Amiloride, a commonly used inhibitor of Na+-H+ exchange, has been shown to exhibit a variety of nonspecific effects. Recently, the more potent amiloride analogs, 5-(N,N-dimethyl)amiloride hydrochloride (DMA) and 5-(N-ethyl-N-isopropyl)amiloride (EIA), have been used to control for the nonspecific effects of the parent compound. In the present study, we have explored the effects of these analogs on Na+/K+-transporting ATPase (Na+/K+-ATPase) and Na+-coupled alanine transport in primary rat hepatocyte cultures and rat liver plasma membranes, and we have compared the effects of these analogs with the effects of amiloride and ouabain. Amiloride, DMA, and EIA increased staedy-state Na+ content and inhibited ouabain-sensitive 86Rb+ uptake in a reversible, concentration-dependent, ouabain-like manner, with estimated 50% inhibitory concentrations (IC50) of 3.0 · 10-3 M, 5.2 · 10-4 M, and 1.2 · 10-4 M, respectively. Amiloride, DMA and EIA also inhibited ouabain-sensitive ATP hydrolysis in rat liver plasma membranes with similar potency (IC50 values of 2.2 · 10-3 M, 2.2 · 10-3 M, and 1.7 · 10-4 M, respectively). In separate experiments, amiloride (5 · 10-3 M), DMA (10-3 M), and EIA (2.5 · 10-4 M) decreased the uptake into hepatocytes of alanine by 20%, 61%, and 59%, respectively, and further studies with DMA (10-3 M) demonstrated that this inhibition was largely due to a decrease in the Na+-dependent fraction of alanine uptake. These findings indicate that amiloride, DMA, and EIA inhibit hepatic Na+/K+-ATPase directly, reversibly, and with a relative rank order potency of EIA > DMA > amiloride. All three compounds also inhibit the hepatic uptake of alanine, and presumably could indirectly inhibit other Na+-coupled transport processes as well.
Bibliographical noteFunding Information:
The excellent technical assistance of Mary Cochran and Michael Wong and the editorial assistance of Diana Fedorchak and Michael Karasik are gratefully acknowledged. We thank Dr. D. Montgomery Bissell and Alan Sato for supplying the cultured hepatocytes, and Dr. Vojtech Licko for help with mathematical analysis of the data. This study was supported in part by NIH grants AM-26270, AM-07453, AM-01254, and UCSF Liver Core Center grant AM-26743, as well as a grant from the American Liver Foundation (JRL). ELR is the recipient of fellowships from the Swiss National Science Foundation and the Swiss Foundation for Biomedical Stipends. Portions of this work have been published in abstract form in Hepatology 6 (1986) 1193, and J. Cell Biol. 103 (1986) 457a.
- (Rat hepatocyte)
- ATPase, Na/K-
- Alanine transport
- Sodium pump