The role of potassium and chloride ions on the Na+/acidic amino acid cotransport system in rat intestinal brush-border membrane vesicles

The Na+/L-glutamate (L-aspartate) cotransport system present at the level of rat intestinal brush-border membrane vesicles is specifically activated by the ions K+ and Cl-. The presence of 100 mM K+ inside the vesicles drastically enhances the uptake rate and the transient intravesicular accumulation (overshoot) of the two acidic amino acids. It has been demonstrated that the activation of the transport system depended only in the intravesicular K+ concentration and that in the absence of any sodium gradient, an outward K+ gradient was unable to influence the Na+/acidic amino acid transport system. It was also found that Cl- could specifically activate the Na+-dependent L-glutamate (L-aspartate) uptake either in the presence or in the absence of K+. Also the effect of Cl- was observed only in the presence of an inward Na+ gradient and it was noted to be higher when chloride ion was present on both sides of the membrane vesicles. No influence (activation or accumulation) was observed in the absence of the Na+ gradient and in the presence of chloride gradient. L-Glutamate uptake measured in the presence of an imposed diffusion potential and in the presence of K+ or Cl- did not show any translocation of net charge.     

Glycine uptake in brush border vesicles isolated from the rat intestinal cells

The uptake of glycine in osmotically active brush border membrane vesicles (obtained by the Mg++ precipitation method) has been studied and a partial characterization of its transport system has been established. The glycine uptake in these vesicles was stimulated by the presence of sodium and in the presence of an inwardly directed Na+ -gradient glycine was accumulated inside the vesicles. The effect of Na+ was specific; other monovalent cation as Li+, K+, Rb+ and choline were uneffective in the stimulation of glycine uptake, under the same experimental conditions. Preliminary experiments show an important role of some anions on the glycine uptake. A strong inhibition in the uptake rate was found when the measurements were carried out in the presence of sodium cyclamate, while in the presence of NaSCN the measured uptake values were similar to those observed in the presence of NaCl.     

Effect of Monovalent Cations on Na+/Ca2+ Exchange and ATP-Dependent Ca2+ Transport in Synaptic Plasma Membranes

Two Ca2+ transport systems were investigated in plasma membrane vesicles isolated from sheep brain cortex synaptosomes by hypotonic lysis and partial purification. Synaptic plasma membrane vesicles loaded with Na+ (Na+i) accumulate Ca2+ in exchange for Na+, provided that a Na+ gradient (in leads to out) is present. Agents that dissipate the Na+ gradient (monensin) prevent the Na+/Ca2+ exchange completely. Ca2+ accumulated by Na+/Ca2+ exchange can be released by A 23187, indicating that Ca2+ is accumulated intravesicularly. In the absence of any Na+ gradient (K+i-loaded vesicles), the membrane vesicles also accumulate Ca2+ owing to ATP hydrolysis. Monovalent cations stimulate Na+/Ca2+ exchange as well as the ATP-dependent Ca2+ uptake activity. Taking the value for Na+/Ca2+ exchange in the presence of choline chloride (external cation) as reference, other monovalent cations in the external media have the following effects: K+ or NH4+ stimulates Na+/Ca2+ exchange; Li+ or Cs+ inhibits Na+/Ca2+ exchange. The ATP-dependent Ca2+ transport system is stimulated by increasing K+ concentrations in the external medium (Km for K+ is 15 mM). Replacing K+ by Na+ in the external medium inhibits the ATP-dependent Ca2+ uptake, and this effect is due more to the reduction of K+ than to the elevation of Na+. The results suggest that synaptic membrane vesicles isolated from sheep brain cortex synaptosomes possess mechanisms for Na+/Ca2+ exchange and ATP-dependent Ca2+ uptake, whose activity may be regulated by monovalent cations, specifically K+, at physiological concentrations.     

