The Na+ gradient hypothesis in cytoplasts derived from Ehrlich ascites tumor cells

The concentration gradients of Na⁺ and the non-metabolizable amino acid, α-aminoisobutyric acid, and the membrane potential were measured in cytoplasts derived from Ehrlich ascites tumor cells in order to test the Na⁺ gradient hypothesis for the active transport of neutral amino acids in animal cells. According to this hypothesis, the Na⁺ electrochemical gradient and the amino acid activity gradient should be equal at the steady state. It has been difficult to measure the Na⁺ electrochemical gradient in intact Ehrlich cells because Na⁺ may be sequestered in the nuclei of these cells. This problem is avoided with cytoplasts derived from Ehrlich cells because they do not contain internal compartments where Na⁺ could be sequestered. Since these cytoplasts also maintain steady state concentrations of Na⁺, K⁺, and α-aminoisobutyric acid similar to those found in whole Ehrlich cells, they are uniquely suited for testing the Na⁺ gradient hypothesis. Assuming the activity coefficients of external and cytoplasmic Na⁺ are equal, the energy in the Na⁺ electrochemical gradient of cytoplasts was 90% of that in the α-aminoisobutyric acid concentration gradient at the steady state. If the Na⁺ gradient hypothesis is correct, the 10% difference between these two gradients cannot be explained in terms of the sequestration of Na⁺ in the nucleus because cytoplasts do not contain internal compartments.     

Glucagon regulation of amino acid transport in hepatocytes: Effect of cell enucleation

Glucagon and cAMP analogs stimulate amino acid transport in freshly isolated hepatocytes by inducing the synthesis of new transport proteins. The role of the cell nucleus in the glucagon regulation of amino acid transport has been studied in rat hepatocytes enucleated by centrifugation through a discontinuous Ficoll gradient in the presence of cytochalasin B. Enucleated hepatocytes take up alpha-aminoisobutyric acid (AIB) through a Na⁺-dependent transport component with kinetic properties similar to those found in intact hepatocytes. Cytoplasts prepared from glucagon-stimulated cells retain the increase AIB transport induced by the hormone in the intact cells. The direct addition of glucagon to cytoplasts has no effect on AIB transport, in spite of the fact that the cytoplasts exhibit a higher capacity to bind glucagon than their nucleated counterparts. These data indicate that the nucleus is required for the glucagon stimulation of amino acid transport in isolated hepatocytes.     

Effects of extremely-law-frequency electromagnetic fields on ion transport in several mammalian cells

We have investigated the effects of sinusoidal electromagnetic fields (EMF) on ion transport (Ca²⁺, Na⁺, K⁺, and H⁺) in several cell types (red blood cells, thymocytes, Ehrlich ascites tumor cells, and HL60 and U937 human leukemia cells). The effects on the uptake of radioactive tracers as well as on the cytosolic Ca²⁺ concentration ([Ca²⁺]_i), the intracellular pH (pH_i), and the transmembrane potentsial (TMP) were studied. Exposure to EMF at 50 Hz and 100–2000 μT (rms) had no significant effects on any of these parameters. Exposure to EMF of 20–1200 μT (rms) at the estimated cyclotron magnetic resonance frequencies for the respective ions had no significant effects except for a 12–32% increase of the uptake of ⁴²K within a window at 14.5–15.5 Hz and 100–200 μT (rms), which was found in U937 and Ehrlich cells but not in the other cell types. © 1994 Wiley-Liss, Inc.     

Ion transport by ouabain resistant and sensitive ehrlich ascites carcinoma cells

Cell division, net Na⁺-K⁺ and amino-acid transport of cultured Ehrlich ascites is reversibly inhibited by Ouabain at a final concentration of 1 × 10^–3M. A line of Ehrlich ascites cells resistant to the growth inhibiting effects of Ouabain has been developed. These cells behave similarly to Ouabain-sensitive cells in the following respects doubling time, S phase time, chromosome number, cell surface charge density, rate of incorporation of C¹⁴ Uridine and ³H-Thymidine, sensitivity to Digoxin and Digitoxin, steady state Na⁺, K⁺ levels and rate of loss of K⁺ and gain of Na⁺ in cold. Ouabain resistant cells differ from sensitive cells only with respect to the effect of ouabain on active Na⁺, K⁺ transport. Although Ouabain inhibits active Na⁺, K⁺ transport in sensitive cells it has no significant effect in resistant cells.     

