CV-1 cell recipients of the mouse ouabain resistance gene express a ouabain-insensitive Na,K-ATPase after growth in cardioactive steroids

The cation-transporting activity and Na,K-ATPase activity of CV-1 cell recipients of the mouse ouabain resistance gene (ouaR6, or OR6 cells; see Levenson, R., Racaniello, V., Albritton, L., and Housman, D. (1984) Proc. Natl. Acad. Sci. U. S. A. 81, 1489-1493) have been further characterized. OR6 cells grown in strophanthidin (a cardiac aglycon which may be removed rapidly from the Na,K-ATPase) possess both ouabain-sensitive and -insensitive 86Rb+ uptake activities. The ouabain-sensitive 86Rb+ uptake activity of these cells (OR6-S cells) exhibits the same Ki for ouabain as that of the CV-1 parent cells (Ki(app) = 3 x 10(-7) M ouabain), but accounts for only approximately 30% of total 86Rb+ uptake into Na+-loaded OR6-S cells, compared to 80% for CV-1 cells. Most of the ouabain-resistant 86Rb+ uptake in OR6-S cells is dependent on internal Na+ and is insensitive to furosemide, suggesting that it is due to an ouabain-resistant Na,K pump. In OR6-S cell lysates, 50% of Na+-dependent ATPase activity is insensitive to 1 mM ouabain, compared to less than 5% in CV-1 cell lysates. In addition, purified plasma membranes from OR6-S cells contain a 100-kDa protein which is transiently phosphorylated by ATP in an Na+-dependent, K+-sensitive manner, like the alpha subunit of the CV-1 Na,K-ATPase and the canine renal Na,K-ATPase, but which is unaffected by preincubation in 1 mM ouabain. All of these data suggest that OR6-S cells possess a ouabain-insensitive Na,K pump with characteristics similar to the ouabain-sensitive pump of CV-1 parent cells. Since the mouse ouabain resistance gene does not encode either subunit of the Na,K-ATPase, these results suggest that the ouabain resistance gene product may modify the ouabain sensitivity of the endogenous CV-1 Na,K pump.     

Combined allosteric and competitive interaction between extracellular Na(+) and K(+) during ion transport by the alpha(1), alpha(2), and alpha(3) isoforms of the Na, K-ATPase

A combined allosteric and competitive model describes the interaction between extracellular Na(+) and Rb(+) during ion transport mediated by the Na, K-ATPase. The model was developed from experiments based on (86)Rb uptake by whole cells transfected with rat isoforms of the enzyme. In the absence of Na(+), only a single transport site for extracellular Rb(+) exists. After the occupation of the Na(+)-specific allosteric site, the Rb(+) transport pocket opens to allow occupation by an additional Rb(+) and the subsequent transport of the two Rb(+) ions into the cells. Na(+) can also directly compete with Rb(+) for binding to at least one of the transport sites. While the model derived here applies to each of the three rat isoforms of the Na, K-ATPase expressed in HeLa cells, subtle differences exist among the isoforms. The alpha(3)* isoform has an increased intrinsic affinity for Rb(+) and a lower affinity for the allosteric Na(+) site than alpha(1) or alpha(2)*. The stimulation of uptake observed according to the best-fit model is due to the displacement by Rb(+) of inhibitory Na(+) bound to the transport site.     

Influence of chronic alterations of salt intake and aging on the kinetic of red cell Na+ and K+ transport in Sprague-Dawley rats

The kinetics of Na+ and K+ (Rb)+ transport mediated by the Na(+)-K+ pump and Na(+)-K+ cotransport system (assessed as a function of Rb+o and Na+i) as well as the magnitude of cation leaks were determined in red cells of young male rats subjected to chronic salt deprivation or salt loading (0.1% and 8% NaCl diet). These salt intake alterations induced moderate kinetic changes of the Na(+)-K+ pump which did not result in significant changes of ouabain-sensitive (OS) Rb+ uptake or Na+ net extrusion at in vivo Na+i and K+o concentrations because a decreased affinity for Na+i in salt-loaded animals was compensated by an increased maximal transport rate. High furosemide-sensitive (FS) Rb+ uptake in red cells of salt-deprived rats was caused by an increase of both the maximal transport rate and the affinity for Rb+o. Cation leaks were also higher in salt-deprived than in salt-loaded rats. In three age groups of rats fed a 1% NaCl diet FS Rb+ uptake (but not FS Na+ net uptake) rose with age due to an increasing maximal transport rate whereas the affinity of the cotransport system for Rbo+ did not change. The age-dependent changes in the kinetics of the Na(+)-K+ pump resulted in a slight decrease of OS Rb+ uptake with age that was not paralleled by corresponding Na+ net extrusion. No major age-related changes of cation leaks were found. Thus some intrinsic properties of red cell transport systems can be altered by salt intake and aging.     

