Amino acid active transport and stimulation by substrates in the absence of a Na+ electrochemical potential gradient

Abstract

Uptake of alpha-aminoisobutyric acid (AIB) was examined in Ehrlich ascites tumor cells treated with the cation-exchange ionophore nigericin (20 microgram/ml). Membrane voltages were measured using the voltage-sensitive dye diethyloxadicarbocyanine (DOCC). In normal phosphate-buffered media, nigericin changed the distribution ratios of Na+ and K+ (the ratio of intra- to extracellular concentrations) nearly to unity, but AIB was still accumulated to a distribution ratio of approximately 9.0. When all but 40 mM Na+ in the medium was replaced by choline, nigericin resulted in K+ loss and Na+ gain and both cation distribution ratios approached 2.8-3.4, as would be expected if both ions were distributing near electrochemical equilibrium with a membrane voltage in the range of -28 to -33 mV. This conclusion was supported by the observation that the addition of 5 X 10(-7) M valinomycin to the nigericin-treated cell suspension produced no change in DOCC absorbance. In spite of the apparent zero electrochemical potential gradients for Na+ and K+, AIB was accumulated to a distribution ratio of 5.4 in the low-Na+ medium. Addition of 0.1 mM oubain or 50 microM vanadate did not alter the extent of AIB accumulation as would have been expected if a large component of the membrane voltage were due to electrogenic operation of the (Na+ + K+)-ATPase. Addition of lactate, pyruvate or glucose increased the AIB distribution ratios to 11.9, 9.4 and 15.3, respectively. The effect of glucose could be explained, at least in part, by an enhanced Na+ electrochemical potential gradient. However, neither lactate nor pyruvate produced any change either in membrane voltage or the intracellular Na+ concentration. Therefore, these results confirm the existence of a metabolic energy source which is coupled to AIB accumulation and operates in addition to the Na+ co-transport mechanism, and which is augmented by metabolic substrates such as lactate and pyruvate.

Ionic dependence of amino-acid transport in the exocrine pancreatic epithelium: Calcium dependence of insulin action

“…It is generally accepted that influx of Na + and the energy from the Na + gradient can drive uphill amino-acid transport [9], although some studies have concluded that active amino-acid transport also occurs in the absence of cation gradients [15,23]. In the pancreas many amino acids evoke a similar depolarization of the acinar cell membrane [33], but it is difficult based on these electrical measurements to quantify what fraction of amino-acid uptake was actually Na + dependent.…”

Plasma membrane potential of Lettré cells does not depend on cation gradients but on pumps

“…Others have failed to observe such an effect of ouabain on Ehrlich ascites cells (Heinz, Sachs & Schafer, 1981a;Heinz et al, 1981b; but see Laris et al, 1978;Philo & Eddy, 1978), possibly because insufficient ouabain was added: the Na + pump is known to have differing affinities for ouabain (Charnock & Simonson, 1977;Kaplan, 1978) in different cells and under different conditions, particularly in Ca>-free media (Mansier & Lelievre, 1982). We have observed considerable variation in the ouabain sensitivity of Lettr6 cells, which might suggest that the Na + pump is variably activated in these cell preparations.…”

Proceedings of the Physiological Society, 11‐12 November 1983, Queen Elizabeth College, London Meeting: Communications

“…The magnitude of these outward currents depended markedly on [Ca2+]1. The outward current associated with a 40 mV depolarizing step, for example, was about 50-100 pA at Hoffmann & Lambert, 1983), a recent report describes circumstances in which mouse ascites tumour cells concentrated 2-aminoisobutyrate extensively when an appropriate sodium gradient appeared to be lacking (Heinz, Jackson, Richey, Sachs & Schafer, 1981). The iso-osmotic Ringer solution contained about 42 mM-Na+ and 24 mM-K+ and the presence ofthe ionophore nigericin (20 ,ug/ml) ensured that the cellular concentrations of Na+ and K+ were up to three times those in the Ringer solution.…”

“…This behaviour suggests that the sodium pump is closely involved in amino acid uptake under these conditions and may hyperpolarize the tumour cells. The assay of membrane voltage used by Hacking & Eddy (1981) cannot be used to test this possibility because it involves deliberately stopping oxidative phosphorylation (see, however, Heinz et al 1981). …”