Ca2+-dependent K+ transport in the Ehrlich ascites tumor cell

The possible presence and properties of the Ca²⁺-dependent K⁺ channel have been investigated in the Ehrlich ascites tumor cell. The treatment with ionophore A23187+Ca²⁺, propranolol or the electron donor system ascorbate-phenazine methosulphate, all of which activate that transport system in the human erythrocyte, produces in the Ehrlich cell a net loss of K⁺ (balanced by the uptake of Na⁺) and a stimulation of both the influx and the efflux of ⁸⁶Rb. These effects were antagonized by quinine, a known inhibitor of the Ca²⁺-dependent K⁺ channel in other cell systems, and by the addition of EGTA to the incubation medium. Ouabain did not have an inhibitory effect. These results suggests that the Ehrlich cell possesses a Ca²⁺-dependent K⁺ channel whose characteristics are similar to those described in other cell systems.     

Lanthanum-induced alterations of cellular electrolytes in Ehrlich ascites tumor cells: a new view

We have shown previously that Ehrlich ascites tumor cells maintained at room temperature under an oxygen atmosphere lose Na⁺, K⁺ and Cl^? isosmotically when exposed to La⁺⁺⁺ (0.1 to 1.0 mM). Concomitant with these changes there is an increase in the recorded membrane potential (increasing intracellular negativity). The present studies further characterize the effect of La⁺⁺⁺ on electrolyte distribution. Ehrlich ascites tumor cells were maintained at 0.5° C to permit Na⁺ gain and K⁺ loss. The addition of 1 mM La⁺⁺⁺ to low temperature cells induces rapid loss of Na⁺, K⁺ and Cl^?. This net loss of cellular electrolytes occurs even in cells depleted of ATP content using 2-deoxyglucose (5 mM) and rotenone (10^?6 M ). Analysis of the appearance of tracer ²²Na in the environment of cells preloaded with the radioisotope shows that La⁺⁺⁺-induced changes in membrane permeability or in active ion transport mechanisms are not responsible for the dramatic loss of electrolytes from experimental cells. The electrolyte loss occurs only when the cells are resuspended mechanically during the washing procedure used to prepare the cells for electrolyte determination. We conclude that the results of La⁺⁺⁺ interaction with Ehrlich ascites tumor cells are twofold. As we have previously reported, La⁺⁺⁺ stabilizes and causes a hyperpolarization of the membrane potential. Secondly, La⁺⁺⁺ predisposes the cell membrane to become highly permeable when subjected to mechanical stress.     

Ca2+ translocation in Ehrlich ascites tumor cells

Summary Ca²⁺ uptake into Ehrlich ascites tumor cells was studied at 0°C in the presence of mitochondrial inhibitors, conditions that minimized complications caused by sequestration of Ca²⁺ into organelles or by excretion. Under these conditions Ruthenium Red inhibited Ca²⁺ uptake, but other previously implicated ions, such as P_i or Mg²⁺, had no effect. Valinomycin either inhibited or slightly stimulated Ca²⁺ uptake depending on the presence of excess K⁺ on the outside or inside of the cell, respectively. Nigericin inhibited Ca²⁺ transport. Based on these data we propose an electrogenic uptake of Ca²⁺, possibly via a Ca²⁺/H⁺ antiport mechanism.The observation that glucose inhibited Ca²⁺ uptake suggested that in Ehrlich ascites tumor cells an energy-driven Ca²⁺ expulsion mechanism is operative, similar to that in erythrocytes. Plasma membrane preparations of ascites tumor cells were found to contain a Ca²⁺-dependent ATPase. These preparations, when incorporated into liposomes in an inside-out orientation, catalyzed an ATP-dependent uptake of Ca²⁺.     

