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.     

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.     

Dicarboxylate transport in human placental brush-border membrane vesicles

Pathways for transport of dicarboxylic acid metabolites by human placental epithelia were investigated using apical membrane vesicles isolated by divalent cation precipitation. The presence of Na+/dicarboxylate cotransport was assessed directly by [14C]succinate tracer flux measurements and indirectly by fluorescence determinations of voltage sensitive dye responses. The imposition of an inwardly directed Na+ gradient stimulated vesicle uptake of succinate achieving levels approximately 5-fold greater than those observed at equilibrium. The increased succinate uptake was specific for Na+ as no stimulation was observed in the presence of Li+, K+ or choline+ gradients. In addition to concentrative accumulation of succinate, a direct coupling of Na+/succinate cotransport was suggested by the absence of a sizeable conductive pathway for succinate uptake and decreased succinate uptake levels associated with a more rapid decay of an imposed Na+ gradient. Na+ gradient-driven succinate uptake was not the result of parallel Na+/H+ and succinate/OH- exchange activities but was reduced by the Na+-coupled inhibitor harmaline. The voltage sensitivity of Na+ gradient-driven succinate uptake suggests Na+/succinate cotransport is electrogenic occurring with net transfer of positive charge. Substrate-specificity studies suggest the tricarboxylic acid cycle intermediates as candidates for transport by the Na+-coupled pathway. Decreasing pH increased the citrate-induced inhibition of succinate uptake suggesting divalent citrate as the preferred substrate for transport. Initial rate determinations of succinate uptake indicate succinate interacts with a single saturable site (Km 33 microM) with a maximal transport rate of 0.5 nmol/mg per min.     

Thermodynamic stoichiometry of Na+-coupled glutathione transport

Ambiguity exists with respect to mechanisms of glutathione (GSH) transport and the molecular identity of GSH transporters. Empirical and theoretical limitations have hindered functional and molecular characterizations. Published literature referring to the isolation and molecular identification of Na+-coupled GSH transporters that mediate the cellular uptake of GSH is highly debated. Whereas a number of functional and kinetic reports of this putative symport mechanism exist, the hypothetical transmembrane Na+-coupled GSH transporter protein or the genetic message encoding it has not been isolated. Theoretical thermodynamic calculations to support the concept of secondary active GSH transport and to rationalize accounts of physical-kinetic measurements describing Na+-coupled cellular GSH uptake were performed. The adequacy of requisite energy and stoichiometric conservation of the separate electrical and chemical components of a Na+ gradient in maintaining a high cellular accumulation gradient for GSH was examined through a purely phenomenological perspective. Dependent on the biological context, the energetic coupling between Na+ and GSH cotransport may occur at ratios from 1:1 to 3:1. Molecular identification of specific transporters responsible for cellular Na+-coupled GSH uptake will facilitate determination of their relative contribution to the overall plasma membrane resting potential. In tissues with a high GSH concentration relative to their extracellular milieu, particularly in pathologies of cystic fibrosis and dry eye syndromes, large energy coupling ratios in cotransport of Na+ and GSH may be expected. Na+-coupled GSH transport may play an important role in disease onset and (or) progression, or treatment modalities thereof.     

Na+- and K+-dependent uridine transport in rat renal brush-border membrane vesicles

The transport of uridine into rat renal brush-border membrane vesicles was investigated using an inhibitor-stop filtration method. Uridine was not metabolized under these conditions. The rapid efflux of intravesicular uridine was prevented by adding 1 mM phloridzin to the ice-cold stop solution. In the presence of inwardly directed gradients of either Na+ or K+, zero-trans uridine uptake exhibited a transient overshoot phenomenon indicating active transport. The overshoot was much more pronounced with Na+ than K+ and it was not observed when either Na+ or K+ was at equilibrium across the membrane. The K+-induced overshoot was not due to the presence of a membrane potential alone, as an inwardly directed gradient of choline chloride failed to produce it. The amplitude of the overshoot was increased by raising either the Na+ or K+ concentration outside the membrane or by using more lipophilic anions (reactive order was NO3- greater than SCN- greater than Cl- greater than SO4(2-). Zero-trans efflux studies showed that the uridine transport is bidirectional. Li+ could substitute poorly for Na+ but not at all for K+. Stoichiometries of 1:1 and greater than 1:1 were observed for Na+: uridine and K+: uridine coupling, respectively. A preliminary analysis of the interactions between Na+ and K+ for uridine uptake showed complex interactions which can best be explained by the involvement of two different systems for nucleoside transport in the rat renal brush-border membrane, one requiring Na+ and the other K+ as transport coupler.     

