Uptake of the cephalosporin, cephalexin, by a dipeptide transport carrier in the human intestinal cell line, Caco-2

The transport of the orally absorbed cephalosporin, cephalexin, was examined in the human epithelial cell line, Caco-2 that possesses intestinal enterocyte-like properties when cultured. In sodium-free buffer, the cells accumulated 1 mM D-[9-14C]cephalexin against a concentration gradient and obtained a distribution ratio of 3.5 within 180 min. Drug uptake was maximal when the extracellular pH was 6.0. Uptake was reduced by metabolic inhibitors and by protonophores indicating that uptake was energy- and proton-dependent. Kinetic analysis of the concentration dependence of the rate of cephalexin uptake showed that a non-saturable component (Kd of 0.18 +/- 0.01 nmol/min per mg protein per mM) and a transport system with a Km of 7.5 +/- 2.8 mM and a Vmax of 6.5 +/- 0.9 nmol/min per mg protein were responsible for drug uptake. Uptake was competitively inhibited by dipeptides. The transport carrier exhibited stereospecificity for the L-isomer of cephalexin. Drug uptake was not affected by the presence of amino acids, organic anions, 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid or 4,4'-diisothiocyano-2,2'-disulfonic stilbene. Therefore, Caco-2 cells take up cephalexin by a proton-dependent dipeptide transport carrier that closely resembles the transporter present in the intestine. Caco-2 cells represent a cellular model for future studies of the dipeptide transporter.     

Kidney epithelial cells of monkey origin (BSC-1) express a sodium bicarbonate cotransport. Characterization by 22Na+ flux measurements

Na movement across the plasma membranes of confluent monolayers of monkey kidney epithelial cells (BSC-1) was studied using 22Na+ uptake and efflux techniques in the presence of 10(-4) M ouabain. In the presence of 28 mM bicarbonate, uptake was inhibited by both 10(-3) M amiloride and 10(-3) M 4,4'diisothiocyanostilbene-2,2'-disulfonic acid (DIDS). In DIDS-pretreated cells, 10(-3) M amiloride led to a further reduction of 22Na+ uptake, while 10(-5) furosemide was ineffective. DIDS also inhibited sodium efflux, indicating that the DIDS-sensitive pathway mediates both influx and efflux of 22Na+. DIDS-sensitive 22Na+ uptake, as studied in the presence of both 10(-4) M ouabain and 10(-3) M amiloride, was abolished by the absence of bicarbonate, which could not be substituted by other plasma membrane-permeable buffers. In 28 mM HCO3-, DIDS-sensitive uptake of 28 mM Na+ was cis-inhibited by 124 mM Na+, but no significant inhibition by K+ or Li+ was found. DIDS-sensitive 22Na+ uptake was a saturable function of both Na+ concentration (apparent Km between 20 and 40 mM at 28 mM HCO3-) and HCO3- concentration (apparent Km between 7 and 14 mM at 151 mM Na+). Intracellular microelectrode measurements showed that net Na+ transport in the presence of HCO3- is electrogenic, i.e. that there is anion cotransport with Na+. This effect is abolished by 1 mM DIDS. It is concluded that monkey kidney epithelial cells possess a stilbene-sensitive, electrogenic sodium bicarbonate symport, which may play an important role in bicarbonate reabsorption in the mammalian kidney.     

Selective inhibition of sodium-linked and sodium-independent bicarbonate/chloride antiport in Vero cells

The effects of compounds previously described to inhibit anion transport were tested for their ability to inhibit anion antiport in Vero cells as measured by uptake of 36Cl- by chloride self-exchange and as bicarbonate-linked uptake of 22Na+. While 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid inhibited both processes, ethacrynic acid and probenecid selectively inhibited the uptake of 36Cl-. Low concentrations of pyridoxal phosphate and picrylsulfonic acid selectively inhibited the bicarbonate linked uptake of 22Na+, while higher concentrations of these compounds also inhibited the uptake of 36Cl-. Measurements of the internal pH indicated that ethacrynic acid inhibits Na+-independent HCO-3/Cl- exchange, while it has no measurable effect on Na+-linked bicarbonate-dependent regulation of the internal pH. Conversely, picrylsulfonic acid selectively inhibits the latter process. The results indicate that anion antiport in Vero cells occurs by two independent processes.     