The efficiency of energetic coupling between Na flow and amino acid transport in ehrlich cells — A revised assessment

The overall efficiency of the coupling between transport of α-aminoisobutyrate and the entry of Na⁺ in Ehrlich cells has previously been determined to be 8–10%. It was concluded that the efficiency is grossly inadequate to account for the energization of amino acid transport by the electrochemical potential gradient of Na⁺, as postulated by the “gradient hypothesis”. This conclusion had, however, not taken into account that a major part of the Na⁺ entry is not coupled to a α-aminoisobutyrate transport. The “intrinsic efficiency”, which relates the amino acid transport to the coupled Na⁺ entry only, has now been evaluated from available experimental data and found to be approximately adequate to account for the highest accumulation ratios for this amino acid reported. It is concluded that the gradient hypothesis cannot be rejected on energetic grounds.     

Regulation of the electrogenic (Na+ + K+)-pump of Ehrlich cells by intracellular cation levels

Ehrlich cells actively accumulate neutral amino acids even if both the Na⁺ and K⁺ gradients are inverted. The seeming contradiction of this observation to the gradient hypothesis is, however, explained by the presence of a powerful electrogenic Na⁺ pump, which stongly raises the electrochemical potential gradient of Na⁺ under these conditions. Since the evidence of this pump has so far been found only during abnormal concentrations of alkali ions (low K⁺, high Na⁺) in these cells, the question arises whether the pump is equally powerful with completely normal cells, when the pump is not ‘needed’ for amino acid transport. Using the initial rate of uptake of the test amino acid (2-aminoisobutyrate) as a sensitive monitor of the electrical potential at constant cation distribution between cell and medium, a procedure has been devised to split the overall electrical potential into the diffusional and the pump component. With this procedure it could be shown that the electrogenic pump per se is most powerful in K⁺-depleted and Na⁺-rich cells but declines to a lower ‘resting’ value according as the electrolyte content of the cell approaches normality. A strong positive correlation between cellular Na⁺ content and the electrogenic pumping activity suggests that the intracellular activity of this ion regulates the rate of the electrogenic pump. The low activity of the pump under normal conditions may explain why the existance of this pump has rarely come to attention previously.     

Shrinkage insensitivity of NKCC1 in myosin II-depleted cytoplasts from Ehrlich ascites tumor cells

Protein phosphorylation/dephosphorylation and cytoskeletal reorganization regulate the Na⁺-K⁺-2Cl^– cotransporter (NKCC1) during osmotic shrinkage; however, the mechanisms involved are unclear. We show that in cytoplasts, plasma membrane vesicles detached from Ehrlich ascites tumor cells (EATC) by cytochalasin treatment, NKCC1 activity evaluated as bumetanide-sensitive ⁸⁶Rb influx was increased compared with the basal level in intact cells yet could not be further increased by osmotic shrinkage. Accordingly, cytoplasts exhibited no regulatory volume increase after shrinkage. In cytoplasts, cortical F-actin organization was disrupted, and myosin II, which in shrunken EATC translocates to the cortical region, was absent. Moreover, NKCC1 activity was essentially insensitive to the myosin light chain kinase (MLCK) inhibitor ML-7, a potent blocker of shrinkage-induced NKCC1 activity in intact EATC. Cytoplast NKCC1 activity was potentiated by the Ser/Thr protein phosphatase inhibitor calyculin A, partially inhibited by the protein kinase A inhibitor H89, and blocked by the broad protein kinase inhibitor staurosporine. Cytoplasts exhibited increased protein levels of NKCC1, Ste20-related proline- and alanine-rich kinase (SPAK), and oxidative stress response kinase 1, yet they lacked the shrinkage-induced plasma membrane translocation of SPAK observed in intact cells. The basal phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK) was increased in cytoplasts compared with intact cells, yet in contrast to the substantial activation in shrunken intact cells, p38 MAPK could not be further activated by shrinkage of the cytoplasts. Together these findings indicate that shrinkage activation of NKCC1 in EATC is dependent on the cortical F-actin network, myosin II, and MLCK. F-actin; Na⁺-K⁺-2Cl^– cotransporter; myosin light chain kinase; protein kinase A     

