Na+-stimulated ATPase activities in basolateral plasma membranes from guinea-pig small intestinal epithelial cells

Two ATPase activities, a Na+-ATPase and a (Na+ + K+)-ATPase, have been found associated with sheets of basolateral plasma membranes from guinea-pig small intestinal epithelial cells. The specific activity of the former is 10-15% of the latter. The two ATPase activities differ in their affinity for Na+, their optimal pH, their K+ requirement and particularly in their behaviour in the presence of some inhibitors and of Ca2+. Thus the Na+-ATPase is refractory to ouabain but it is strongly inhibited by ethacrynic acid and furosemide, whilst the (Na+ + K+)-ATPase is totally suppressed by ouabain, partially by ethacrynic acid and refractory to furosemide. In addition, the Na+-ATPase is activated by micromolar concentrations of calcium and by resuspension of the membrane preparation at pH 7.8. The Na+-ATPase is only stimulated by sodium and to a lesser extent by lithium; however, this stimulation is independent of the anion accompanying Na+. The latter rules out the participation of an anionic ATPase. The relation between the characteristics of the sodium transport mechanism in basolateral membrane vesicles (Del Castillo, J.R. and Robinson, J.W.L. (1983) Experientia 39,631) and those of the two ATPase activities present in the same membranes, allow us to postulate the existence of two separate sodium pumps in this membranes. Each pump would derive the necessary energy for active ion transport from the hydrolysis of ATP, catalyzed by different ATPase systems.     

Active sodium transport in basolateral plasma membrane vesicles from rat kidney proximal tubular cells

Inside-out vesicles prepared with basolateral plasma membranes from rat kidney proximal tubular cells can accumulate Na+ actively in two ways. Mode 1, which is K+-independent, is ouabain-insensitive and is inhibited by furosemide and mode 2, which is K+-dependent, is inhibited by ouabain and is insensitive to furosemide. The presence of Mg2+ and ATP in the incubation medium is essential for both modes of Na+ uptake to proceed and in both cases, the nucleotide is hydrolyzed during the process. These results are consistent with the idea of the existence, in these membranes, of two Na+ pumps: one, which can work in the absence of K+ (Na+ pump) and another, which needs K+ to work (Na+ + K+ pump).     

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.     

Effect of Ca2+ on the ouabain-insensitive, active Na+ uptake in inside-out basolateral plasma membrane vesicles from rat kidney proximal tubular cells

The ouabain-insensitive, active Na+ uptake of inside-out vesicles prepared with basolateral plasma membranes from rat kidney proximal tubular cells can be increased by the presence of micromolar concentrations of Ca2+ in the assay medium. The concomitant ATP hydrolysis associated with the Na+ uptake is also increased by the presence of Ca2+. The Na+ uptake and the concomitant ATP hydrolysis are inhibited by 2 mM furosemide. The effect of Ca2+ is not due to the activity of an Na+-Ca2+ exchanger. The present results are in accordance with our previous model (Proverbio, F., Proverbio, T. and Marín, R. (1982) Biochim. Biophys. Acta 688, 757-763) in which we proposed that Ca2+ seems to modulate the activity of the ouabain-insensitive Na+ pump, in two different ways: (1) in a strong association with the membranes in which Ca2+ (stable component) is essential for the pump activity and (2) in a weak association with the membranes in which Ca2+ (labile component) can be quickly and easily removed by reducing the free Ca2+ concentration of the assay medium to values lower than 1 microM. The Ka for Ca2+ (for the labile component) is around 5 microM. The Ca2+ modulation of the ouabain-insensitive Na+ pump is an indication that Ca2+ could regulate the magnitude of the Na+ extrusion accompanied by Cl- and water present in rat kidney proximal tubular cells.     

