Proton-lactate co transport in basolateral membrane vesicles from rat jejunum

Proton-coupled lactate transport across the basolateral membrane of rat jejunal enterocyte was studied using well purified membrane vesicles. L-lactate uptake is stimulated by an inwardly directed H⁺ gradient; the effect of the pH difference is drastically reduced by FCCP and by pCMBS; unlabelled L-lactate causes a strong inhibition, whilst furosemide is uneffective. The H⁺ gradient-dependent stimulation of L-lactate uptake is significantly inhibited also by SCN^–: this finding could explain results recently reported in the literature in which H⁺-lactate symport was not evidenced in basolateral membranes from rat jejunum.     

Uridine transport in basolateral plasma membrane vesicles from rat liver

Summary The characteristics of uridine transport were studied in basolateral plasma membrane vesicles isolated from rat liver. Uridine was not metabolized under transport measurement conditions and was taken up into an osmotically active space with no significant binding of uridine to the membrane vesicles. Uridine uptake was sodium dependent, showing no significant stimulation by other monovalent cations. Kinetic analysis of the sodium-dependent component showed a single system with Michaelis-Menten kinetics. Parameter values were K _M 8.9 m and V _max 0.57 pmol/mg prot/sec. Uridine transport proved to be electrogenic, since, firstly, the Hill plot of the kinetic data suggested a 1 uridine: 1 Na⁺ stoichiometry, secondly, valinomycin enhanced basal uridine uptake rates and, thirdly, the permeant nature of the Na⁺ counterions determined uridine transport rates (SCN^– > NO ₃ ^– > Cl^– > SO ₄ ^2– ). Other purines and pyrimidines cis-inhibited and trans-stimulated uridine uptake.This work has been partially supported by grant PM90-0162 from D.G.I.C.Y.T. (Ministerio de Educación y Ciencia, Spain). B.R.-M. is a research fellow supported by the Nestlé Nutrition Research Grant Programme.     

Osmotic water permeabilities of brush border and basolateral membrane vesicles from rat renal cortex and small intestine

Summary The osmotic water permeabilityP _f of brush border (BBM) and basolateral (BLM) membrane vesicles from rat small intestine and renal cortex was studied by means of stopped-flow spectrophotometry. Scattered light intensity was used to follow vesicular volume changes upon osmotic perturbation with hypertonic mannitol solutions. A theoretical analysis of the relationship of scattered light intensity and vesicular volume justified a simple exponential approximation of the change in scattered light intensity. The rate constants extracted from fits to an exponential function were proportional to the final medium osmolarity as predicted by theory. For intestinal membranes, computer analysis of optical responses fitted well with a single-exponential treatment. For renal membranes a double-exponential treatment was needed, implying two distinct vesicle populations.P _f values for BBM and BLM preparations of small intestine were equal and amount to 60 m/sec. For renal preparations,P _f values amount to 600 m/sec for the fast component, BBM as well as BLM, and to 50 (BBM) and 99 (BLM) m/sec for the slow component. The apparent activation energy for water permeation in intestinal membranes was 13.3±0.6 and in renal membranes, 1.0±0.3 kCal/mole, between 25 and 35°C. The mercurial sulfhydryl reagentpCMBS inhibited completely and reversibly the highP _f value in renal brush border preparations. These observations suggest that in intestinal membranes water moves through the lipid matrix but that in renal plasma membranes water channels may be involved. From the highP _f values of renal membrane vesicles a transcellular water permeability for proximal tubules can be calculated which amounts to 1 cm/sec. This value allows for an entirely transcellular route for water flow during volume reabsorption.     