Electrically silent cotransport of Na+, K+ and Cl− in ehrlich cells

A cotransport system for Na⁺, K⁺ and Cl^? in Ehrlich cells is described. It is insensitive towards ouabain but specifically inhibited by furosemide and other ‘high ceiling’ diuretics at concentrations which do not affect other pathways of the ions concerned. As the furosemide-sensitive fluxes of these ions are not affected by changes in membrane potential, and as their complete inhibition by furosemide does not appreciably alter the membrane potential, they appear to be electrically silent. Application of the pulse-response methods in terms of irreversible thermodynamics reveals tight coupling between the furosemide-sensitive flows of Na⁺, K⁺ and Cl^? ($\text{q}$ close to unity for all three combinations) at a stoichiometry of 1 : 1 : 2. The site for each of the ions appears to be rather specific: K⁺ can be replaced by Rb⁺ but not by other cations tested whereas Cl^? can be poorly replaced by Br^? but not by NO₃^?, in contradistinction to the Cl^?-OH^? exchange system. The cotransport system appears to function in cell volume regulation as it tends to make the cell swell, thus counteracting the shrinking effect of the ouabain-sensitive (Na⁺, K⁺) pump.The experiments presented could not clarify whether the cotransport process is a primary or secondary active one; while incongruence between transport and conjugated driving force seems to indicate primary active transport, it is very unlikely that hydrolysis of ATP supplies energy for the transport process, since there is no stimulation of ATP turnover observable under operation of the cotransport system.     

Intracellular compartmentation of Na+, K+ and Cl− in the Ehrlich ascites tumor cell: Correlation with the membrane potential

Summary The intracellular distribution of Na⁺, K⁺, Cl^– and water has been studied in the Ehrlich ascites tumor cell. Comparison of the ion and water contents of whole cells with those of cells exposed to La³⁺ and mechanical stress indicated that La³⁺ treatment results in selective damage to the cell membrane and permits evaluation of cytoplasmic and nuclear ion concentrations. The results show that Na⁺ is sequestered within the nucleus, while K⁺ and Cl^– are more highly concentrated in the cell cytoplasm. Reduction of the [Na⁺] of the incubation medium by replacement with K⁺ results in reduced cytoplasmic [Na⁺], increased [Cl^–] and no change in [K⁺]. Nuclear concentrations of these ions are virtually insensitive to the cation composition of the medium. Concomitant measurements of the membrane potential were made. The potential in control cells was –13.7 mV. Reduction of [Na⁺] in the medium caused significant depolarization. The measured potential is describable by the Cl^– equilibrium potential and can be accounted for in terms of cation distributions and permeabilities. The energetic implications of the intracellular compartmentation of ions are discussed.     

Volume changes of mammalian cells subjected to hypotonic solutions in vitro: evidence for the requirement of a sodium pump for the shrinking phase

A Coulter-orifice pulse-height analyzer system was used to measure volume spectra of mammalian cells in suspension at different times after the addition of an equal volume of water. In appropriate hypotonic medium, cultured mammalian cells rapidly increase in volume and then shrink, more slowly, approaching their initial volumes within 20 to 30 minutes at 37.5°C. The shrinking phase was found to be reversibly inhibited by ouabain and inhibited in both K⁺-free and Na⁺-free solutions; neither choline⁺ nor Li⁺ could substitute for extracellular Na⁺ in supporting the shrinking phenomenon but Rb⁺ and Cs⁺ were fairly good substitutes for K⁺. Under conditions similar to those with which the shrinking phenomenon was observed with cultured cells, it was not found with either human or mouse red blood cells. Two methods were used to determine intracellular Na⁺ and K⁺ content in osmotically shocked cells and in unshocked controls. An isotope equilibration method was employed with L5178-Y mouse lymphoblasts and a chemical determination by flame photometry was used with Ehrlich ascites tumor cells. The K⁺ content was significantly reduced and the Na⁺ content was unchanged or somewhat increased in cells which had returned to their original volumes in hypotonic medium. The K⁺ content was even more reduced but the Na⁺ content was greatly increased in cells which were osmotically shocked in the presence of ouabain.     

