Direct evidence for the role of the membrane potential in glutathione transport by renal brush-border membranes

Transport of GSH was studied in isolated rat kidney cortical brush-border membrane vesicles in which gamma-glutamyltransferase had been inactivated by a specific affinity labeling reagent, L-(alpha S,5S)-alpha-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid (AT-125). Transport of intact 2-3H-glycine-labeled GSH occurred into an osmotically active intravesicular space of AT-125-treated membranes. The initial rate of transport followed saturation kinetics with respect to GSH concentrations; an apparent Km of 0.21 mM and Vmax of 0.23 nmol/mg protein X 20 were calculated at 25 degrees C with a 0.1 M NaCl gradient (vesicle inside less than vesicle outside). Sodium chloride in the transport medium could be replaced with KCl without affecting transport activity. The rate of GSH uptake was enhanced by replacing KCl in the transport medium with K2SO4, providing a less permeant anion, and was reduced by replacing KCl with KSCN, providing a more permeant anion. The rate of GSH transport markedly decreased in the absence of a K+ gradient across the vesicular membranes and was enhanced by a valinomycin-induced K+ diffusion potential (vesicle-inside-positive). These results indicate that GSH transport is dependent on membrane potential and involves the transfer of negative charge. The rate of GSH transport was inhibited by S-benzyl glutathione but not by glycine, glutamic acid, and gamma-glutamyl-p-nitroanilide. When incubated with [2-3H]glycine-labeled GSH, intact untreated vesicles also accumulated radioactivity; the rate of uptake was significantly higher in a Na+ gradient than in a K+ gradient. Sodium-dependent transport, but not sodium-independent uptake, was almost completely inhibited by a high concentration of unlabeled glycine. At equilibrium, most of the radioactivity which accumulated in the intravesicular space was accounted for by free glycine. These results suggest that GSH which is secreted into the tubular lumen by a specific translocase in the lumenal membranes or filtered by the glomerulus may be degraded in situ by membranous gamma-glutamyltransferase and peptidase activities which hydrolyze peptide bonds of cysteinylglycine and its derivatives. The resulting free amino acids can be reabsorbed into tubule cells by sodium-dependent transport systems in renal cortical brush-border membranes.     

The mechanism of biliary secretion of reduced glutathione. Analysis of transport process in isolated rat-liver canalicular membrane vesicles

Transport of reduced glutathione (GSH) was studied in isolated rat liver canalicular membrane vesicles by a rapid filtration technique. The membrane vesicles exhibit uptake of [2-3H]glycine--labeled GSH into an osmotically reactive intravesicular space. Although the canalicular membrane vesicles possess gamma-glutamyltransferase and aminopeptidase M, enzymes that hydrolyze glutathione into component amino acids, inactivation of the vesicle-associated transferase by affinity labeling with L-(alpha S,5S)-alpha-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid (AT-125) had no effect on the initial rate of GSH transport. Chemical analysis revealed that intact GSH accounted for most of vesicle-associated radioactivity. The initial rate of transport followed saturation kinetics with respect to GSH concentration; an apparent Km of 0.33 mM and V of 1.47 nmol/mg protein in 20 s were calculated. These results indicate that transport of GSH across the canalicular membranes is a carrier-mediated process. Replacement of NaCl in the transport medium by KCl, LiCl or choline chloride had no effect on the transport activity of the vesicles. The rate of GSH uptake by the vesicles was enhanced by valinomycin-induced K+-diffusion potential (vesicle inside-positive) and was inhibited by probenecid, indicating that GSH transport across the canalicular membranes is electrogenic and involves the transfer of negative charge. The transport of GSH was inhibited by oxidized glutathione or S-benzyl-glutathione. This transport system in canalicular plasma membranes may function in biliary secretion of GSH and its derivatives which are synthesized in hepatocytes by oxidative processes or glutathione S-transferase.     

