Advertisement

The Journal of Membrane Biology

, Volume 65, Issue 3, pp 227–234 | Cite as

Effect of amiloride, ouabain and Ba++ on the nonsteady-state Na−K pump flux and short-circuit current in isolated frog skin epithelia

  • Robert Nielsen
Articles

Summary

Effect of amiloride, ouabain, and Ba++ on the nonsteady-state Na−K pump flux and short-circuit current in isolated frog skin epithelia.

The active Na+ transport across isolated frog skin occurs in two steps: passive diffusion across the apical membrane of the cells followed by an active extrusion from the cells via the Na+−K+ pump at the basolateral membrane. In isolated epithelia with a very small Na+ efflux, the appearing Na+-flux in the basolateral solution is equal to the rate of the pump, whereas the short-circuit current (SCC) is equal to the active transepithelial Na+ transport. It was found that blocking the passive diffusion of Na+ across the apical membrane (addition of amiloride) resulted in an instantaneous inhibition of the SCC (the transepithelial Na+ transport, whereas the appearing flux (the rate of the Na+−K+ pump) decreased with a halftime of 1.9 min. Addition of the Na+−K+ pump inhibitor ouabain (0.1mm) resulted in a faster and bigger inhibition of the appearing flux than of the SCC. Thus, by simultaneous measurement of the SCC and the appearing Na+ flux one can elucidate whether an inhibitor exerts its effect by inhibiting the pump or by decreasing the passive permeability. Addition of the K+ channel inhibitor Ba++, in a concentration which gave maximum inhibition of the SCC, had no effect on the appearing flux (the rate of the Na−K pump) in the first 2 min, although the inhibition of the SCC was already at its maximum.

It is argued that in the short period, where the Ba++-induced inhibition of SCC is at its maximum and the appearing flux in unchanged, the decrease in the SCC (ΔSCC) is equal to the net K+ flux via the Na+−K+ pump, and the coupling ratio (β) of the Na+−K+ pump can be calculated from the following equation β=SCCt=0/ΔSCC where SCCt=0 is the steady-state SCC before the addition of Ba++.

