Sodium-alanine cotransport in oocytes ofXenopus laevis: Correlation of alanine and sodium fluxes with potential and current changes

Summary The sodium-dependentl-alanine transport across the plasma membrane of oocytes ofXenopus laevis was studied by means of [¹⁴C]-l-alanine,²²Na⁺ and electrophysiological measurements. At fixed sodium concentrations, the dependence of alanine transport on alanine concentration follows Michaelis-Menten kinetics; at fixed alanine concentrations, the transport varies with sodium concentration with a Hill coefficient of 2. In the presence of sodium the uptake of alanine is accompanied by a depolarization of the membrane. Under voltage-clamp conditions this depolarization can be compensated by an inward-directed current. Assuming that this current is carried by sodium we arrive at a 21 stoichiometry for the sodium-alanine cotransport. The assumption was confirmed by direct measurements of both sodium and alanine fluxes at saturating concentrations of the two substrates, which also yielded a stoichiometry close to 21. The sodium-l-alanine cotransport is neither inhibited by furosemide (0.5 mmol/liter) nor by N-methyl amino isobutyric acid (5 mmol/liter). A 20-fold excess ofd-alanine overl-alanine caused about 60% inhibition.     

Delineation of sodium-stimulated amino acid transport pathways in rabbit kidney brush border vesicles

Summary We have confirmed previous demonstrations of sodium gradient-stimulated transport ofl-alanine, phenylalanine, proline, and -alanine, and in addition demonstrated transport of N-methylamino-isobutyric acid (MeAIB) and lysine in isolated rabbit kidney brush border vesicles. In order to probe the multiplicity of transport pathways available to each of these¹⁴C-amino acids, we measured the ability of test amino acids to inhibit tracer uptake. To obtain a rough estimate of nonspecific effects, e.g., dissipation of the transmembrane sodium electrochemical potential gradient, we measured the ability ofd-glucose to inhibit tracer uptake.l-alanine and phenylalanine were completely mutually inhibitory. Roughly 75% of the¹⁴C-l-alanine uptake could be inhibited by proline and -alanine, while lysine and MeAIB were no more effective thand-glucose. Roughly 50% of the¹⁴C-phenylalanine uptake could be inhibited by proline and -alanine; lysine was as effective as proline and -alanine, and the effects of pairs of these amino acids at 50mm each were not cumulative. MeAIB was no more effective thand-glucose. We conclude that three pathways mediate the uptake of neutral,l, -amino acids. One system is inaccessible to lysine, proline, and -alanine. The second system carries a major fraction of thel-alanine flux; it is sensitive to proline and -alanine, but not to lysine. The third system carries half the¹⁴C-phenylalanine flux, and it is sensitive to proline, lysine, and -alanine. Since the neutral,l, -amino acid fluxes are insensitive to MeAIB, we conclude that they are not mediated by the classicalA system, and since all of thel-alanine flux is inhibited by phenylalanine, we conclude that it is not mediated by the classicalASC system.l-alanine and phenylalanine completely inhibit uptake of lysine. MeAIB is no more effective thand-glucose in inhibiting lysine uptake, while proline and -alanine appear to inhibit a component of the lysine flux. We conclude that the¹⁴C-lysine fluxes are mediated by two systems, one, shared with phenylalanine, which is inhibited by proline, -alanine, andl-alanine, and one which is inhibited byl-alanine and phenylalanine but inaccessible to proline, -alanine, and MeAIB. Fluxes of¹⁴C-proline and¹⁴C-MeAIB are completely inhibited byl-alanine, phenylalanine, proline, and MeAIB, but they are insensitive to lysine. Proline and MeAIB, as well as alanine and phenylalanine, but not lysine, inhibit¹⁴C--alanine uptake. However, -alanine inhibits only 38% of the¹⁴C-proline uptake and 57% of the MeAIB uptake. We conclude that two systems mediate uptake of proline and MeAIB, and that one of these systems also transports -alanine.     