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.     

Effects of sodium chloride stress and calcium supply on growth, potassium uptake, and internal chloride and sodium levels of winter wheat seedlings

The effects of saline conditions on the K+ (86Rb), Na+ and Cl- uptake and growth of 6-day-old wheat (Triticum aestivum L. cv. GK Szeged) seedlings were studied in the absence and presence of Ca2+. It was found that on direct NaCl treatment the K+ uptake of the roots in the absence of Ca2+ declined significantly with increasing salinity. The reverse was true, however, in the case of NaCl pretreatment: seedlings grown under highly saline conditions (50 mM NaCl) absorbed more K+ than those pretreated with low levels of NaCl (1 or 10 mM NaCl). The data indicate a definite Na(+)-induced K+ uptake inhibition and/or feed-back regulation in the K+ uptake of roots under the above-mentioned growth conditions. As regards the Ca2+ effect, it was established that supplemental Ca2+ counteracts the unfavourable effect of saline conditions as concerns both the K+ uptake of the roots and the dry matter yield of the seedlings. The internal concentrations of Na+ and Cl- in the seedlings increased in proportion to increasing salinity. Marked differences were experienced, however, in the internal concentrations of Na+ and Cl- in the roots and shoots, respectively. It was concluded that under these experimental conditions the salt tolerance of wheat could be related to its capability of restricting the transport of Na+ at low and moderate levels to the shoots, where it is highly toxic.     

(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.     

Proline uptake through the major transport system of Salmonella typhimurium is coupled to sodium ions.

Strains of Salmonella typhimurium deficient in one or more of the proline transport systems have been constructed and used to study the mechanism of energy coupling to transport. Proline uptake through the major proline permease (PP-I, putP) is shown to be absolutely coupled to Na+ ions and not to H+ ions as has previously been assumed. Transport through the minor proline permease (PP-II, proP), however, is unaffected by the presence or absence of Na+. The effect of Na+ on the kinetics of proline uptake shows that external Na+ increases the Vmax for transport. It seems probable that proline transport through PP-I is also coupled to Na+ ions in Escherichia coli.     

K+ currents through SV-type vacuolar channels are sensitive to elevated luminal sodium levels

Non-selective slow vacuolar (SV) channels mediate uptake of K+ and Na+ into vacuolar compartment. Under salt stress plant cells accumulate Na+ in the vacuole and release vacuolar K+ into the cytoplasm. It is, however, unclear how plants mediate transport of K+ from the vacuole without concomitant efflux of toxic Na+. Here we show by patch-clamp studies on isolated Arabidopsis thaliana cell culture vacuoles that SV channels do not mediate Na+ release from the vacuole as luminal Na+ blocks this channel. Gating of the SV channel is dependent on the K+ gradient across the vacuolar membrane. Under symmetrical K+ concentrations on both sides of the vacuolar membrane, SV channels mediate potassium uptake. When cytoplasmic K+ decreases, SV channels allow K+ release from the vacuole. In contrast to potassium, Na+ can be taken up by SV channels, but not released even in the presence of a 150-fold gradient (lumen to cytoplasm). Accumulation of Na+ in the vacuole shifts the activation potential of SV channels to more positive voltages and prevents gradient-driven efflux of K+. Similar to sodium, under physiological conditions, vacuolar Ca2+ is not released from vacuoles via SV channels. We suggest that a major Arabidopsis SV channel is equipped with a positively charged intrinsic gate located at the luminal side, which prevents release of Na+ and Ca2+, but permits efflux of K+. This property of the SV channel guarantees that K+ can shuttle across the vacuolar membrane while maintaining Na+ and Ca2+ stored in this organelle.     

Effect of Monovalent Cations on Na+/Ca2+ Exchange and ATP-Dependent Ca2+ Transport in Synaptic Plasma Membranes