Uptake of [3H]serotonin into plasma membrane vesicles from mouse cerebral cortex

Preparations of plasma membrane vesicles were used as a tool to study the properties of the serotonin transporter in the central nervous system. The vesicles were obtained after hypotonic shock of synaptosomes purified from mouse cerebral cortex. Uptake of [3H]serotonin had a Na+-dependent and Na+-independent component. The Na+-dependent uptake was inhibited by classical blockers of serotonin uptake and had a Km of 63-180 nM, and a Vmax of 0.1-0.3 pmol mg-1 s-1 at 77 mM Na+. The uptake required the presence of external Na+ and internal K+. It required a Na+ gradient ([Na+]out greater than [Na+]in) and was stimulated by a gradient of K+ ([K+]in greater than [K+]out). Replacement of Cl- by other anions (NO2-, S2O3-(2-)) reduced uptake appreciably. Gramicidin prevented uptake. Although valinomycin increased uptake somewhat, the membrane potential per se could not drive uptake because no uptake was observed when a membrane potential was generated by the SCN- ion in the absence of internal K+ and with equal [Na+] inside and outside. The increase of uptake as a function of [Na+] indicated a Km for Na+ of 118 mM and a Hill number of 2.0, suggesting a requirement of two sodium ions for serotonin transport. The present results are accommodated very well by the model developed for porcine platelet serotonin transport (Nelson, P. J., and Rudnick, G. (1979) J. Biol. Chem. 254, 10084-10089), except for the number of sodium ions that are required for transport.     

A proton gradient, not a sodium gradient, is the driving force for active transport of lactate in rabbit intestinal brush-border membrane vesicles.

An inward-directed H+ gradient markedly stimulated lactate uptake in rabbit intestinal brush-border membrane vesicles, and uphill transport against a concentration gradient could be demonstrated under these conditions. Uptake of lactate was many-fold greater in the presence of a H+ gradient than in the presence of a Na+ gradient. Moreover, there was no evidence for uphill transport of lactate in the presence of a Na+ gradient. The H+-gradient-dependent stimulation of lactate uptake was not due to the effect of a H+-diffusion potential. The uptake process in the presence of a H+ gradient was saturable [Kt (concn. giving half-maximal transport) for lactate 12.7 +/- 4.5 mM] and was inhibited by many monocarboxylates. It is concluded that a H+ gradient, not a Na+ gradient, is the driving force for active transport of lactate in rabbit intestinal brush-border membrane vesicles.     

Sodium gradient-dependent L-glutamate transport is localized to the canalicular domain of liver plasma membranes. Studies in rat liver sinusoidal and canalicular membrane vesicles

The driving forces for L-glutamate transport were determined in purified canalicular (cLPM) and basolateral (i.e. sinusoidal and lateral; blLPM) rat liver plasma membrane vesicles. Initial rates of L-glutamate uptake in cLPM vesicles were stimulated by a Na+ gradient (Na+o greater than Na+i), but not by a K+ gradient. Stimulation of L-glutamate uptake was specific for Na+, temperature sensitive, and independent of nonspecific binding. Sodium-dependent L-glutamate uptake into cLPM vesicles exhibited saturation kinetics with an apparent Km of 24 microM, and a Vmax of 21 pmol/mg X min at an extravesicular sodium concentration of 100 mM. Specific anionic amino acids inhibited L-[3H]glutamate uptake and accelerated the exchange diffusion of L-[3H]glutamate. An outwardly directed K+ gradient (K+i greater than K+o) further increased the Na+ gradient (Na+o greater than Na+i)-dependent uptake of L-glutamate in cLPM vesicles, resulting in a transient accumulation of L-glutamate above equilibrium values (overshoot). The K+ effect had an absolute requirement for Na+. In contrast, in blLPM the initial rates of L-glutamate uptake were only minimally stimulated by a Na+ gradient, an effect that could be accounted for by contamination of the blLPM vesicles with cLPM vesicles. These results indicate that hepatic Na+ gradient-dependent transport of L-glutamate occurs at the canalicular domain of the plasma membrane, whereas transport of L-glutamate across sinusoidal membranes results mainly from passive diffusion. These findings provide an explanation for the apparent discrepancy between the ability of various in vitro liver preparations to transport glutamate and suggest that a canalicular glutamate transport system may serve to reabsorb this amino acid from bile.     

Functional reconstitution of the gamma-aminobutyric acid transporter from synaptic vesicles using artificial ion gradients.