Electrogenic properties of the sodium-alanine cotransporter in pancreatic acinar cells: II. Comparison with transport models

Summary In this paper, the results of the preceding electrophysiological study of sodium-alanine cotransport in pancreatic acinar cells are compared with kinetic models. Two different types of transport mechanisms are considered. In the simultaneous mechanism the cotransporterC forms a ternary complexNCS with Na⁺ and the substrateS; coupled transport of Na⁺ andS involves a conformational transition between statesNCS andNCS with inward- and outward-facing binding sites. In the consecutive (or ping-pong) mechanism, formation of a ternary complex is not required; coupled transport occurs by an alternating sequence of association-dissociation steps and conformational transitions. It is shown that the experimentally observed alanine- and sodium-concentration dependence of transport rates is consistent with the predictions of the simultaneous model, but incompatible with the consecutive mechanism. Assuming that the association-dissociation reactions are not rate-limiting, a number of kinetic parameters of the simultaneous model can be estimated from the experimental results. The equilibrium dissociation constants of Na⁺ and alanine at the extracellular side are determined to beK _N <-64mm andK _S <-18mm. Furthermore, the ratioK _N /K _N ^S of the dissociation constants of Na⁺ from the binary (NC) and the ternary complex (NCS) at the extracellular side is estimated to be <-6. This indicates that the binding sequence of Na⁺ andS to the transporter is not ordered. The current-voltage behavior of the transporter is analyzed in terms of charge translocations associated with the single-reaction steps. The observed voltage-dependence of the half-saturation concentration of sodium is consistent with the assumption that a Na⁺ ion that migrates from the extracellular medium to the binding site has to traverse part of the transmembrane voltage.     

The mechanistic nature of the membrane potential dependence of sodium-sugar cotransport in small intestine

Summary Methods are described which demonstrate the use of unidirectional influx of¹⁴C-tetraphenylphosphonium (¹⁴C-TPP⁺) into isolated intestinal epithelial cells as a quantitative sensor of the magnitude of membrane potentials created by experimentally imposed ion gradients. Using this technique the quantitative relationship between membrane potential () and Na⁺-dependent sugar influx was determined for these cells at various Na⁺ and -methylglucoside (-MG) concentrations. The results show a high degree of dependence for the transport Michaelis constant but a maximum velocity for transport which is independent of . No transinhibition by intracellular sugar (40mm) can be detected. Sugar influx in the absence of Na⁺ is insensitive to 1.3mm phlorizin and independent of . The mechanistic implications of these results were evaluated using the quality of fit between calculated and experimentally observed kinetic constants for rate equations derived from several transport models. The analysis shows that for models in which translocation is the potential-dependent step the free carrier cannot be neutral. If it is anionic, the transporter must be functionally asymmetric. A model in which Na⁺ binding is the potential-dependent step (Na⁺ well concept) also provides an appropriate kinetic fit to the experimental data, and must be considered as a possible mechanistic basis for function of the system.     

Uptake of lysine and prolinevia separate α-neutral amino acid transport pathways inMytilus gill brush border membranes

Summary Brush border membrane vesicles (BBMV) were prepared from the gills of the marine mussel,Mytilus edulis. These membranes contained two distinct pathways for cotransport of Na⁺ and -neutral amino acids. The major pathway in mussel gill BBMV was the alanine-lysine (AK) pathway, which had a high affinity for alanine and for the cationic amino acid, lysine. The AK pathway was inhibited by nonpolar -neutral amino acids and cationic amino acids, but was not affected by -neutral amino acids or imino acids. The kinetics of lysine transport were consistent with a single saturable process, with aJ _max of 550 pmol/mg-min and aK _t of 5 m. The AK pathway did not have a strict requirement for Na⁺, and concentrative transport of lysine was seen in the presence of inwardly directed gradients of Li⁺ and K⁺, as well as Na⁺. Harmaline inhibited the transport of lysine in solutions containing either Na⁺ or K⁺. The alanine-proline (AP) pathway transported both alanine and proline in mussel gill BBMV. The AP pathway was strongly inhibited by nonpolar -neutral amino acids, proline, and -(methylamino)isobutyric acid (Me-AIB). The kinetics of proline transport were described by a single saturable process, with aJ _max of 180 pmol/mg-min andK _t of 4 m. In contrast to the AK pathway, the AP pathway appeared to have a strict requirement for Na⁺. Na⁺-activation experiments with lysine and proline revealed sigmoid kinetics, indicating that multiple Na⁺ ions are involved in the transport of these substrates. The transport of both lysine and proline was affected by membrane potential in a manner consistent with electrogenic transport.     

