GSH Transport in Immortalized Mouse Brain Endothelial Cells

We have previously shown GSH transport across the blood-brain barrier in vivo and expression of transport in Xenopus laevis oocytes injected with bovine brain capillary mRNA. In the present study, we have used MBEC-4, an immortalized mouse brain endothelial cell line, to establish the presence of Na+-dependent and Na+-independent GSH transport and have localized the Na+-dependent transporter using domain-enriched plasma membrane vesicles. In cells depleted of GSH with buthionine sulfoximine, a significant increase of intracellular GSH could be demonstrated only in the presence of Na+. Partial but significant Na+ dependency of [35S]GSH uptake was observed for two GSH concentrations in MBEC-4 cells in which gamma-glutamyltranspeptidase and gamma-glutamylcysteine synthetase were inhibited to ensure absence of breakdown and resynthesis of GSH. Uniqueness of Na+-dependent uptake in MBEC-4 cells was confirmed with parallel uptake studies with Cos-7 cells that did not show this activity. Molecular form of uptake was verified as predominantly GSH, and very little conversion of [35S]cysteine to GSH occurred under the same incubation conditions. Poly(A)+ RNA from MBEC expressed GSH uptake with significant (approximately 40-70%) Na+ dependency, whereas uptake expressed by poly(A)+ RNA from HepG2 and Cos-1 cells was Na+ independent. Plasma membrane vesicles from MBEC were separated into three fractions (30, 34, and 38% sucrose, by wt) by density gradient centrifugation. Na+-dependent glucose transport, reported to be localized to the abluminal membrane, was found to be associated with the 38% fraction (abluminal). Na+-dependent GSH transport was present in the 30% fraction, which was identified as the apical (luminal) membrane by localization of P-glycoprotein 170 by western blot analysis. Localization of Na+-dependent GSH transport to the luminal membrane and its ability to drive up intracellular GSH may find application in the delivery of supplemented GSH to the brain in vivo.     

Identification of proximal tubular transport functions in the established kidney cell line, OK

OK cells, derived from an American opossum kidney, were analyzed for proximal tubular transport functions. In monolayers, L-glutamate, L-proline, L-alanine, and alpha-methyl-glucopyranoside (alpha-methyl D-glucoside) were accumulated through Na+-dependent and Na+-independent transport pathways. D-Glucose and inorganic sulfate were accumulated equally well in the presence or absence of Na+. Influx of inorganic phosphate was only observed in the presence of Na+. Na+/alpha-methyl D-glucoside uptake was preferentially inhibited by phlorizin and D-glucose uptake by cytochalasin B. An amiloride-sensitive Na+-transport was also identified. In isolated apical vesicles (enriched 8-fold in gamma-glutamyltransferase), L-glutamate, L-proline, L-alanine, alpha-methyl D-glucoside and inorganic phosphate transport were stimulated by an inwardly directed Na+-gradient as compared to an inwardly directed K+-gradient. L-Glutamate transport required additionally intravesicular K+. D-Glucose transport was similar in the presence of a Na+- and a K+-gradient. Na+/alpha-methyl D-glucoside uptake was inhibited by phlorizin whereas cytochalasin B had no effect on Na+/D-glucose transport. An amiloride-sensitive Na+/H+ exchange mechanism was also found in the apical vesicle preparation. It is concluded that the apical membrane of OK cells contains Na+-coupled transport systems for amino acids, hexoses, protons and inorganic phosphate. D-Glucose appears a poor substrate for the Na+/hexose transport system.     

Coupling of Li+ distribution to the plasma membrane potential of rat cortical synaptosomes