Two Ca2+ transport systems were investigated in plasma membrane vesicles isolated from sheep brain cortex synaptosomes by hypotonic lysis and partial purification. Synaptic plasma membrane vesicles loaded with Na+ (Na+i) accumulate Ca2+ in exchange for Na+, provided that a Na+ gradient (in leads to out) is present. Agents that dissipate the Na+ gradient (monensin) prevent the Na+/Ca2+ exchange completely. Ca2+ accumulated by Na+/Ca2+ exchange can be released by A 23187, indicating that Ca2+ is accumulated intravesicularly. In the absence of any Na+ gradient (K+i-loaded vesicles), the membrane vesicles also accumulate Ca2+ owing to ATP hydrolysis. Monovalent cations stimulate Na+/Ca2+ exchange as well as the ATP-dependent Ca2+ uptake activity. Taking the value for Na+/Ca2+ exchange in the presence of choline chloride (external cation) as reference, other monovalent cations in the external media have the following effects: K+ or NH4+ stimulates Na+/Ca2+ exchange; Li+ or Cs+ inhibits Na+/Ca2+ exchange. The ATP-dependent Ca2+ transport system is stimulated by increasing K+ concentrations in the external medium (Km for K+ is 15 mM). Replacing K+ by Na+ in the external medium inhibits the ATP-dependent Ca2+ uptake, and this effect is due more to the reduction of K+ than to the elevation of Na+. The results suggest that synaptic membrane vesicles isolated from sheep brain cortex synaptosomes possess mechanisms for Na+/Ca2+ exchange and ATP-dependent Ca2+ uptake, whose activity may be regulated by monovalent cations, specifically K+, at physiological concentrations.     

Cation transport mechanisms in Mycoplasma mycoides var. Capri cells. Na+-dependent K+ accumulation

We have studied some features of K+ accumulation by glycolysing Mycoplasma mycoides var. Capri cells. We report that when Na+ is absent from the external medium, K+ accumulates up to the level predicted by the amplitude of the transmembrane electrical potential, delta psi m, measured by Rb+ and methyltriphenylphosphonium cation (TPMP+) distribution. Therefore, under these experimental conditions, the coupling mechanism of K+ uptake consists of a delta psi m-driven uniport. More important, when Na+ is present in the external medium, the level of K+ accumulation by glycolysing Mycoplasma cells is far too steep to be equilibrium with delta psi m (-120 mV for delta muK+ compared with -90mV for delta muRb+ or delta muTPMP+). Our results clearly indicate the presence in Mycoplasma of an active K+-transport system specifically stimulated by Na+. Furthermore, by controlling the amplitude of the energy-dependent delta muH+, we obtain strong evidence that this specific Na+-stimulated K+ transport is modulated by the transmembrane electrical potential. Finally, we show that ATP is consumed when such a transport system is in activity.     

Acetyl phosphate can act as a substrate for Na+ transport by (Na+ + K+)-ATPase

Experiments using liposomes with (Na+ + K+)-ATPase incorporated showed that in the presence of extravesicular Mg2+, acetyl phosphate was able to stimulate Na+ uptake when the liposomes contained Na+ or choline and were K+-free; this acetyl phosphate-dependent Na+ transport was similar to the ATP-dependent transport observed with 0.003 mM or 3 mM ATP. When the intravesicular solution contained K+, there was an ATP-dependent Na+ uptake which was large with 3 mM ATP and small (about the size seen in K+-free liposomes) with 0.003 mM ATP; in this case, although acetyl phosphate produced a slight activation of Na+ transport, the effect was not statistically significant. All ATP and acetyl phosphate-stimulated Na+ transport disappeared in the absence of extravesicular Mg2+ or in the presence of ouabain in the intravesicular solution. These results are consistent with the hypothesis that, at the concentration used, acetyl phosphate can replace ATP in the catalytic but not in the regulatory site of the (Na+ + K+)-ATPase and active Na+ transport system. This suggests that as far as the early stages of the pump cycle are concerned the role of ATP is simply to phosphorylate.     

The broadly selective human Na+/nucleoside cotransporter (hCNT3) exhibits novel cation-coupled nucleoside transport characteristics

The concentrative nucleoside transporter (CNT) protein family in humans is represented by three members, hCNT1, hCNT2, and hCNT3. hCNT3, a Na+/nucleoside symporter, transports a broad range of physiological purine and pyrimidine nucleosides as well as anticancer and antiviral nucleoside drugs, and belongs to a different CNT subfamily than hCNT1/2. H+-dependent Escherichia coli NupC and Candida albicans CaCNT are also CNT family members. The present study utilized heterologous expression in Xenopus oocytes to investigate the specificity, mechanism, energetics, and structural basis of hCNT3 cation coupling. hCNT3 exhibited uniquely broad cation interactions with Na+, H+, and Li+ not shared by Na+-coupled hCNT1/2 or H+-coupled NupC/CaCNT. Na+ and H+ activated hCNT3 through mechanisms to increase nucleoside apparent binding affinity. Direct and indirect methods demonstrated cation/nucleoside coupling stoichiometries of 2:1 in the presence of Na+ and both Na+ plus H+, but only 1:1 in the presence of H+ alone, suggesting that hCNT3 possesses two Na+-binding sites, only one of which is shared by H+. The H+-coupled hCNT3 did not transport guanosine or 3'-azido-3'-deoxythymidine and 2',3'-dideoxycytidine, demonstrating that Na+- and H+-bound versions of hCNT3 have significantly different conformations of the nucleoside binding pocket and/or translocation channel. Chimeric studies between hCNT1 and hCNT3 located hCNT3-specific cation interactions to the C-terminal half of hCNT3, setting the stage for site-directed mutagenesis experiments to identify the residues involved.     