Renal handling of taurine,l-alanine,l-glutamate andd-glucose inOpsanus tau: studies on isolated brush border membrane vesicles

Summary Renal brush border membrane vesicles (bbmv) from the aglomerular toadfish (Opsanus tau), isolated by differential precipitation, were tested for their ability to actively translocate (i) taurine, known to be secreted by the kidney of several marine teleosts, and (ii)l-alanine,l-glutamic acid, andd-glucose, solutes that are normally reabsorbed in the filtering nephron. Vesicular taurine uptake displayed a Na⁺ dependence. Transport was greatest under conditions of an inward-directed Na⁺ gradient, but a significant stimulation by Na⁺ over K⁺ could also be observed in the absence of a salt gradient. At high extravesicular K⁺, the addition of valinomycin reduced taurine uptake. Na⁺-dependent³H-taurine flux was almost completely inhibited by non-labeled taurine (tracer replacement) or -alanine, but was unaffected byl-alanine. Replacement of medium chloride by SCN^– or NO ₃ ^– in the presence of Na⁺ resulted in significantly lower uptake rates under both anion gradient and anion equilibrium conditions, whereas Br^– could almost fully substitute for the stimulatory Cl^– action. These results indicate the presence of an electrogenic Na⁺-cotransport mechanism with specificity for -amino acids in the toadfish renal brush border. Whether the system under physiological conditions mediates reabsorption or secretion of taurine remains to be determined. Toadfish bbmv also translocatedl-alanine andl-glutamic acid in a Na⁺-dependent manner. Possible roles for these most likely reabsorptive transport systems in a non-filtering kidney are discussed.d-glucose uptake, however, appeared to occur via Na⁺-independent pathways, since it was not affected by phlorizin in the presence of Na⁺, or by Na⁺ replacement.Abbreviation bbmv brush border membrane vesicles     

l-Alanine uptake in brush border membrane vesicles from the gill of a marine bivalve

Summary Brush border membrane vesicles were prepared from mussel gills using differential and sucrose density gradient centrifugation. These vesicles contained both the maximal Na⁺-dependent alanine transport activity found in the gradient and the maximal activities of -glutamyl transpeptidase and alkaline phosphatase. Electron micrographs showed closed vesicles of approximately 0.1–0.5 m diameter. Transport experiments using these vesicles demonstrated a transient 18-fold overshoot in intravesicular alanine concentration in the presence of an inwardly directed Na⁺ gradient, but not under Na⁺ equilibrium conditions. A reduced overshoot (10-fold) was seen with an inwardly directed K⁺ gradient. Further studies revealed a broad cation selectivity, with preference for Na⁺, which was characteristic of alanine transport but not glucose transport in these membranes. The apparent amino acid specificity of the uptake pathway(s) was similar to that of intact gills and supported the idea of at least four separate pathways for amino acid transport in mussel gill brush border membranes. The apparent Michaelis constant for alanine uptake was approximately 7m, consistent with values forK _t determined with intact tissue.     

A re-examination of the Na+-independent binding of [3H]β-alanine to rat brain stem-spinal cord

The Na⁺-independent binding of [³H]-alanine to rat brain stem plus spinal cord was reinvestigated, in order to study in more detail the characteristics of previously described -alanine binding processes. Binding was absent when amino acid-free postnuclear supernatants or crude synaptic membranes were used. Experiments performed with several other Na⁺-free preparations showed a sole binding component, irrespective of the preparation used. Biochemical characterization of this Na⁺-independent binding, using frozen/thawed/washed synaptosomal-mitochodrial fractions, showed that binding reached a plateau between 7 min and 13 min, increasing thereafter. Binding was linear with fraction protein over a range of 200–415 g/ml incubation medium. Binding was completely inhibited by glycine, alanine, -aminobutyric acid, -aminoisobutyric acid, hypotaurine and strychnine, and to a lesser extent by 2,2-dimethyl--alanine, brucine and gelsemine. It was insensitive to taurine, -aminobutyric acid (GABA), 2-guanidinoethanesulfonic acid (GES), carnosine, and bicuculline methiodide. Binding was reversible, saturable (K _D 20 M), and heat sensitive.     