The effect of the plasma membrane potential delta psi p on the transport rate and steady state distribution of Li+ was assessed in rat cortical synaptosomes. Up to 15 mM Li+ failed to saturate Li+ influx into polarized synaptosomes in a Na+-based medium with 3 mM external K+. Veratridine increased and tetrodotoxin, ouabain, or high external K+ decreased the rate of Li+ influx. At steady state, Li+ was concentrated about 3-fold in resting synaptosomes at 0.3 to 1 mM Li+ externally. Subsequent depolarization of the plasma membrane by veratridine or high external K+ induced an immediate release of Li+. When graded depolarizations were imposed onto the plasma membrane by varying concentrations of ouabain, veratridine, or external K+, steady state distribution of Li+ was linearly related with K+ distribution or electrochemical activity coefficients. It was concluded that uptake rate and steady state distribution of Li+ depend significantly on delta psi p. However, Li+ gradients were lower than predicted from delta psi p, suggesting that (secondary) active transport systems counteracted passive equilibration by uphill extrusion of Li+. The electrochemical potential difference delta mu Li+ maintained at a delta psi p of -72 mV was calculated to 4.2 kJ/mol of Li+. At physiological external K+, Li+ was not actively transported by the sodium pump. The ouabain sensitivity resulted from the coupling of Li+ uptake to the pump-dependent K+ diffusion potential. In low K+ and K+-free media, however, active transport of Li+ by the sodium pump contributed to total uptake. In the absence of K+, Li+ substituted for K+ in generating a delta psi p of -64 mV maximally, as calculated from TPMP+ distribution at 40 mM external Li+. Since Li+ gradients were far too low to account for a diffusion potential, it was assumed that Li+ gave rise to an electrogenic pump potential.     

The Physiological Relevance of Na+-Coupled K+-Transport

Plant roots utilize at least two distinct pathways with high and low affinities to accumulate K+. The system for high-affinity K+ uptake, which takes place against the electrochemical K+ gradient, requires direct energization. Energization of K+ uptake via Na+ coupling has been observed in algae and was recently proposed as a mechanism for K+ uptake in wheat (Triticum aestivum L.). To investigate whether Na+ coupling has general physiological relevance in energizing K+ transport, we screened a number of species, including Arabidopsis thaliana L. Heynh. ecotype Columbia, wheat, and barley (Hordeum vulgare L.), for the presence of Na+-coupled K+ uptake. Rb+-flux analysis and electrophysiological K+-transport assays were performed in the presence and absence of Na+ and provided evidence for a coupling between K+ and Na+ transport in several aquatic species. However, all investigated terrestrial species were able to sustain growth and K+ uptake in the absence of Na+. Furthermore, the addition of Na+ was either without effect or inhibited K+ absorption. The latter characteristic was independent of growth conditions with respect to Na+ status and pH. Our results suggest that in terrestrial species Na+-coupled K+ transport has no or limited physiological relevance, whereas in certain aquatic angiosperms and algae this type of secondary transport energization plays a significant role.     

Direct evidence for the role of the membrane potential in glutathione transport by renal brush-border membranes

Transport of GSH was studied in isolated rat kidney cortical brush-border membrane vesicles in which gamma-glutamyltransferase had been inactivated by a specific affinity labeling reagent, L-(alpha S,5S)-alpha-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid (AT-125). Transport of intact 2-3H-glycine-labeled GSH occurred into an osmotically active intravesicular space of AT-125-treated membranes. The initial rate of transport followed saturation kinetics with respect to GSH concentrations; an apparent Km of 0.21 mM and Vmax of 0.23 nmol/mg protein X 20 were calculated at 25 degrees C with a 0.1 M NaCl gradient (vesicle inside less than vesicle outside). Sodium chloride in the transport medium could be replaced with KCl without affecting transport activity. The rate of GSH uptake was enhanced by replacing KCl in the transport medium with K2SO4, providing a less permeant anion, and was reduced by replacing KCl with KSCN, providing a more permeant anion. The rate of GSH transport markedly decreased in the absence of a K+ gradient across the vesicular membranes and was enhanced by a valinomycin-induced K+ diffusion potential (vesicle-inside-positive). These results indicate that GSH transport is dependent on membrane potential and involves the transfer of negative charge. The rate of GSH transport was inhibited by S-benzyl glutathione but not by glycine, glutamic acid, and gamma-glutamyl-p-nitroanilide. When incubated with [2-3H]glycine-labeled GSH, intact untreated vesicles also accumulated radioactivity; the rate of uptake was significantly higher in a Na+ gradient than in a K+ gradient. Sodium-dependent transport, but not sodium-independent uptake, was almost completely inhibited by a high concentration of unlabeled glycine. At equilibrium, most of the radioactivity which accumulated in the intravesicular space was accounted for by free glycine. These results suggest that GSH which is secreted into the tubular lumen by a specific translocase in the lumenal membranes or filtered by the glomerulus may be degraded in situ by membranous gamma-glutamyltransferase and peptidase activities which hydrolyze peptide bonds of cysteinylglycine and its derivatives. The resulting free amino acids can be reabsorbed into tubule cells by sodium-dependent transport systems in renal cortical brush-border membranes.     