Properties of the Na+-dependent Cl-/HCO3- exchange system in U937 human leukemic cells

U937 cell possess two mechanisms that allow them to recover from an intracellular acidification. The first mechanism is the amiloride-sensitive Na+/H+ exchange system. The second system involves bicarbonate ions. Its properties have been defined from intracellular pH (pHi) recovery experiments, 22Na+ uptake experiments, 36Cl- influx and efflux experiments. Bicarbonate induced pHi recovery of the cells after a cellular acidification to pHi = 6.3 provided that Na+ ions were present in the assay medium. Li+ or K+ could not substitute for Na+. The system seemed to be electroneutral. 22Na+ uptake experiments showed the presence of a bicarbonate-stimulated uptake pathway for Na+ which was inhibited by 4,4'-diisothiocyanostilbene-2,2'-disulfonate. The bicarbonate-dependent 22Na+ uptake component was reduced by depleting cells of their internal Cl- and increased by removal of external Cl-. 36Cl- efflux experiments showed that the presence of both external Na+ and bicarbonate stimulated the efflux of 36Cl- at a cell pHi of 6.3. Finally a 36Cl- uptake pathway was documented. It was inhibited by 4,4'-diisothiocyanostilbene-2,2'-disulfonate (K0.5 = 10 microM) and bicarbonate (K0.5 = 2 mM). These results are consistent with the presence in U937 cells of a coupled exchange of Na+ and bicarbonate against chloride. It operates to raise the intracellular pH. Its pHi and external Na+ dependences were defined. No evidence for a Na+-independent Cl-/HCO3- exchange system could be found. The Na+-dependent Cl-/HCO3- exchange system was relatively insensitive to (aryloxy)alkanoic acids which are potent inhibitors of bicarbonate-induced swelling of astroglia and of the Li(Na)CO3-/Cl- exchange system of human erythrocytes. It is concluded that different anionic exchangers exist in different cell types that can be distinguished both by their biochemical properties and by their pharmacological properties.     

Osmotic Swelling Stimulates Ascorbate Efflux from Cerebral Astrocytes

Abstract: Ascorbate (reduced vitamin C) is an important enzyme cofactor, neuromodulator, and antioxidant that is stored at millimolar concentrations in the cytosol of cerebral astrocytes. Because these cells swell during hyponatremia, cerebral ischemia, and trauma, we investigated the effects of osmotic stress on astrocytic transport of ascorbate. Ascorbate efflux from primary cultures of rat astrocytes was rapidly (within 1 min) increased by incubation in hypotonic medium. Efflux also increased when astrocytes, which had been adapted to a hypertonic environment, were swollen by transfer to isotonic medium. Swelling-induced ascorbate efflux was inhibited by the anion-transport inhibitors 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) and 4,4'-dinitrostilbene-2,2'-disulfonic acid (DNDS). The pathway that mediates ascorbate efflux was found to be selective because a larger anion, 2',7'-bis(carboxyethyl)-5-(or -6)-carboxyfluorescein (BCECF), was retained in the swollen astrocytes. Na⁺-dependent ascorbate uptake into astrocytes was inhibited slightly during the first minute of hypotonic stress, indicating that the sodium ascorbate cotransporter does not mediate swelling-induced efflux. Cell concentration of authentic ascorbate was measured by HPLC with electrochemical detection. When astrocytes were incubated in ascorbate-free medium, hypotonicity decreased cell ascorbate concentration by 50% within 3 min. When astrocytes were incubated in ascorbate-supplemented hypotonic medium, intracellular ascorbate concentration was restored within 10 min because uptake remained effective. Many pathological conditions cause brain cell swelling and formation of reactive oxygen species. Ascorbate release during astrocytic swelling may contribute to cellular osmoregulation in the short-term and the scavenging of reactive oxygen species.     

Expression of rat liver canalicular sulfate carrier in Xenopus laevis oocytes

Poly(A)+ RNA (mRNA)extracted from rat liver was injected into Xenopus laevis oocytes and the expression of sulfate transport was determined by measuring [35S] sulfate uptake. Compared to water-injected oocytes, which exhibited virtually no sulfate uptake, injection of rat liver mRNA resulted in a time- and dose-dependent increase in uptake of sulfate. Depending on the method used for the isolation of the mRNA, sulfate uptake was stimulated after injection (40 ng after 6 days) between 8- and 72-fold compared to water-injected oocytes. Sulfate uptake of oocytes injected with mRNA was found to be sensitive to 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (IC50 less than 20 microM) and could also be inhibited by thiosulfate. Sulfate uptake of injected oocytes showed Michaelis-Menten kinetics (apparent Km, 0.31 mM) which is similar to the Km of the sulfate/bicarbonate antiporter of rat liver canalicular plasma membranes. After fractionation by a sucrose density gradient, the mRNA encoding for the expressed rat liver sulfate carrier was found in fractions containing messages of 3.5-4.0 kilobases in length.     