Taurine indirectly increases [Ca]i by inducing Ca2+ influx through the Na+-Ca2+ exchanger

Recent studies in heart cells have shown taurine to induce a sustained increase of both intracellular Ca²⁺ and Na⁺. These results led us to believe that the increase in Na⁺ by taurine could be due to Na⁺ entry through the taurine-Na⁺ cotransporter which in turn favours transarcolemmal Ca²⁺ influx through Na⁺-Ca²⁺ exchange. Therefore, we investigated the effect of -alanine, a blocker of the taurine-Na⁺ cotransporter and low concentrations of CBDMB (a pyrazine derivative, 5-(N-4chlorobenzyl)-2,4-dimethylbenzamil), a Na⁺-Ca²⁺ exchanger blocker on taurine-induced [Ca]_i increase in embryonic chick heart cells. Using Fura-2 Ca²⁺ imaging and Fluo-3 Ca²⁺ confocal microscopy techniques, taurine (20 mM) as expected, induced a sustained increase in [Ca]_i at both the cytosolic and the nuclear levels. Preexposure to 500 M of the blocker of the taurine-Na⁺ cotransporter, -alanine, prevented the amino acid-induced increase of total [Ca]_i. On the other hand, application of -alanine did not reverse the action of taurine on total [Ca]_i. However, low concentrations of the Na⁺-Ca²⁺ exchanger blocker, CBDMB, reversed the taurine-induced sustained increase of cytosolic and nuclear free calcium (in presence or absence of -alanine). Thus, the effect of taurine on [Ca]_i in heart cells appears to be due to Na⁺ entry through the taurine-Na⁺ cotransporter which in turn favours transarcolemmal Ca²⁺ influx through the Na⁺-Ca²⁺ exchanger.     

The role of the proton electrochemical gradient in the transepithelial absorption of amino acids by human intestinal Caco-2 cell monolayers

We determined the extent of Na⁺-independent, proton-driven amino acid transport in human intestinal epithelia (Caco-2). In Na⁺-free conditions, acidification of the apical medium (apical pH 6.0, basolateral pH 7.4) is associated with a saturable net absorption of glycine. With Na⁺-free media and apical pH set at 6.0, (basolateral pH 7.4), competition studies with glycine indicate that proline, hydroxyproline, sarcosine, betaine, taurine, -alanine, -aminoisobutyric acid (AIB), -methylaminoisobutyric acid (MeAIB), -amino-n-butyric acid and l-alanine are likely substrates for pH-dependent transport in the brush border of Caco-2 cells. Both d-serine and d-alanine were also substrates. In contrast leucine, isoleucine, valine, phenylalanine, methionine, threonine, cysteine, asparagine, glutamine, histidine, arginine, lysine, glutamate and d-aspartate were not effective substrates. Perfusion of those amino acids capable of inhibition of acid-stimulated net glycine transport at the brush-border surface of Caco-2 cell monolayers loaded with the pH-sensitive dye 2,7-bis(2-carboxyethyl-5(6)-carboxyfluorescein) (BCECF) caused cytosolic acidification consistent with proton/amino acid symport. In addition, these amino acids stimulate an inward short-circuit current (I _sc) in voltage-clamped Caco-2 cell monolayers in Na⁺-free media (pH 6.0). Other amino acids such as leucine, isoleucine, phenylalanine, tryptophan, methionine, valine, serine, glutamine, asparagine, d-aspartic acid, glutamic acid, cysteine, lysine, arginine and histidine were without effect on both pH_i and inward I _sc. In conclusion, Caco-2 cells express a Na⁺-independent, H⁺-coupled, rheogenic amino acid transporter at the apical brush-border membrane which plays an important role in the transepithelial transport of a range of amino acids across this human intestinal epithelium.This study was supported by a Wellcome Trust Fellowship (to DTT). Charlotte Ward, Maureen Sinclair and Ken Elliott provided excellent technical assistance.     

Sodium channels contribute to action potential generation in canine and human pancreatic islet B cells

Summary Pancreatic islet B cells depolarize and display trains of action potentials in response to stimulatory concentrations of glucose. Based on data from rodent islets these action potentials are considered to be predominantly Ca²⁺ dependent. Here we describe Na⁺-dependent action potentials and Na⁺ currents recorded from canine and human pancreatic islet B cells. Current-clamp recording using the nystatin perforated-patch technique demonstrates that B cells from both species display tetrodotoxin-sensitive Na⁺ action potentials in response to modest glucose-induced depolarization. In companion whole-cell voltage-clamp experiments on canine B cells, the underlying Na⁺ current displays steep voltage-dependent activation and inactivation over the range of –50 to –40 mV. The Na⁺ current is sensitive to tetrodotoxin block with aK ₁=3.2nm and has a reversal potential which changes with [Na⁺] _o as predicted by the Nernst equation. These results suggest that a voltage-dependent Na⁺ current may contribute significantly to action potential generation in some species outside the rodent family.     