Characterization of a Na(+)-K(+)-2Cl- cotransport system in oocytes from Xenopus laevis

In order to characterize the transport systems mediating K+ uptake into oocytes, flux studies employing 86Rb were performed on Xenopus oocytes stripped of follicular cells by pretreatment with Ca2(+)-Mg2(+)-free Barth's medium. Total Rb+ uptake consisted of an ouabain-sensitive and an ouabain-insensitive flux. In the presence of 100 mmol/l NaCl and 0.1 mmol/l ouabain the ouabain-insensitive flux amounted to 754.7 +/- 59.9 pmol/oocyte per h (n = 30 cells, i.e., 10 cells each from three different animals). In the absence of Na+ (Na+ substituted by N-methylglucamine) or when Cl- was replaced by NO3- the ouabain-insensitive flux was reduced to 84.4 +/- 42.9 and 79.2 +/- 12.1 pmol/oocyte per h, respectively (n = 50 cells). Furthermore, this Na(+)- and Cl(-)-dependent flux was completely inhibited by 10(-4) mol/l bumetanide, a specific inhibitor of the Na(+)-K(+)-2Cl- cotransport system. These results suggest that K+ uptake via a bumetanide-sensitive Na(+)-K(+)-2Cl- cotransport system represents a major K+ pathway in oocytes.     

Interaction of phosphate with monovalent cation uptake in yeast.

The uptake of monovalent cations by yeast via the monovalent cation uptake mechanism is inhibited by phosphate. The inhibition of Rb+ uptake shows saturation kinetics and the phosphate concentration at which half-maximal inhibition is observed is equal to the Km of phosphate for the sodium-independent phosphate uptake mechanism. The kinetic coefficients of Rb+ and TI+ uptake are affected by phosphate: the maximal rate of uptake is decreased and the apparent affinity constants for the translocation sites are increased. In the case of Na+ uptake, the inhibition by phosphate may be partly or completely compensated by stimulation of Na+ uptake via a sodium-phosphate cotransport mechanism. Phosphate effects a transient stimulation of the efflux of the lipophilic cation dibenzyldimethylammonium from preloaded yeast cells and a transient inhibition of dibenzyldimethylammonium uptake. Possibly, the inhibition of monovalent cation uptake in yeast can be explained by a transient depolarization of the cell membrane by phosphate.     

Isolation, properties and tissue distribution of rat glutathione transferase E

Uptake of Li+ induced by the addition of proline to a cell suspension of Escherichia coli was detected using an Li+-selective electrode. This Li+ uptake was inhibited by L-azetidine 2-carboxylic acid, a competitive inhibitor of the proline transport system. Thus, direct evidence for Li+-proline cotransport via the proline transport system was obtained. Kinetic parameters of the Li+ uptake were determined.     

Cyclic AMP increases cell surface expression of functional Na,K-ATPase units in mammalian cortical collecting duct principal cells

Cyclic AMP (cAMP) stimulates the transport of Na(+) and Na,K-ATPase activity in the renal cortical collecting duct (CCD). The aim of this study was to investigate the mechanism whereby cAMP stimulates the Na,K-ATPase activity in microdissected rat CCDs and cultured mouse mpkCCD(c14) collecting duct cells. db-cAMP (10(-3) M) stimulated by 2-fold the activity of Na,K-ATPase from rat CCDs as well as the ouabain-sensitive component of (86)Rb(+) uptake by rat CCDs (1.7-fold) and cultured mouse CCD cells (1.5-fold). Pretreatment of rat CCDs with saponin increased the total Na,K-ATPase activity without further stimulation by db-cAMP. Western blotting performed after a biotinylation procedure revealed that db-cAMP increased the amount of Na,K-ATPase at the cell surface in both intact rat CCDs (1.7-fold) and cultured cells (1.3-fold), and that this increase was not related to changes in Na,K-ATPase internalization. Brefeldin A and low temperature (20 degrees C) prevented both the db-cAMP-dependent increase in cell surface expression and activity of Na,K-ATPase in both intact rat CCDs and cultured cells. Pretreatment with the intracellular Ca(2+) chelator bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid also blunted the increment in cell surface expression and activity of Na,K-ATPase caused by db-cAMP. In conclusion, these results strongly suggest that the cAMP-dependent stimulation of Na,K-ATPase activity in CCD results from the translocation of active pump units from an intracellular compartment to the plasma membrane.     

