Active transport of dimethialium in isolated rat hepatocytes

The uptake of dimethialium, a thiamine analog having a methyl group in place of the hydroxyethyl group in the thiazole moiety, was studied in freshly isolated rat hepatocytes. In an Na+-medium, dimethialium at 10 microM was accumulated rapidly by the cells and an almost steady intra- to extracellular distribution ratio of 4.2 was attained in 5 min of incubation. The Kt and the Vmax for the saturable component were estimated to be 27 microM and 19 pmol/10(5) cells per min, respectively. In a K+ medium, the uptake of dimethialium was decreased to 58% of that of control. Ouabain and 2,4-dinitrophenol significantly lowered the rate of dimethialium uptake. Both phenylthiazinothiamine and oxythiamine were inhibitory on the uptake of dimethialium, which uptake was also inhibited by choline. These data indicate that dimethialium transport in liver cells proceeds via a carrier-mediated active process dependent on Na+ and biological energy. Furthermore, these results also suggest that thiamine transport in liver is dissociable from thiamine phosphorylation.     

The elementary reactions of the pig heart pyruvate dehydrogenase complex. A study of the inhibition by phosphorylation.

1. A method was devised for preparing pig heart pyruvate dehydrogenase free of thiamin pyrophosphate (TPP), permitting studies of the binding of [35S]TPP to pyruvate dehydrogenase and pyruvate dehydrogenase phosphate. The Kd of TPP for pyruvate dehydrogenase was in the range 6.2-8.2 muM, whereas that for pyruvate dehydrogenase phosphate was approximately 15 muM; both forms of the complex contained about the same total number of binding sites (500 pmol/unit of enzyme). EDTA completely inhibited binding of TPP; sodium pyrophosphate, adenylyl imidodiphosphate and GTP, which are inhibitors (competitive with TPP) of the overall pyruvate dehydrogenase reaction, did not appreciably affect TPP binding. 2. Initial-velocity patterns of the overall pyruvate dehydrogenase reaction obtained with varying TPP, CoA and NAD+ concentrations at a fixed pyruvate concentration were consistent with a sequential three-site Ping Pong mechanism; in the presence of oxaloacetate and citrate synthase to remove acetyl-CoA (an inhibitor of the overall reaction) the values of Km for NAD+ and CoA were 53+/- 5 muM and 1.9+/-0.2 muM respectively. Initial-velocity patterns observed with varying TPP concentrations at various fixed concentrations of pyruvate were indicative of either a compulsory order of addition of substrates to form a ternary complex (pyruvate-Enz-TPP) or a random-sequence mechanism in which interconversion of ternary intermediates is rate-limiting; values of Km for pyruvate and TPP were 25+/-4 muM and 50+/-10 nM respectively. The Kia-TPP (the dissociation constant for Enz-TPP complex calculated from kinetic plots) was close to the value of Kd-TPP (determined by direct binding studies). 3. Inhibition of the overall pyruvate dehydrogenase reaction by pyrophosphate was mixed non-competitive versus pyruvate and competitive versus TPP; however, pyrophosphate did not alter the calculated value for Kia-TPP, consistent with the lack of effect of pyrophosphate on the Kd for TPP. 4. Pyruvate dehydrogenase catalysed a TPP-dependent production of 14CO2 from [1-14C]pyruvate in the absence of NAD+ and CoA at approximately 0.35% of the overall reaction rate; this was substantially inhibited by phosphorylation of the enzyme both in the presence and absence of acetaldehyde (which stimulates the rate of 14CO2 production two- or three-fold). 5. Pyruvate dehydrogenase catalysed a partial back-reaction in the presence of TPP, acetyl-CoA and NADH. The Km for TPP was 4.1+/-0.5 muM. The partial back-reaction was stimulated by acetaldehyde, inhibited by pyrophosphate and abolished by phosphorylation. 6. Formation of enzyme-bound [14C]acetylhydrolipoate from [3-14C]pyruvate but not from [1-14C]acetyl-CoA was inhibited by phosphorylation. Phosphorylation also substantially inhibited the transfer of [14C]acetyl groups from enzyme-bound [14C]acetylhydrolipoate to TPP in the presence of NADH. 7...     

Relationship between L-alanine and sodium ion transport in isolated renal tubules.