Glutathione transport across intestinal brush-border membranes: effects of ions, pH, delta psi, and inhibitors

We characterized glutathione transport in brush-border membrane vesicles (BBMV) that were prepared from rabbit small intestine in which gamma-glutamyl transpeptidases (gamma-glutamyltransferases, EC 2.3.2.2) had been inactivated by a specific affinity-labeling reagent (AT125). Intact GSH transport was strongly increased by the presence of Na+, K+, LI+, Ca2+ and Mn2+ and, of all these, the Ca2+ activation effect was prevalent. This cation effect was selective and catalytic but not energetic; Vmax obtained in the presence of both Na+ and Ca2+ was about 6-times higher than it was in their absence, while Km did not change. Moreover, these cations almost completely eliminated GSH binding on the membrane surface. Na+ activation cannot be explained as a stimulation effect on the Na+-H+ antiport system, since a GSH proton-driven transport was excluded. We determined a pH optimum (7.5), while low or high extravesicular pH values diminished the GSH uptake rate. The Ca2+ effect on GSH transport, when an electrical potential difference was imposed across BBMV, was different from that of monovalent cations. Indeed, experiments performed by valinomycin-induced K+ diffusion potential or by anion substitution showed that the GSH transport system was an electroneutral process in the presence of Na+ or K+, but that it was electrogenic in the presence of Ca2+ or in the absence of extravesicular cations. These results suggest that GSH is also cotransported with these cations, without its accumulation inside vesicles. Moreover, since GSH is negatively charged, the effect of pH changes and of cation activation on GSH transport is arguably mediated by changes in the ionization state of certain groups as the carrier site and of GSH itself, indicating the electrostatic nature of GSH binding sites on the transporter. The high Ca2+ activation effect is perhaps also partly due to fluidity changes in the lipoproteic microenvironment of the GSH transporter. Moreover, this transport system has high affinity with GSH, given the low Km value (17 microM) and the fact that it was only inhibited by GSH S-derivatives and by GSH monoethyl ester, which probably share the same transport system.     

Characterization of ammonium (methylammonium)/potassium antiport in Escherichia coli

The energetics of ammonium ion transport by Escherichia coli have been studied using [14C]methylammonium as a substrate. Rapid assays for uptake allowed kinetic parameters (CH3NH3+ Km = 36 microM; Vmax = 4 nmol X s-1 X mg-1 to be determined in the absence of CH3NH3+ metabolism. Cells cultured in media containing 1 mM NH4+ failed to express CH3NH3+ transport activity. Methylammonium accumulated at levels which were 100-fold higher than those of the medium. This accumulation was dependent upon the addition of glucose or pyruvate. The entry of CH3NH3+ supported by glucose oxidation in an F1F0-ATPase-deficient mutant was blocked by uncoupler. Transport by wild-type cells under similar conditions was significantly inhibited by arsenate. Thus, CH3NH3+ uptake requires both ATP and an electrochemical H+ gradient. This transport activity was lost upon exposure of E. coli to osmotic shock, but could be recovered by incubation of shocked cells with boiled shock fluid or with glucose plus K+ in the presence of chloramphenicol. Similar reconstitution was observed in K+-depleted parental strains, but not in a mutant defective in K+ transport, demonstrating a requirement for internal K+. However, external K+ proved to be a noncompetitive inhibitor (Ki = 1 mM) of CH3NH3+ uptake by K+ -replete bacteria. External Na+ had no effect on transport. The addition of NH4+ or CH3NH3+ induced a rapid exodus of intracellular 86Rb+, an analog which was able to substitute for K+. The molar ratio of CH3NH3+ uptake to Rb+ exit was 1.12 +/- 0.11. These findings support a mechanism for CH3NH3+ (NH4+) accumulation which requires K+ antiport (exchange) and is driven by the electrochemical K+ gradient.     