Kinetic analysis of Na,K-activated adenosine triphosphatase induced by low external K+ in a rat liver cell line

Exposure of ARL 15 cells to medium containing reduced concentrations of K+ (0.65 mM) elicited a 50-100% increase in Na,K-ATPase activity. The inhibition by ouabain of both the basal and the induced enzyme conformed to a single-site model (KI = 1 x 10(-4) M). The low K+-induced increment in Na,K-ATPase activity was accompanied by an equivalent increase in the abundance of Na,K-pump sites estimated by ouabain-stabilized ("back-door") phosphorylation, such that the calculated catalytic turnover number of approximately 8000/min was minimally changed. Comparison of the dependence of ouabain-inhibitable K+ uptake on intracellular Na+ and on extracellular K+ concentrations in control and low K+-treated cells revealed no change in the respective half-maximal stimulatory concentrations for these cations, whereas the maximal rate of active K+ uptake in cells exposed to low external K+ increased by nearly 100%. The derived Hill coefficients for active K+ transport rate were also unchanged by the low K+ treatment (i.e. approximately 1.4 for extracellular K+ and 2.6 for intracellular Na+). Na,K-ATPase activity of basal and low K+-induced cells calculated from the measured maximal Na,K transport rate closely approximated the Na,K-ATPase activity measured enzymatically in unfractionated cell lysates under Vmax conditions, suggesting that all or most of the Na,K-ATPase enzymatic units present in both basal and stimulated states are functionally active. Northern blot analysis of RNA isolated from control cells indicated the presence of the Na,K-ATPase alpha-I isoform of the enzyme which increased by nearly 200% following incubation of the cells in low-K+ medium. By contrast, the alpha-II and alpha-III mRNAs were undetectable in either the basal or low K+-stimulated state. These results indicate that the Na,K-ATPase induced by incubation of ARL 15 cells in low-K+ medium is kinetically and functionally indistinguishable from the basal enzyme, and that only the alpha-I isoform is expressed under control and low-K+ conditions.     

Sodium-dependent glucose transport by cultured proximal tubule cells

The cotransport of sodium ion and alpha-methyl glucose, a non-metabolized hexose, was studied in rabbit proximal tubule cells cultured in defined medium. The rate of uptake of alpha-methyl glucose shows saturation kinetics, in which Km, but not Vmax, is dependent upon the Na+ concentration in the medium. The transport system was found to be of the high-affinity type, characteristic of the straight portion of the proximal tubule. Analysis of the rates of initial uptake within the context of a generalized cotransport model, suggests that two Na+ ions are bound in the activation of the hexose transport. The steady-state level of accumulation of alpha-methyl glucose also depends upon sodium concentration, consistent with the initial rate findings. The uptake of alpha-methyl glucose is inhibited by other sugars with the relative potencies of D-glucose greater than alpha-methyl glucose greater than D-galactose = 3-O methylglucose. L-Glucose, D-fructose, and D-mannose show no inhibition. Phlorizin inhibits the alpha-methyl glucose uptake with a Ki of 9 X 10(-6) M. Ouabain (10(-3) M) decreases the steady-state alpha-methyl glucose accumulation by 60%. In the absence of sodium, the accumulation of alpha-methyl glucose is 7-fold less than at 142 mM Na+, reaching a level comparable to the sodium-independent accumulation of 3-O-methyl-D-glucose. These findings are similar to those observed in the proximal tubule of the intact kidney.     

Sodium-phosphate cotransport in human red blood cells. Kinetics and role in membrane metabolism