1. Rat renal tubules were isolated by incubation with collagenase. The Na+ concentration in the tubules at 37 degrees C was increased by additions of g-strophantin and L-alanine. The increase of Na+ in the presence of both g-strophantin and L-alanine was stronger than with either alone. 2. Radioactive sodium (22-Na), which was taken up by the tubules at 0 degrees C in K+-free medium, was more slowly washed out in the buffer with added g-strophantin than in the control buffer, but L-alanine had no effect. 3. At 0 degrees C incubation without K+, g-strophantin did not affect the 22-Na transport of the tubules. But under the same conditions, L-alanine increased Na+ uptake significantly, and in conjunction with it, L-alanine uptake was also increased. 4. The relationship between L-alanine uptake and intra- extracellular Na+ concentration gradients was linear. The ration of L-alanine to Na+ uptake at 0 degrees C was about 1:2. 5. In the incubation without K+ at 0 degrees C, L-alanine could be accumulated in tubules against the chemical concentration gradient (about 1.5-fold). 6. In the incubation without K+ at 37 degrees C, the L-alanine concentration in tubules after 5 min was already steady (Ci/Ce = 2.2), but with K+ it was not stabilized after 10 min. The ration Ci/Ce with K+ WAS HIGHER THAN WITHOUT K+. 7. G-Strophantin, p-hydroxymercuribenzoate, amiloride, and 2,4-dinitrophenol inhibited L-alanine uptake in the tubules and at the same time increased Na+ concentration. The relationship between the L-alanine uptakes inhibited by g-strophantin, amiloride and dinitrophenol, and the respective intra- extracellular Na+ concentration gradients was strikingly linear. But in the case of p-hydroxymercuribenzoate there was no correlation. 8. The results indicate that L-alanine transport into the renal tubules might be regulated mainly by the intra- extracellular Na+ concentration gradient and that inhibitors such as g-strophantin, amiloride, and dinitrophenol could have a secondary effect on the L-alanine transport which follows the change of Na+ concentration in cells. p-Hydroxymercuribenzoate might have an inhibiting effect on the binding of carrier with Na+ and/or L-alanine.     

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.     

Monensin Inhibition of Na+-Dependent HCO3- Transport Distinguishes It from Na+-Independent HCO3- Transport and Provides Evidence for Na+/HCO3- Symport in the Cyanobacterium Synechococcus UTEX 625

The effect of monensin, an ionophore that mediates Na+/H+ exchange, on the activity of the inorganic carbon transport systems of the cyanobacterium Synechococcus UTEX 625 was investigated using transport assays based on the measurement of chlorophyll a fluorescence emission or 14C uptake. In Synechococcus cells grown in standing culture at about 20 [mu]M CO2 + HCO3-, 50 [mu]M monensin transiently inhibited active CO2 and Na+-independent HCO3- transport, intracellular CO2 and HCO3- accumulation, and photosynthesis in the presence but not in the absence of 25 mM Na+. These activities returned to near-normal levels within 15 min. Transient inhibition was attributed to monensin-mediated intracellular alkalinization, whereas recovery may have been facilitated by cellular mechanisms involved in pH homeostasis or by monensin-mediated H+ uptake with concomitant K+ efflux. In air-grown cells grown at 200 [mu]M CO2 + HCO3- and standing culture cells, Na+-dependent HCO3- transport, intracellular HCO3- accumulation, and photosynthesis were also inhibited by monensin, but there was little recovery in activity over time. However, normal photosynthetic activity could be restored to air-grown cells by the addition of carbonic anhydrase, which increased the rate of CO2 supply to the cells. This observation indicated that of all the processes required to support photosynthesis only Na+-dependent HCO3- transport was significantly inhibited by monensin. Monensin-mediated dissipation of the Na+ chemical gradient between the medium and the cells largely accounted for the decline in the HCO3- accumulation ratio from 751 to 55. The two HCO3- transport systems were further distinguished in that Na+-dependent HCO3- transport was inhibited by Li+, whereas Na+-independent HCO3- transport was not. It is suggested that Na+-dependent HCO3- transport involves an Na+/HCO3- symport mechanism that is energized by the Na+ electrochemical potential.     