The Relationship between Cell Membrane Potassium Ion Transport and Glycolysis : The effect of ethacrynic acid

Cell membrane transport of K⁺ stimulates the rate of glycolysis in Ehrlich ascites tumor cells. A study of the characteristics of this relationship indicates that the stimulation occurs under anaerobic as well as under aerobic conditions. The data suggest that glycolysis is stimulated by a K⁺ transport mechanism that is coupled to Na⁺ transport because the effect is blunted or abolished when the principal intracellular ion is lithium or choline. This stimulus to glycolysis is blocked by ouabain and ethacrynic acid, agents that have been shown to inhibit monovalent cation transport in erythrocytes. In contrast to the action of ouabain, glycolysis is inhibited by ethacrynic acid in Ehrlich ascites tumor cells in the absence of cell membrane K⁺ transport. In studies with ghost-free hemolysates of human erythrocytes and with cytosol prepared from Ehrlich ascites tumor cells, ethacrynic acid significantly blocks lactate formation from fructose diphosphate demonstrating the direct inhibitory effect of this agent on one or more enzymes of the Embden-Meyerhof pathway. Since ethacrynic acid has no influence on lactate formation in intact erythrocytes utilizing an endogenous substrate, the presumptive site of inhibition is proximal to the 3-phosphoglycerate level.     

Furosemide-sensitive K+ channel in glioma cells but not neuroblastoma cells in culture

A furosemide-sensitive, ouabain-insensitive [⁸⁶Rb⁺] uptake is described in glioma cells in culture which is dependent upon external Na⁺, K⁺, and Cl^? concentrations. This transport activity was also inhibited by bumetanide at 100-fold lower concentrations than furosemide. Furosemide-sensitive swelling of glioma cells is demonstrated and this activity is dependent upon external Na⁺ and K⁺ in a manner similar to [⁸⁶Rb⁺] uptake. This transport activity was not detected in neuroblastoma cells and the possible relevance of these findings to extracellular K⁺ buffering by glia is discussed.     

Na+, K+, Cl− cotransport and its regulation in Ehrlich ascites Tumor cells. Ca2+/Calmodulin and protein kinase C dependent pathways

Summary Net Cl^– uptake as well as unidirectional³⁶Cl influx during regulatory volume increase (RVI) require external K⁺. Half-maximal rate of bumetanide-sensitive³⁶Cl uptake is attained at about 3.3mm external K⁺. The bumetanide-sensitive K⁺ influx found during RVI is strongly dependent on both Na⁺ and Cl^–. The bumetanide-sensitive unidirectional Na⁺ influx during RVI is dependent on K⁺ as well as on Cl^–. The cotransporter activated during RVI in Ehrlich cells, therefore, seems to transport Na⁺, K⁺ and Cl^–. In the presence of ouabain and Ba⁺ the stoichiometry of the bumetanide-sensitive net fluxes can be measured at 1.0 Na⁺, 0.8 K⁺, 2.0 Cl^– or approximately 1 : Na, 1 : K, 2 : Cl. Under these circumstances the K⁺ and Cl^– flux ratios (influx/efflux) for the bumetanide-sensitive component were estimated at 1.34 ±0.08 and 1.82 ± 0.15 which should be compared to the gradient for the Na⁺, K⁺, 2Cl^– cotransport system at 1.75 ± 0.24.Addition of sucrose to hypertonicity causes the Ehrlich cells to shrink with no signs of RVI, whereas shrinkage with hypertonic standard medium (all extracellular ion concentrations increased) results in a RVI response towards the original cell volume. Under both conditions a bumetanide-sensitive unidirectional K⁺ influx is activated. During hypotonic conditions a small bumetanide-sensitive K⁺ influx is observed, indicating that the cotransport system is already activated.The cotransport is activated 10–15 fold by bradykinin, an agonist which stimulates phospholipase C resulting in release of internal Ca²⁺ and activation of protein kinase C.The anti-calmodulin drug pimozide inhibits most of the bumetanide-sensitive K⁺ influx during RVI. The cotransporter can be activated by the phorbol ester TPA. These results indicate that the stimulation of the Na⁺, K⁺, Cl^– cotransport involves both Ca²⁺/calmodulin and protein kinase C.     

