title: Effects of N-ethylmaleimide on ouabain-insensitive cation fluxes in human red cell ghosts.
abstract: In red cells of several species, the sulfhydryl reagent N-ethylmaleimide activates a Cl- -dependent, ouabain-resistant K+ transport pathway. Here we report our attempts to demonstrate ouabain-resistant Cl- -dependent K+ fluxes stimulated by N-ethylmaleimide in resealed human red cell ghosts using Rb+ as a K+ analogue. In contrast to intact cells, the rate constants of the base level Rb+ efflux in ghosts were similar in NaNO3 and NaCl (okRb = 0.535 +/- 0.079 h-1 and 0.534 +/- 0.085 h-1, respectively), while 1 mM N-ethylmaleimide stimulated Rb+ efflux strongly in NaNO3 (okRb = 14.26 +/- 1.32 h-1) and moderately in NaCl (okRb = 2.73 +/- 0.54 h-1). This effect was dependent on the presence of internal ATP. Stimulation of Rb+ efflux was observed in the presence of greater than or equal to 0.2 mM N-ethylmaleimide and increased at pH values approaching 8.0, consistent with titration of SH groups. N-Ethylmaleimide-stimulated Rb+ efflux was approx. 50% inhibited by 100 microM quinine sulfate whereas 1 microM bumetanide had no effect. In NaCl the N-ethylmaleimide-stimulated efflux saturated with initial internal ghost Rb+ concentration, but rates increased linearly in NaNO3. Replacement of external Na+ with glucamine or choline decreased the N-ethylmaleimide-stimulated Rb+ efflux, suggesting a role for external Na+. N-Ethylmaleimide-stimulated Rb+ efflux was greater in buffers with lipophilic anions such as SCN- or NO3- than in solutions with Cl- or acetate. However, the cation selectivity of the pathway studied was low, as Li+ efflux was also stimulated by N-ethylmaleimide. We conclude that the effect of N-ethylmaleimide on ouabain-resistant cation effluxes of human red cell ghosts is very different from the selective action of N-ethylmaleimide on Rb+ influxes in intact red cells.
mesh_terms: D000085:Acetates / Q000097:blood; D019342:Acetic Acid; D000255:Adenosine Triphosphate / Q000502:physiology; D001692:Biological Transport / Q000187:drug effects; D002254:Carbonates / Q000494:pharmacology; D004910:Erythrocyte Membrane / Q000187:drug effects / Q000378:metabolism*; D005033:Ethylmaleimide / Q000494:pharmacology*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D008094:Lithium / Q000097:blood; D009566:Nitrates / Q000097:blood; D010042:Ouabain / Q000494:pharmacology*; D011188:Potassium / Q000097:blood*; D012413:Rubidium / Q000097:blood; D012965:Sodium Chloride / Q000097:blood; D013696:Temperature; D013861:Thiocyanates / Q000097:blood
chemical_list: D000085:Acetates; D002254:Carbonates; D009566:Nitrates; D013861:Thiocyanates; D012965:Sodium Chloride; C005686:sodium carbonate; D010042:Ouabain; C024553:sodium thiocyanate; D000255:Adenosine Triphosphate; C031618:sodium nitrate; D008094:Lithium; D012413:Rubidium; D005033:Ethylmaleimide; D019342:Acetic Acid; D011188:Potassium