The gamma-aminobutyric acid transporter of rat brain synaptic vesicles was reconstituted in proteoliposomes, and its activity was studied in response to artificially created membrane potentials or proton gradients. Changes of the membrane potential were monitored using the dyes oxonol VI and 3,3'-diisopropylthiodicarbocyanine iodide, and changes of the H+ gradient were followed using acridine orange. An inside positive membrane potential was generated by the creation of an inwardly directed K+ gradient and the subsequent addition of valinomycin. Under these conditions, valinomycin evoked uptake of [3H]GABA which was saturable. Similarly, [3H]glutamate uptake was stimulated by valinomycin, indicating that both transporters can be driven by the membrane potential. Proton gradients were generated by the incubation of K(+)-loaded proteoliposomes in a buffer free of K+ or Na+ ions and the subsequent addition of nigericin. Proton gradients were also generated via the endogenous H+ ATPase by incubation of K(+)-loaded proteoliposomes in equimolar K+ buffer in the presence of valinomycin. These proton gradients evoked nonspecific, nonsaturable uptake of GABA and beta-alanine but not of glycine in proteoliposomes as well as protein-free liposomes. Therefore, transporter activity was monitored using glycine as an alternative substrate. Proton gradients generated by both methods elicited saturable glycine uptake in proteoliposomes. Together, our data confirm that the vesicular GABA transporter can be energized by both the membrane potential and the pH gradient and show that transport can be achieved by artificial gradients independently of the endogenous proton ATPase.     

Proton gradient-dependent transport of glycine in rabbit renal brush-border membrane vesicles

This study describes evidence for the existence of a H+/glycine symport system in rabbit renal brush-border membrane vesicles. An inward proton gradient stimulates glycine transport across the brush-border membrane, and this H+-driven glycine uptake is attenuated by the protonophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone. It is a positive rheogenic process, i.e. the H+-dependent glycine uptake is further enhanced by an intravesicular negative potential. Glycine uptake is stimulated to a lesser degree by an inward Na+ gradient. H+-dependent glycine uptake is inhibited by sarcosine (69%), an analog amino acid, imino acids (proline 81%, hydroxy proline 67%), and beta-alanine (31%), but not by neutral (L-leucine) or basic (L-lysine) amino acids. The results demonstrate that H+ glycine co-transport system in rabbit renal brush-border membrane vesicles is a carrier-mediated electrogenic process and that transport is shared by imino acids and partially by beta-alanine.     

Modulation of salt permeabilities of intestinal brush-border membrane vesicles by micromolar levels of internal calcium

A possible modulation of ion permeabilities of rat intestinal brush-border membrane vesicles by Ca2+, a putative second messenger of salt secretion, was explored by three independent methods: (1) measurements of [3H]glucose accumulation driven by a Na+ gradient; (2) stopped-flow spectrophotometry of salt-induced osmotic swelling; (3) 86Rb+, 22Na+ and 36Cl- flux measurements. Cytoskeleton-deprived membrane vesicles were prepared from isolated brushborders by thiocyanate treatment. Intravescicular Ca2+ levels were varied by preincubating vesicles in Ca-EGTA buffers in the presence of the Ca2+-ionophore A23187. At Ca2+free greater than 10(-5) M, initial Na+-dependent glucose uptake in the presence of a 0.1 M NaSCN gradient (but not in its absence) was inhibited by about 50 per cent as compared to EGTA alone (ED50 approximately equal to 10(-6) M Ca2+). By contrast, initial rates of 22Na+ uptake and reswelling rates of vesicles exposed to a NaSCN gradient were increased at least 2-fold by 10(-5) M Ca2+free. Both observations are compatible with a Ca2+-induced increase of the Na+-permeability of the vesicle membrane. The modulation of ion transport was fully reversible and critically dependent on internal Ca2+, suggesting a localization of Ca2+-sensor sites at the inner surface of the microvillous membrane. As shown by radiotracer and osmotic swelling measurements, micromolar Ca2+ additionally increased the flux rate of K+, Rb+, Cl- and NO-3 but did not change the membrane permeability for small uncharged molecules, including glucose and mannitol. The effect of Ca2+ on ion permeabilities could be blocked by Ba2+ (10(-3) M) or Mg2+ (10(-2) M), but not by amiloride (10(-3) M), apamin (2 X 10(-7) M), trifluoperazine (10(-4) M) or quinine (5 X 10(-4) M). At present it is unclear whether Ca2+ activates a nonselective cation and anion channel or multiple highly selective channels in the vesicle membrane.     