Amino acid-dependent sodium transport in plasma membrane vesicles from rat liver

Summary Thel-alanine-dependent transport of sodium ions across the plasma membrane of rat-liver parenchymal cells was studied using isolated plasma membrane vesicles. Sodium uptake is stimulated specifically by thel-isomer of alanine and other amino acids, whose transport is sodium-dependent in rat-liver plasma membrane vesicles. Thel-alanine-dependent sodium flux across the membrane is inhibited by an excess of Li⁺ ions, but not by K⁺ or choline ions. Sodium transport is sensitive to-SH reagents and ionophores, and is an electrogenic process: a membrane potential (negative inside) can enhancel-alanine-dependent sodium accumulation. The data presented provide further evidence for a sodium-alanine cotransport mechanism.     

Interaction between Na ions and the Cl/HCO3 exchanger in basolateral membranes from rat jejunum

The jejunal basolateral Cl/HCO₃ exchanger is modulated by two Na-dependent regulatory sites located on the inner and outer membrane surfaces. The aim of this work was to focus on the interaction between the anion exchanger and intracellular or extracellular sodium. Uptake studies, performed using basolateral membrane vesicles, provided kinetic parameters as a function of outside or inside Na concentration. The intracellular Na-sensitive modifier site seems to be primarily involved in the modulation of the Cl/HCO₃ exchanger.     

ATP-dependent strontium uptake by basolateral membrane vesicles from rat renal cortex in the absence or presence of calcium

ATP-dependent Sr²⁺ transport was examined in vitro using basolateral membrane (BLM) vesicles isolated from rat renal cortex to clarify the discrimination mechanisms between strontium (Sr) and calcium (Ca) in renal tubules during reabsorption. ATP-dependent Sr²⁺ uptake and Ca²⁺ uptake were observed in renal BLM vesicles and were inhibited by vanadate. Hill plots indicate similar kinetic behavior for Ca²⁺ and Sr²⁺ uptake. The apparentK _m andV _max of ATP-dependent Sr²⁺ uptake were both higher than those for Ca²⁺ uptake. ATP-dependent Sr²⁺ uptake by BLM vesicles diminished in the presence of 0.1 μM Ca²⁺ and was more markedly inhibited by 1 μM Ca²⁺. Hill plots of Sr²⁺ uptake data with and without 0.1 μM Ca²⁺ showed that the cooperative behavior of Sr²⁺ uptake was not changed by Ca²⁺. In the presence of 0.1 μM Ca²⁺, the affinity of the transport system for Sr²⁺ and the velocity of Sr²⁺ uptake in the BLM were both decreased. However, the rate of Ca²⁺ uptake was not diminished by Sr²⁺ concentrations of <1.6 μM. These results suggest that Ca²⁺ is preferentially transported in the renal cortex BLM when Ca²⁺ and Sr²⁺ are present at the same time.     

Kinetic characterization of Zinc transport process and its inhibition by Cadmium in isolated rat renal basolateral membrane vesicles: In vitro and In vivo studies

We firstly characterized zinc uptake phenomenon across basolateral membrane vesicles (BLMVs) isolated from normal rat kidney. The process was found to be time, temperature, and substrate concentration dependent, and displayed saturability. Zn²⁺ uptake was competitively inhibited in the presence of 2 mM Cd with Ki of 3.9 mM. Zinc uptake was also inhibited in the presence of sulfhydryl reacting compound suggesting involvement of {–}SH groups in the transport process. Further, to elucidate the effect of in vivo Cd on zinc transport in BLMVs, Cd nephrotoxicity was induced by subcutaneous administration of CdCl₂ at dose of 0.6 mg/kg/d for 5 days in a week for 12 weeks. An indolent renal failure developed in Cd exposed rats was accompanied with a significantly high urinary excretion of Cd²⁺, Zn²⁺ and proteins. The histopathology and electron microscopy of kidneys of Cd exposed rats documented changes of proximal tubular degeneration. Notably, Cd content in renal cortex of Cd exposed rats was 215 μg/g tissue that was higher than the critical concentration of Cd in kidneys which was associated with significantly higher Zn and metallothionein (MT) contents. Zinc uptake in BLMVs isolated from kidneys of Cd exposed rats was significantly reduced. Further, kinetic studies revealed that decrease in zinc uptake synchronized with decrease in maximal velocity (V_max) and increase in affinity constant which is suggestive of decreased number of active zinc transporters. Furthermore, conformational modulation of Zn transporter in BLM was further supported by observed variation in transition temperature for zinc transport in BLMVs isolated from Cd-exposed kidney.     