Cell detachment in ouabain-sensitive and resistant ehrlich ascites cells

Ehrlich ascites tumor cells selected for resistance (OR) to the toxic effects of ouabain, a specific inhibitor of (Na⁺, K⁺)-activated ATPase, differ from wild-type ouabain-sensitive (OS) ascites cells in their detachment behavior from protein-coated glass surfaces in the absence of ouabain. In the presence of ouabain OS cells are more easily detached from cultured vessel surfaces, but ouabain is either without effect (10⁻⁴ M) or inhibits detachment of OR cells (10⁻³ M) under similar conditions. The results are discussed in terms of the possible relationship between membrane ATPases and cell behavior.     

KCl loss and cell shrinkage in the ehrlich ascites tumor cell induced by hypotonic media, 2-deoxyglucose and propranolol

Ehrlich ascites tumor cells lose KCl and shrink after swelling in hypotonic media and in response to the addition of 2-deoxyglucose, propranolol, or the Ca²⁺ ionophore, A23187, plus Ca²⁺ in isotonic media. All of these treatments activate cell shrinkage via a pathway with the following characteristics: (1) the KCl loss responsible for cell shrinkage does not alter the membrane potential; (2) NO₃^? does not substitute for Cl^?; (3) the net KCl movements are not inhibited by quinine or DIDS; and (4) early in this study furosemide was effective in inhibiting cell shrinkage but this sensitivity was subsequently lost. This evidence suggests that the KCl loss in these cells occurs via a cotransport mechanism. In addition, hypotonic media and the other agents used here stimulate a Cl^? -Cl^? exchange, a net loss of K⁺ and a net gain of Na⁺ which are not responsible for cell shrinkage. The Ehrlich cell also appears to have a Ca²⁺-activated, quinine-sensitive K⁺ conductive pathway but this pathway is not part of the mechanism by which these cells regulate their volume following swelling or shrink in isotonic media in response to 2-deoxyglucose or propranolol. Shrinkage by the loss of K⁺ through the Ca²⁺ stimulated pathway appears to be limited by Cl^? conductive movements; for when NO₃^?, an anion demonstrated here to have a higher conductive movement than Cl^?, is substituted for Cl^?, the cells will shrink when the Ca²⁺-stimulated K⁺ pathway is activated.     

The Na-K-2Cl cotransporter is in a permanently activated state in cytoplasts from Ehrlich ascites tumor cells

Brief incubation of Ehrlich ascites tumor cells with cytochalasin B causes the formation of blebs in the surface membrane. Gentle homogenization removes the blebs as intact cytoplasts which contain neither mitochondrian or nucleus, nor other cytoplasmic membranous organelles. The Na-K-2Cl cotransporter is present in the cytoplasts in a permanently activated state, whereas the Na-K-2Cl transport system in unperturbed intact cells is silent. Pretreatment of intact cells with cytochalasin B for l min stimulates the bumetanide-inhibitable K⁺ influx fivefold. The influx into purified cytoplasts when expressed per g protein is three- to fourfold higher than the influx into cytochalasin B-treated intact cells. Thus, the membrane vesicles are enriched with the cotransporter, and the cotransporter is present in an activated state. The K influx into cytoplasts is inhibited about 40% by Na-free, Cl-free or bumetanide-containing media and to a similar extent by Fab fragments prepared from antiserum against purified proteins of the cotransporter. The K _I for bumetanide was 0.19±0.06 m for the cytoplasts as compared to 0.67±0.11 m for the intact cells. SDS gel electrophoresis of membrane proteins from the cytoplast membranes compared to the membranes of intact cells shows a reduced number of bands and a majority of bands showing reduced staining, whereas a few bands are stained more intensely. Particularly notable is a band at 80 kD, which is similar to the molecular weight previously reported for the main membrane protein isolated from intact cells using a bumetanide-Sepharose affinity column. An immunoblot of the cytoplast preparation using antibodies against the purified bumetanide binding proteins showed strong immunodetection of the 80 kD protein.We are grateful to Marianne Schiødt, Birgit Blytmann Jørgensen, Thomas Krarup and Beverley Dyer for expert assistance. This work was supported by grants from the Danish Natural Science Research Council (11-6835 to E.K.H.) and the National Institutes of Health (DK 33640 to P.B.D.) and by a Carlsberg Foundation research fellowship (to F.J.).     