On the Mechanism of Ouabain-Induced Release of Acetylcholine from Synaptosomes

Ouabain (5 x 10(-8)-5 x 10(-4) M) was confirmed to cause a dose-dependent increase in [3H]acetylcholine ([3H]ACh) release, cytosolic free Ca2+ concentration ([Ca2+]i), and 22Na+ uptake in cerebrocortical synaptosomes of rats in the presence of extracellular Ca2+. Ouabain also caused a dose-dependent decrease in membrane potential. In a low-Na+ (10 mM) medium, ouabain failed to increase [3H]ACh release and [Ca2+]i. Tetrodotoxin (10(-6) M) had no effect on the ouabain-induced increase in both [3H]ACh release and [Ca2+]i but abolished the increase in 22Na+ uptake and partially inhibited the depolarizing effect. Verapamil (10(-6)-5 x 10(-4) M) inhibited the ouabain-induced increase in both [3H]ACh release and [Ca2+]i in a dose-dependent manner. Removal of extracellular Ca2+ abolished the effect of ouabain on [Ca2+]i but not on [3H]ACh release and 22Na+ uptake, regardless of the presence or absence of EGTA. In the absence of extracellular Ca2+, 10 mM Mg2+ blocked ouabain-induced [3H]ACh release, which was resistant to verapamil. These results suggest that ouabain can increase ACh release from synaptosomes without the preceding increases in intracellular Ca2+ and/or Na+ content. It seems likely that the removal of extracellular Ca2+ unmasks mechanisms of ouabain action different from those operating in the presence of Ca2+.     

Transport, nutritional and metabolic studies of taurine in staphylococci

A specific, Na+-dependent, energy-requiring transport system for taurine has been reported recently in the Staphylococcus aureus M strain. Taurine was taken up vigorously by all S. aureus strains tested. The system was Na+-dependent, and Na+ decreased the Km but had no effect on the Vmax of the transport system. Among coagulase-negative staphylococci, the Staphylococcus epidermidis group (a taxonomically related group of species associated with humans or other primates) and the free-living, wide-ranging species Staphylococcus sciuri showed vigorous taurine uptake. Somewhat lower rates were found in the Staphylococcus saprophyticus group. Low or barely detectable uptake rates were noted in other staphylococcal species that were primarily of animal origin. No taurine uptake was detected in a variety of other bacterial species tested. Taurine uptake, which was not Na+-dependent, occurred in a Pseudomonas aeruginosa strain grown on taurine as sole energy, carbon, nitrogen, and sulphur source, but not when it was grown in a gluconate/salts medium. In nutritional studies we were unable to demonstrate a role for taurine as a sulphur source for S. aureus. [1,2-14C]- and [35S]taurine were taken up during overnight growth of cells, and radioactivity was distributed similarly among cellular fractions, indicating that the carbon and sulphur atoms of taurine were not cleaved and had the same fate. We were unable to demonstrate any catabolism of taurine in radiorespirometric experiments to detect evolution of 14CO2 by cells incubated with [1,2-14C]taurine. Thus, we found no evidence for a role of taurine in the energy, carbon and sulphur metabolism of S. aureus.     

Bidirectional mechanism of plasma membrane transport of reduced glutathione in intact rat hepatocytes and membrane vesicles.

We determined the trans effects of extracellular reduced glutathione (GSH) on the rate of efflux of endogenous labeled GSH from freshly isolated rat hepatocytes. The presence of GSH (10 mM) in the medium significantly stimulated the fractional rate of efflux of [35S]GSH from 5.2 to 12.6%/15 min (p < 0.01). This effect was concentration-dependent, had sigmoid type of kinetics (D50 of 0.32 mM), and was reversible upon removal of external GSH. trans-Stimulation (counter-transport) was also observed with 5 mM oxidized glutathione (GSSG) and ophthalmic acid (fractional [35S] GSH efflux: 13.4% +/- 4.1 and 8.8% +/- 2.3 in 15 min, respectively, compared with control: 4.7 +/- 2.5/15 min). Bromosulphthalein-glutathione (BSP-GSH, 5 mM) in Krebs buffer inhibited the fractional [35S]GSH efflux (1.1%/15 min), whereas in Cl(-)-free buffer, GSH efflux was stimulated (14.2%/15 min) compared with control. trans-Stimulation was independent of chloride. BSP-GSH cis-inhibited and trans-stimulated the initial rate of GSH transport in basolateral-enriched membrane vesicles (bLPM) but not in canalicular-enriched membrane vesicles (cLPM). gamma-Glutamyl compounds also cis-inhibited and trans-stimulated GSH transport in bLPM vesicles. GSH-depleted hepatocytes incubated with 10 mM [35S]GSH accumulated more GSH than repleted cells, but the initial rate of uptake of radioactivity was faster in repleted cells. In contrast, repleted hepatocytes incubated with tracer or 50 microM [35S]GSH did not take up GSH. Thus, the sinusoidal membrane GSH transporter exhibits low affinity kinetics with sigmoid features for both GSH uptake and trans-stimulation of efflux, explaining the lack of uptake of GSH at low physiologic extracellular concentrations. Therefore, our findings support and explain the widely held view that GSH transport is unidirectional under physiologic conditions. However, the efflux of GSH may also occur in exchange for the uptake of organic anions and gamma-glutamyl compounds.     

