The use of HgCl2 to evaluate the cosubstrate: amino acid transport stoichiometry in Ehrlich ascites tumor cells.
Recent investigations have indicated that cellular rheogenic properties may interfere with the correct estimation of Na+ and amino transport stoichiometry. We have reevaluated the stoichiometry of Na+ and alpha-aminoisobutyric acid (alpha-AIB) cotransport in Ehrlich ascites tumor cells depleted of Na+ and ATP by incubation in Na+-free HEPES-buffered medium (pH 7.2) containing 160 mM K+ and 2.5 microM valinomycin. Transfer of the cells to a medium with 10 mM 22Na+, 10 mM 3H-AIB, and 150 mM K+ resulted in an enhancement of Na+ flux above basal levels, which represents 0.6 of the AIB uptake. Under these conditions the membrane potential, -7.0 +/- 0.1 mV (SEM), does not change with the addition of AIB, -7.3 +/- 0.6 mV (SEM). HgCl2 (10 microM) added to the medium inhibited AIB flux and AIB-stimulated Na+ flux by 45-50% but did not change the coupling ratio. HgCl2 (10 microM) does not inhibit the basal Na+ flux nor does it affect cellular Na+ or K+ content. In physiological medium cotransport is electrogenic. The membrane potential of Ehrlich cells in physiological medium is -22.3 +/- 0.8 mV (SEM) and depolarizes to -16.7 +/- 0.7 mV (SEM) upon addition of AIB. Under these conditions the coupling ratio was highly variable but the ratio of codepression is 0.90 +/- 0.02 (SEM) in the presence of HgCl2 (10 microM). These results are consistent with a model (Smith and Robinson, 1981) in which the stoichiometry is one cosubstrate molecule per molecule of alpha-AIB. We suggest that H+ provides the alternative cosubstrate in this low Na+ environment and that in high Na+ medium the Na+:AIB stoichiometry approaches 1:1.
D000596:Amino Acids / Q000378:metabolism*; D000621:Aminoisobutyric Acids / Q000494:pharmacology; D000818:Animals; D001693:Biological Transport, Active; D002286:Carcinoma, Ehrlich Tumor / Q000378:metabolism*; D066298:In Vitro Techniques; D007700:Kinetics; D008564:Membrane Potentials / Q000187:drug effects; D008627:Mercuric Chloride; D008628:Mercury / Q000494:pharmacology; D051379:Mice; D012964:Sodium / Q000502:physiology
D000596:Amino Acids; D000621:Aminoisobutyric Acids; D008627:Mercuric Chloride; D012964:Sodium; D008628:Mercury