Key words

frog skin sodium flux ouabain amiloride barium 

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. Baba, W.I., Lant, A.F., Smith, A.J., Townshend, M.M., Wilson, G.M. 1968. Pharmacological effects in animals and normal human subjects of the diuretic amiloride hydrochloride (mK 870).Clin. Pharmacol. Ther. 9:318–327PubMedGoogle Scholar
  2. Cala, P.M., Cogswell, N., Mandel, L.J. 1978. Binding of [3H] ouabain to split frog skin. The role of the Na, K-ATPase in the generation of short circuit current.J. Gen. Physiol. 71:347–367PubMedGoogle Scholar
  3. Candia, O.A., Reinach, P.S. 1977. Sodium washout kinetics across inner and outer barriers of the isolated frog skin epithelium.Biochim. Biophys. Acta 468:341–352PubMedGoogle Scholar
  4. Carasso, N., Favard, P., Jard, S., Rajerison, R.M. 1971. The isolated frog skin epithelium. I. Preparation and general structure in different physiological states.J. Microsc. 10:315–330Google Scholar
  5. Henderson, E.C. 1974. Strophanthidin sensitive electrogenic mechanisms in frog sartorius muscles exposed to barium.Pfluegers Arch. 350:81–95Google Scholar
  6. Hermsmeyer, K., Sperelakis, N. 1970. Decrease in K+ conductance and depolarization of frog cardiac muscle produced by Ba++.Am. J. Physiol. 219:1108–1114Google Scholar
  7. Hodgkin, A.L., Katz, B. 1949. The effect of sodium ions on the electrical activity of the giant axon of the squid.J. Physiol. (London) 108:37–77Google Scholar
  8. Johnsen, A.H., Nielsen, R. 1978. Effects of the antidiuretic hormone, arginine vasotocin, theophylline, filipin and A23187 on cyclic AMP in isolated frog skin epithelium (Rana temporaria).Acta Physiol. Scand. 102:281–289PubMedGoogle Scholar
  9. Koefoed-Johnsen, V., Ussing, H.H. 1958. The nature of the frog skin potential.Acta Physiol. Scand. 42:298–308PubMedGoogle Scholar
  10. Lewis, S.A., Wills, N.K., Eaton, D.C. 1978. Basolateral membrane potential of a tight epithelium: Ionic diffusion and electrogenic pumps.J. Membrane Biol. 41:117–148Google Scholar
  11. Macknight, A.D.C., Leaf, A. 1978. Epithelial cell electrolyte in relation to transepithelial sodium transport across toad urinary bladder.J. Membrane Biol. Special Issue:247–260Google Scholar
  12. Nagel, W. 1979. Inhibition of potassium conductance by barium in frog skin epithelium.Biochim. Biophys. Acta 552:346–357PubMedGoogle Scholar
  13. Nielsen, R. 1979a. Coupled transepithelial sodium and potassium transport across isolated frog skin: Effect of ouabain, amiloride and the polyene antibiotic filipin.J. Membrane Biol. 51:161–184Google Scholar
  14. Nielsen, R. 1979b. A 3 to 2 coupling of the Na−K pump responsible for the transepithelial Na transport in frog skin disclosed by the effect of Ba.Acta Physiol. Scand. 107:189–191PubMedGoogle Scholar
  15. Nielsen, R. 1981. Determination of the coupling ratio of the Na−K pump reponsible for transepithelial Na transport by blockade of K channels.In: Advances in Physiological Sciences. Vol. 3 Physiology of Non-excitable Cells. J. Salánki, editor. 28th International Congress of Physiological Sciences (Budapest, 1980). Akadémiai Kiadó, BudapestGoogle Scholar
  16. Nielsen, R. 1982. Effect of ouabain, amiloride, and antidiuretic hormone on the sodium-transport pool in isolated epithelia from frogskin (Rana temporaria).J. Membrane Biol. 65:221–226Google Scholar
  17. Nielsen, R., Tomlinson, R.W.S. 1970. The effect of amiloride on sodium transport in the normal and moulting frog skin.Acta Physiol. Scand. 79:238–243PubMedGoogle Scholar
  18. Parsons, R.H., Hoshiko, T. 1971. Separation of epithelium from frog skin and rapid washout of22Na.J. Gen. Physiol. 57:254–255Google Scholar
  19. Rick, R., Dörge, A., Arnim, E. von, Thurau, K. 1978. Electron microprobe analysis of frog skin epithelium: Evidence for a syncytial sodium transport compartment.J. Membrane Biol. 39:313–331Google Scholar
  20. Schultz, S.C. 1978. Is a coupled Na−K exchange pump involved in active transepithelial Na transport? A status reportIn: Membrane Transport Processes. J.F. Hoffman, editor. Vol. 1, pp. 213–227. Raven, New YorkGoogle Scholar
  21. Ussing, H.H., Zerahn, K. 1951. Active transport of sodium as the source of electric current in the short-circuited isolated frog skin.Acta Physiol. Scand. 23:110–127PubMedGoogle Scholar
  22. Voûte, C.L., Møllgård, K., Ussing, H.H. 1975. Quantitative relationship between active sodium transport expansion of endoplasmic reticulum and specialized vacuoles (“scalloped sacs”) in the outermost living cell layer of the frog skin epithelium (Rana temporaria).J. Membrane Biol. 21:273–289Google Scholar
  23. Voûte, C.L., Ussing, H.H. 1970a. The morphological aspects of shunt-path in the epithelium of the frog skin (Rana temporaria).Exp. Cell Res. 61:133–140PubMedGoogle Scholar
  24. Voûte, C.L., Ussing, H.H. 1970b. Quantitative relation between hydrostatic pressure gradient, extracellular volume and active sodium transport in the epithelium of the frog skin (Rana temporaria).Exp. Cell Res. 62:375–383.PubMedGoogle Scholar

Copyright information

© Springer-Verlag New York Inc. 1982

Authors and Affiliations

  • Robert Nielsen
    • 1
  1. 1.Institute of Biological Chemistry AUniversity of CopenhagenCopenhagenDenmark

Personalised recommendations