Effects of hyperthermia on the membrane potential and Na+ transport of V79 fibroblasts

The effects of hyperthermia (41-43 degrees C) on the membrane potential (calculated from the transmembrane distribution of [3H]tetraphenylphosphonium) and Na+ transport of Chinese hamster V79 fibroblasts were studied. At 41 degrees C, hyperthermia induced a membrane hyperpolarization of log phase cells (5 to 26 mV) that was reversible upon returning to 37 degrees C. The hyperpolarization was inhibited 50% by 1 mM ouabain or 0.25 mM amiloride, an inhibitor of Na+:H+ exchange. Shifting temperature to 41 degrees C increased ouabain-sensitive Rb+ uptake indicating activation of the electrogenic Na+ pump. At 43 degrees C for 60 min, the membrane potential of log phase cells depolarized (20-35 mV). Parallel studies demonstrated enhanced Na+ uptake at 41 degrees C only in the presence of ouabain. At 43 degrees C, Na+ uptake was increased relative to controls with or without ouabain present. At both 41 and 43 degrees C, 0.25 mM amiloride inhibited heat-stimulated Na+ uptake. Na+ efflux was enhanced at 41 degrees C in a process inhibited by ouabain. Thus, one consequence of heat treatment at 41 degrees C is activation of Na+:H+ exchange with the resultant increase in cytosolic [Na+] activating the electrogenic Na+ pump. At temperatures greater than or equal to 43 degrees C, the Na+ pump is inhibited.     

Analysis of the kinetics of Ca2+ signals in Ehrlich ascites tumor cells upon the inhibition of the mitochondrial Na+/Ca2+ exchanger

Signals of cytosolic Ca2+ (Ca2+c) and mitochondrial Ca2+ (Ca2+m) evoked by the activation of purinoreceptors of Ehrlich ascites tumor cells at different extents of inhibition of the mitochondrial Na+/Ca2+ exchanger by tetraphenylphosphonium (TPP+) were investigated. [Ca2+c] was measured by Fura-2 fluorescence, and [Ca2+m] changes were inferred from NAD(P)H fluorescence. The addition of ATP to the cell suspension induced a NAD(P)H response, which replicated Ca2+c signal with some retardation of the peak and a slower decay. In the presence of increasing TPP+ concentrations, NAD(P)H responses evidenced that the rate of [Ca2+m] decay strongly decreases, while the phase of initial rise does not change. The maximal TPP+ dose did not affect [Ca2+c] and NAD(P)H fluorescence in the resting state, as well as ATP-induced [Ca2+c] responses. These data are described in a mathematical model, which accounts for Ca2+ transport through the membranes of endoplasmic reticulum and mitochondria, as well as through the plasma membrane. The model indicates a low rate of the mitochondrial cycle of Ca2+ uptake/efflux at rest and a strong activation of the uptake with increasing [Ca2+c] to which a Hill coefficient of no less than 4 corresponds. Furthermore, the rise of the uptake rate changes in a short time to a decline, and the peak of the rate is markedly ahead of the peak of [Ca2+c].     

