Summary
The cultured renal amphibian cell line A6 has proven advantageous for studies of Na+ transport regulation. In the present study, the effects of aldosterone action on the transepithelial electrical properties of this epithelium were assessed. Specifically, the time course of aldosterone action was determined and the effects of chronic (10–18 day) aldosterone elevation were assessed using transepithelial equivalent circuit methods and impedance analysis techniques.
Short-term (<4 hr) exposure to aldosterone (0.1 μ m) stimulated the amiloride-sensitive short-circuit current (I sc) by over twofold and increased the transepithelial conductance (G T) by approximately 12%. The increases in I scand G Twere maintained in epithelia subjected to chronic aldosterone exposure. In contrast to previous reports, paracellular resistance (R j) was not altered by aldosterone. This difference may be related to the longer time of exposure or different basal Na+ transport rates in the present study.
The apical membrane conductance was significantly increased for aldosterone-treated epithelia compared to aldosterone-depleted (i.e., serum-deprived) controls. Apical membrane area (capacitance) was not significantly affected. This finding is consistent with a higher density (number of channels per membrane area) of conducting Na+ channels in this membrane following aldosterone stimulation. Basolateral membrane properties were not significantly altered for aldosteronetreated tissues compared to serum-treated control tissues. In contrast, basolateral membrane-specific conductance (i.e., basolateral membrane conductance normalized to basolateral membrane capacitance) was significantly lower for serum-deprived epithelia than for serum-treated controls or aldosterone-treated tissues.
The effects of chronic aldosterone exposure were also evaluated for the A6 subclonal cell line, 2F3. Similar to A6 epithelia, I scwas essentially doubled following aldosterone stimulation while R jand cellular driving force (E c) were not affected. Apical membrane conductances under control conditions for 2F3 epithelia were higher than those for A6, but were not significantly different from A6 following aldosterone exposure or serum deprivation. These findings suggest possible differences in the regulation of apical membrane Na+ channels for 2F3 and A6 epithelia.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Bindels, R.J.M., Schafer, J.A., Reif, M.C. 1988. Stimulation of sodium transport by aldosterone and arginine vasotocin in A6 cells. Biochim. Biophys. Acta 972:320–330
Claire, M., Machard, B., Lombes, M., Oblin, M.E., Bonvalet, J.P., Farman, N. 1989. Aldosterone receptors in A6 cells: physicochemical characterization and autoradiographic study. Am. J. Physiol. 257 (26):C665-C677
Clausen, C., Lewis, S.A., Diamond, J.M. 1979. Impedance analysis of a tight epithelium using a distributed resistance model. Biophys. J. 26:291–318
Cole, K.S. 1972. Membranes, Ions and Impulses. University of California, Berkeley, CA
Fidelman, M.L., Watlington, C.O. 1984. Insulin and aldosterone interaction on Na+ and K+ transport in cultured kidney cells (A6). Endocrinology 115(3):1171–1178
Fidelman, M.L., Watlington, C.O. 1987. Effect of aldosterone and insulin on mannitol Na+ and Cl− Fluxes in cultured epithelia of renal origin (A6): Evidence for increased permeability in the paracellular pathway. Biochim. Biophys. Acta 931:205–214
Garty, H. 1986. Mechanisms of aldosterone action in tight epithelia. J. Membrane Biol. 90:193–205
Gnionsahe, A., Claire, M. Koechlin, N., Bonvalet, J.P., Farman, N. 1989. Aldosterone binding sites along nephron of Xenopus and rabbit. Am. J. Physiol. 257:R87-R95
Granitzer M., Nagel, W., Crabbe J. 1992. Basolateral membrane conductance in A6 cells: effect of high sodium transport rate. Pfluegers Arch. 420:559–565
Hamilton, K.L., Eaton, D.C. 1985. Single-channel recordings from two types of amiloride-sensitive epithelial Na+ channels. Membr. Biochem. 6(2):149–171
Kashgarian, M., Taylor, C.R., Binder, H.J., Hayslett, J.P. 1980. Amplification of cell membrane surface in potassium adaptation. Lab. Invest. 42:581–588
Kemendy, A.E., Kleyman, T., Eaton, D.C. 1992. The effects of aldosterone on single amiloride-blockable channels in A6 epithelia. Am. J. Physiol. 263:C825-C837