Regulation of amino acid and glucose transport activity expressed in isolated membranes from untransformed and SV 40-transformed mouse fibroblasts.

Membrane vesicles isolated from untransformed Balb/c and Swiss mouse fibroblasts and their SV 40-transformed derivatives were shown to catalyze carrier-mediated, intravesicular uptake of alpha-aminoisobutyric acid and D-glucose. Concentrative uptake of alpha-aminoisobutyric acid required the presence of a Na+-gradient (external greater than internal) and could occur independently of endogenous (Na+ + K+)ATPase activity. A K+ diffusion gradient (internal greater than external) in the presence of valinomycin, or the addition of the Na+ salt of a highly permeant anion, conditions expected to create an interior-negative membrane potential stimulated Na+-gradient-dependent uptake, suggesting this process is electrogenic. D-Glucose uptake was nonconcentrative and did not require ion gradients or metabolic conversion. Na+ gradient-dependent transport of alpha-aminoisobutyric acid was reduced both in initial rate and extent of uptake in vesicles from confluent untransformed cells and increased in those from SV 40-transformed cells, compared with activities observed in vesicles from proliferating untransformed cells. No changes in D-glucose carrier activity were observed when assayed at low glucose concentrations.     

Evidence for tripeptide-proton symport in renal brush border membrane vesicles. Studies in a novel rat strain with a genetic absence of dipeptidyl peptidase IV

We have investigated the transport characteristics of L-phenylalanyl-L-prolyl-L-alanine in renal brush-border membrane vesicles isolated from Japan Fisher 344 rats. This particular rat strain genetically lacks dipeptidyl peptidase IV. Owing to the absence of this enzyme, the tripeptide was found to be completely resistant to hydrolysis by the renal brush-border membrane vesicles. Uptake of the tripeptide into these membrane vesicles in the presence of an inwardly directed Na+ gradient was slightly greater than in the presence of a K+ gradient, but there was no evidence for active transport. On the contrary, uptake was very rapid in the presence of an inside-alkaline transmembrane pH gradient, and accumulation of the tripeptide inside the vesicles against a concentration gradient could be demonstrated under these conditions. The uptake was drastically reduced by dissipation of the pH gradient. The uptake was stimulated by an inside-negative membrane potential and inhibited by an inside-positive membrane potential. Moreover, the uptake was greater in voltage-clamped membrane vesicles than in control vesicles. Many di- and tripeptides inhibited this pH gradient-stimulated uptake of Phe-Pro-Ala. The apparent dissociation constant for the tripeptide was 48 microM. High performance liquid chromatography analysis of the intravesicular content at the peak of the overshoot revealed that the tripeptide was transported across the membrane almost entirely in the intact form. These data provide the first direct evidence for the presence of an electrogenic tripeptide-proton symport in renal brush-border membranes.     

Phenylalanine uptake in isolated renal brush border vesicles.

The uptake of L-phenylalanine into brush border microvilli vesicles and basolateral plasma membrane vesicles isolated from rat kidney cortex by differential centrifugation and free flow electrophoresis was investigated using filtration techniques. Brush border microvilli but not basolateral plasma membrane vesicles take up L-phenylalanine by an Na+-dependent, saturable transport system. The apparent affinity of the transport system for L-phenylalanine is 6.1 mM at 100 mM Na+ and for Na+ 13mM at 1 mM L-phenylalanine. Reduction of the Na+ concentration reduces the apparent affinity of the transport system for L-phenylalanine but does not alter the maximum velocity. In the presence of an electrochemical potential difference of Na+ across the membrane (etaNao greater than etaNai) the brush border microvilli accumulate transiently L-phenylalanine over the concentration in the incubation medium (overshoot pheomenon). This overshoot and the initial rate of uptake are markedly increased when the intravesicular space is rendered electrically more negative by membrane diffusion potentials induced by the use of highly permeant anions, of valinomycin in the presence of an outwardly directed K+ gradient and of carbonyl cyanide p-trifluoromethoxyphenylhydrazone in the presence of an outward-directed proton gradient. These results indicate that the entry of L-phenylalanine across the brush border membrane into the proximal tubular epithelial cells involves cotransport with Na+ and is dependent on the concentration difference of the amino acid, on the concentration difference of Na+ and on the electrical potential difference. The exit of L-phenylalanine across the basolateral plasma membranes is Na+-independent and probably involves facilitated diffusion.     