Neutral amino acid transport in surface membrane vesicles isolated from mouse fibroblasts: Intrinsic and extrinsic models of regulation

Membrane transport carrier function, its regulation and coupling to metabolism, can be selectively investigated dissociated from metabolism and in the presence of a defined electrochemical ion gradient driving force, using the single internal compartment system provided by vesiculated surface membranes. Vesicles isolated from nontransformed and Simian virus 40-transformed mouse fibroblast cultures catalyzed carrier-mediated transport of several neutral amino acids into an osmotically-sensitive intravesicular space without detectable metabolic conversion of substrate. When a Na⁺ gradient, external Na⁺ > internal Na⁺, was artifically imposed across vesicle membranes, accumulation of several neutral amino acids achieved apparent intravesicular concentrations 6- to 9-fold above their external concentrations. Na⁺-stimulated alanine transport activity accompanied plasma membrane material during subcellular fractionation procedures. Competitive interactions among several neutral amino acids for Na⁺-stimulated transport into vesicles and inactivation studies indicated that at least 3 separate transport systems with specificity properties previously defined for neutral amino acid transport in Ehrlich ascites cells were functional in vesicles from mouse fibroblasts: the A system, the L system and a glycine transport system. The pH profiles and apparent K_m values for alanine and 2-aminoisobutyric acid transport into vesicles were those expected of components of the corresponding cellular uptake system. Several observations indicated that both a Na⁺ chemical concentration gradient and an electrical membrane potential contribute to the total driving force for active amino acid transport via the A system and the glycine system. Both the initial rate and quasi-steady-state of accumulation were stimulated as a function of increasing concentrations of Na⁺ applied as a gradient (external > internal) across the membrane. This stimulation was independent of endogenous Na⁺, K⁺-ATPase activity in vesicles and was diminished by monensin or by preincubation of vesicles with Na⁺. The apparent K_m for transport of alanine and 2-aminoisobutyric acid was decreased as a function of Na⁺ concentration. Similarly, in the presence of a standard initial Na⁺ gradient, quasi-steady-state alanine accumulation in vesicles increased as a function of increasing magnitudes of interior-negative membrane potential imposed across the membrane by means of K⁺ diffusion potentials (internal > external) in the presence of valinomycin; the magnitude of this electrical component was estimated by the apparent distributions of the freely permeant lipophilic cation triphenylme thylphosphonium ion. Alanine transport stimulation by charge asymmetry required Na⁺ and was blocked by the further addition of either nigericin or external K⁺. As a corollary, Na⁺-stimulated alanine transport was associated with an apparent depolarization, detectable as an increased labeled thiocyanate accumulation. Permeant anions stimulated Na⁺-coupled active transport of these amino acids but did not affect Na⁺-independent transport. Translocation of K⁺, H⁺, or anions did not appear to be directly involved in this transport mechanism. These characteristics support an electrogenic mechanism in which amino acid translocation is coupled t o an electrochemical Na⁺ gradient by formation of a positively charged complex, stoichiometry unspecified, of Na⁺, amino acid, and membrane component. Functional changes expressed in isolated membranes were observed t o accompany a change in cellular proliferative state or viral transformation. Vesicles from Simian virus 40-transformed cells exhibited an increased Vmax of Na⁺-stimulated 2-aminoisobutyric acid transport, as well as an increased capacity for steady-state accumulation of amino acids in response t o a standard Na⁺ gradient, relative t o vesicles from nontransformed cells. Density-inhibition of nontransformed cells was associated with a marked decrease in these parameters assayed in vesicles. Several possibilities for regulatory interactions involving gradient-coupled transport systems are discussed.