Growth-dependent AIB and meAIB uptake in LLC-PK1 cells: effects of differentiation inducers and of TPA

Cultured pig kidney cells designated LLC-PK1, previously shown to acquire Na+-dependent concentrative transport of hexoses as the cells become growth arrested, also show Na+-dependent concentrative uptake of the amino acid analogs alpha-aminoisobutyric acid (AIB) and (methyl) meAIB. This A system-like transport is most active in sparse, growing cultures and becomes stepped down at confluence. The cell/medium equilibrium distribution ratio of the lipophilic cation tetraphenylphosphonium ion (TPP+) decreases in parallel fashion, suggesting that a decrease in membrane potential may be a major factor in the stepdown. Differentiation inducers (hexamethylene bisacetamide) and phosphodiesterase inhibitors (theophylline, methylisobutyl xanthine) accelerate the stepdown, but even in the presence of these compounds addition of the tumor promoter 12-0-tetradecanoylphorbol-13-acetate (TPA) results in the maintenance of a high level of AIB and meAIB uptake. In all these respects the changes in A system-like amino acid transport are the reciprocal of those seen for concentrative hexose transport, although the driving force appears to be the same for both systems. The TPA analogs phorbol and 4-0-methyl TPA which are inactive in tumor promotion are inactive in this system as well. In confluent, already stepped-down cultures, addition of TPA leads to a rapid (2-6 hour) stimulation of AIB and meAIB uptake. The enhancement is sensitive to cycloheximide and actinomycin D. The ouabain-sensitive fraction of meAIB uptake is not markedly changed in the TPA-enhanced uptake, nor is the TPP+ distribution ratio elevated in TPA-treated cells, making it unlikely that the TPA effect is through an alteration in the membrane potential.     

Changes in 45Ca and 109Cd uptake, membrane potential and cell pH in Saccharomyces cerevisiae provoked by Cd2+

The effect of Cd2+ poisoning of Saccharomyces cerevisiae on 45Ca, 109Cd and [14C]tetraphenylphosphonium (TPP) uptake and cell pH was examined. At Cd2+ concentrations that produced substantial K+ efflux the rates of uptake of 45Ca, 109Cd and [14C]TPP increased progressively during incubation of the cells with Cd2+, and the cell pH was lowered concomitantly. The initial rates of uptake of the divalent cations and of TPP were increased in cells pre-loaded with Cd2+, which shows that stimulation of the ion fluxes was exerted by the Cd2+ that accumulated in the cells. The distribution ratio of TPP between cells and medium, however, was decreased by Cd2+. Although hyperpolarization of the cell membrane by Cd2+ cannot be excluded, it is argued that Cd2+ primarily stimulated divalent cation uptake by increasing the cation permeability of the cell membrane allowing the cations to enter the cells more easily.     

Functional and molecular identification of sodium-coupled dicarboxylate transporters in rat primary cultured cerebrocortical astrocytes and neurons

Na+-coupled carboxylate transporters (NaCs) mediate the uptake of tricarboxylic acid cycle intermediates in mammalian tissues. Of these transporters, NaC3 (formerly known as Na+-coupled dicarboxylate transporter 3, NaDC3/SDCT2) and NaC2 (formerly known as Na+-coupled citrate transporter, NaCT) have been shown to be expressed in brain. There is, however, little information available on the precise distribution and function of both transporters in the CNS. In the present study, we investigated the functional characteristics of Na+-dependent succinate and citrate transport in primary cultures of astrocytes and neurons from rat cerebral cortex. Uptake of succinate was Na+ dependent, Li+ sensitive and saturable with a Michaelis constant (Kt) value of 28.4 microM in rat astrocytes. Na+ activation kinetics revealed that the Na+ to succinate stoichiometry was 3:1 and the concentration of Na+ necessary for half-maximal transport was 53 mM. Although uptake of citrate in astrocytes was also Na+ dependent and saturable, its Kt value was significantly higher (approximately 1.2 mM) than that of succinate. Unlabeled succinate (2 mM) inhibited Na+-dependent [14C]succinate (18 microM) and [14C]citrate (4.5 microM) transport completely, whereas unlabeled citrate inhibited Na+-dependent [14C]succinate uptake more weakly. Interestingly, N-acetyl-L-aspartate, which is the second most abundant amino acid in the nervous system, also completely inhibited Na+-dependent succinate transport in rat astrocytes. The inhibition constant (Ki) for the inhibition of [14C]succinate uptake by unlabeled succinate, N-acetyl-L-aspartate and citrate was 15.9, 155 and 764 microM respectively. In primary cultures of neurons, uptake of citrate was also Na+ dependent and saturable with a Kt value of 16.2 microM, which was different from that observed in astrocytes, suggesting that different Na+-dependent citrate transport systems are expressed in neurons and astrocytes. RT-PCR and immunocytochemistry revealed that NaC3 and NaC2 are expressed in cerebrocortical astrocytes and neurons respectively. These results are in good agreement with our previous reports on the brain distribution pattern of NaC2 and NaC3 mRNA using in situ hybridization. This is the first report of the differential expression of different NaCs in astrocytes and neurons. These transporters might play important roles in the trafficking of tricarboxylic acid cycle intermediates and related metabolites between glia and neurons.     