Glutathione transport across intestinal brush-border membranes: effects of ions, pH, delta psi, and inhibitors

We characterized glutathione transport in brush-border membrane vesicles (BBMV) that were prepared from rabbit small intestine in which gamma-glutamyl transpeptidases (gamma-glutamyltransferases, EC 2.3.2.2) had been inactivated by a specific affinity-labeling reagent (AT125). Intact GSH transport was strongly increased by the presence of Na+, K+, LI+, Ca2+ and Mn2+ and, of all these, the Ca2+ activation effect was prevalent. This cation effect was selective and catalytic but not energetic; Vmax obtained in the presence of both Na+ and Ca2+ was about 6-times higher than it was in their absence, while Km did not change. Moreover, these cations almost completely eliminated GSH binding on the membrane surface. Na+ activation cannot be explained as a stimulation effect on the Na+-H+ antiport system, since a GSH proton-driven transport was excluded. We determined a pH optimum (7.5), while low or high extravesicular pH values diminished the GSH uptake rate. The Ca2+ effect on GSH transport, when an electrical potential difference was imposed across BBMV, was different from that of monovalent cations. Indeed, experiments performed by valinomycin-induced K+ diffusion potential or by anion substitution showed that the GSH transport system was an electroneutral process in the presence of Na+ or K+, but that it was electrogenic in the presence of Ca2+ or in the absence of extravesicular cations. These results suggest that GSH is also cotransported with these cations, without its accumulation inside vesicles. Moreover, since GSH is negatively charged, the effect of pH changes and of cation activation on GSH transport is arguably mediated by changes in the ionization state of certain groups as the carrier site and of GSH itself, indicating the electrostatic nature of GSH binding sites on the transporter. The high Ca2+ activation effect is perhaps also partly due to fluidity changes in the lipoproteic microenvironment of the GSH transporter. Moreover, this transport system has high affinity with GSH, given the low Km value (17 microM) and the fact that it was only inhibited by GSH S-derivatives and by GSH monoethyl ester, which probably share the same transport system.     

Evidence for an essential sulfhydryl group at the substrate binding site of the A-system transporter of Ehrlich cell plasma membranes

Plasma membrane suspensions of Ehrlich ascites cells solubilized with cholic acid were used to study the effects of sulfhydryl reagents on Na(+)-dependent amino acid transport. These suspensions were treated with the sulfhydryl binding agents p-chloromercuribenzenesulfonic acid or N-ethylmaleimide prior to reconstitution for the assay of transport activity. The proteoliposomes formed from dissolved membranes treated with p-chloromercuribenzenesulfonic acid showed no Na(+)-dependent alpha-aminoisobutyric acid transport, while N-ethylmaleimide pretreated membranes retained approximately 90% of the original activity. To avoid interference by the N-ethylmaleimide component, further studies were carried out with membranes pretreated with 200 microM N-ethylmaleimide prior to p-chloromercuribenzenesulfonic acid treatment. A concentration of 25 microM p-chloromercuribenzenesulfonic acid inhibited Na(+)-dependent alpha-aminoisobutyric acid transport by 50%. The degree of inhibition was dramatically reduced in the presence of substrates specific for the A transport system. Using an inhibition index to address the efficacy of inhibition in presence and absence of substrates, it could be shown that an index of 1.0 in presence of p-chloromercuribenzenesulfonic acid was reduced to 0.84 with (methylamino)isobutyric acid alone and 0.05 in the presence of 100 mM Na+ and 5 mM (methylamino)isobutyric acid. Na+ alone offered no protection. The results show that sulfhydryl group(s) on the amino acid carrier may be directly involved in substrate binding and that substrate binding sites are functional in the disaggregated membrane state. Furthermore, Na+ directly affects (methylamino)isobutyrate binding, since the degree of protection by the amino acid analogue against p-chloromercuribenzenesulfonic acid inhibition was influenced by the presence of Na+.(ABSTRACT TRUNCATED AT 250 WORDS)     

