Structural requirement of monophosphates for inhibition of Na+-Pi cotransport in renal brush border membrane.

Using the chemical structural analogs of phosphonoacetic acid (PAA) and related phosphonate compounds, we investigated which structural features are required for competitive inhibition of Na+-Pi cotransport in rat renal cortical brush border membrane (BBM) vesicles (BBMV). The effects of compounds on [Nao+ greater than Nai+]-gradient-dependent 32Pi uptake by BBMV were examined using various inhibitor-to-32Pi concentration ratios in the transport assay medium. The replacement of a phosphono-group with an arsono-group in PAA, or the substitution of a carboxylic group in PAA by an amino or hydroxyl group, totally abolished the inhibitory action on Na+-Pi cotransport. Decreased electronegativity of carboxyl in PAA by coupling with hydrazine or hydroxylamine lowered the inhibitory potenty of PAA. Substitution of H at the alpha-carbon of PAA with ethyl or p-Cl-phenyl groups completely abolished the inhibitory activity, whereas alpha-halogenation with Br greatly increased the inhibitory potency of PAA, close to that of phosphonoformic acid (PFA). The inhibition by all the active tested monophosphates was strictly competitive. The tested compounds displaced [14C]PFA pre-bound onto BBMV in the presence of 100 mM NaCl. The ability of monophosphates to inhibit Na+-Pi cotransport across BBM and the binding of [14C]PFA were closely correlated (r = 0.925; P greater than 0.001). These results show that: (a) strong electronegativity at both ends of the PAA molecule is needed for inhibitory action, (b) an alpha-aliphatic or aromatic substituent at the alpha-carbon probably hinders the access of the inhibitor to the Pi-binding site of the Na+-Pi cotransporter in BBM, whereas (c) an alpha-electrophilic substituent--Br--enhances the inhibitory potency of PAA. The tested compounds inhibited Na+-Pi cotransport by binding, in the presence of Na+, on the same site on the luminal surface of BBM as did PFA and, by extension, Pi.