INHIBITION OF AMINO ACID UPTAKE BY THE ABSENCE OF Na+ IN SLICES OF BRAIN

—The Na+ requirement of amino acid transport was measured in brain slices. The tissue was first washed free of Na+ and then Na+ was replaced by one of the following: choline, Li+, Rb+, or mannose. Amino acid uptake was measured at different times (5–120 min) and at low (10^-7–10^-5m ) and high (10^-3m ) concentrations. Most of the Na+ could be washed out of the tissue; this also decreased K+ levels despite increased K+ in the medium. K+ tissue levels were partially restored when Na+ was added. The absence of Na+ abolished the uptake of Glu, Asp, GABA, Gly, Tau and Pro. Most of the neutral amino acids (Ala, Val, Trp, His) were very strongly inhibited by the absence of Na+ under most experimental conditions. Basic amino acids (Arg, Lys) were not completely inhibited, in that 30 per cent of the equilibrium uptake remained and some of the basic amino acid influx was independent of the Na+ tissue level. The uptake of amines (tyramine, cadaverine, putrescine) did not require Na+, and often was greater in the absence of Na+. We conclude that amino acid uptake in brain slices is Na+ dependent, although the absence of Na+ may affect transport indirectly.     

Two types of HKT transporters with different properties of Na+ and K+ transport in Oryza sativa

It is thought that Na+ and K+ homeostasis is crucial for salt-tolerance in plants. To better understand the Na+ and K+ homeostasis in important crop rice (Oryza sativa L.), a cDNA homologous to the wheat HKT1 encoding K+-Na+ symporter was isolated from japonica rice, cv Nipponbare (Ni-OsHKT1). We also isolated two cDNAs homologous to Ni-OsHKT1 from salt-tolerant indica rice, cv Pokkali (Po-OsHKT1, Po-OsHKT2). The predicted amino acid sequence of Ni-OsHKT1 shares 100% identity with Po-OsHKT1 and 91% identity with Po-OsHKT2, and they are 66-67% identical to wheat HKT1. Low-K+ conditions (less than 3 mM) induced the expression of all three OsHKT genes in roots, but mRNA accumulation was inhibited by the presence of 30 mM Na+. We further characterized the ion-transport properties of OsHKT1 and OsHKT2 using an expression system in the heterologous cells, yeast and Xenopus oocytes. OsHKT2 was capable of completely rescuing a K+-uptake deficiency mutation in yeast, whereas OsHKT1 was not under K+-limiting conditions. When OsHKTs were expressed in Na+-sensitive yeast, OsHKT1 rendered the cells more Na+-sensitive than did OsHKT2 in high NaCl conditions. The electrophysiological experiments for OsHKT1 expressed in Xenopus oocytes revealed that external Na+, but not K+, shifted the reversal potential toward depolarization. In contrast, for OsHKT2 either Na+ or K+ in the external solution shifted the reversal potential toward depolarization under the mixed Na+ and K+ containing solutions. These results suggest that two isoforms of HKT transporters, a Na+ transporter (OsHKT1) and a Na+- and K+-coupled transporter (OsHKT2), may act harmoniously in the salt tolerant indica rice.     

Inhibition by K+ of Na+-Dependent D-Aspartate Uptake into Brain Membrane Saccules

Na+-dependent uptake of dicarboxylic amino acids in membrane saccules, due to exchange diffusion and independent of ion gradients, was highly sensitive to inhibition by K+. The IC50 was 1-2 mM under a variety of conditions (i.e., whole tissue or synaptic membranes, frozen/thawed or fresh, D-[3H]aspartate (10-1000 nM) or L-[3H]glutamate (100 nM), phosphate or Tris buffer, NaCl or Na acetate, presence or absence of Ca2+ and Mg2+). The degree of inhibition by K+ was also not affected on removal of ion gradients by ionophores, or by extensive washing with H2O and reloading of membrane saccules with glutamate and incubation medium in the presence or absence of K+ (3 mM, i.e., IC70). Rb+, NH4+, and, to a lesser degree Cs+, but not Li+, could substitute for K+. [K+] showed a competitive relationship to [Na+]2. Incubation with K+ before or after uptake suggested that the ion acts in part by allowing net efflux, thus reducing the internal pool of amino acid against which D-[3H]aspartate exchanges, and in part by inhibiting the interaction of Na+ and D-[3H]aspartate with the transporter. The current model of the Na+-dependent high-affinity acidic amino acid transport carrier allows the observations to be explained and reconciled with previous seemingly conflicting reports on stimulation of acidic amino acid uptake by low concentrations of K+. The findings correct the interpretation of recent reports on a K+-induced inhibition of Na+-dependent "binding" of glutamate and aspartate, and partly elucidate the mechanism of action.     