Taurine and β-alanine uptake in primary astrocytes differentiating in culture: Effects of ions

The effects of ions on taurine and -alanine uptake were studied in astrocytes during cellular differentiation in primary cultures. The uptakes were strictly Na⁺-dependent and also inhibited by the omission of K⁺ and in the presence of ouabain suggesting that their transport is fuelled mainly by these cation gradients. Two sodium ions were associated in the transport of one taurine and -alanine molecule across cell membranes. A reduction in Cl^– concentration also markedly inhibited the uptake of both amino acids, indicating that this anion is of importance in the transport processes. The similar ion dependency profiles of taurine and -alanine uptake corroborate the assumption that the uptake of these amino acids in astrocytes is mediated by the same carrier. In Na⁺- and K⁺-free media both taurine and -alanine uptakes were reduced significantly more in 14-day-old or older than in 7-day-old cultures. No significant changes occurred in the coupling ratio between Na⁺ and taurine or -alanine as a function of spontaneous cellular differentiation or upon dBcAMP treatment. These results suggest that the uptake systems of these structurally related amino acids in astrocytes have reached a relatively high degree of functional maturity by two weeks in culture.     

Sodiumd-glucose cotransport in the gill of marine mussels: Studies with intact tissue and brush-border membrane vesicles

Summary Glucose transport was studied in marine mussels of the genusMytilus. Initial observations, with intact animals and isolated gills, indicated that net uptake of glucose occurred in mussels by a carrier-mediated, Na⁺-sensitive process. Subsequent studies included use of brush-border membrane vesicles (BBMV) in order to characterize this transport in greater detail. The highest activity of Na⁺-dependent glucose transport was found in the brush-border membrane fractions used in this study, while basal-lateral membrane fractions contained the highest specific binding of ouabain. Glucose uptake into BBMV showed specificity for Na⁺, and concentrative glucose transport was observed in the presence of an inwardly directed Na⁺ gradient. There was a single saturable pathway for glucose uptake, with an apparentK _t of 3 m in BBMV and 9 m in intact gills. The kinetics of Na⁺ activation of glucose uptake were sigmoidal, with apparent Hill coefficients of 1.5 in BBMV and 1.2 in isolated gills, indicating that more than one Na⁺ may be involved in the transport of each glucose. Harmaline inhibited glucose transport in mussel BBMV with aK _i of 44 m. The uptake of glucose was electrogenic and stimulated by an inside-negative membrane potential. The substrate specificity in intact gills and BBMV resembled that of Na⁺-glucose cotransporters in other systems;d-glucose and -methyl glucopyranoside were the most effective inhibitors of Na⁺-glucose transport,d-galactose was intermediate in its inhibition, and there was little or no effect ofl-glucose,d-fructose, 2-deoxy-glucose, or 3-O-methyl glucose. Phlorizin was an effective inhibitor of Na⁺-glucose uptake, with an apparentK _i of 154nm in BBMV and 21nm in intact gills. While the qualitative characteristics of glucose transport in the mussel gill were similar to those in other epithelia, the quantitative characteristics of this process reflect adaptation to the seawater environment of this animal.     

d-Alanine dehydrogenase

Pseudomonas aeruginosa PA01 was found to utilise both thed- andl-isomers of -alanine and also -alanine as sole sources of carbon and energy for growth. Enzymological studies of wild-type cultures and comparison with mutants deficient in growth upon one or more isomers of alanine led to the following conclusions: (i) utilisation ofd-alanine involved its direct oxidation by an inducible, membrane-bound, cytochrome-linked dehydrogenase; (ii) utilisation ofl-alanine required its conversion to the directly oxidisabled-form by a soluble racemase; (iii) utilisation of -alanine, likel-alanine, involves both the racemase andd-alanine dehydrogenase enzymes, but in addition must involve other enzymes the identity, of which is still speculative; (iv)P. aeruginosa, likeEscherichia coli, appears to take upd-alanine andl-alanine by means of two specific permeases.Abbreviation DCPIP 2,6-dichlorophenol-indophenol     