Mechanism for electrosilent Ca2+ transport to cause calcification of spicules in sea urchin embryos

Embryos of the sea urchin, Hemicentrotus pulcherrimus, kept in sea water containing the calcium antagonists, diltiazem and verapamil, or an anion transport inhibitor, 4,4'-diisothiocyano-2,2'-disulfonic acid stilbene (DIDS), during a developmental period between the mesenchyme blastula and the pluteus corresponding stage, became abnormal plutei with poorly developed arms and quite small spicules. Treatment with ethacrynic acid and furosemide, inhibitors of chloride transport, during the same period of development yielded quasi-normal plutei with poor spicules and somewhat developed arms. In late gastrulae, the inhibitory effects of these calcium antagonists and DIDS on the uptake of 45Ca2+ in whole embryos were as strong as those on 45Ca deposition in spicules, whereas the effects of chloride transport inhibitors on calcium deposition in the spicules were markedly stronger than on its uptake in whole embryos. Electrosilent uptake of Ca2+ seems to be established mainly by coupled influx of chloride in the cells which mediate spicule calcification, and by concomitant influx of anions in the other cells. In swimming blastulae, 45Ca2+ uptake was inhibited by calcium antagonists and DIDS, but not by chloride transport inhibitors. Ca2+ uptake probably becomes coupled with chloride influx only in embryos in which spicule calcification occurs.     

Role of ABH blood group antigens in the stimulation of a DIDS-sensitive Ca2+ influx pathway in human erythrocytes by Ulex europaeus agglutinin I and a monoclonal anti A1 antibody

Of eleven agglutinating lectins tested, only one, Ulex europaeus agglutinin I (UEA1), stimulated Ca2+ uptake in quin2-loaded erythrocytes by about 2-fold. UEA1 is known to be an alpha-L-fucose and ABH blood group specific lectin. The 45Ca2+ influx induced by UEA1 was absent in the presence of extracellular fucose (5 and 15 mM) and depended on the ABH blood group of the donor, the stimulatory potency of the lectin decreasing in the order H greater than A2 greater than A1. Ca2+ entry blockers, such as cobalt and verapamil, did not affect the 45Ca2+ influx induced by UEA1. 4,4'-Diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) inhibited dose-dependently with a Ki of 1-2 microM. 10 microM DIDS, 10 microM 4,4'-dinitrostilbene-2,2'-disulfonic acid (DNDS) and 20 microM dipyridamole fully blocked the 45Ca2+ influx induced by UEA1. The effect of UEA1 on 45Ca2+ influx was absent in K+ and Mg2+ media and was less pronounced in choline than in Na+ media. The 45Ca2+ influx induced by the lectin was abolished by preincubation with 12-O-tetradecanoylphorbol 13-acetate (TPA, 60 ng/ml). A monoclonal antibody raised against A1 erythrocytes (Bric 54) accelerated 45Ca2+ influx in quin2 loaded A1 erythrocytes by about 2-fold. No effect was seen in A2 and H erythrocytes. The 45Ca2+ influx elicited by Bric 54 exhibited a sensitivity towards inhibition by DIDS and TPA, as well as a dependence on the cation composition of the incubation medium similar to that observed with UEA1. The effects of UEA1 and Bric 54 were not additive. These observations suggest that the Ca2+ influx induced by UEA1 and Bric 54 is mediated by the same transport pathway. Since both the lectin and the antibody exhibit ABH blood group specificity, it appears reasonable to conclude that ABH antigens can serve as recognition sites for activation of a Ca2+ influx pathway in human erythrocytes, which is sensitive to inhibitors of the band 3 anion-exchanger.     