K/Na selectivity at the plasmalemma of cortical root cells and preferential release of sodium to the xylem vessels in roots of Atriplex hortensis

Using excised roots of Atriplex hortensis L., cv. Gelbe Gartenmelde, the uptake, accumulation and xylem transport of K⁺ and Na⁺ have been measured. Influx as well as xylem transport proved to discriminate little between K⁺ and Na⁺, when considered in relation to the external solution. Both K⁺ and Na⁺ inhibited the uptake and xylem transport of each other to about the same degree. Measurements of intracel-lular Na⁺ fluxes by means of compartment analysis indicated that the low degree of K/Na discrimination during uptake was due to low influx selectivity. Moreover, K⁺/Na⁺ exchange at the plasmalemma was not very efficient in Atriplex roots. In order to establish the basis of the low K/Na discrimination in xylem transport, the rates of K⁺ and Na⁺ transport were related to the cytoplasmic K⁺ and Na⁺ concentrations to yield the selectivity ratio of transport, S(transport) = (φ_cx(K) × [Na⁺]_c)/(φ_cx(Na) × [K⁺]_c). Under all conditions this ratio was far below one indicating that Na⁺ was favoured during xylem release in excised roots of Atriplex at low external concentrations. The implications of this discrimination in favour of Na+ are discussed with respect to salt tolerance of A. hortensis .     

Reconstitution into liposomes of the B degrees -like glutamine-neutral amino acid transporter from renal cell plasma membrane

Na+ dependent [3H]glutamine uptake was found in liposomes reconstituted with solubilized rat kidney brush border in the presence of intraliposomal K+. The reconstituted system was optimised with respect to the critical parameters of the cyclic detergent removal procedure, i.e., the detergent used for the solubilization, the protein concentration, the detergent/phospholipid ratio and the number of passages through a single Amberlite column. Time dependent [3H]glutamine accumulation in proteoliposomes occurred only in the presence of external Na+ and internal K+. The transporter showed low if there is any tolerance towards the substitution of Na+ or K+ for other cations. Valinomycin strongly stimulated the transport indicating that it is electrogenic. Intraliposomal glutamine had no effect. From the dependence of the transport rate on the Na+ concentration cooperativity index close to 1 was derived, indicating that 1 Na+ should be involved in the cotransport with glutamine. The electrogenicity of the transport originated from the Na+ transport. Optimal rate of 0.1 mM [3H]glutamine uptake was found in the presence of 50 mM intraliposomal K-gluconate. At higher K-gluconate concentrations the transport rate decreased. The activity of the reconstituted transporter was pH dependent with optimal function in the range pH 6.5-7.0. [3H]glutamine (and [3H]leucine) uptake was inhibited by all the neutral but not by the positively or negatively charged amino acids. The sulfhydryl reagents HgCl2, mersalyl, p-hydroxymercuribenzoate and the substrate analogue 2-aminobicyclo[2,2,1]heptane-2-carboxylate strongly inhibited the transporter, whereas the amino acid analogue alpha-(methylamino)isobutyrate had no effect. The inhibition by mersalyl was protected by the presence of the substrate. On the basis of the Na+ dependence, the electrogenic transport mode and the specificity towards the amino acids, the reconstituted transporter was classified as B degrees-like.     