The Na+-independentd-glucose transporter in the enterocyte basolateral membrane: Orientation and cytochalasin B binding characteristics

Summary Phloridzin-insensitive, Na⁺-independentd-glucose uptake into isolated small intestinal epithelial cells was shown to be only partially inhibited by trypsin treatment (maximum 20%). In contrast, chymotrypsin almost completely abolished hexose transport. Basolateral membrane vesicles prepared from rat small intestine by a Percoll^® gradient procedure showed almost identical susceptibility to treatment by these proteolytic enzymes, indicating that the vesicles are predominantly oriented outside-out. These vesicles with a known orientation were employed to investigate the kinetics of transport in both directions across the membrane. Uptake data (i.e. movement into the cell) showed aK _t of 48mm and aV _max of 1.14 nmol glucose/mg membrane protein/sec. Efflux data (exit from the cell) showed a lowerK _t of 23mm and aV _max of 0.20 nmol glucose/mg protein/sec.d-glucose uptake into these vesicles was found to be sodium independent and could be inhibited by cytochalasin B. TheK _t for cytochalasin B as an inhibitor of glucose transport was 0.11 m and theK _D for binding to the carrier was 0.08 m.d-glucose-sensitive binding of cytochalasin B to the membrane preparation was maximized withl- andd-glucose concentrations of 1.25m. Scatchard plots of the binding data indicated that these membranes have a binding site density of 8.3 pmol/mg membrane protein. These results indicate that the Na⁺-independent glucose transporter in the intestinal basolateral membrane is functionally and chemically asymmetric. There is an outward-facing chymotrypsin-sensitive site, and theK _t for efflux from the cell is smaller than that for entry. These characteristics would tend to favor movement of glucose from the cell towards the bloodstream.     

l- andd-alanine transport in brush border membrane vesicles from lepidopteran midgut: Evidence for two transport systems

Summary In brush border membrane vesicles from the midgut ofPhilosamia cynthia larvae (Lepidoptera) thel- andd-alanine uptake is dependent on a potassium gradient and on transmembrane electrical potential difference. Each isomer inhibits the uptake of the other form: inhibition ofl-alanine uptake byd-alanine is competitive, whereas inhibition ofd-alanine uptake byl-alanine is noncompetitive. Transstimulation experiments as well as the different pattern of specificity to cations suggest the existence of two transport systems. Kinetic parameters for the two transporters have been calculated both when K_out>K_in and K_out=K_in.d-alanine is actively transported also by the whole midgut, but it is not metabolized by the intestinal tissue.     

ATP-dependent calcium transport in rat parotid basolateral membrane vesicles is modulated by membrane potential