Conformational change of band 3 protein induced by diethyl pyrocarbonate modification in human erythrocyte ghosts

Diethyl pyrocarbonate inhibited the phosphate exchange across the human erythrocyte membrane. The exchange rate was inhibited only when the membranes were modified with the reagent from the cytosolic surface of resealed ghosts. The intracellular modification by diethyl pyrocarbonate inhibited the extracellular binding of [3H]dihydro-4,4'-diisothiocyanostilbene-2,2'-disulfonic acid to band 3 protein. Furthermore, the extracellular 4,4'-dinitrostilbene-2,2'-disulfonic acid protected the membranes from the intracellular modification by diethyl pyrocarbonate. These results suggest that the extracellular binding of 4,4'-dinitrostilbene-2,2'-disulfonic acid to band 3 protein induces the conformational change of the intracellular counterpart of band 3 protein and the diethyl pyrocarbonate susceptible residue(s) is (are) hidden from the cytosolic surface of the cell membrane in connection with the conformational change. Conversely, under the conditions where the diethyl pyrocarbonate modification is confined to the intracellular side of the membrane, the extracellular binding site of [3H]dihydro-4,4'-diisothiocyanostilbene-2,2'-disulfonic acid is hidden from the cell surface.     

Transport of Lead-203 at the Blood-Brain Barrier During Short Cerebrovascular Perfusion with Saline in the Rat

Lead transport at the blood-brain barrier has been studied by short (less than 1.5 min) vascular perfusion of one cerebral hemisphere of the rat with a buffered physiological salt solution at pH 7.4 without calcium, magnesium, or bicarbonate and containing 203 Pb-labelled lead chloride. In the absence of complexing agents, 203Pb uptake was rapid, giving a space of 9.7 ml/100 g of wet frontal cortex at 1 min. Lead-203 influx was linear with lead concentration up to 4 microM. Five percent albumin, 200 microM cysteine, or 1 mM EDTA almost abolished 203Pb uptake. Lead-203 entry into brain was uninfluenced by varying the calcium concentration or by magnesium or the calcium blocker methoxyverapamil. Similarly, 1 mM bicarbonate or 50 microM 4,4'-diisothiocyanostilbene-2,2'-disulphonic acid was without effect. Increasing the potassium concentration reduced 203Pb uptake. Vanadate at 2 mM, 2 microM carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (a metabolic uncoupler), or 2 microM stannic chloride all markedly enhanced lead entry into brain, as did a more alkaline pH (7.80). In conclusion, there is a mechanism allowing rapid passive transport of 203Pb at the brain endothelium, perhaps as PbOH+. Lead uptake into brain via this system is probably made less important by active transport of lead back into the capillary lumen by the calcium-ATP-dependent pump.     

Fluorescein-methotrexate transport in brush border membrane vesicles from rat small intestine

The transport characteristics of fluorescein-methotrexate (F-MTX) in isolated brush border membrane vesicles (BBMVs) from rat small intestine were studied. F-MTX uptake in BBMVs was measured by a rapid filtration technique. Our results demonstrated that F-MTX uptake into vesicles was 1) significantly increased under the experimental conditions of an outwardly directed OH(-) gradient or an inwardly directed H(+)gradient, 2) sensitive to temperature, 3) increased with decreasing pH of the incubation buffer, 4) significantly inhibited by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) at the early stage of the uptake, and 5) significantly inhibited by methotrexate (MTX). Thus, the transport of F-MTX in BBMVs was shown to be mediated in part by the reduced folate transporter (RFC) which was known to transport MTX through the epithelium of small intestine.     

Uptake of l-Glutamate into Rat Brain Synaptic Vesicles: Effect of Inhibitors that Bind Specifically to the Glutamate Transporter

Abstract: In this study we have described a series of new and potent inhibitors of the vesicular uptake of glutamate. The two most efficient inhibitors were the dyes Evans blue and Chicago Skye Blue 6B, which are structurally related to glutamate and were competitive inhibitors in the nanomolar range. The anion channel blocker 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (SITS) and the diuretics furosemide and bumetanide are inhibitors of chloride transport in other organs but were competitive inhibitors of glutamate and noncompetitive with respect to chloride ions. Evans blue, Chicago Skye Blue 6B, SITS, furosemide, and bumetanide are all large organic acids with two centers of negative charge and an electron-donating group at close vicinity of the negative charge at physiological pH. The inhibition of the glutamate uptake with these inhibitors was noncompetitive with respect to Cl⁻. The inhibitors, therefore, probably interact directly with the glutamate carrier. Bafilomycin A₁, which is a specific vacuolar ATPase inhibitor, was used as a control and inhibited the vesicular dopamine, glutamate, and GABA uptake to the same extent. None of the inhibitors had any effect on the plasma membrane carrier, which is therefore clearly different from the vesicular carrier.     