Bioenergetic response of isolated nerve terminals of rat brain to osmotic swelling

The swelling of nerve terminals of rat brain in a hypotonic medium (230 mOsm) induced the potential-independent entrance of 45Ca2+ into synaptosomes and intrasynaptosomal mitochondria that changed the energy status of synaptosomes, the rate of O2 consumption and the content of ATP being decreased. The ratio ATP/ADP decreased from 6.5 +/- 0.26 (310 mOsm medium) to 3.1 +/- 0.18 (the medium 230 mOsm). Studies on the equilibrium distribution of K+ (86Rb+) and [3H]TPP+ showed that contents of these cations in the nerve terminals were virtually the same on incubation in both iso- and hypotonic media. This indicated that the swelling did not damage intrasynaptosomal mitochondria and plasma membranes of the synaptosomes. The inhibition of oxidative phosphorylation increased twofold the rate of glycolysis. The incubation of synaptosomes in calcium-free medium (230 mOsm) in the presence of EGTA (1 mM) prevented the inhibition of oxidative phosphorylation and synthesis of ATP by the osmotic swelling. Ruthenium Red (10 microM) in the medium 230 mOsm inhibited the entrance of 45Ca2+ into the intrasynaptosomal mitochondria and normalized the oxidative phosphorylation to the control level (310 mOsm medium). The decrease in the energy potential of synaptosomes induced by the hypoosmotic shock is suggested to be associated with the increase in Ca2+ content in the cytoplasm, its transport into the mitochondria, and the inhibitory effect on oxidative phosphorylation.     

Na+-Independent Transporters, LAT-2 and b0,+, Exchange L-DOPA with Neutral and Basic Amino Acids in Two Clonal Renal Cell Lines

The present study examined the functional characteristics of L-DOPA transporters in two functionally different clonal subpopulations of opossum kidney (OKLC and OKHC) cells. The uptake of L-DOPA was largely Na+-independent, though in OKHC cells a minor component (approximately 15%) required extracellular Na+. At least two Na+-independent transporters appear to be involved in L-DOPA uptake. One of these transporters has a broad specificity for small and large neutral amino acids, is stimulated by acid pH and inhibited by 2-aminobicyclo(2,2,l)-heptane-2-carboxylic acid (BCH; OKLC, Ki = 291 mM; OKHC, Ki = 380 mM). The other Na+-independent transporter binds neutral and basic amino acids and also recognizes the di-amino acid cystine. [14C]-L-DOPA efflux from OKLC and OKHC cells over 12 min corresponded to a small amount of intracellular [14C]-L-DOPA. L-Leucine, nonlabelled L-DOPA, BCH and L-arginine, stimulated the efflux of [14C]-L-DOPA in a Na+-independent manner. It is suggested that L-DOPA uses at least two major transporters, systems LAT-2 and b0,+. The transport of L-DOPA by LAT-2 corresponds to a Na+-independent transporter with a broad specificity for small and large neutral amino acids, stimulated by acid pH and inhibited by BCH. The transport of L-DOPA by system b0,+ is a Na+-independent transporter for neutral and basic amino acids that also recognizes cystine. LAT-2 was found equally important at the apical and basolateral membranes, whereas system b0,+ had a predominant distribution in apical membranes.     

alpha-aminoisobutyrate transport into cells from R3230AC mammary adenocarcinoma. Evidence for sodium ion-dependent and -independent carrier-mediated entry and effects of diabetes.