Orthophosphate (Pi) uptake was examined in human red blood cells at 37 degrees C in media containing physiological concentrations of Pi (1.0- 1.5 mM). Cells were shown to transport Pi by a 4,4'-dinitro stilbene- 2,2'-disulfonate (DNDS) -sensitive pathway (75%), a newly discovered sodium-phosphate (Na/Pi) cotransport pathway (20%), and a pathway linearly dependent on an extracellular phosphate concentration of up to 2.0 mM (5%). Kinetic evaluation of the Na/Pi cotransport pathway determined the K1/2 for activation by extracellular Pi ([Na]o = 140 mM) and extracellular Na [( Pi]o = 1.0 mM) to be 304 +/- 24 microM and 139 +/- 8 mM, respectively. The phosphate influx via the cotransport pathway exhibited a Vmax of 0.63 +/- 0.05 mmol Pi (kg Hb)-1(h)-1 at 140 mM Nao. Activation of Pi uptake by Nao gave Hill coefficients that came close to a value of 1.0. The Vmax of the Na/Pi cotransport varied threefold over the examined pH range (6.90-7.75); however, the Na/Pi stoichiometry of 1.73 +/- 0.15 was constant. The membrane transport inhibitors ouabain, bumetanide, and arsenate had no effect on the magnitude of the Na/Pi cotransport pathway. No difference was found between the rate of incorporation of extracellular Pi into cytosolic orthophosphate and the rate of incorporation into cytosolic nucleotide phosphates, but the rate of incorporation into other cytosolic organic phosphates was significantly slower. Depletion of intracellular total phosphorus inhibited the incorporation of extracellular Pi into the cytosolic nucleotide compartment; and this inhibition was not reversed by repletion of phosphorus to 75% of control levels. Extracellular 32Pi labeled the membrane-associated compounds that migrate on thin-layer chromatography (TLC) with the Rf values of ATP and ADP, but not those of 2,3-bisphosphoglycerate (2,3-DPG), AMP, or Pi. DNDS had no effect on the level of extracellular phosphate incorporation or on the TLC distribution of Pi in the membrane; however, substitution of extracellular sodium with N-methyl-D-glucamine inhibited phosphorylation of the membranes by 90% and markedly altered the chromatographic pattern of the membrane-associated phosphate.(ABSTRACT TRUNCATED AT 400 WORDS)     

Red cell sodium in DOCA-salt hypertension: a Brattleboro study.

The alteration of red cell Na+ content (Na+i), its causes and the possible relationship to the development of DOCA-salt hypertension were studied in Brattleboro rats. A pronounced hypertension developed in heterozygous (non-DI) animals that synthesize vasopressin (VP) although no substantial Na+i elevation was observed in their erythrocytes. In contrast, Na+i rose progressively in red cells of homozygous VP-deficient (DI) rats in which only marginal increase of systolic blood pressure was found after six weeks of DOCA-salt regimen. DOCA-salt treatment of non-DI rats did not cause major alterations in ouabain-resistant (OR) net Na+ uptake or ouabain-sensitive (OS) net Na+ extrusion but moderately increased furosemide-sensitive (FS) Rb+ uptake. The same treatment of DI rats doubled Na+i by an increased OR net Na+ uptake (due to a major elevation in both Na(+)-K+ cotransport and Na+ leak). Consequently, OS net Na+ extrusion was augmented in red cells of these animals. This was accompanied by an about threefold elevated FS Rb+ uptake. It can be concluded that a) the alterations of OR and/or OS Na+ or K+ transport observed in erythrocytes of Brattleboro DI rats are not essential for the development of severe DOCA-salt hypertension, b) red cell ion transport abnormalities revealed in DOCA-salt treated DI rats might be rather ascribed to cell potassium depletion, and c) increased inward Na(+)-K+ cotransport and Na+ leak causes red cell Na+i elevation that stimulates Na(+)-K+ pump activity.     

Relationships between membrane lipids and ion transport in red blood cells of Dahl rats

Distinct changes of membrane lipid content could contribute to the abnormalities of ion transport that take part in the development of salt hypertension in Dahl rats. The relationships between lipid content and particular ion transport systems were studied in red blood cells (RBC) of Dahl rats kept on low- and high-salt diets for 5 weeks since weaning. Dahl salt-sensitive (SS/Jr) rats on high-salt diet had increased blood pressure, levels of plasma triacylglycerols and total plasma cholesterol compared to salt-resistant (SR/Jr) rats. Furthermore, RBC of SS/Jr rats differed from SR/Jr ones by increased content of total membrane phospholipids, but membrane cholesterol was not changed significantly. SS/Jr rats had higher RBC intracellular Na+ (Na(i)+) content and enhanced bumetanide-sensitive Rb+ uptake. RBC membrane content of cholesterol and phospholipids correlated positively with RBC Na(i)+ content, with the activity of Na+-K+ pump and Na+-K+-2Cl- cotransport and also with Rb+ leak. The content of phosphatidylserines plus phosphatidylinositols was positively associated with RBC Na(i)+ content, with the activity of Na+-K+ pump and Na+-K+-2Cl- cotransport and with Rb+ leak. The content of sphingomyelins was positively related to Na+-K+-2Cl- cotransport activity and negatively to ouabain-sensitive Rb+-K+ exchange. We can conclude that observed relationships between ion transport and the membrane content of cholesterol and/or sphingomyelins, which are known to regulate membrane fluidity, might participate in the pathogenesis of salt hypertension in Dahl rats.     

(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.     

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.     

Steady-state physiological variations across a graded series of Na,K-ATPase-amplified cells.