Characterization of a proton-driven carrier for sialic acid in the lysosomal membrane. Evidence for a group-specific transport system for acidic monosaccharides

Highly purified lysosomal membrane vesicles, obtained from rat liver lysosomes, were used to study characteristics of NeuAc transport across the lysosomal membrane. Uptake of [14C]NeuAc was found to be strongly influenced by a pH gradient across the membrane. When a proton gradient (pHin greater than pHout) was generated by impermeable buffers, NeuAc uptake above equilibrium level (overshoot) was observed. The influence of membrane diffusion potentials was ruled out by experiments where K+ and valinomycin were present. The overshoot appeared to be specifically produced by protons, since gradients of other cations (Na+ and K+) did not give stimulation. Proton-driven uptake was saturable (Kt = 0.24 mM) and mediated by a single system, as shown by linearity of the Scatchard plot. Stimulation of transport was also obtained by preincubation of vesicles with MgATP and the effect was blocked by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, but not by the protonophore carbonyl cyanide p-trifluoromethoxyphenyl hydrazone. Monocarboxylic sugars like glycuronic acids were competitive inhibitors of sialic acid transport. Transstimulation of [14C] NeuAc uptake was observed when vesicles were preloaded either with unlabeled NeuAc or with glucuronic acid. The data demonstrate that lysosomal membrane vesicles from rat liver are a suitable system for kinetic studies of solute transport events. The presence of a proton-driven carrier in the lysosomal membrane specific for sialic acid and other acidic sugars, including glucuronic acid, is shown. The possible physiological significance of these findings for the human lysosomal carrier and the patients with a sialic acid transport defect is discussed.     

Regulation of calcium transport in bovine spermatozoa

Calcium uptake into bovine epididymal spermatozoa is enhanced by introducing phosphate in the suspending medium (Babcock et al. (1975) J. Biol. Chem. 250, 6488-6495). This effect of phosphate is found even at a low extracellular Ca2+ concentrations (i.e., 5 microM) suggesting that phosphate is involved in calcium transport via the plasma membrane. Bicarbonate (2 mM) cannot substitute for phosphate, and a relatively high bicarbonate concentration (20 mM) causes partial inhibition of calcium uptake in absence of Pi. In the presence of 1-2 mM phosphate, 20 mM bicarbonate enhances Ca2+ uptake. The data indicate that the plasma membrane of bovine spermatozoa contains two carriers for Ca2+ transport: a phosphate-independent Ca2+ carrier that is stimulated by bicarbonate and a phosphate-dependent Ca2+ carrier that is inhibited by bicarbonate. Higher phosphate concentrations (i.e., 10 mM) inhibit Ca2+ uptake into intact cells (compared to 1.0 mM phosphate) and this inhibition can be relieved partially by 20 mM bicarbonate. This effect of bicarbonate is inhibited by mersalyl. Calcium uptake into the cells is enhanced by adding exogenous substrates to the medium. There is no correlation between ATP levels in the cells and Ca2+ transport into the cell. ATP levels are high even without added exogenous substrate and this ATP level is almost completely reduced by oligomycin, suggesting that ATP can be synthesized in the mitochondria in the absence of exogenous substrate. Calcium transport into the sperm mitochondria (washed filipin-treated cells) is absolutely dependent upon the presence of phosphate and mitochondrial substrate. Bicarbonate cannot support Ca2+ transport into sperm mitochondria. There is good correlation between Ca2+ uptake into intact epididymal sperm and into sperm mitochondria with the various substrates used. This indicates that the rate of calcium transport into the cells is determined by the rate of mitochondrial Ca2+ uptake and respiration with the various substrates.     