GSH Transport in Immortalized Mouse Brain Endothelial Cells

We have previously shown GSH transport across the blood-brain barrier in vivo and expression of transport in Xenopus laevis oocytes injected with bovine brain capillary mRNA. In the present study, we have used MBEC-4, an immortalized mouse brain endothelial cell line, to establish the presence of Na+-dependent and Na+-independent GSH transport and have localized the Na+-dependent transporter using domain-enriched plasma membrane vesicles. In cells depleted of GSH with buthionine sulfoximine, a significant increase of intracellular GSH could be demonstrated only in the presence of Na+. Partial but significant Na+ dependency of [35S]GSH uptake was observed for two GSH concentrations in MBEC-4 cells in which gamma-glutamyltranspeptidase and gamma-glutamylcysteine synthetase were inhibited to ensure absence of breakdown and resynthesis of GSH. Uniqueness of Na+-dependent uptake in MBEC-4 cells was confirmed with parallel uptake studies with Cos-7 cells that did not show this activity. Molecular form of uptake was verified as predominantly GSH, and very little conversion of [35S]cysteine to GSH occurred under the same incubation conditions. Poly(A)+ RNA from MBEC expressed GSH uptake with significant (approximately 40-70%) Na+ dependency, whereas uptake expressed by poly(A)+ RNA from HepG2 and Cos-1 cells was Na+ independent. Plasma membrane vesicles from MBEC were separated into three fractions (30, 34, and 38% sucrose, by wt) by density gradient centrifugation. Na+-dependent glucose transport, reported to be localized to the abluminal membrane, was found to be associated with the 38% fraction (abluminal). Na+-dependent GSH transport was present in the 30% fraction, which was identified as the apical (luminal) membrane by localization of P-glycoprotein 170 by western blot analysis. Localization of Na+-dependent GSH transport to the luminal membrane and its ability to drive up intracellular GSH may find application in the delivery of supplemented GSH to the brain in vivo.     

Studies on sodium transport in rat brain nerve-ending particles

(1) Nerve-ending particles isolated from crude mitochondrial preparations from rat brain by discontinuous Ficoll density gradient ultracentrifugation were shown to possess a Mg2+ and energy-dependent transport system for Na+. (2) Ouabain or iodoacetate plus cyanide exerted an inhibitory effect on the outflux but not the influx of Na+. (3) When K+ was added to a medium containing particles loaded with Na+ (22Na), an immediate release of Na+ from these particles was observed; this suggests the existence of a Na+-K+ exchange transport system. (4) The K+ effect was inhibited by 10(-4) M-ouabain only at low (about 3.3 mM), but not at high (20 mM), K+ concentrations. (5) The uptake and release of Na+ by the nerve-ending particles were found to be temperature-dependent. (6) Only nerve-ending particles with intact synaptosomal membranes were found to transport Na+ actively.     

H+ extrusion by an apical vacuolar-type H+-ATPase in rat renal proximal tubules

The activity of Na+/H(+)-exchange and H(+)-ATPase was measured in the absence of CO2/HCO3 by microfluorometry at the single cell level in rat proximal tubules (superficial S1/S2 segments) loaded with BCECF [2'7'-bis(carboxyethyl)5-6-carboxyfluorescein- acetoxymethylester]. Intracellular pH (pHi) was lowered by a NH4Cl-prepulse technique. In the absence of Na+ in the superfusion solutions, pHi recovered from the acid load by a mechanism inhibited by 0.1 microM bafilomycin A1, a specific inhibitor of a vacuolar-type H(+)-ATPase. Readdition of Na+ in the presence of bafilomycin A1 produced an immediate recovery of pHi by a mechanism sensitive to the addition of 10 microM EIPA (ethylisopropylamiloride), a specific inhibitor of Na+/H+ exchange. The transport rate of the H(+)-ATPase is about 40% of Na+/H(+)-exchange activity at a similar pHi (0.218 +/- 0.028 vs. 0.507 +/- 0.056 pH unit/min. Pre-exposure of the tubules to 30 mM fructose, 0.5 mM iodoacetate and 1 mM KCN (to deplete intracellular ATP) prevented a pHi recovery in Na(+)-free media; readdition of Na+ led to an immediate pHi recovery. Tubules pre-exposed to Cl(-)-free media for 2 hr also reduced the rate of Na(+)-independent pHi recovery. In free-flow electrophoretic separations of brush border membranes and basolateral membranes, a bafilomycin A1-sensitive ATPase activity was found to be associated with the brush border membrane fraction; half maximal inhibition is at 6 x 10(-10) M bafilomycin A1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Smooth muscle cell sarcolemma. Purification and properties of plasma membranes from the rat uterus.

