Chemical modification of adenosine A1 receptors. Implications for the interaction with R-PIA, DPCPX and amiloride

Amiloride, a potassium sparing diuretic, inhibits the specific binding of [3H]8-cyclopentyl-1,3-dipropylxanthine (DPCPX) and [3H]N6-R-1-phenyl-2-propyladenosine (PIA) to adenosine A1 receptors in calf brain. This interaction is different from the agonist-receptor or the antagonist-receptor interaction as Na+ and H+ counteract the inhibitory effect of amiloride whereas these ions hardly affect the binding of the classic A1 receptor ligands. In the present study, the effects of protein modifiers on the equilibrium inhibition constant of amiloride are compared with effects of these reagents on the affinities of DPCPX and PIA. It is demonstrated that the affinities of amiloride and [3H]DPCPX are changed after treatment with a carboxyl-modifying reagent but unaffected by modification of histidyl, arginyl and cystein residues. The maximal binding capacity of [3H]DPCPX is enhanced by sulfhydryl modification, whereas the number of [3H]DPCPX binding sites is reduced by treatment with a histidine-modifying reagent. The histidyl residues of the [3H]DPCPX binding site can be partially protected against modification by 300 microM amiloride, present during treatment of the membranes. An equivalent concentration of 8-phenyltheophylline results in complete protection. The apparent affinity of PIA is altered by modification of histidyl, carboxyl, arginyl and cystein residues. In the latter two cases, uncoupling of the G protein seems to be the major reason for the decrease in affinity of PIA. The results suggest that amiloride is an A1 antagonist with binding characteristics that differ from the classic A1 antagonists such as DPCPX.