Sodium-dependent D-glucose transport in brush-border membrane vesicles from isolated rat small intestinal villus and crypt epithelial cells

Differentiation and maturation of enterocytes occur with migration from the crypt to villus compartments. To investigate the effect of epithelial cell differentiation on sodium-dependent D-glucose transport, brush-border membrane vesicles were prepared from small intestinal epithelial cell suspensions selectively isolated from villus and crypt populations. Enterocytes were isolated with a morphologically monitored sequential cell dissociation method. Thymidine kinase, sucrase, and alkaline phosphatase activities were measured as differentiation markers of specific cell populations. Brush-border membrane vesicles were purified and their kinetic characteristics defined with a rapid filtration method under conditions of a zero-trans, 100 mM cis-NaSCN gradient. Typical "overshoot" phenomena characteristic of sodium D-glucose cotransport were observed for both villus (five- to eight-fold equilibrium values) and crypt brush-border membrane vesicles (two- to four-fold equilibrium values). Kinetics analyses of the initial D-glucose flux in brush-border membrane vesicles suggested the presence of at least two sodium-dependent D-glucose carriers in the villus and only a single carrier in the crypt compartments. These data indicate that sodium D-glucose cotransport occurs in brush-border membranes of both villus and crypt populations. Moreover, quantitative and qualitative differences between these two membrane populations suggest that epithelial D-glucose transport processes are differentiation dependent and reflect the degree of enterocyte development.     

Ca2+ transport and permeability in inside-out red cell membrane vesicles after freezing

To evaluate the effects of freezing and thawing on Ca2+ transport and permeability, inside-out red cell membrane vesicles (IORCMV) are examined. Exposure to the cryoprotectant Me2SO as well as different cooling regimes on unprotected and cryoprotected vesicles do not affect the membrane Ca2+ transport. However, freezing and thawing increase the membrane permeability to sucrose.     

Mechanisms of Ca2+ transport in plasma membrane vesicles prepared from cultured pituitary cells. II. (Ca2+ + Mg2+)-ATPase-dependent Ca2+ transport activity

Purified plasma membrane vesicles from GH3 rat anterior pituitary cells exhibit a Mg2+-ATP-dependent Ca2+ transport activity. Concentrative uptake of Ca2+ is abolished by exclusion of either Mg2+ or ATP or by inclusion of the Ca2+ ionophore A23187. Furthermore, addition of A23187 to vesicles which have reached a steady state of ATP-supported Ca2+ accumulation rapidly and completely discharges accumulated cation. Ca2+ uptake is unaffected by treatment of vesicles with oligomycin, the uncoupler CCCP, or valinomycin and is greatly reduced in non-plasma membrane fractions. Likewise, Ca2+ accumulation is not stimulated by oxalate, consistent with the plasma membrane origin of this transport system. (Na+, K+)-ATPase participation in the Ca2+ transport process (i.e. via coupled Na+/Ca2+ exchange) was eliminated by omitting Na+ and including ouabain in the reaction medium. Ca2+ transport activity in GH3 vesicles has a similar pH dependence as that seen in a number of other plasma membrane systems and is inhibited by orthovanadate in the micromolar range. Inhibition is enhanced if the membranes are preincubated with vanadate for a short time. A kinetic analysis of transport indicates that the apparent Km for free Ca2+ and ATP are 0.7 and 125 microM, respectively. The average Vmax is 3.6 nmol of Ca2+/min/mg of protein at 37 degrees C. Addition of exogenous calmodulin or calmodulin antagonists had no significant effect on these kinetic properties. GH3 plasma membranes also contain a Na+/Ca2+ exchange system. The apparent Km for Ca2+ is almost 10-fold higher in this system than that for ATP-driven Ca2+ uptake. When both processes are compared under similar conditions, the Vmax of the exchanger is approximately 2-3 times that of ATP-dependent Ca2+ accumulation. Similar results are obtained when purified plasma membranes from bovine anterior pituitary glands were investigated. It is suggested that both Na+/Ca2+ exchange and the (Ca2+ + Mg2+)-ATPase are important in controlling intracellular levels of Ca2+ in anterior pituitary cells.     

Inside-out basolateral plasma membrane vesicles from rat kidney proximal tubular cells

A method for preparation of highly purified basolateral plasma membranes from rat kidney proximal tubular cells is reported. These membranes were assayed for the presence of vesicles as well as for their orientation. (Na+ + K+)-ATPase activity and [3H]ouabain binding studies with membranes treated with or without SDS revealed that the preparation consisted of almost 100% vesicles. The percentage of inside-out vesicles was found to be approx. 70%. This percentage was determined measuring the (Na+ + K+)-ATPase activity in K+-loaded vesicles and in membranes treated with or without trypsin and SDS. These membranes represent a very efficient tool to assay the correlation between active transport and ATPase activities in basolateral plasma membranes from rat kidney proximal tubular cells.     

