (ASA-EIA), does not foster an accelerated rate of dissociation of [3H]yohimbine binding from the alpha 2 receptor but does block the ability of A-EIA-AS to do so, suggesting that ASA-EIA and A-EIA-AS interact at a common binding site. Interestingly, the ability of EIA to accelerate [3H]yohimbine dissociation is not blocked by ASA-EIA, a finding that may indicate that EIA and A-EIA-AS allosterically modulate alpha 2 receptor-ligand interactions via distinct or nonoverlapping binding sites.