Activation by N-ethylmaleimide of a Cl--dependent K+ flux in isolated trout hepatocytes.
Isolated trout hepatocytes when swollen in hypotonic medium undergo a regulatory volume decrease (RVD), which occurs via KCl loss. The system shows characteristics similar to those of the transporter described in red cells. This led us to investigate, in trout hepatocytes, the effect of another signal known to activate this flux in red cells, i.e. treatment with the sulphhydryl-group reagent N-ethylmaleimide (NEM). NEM treatment resulted in a striking increase in ouabain-resistant K+ uptake measured by an isotope pulse uptake technique. The time course of the response to NEM was similar to that obtained with a hypotonic shock, indicating that the effect of NEM was immediate and transient. The NEM-stimulated K+ influx demonstrated the same anion sensitivity as the volume-induced K+ influx, i.e. a specific requirement for Br- or Cl-. Efflux experiments showed that NEM treatment produced a stimulation of both K+ and Cl- effluxes leading to a substantial net loss (10%) of cellular KCl, as confirmed by analysis of ionic contents. This KCl loss is consistent with the rapid cell shrinkage observed after addition of NEM. The Cl--dependent K+ influx was found to be independent of external Na+; in addition, NEM had no effect on Na+ content, indicating that Na+ is not implicated in this process. The effect of loop diuretics was tested on the NEM-stimulated K+ influx. As observed for the volume-induced K+ flux, a high concentration of furosemide (10(-3) mol l-1) is required for full inhibition of this flux; no effect was obtained with bumetanide (10(-4) mol l-1). Consequently, NEM appears to activate a KCl cotransport similar to the one induced in hypotonically swollen cells. Finally, the combination of the two treatments, NEM and hypotonic shock, was found to increase the K+ fluxes even further, suggesting additivity of the two stimuli by mutual positive interaction.
Bianchini
L
L
Laboratoire de Physiologie Cellulaire et Comparée, Faculté des Sciences, Université de Nice-Sophia Antipolis, France.
Fossat
B
B
Porthé-Nibelle
J
J
Lahlou
B
B
D000818:Animals; D001834:Body Water / Q000378:metabolism; D002034:Bumetanide / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D002478:Cells, Cultured; D005033:Ethylmaleimide / Q000494:pharmacology*; D005665:Furosemide / Q000494:pharmacology; D008099:Liver / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D011188:Potassium / Q000378:metabolism*; D014337:Trout
D002034:Bumetanide; D005665:Furosemide; D005033:Ethylmaleimide; D011188:Potassium; D002118:Calcium