Measurements of internal ion concentrations, amino acid pools, and membrane potential were made across a series of HeLa subclones which are amplified for the genes for the sodium- and potassium-activated ATPase (Na,K-ATPase). These subclones expressed heterogeneous levels of ouabain-binding sites, allowing us to construct a graded amplification series. While [K+]i levels did not vary systematically across the series studied, [Na+]i ranged from 9 to 20 mM as a function of Na,K-ATPase expression. Steady-state accumulation of tetraphenylphosphonium in low versus high potassium was used to measure membrane potential. Values for [Na+]i and the membrane potential were used to calculate the sodium electrochemical potential, which was also found to be a function of Na,K-ATPase expression. Measurements of acid-soluble amino acid pools in cell lysates demonstrated that amino acids which are substrates for sodium-dependent transport systems, or which can potentially exchange through system L for a substrate of a sodium-dependent system, varied as a function of the sodium electrochemical potential. This confirmed our prediction of increased amino acid pool sizes in Na,K-ATPase-amplified lines based on observations of elevated flux through the sodium-independent system L. Finally, we measured lactate production and glycolytic potential in a subset of clones and found that both were reduced in subclones with elevated Na,K-ATPase.     

Pantothenate-sodium cotransport in renal brush-border membranes

The mechanism of pantothenate transport into rabbit renal brush-border membrane vesicles was studied. Under voltage-clamped conditions, an inward NaCl gradient induced the transient accumulation of pantothenate against its concentration gradient, indicating Na+/pantothenate cotransport. K+, Rb+, Li+, NH4+, and choline+ were ineffective in replacing Na+. Pantothenate analogs, D-glucose, and various carboxylic acids did not inhibit Na+-dependent pantothenate transport, suggesting that this system is specific for pantothenate. Kinetic analysis of the Na+-dependent pantothenate uptake revealed a single transport system which obeyed Michaelis-Menten kinetics (Km = 16 microM and Vmax = 6.7 pmol X mg-1 X 10 s-1). Imposition of an inside-negative membrane potential caused net uphill pantothenate accumulation in the presence of Na+ but absence of a Na+ gradient, indicating that Na+/pantothenate cotransport is electrogenic. The relationship between extravesicular Na+ concentration and pantothenate transport measured under voltage-clamped conditions was sigmoidal: a Hill coefficient (napp) of 2 and a [Na+]0.5 of 55 mM were calculated. It is suggested that an anionic pantothenate1- molecule is cotransported with two Na+ to give a net charge of +1. The coupling of pantothenate transport to the Na+ electrochemical gradient may provide an efficient mechanism for reabsorption of pantothenate in the kidney.     

Sodium-chloride transport in the medullary thick ascending limb of henle's loop: Evidence for a sodium-chloride cotransport system in plasma membrane vesicles

Sodium transport mechanisms were investigated in plasma membrane vesicles prepared from the medullary thick ascending limb of Henle's loop (TALH) of rabbit kidney. The uptake of 22Na into the plasma membrane vesicles was investigated by a rapid filtration technique. Sodium uptake was greatest in the presence of chloride; it was reduced when chloride was replaced by nitrate, gluconate or sulfate. The stimulation of sodium uptake by chloride was seen in the presence of a chloride gradient directed into the vesicle and when the vesicles were equilibrated with NaCl, KCl plus valinomycin so that no chemical or electrical gradients existed across the vesicle (tracer exchange experiments). Furosemide decreased sodium uptake into the vesicles in a dose-dependent manner only in the presence of chloride, with a Ki of around 5 X 10(-6) M. Amiloride, at 2 mM, had no effect on the chloride-dependent sodium uptake. Similarly, potassium removal had no effect on the chloride-dependent sodium uptake and furosemide was an effective inhibitor of sodium uptake in a potassium-free medium. The results show the presence of a furosemide-sensitive sodium-chloride cotransport system in the plasma membranes of the medullary TALH. There is no evidence for a Na+/H+ exchange mechanism or a Na+ -K+ -Cl- cotransport system. The sodium-chloride cotransport system would effect the uphill transport of chloride against its electrochemical potential gradient at the luminal membrane of the cell.     

Localization of the phlorizin site on Na, K-ATPase in red cell membranes.

Phlorizin at 2 X 10(-4) M inhibited Na+ and Rb+-activated ATPase activities in human red cell membranes by 43%. It inhibited the 86Rb uptake activity of erythrocytes by only 15%. 86Rb uptake into resealed ghosts was inhibited strongly when phlorizin and ATP were preloaded in the ghosts before resealing. Na,K-ATPase activity in the resealed ghosts was also inhibited in the presence of phlorizin inside but not outside the ghosts. These findings suggested that the phlorizin site is located inside the cell.