A membrane fraction with sarcolemmal properties was purified from the smooth muscle layers (myometrium) of rat uterus by successive differential and equilibrium centrifugation in sucrose. The putative sarcolemmal fraction was identified by iodination with [125I]iodosulfanilic acid, had an equilibrium density of 1.15, and was enriched in enzyme activities usually associated with the plasma membrane including 5'-nucleotidase (EC 3.1.3.5) and (Na+ + K+) ATPase (EC 3.6.1.3). These membranes were free of mitochondrial or nuclear membrane contamination, suggesting the relative enrichment of sarcolemmal membranes in the fraction. Proteins of the membranes were heterogeneous with respect to molecular weight, but only a few were labelled when intact muscle was radioiodinated. Uniform resistance of sarcolemmal proteins to trypsin digestion and salt extraction suggested many are tightly bound or intrinsic membrane proteins and was a further indication of the homogeneity of membranes in this fraction.     

Reconstitution of the partially purified renal phosphate (Pi) transporter.

Proteins from rabbit kidney brush border membranes were solubilized with 1% Nonidet P-40 (crude membrane proteins) and fractionated according to their isoelectric points (pI) by chromatofocusing. The eluate was pooled into three fractions according to the pI of the samples (1, greater than 6.8; 2, 6.8-5.4; 3, 5.4-4.0). The crude membrane proteins as well as the three fractions were reconstituted into liposomes and transport of Pi was measured by a rapid filtration technique in the presence of an inwardly directed K+ or Na+ gradient. Arsenate-inhibitable Na+-dependent transport of Pi was reconstituted into an osmotically active intravesicular space from both the crude membrane proteins and Fraction 1. In contrast, Fractions 2 and 3 were inactive. Treatment of the crude membrane proteins and the three fractions with the method for extracting phosphorin (a Pi-binding proteolipid found in brush border membranes) yielded Mn2+-dependent binding of Pi characteristic of phosphorin only in the extracts from crude membrane proteins and Fraction 1, the same fractions in which Na+-dependent transport of Pi was found in the reconstituted system. When reconstituted into liposomes, phosphorin was, however, unable to yield Na+-dependent transport of Pi. Moreover, we cannot eliminate the possibility that Na+-Pi transport can occur in the absence of phosphorin, since complete recovery of Na+-Pi transport was not achieved. However, the present data showing localization of the recovered binding and transport systems for Pi in the same protein fraction lend support to the hypothesis that phosphorin might be a constituent of the renal Pi transport system. Whether the presence of phosphorin is necessary or accessory for Na+-dependent Pi transport in intact brush border membrane vesicles or in liposomes reconstituted with crude or purified membrane proteins requires further investigation.     

Reconstitution of the voltage-sensitive sodium channel of rat brain from solubilized components

