melitten and methylisopropylamiloride

Amiloride inhibits the mitogen-stimulated Na+ influx of cultured fibroblasts with very low affinity. We have therefore analyzed eight new derivatives of amiloride for their efficacies of inhibiting the Na+ flux in Chinese hamster ovary cells (CHO-K1). Four of these analogs demonstrate markedly enhanced potencies relative to amiloride. One of the derivatives, methylisopropyl-amiloride (MIA), is approximately 900-fold more potent (ID50 = 42 nM) than amiloride in inhibiting Na+ uptake of these cells. Inasmuch as external Na+ ions antagonize amiloride inhibition of Na+ influx competitively, we investigated the ability of MIA to inhibit Na+ flux in the absence of external Na+ ions. The ID50 value determined for MIA inhibition of CHO-K1 cell Na+ efflux from Na+-loaded cells into Na+-free media is 15 nM. We also examined the efficacy of MIA for Na+ efflux inhibition in a nontransformed fibroblast cell line, Swiss 3T3. The ID50 value for inhibition of Na+ efflux from 3T3 cells is comparable to that for CHO-K1 cells. Thus, we have identified a highly potent derivative of amiloride, MIA, that inhibits Na+ flux at nanomolar concentrations in both a transformed and a nontransformed cell line. It is possible that MIA may serve as a useful tool for biochemical characterization of the mitogen-stimulated Na+ transporter and for assessment of the role of this transporter in mitogenesis.

Topics: Amiloride; Animals; Biological Transport; Blood Physiological Phenomena; Cells, Cultured; Cricetinae; Cricetulus; Depression, Chemical; Dose-Response Relationship, Drug; Female; Melitten; Nigericin; Ovary; Protons; Sodium