Pathways for alanine transport in intestinal basal lateral membrane vesicles

Summary Membrane vesicles obtained from the basal lateral membranes of the rat intestinal epithelium were used to study the pathways for neutral amino acid transport.In the absence of sodium there was a stereospecific uptake ofl-alanine which exhibited saturation kinetics (K _m 0.73mm andV _max 5.3 nmol/mg min at 22°C). The activation energy for this process was 8.1 kcal/mole between 5 and 25°C. Preloading the vesicles with alanine increased the unidirectional influx of alanine into the vesicle. Competition experiments indicated that the affinity of the sodium-independent transport system was glutamine > threonine > alanine > phenylalanine > valine > methionine > glycine > histidine > proline, N-MeAIB. These are the characteristics of the classical L transport system.External sodium increased the rate of the stereospecificl-alanine uptake. The Na-dependent flux had aK _m of 0.04mm and aV _max of 0.26 nmol/mg min at 22°, and an activation energy of 9.1 kcal/mole between 5 and 25°C. Competition experiments suggest the existence of three separate pathways for alanine transport in the presence of sodium. A major pathway is shared by all other amino acids tested (i.e., threonine, glutamine, methionine, phenylalanine, valine, proline and N-MeAIB). This resembles the classical A system. A second pathway is unavailable to either phenylalanine or N-MeAIB; this is reminiscent of the classical ASC system; and the third is a novel pathway which is shared by N-MeAIB but not phenylalanine.The sodium-independent and the sodium-dependent transport ofl-alanine was blocked by PCMBS and significantly inhibited by DTP and NEM. It is concluded that the sodium-independent system (the L-like system) accounts for the efflux of neutral amino acids from the epithelium to the blood during the absorption of amino acids from the gut, and that the sodium-dependent transport processes may play an important role in the supply of amino acids to the epithelium in the absence of amino acids from the gut lumen.     

Uptake of acetyl-l-carnitine in the brain

Analysis in mouse brain slices of the uptake of acetyl-l-[N-methyl-¹⁴C]carnitine with time showed it to be concentrative, and kinetic analysis gave aK _m of 1.92 mM and aV _max of 1.96 mol/min per ml, indicating the presence of a low-affinity carrier system. The uptake was energy-requiring and sodium-dependent, being inhibited in the presence of nitrogen (absence of O₂), sodium cyanide, low temperature (4°C), and ouabain, and in the absence of Na⁺. The uptake of acetyl-l-carnitine was not strictly substrate-specific; -butyrobetaine,l-carnitine,l-DABA, and GABA were potent inhibitors, hypotaurine andl-glutamate were moderate inhibitors, and glycine and -alanine were only weakly inhibitory. In vivo, acetyl-l-carnitine transport across the blood-brain barrier had a brain uptake index of 2.4±0.2, which was similar to that of GABA. These results indicate an affinity of acetyl-l-carnitine to the GABA transport system.     