The voltage-sensitive sodium channel of rat brain synaptosomes was solubilized with sodium cholate. The solubilized sodium channel migrated on a sucrose density gradient with an apparent S20,w of approximately 12 S, retained [3H]saxitoxin ([3H]STX) binding activity that was labile at 36 degrees C but no longer bound 125I-labeled scorpion toxin (125I-ScTX). Following reconstitution into phosphatidylcholine vesicles, the channel regained 125I-ScTX binding and thermal stability of [3H]STX binding. Approximately 50% of the [3H]STX binding activity and 58% of 125I-ScTX binding activity were recovered after reconstitution. The reconstituted sodium channel bound STX and ScTX with KD values of 5 and 10 nM, respectively. Under depolarized conditions, veratridine enhanced the binding of 125I-ScTX with a K0.5 of 20 microM. These KD and K0.5 values are similar to those of the native synaptosome sodium channel. 125I-ScTX binding to the reconstituted sodium channel, as with the native channel, was voltage dependent. The KD for 125I-ScTX increased with depolarization. This voltage dependence was used to demonstrate that the reconstituted channel transports Na+. Activation of sodium channels by veratridine under conditions expected to cause hyperpolarization of the reconstituted vesicles increased 125I-ScTX binding 3-fold. This increased binding was blocked by STX with K0.5 = 5 nM. These data indicate that reconstituted sodium channels can transport Na+ and hyperpolarize the reconstituted vesicles. Thus, incorporation of solubilized synaptosomal sodium channels into phosphatidylcholine vesicles results in recovery of toxin binding and action at each of the three neurotoxin receptor sites and restoration of Na+ transport by the reconstituted channels.     

Efflux of preloaded iodide from the thyroid induced by externally added iodide. A study using a biological model of the thyroid iodide transport system

Efflux of preloaded I- from the thyroid induced by externally added I- was studied using a biological model of the thyroid I- transport system. Phospholipid vesicles (P-vesicles) made from thyroid plasma membranes and soybean phospholipids were capable of accumulating I- in the presence of external Na+. P-vesicles incubated in 136 mM Na+ containing 0.9 microM I- with 125I- for 2 min accumulated I- so that the I- concentration in the vesicles became about 2 microM. Addition of 5-20 microM stable I- to the incubation mixture at 2 min incubation resulted in a dose-dependent decrease in previously loaded 125I- in the vesicles. In other words, a dose-dependent increase in efflux of preloaded 125I- was observed. While the efflux occurred, Na+-dependent I- influx into P-vesicles was preserved. When 2 mM ClO4-, a specific inhibitor of Na+-dependent I- influx, was added together with 10 microM I-, the external I- failed to diminish preloaded 125I- in P-vesicles. The 125I- efflux did not occur when a large amount of stable I- entered P-vesicles independently of Na+ in the presence of ClO4-. Similar 125I- efflux induced by externally added 5 microM SCN- was also blocked by simultaneously added ClO4-. These observations suggest that such I- efflux from the thyroid is a certain type of uphill I- transport which is closely related to Na+-dependent I- transport and that ClO4- and SCN- act on a common site of the I- transport system.     

Cl- permeability of the basolateral membrane of the Rana esculenta epithelium: activation of Cl-/HCO3- exchange by alkaline intracellular pH.

We have investigated Cl- transport mechanism(s) located in the basolateral membranes of the frog skin epithelium and in particular activation of Cl-/HCO3- exchange following an alkaline load. We found that 87% of the total 36Cl uptake by the epithelial cells occurs across the basolateral membranes (JbCl-) and submitting the epithelium to an alkaline load (HCO3(-)-Ringer solution, pH 8.1) increased JbCl-. Intracellular Cl- activity (aiCl-), measured with ion-sensitive microelectrodes, increased when the Ringer solution bathing the basolateral membranes was changed from a Ringer solution equilibrated in air (pH 7.4) to one containing CO2/HCO3- (pH 7.4). pHi recovery following an alkaline load was dependent on Cl- since it did not occur in serosal Cl(-)-free media, indicating the presence of a Cl(-)-dependent regulatory mechanism. Acid loading of the epithelial cells (5% CO2, HCO3(-)-free Ringer) produced no change in JbCl- but stimulated an amiloride-sensitive 22Na uptake across the basolateral membranes of the epithelium, compatible with an activation of a Na+/H+ exchanger, previously described in this tissue. JbCl- was partially blocked by SITS (5 x 10(-4) mmol/I), niflumic acid (5 x 10(-5) mmol/I), furosemide or bumetanide. Simultaneous addition of furosemide and niflumic acid produced an inhibition of JbCl- which was not different with furosemide alone. Substitution of Na+ by choline had no effect on JbCl- and furosemide did not block the 22Na+ uptake, suggesting that JbCl- is not a Na(+)-dependent process (cotransport). We conclude that a significant Cl- permeability at the basolateral membranes of the epithelial cells is due to the presence of a Cl-/HCO3- exchanger which is essential for the recovery of pHi following an alkaline load.     