Amino acid transport in membrane vesicles from CHO-K1 and alanine-resistant transport mutants

Membrane vesicles were prepared from CHO-K1 and alanine-resistant transport mutants, alar4 and alar4-H3.9. Alar4 is a constitutive mutant of the A system, and alar4-H3.9, derived from alar4, may be the result of amplification of a gene coding for an A-system transporter. Under conditions in which the same membrane potential (interior negative) and Na+ gradient were employed, the mutant vesicles show increases in the A system over that of the parental CHO-K1 cell line, paralleling, but not equivalent to, that found in whole cells. L-system and 5'-nucleotidase activities of these vesicles were similar, indicating that the increased A-system activity of the mutant vesicles is not due to the differential enrichment of the A system in these vesicles. The membrane potential was produced by a K+ diffusion gradient (internal greater than external) in the presence of valinomycin or by the addition of a Na+ salt of a highly permeant anion such as SCN-. Monensin was employed to study the effect of the Na+ gradient on transport and membrane potential. The latter was determined by measuring the uptake of tetraphenylphosphonium ion. A negative membrane potential determines the concentrative ability and the initial velocity of the A system in these vesicles. The concentration of external Na+ has a stimulatory effect on the initial velocity of this system. However, the Na+ gradient (external greater than internal) has no effect on the initial velocity or the membrane potential when the potential is set by valinomycin and high internal K+. Little if any ASC system could be detected in vesicles from CHO-K1.     

Energetics of alanine, lysine, and proline transport in cytoplasmic membranes of the polyphosphate-accumulating Acinetobacter johnsonii strain 210A.

Amino acid transport in right-side-out membrane vesicles of Acinetobacter johnsonii 210A was studied. L-Alanine, L-lysine, and L-proline were actively transported when a proton motive force of -76 mV was generated by the oxidation of glucose via the membrane-bound glucose dehydrogenase. Kinetic analysis of amino acid uptake at concentrations of up to 80 microM revealed the presence of a single transport system for each of these amino acids with a Kt of less than 4 microM. The mode of energy coupling to solute uptake was analyzed by imposition of artificial ion diffusion gradients. The uptake of alanine and lysine was driven by a membrane potential and a transmembrane pH gradient. In contrast, the uptake of proline was driven by a membrane potential and a transmembrane chemical gradient of sodium ions. The mechanistic stoichiometry for the solute and the coupling ion was close to unity for all three amino acids. The Na+ dependence of the proline carrier was studied in greater detail. Membrane potential-driven uptake of proline was stimulated by Na+, with a half-maximal Na+ concentration of 26 microM. At Na+ concentrations above 250 microM, proline uptake was strongly inhibited. Generation of a sodium motive force and maintenance of a low internal Na+ concentration are most likely mediated by a sodium/proton antiporter, the presence of which was suggested by the Na(+)-dependent alkalinization of the intravesicular pH in inside-out membrane vesicles. The results show that both H+ and Na+ can function as coupling ions in amino acid transport in Acinetobacter spp.     

Demonstration of a Na+/H+ exchange activity in purified canine cardiac sarcolemmal vesicles