Kottra, G., Fromter, E. 1984. Rapid determination of intraepithelial resistance barriers by alternating current spectroscopy. II. Test of model circuits and quantification of results. Pfluegers Arch. 402:421–432
Paccolat, M.P., Geering, K., Gaeggeler, H.P., Rossier, B.C. 1987. Aldosterone regulation of Na+-K+-ATPase in A6 cells: role of growth conditions. Am. J. Physiol. 252(21):C468-C476
Palmer, L.R., Pacha, J., Frindt, G. 1991. Regulation of apical sodium channels in the rat cortical collecting tubule by aldosterone. In: Aldosterone Fundamental Aspects J.P. Bonvalet, N. Farman, M. Lombes, and Rafestin-Oblin, editors, pp. 285–294. John Libber Eurotext and INSERM, Paris
Pellanda, A.M., Gaeggeler, H.P., Horisberger, J.D., Rossier, B.C. 1992. Sodium-independent effect of aldosterone on initial rate of ouabain binding in A6 cells. Am. J. Physiol. 262:C899-C906
Perkins, F.M., Handler, J.S. 1981. Transport properties of toad kidney epithelia in culture. Am. J. Physiol. 241:C154-C159
Rossier, B.C., Pellands, A., Jaisser, F. 1991. Dual sodium independent effects of aldosterone on Na,K ATPase in amphibian cell lines. In: Aldosterone Fundamental Aspects J.P. Bonvalet, N. Farman, M. Lombes, and Rafestin-Oblin, editors, pp. 123–130. John Libber Eurotext and INSERM, Paris
Sansom, S.C., Muto, S., Giebisch, G. 1987. Na-dependent effects of DOCA on cellular transport properties of CCDs from ADX rabbits. Am. J. Physiol. 253:F753-F759
Sansom, S.C., O'Neil, R.G. 1986. Effects of mineralocorticoids on transport properties of cortical collecting duct basolateral membrane. Am. J. Physiol. 251(20):F743-F757
Spooner, P.M., Edelman, I.S. 1975. Further studies on the effect of aldosterone on electrical resistance of toad bladder. Biochim. Biophys. Acta 406:304–314
Verrey, F., Schaerer, E., Zoerkler, P., Paccolat, M.P., Geering, K., Kraehenbuhl, J.P., Rossier, B.C. 1987. Regulation by aldosterone of Na+, K+-ATPase mRNAs, protein synthesis, and sodium transport in cultured kidney cells. J. Cell Biol. 104:1231–1237
Wade, J.B., O'Neil, R.G., Pryor, J.L., Boulpaep, E.L. 1979. Modulation of cell membrane area in renal collecting tubules by corticosteroid hormones. J. Cell Biol. 81:439–445
Watlington, C.O., Perkins, F.M., Munson, P.J., Handler, J.S. 1982. Aldosterone and corticosterone binding and effects on Na+ transport in cultured kidney cells. Am. J. Physiol. 242:F610-F619
Wills, N.K., Clausen, C. 1987. Transport-dependent alterations of membrane properties of mammalian colon measured using impedance analysis. J. Membrane Biol. 95:21–35
Wills, N.K., Eaton, D.C., Lewis, S.A., Ifshin, M.S. 1979. Current-voltage relationship of the basolateral membrane of a tight epithelium. Biochim. Biophys. Acta 555:519–523
Wills, N.K., Lewis, S.A. 1980. Intracellular Na+ activity as a function of Na+ transport rate across a tight epithelium. Biophys. J. 30:181–186
Wills, N.K., Milinoff, L.P. 1990. Amiloride-sensitive Na+ transport across cultured renal (A6) epithelium: Evidence for large currents and high Na:K selectivity. Pfluegers Arch. 416:481–492
Wills, N.K., Millinoff, L.P., Crowe, W.E. 1991. Na+ channel activity in cultured renal (A6) epithelium: regulation by solution osmolarity. J. Membrane Biol. 121:79–90
Wills, N.K., Purcell, R.K., Clausen, C. 1992. Na+ transport and impedance properties of cultured renal (A6 and 2F3) epithelia. J. Membrane Biol. 125:273–285
Yonath, J., Civan, M.M. 1971. Determination of the driving force of the Na+ pump in toad bladder by means of vasopressin. J. Membrane Biol. 5:366–385
Author information
Authors and Affiliations
Additional information
We wish to thank Dr. Simon Lewis for his comments on this manuscript, Dr. B. Rossier for his generous donation of the 2F3 cell line, and Dr. E. Fulep for her technical support. This work was supported by NIH grant DK-29962 (to N.K.W.).
Rights and permissions
About this article
Cite this article
Wills, N.K., Purcell, R.K., Clausen, C. et al. Effects of aldosterone on the impedance properties of cultured renal amphibian epithelia. J. Membarin Biol. 133, 17–27 (1993). https://doi.org/10.1007/BF00231874
Received:
Revised:
Issue Date:
DOI: https://doi.org/10.1007/BF00231874