Molecular size of a Na(+)-dependent amino acid transporter in Ehrlich ascites cell plasma membranes estimated by radiation inactivation

Radiation inactivation was used to estimate the molecular size of a Na(+)-dependent amino acid transport system in Ehrlich ascites cell plasma membrane vesicles. Na(+)-dependent alpha-aminoisobutyric acid uptake was measured after membranes were irradiated at -78.5 degrees C in a cryoprotective medium. Twenty-five percent of the transport activity was lost at low radiation doses (less than 0.5 Mrad), suggesting the presence of a high molecular weight transport complex. The remaining activity (approximately 75% of total) decreased exponentially with increasing radiation dose, and a molecular size of 347 kDa was calculated for the latter carrier system. Vesicle permeability and intravesicular volume were measured to verify that losses in transport activity were due to a direct effect of radiation on the transporter and not through indirect effects on the structural integrity of membrane vesicles. Radiation doses 2-3-fold higher than those required to inactivate amino acid transport were needed to cause significant volume changes (greater than 15%). Vesicle permeability was unchanged by the irradiation. The structural integrity of plasma membrane vesicles was therefore maintained at radiation doses where there was a dramatic decrease in amino acid transport. The relationship between the fragmentation of a 120-130-kDa peptide, a putative component of the Na(+)-dependent amino acid carrier [McCormick, J. I., & Johnstone, R. M. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 7877-7881], and loss of transport activity in irradiated membranes was also examined. Peptide loss was quantitated by Western blot analysis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of thallium ions on sodium and potassium transport in frog skin

It has been shown that Tl+ accumulated in the frog skin cells (Rana temporaria) inhibits irreversibly the unidirectional transport of Na+ estimated by the short circuit current (SCC). The inhibiting effect of Tl+ cannot be attributed to a decrease of Na+ penetration through the apical membranes. The influx of 22Na+ from mucosal bathing solution into the skin poisoned with Tl+ was about 50% of that observed in the intact skin, while the SCC was completely inhibited. The activity of the ouabain-sensitive Na+/K+ pumps located in the basolateral cell membranes was estimated by studying the uptake of 86Rb+ as a tracer for K+. This activity was high enough to maintain the ion composition of epithelial cells in spite of their ability to accomplish the undirectional transport of Na+. Tl+ seems to inhibit the production of respiration energy utilized in the undirectional Na+ transport, while the ion homeostasis of epithelial cells may be supported by the Na+/K+ pumps consuming energy of glycolytic reactions.     

Development of an AT<Subscript>2</Subscript>-deficient proximal tubule cell line for transport studies