Summary The ATP-dependent Ca²⁺ transport activity (T. Takuma, B.L. Kuyatt and B.J. Baum,Biochem. J. 227:239–245, 1985) exhibited by inverted basolateral membrane vesicles isolated from rat parotid gland was further characterized. The activity was dependent on Mg²⁺. Phosphate (5mm), but not oxalate (5mm), increased maximum Ca²⁺ accumulation by 50%. Half-maximal Ca²⁺ transport was achieved at 70nm Ca²⁺ in EGTA-buffered medium while maximal activity required >1 m Ca²⁺ (V _max=54 nmol/mg protein/min). Optimal rates of Ca²⁺ transport were obtained in the presence of KCl, while in a KCl-free medium (mannitol or sucrose) 40% of the total activity was achieved, which could not be stimulated by FCCP. The initial rate of Ca²⁺ transport could be significantly altered by preimposed membrane potentials generated by K⁺ gradients in the presence of valinomycin. Compared to the transport rate in the absence of membrane potential, a negative (interior) potential stimulated uptake by 30%, while a positive (interior) potential inhibited uptake. Initial rates of Ca²⁺ uptake could also be altered by imposing pH gradients, in the absence of KCl. When compared to the initial rate of Ca²⁺ transport in the absence of a pH gradient, pH _i =7.5/pH _o =7.5; the activity was 60% higher in the presence of an outwardly directed pH gradient, pH _i =7.5/pH _o =8.5; while it was 80% lower when an inwardly directed pH gradient was imposed, pH _i =7.5/pH _o =6.2. The data show that the ATP-dependent Ca²⁺ transport in BLMV can be modulated by the membrane potential, suggesting therefore that there is a transfer of charge into the vesicle during Ca²⁺ uptake, which could be compensated by other ion movements.     

Ionic requirements for taurocholate transport in rat liver plasma membrane vesicles

As part of the enterohepatic circulation, taurocholate is taken up by hepatocytes by a Na⁺-gradient-dependent, carrier-mediated process. The dependence of taurocholate uptake on the presence of a Na⁺ gradient, outside greater than inside, has been studied in isolated rat liver plasma membranes. The uptake is specific for sodium, and a cotransport stoichiometry of 2 Na⁺ per taurocholate taken up was found. The presence of K⁺ ions inside the vesicles was also found to be essential for maximum Na⁺-stimulated uptake of taurocholate, although a K⁺ gradient is not required. Mg²⁺ was almost as effective as K⁺ in this regard. The symport of Na⁺ and taurocholate during uptake was shown to be electrogenic, so that K⁺ may act as an exchange counterion preventing the accumulation of positive charge within the vesicles.Dedicated to the memory of Prof. David E. Green, friend, mentor, and colleague.     

The kinetic mechanism of the glutamate-aspartate carrier in rat intestinal brush-border membrane vesicles: the role of potassium

The sodium dependent transport system for L-glutamate and L-aspartate localized in the apical part of rat enterocytes has previously been kinetically characterized (Prezioso, G., and Scalera, V. (1996). Biochim. Biophys. Acta 1279, 144–148). In this paper the mechanism by which the potassium cation specifically activates the L-glutamate–sodium cotransport process is investigated. Potassium has been found to act as an activator when it is present inside the membrane vesicles, while its presence outside is ineffective, and the effect is saturable. The kinetic parameters with respect to sodium and glutamate have been compared in the presence and in the absence of the activator. The results indicate that the ordered sodium–sodium glutamate mechanism is not altered by potassium, and that the activation is probably exerted on both the rate determining steps of the transport process. It is proposed that (1) a specific binding site for potassium is present on the inside hydrophilic part of the membrane carrier, (2) the binding of the effector accelerates the intramembrane rearrangement steps of both the disodium glutamate–carrier complex and the free carrier, (3) the affinity of the carrier is lowered with respect to sodium whereas it is increased for glutamate, and (4) K⁺ antiport is not performed by this carrier.     

Studies of the kinetics of Na+ gradient-coupled glucose transport as found in brush-border membrane vesicles from rabbit jejunum

The Na⁺-dependent d-glucose transport reaction in rabbit jejunal brush-border vesicles was studied. Initial rate data were obtained by fitting a polynomial equation to progress curves at different d-glucose concentrations and extracting the slope of the tangent at zero-time. Kinetic replots of the initial rate values produced biphasic Hofstee patterns indicative of two pathways for transport distinguished by their $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ values for glucose. Neither was dependent on the presence of a membrane potential. Both were dependent on Na⁺ and both were inhibited by phlorizin. Increasing external sodium was found to elevate the apparent $\text{V}{}_{\mathrm{<mtext>max</mtext>}}$ for both pathways. Internal sodium was inhibitory. Pulsed progress curve analysis indicated that the effect of internal sodium was best characterized as carrier sequestration by a sodium-carrier binary complex. Inhibition by internal sodium was completely reversed by the presence, internally, of d-glucose. The presence of two pathways and the kinetic constants for these pathways do not agree with the conclusions of Hopfer and Groseclose (1980) J. Biol. Chem. 255, 4453–4462). Experiments are presented which bear on the reason for the disagreement.     