The Na+ gradient-dependent transport of D-glucose in renal brush border membranes.

The Na+-dependent transport of D-glucose was studied in brush border membrane vesicles isolated from the rabbit renal cortex. The presence of a Na+ gradient between the external incubation medium and the intravesicular medium induced a marked stimulation of D-glucose uptake. Accumulation of the sugar in the vesicles reached a maximum and then decreased, indicating efflux. The final level of uptake of the sugar in the presence of the Na+ gradient was identical with that attained in the absence of the gradient, suggesting that equilibrium was established. At the peak of the overshoot the uptake of D-glucose was more than 10-fold the equilibrium value. These results suggest that the imposition of a large extravesicular to intravesicular gradient of Na+ effects the transient movement of D-glucose into renal brush border membranes against its concentration gradient. The stimulation of D-glucose uptake into the membranes was specific for Na+. The rate of uptake was enhanced with increased concentration of Na+. Increasing Na+ in the external medium lowered the apparent Km for D-glucose. The Na+ gradient effect on D-glucose transport was dissected into a stimulatory effect when Na+ and sugar were on the same side of the membrane (cis stimulation) and an inhibitory effect when Na+ and sugar were on opposite sides of the membrane (trans inhibition). The uptake of D-glucose, at a given concentration of sugar, reflected the sum of the contributions from a Na+-dependent transport system and a Na+-independent system. The relative stimulation of D-glucose uptake by Na+ decreased as the sugar concentration increased. It is suggested, however, that at physiological concentrations of D-glucose the asymmetry of Na+ across the brush border membrane might fully account for uphill D-glucose transport. The physiological significance of the findings is enhanced additionally by observations that the Na+-dependent D-glucose transport system in the membranes in vitro possessed the sugar specificities and higg phlorizin sensitivity characteristic of more intact preparations. These results provide strong experimental evidence for the role of Na+ in transporting D-glucose across the renal proximal tubule luminal membrane.     

Characteristics of glutamic acid transport by rabbit intestinal brush-border membrane vesicles. Effects of Na+-, K+- and H+-gradients

In the presence of a Na+-gradient (out greater than in), L-glutamic acid and L-and D-aspartic acids were equally well concentrated inside the vesicles, while no transport above simple diffusion levels was seen by replacement of Na+ by K+. Equilibrium uptake values were found inversely proportional to the medium osmolarity, thus demonstrating uptake into an osmotically sensitive intravesicular space. The extrapolation of these lines to infinite medium osmolarity (zero space) showed only a small binding component in acidic amino-acid transport. When the same experiment was performed at saturating substrate concentrations, linear relationships extrapolating through the origin but showing smaller slope values were recorded, thus indicating that the binding component could be more important than suspected above. However, binding to the membrane was neglected in our studies as it was absent from initial rate measurements. Na+-dependent uphill transport of L-glutamic acid was stimulated by K+ present on the intravesicular side only but maximal stimulation was recorded under conditions of an outward K+-gradient (in greater than out). Quantitative and qualitative differences in the K+ effect were noted between pH 6.0 and 8.0. Initial uptake rates showed pH dependency in Na+-(out greater than in) + K+-(in greater than out) gradient conditions only with a physiological pH optimum between 7.0 and 7.5. It was also found that a pH-gradient (acidic outside) could stimulate both the Na+-gradient and the Na+ + K+-gradient-dependent transport of L-glutamic acid. However, pH- or K+-gradient alone were ineffective in stimulating uptake above simple diffusion level. Finally, it was found that increased rates of efflux were always observed with an acidic pH outside, whatever the conditions inside the vesicles. From these results, we propose a channel-type mechanism of L-glutamic acid transport in which Na+ and K+ effects are modulated by the surrounding pH. The model proposes a carrier with high or low affinity for Na+ in the protonated or unprotonated forms, respectively. We also propose that K+ binding occurs only to the unprotonated carrier and allows its fast recycling as compared to the free form of the carrier. Such a model would be maximally active and effective in the intestine in the in vivo physiological situations.