Angiotensin II is a major regulatory peptide for proximal tubule Na(+) reabsorption acting through two distinct receptor subtypes: AT(1) and AT(2). Physiological or pathological roles of AT(2) have been difficult to unravel because angiotensin II can affect Na(+) transport either directly via AT(2) on luminal or peritubular plasma membranes of proximal tubule cells or indirectly via the renal vasculature. Furthermore, separate systemic and intratubular renin-angiotensin systems impart considerable complexity to angiotensin's regulation. A transport-competent, proximal tubule cell model that lacks AT(2) is a potentially useful tool to assess cellular angiotensin II regulation. To this end, AT(2)-receptor-deficient mice were bred with an Immortomouse, which harbors the thermolabile immortalization gene SV40 large-T antigen (Tag), and AT(2)-receptor-deficient [AT(2) (-/-)], Tag heterozygous [Tag (+/-)] F(2) offspring were selected for cell line generation. S1 proximal tubule segments were microdissected, and epithelial cell outgrowth was expanded in culture. Cells that formed confluent, electrically resistive monolayers were selected for cryopreservation, and one isolate was extensively characterized for conductance (2 mS/cm(2)), short-circuit current (Isc; 0.2 microA/cm(2)), and proximal tubule-specific Na3(+) - succinate (DeltaIsc = 0.8 microA/cm(2) at 2 mM succinate) and Na3(+) - phosphate cotransport (DeltaIsc = 3 microA/cm(2) at 1 mM phosphate). Light microscopy showed a uniform, cobblestone-shaped monolayer with prominent cilia and brush borders. AT(2) receptor functionality, as demonstrated by angiotensin II inhibition of ANF-stimulated cGMP synthesis, was absent in AT(2)-deficient cells but prominent in wild-type cells. This transport competent cell line in conjunction with corresponding wild type and AT(1)-deficient lines should help explain angiotensin II signaling relevant to Na(+) transport.     

Thermodynamic stoichiometry of Na+-coupled glutathione transport

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

Amino acid transport in the thermophilic anaerobe Clostridium fervidus is driven by an electrochemical sodium gradient.

Amino acid transport was studied in membranes of the peptidolytic, thermophilic, anaerobic bacterium Clostridium fervidus. Uptake of the negatively charged amino acid L-glutamate, the neutral amino acid L-serine, and the positively charged amino acid L-arginine was examined in membrane vesicles fused with cytochrome c-containing liposomes. Artificial ion diffusion gradients were also applied to establish the specific driving forces for the individual amino acid transport systems. Each amino acid was driven by the delta psi and delta mu Na+/F and not by the Z delta pH. The Na+ stoichiometry was estimated from the amino acid-dependent 22Na+ efflux and Na(+)-dependent 3H-amino acid efflux. Serine and arginine were symported with 1 Na+ and glutamate with 2 Na+. C. fervidus membranes contain Na+/Na+ exchange activity, but Na+/H+ exchange activity could not be demonstrated.     

Na+ stimulates binding of dopamine to the dopamine transporter in cells but not in cell-free preparations

Although Na+ is crucial for the function of the dopamine (DA) transporter (DAT), its role in the substrate binding step has been questioned. To address this issue, we investigated the effect of Na+ on DA binding by measuring the potency of DA in inhibiting the binding of the cocaine analogue [3H]2beta-carbomethoxy-3beta-(4-fluorophenyl)tropane (CFT) in intact cells expressing DAT in their plasma membranes and in membranes isolated from these cells. In cells, Na+ substantially enhanced the potency of DA in inhibiting CFT binding. This effect of Na+ was independent of buffer compositions and substitutes (sucrose vs. NMDG), more pronounced at 4 degrees C than 25 degrees C, and correlated with its stimulatory effect on DA uptake Km. Removing extracellular Na+ had little effect on intracellular concentrations of Na+ and K+, or on membrane potential. These data suggest that extracellular Na+ most likely acts at the transporter level to enhance the binding of external DA during the transport cycle. In contrast, in cell-free membrane preparations the Na+ stimulation was abolished without impairment of the potency of DA in inhibiting CFT binding, regardless of whether sucrose was used to maintain the buffer osmolarity. The difference in Na+ dependence for DA to inhibit CFT binding between plasma membranes of intact cells and isolated membranes raises the possibility that intracellular ion environment, alone or in combination with other cellular factors, plays a critical role in determining DA-DAT interaction and the integration of Na+ modulation in this interaction.     