Uptake of [3H]serotonin into plasma membrane vesicles from mouse cerebral cortex

Preparations of plasma membrane vesicles were used as a tool to study the properties of the serotonin transporter in the central nervous system. The vesicles were obtained after hypotonic shock of synaptosomes purified from mouse cerebral cortex. Uptake of [3H]serotonin had a Na+-dependent and Na+-independent component. The Na+-dependent uptake was inhibited by classical blockers of serotonin uptake and had a Km of 63-180 nM, and a Vmax of 0.1-0.3 pmol mg-1 s-1 at 77 mM Na+. The uptake required the presence of external Na+ and internal K+. It required a Na+ gradient ([Na+]out greater than [Na+]in) and was stimulated by a gradient of K+ ([K+]in greater than [K+]out). Replacement of Cl- by other anions (NO2-, S2O3-(2-)) reduced uptake appreciably. Gramicidin prevented uptake. Although valinomycin increased uptake somewhat, the membrane potential per se could not drive uptake because no uptake was observed when a membrane potential was generated by the SCN- ion in the absence of internal K+ and with equal [Na+] inside and outside. The increase of uptake as a function of [Na+] indicated a Km for Na+ of 118 mM and a Hill number of 2.0, suggesting a requirement of two sodium ions for serotonin transport. The present results are accommodated very well by the model developed for porcine platelet serotonin transport (Nelson, P. J., and Rudnick, G. (1979) J. Biol. Chem. 254, 10084-10089), except for the number of sodium ions that are required for transport.     

(Na+,K+)-cotransport in the Madin-Darby canine kidney cell line. Kinetic characterization of the interaction between Na+ and K+

Confluent monolayer cultures of the differentiated kidney epithelial cell line, Madin-Darby canine kidney cells (MDCK), have been used to study ion transport mechanisms involved in transepithelial transport. We have investigated the previously reported K+-stimulation of 22Na+ uptake by confluent monolayers of Na+ depleted cells (Rindler, M. J., Taub, M., and Saier, M. H., Jr. (1979) J. Biol. Chem. 254, 11431-11439). This component of Na+ uptake was insensitive to ouabain and amiloride, but was strongly inhibited by furosemide or bumetanide. Ouabain-insensitive 86Rb+ uptake was also inhibitable by furosemide or bumetanide and stimulated by extracellular Na+. The synergistic effect of Na+ and 86Rb+ uptake and K+ on 22Na+ uptake was reflected by an increase in the apparent Vmax and a decrease in the apparent Km as the concentration of the other cation was increased. The extrapolated Km for either 86Rb+ or 22Na+ uptake in the absence of the other cation was 30 mM while the Km in the presence of a saturating concentration of the other cation was 9 mM. The absolute Vmax values for 22Na+ and 86Rb+ uptake suggest a cotransport system with a stoichiometry of 2Na+:3K+. However, because of the experimental design, the actual ratio may be closer to 1:1. Competition with, and stimulation by, a variety of unlabeled cations indicated that Na+ could be partially replaced by Li+, while K+ could be fully replaced by Rb+ and partially replaced by NH4+ and CS+. Uptake by this system was dependent upon cellular ATP. Reduction of intracellular ATP to 3% of normal abolished both K+-stimulated 22Na+ uptake and Na+-stimulated 86Rb+ uptake.     

Studies on sodium transport in rat brain nerve-ending particles

(1) Nerve-ending particles isolated from crude mitochondrial preparations from rat brain by discontinuous Ficoll density gradient ultracentrifugation were shown to possess a Mg2+ and energy-dependent transport system for Na+. (2) Ouabain or iodoacetate plus cyanide exerted an inhibitory effect on the outflux but not the influx of Na+. (3) When K+ was added to a medium containing particles loaded with Na+ (22Na), an immediate release of Na+ from these particles was observed; this suggests the existence of a Na+-K+ exchange transport system. (4) The K+ effect was inhibited by 10(-4) M-ouabain only at low (about 3.3 mM), but not at high (20 mM), K+ concentrations. (5) The uptake and release of Na+ by the nerve-ending particles were found to be temperature-dependent. (6) Only nerve-ending particles with intact synaptosomal membranes were found to transport Na+ actively.     

Energy coupling of L-glutamate transport and vacuolar H(+)-ATPase in brain synaptic vesicles