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.     

Altered Mg2+ transport across liver plasma membrane from streptozotocin-treated rats

Type-I diabetes is associated with a decrease in magnesium content in various tissues, including liver. We have reported that hepatocytes from streptozotocin-injected rats have lost the ability to accumulate Mg2+ following hormonal stimulation. To assess whether the defect is inherent to the Mg2+ transport mechanism located in the hepatocyte cell membrane, plasma membrane vesicles were purified from diabetic livers. Diabetic plasma membranes do not retain intravesicular Mg2+ as tightly as vesicles purified from livers of age-matched non-diabetic rats. In addition, the amount of intravesicular Mg2+ these vesicles exchange for extravesicular Na+ or Ca2+ is 2-3-fold larger than in non-diabetic vesicles. The partition of Ca2+/Mg2+ and Na+/Mg2+ exchange mechanisms in the apical and basolateral domains of liver plasma membrane is maintained under diabetic conditions, although the Na+/Mg2+ exchanger in diabetic basolateral membranes has lost the ability to operate in reverse and favor an accumulation of extravesicular Mg2+ within the vesicles in exchange for entrapped Na+. These data indicate the occurrence of a major alteration in Mg2+ transport across the hepatocyte membrane, which can explain, at least in part, the decrease in liver magnesium content observed in diabetic animals and patients.     

Studies on the Ca2+ transport mechanism of human erythrocyte inside-out plasma membrane vesicles V. Chlortetracycline fluorescence

The measurement of chlortetracycline fluorescence was employed as a probe for measuring the process to calcium transport by human erythrocyte inside-out vesicles. Chlortetracycline is a divalent metal chelator which increases its fluorrescence when bound to calcium in the presence of a membrane. Addition of calcium and ATP to inside out vesicles in the presence of chlortetracycline increased the chlortetracycline fluorescence as a function of time following an initial delay. Only after a threshold level of calcium had been accumulated did the fluorescence increase. The presence of both ATP and calcium were required. The addition of calmodulin increased the rate and absolute magnitude of the chlortetracycline fluorescence change. Similarly, calmodulin stimulated the rate and extent of ⁴⁵Ca transport by inside-out vesicles. Moreover, the presence of saponin abolished both chlortetracycline fluorescence change and ⁴⁵Ca uptake; a non-hydrolyzable ATP analog would not substitute for ATP in either ⁴⁵Ca transport or chlortetracycline fluorescence experiments. Comparison between the slopes of the linear portions of chlortetracycline fluorescence change and calcium transport time courses at varied free calcium concentrations showed a consistent ratio between the slopes. This suggests that calcium transport change can be calibrated by employing chlortetracycline fluorescence. Based on this data, it is concluded that chlortetracycline fluorescence is a rapid and accurate method for monitoring calcium transport by human erythrocyte inside-out vesicles.     

Prostaglandin E2 transport in rabbit renal basolateral membrane vesicles

We examined the mechanism of prostaglandin E2 transport in rabbit renal basolateral membrane vesicles which were predominantly oriented right-side-out. In the presence of an inwardly directed H+ gradient, the initial rate of uptake was markedly accelerated and the influx of prostaglandin E2 resulted in a transient accumulation (overshoot) above the equilibrium value. Both H+-independent and H+-stimulated prostaglandin E2 uptake were shown to be insensitive to valinomycin-induced K+ diffusion potentials. Intravesicular probenecid inhibited the pH gradient-stimulated uptake of prostaglandin E2 but did not affect the pH-stimulated uptake of thiocyanate and acetate which enter membranes via ionic and nonionic diffusion, respectively. Finally, the existence of a Na+ cotransport or of a K+ antiport pathway for prostaglandin E2 could not be demonstrated. Thus, these data demonstrate the presence of an electrically neutral H+-prostaglandin E2 cotransport or OH- -prostaglandin E2 antiport mechanism in the basolateral membrane of the rabbit proximal tubule.     

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.     