A membrane transporter mediates access of uridine 5'-diphosphoglucuronic acid from the cytosol into the endoplasmic reticulum of rat hepatocytes: implications for glucuronidation reactions

Hepatic glucuronidation of a wide variety of substrates is catalyzed by the membrane-bound UDP-glucuronosyltransferases. Uridine 5'-diphosphoglucuronic acid (UDP-GlcUA) is the essential cosubstrate for all UDP-glucuronosyltransferase-mediated reactions. The mechanism by which this bulky, hydrophilic nucleotide-sugar is transported from the cytosol (where it is synthesized) to its binding site(s) on the enzyme is unknown. To determine whether a membrane carrier mediates the access of UDP-GlcUA into the endoplasmic reticulum, the transport of uridine 5'-diphospho-D-[U-14C]glucuronic acid into vesicles of rough and smooth endoplasmic reticulum isolated from rat liver was investigated at 38 degrees C using a rapid filtration technique. Uptake of UDP-GlcUA by both rough and smooth vesicles was extremely rapid (linear for only 10-20 s) and temperature-dependent (negligible at 4 degrees C). UDP-GlcUA uptake was saturable, and similar kinetic parameters were obtained for rough and smooth vesicles (Km 1.9 microM, Vmax 443 pmol/mg protein per min, and Km 1.3 microM, Vmax 503 pmol/mg protein per min, respectively). The uptake of UDP-GlcUA also exhibited a high degree of specificity, since many related compounds, including UMP, UDP and UDP-Glc, did not influence uptake. In addition, the non-penetrating inhibitors of anion transport, 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS), 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), and probenecid, markedly inhibited UDP-GlcUA uptake. Finally, osmotic modulation of the intravesicular volume did not affect total uptake of UDP-GlcUA by membrane vesicles at equilibrium, indicating that this nucleotide-sugar is transported into the membrane rather than the intravesicular space. Collectively, these data provide direct evidence for a specific, carrier-mediated uptake process, which transports UDP-GlcUA from the cytosol into the endoplasmic reticulum of hepatocytes. This UDP-GlcUA transporter may be involved in the regulation of hepatic glucuronidation reactions.     

The Cyanelles (Organelles of a Low Evolutionary Scale) Possess a Phosphate-Translocator and a Glucose-Carrier in Cyanophora paradoxa

Cyanelles from Cyanophora paradoxa can easily be isolated and assayed for their carrier composition by the silicone oil filtering technique. The present investigation demonstrates a P_i-translocator transferring phosphate, dihydroxyacetone phosphate and 3-phosphoglycerate in a counter exchange mode in cyanelles as in chloroplasts of higher plants. The uptake of P_i is inhibited by dihydroxyacetone phosphate, phosphoglycerate and glucose-6-P, only poorly by phosphoenolpyruvate and not by 2-phosphoglycerate. The inhibitors pyridoxalphosphate and 4,4′diisothiocyanostilbene-2,2K'disulfonic acid at low concentration also affect P_i-uptake. Cyanelles probably transport photosynthate (reductant and ATP) by triosephosphates. This is the first demonstration of a phosphate translocator in an organism of a low evolutionary scale. Cyanelles also transport glucose which proceeds in two phases. In the lower concentration range (≤ 2.5 mM), glucose penetrates by facilitated diffusion, whereas transport follows first-order kinetics at higher amounts (> 2.5 mM). In the low concentration range, glucose-transport is affected by high concentrations of 3-O-methylglucose and fructose. The physiological role of the glucose-transport carrier in Cyanophora is doubtful. It may function in transporting glucose into cyanelles if the carbon level inside them becomes limiting, e.g. in dark periods.     

Na+/HCO3-co-transport in basolateral membrane vesicles isolated from rabbit renal cortex

Recent studies suggest that the major pathway for exit of HCO3- across the basolateral membrane of the proximal tubule cell is electrogenic Na+/HCO3- co-transport. We therefore evaluated the possible presence of Na+/HCO3- co-transport in basolateral membrane vesicles isolated from the rabbit renal cortex. Imposing an inward HCO3- gradient induced the transient uphill accumulation of Na+, and imposing an outward Na+ gradient caused HCO3- -dependent generation of an inside-acid pH gradient as monitored by quenching of acridine orange fluorescence, findings consistent with the presence of Na+/HCO3- co-transport. In the absence of other driving forces, generating an inside-positive membrane potential by imposing an inward K+ gradient in the presence of valinomycin caused net Na+ uptake via a HCO3- -dependent pathway, indicating that Na+/HCO3- co-transport is electrogenic and associated with a flow of negative charge. Imposing transmembrane Cl- gradients did not appreciably affect HCO3- gradient-stimulated Na+ influx, suggesting that Na+/HCO3- co-transport is not Cl- -dependent. The rate of HCO3- gradient-stimulated Na+ influx was a simple, saturable function of the Na+ concentration (Km = 9.7 mM, Vmax = 160 nmol/min/mg of protein), was inhibited by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (I50 = 100 microM), but was inhibited less than 10% by up to 1 mM amiloride. We could not demonstrate a HCO3- -dependent or 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid-sensitive component of Na+ influx in microvillus membrane vesicles. This study thus indicates the presence of a transport system mediating electrogenic Na+/HCO3- co-transport in basolateral, but not luminal, membrane vesicles isolated from the rabbit renal cortex. Analogous to the use of renal microvillus membrane vesicles to study Na+/H+ exchange, renal basolateral membrane vesicles may be a useful model system for examining the kinetics and possible regulation of Na+/HCO3- co-transport.     