In the presence of Na+, alpha-aminoisobutyrate was transported by saturable and non-saturable processes into R3230AC mammary tumour cells isolated by enzymic treatment. Eadie-Hofstee analysis for the saturable process gave a curvilinear plot, suggesting that transport occurred by more than one carrier. In the absence of Na+, alpha-aminoisobutyrate was also transported by both saturable and non-saturable processes. This Na+-independent saturable process gave a linear plot according to Eadie-Hofstee analysis: V, 708 +/- 105 pmol/min per 5 X 10(6) cells; Km, 0.36 +/- 0.33 mM (mean +/- S.E.M.). Subtracting alpha-aminoisobutyrate entry in the absence of Na+ from total alpha-aminoisobutyrate uptake (in the presence of Na+) showed the presence of another saturable process (Na+-dependent), accounting for 75% of total alpha-aminoisobutyrate uptake. This component gave a linear Eadie-Hofstee plot: V, 2086 +/- 213; Km, 1.75 +/- 0.16 alpha-(Methylamino)isobutyrate, a substrate specifically taken up by the A system, inhibited 80% of alpha-aminoisobutyrate entry. The presence of both alhpa-(methylamino)isobutyrate and phenylalanine inhibited alpha-aminoisobutyrate entry completely. 2-Aminobicyclo[2.2.1]heptane-2-carboxylate, an analogue specifically taken up by the Na+-independent system, inhibited completely the Na+-independent entry of alpha-aminoisobutyrate. In the presence of Na+, the distribution ratio, which is defined as the amino acid concentration in the intracellular space divided by that in the incubation medium for alpha-aminoisobutyrate, at 90 min was 19, and in the absence of Na+ at 60 min was 5. These concentrative processes were sensitive to the metabolic inhibitor pentachlorophenol. The Na+-dependent, but not the Na+-independent, alpha-aminoisobutyrate uptake was increased in cells from diabetic rats. This was primarily due to an increase in the V for the Na+-dependent component (164%) with no effect on the Km. We conclude, therefore, that alpha-aminoisobutyrate entry into cells from this mammary tumour is mediated by two transport systems, one Na+-dependent and another Na+-independent. Furthermore, the Na+-dependent component of alpha-aminoisobutyrate is sensitive to alterations of insulin in vivo.     

The Na+/K+-ATPase reaction of human erythrocytes is not near equilibrium. A 31P-NMR study

We have addressed the question of whether the Na/K+-ATPase in the human erythrocyte is in a state of near-equilibrium by varying the extracellular ratio of Na+ and K+ and following the cytosolic phosphorylation potential by 31P-NMR and by combined enzymatic colorimetric measurements. There was no correlation at room temperature between the extracellular Na+/K+ ratio and the cytosolic phosphorylation potential measured either by NMR or alternative methods. The cytosolic phosphorylation potential measured by NMR was 4100 +/- 1300 (S.E.) M-1 at an extracellular K+ concentration of 5.9 mM (Na+/K+ ratio of 24.3) and 2800 +/- 700 (S.E.) M-1 at 75 mM extracellular K+ (Na+/K+ ratio of 0.99). The chemically determined phosphorylation potential was 6400 +/- 1200 (S.E.) and 5000 +/- 700 (S.E.) M-1 at 5.9 and 75 mM extracellular K+, respectively. Omission of Ca2+ from the buffer solutions did not affect the results. A consistent finding in this study was that the NMR-determined value of ATP was about 10-20% lower than the value determined enzymatically on perchloric acid extracts. The inorganic phosphate (Pi) was fully NMR visible.     

The electrochemical proton potential and the proton/electron ratio of the electron transport with fumarate in Wolinella succinogenes

The electrochemical proton potential across the cytoplasmic membrane ( ) as well as the H⁺ / e ratio, which were brought about by the electron transport of Wolinella succinogenes, was measured with the aim of understanding the mechanism of electron-transport-coupled phosphorylation in this anaerobic bacterium. (1) Inverted vesicles derived from the bacterial membrane were found to take up protons from the external medium on initiation of fumarate reduction by H₂. Proton uptake was dependent on the presence of K⁺ within the vesicles, was enhanced by the presence of valinomycin and DCCD and high internal buffer concentration, and was abolished by protonophores. The maximum H⁺ / e ratio slightly exceeded 1. (2) The vesicles accumulated thiocyanate in the steady state of fumarate reduction by H₂. The concentration ratio (internal / external) was close to 1000 at an external thiocyanate concentration below 10 μM. Under the same conditions the uptake of methylamine was negligible. Thiocyanate uptake was abolished by the presence of a protonophore. (3) Cells of W. succinogenes accumulated tetraphenylphosphonium cation (TPP) in the steady state of fumarate reduction with H₂ or formate. Under the same conditions the uptake of benzoic acid was negligible. From the amount of TPP taken up by the bacteria, the free internal concentration of TPP was evaluated according to the procedure of Zaritsky et al. (Zaritsky, A., Kihara, M. and MacNab, R.M. (1981) J. Membrane Biol. 63, 215–231). The concentration ratio (internal / external) was 700 in the absence and close to 1 in the presence of a protonophore or in the absence of external Na⁺. (4) The experimental results are consistent with the view that the energy transduction from electron transport to phosphorylation is done by means of the across the bacterial membrane.