Ca2+-stimulated, Mg2+-dependent ATPase activity in neutrophil plasma membrane vesicles. Coupling to Ca2+ transport

Low concentrations of free Ca2+ stimulated the hydrolysis of ATP by plasma membrane vesicles purified from guinea pig neutrophils and incubated in 100 mM HEPES/triethanolamine, pH 7.25. In the absence of exogenous magnesium, apparent values obtained were 320 nM (EC50 for free Ca2+), 17.7 nmol of Pi/mg X min (Vmax), and 26 microM (Km for total ATP). Studies using trans- 1,2-diaminocyclohexane- N,N,N',N',-tetraacetic acid as a chelator showed this activity was dependent on 13 microM magnesium, endogenous to the medium plus membranes. Without added Mg2+, Ca2+ stimulated the hydrolysis of several other nucleotides: ATP congruent to GTP congruent to CTP congruent to ITP greater than UTP, but Ca2+-stimulated ATPase was not coupled to uptake of Ca2+, even in the presence of 5 mM oxalate. When 1 mM MgCl2 was added, the vesicles demonstrated oxalate and ATP-dependent calcium uptake at approximately 8 nmol of Ca2+/mg X min (based on total membrane protein). Ca2+ uptake increased to a maximum of approximately 17-20 nmol of Ca2+/mg X min when KCl replaced HEPES/triethanolamine in the buffer. In the presence of both KCl and MgCl2, Ca2+ stimulated the hydrolysis of ATP selectively over other nucleotides. Apparent values obtained for the Ca2+-stimulated ATPase were 440 nM (EC50 for free Ca2+), 17.5 nmol Pi/mg X min (Vmax) and 100 microM (Km for total ATP). Similar values were found for Ca2+ uptake which was coupled efficiently to Ca2+-stimulated ATPase with a molar ratio of 2.1 +/- 0.1. Exogenous calmodulin had no effect on the Vmax or EC50 for free Ca2+ of the Ca2+-stimulated ATPase, either in the presence or absence of added Mg2+, with or without an ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N',-tetraacetic acid pretreatment of the vesicles. The data demonstrate that calcium stimulates ATP hydrolysis by neutrophil plasma membranes that is coupled optimally to transport of Ca2+ in the presence of concentrations of K+ and Mg2+ that appear to mimic intracellular levels.     

L-Carnitine transport in mouse renal and intestinal brush-border and basolateral membrane vesicles.

We characterized the uptake of carnitine in brush-border membrane (BBM) and basolateral membrane (BLM) vesicles, isolated from mouse kidney and intestine. In kidney, carnitine uptake was Na(+)-dependent, showed a definite overshoot and was saturable for both membranes, but for intestine, it was Na(+)-dependent only in BLM. The uptake was temperature-dependent in BLM of both kidney and intestine. The BBM transporter in kidney had a high affinity for carnitine: apparent K(m)=18.7 microM; V(max)=7.85 pmol/mg protein/s. In kidney BLM, similar characteristics were obtained: apparent K(m)=11.5 microM and V(max)=3.76 pmol/mg protein/s. The carnitine uptake by both membranes was not affected within the physiological pH 6.5-8.5. Tetraethylammonium, verapamil, valproate and pyrilamine significantly inhibited the carnitine uptake by BBM but not by BLM. By Western blot analysis, the OCTN2 (a Na(+)-dependent high-affinity carnitine transporter) was localized in the kidney BBM, and not in BLM. Strong OCTN2 expression was observed in kidney and skeletal muscle, with no expression in intestine in accordance with our functional study. We conclude that different polarized carnitine transporters exist in kidney BBM and BLM. L-Carnitine uptake by mouse renal BBM vesicles involves a carrier-mediated system that is Na(+)-dependent and is inhibited significantly by specific drugs. The BBM transporter is likely to be OCTN2 as indicated by a strong reactivity with the anti-OCTN2 polyclonal antibody.     

Differences in neutral amino acid and glucose transport between brush border and basolateral plasma membrane of intestinal epithelial cells.