Purified canine cardiac sarcolemmal membrane vesicles exhibit a sodium ion for proton exchange activity (Na+/H+ exchange). Na+/H+ exchange was demonstrated both by measuring rapid 22Na uptake into sarcolemmal vesicles in response to a transmembrane H+ gradient and by following H+ transport in response to a transmembrane Na+ gradient with use of the probe acridine orange. Maximal 22Na uptake into the sarcolemmal vesicles (with starting intravesicular pH = 6 and extravesicular pH = 8) was approximately 20 nmol/mg protein. The extravesicular Km of the Na+/H+ exchange activity for Na+ was determined to be between 2 and 4 mM (intravesicular pH = 5.9, extravesicular pH = 7.9), as assessed by measuring the concentration dependence of the 22Na uptake rate and the ability of extravesicular Na+ to collapse an imposed H+ gradient. All results suggested that Na+/H+ exchange was reversible and tightly coupled. The Na+/H+ exchange activity was assayed in membrane subfractions and found most concentrated in highly purified cardiac sarcolemmal vesicles and was absent from free and junctional sarcoplasmic reticulum vesicles. 22Na uptake into sarcolemmal vesicles mediated by Na+/H+ exchange was dependent on extravesicular pH, having an optimum around pH 9 (initial internal pH = 6). Although the Na+/H+ exchange activity was not inhibited by tetrodotoxin or digitoxin, it was inhibited by quinidine, quinacrine, amiloride, and several amiloride derivatives. The relative potencies of the various inhibitors tested were found to be: quinacrine greater than quinidine = ethylisopropylamiloride greater than methylisopropylamiloride greater than dimethylamiloride greater than amiloride. The Na+/H+ exchange activity identified in purified cardiac sarcolemmal vesicles appears to be qualitatively similar to Na+/H+ exchange activities recently described in intact cell systems. Isolated cardiac sarcolemmal vesicles should prove a useful model system for the study of Na+/H+ exchange regulation in myocardial tissue.     

Adriamycin-liposome interactions. A magnetic resonance study of the differential effects of cardiolipin on drug-induced fusion and permeability

L-Glutamate and L-aspartate transport into osmotically active intestinal brush border membrane vesicles is specifically increased by Na+ gradient (extravesicular greater than intravesicular) which in addition energizes the transient accumulation (overshoot) of the two amino acids against their concentration gradients. The "overshoot" is observed at minimal external Na+ concentration of 100 mM for L-glutamate and 60 mM for L-aspartate; saturation with respect to [Na+] was observed at a concentration near 100 mM for both amino acids. Increasing amino acid concentration, saturation of the uptake rate was observed for L-glutamate and L-aspartate in the concentration range between 1 and 2 mM. Experiments showing mutual inhibition and transtimulation of the two amino acids indicate that the same Na+ -dependent transport system is shared by the two acidic amino acids. The imposition of diffusion potentials across the membrane vesicles artificially induced by addition of valinomycin in the presence of a K+ gradient supports the conclusion that the cotransport Na+/dicarboxylic amino acid in rat brush border membrane vesicles is electroneutral.     

Chloride transport across rat ileal basolateral membrane vesicles.

The present study was designed to investigate Cl- transport across rat ileal basolateral membranes. Basolateral membrane vesicles were prepared by a well-validated technique. The purity of the basolateral membrane vesicles was verified by marker enzyme studies and by studies of d-glucose and calcium uptake. Cl- uptake was studied by a rapid filtration technique. Neither an outwardly directed pH gradient, nor a HCO3- gradient, or their combination could elicit any stimulation of Cl- transport when compared with no gradient. 4,4-Diisothiocyanostilbene-2,2-disulfonic acid at 5 mM concentration did not inhibit Cl- uptake under gradient condition. Similarly, the presence of the combination of outwardly directed Na+ and HCO3- gradients did not stimulate Cl- uptake compared with the combination of K+ and HCO3- gradients or no HCO3- gradient. This is in contrast to our results in the brush border membranes, where an outwardly directed pH gradient caused an increase in Cl- uptake. Cl- uptake was stimulated in the presence of combined Na+ and K+ gradient. Bumetanide at 0.1 mM concentration inhibited the initial rate of Cl- uptake in the presence of combined Na+ and K+ gradients. Kinetic studies of bumetanide-sensitive Cl- uptake showed a Vmax of 5.6 +/- 0.7 nmol/mg protein/5 sec and a Km of 30 +/- 8.7 mM. Cl- uptake was stimulated by an inside positive membrane potential induced by the ionophore valinomycin in the setting of inwardly directed K+ gradient compared with voltage clamp condition. These studies demonstrate two processes for Cl- transport across the rat ileal basolateral membrane: one is driven by an electrogenic diffusive process and the second is a bumetanide-sensitive Na+/K+/2 Cl- process. Cl- uptake is not enhanced by pH gradient, HCO3- gradient, their combination, or outwardly directed HCO3- and Na+ gradients.     

