Role of Na ⁺ in α-aminoisobutyric acid uptake by membrane vesicles from mouse fibroblasts transformed by simian virus 40

Abstract

The uptake of a-amino[3H]isobutyric acid (AIB) was studied in membrane vesicles from mouse fibroblasts transformed by simian virus 40 to examine the features of the Na+-stimulated and Na+-dependent AIB transport process. The simultaneous addition of NaCI and AIB to these vesicles produced a transient accumulation, or "overshoot," of amino acid 3-4 times the equilibrium value. Both the initial rate of uptake and the rate of fall of intravesicular AIB after maximal accumulation were sensitive to the temperature of incubation.The overshoot of AIB uptake was enhanced with Na+ salts of highly permeant lipophilic anions, such as SCN-and NO3-, and was decreased by the addition of S042' a relatively impermeant ion. Gramicidin D, which enhances the membrane conductance of Na+ electrogenically, decreased the overshoot, while a potassium diffusion potential, induced by valinomycin (in K+-preloaded membrane vesicles), produced a Na+-dependent overshoot of AIB uptake.When vesicles were preincubated with both Na+ and AIB, followed by the generation of an interior negative membrane potential (by the addition of SCN-), an overshoot of AIB uptake resulted. However, this did not occur in the absence of Na+. It is concluded that, apart from its role in the generation of a transmembrane electrochemical potential, Na+ is essential for the overshoot of AIB uptake.The recent development of techniques for the isolation of cell plasma membrane vesicles that retain transport activity has permitted a more detailed study of the possible mechanisms involved in the transport of nutrients across cell membranes. The use of membrane vesicles to study transport allows for a separation of membrane events from subsequent intracellular metabolic processes. Membrane vesicles were originally used to study bacterial transport systems (1); recently they have also been used to investigate the uptake of nutrients by mammalian cells. Several laboratories have reported that Na+ stimulates the uptake of amino acids, such as L-leucine, a-aminoisobutyric acid (AIB), and L-alanine, in membrane vesicles from virally transformed and nontransformed mouse fibroblasts (2-9). Studies in this laboratory have demonstrated that mediated uptake of L-leucine and AIB by membrane vesicles from mouse fibroblasts transformed by simian virus 40 is stimulated by a NaCl gradient (external > internal) (2,5). In those studies it was shown that this enhancement is Na+ specific and includes a prominent "overshoot," namely, a transient intravesicular accumulation above the equilibrium level (i.e., active transport) during the initial uptake phase. However, a detailed understanding of the characteristics and mechanism of this stimulatory effect of NaCl (overshoot phenomenon) was not obtained. To investigate this mechanism, we have examined the effects on AIB uptake of (i) temperature of incubation, (ii) specific

Amino acid and22Na+ uptake in membrane vesicles from confluent simian virus 40 transformed Balb/c3T3 and Balb/c3T3

“…8, 9 and 10) reveals that the maximal level of transient, intravesicular concentration of AIB varies with the magnitude of an externally imposed Na + gradient. These data are consistent with the possibility that the overshoot is energized by the chemical potential difference for Na + and are in agreement with findings of other investigators using vesicles from SV3T3 and other sources [14,16,19].…”

“…Overshooting in SV3T3 vesicles in response to an initial Na + gradient can be greatly attenuated, or abolished by the Na + ionophore, gramicidin [19]. This effect is presumably produced by rapid collapse of the initial Na + gradient.…”

Amino Acid Transport by Membrane Vesicles from VirallyTransformed and Nontransformed Cells

“…Therefore, the effect of SCN-and NO3-on AIB uptake by native membrane vesicles from 3T3 cells was tested. The transient concentrative uptake of AIB by native membrane vesicles from nontransformed 3T3 cells took place in the presence of 100 mM NaSCN or NaNO3, although the overshoot pattern itself differed from that of SV3T3 cell membranes under the same conditions of transport assay (8) (Fig. 2).…”

“…Furthermore, the overshoot may be generated by native membrane vesicles from nontransformed cells under adequate conditions. In SV3T3 membrane vesicles a greater overshoot of AIB uptake has been reported in the presence of the Na+ salts of the permeant lipophilic anions, SCN-and NO3-, in comparison with NaCl at the same concentration (8). Therefore, the effect of SCN-and NO3-on AIB uptake by native membrane vesicles from 3T3 cells was tested.…”

“…2). Interestingly, in SV3T3 membrane vesicles changes in the shape of the overshoot have been reported when the temperature of the transport assay was lowered (8). Therefore, the shape of the overshoot at a low temperature of transport assay was analyzed in native membrane vesicles from SV3T3 cells and compared with that generated by vesicles from nontransformed 3T3 cells.…”