Intrabiliary glutathione hydrolysis. A source of glutamate in bile

High concentrations of glutathione (GSH) and two of its constituent amino acids, glutamate and glycine, are normally found in rat bile. To examine the role of intrabiliary GSH hydrolysis as a source of these amino acids, as well as of cystine in bile, the biliary excretion of GSH and free amino acids was measured in normal male Sprague-Dawley rats; in animals given either phenol 3,6-dibromphthalein disulfonate or diethyl maleate, inhibitors of GSH secretion into bile; and after a retrograde intrabiliary infusion of (alpha S, 5S)-alpha-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid (AT-125), an irreversible inhibitor of gamma-glutamyl transferase activity. Total concentration of amino acids in normal rat bile ranged from 4 to 7 mM and was more than double the concentration in plasma (2-3 mM). Although most amino acids were detected in bile, glutamate and glycine were the most prevalent (1.2 and 1.0 mM, respectively), followed by the branched chain amino acids valine and leucine. The administration of phenol 3,6-dibromphthalein disulfonate (180 mumol/kg, intravenous), or of diethyl maleate (1 mmol/kg, intraperitoneal), resulted in a marked decrease in the biliary excretion of GSH, as well as a decrease in the excretion of glutamate, cystine, and glycine; however, the effects of these agents were not specific for the amino acid constituents of GSH. Following retrograde intrabiliary infusion of AT-125 (10 mumol/kg), there was an immediate and sustained doubling in the rate of biliary excretion of both GSH and glutathione disulfide and a marked decrease in the rate of excretion of glutamate. Varying the dose of AT-125 (0-20 mumol/kg) resulted in an inverse linear relation between hepatic gamma-glutamyl transferase activity and the biliary excretion of intact GSH. These findings suggest that most, if not all, of the free glutamate in excreted bile is formed from the intrabiliary hydrolysis of GSH. Prior to hydrolysis within the biliary tree, substantial concentrations of GSH must be transported from liver cells into bile; minimal canalicular concentrations of this tripeptide are estimated at 5 mM.     

Na+- and cGMP-induced Ca2+ fluxes in frog rod photoreceptors

We have examined the Ca2+ content and pathways of Ca2+ transport in frog rod outer segments using the Ca2+-indicating dye arsenazo III. The experiments employed suspensions of outer segments of truncated, but physiologically functional, frog rods (OS-IS), intact isolated outer segments (intact OS), and leaky outer segments (leaky OS with a plasma membrane leaky to small solutes, but with sealed disk membranes). We observed the following. Intact OS or OS-IS isolated and purified in Percoll-Ringer's solution contained an average of 2.2 mM total Ca2+, while leaky OS contained 2.0 mM total Ca2+. This suggests that most of the Ca2+ in OS-IS is contained inside OS disks. Phosphodiesterase inhibitors increased the Ca2+ content to approximately 4.2 mM in intact OS or OS-IS, whereas the Ca2+ content of leaky OS was not altered. Na-Ca exchange was the dominant pathway for Ca2+ efflux in both intact and leaky OS/OS-IS. The rate of Na-Ca exchange in intact OS/OS-IS was half-maximal between 30 and 50 mM Na+; at 50 mM Na+, this amounted to 5.8 X 10(7) Ca2+/OS X s or 0.05 mM total Ca2+/s. This is much larger than the Ca2+ component of the dark current. Other alkali cations could not replace Na+ in Na-Ca exchange in either OS-IS or leaky OS. They inhibited the rate of Na-Ca exchange (K greater than or equal to Rb greater than Cs greater than or equal to Li greater than TMA) and, as the inhibition became greater, a delay developed in the onset of Na-Ca exchange. The inhibition of Na-Ca exchange by alkali cations correlates with the prolonged duration of the photoresponse induced by these cations (Hodgkin, A. L., P. A. McNaughton, and B. J. Nunn. 1985. Journal of Physiology. 358:447-468). In addition to Na-Ca exchange, disk membranes in leaky OS showed a second pathway of Ca2+ transport activated by cyclic GMP (cGMP). The cGMP-activated pathway required the presence of alkali cations and had a maximal rate of 9.7 X 10(6) Ca2+/OS X s. cGMP caused the release of only 30% of the total Ca2+ from leaky OS. The rate of Na-Ca exchange in leaky OS amounted to 1.9 X 10(7) Ca2+/OS X s.(ABSTRACT TRUNCATED AT 400 WORDS)