Uptake of selenite,selenomethionine and selenate by brush border membrane vesicles isolated from rat small intestine

The uptake of selenite, selenate and selenomethionine (SeMet) was performed with brush border membrane vesicles (BBMV) prepared from rats fed selenium-deficient and supplemented diets. At equilibrium (60 min), the uptake of ⁷⁵Se from [⁷⁵Se]selenite ranged from 16.5 to 18.9 nmol mg^-1 protein. There was a curvilinear relationship in the uptake of selenite over a concentration range of 10–1000 m. About 2 nmol mg^-1 protein was obtained with selenomethionine (SeMet) which occurred between 90 and 180 s. In contrast to selenite, there was a linear relationship in the initial uptake of SeMet over a concentration range of 10–1000 m. The uptake of selenate was approximately 50-fold lower than selenite, reaching 350 pmol mg^-1 protein. Dietary selenium level had no effect on the rate of ⁷⁵Se accumulation by BBMV. Dramatic differences are found in the uptake and binding of selenium by BBMV incubated with different selenocompounds.     

Autocatalytic cooperativity and self-regulation of ATPase pumps in membrane active transport

We investigate the effect of autocatalysis on the conformational changes of membrane pumps during active transport driven by ATP. The translocation process is described by means of an alternating access model. The usual kinetic scheme is extended by introducing autocatalytic steps and allowing for dynamic formation of enzyme complexes. The usual features of cooperative models are recovered, i.e., sigmoid shapes of flux versus concentration curves. We show also that two autocatalytic steps lead to a mechanism of inhibition by the substrate as experimentally observed for some ATPase pumps. In addition, when the formation of enzyme complexes is allowed, the model exhibits a multiple stationary states regime, which can be related to a self-regulation mechanism of the active transport in biological systems. Correspondence to: G. Weissmüller     

Effects of membrane potential on Na cotransports in eel intestinal brush-border membrane vesicles: Studies with a fluorescent dye

Summary The Na-dependent transport of a number of organic molecules (d-glucose,l-proline,l-alanine,l-phenylalanine) in brush-border membrane vesicles isolated from the intestine of the eel (Anguilla anguilla) was monitored by recording the fluorescence quenching of the voltage-sensitive cyanine dye 3,3-diethylthiacarbocyanine iodide (DiS-C₂(5)). The experimental approach consisted of: a) generating an inside-negative membrane potential mimicking in vivo conditions: b) measuring the rate of membrane potential decay (i.e., the rate of fluorescence quenching decay) due to Na-neutral substrate cotransport. Rates of membrane potential decay showed saturation on substrate concentration andK _app values (the substrate concentration giving 50% of the maximal rate) were estimated for Na-dependent transport ofd-glucose (0,099mm),l-alanine (0.516mm),l-proline (0.118mm) andl-phenylalanine (2.04mm). The influence of an inside-negative membrane potential on the affinity of the transporter for glucose and for sodium is discussed.     

A simple method for the isolation of basolateral plasma membrane vesicles from rat kidney cortex Enzyme activities and some properties of glucose transport

A procedure for preparing basolateral membrane vesicles from rat renal cortex was developed by differential centrifugation and Percoll density gradient centrifugation, and the uptake of d-[³H]glucose into these vesicles was studied by a rapid filtration technique. (Na⁺ + K⁺)-ATPase, the marker enzyme for basolateral membranes, was enriched 22-fold compared with that found in the homogenate. The rate of d-glucose uptake was almost unaffected by Na⁺ gradient (no overshoot).     