Energy coupling of L-glutamate transport in brain synaptic vesicles has been studied. ATP-dependent acidification of the bovine brain synaptic vesicles was shown to require CI-, to be accelerated by valinomycin and to be abolished by ammonium sulfate, nigericin or CCCP plus valinomycin, and K+. On the other hand, ATP-driven formation of a membrane potential (positive inside) was found to be stimulated by ammonium sulfate, not to be affected by nigericin and to be abolished by CCCP plus valinomycin and K+. Like formation of a membrane potential, ATP-dependent L-[3H]glutamate uptake into vesicles was stimulated by ammonium sulfate, not affected by nigericin and abolished by CCCP plus valinomycin and K+. The L-[3H]glutamate uptake differed in specificity from the transport system in synaptic plasma membranes. Both ATP-dependent H+ pump activity and L-glutamate uptake were inhibited by bafilomycin and cold treatment (common properties of vacuolar H(+)-ATPase). ATP-dependent acidification in the presence of L-glutamate was also observed, suggesting that L-glutamate uptake lowered the membrane potential to drive further entry of H+. These results were consistent with the notion that the vacuolar H(+)-ATPase of synpatic vesicles formed a membrane potential to drive L-glutamate uptake. ATPase activity of the vesicles was not affected by the addition of Cl-, glutamate or nigericin, indicating that an electrochemical H+ gradient had no effect on the ATPase activity.     

Passive transport of Rb+ by hog gastric (H+,K+)-ATPase

Gastric vesicles enriched in (H+,K+)-ATPase were prepared from hog fundic mucosa and studied for their ability to transport K+ using 86Rb+ as tracer. In the absence of ATP, the vesicles elicited a rapid uptake of 86Rb+ (t 1/2 = 45 +/- 9 s at 30 degrees C) which accounted for both transport and binding. Transport was osmotically sensitive and was the fastest phase. It was not limited by anion permeability (C1- was equivalent to SO2-4) but rather by availability of either H+ or K+ as intravesicular countercation suggesting a Rb+-K+ or a Rb+-H+ exchange. Selectivity was K+ greater than Rb+ greater than Cs+ much greater than Na+,Li+. The capacity of vesicles which catalyzed the fast transport of K+ was 83 +/- 4% of maximal vesicular capacity of the fraction. Addition of ATP decreased both rate and extent of 86Rb+ uptake (by 62 and 43%, respectively with 1 mM ATP) with an apparent Ki of 30 microM. Such an effect was not seen on 22Na+ transport. ATP inhibition of transport did not require the presence of Mg2+, and inhibition was also produced by ADP even in the presence of myokinase inhibitor. On the other hand, 86Rb+ uptake was as strongly inhibited by 200 microM vanadate in the presence of Mg2+. Efflux studies suggested that ATP inhibition was originally due to a decrease of vesicular influx with little or no modification of efflux. Since ATP, ADP, and vanadate are known modulators of the (H+,K+)-ATPase, we propose that, in the absence of ATP, (H+,K+)-ATPase passively exchanges K+ for K+ or H+ and that ATP, ADP, and vanadate regulate this exchange.     

AtKuP1: a dual-affinity K+ transporter from Arabidopsis.

Plant roots contain both high- and low-affinity transport systems for uptake of K+ from the soil. In this study, we characterize a K+ transporter that functions in both high- and low-affinity uptake. Using yeast complementation analysis, we isolated a cDNA for a functional K+ transporter from Arabidopsis (referred to as AtKUP1 for Arabidopsis thaliana K+ uptake). When expressed in a yeast mutant, AtKUP1 dramatically increased K+ uptake capacity at both a low and high [K+] range. Kinetic analyses showed that AtKUP1-mediated K+ uptake displays a "biphasic" pattern similar to that observed in plant roots. The transition from the high-affinity phase (K(m) of 44 microM) to the low-affinity phase (K(m) of 11 mM) occurred at 100 to 200 microM external K+. Both low- and high-affinity K+ uptake via AtKUP1 were inhibited by 5 mM or higher concentrations of NaCl. In addition, AtKUP1-mediated K+ uptake was inhibited by K+ channel blockers, including tetraethylammonium, Cs+, and Ba2+. Consistent with a possible function in K+ uptake from the soil, the AtKUP1 gene is primarily expressed in roots. We conclude that the AtKUP1 gene product may function as a K+ transporter in Arabidopsis roots over a broad range of [K+] in the soil.     

