Regulation of active alpha-aminoisobutyric acid transport expressed in membrane vesicles from mouse fibroblasts.

Abstract

Membrane vesicles isolated from untransformed Balb/c and Swiss mouse fibroblasts and from those transformed by simian virus 40 catalyzed carrier-mediated uptake of L-a-aminoisobutyric acid. Concentrative uptake required the presence of a Na+ gradient (external Na+ > internal Na+) and occurred independently of endogenous (Na+ + K+) ATPase activity. This process is electrogenic, since uptake was stimulated by a K+ diffusion gradient (internal > external) in the presence of valinomycin or by the addition of the Na+ salt of a permeant ion, conditions expected to create an interiornegative membrane potential. Both the initial rate of concentrative uptake of L-a-aminoisobutyric acid and its maximal accumulation, driven by a standard Na+ gradient, were decreased in vesicles from density-inhibited, untransformed cells and increased in those from cells transformed by simian virus 40 compared with vesicles from proliferating untransformed cells. An increased maximal velocity ( Vm) of uptake stimulated by Na+ gradient was observed in vesicles from transformed cells compared with those from untransformed cells, suggesting an increase in the number of carriers or in their mobility. Since the relative extent of accumulation of this model amino acid driven by a standard Na+ gradient also differed with growth or transformed status, an additional possibility for cellular regulation of this process could be alteration of membrane Na+ permeability or carrier response to Na+. Alterations in nutrient uptake activity that accompany changes in cellular proliferation rate of untransformed mouse fibroblasts induced by serum, hormones, or viral transformation (1) have been implicated in models of cell cycle control involving regulation of surface membrane functions (2, 3). However, studies of regulation of nutrient uptake using intact cells have been unable to distinguish unambiguously between effects of intracellular metabolic changes, changes associated with the plasma membrane, and effects of surface area and cell overlap on uptake rates. In certain cases, even the use of nonmetabolizable analogues does not permit specific assessment of the locus of regulation of uptake activity. For example, the activity of the Na+ and K+ transport system in fibroblasts, expressed enzymatically as a (Na+ + K+)ATPase, is altered by cell density and proliferation rate (4), serum and purified growth-promoting agents (5), and viral transformation (6). Thus, the rate of Na+-dependent, ouabain-sensitive a-aminoisobutyric acid (iso-Abu) uptake (7) could reflect this observed regulation of the Na+ pump activity rather than alteration of the amino acid carriers.In this report, these possibilities are investigated using sealed membrane vesicles with transport catalytic activity, isolated from mouse fibroblasts. Evidence for Na+ gradient-stimulated, electrogenic, concentrative iso-Abu uptake was obtained, as recently described for intestinal brush border membranes (8) and Ehrlich ascites cell membranes (9). By use of transvesicular Na+ gradients maintai...

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

“…Using vesicles from SV3T3, we have previously demonstrated Na +-gradient stimulated, mediated transport of L-leucine and AIB into an osmotically sensitive space [23]. The results with AIB have been confirmed in several subsequent reports [4,12]. We have also examined uptake of Lleucine and AIB into vesicles prepared from the untransformed parent line, Balb/c3T3, and reported that the transient, concentrative accumulation of L-leucine and AIB in response to an initial Na+-gradient produced by NaC1, characteristic of vesicles from SV3T3, is much less prominent in 3T3 vesicles [21].…”

“…4 Previously reported experiments with SV3T3 in which the osmolarity of the extra-vesicular medium was systematically varied with sucrose also produced evidence of significant binding [221. Replots of data derived from studies of Na+-independent AIB uptake in SV3T3 vesicles reported by other investigators [ 12,18] according to Eq. (4) also give nonzero intercepts similar in magnitude to those in Fig.…”

Evidence for Amino Acid-H+ Co-Transport in Oat Coleoptiles

“…These depolarization data can be compared with the AIB transport data in Figure 3, which show a similar trend. (10) 3.5 + 1 (12) All solutions were S1 with 25 mM NaCl added for a total NaCl concentration of 29 mM. The pH was 6.5 and the AIB concentration was 4 mM.…”

“…Many workers propose that these energy-requiring transport processes are mediated by membrane carrier mechanisms that use the inwardly directed free energy (chemical potential) gradients of either sodium ions in animal cells (2,12,19) or hydrogen ions in bacteria, fungi, algae, and higher plants (6,7,8,14,22). This process, called co-transport (21) or symport (14), is essentially a means for coupling the active transport of one substrate to the passive movement of another substance-both moving in the same direction.…”

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

“…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)(3)(4)(5)(6)(7)(8)(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).…”