Amino acid uptake in plasma membrane vesicles isolated from proliferating tumor cells and tissues

Summary The transport of L-alanine, a natural substrate of system A, across plasma membrane vesicle preparations has been studied in the early stages of rat DENA-PH hepato-carcinogenesis and in a very undifferentiated rat ascites hepatoma cell line (Yoshida AH-130) in the exponential and stationary phase of growth.Kinetic analyses indicated an increase of the V_max value in DENA-PH-treated rats 30 h after partial hepatectomy as well as in exponential growing Yoshida ascites cells. In DENA-PH-treated rats the Km value was drastically reduced 7 and 60 days after surgery, when enzyme-altered hyperplastic and preneoplastic lesions were present in rat liver. Drastically reduced Km values were also found in Yoshida ascites cells.The results suggest that an altered alanine transporter might take place in liver plasma membranes from carcinogen-treated rats. This appears to occur also in an established tumor cell line, grown in vivo.Abbreviations AAF 2-acetylaminofluorene - DENA diethylnitrosamine - PH partial hepatectomy - PMSF phenylmethanesulfonyl fluoride     

Na+ -dependent proline transport in isolated membrane vesicles from the L6 muscle cell line. Stimulation of uptake by intravesicular proline

Membrane vesicles of L6 myoblasts were prepared in order to study the amino acid transport system A. The role of the membrane in the adaptive response of transport to amino acid-supplementation was assessed. The membranes, prepared by N2 cavitation, displayed Na+ (but not K+)-dependent L-proline uptake. An overshoot of L-[3H]proline uptake was observed after exposure of the vesicles to an inward Na+ gradient. Isolated membrane vesicles loaded with 50 microM proline displayed countertransport (stimulation of proline uptake). It is concluded that the adaptive decrease of proline uptake observed in amino acid-supplemented cells cannot be accounted for by trans-inhibition of transport.     

Ca2+ transport activities of inside-out vesicles prepared from density-separated erythrocytes from rat and human

The plasma membrane Ca²⁺ ATPase in erythrocytes is vital for the maintenance of intracellular Ca²⁺ levels. Since the cytoplasmic Ca²⁺ concentration is elevated in older erythrocytes, the properties of the Ca²⁺ transport ATPase were examined during cell aging using inside-out vesicles (IOVs) prepared from density-separated, young (less dense, Ey) and old (more dense, Eo) rat and human erythrocytes. The transport of Ca²⁺ and the coupled hydrolysis of ATP were measured using radiolabeled substrates. The calmodulin-independent Ca²⁺ transport activity (Ey, 38.8 vs. Eo, 23.3 nmols/min/mg IOV protein) and the Ca²⁺ dependent ATP phosphohydrolase activity (Ey, 53.5 vs. Eo, 48.8 nmols/min/mg protein) were greater in IOVs prepared from younger (less dense) rat erythrocytes. The calmodulin-independent Ca²⁺ transport activity in IOVs from human erythrocytes was 12.9 nmols/min/mg IOV protein for Ey and 10.7 nmols/min/mg IOV protein for Eo. Inside-out vesicles from older (more dense) cells exhibited a lower pumping efficiency as determined by the calculated stoichiometry, molecule of Ca²⁺ transported per molecule of ATP hydrolyzed (rat: Ey, 0.74 vs. Eo, 0.49; human: Ey, 1.22 vs. Eo, 0.77). The enzymatic activity of rat and human Ey IOVs was labile. The Ca²⁺ transport activity in Ey but not Eo IOVs rapidly declined during cold storage (4°C). The decrease in Ca²⁺ transport activity during aging may accentuate the age-related decline in several erythrocytic properties.Abbreviations IOV Inside-Out Vesicles - Ey Erythrocytes enriched with young (less dense) cells - Eo Erythrocytes enriched with old (more dense) cells - ACEase Acetylcholinesterase