Functional reconstitution of the malate carrier of barley mesophyll vacuoles in liposomes

The malate carrier of barley (Hordeum vulgare L.) mesophyll vacuoles was highly purified by chromatography on hydroxyapatite followed by affinity-chromatography using 5-amino-1,2,3-benzenetricarboxylic acid as ligand. The carrier, reconstituted in asolectin liposomes, had properties similar to those described previously for the carrier in intact vacuoles (Martinoia, E., Flügge, U.I., Kaiser, G., Heber, U. and Heldt, H.W. (1985) Biochim. Biophys. Acta 806, 311-319). The apparent Km for malate uptake was 2-3 mM, and the uptake was inhibited by other carboxylic acids (preferentially tricarboxylic). The sulfhydryl reagent, p-chloromercuribenzenesulfonate, as well as the anion transport inhibitor 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid, also inhibited malate uptake. The transport was dependent on the membrane potential with an optimum at about 35 mV.     

Anion transport activity in the human erythrocyte membrane modulated by proteolytic digestion of the 38,000-dalton fragment in Band 3

Extracellular treatment of human erythrocytes with papain completely converted the chymotryptic 38,000-dalton fragment of Band 3 to the 29,000-dalton fragment and inhibited the transport of inorganic phosphate in the cells. The inhibition, however, was not complete, indicating the presence of two components in the anion-transport system: the one resistant to papain digestion and the other sensitive to the digestion. The latter activity is well correlated with the degradation of the 38,000-dalton fragment. The activity remaining in the cells treated with papain was markedly different from that of the control cells. The remaining activity was not inhibited by pyridoxal phosphate and dinitrostilbene-2,2'-disulfonic acid, potent inhibitors to the anion transport, whereas phenyl phosphate inhibited the activities of both papain-treated and control cells. The results indicate that the anion-transport system consists of multiple anion-binding sites and a part of the system which is sensitive to pyridoxal phosphate and dinitrostilbene-2,2'-disulfonic acid was located in the papain-sensitive portion of 38,000-dalton fragment. A possible model of the anion-transport system was presented.     

Effects of bicarbonate on lithium transport in human red cells

Lithium influx into human erythrocytes increased 12-fold, when chloride was replaced with bicarbonate in a 150 mM lithium medium (38 degrees C. pH 7.4). The increase was linearly related to both lithium- and bicarbonate concentration, and was completely eliminated by the amino reagent 4, 4'- diisothiocyanostilbene-2,2'-disulfonic acid (DIDS). DIDS binds to an integral membrane protein (mol wt approximately 10(5) dalton) involved in anion exchange. Inhibition of both anion exchange and of bicarbonate-stimulated lithium influx was linearly related to DIDS binding. 1.1 X 10(6) DIDS molecules per cell caused complete inhibition of both processes. Both Cl- and Li+ can apparently be transported by the anion transport mechanism. The results support our previous proposal that bicarbonate-induced lithium permeability is due to transport of lithium-carbonate ion pairs (LiCO-3). DIDS-sensitive lithium influx had a high activation energy (24 kcal/mol), compatible with transport by the anion exchange mechanism. We have examined how variations of passive lithium permeability, induced by bicarbonate, affect the sodium-driven lithium counter-transport in human erythrocytes. The ability of the counter-transport system to establish a lithium gradient across the membrane decrease linearly with bicarbonate concentration in the medium. The counter-transport system was unaffected by DIDS treatement. At a plasma bicarbonate concentration of 24 mM, two-thirds of the lithium influx is mediated by the bicarbonate-stimulated pathway, and the fraction will increase significantly in metabolic alkalosis.