Effect of H+ on the kinetics of Na+‐dependent amino acid transport in ehrlich ascites tumor cells: Evidence for H+ as an alternative substrate

Smith, Thomas C.; Robinson, Susan C.

doi:10.1002/jcp.1041090317

Cited by 7 publications

(3 citation statements)

References 24 publications

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“…8 and 9). A similar observation has been previously reported (Heinz et al, 1981;Smith & Robinson, 1981a). It is important to note that, although the cells continuously removed H +, pHi did not change.…”

Section: Discussionsupporting

confidence: 78%

H+ transport and the regulation of intracellular pH in ehrlich ascites tumor cells

Bowen

Levinson

1984

J. Membrain Biol.

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The intracellular pH (pHi) of Ehrlich ascites tumor cells, both in the steady state and under conditions of acid loading or recovery from acid loading, was investigated by measuring the transmembrane flux of H+ equivalents and correlating this with changes in the distribution ratio of dimethyloxazolidine-2,4-dione (DMO). The pHi of cells placed in an acidic medium (pHo below 7.15) decreases and reaches a steady-state value that is more alkaline than the outside. For example when pHo is acutely reduced to 5.5, pHi falls exponentially from 7.20 +/- 0.06 to 6.29 +/- 0.04 with a halftime of 5.92 +/- 1.37 min, suggesting a rapid influx of H+. The unidirectional influx of H+ exhibits saturation kinetics with respect to extracellular [H+]; the maximal flux is 15.8 +/- 0.05 mmol/(kg dry wt X min) and Km is 0.74 +/- 0.09 X 10(-6) M. Steady-state cells with pHi above 6.8 continuously extrude H+ by a process that is not dependent on ATP but is inhibited by anaerobiosis. Acid-loaded cells (pHi 6.3) when returned to pHo 7.3 medium respond by transporting H+, resulting in a rapid rise in pHi. The halftime for this process is 1.09 +/- 0.22 min. The H+ efflux measured under similar conditions increases as the intracellular acid load increases. An ATP-independent as well as an ATP-dependent efflux contributes to the restoration of pHi to its steady-state value.

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Section: Discussionsupporting

confidence: 78%

H+ transport and the regulation of intracellular pH in ehrlich ascites tumor cells

Bowen

Levinson

1984

J. Membrain Biol.

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“…This could result in a lower rate of carrier cycling. Such a mechanism has been proposed to account for inhibition of amino acid transport by H' (Smith and Robinson, 1981).…”

Section: Discussionmentioning

confidence: 99%

Evidence for monovalent phosphate transport in Ehrlich ascites tumor cells

Bowen

Levinson

1983

Journal Cellular Physiology

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In an effort to determine whether the Na+-dependent Pi transport system of Ehrlich ascites tumor cells exhibits specificity for H2PO4- or HPO4(-2), Pi fluxes were determined by measuring 32Pi-Pi self-exchange. Three experimental approaches were employed. First, the effect of pH on steady-state Pi transport at 0.5 and 5 mM was studied. Second, the relationship between Pi transport and Pi concentration (0.25-9.2 mM) at pH 5.6 and 7.9 was determined. Third, the dependence of Pi transport on [H2PO4-] (0.05-4.2 mM) at constant [HPO4(-2)] (0.5 mM), and the converse, [HPO4(-2)] (0.06-4.5 mM) at constant [H2PO4-] (0.5 mM), was evaluated. Ks (apparent half-saturation constant) and Jmax (maximal transport rate) were calculated by two methods: weighted linear regression (WLR) and a nonparametric procedure. The dependence of Pi flux on pH indicates that optimum transport occurs at pH 6.9. Pi transport decreases as pH is reduced when extracellular Pi is either 0.5 or 5 mM. However, at pH 7.9, Pi flux is reduced only in 0.5 mM Pi. At pH 5.6, H2PO4- comprises 93% of the total Pi present, and the calculated Ks is 0.055 +/- 0.026 mM (WLR). This is the same as the Ks determined from the initial phase of the flux vs. [H2PO4-] relationship (0.056 +/- 0.020 mM). However, at pH 7.9 (where 94% of Pi is HPO4(-2)), the measured Ks is 0.58 +/- 0.11 mM (WLR), which is ten times higher than at pH 5.6. This value is also five times greater than the Ks calculated from the flux vs. [HPO4(-20)] curve (0.106 +/- 0.16 mM). Kinetic parameters calculated by the nonparametric method, though somewhat different, gave similar relative results. Taken together, these results support two conclusions: (1) H2PO4- is the substrate for the Na+-dependent Pi transport system of the Ehrlich cell, and (2) H+ can inhibit Pi transport.

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“…Garcia-Sancho et al (1977), have suggested that a portion of the energy supply for active transport through System A is de-rived from intracellular reducing equivalents. Smith and Robinson (1981) have proposed that, under certain conditions (low "a+],), amino acid transport could be driven by a proton gradient. Recently, Dawson and Smith (1987) reported that the membrane potential and/or a change in the intracellular Na+ activity, as measured by ion-selective microelectrodes, could provide the needed energy to drive amino acid uptake.…”

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confidence: 99%

Energetic mechanism of system a amino acid transport in normal and transformed mouse fibroblasts

Leister

Schenerman

Racker

1988

Journal Cellular Physiology

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Ouabain treatment (0.4 mM) of normal and transformed C3H-10T1/2 cells caused a progressive increase in 2-aminoisobutyrate (AIB) transport reaching a maximum after 16 to 18 h exposure. There was a virtually complete blockage of this stimulated rate when 3 microM cycloheximide (CHX) was added together with ouabain at T = 0. In the transformed cell, addition of CHX after 14 h had no effect; in the normal cell, it inhibited (ca. 50%) the final AIB transport rate achieved after 24 h. The t1/2 for reaching maximal activity (insensitive to CHX exposure) was thus shifted from 8 h in the transformed cell to 15 h in the normal cell. Since the rate of achieving maximal activity in the absence of CHX was about the same in the two cells, the shift in t1/2 in the presence of CHX suggests that the rate of degradation is more rapid in the normal cell. Following ouabain treatment, the apparent Km for Na+ was decreased in both cells. The Km returned to the basal level 1 h after ouabain removal in the normal cell, but remained low in the transformed cell during this time period. The stimulation of AIB transport following ouabain removal was largely abolished by a proton ionophore (1799), a lipophilic cation (tetraphenyl-phosphonium), or ouabain. These results suggest that, under the conditions of ouabain stress, there is a switch in the bioenergetic mechanism. The Na+/K+ pump and System A transporter appear to be linked and the membrane potential generated by the Na+/K+ pump activity becomes a major driving force for AIB uptake.

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Effect of H⁺ on the kinetics of Na⁺‐dependent amino acid transport in ehrlich ascites tumor cells: Evidence for H⁺ as an alternative substrate

Cited by 7 publications

References 24 publications

H+ transport and the regulation of intracellular pH in ehrlich ascites tumor cells

H+ transport and the regulation of intracellular pH in ehrlich ascites tumor cells

Evidence for monovalent phosphate transport in Ehrlich ascites tumor cells

Energetic mechanism of system a amino acid transport in normal and transformed mouse fibroblasts

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