A comparison of L-valine and D-glucose transport was carried out with vesicles of plasma membrane isolated either from the luminal (brush border) or from the contra-luminal (basolateral) region of small intestinal epithelial cells. The existence of transport systems for both non-electrolytes was demonstrated by stereospecificity and saturability of uptake, as well as tracer coupling. Transport of L-valine and D-glucose differs markedly in the two types of plasma membrane with respect to stimulation by Na+. The presence of Na+ stimulated initial L-valine and D-glucose uptake in brush border, but not in basolateral membrane. Moreover, an electro-chemical Na+ gradient, oriented with the lower potential on the inside, supported accumulation of the non-electrolytes above medium concentration only in the brush border membrane. L-Valine and D-glucose transport also were saturated at lower concentrations in brush border (10-20 mM) than in basolateral plasma membranes (30-50 mM). A third difference between the two membranes was found in the effectiveness of known inhibitors of D-glucose transport. In brush border membranes phlorizin was more potent than phloretin and 2', 3', 4'-trihydroxy-4-methoxy chalcone and cytochalasin B did not inhibit at all. In contrast, with the basolateral plasma membranes the order of potency was changed to phloretin = 2',3',4'-trihydroxy-4-methoxy chalcone greater than cytochalasin B greater than phlorizin. These results indicate the presence of different types of transport systems for monosaccharides and neutral amino acids in the luminal and contra-luminal region of the plasma membrane. Active transepithelial transport can be explained on the basis of the different properties of the non-electrolyte transport systems in the two cellular regions and an electro-chemical Na+ gradient that is dependent on cellular metabolism.     

Effects of electron donors on Ca2+-dependent K+ transport in one-step inside-out vesicles from the human erythrocyte membrane

The interactions between reducing agents and Ca2+ in the activation of Ca2+-dependent K+ transport have been studied in one-step inside-out vesicles. The artificial electron donor system ascorbate + phenazine methosulphate increases the apparent sensitivity to Ca2+ by about 5-times over control values (half activation constant, about 5 X 10(-8) M) while oxidized cytochrome c decreases the sensitivity to about 1/3 of the controls. Using redox buffers at a fixed pCa it is shown that the shift from the low to the high-affinity state can be accounted by the reduction of a membrane component accepting two electrons and with an apparent standard redox potential (pH 7.5) of 47 mV. The electrons can be transferred directly from reduced PMS or to oxidized cytochrome c, but not from ascorbate, NADH or reduced glutathione.     

Cytoskeleton components of inside-out and right-side-out plasma membrane vesicles from plants

Summary Isolated plasma membrane vesicles purified by aqueous polymer two-phase partitioning were used as a model system for studies on the membrane-associated (cortical) cytoskeleton in plants. Actin, as identified by immunoblotting, was found to be specifically attached to plasma membrane vesicles from cauliflower (Brassica oleracea L.). The actin was not washed off as the vesicles were turned inside-out, indicative of a fairly strong attachment. Triton X-100 extraction of plasma membrane vesicles resulted in an insoluble and hence pelletable fraction where actin could be found together with several other proteins. Our results show that the cortical cytoskeleton is to some extent co-purified with the plasma membrane, and we believe that well defined, inside-out and right-side-out plasma membrane vesicles can be used to study the structure and dynamics of the plant cortical cytoskeleton.Abbreviations ATP adenosine 5-triphosphate - BCIP 5-bromo-4-chloro-3-indolyl phosphate - BSA bovine serum albumin - CCD counter-current distribution - DTT dithiothreitol - EDTA ethylene-diamine-tetraacetic acid - EGTA ethylene glycol-bis(-aminoethyl ether) - GSII 1,3--glucan synthase - HEPES N-[2-hydroxyethyl]-piperazine-N-[2-ethane sulfonic acid] - MES 2-(N-morpholino)ethane sulfonic acid - NBT p-nitro blue tetrazolium chloride - IDP inosine 5-diphosphate - PAGE polyacrylamide gel electrophoresis - PBS phosphate buffered saline - PIPES piperazine-N,N-bis(2-ethane-sulfonic acid) - PPB potassium phosphate buffer - PM plasma membrane - PMSF phenylmethylsulfonyl fluoride - PVDF polyvinylidene difluoride - PVPP polyvinylpolypyrrolidone - SDS sodium dodecyl sulfate - TBS Tris-buffered saline - TTBS Tris-buffered saline with Tween 20 - Tris tris(hydroxymethyl) aminomethane