Active amino acid transport in plasma membrane vesicles from Simian virus 40-transformed mouse fibroblasts. Characteristics of electrochemical Na+ gradient-stimulated uptake.

Selectively permeable membrane vesicles isolated from Simian virus 40-transformed mouse fibroblasts catalyzed Na+ gradient-coupled active transport of several neutral amino acids dissociated from intracellular metabolism. Na+-stimulated alanine transport activity accompanied plasma membrane material during centrifugation in discontinuous dextran 110 gradients. Carrier-mediated transport into the vesicle was demonstrated. When Na+ was equilibrated across the membrane, countertransport stimulation of L-[3H]alanine uptake occurred in the presence of accumulated unlabeled L-alanine, 2-aminoisobutyric acid, or L-methionine. Competitive interactions among neutral amino acids, pH profiles, and apparent Km values for Na+ gradient-stimulated transport into vesicles were similar to those previously described for amino acid uptake in Ehrlich ascites cells, which suggests that the transport activity assayed in vesicles is a component of the corresponding cellular uptake process. Both the initial rate and quasi-steady state of uptake were stimulated as a function of a Na+ gradient (external Na+ greater than internal Na+) applied artificially across the membrane and were independent of endogenous (Na+ + K+)-ATPase activity. Stimulation by Na+ was decreased when the Na+ gradient was dissipated by monensin, gramicidin D or Na+ preincubation. Na+ decreased the apparent Km for alanine, 2-aminoisobutyric acid, and glutamine transport. Na+ gradient-stimulated amino acid transport was electrogenic, stimulated by conditions expected to generate an interior-negative membrane potential, such as the presence of the permeant anions NO3- and SCN-. Na+-stimulated L-alanine transport was also stimulated by an electrogenic potassium diffusion potential (K+ internal greater than K+ external) catalyzed by valinomycin; this stimulation was blocked by nigericin. These observations provide support for a mechanism of active neutral amino acid transport via the "A system" of the plasma membrane in which both a Na+ gradient and membrane potential contribute to the total driving force.     

Na+- and K+-dependent uridine transport in rat renal brush-border membrane vesicles

The transport of uridine into rat renal brush-border membrane vesicles was investigated using an inhibitor-stop filtration method. Uridine was not metabolized under these conditions. The rapid efflux of intravesicular uridine was prevented by adding 1 mM phloridzin to the ice-cold stop solution. In the presence of inwardly directed gradients of either Na+ or K+, zero-trans uridine uptake exhibited a transient overshoot phenomenon indicating active transport. The overshoot was much more pronounced with Na+ than K+ and it was not observed when either Na+ or K+ was at equilibrium across the membrane. The K+-induced overshoot was not due to the presence of a membrane potential alone, as an inwardly directed gradient of choline chloride failed to produce it. The amplitude of the overshoot was increased by raising either the Na+ or K+ concentration outside the membrane or by using more lipophilic anions (reactive order was NO3- greater than SCN- greater than Cl- greater than SO4(2-). Zero-trans efflux studies showed that the uridine transport is bidirectional. Li+ could substitute poorly for Na+ but not at all for K+. Stoichiometries of 1:1 and greater than 1:1 were observed for Na+: uridine and K+: uridine coupling, respectively. A preliminary analysis of the interactions between Na+ and K+ for uridine uptake showed complex interactions which can best be explained by the involvement of two different systems for nucleoside transport in the rat renal brush-border membrane, one requiring Na+ and the other K+ as transport coupler.     

Na+/H+ exchange mechanism in the basolateral membrane of the rat enterocyte

Basolateral membrane vesicles from rat jejunal enterocytes, especially purified of brush-border contamination, were used for Na+ uptake. The basolateral membrane vesicles are osmotically active and under our experimental conditions Na+ binding is much lower than transport. An outwardly directed proton gradient stimulates Na+ uptake at both 5 microM and 5 mM concentrations. The proton gradient effect can be inhibited completely by 2 mM amiloride and partially by either FCCP or NH4Cl (NH3 diffusion). Membrane potential effects can be excluded by having valinomycin plus K+ on both sides of the vesicles. These results suggest that there is an Na+/H+ exchanger in the basolateral membrane of rat enterocytes.