Inward-Rectifying K+ Channels in Root Hairs of Wheat (A Mechanism for Aluminum-Sensitive Low-Affinity K+ Uptake and Membrane Potential Control)

K+ is the most abundant cation in cells of higher plants, and it plays vital roles in plant growth and development. Extensive studies on the kinetics of K+ uptake in roots have shown that K+ uptake is mediated by at least two transport mechanisms, one with a high and one with a low affinity for K+. However, the precise molecular mechanisms of K+ uptake from soils into root epidermal cells remain unknown. In the present study we have pursued the biophysical identification and characterization of mechanisms of K+ uptake into single root hairs of wheat (Triticum aestivum L.), since root hairs constitute an important site of nutrient uptake from the soil. These patch-clamp studies showed activation of a large inward current carried by K+ ions into root hairs at membrane potentials more negative than -75 mV. This K+ influx current was mediated by hyperpolarization-activated K+-selective ion channels, with a selectivity sequence for monovalent cations of K+ > Rb+ [almost equal to] NH4+ >> Na+ [almost equal to] Li+ > Cs+. Kinetic analysis of K+ channel currents yielded an apparent K+ equilibrium dissociation constant (Km) of [almost equal to]8.8 mM, which closely correlates to the major component of low-affinity K+ uptake. These channels did not inactivate during prolonged stimulation and would thus enable long-term K+ uptake driven by the plasma membrane proton-extruding pump. Aluminum, which is known to inhibit cation uptake at the root epidermis, blocked these inward-rectifying K+ channels with half-maximal current inhibition at [almost equal to]8 [mu]M free Al3+. Aluminum block of K+ channels at these Al3+ concentrations correlates closely to Al3+ phytotoxicity. It is concluded that inward-rectifying K+ channels in root hairs can function as both a physiologically important mechanism for low-affinity K+ uptake and as regulators of membrane potential. The identification of this mechanism is a major step toward a detailed molecular characterization of the multiple components involved in K+ uptake, transport, and membrane potential control in root epidermal cells.     

Relationship of hepatic cholate transport to regulation of intracellular pH and potassium.

Modulation of hepatic cholate transport by transmembrane pH-gradients and during interferences with the homeostatic regulation of intracellular pH and K+ was studied in the isolated perfused rat liver. Within the concentration range studied uptake into the liver was saturable and appeared to be associated with release of OH- and uptake of K+. Perfusate acidification ineffectually stimulated uptake. Application of NH4Cl caused intracellular alkalinization, release of K+ and stimulation of cholate uptake, withdrawal of NH4Cl resulted in intracellular acidification, regain of K+ and inhibition of cholate uptake. Inhibition of Na+/H(+)-exchange with amiloride reduced basal release of acid equivalents into the perfusate, initiated K(+)-release, and inhibited both, control cholate uptake and its recovery following intracellular acidification. K(+)-free perfusion caused K(+)-release and inhibited cholate uptake. K(+)-readmission resulted in brisk K(+)-uptake and recovery of cholate transport. Both effects were inhibited by amiloride. Interference with cholate transport through modulation of pH homeostasis by diisothiocyanostilbenedisulfonate (DIDS) could not be demonstrated because DIDS affected bile acid transport directly. Biliary bile acid secretion was stimulated by intracellular alkalinization and by activation of K(+)-transport. Uncoupling of the mutual interference between pH-dependent cholate uptake and K(+)-transport by amiloride indicates tertiary active transport of cholate. In this, Na+/K(+)-ATPase provides the transmembrane Na(+)-gradient to sustain Na+/H(+)-exchange which maintains the transmembrane pH-gradient and thus supports cholate uptake. Effects of canalicular bile acid secretion are consistent with a saturable, electrogenic transport.     

K+-independent active transport of Na+ by the (Na+ and K+)-stimulated adenosine triphosphatase

The (Na+ and K+)-stimulated adenosine triphosphatase (Na+,K+)-ATPase) from canine kidney reconstituted into phospholipid vesicles showed an ATP-dependent, ouabain-inhibited uptake of 22Na+ in the absence of added K+. This transport occurred against a Na+ concentration gradient, was not affected by increasing the K+ concentration to 10 microM (four times the endogenous level), and could not be explained in terms of Na+in in equilibrium Na+out exchange. K+-independent transport occurred with a stoichiometry of 0.5 mol of Na+ per mol of ATP hydrolyzed as compared with 2.9 mol of Na+ per mol of ATP for K+-dependent transport.     

Potassium transport system of Rhodopseudomonas capsulata

Rhodopseudomonas capsulata required potassium (or rubidium or cesium as analogs of potassium) for growth. These cations were actively accumulated by the cells by a process following Michaelis-Menten saturation kinetics. The monovalent cation transport system had Km's of 0.2 mM K+, 0.5 mM Rb+, and 2.6 mM Cs+. The rates of uptake of substrates by the potassium transport system varied with the age of the culture, although the affinity constant for the substrates remained constant. The maximal velocity of uptake of K+ was lower in aerobically grown cells than in photosynthetically grown cells, although the Km's for K+ and for Rb+ were about the same.     

Insulin stimulation of (Na+,K+)-adenosine triphosphatase-dependent 86Rb+ uptake in rat adipocytes

Insulin stimulated the uptake of 86Rb+ (a K+ analog) in rat adipocytes and increased the steady state concentration of intracellular potassium. Half-maximal stimulation occurred at an insulin concentration of 200 pM. Both basal- and insulin-stimulated 86Rb+ transport rates depended on the concentration of external K+, external Na+, and were 90% inhibited by 10(-3) M ouabain and 10(-3) M KCN, indicating that the hormone was activating the (Na+,K+)-ATPase. Insulin had no effect on the entry of 22Na+ or exit of 86Rb+. Kinetic analysis demonstrated that insulin acted by increasing the maximum velocity, Vmax, of 86Rb+ entry. Inhibition of the rate of Rb+ uptake by ouabain was best described by a biphasic inhibition curve. Scatchard analysis of ouabain binding to intact cells indicated binding sites with multiple affinities. Only the rubidium transport sites which exhibited a high affinity for ouabain were stimulated by insulin. Stimulation required insulin binding to an intact cell surface receptor, as it was reversible by trypsinization. We conclude that the uptake of 86Rb+ by the (Na+,K+)-ATPase is an insulin-sensitive membrane transport process in the fat cell.     

Stimulation of bumetanide-sensitive K+ transport in Swiss 3T3 fibroblasts by serum and mitogenic hormones

Rapidly growing Swiss 3T3 fibroblasts possess a bumetanide-sensitive K+ transport system that is dependent on both Na+ and Cl- ions; a smaller bumetanide-insensitive component of K+ transport is also present. In cells brought to the quiescent state by 8-11 days of incubation without a medium change, the bumetanide-sensitive rate of transport was reduced by 63%; the bumetanide-insensitive rate did not change. Removal of dialyzed fetal calf serum from the uptake medium resulted in a substantial reduction in bumetanide-sensitive uptake in both rapidly growing cells (33% reduction) and quiescent cells (68% reduction) but had no effect on bumetanide-insensitive uptake. Insulin was almost as effective as dialyzed fetal calf serum in stimulating bumetanide-sensitive uptake; insulin was maximally stimulatory at 2.5 micrograms/ml. The combination of insulin, epidermal growth factor, and arginine-vasopressin was maximally effective in stimulating both bumetanide-sensitive K+ uptake and 3H-thymidine incorporation in quiescent cells; bumetanide, however, did not interfere with the hormonal stimulation of DNA synthesis. Thus, the bumetanide-sensitive K+ transport system is not necessary for such stimulation to occur. Furthermore, concentrations of hormones which stimulated significant levels of DNA synthesis produced no elevation in the intracellular concentration of K+. We conclude that the bumetanide-sensitive pathway of K+ transport is modulated by serum and by mitogenic hormones, but does not play a role in the stimulation of DNA synthesis by these factors.     

Relationship between ion requirements for respiration and membrane transport in a marine bacterium.

Intact cells of the marine bacterium Alteromonas haloplanktis 214 oxidized NADH, added to the suspending medium, by a process which was stimulated by Na+ or Li+ but not K+. Toluene-treated cells oxidized NADH at three times the rate of untreated cells by a mechanism activated by Na+ but not by Li+ or K+. In the latter reaction, K+ spared the requirement for Na+. Intact cells of A. haloplanktis oxidized ethanol by a mechanism stimulated by either Na+ or Li+. The uptake of alpha-aminoisobutyric acid by intact cells of A. haloplanktis in the presence of either NADH or ethanol as an oxidizable substrate required Na+, and neither Li+ nor K+ could replace it. The results indicate that exogenous and endogenous NADH and ethanol are oxidized by A. haloplanktis by processes distinguishable from one another by their requirements for alkali metal ions and from the ion requirements for membrane transport. Intact cells of Vibrio natriegens and Photobacterium phosphoreum oxidized NADH, added externally, by an Na+-activated process, and intact cells of Vibrio fischeri oxidized NADH, added externally, by a K+-activated process. Toluene treatment caused the cells of all three organisms to oxidize NADH at much faster rates than untreated cells by mechanisms which were activated by Na+ and spared by K+.