Na+-dependent medium-affinity uptake of L-glutamate in the insect epidermis

It is proposed that the activity of an epidermal cotransport system for Na⁺ and dicarboxylic amino acids accounts for the small amounts of L-glutamate and L-aspartate in the otherwise amino-acid-rich blood plasma of insects. This Na⁺-dependent transport system is responsible for more than 95% of the uptake of these amino acids into the larval epidermis of the beetle Tenebrio molitor. Kinetic analysis of uptake showed that the Na⁺-dependent co-transporter has medium affinity for L-glutamate and L-aspartate. The K _m for L-glutamate uptake was 146 mol·l^-1, and the maximum velocity of uptake (V _max) was 12.1 pmol·mm^-2 of epidermal sheet per minute. The corresponding values for L-aspartate were 191 mol·l^-1 and 8.4 pmol·mm^-2·min^-1. The Na⁺/L-glutamate co-transporter has a stoichiometry of at least two Na⁺ ions for each L-glutamate-ion transported (n=217). The co-transporter has an affinity for Na⁺ equivalent to a K _m of 21 mmol · l^-1 Na⁺. Na⁺ is the only external ion apparently required to drive L-glutamate uptake. Li⁺ substitutes weakly for Na⁺. Removal of external K⁺ or addition of ouabain decreases uptake slowly over 1 h, suggesting that these treatments dissipate the Na⁺/K⁺ gradient by inhibiting epidermal Na⁺/K⁺ ATPase. Several structural analogues of L-glutamate inhibit the medium-affinity uptake of L-glutamate. The order of potency with which these competitive inhibitors block glutamate uptake is L-cysteatethreo-3-hydroxy-Dl-aspartate > D-aspartateL-aspartate> L-cysteine sulphinate > L-homocysteateD-glutamate. L-trans-Pyrrolidine-2,4-dicarboxylate, a potent inhibitor of L-glutamate uptake in mammalian synaptosomes, is a relatively weak blocker of epidermal uptake. The epidermis takes up substantially more L-glutamate by this Na⁺-dependent system than tissues such as skeletal muscle and ventral nerve cord. The epidermis may be a main site regulating blood L-glutamate levels in insects with high blood [Na⁺]. Because L-glutamate and L-aspartate stimulate skeletal muscle in insects, a likely role for epidermal L-glutamate/L-aspartate transporter is to keep the level of these excitatory amino acids in the blood below the postsynaptic activation thresholds.Abbreviation ac acetate - Ch choline - CNS central nervous system - cpm counts per minute - CDTA trans-1,2-diaminocyclohexane-N,N,N,N-tetraacetic acids - HPLC high performance liquid chromatography - K _m Michaelis constant - n _app apparent number - NMG N-methyl-D-glucamine - Pipes Piperazine-N,N-bis-[2-ethanesulfonic acid] - SD standard deviation - TEA tetraethyl-ammonium - V velocity of uptake - V _max maximum velocity of uptake     

Stereoselective aminoacylation of a dinucleoside monophosphate by the imidazolides ofDl-alanine and N-(tert-butoxycarbonyl)-Dl-alanine

Summary The aminoacylation of diinosine monophosphate (IpI) was studied. When the acylating agent was the imidazolide of N-(tert-butoxycarbonyl)-Dl-alanine, a 40% enantiomeric excess of thel isomer was incorporated at the internal 2 site and the positions of equilibrium for the 23 migration reaction differed for theD andl enantiomers. The reactivity of the nucleoside hydroxyl groups decreased in the order 2(3)>internal 2>5, and the extent of reaction was affected by the concentration of the imidazole buffer (pH 7.1). In contrast, reaction of IpI with the imidazolide of unprotectedDl-alanine led to an excess of theD isomer at the internal 2 site, while reaction with the N-carboxy anhydride ofDl-alanine proceeded without detectable stereoselection. The relevance of these results to the evolution of optical activity and the origin of genetically directed protein synthesis is discussed.     

The sodium concentration of the lateral intercellular spaces of MDCK cells: A microspectrofluorimetric study

MDCK cell monolayers grown on glass coverslips were used to examine the Na⁺ concentration in individual lateral intercellular spaces (LIS) by video fluorescence microscopy. The LIS was filled with the Na⁺-sensitive fluorescent dye SBFO by incubation of the monolayers for 75–90 min with 250 m of the membrane impermeant form of the dye. After dye loading, the monolayers were perfused at 37°C with solutions buffered with HEPES or bicarbonate/CO₂ containing 142 mm Na⁺. Ratios of the fluorescence images after sequential excitation with 340 nm and 380 nm light were performed and in situ calibration of LIS Na⁺ was accomplished after blocking the Na⁺ pump with 5 × 10^–4 m ouabain. Measurements of Na⁺ along the basolateral-to-apical axis of the LIS at 1.0 or 1.5 m intervals did not reveal a Na⁺ gradient when the perfusate was either HEPES or bicarbonate/CO₂ solutions. In bicarbonate solutions, the mean Na⁺ concentration (mm) was 157.2 ± 2.3, 15 mm higher than the bath Na⁺ concentration. In HEPES solutions, however, the Na⁺ concentration was not different from the bath concentration (142.7 ± 3.1 mm). The time course of Na⁺ changes in LIS was investigated by rapidly switching the perfusate from 142 to 80 mm Na⁺ and measuring the Na⁺ changes at one focal plane.We would like to thank P.H. Tran and C. Gibson for their technical and computational assistance as well as Dr. B.-E. Persson (University of Uppsala, Sweden) for his contribution in the early phases of the study.     

Alanine and taurine transport by the gill epithelium of a marine bivalve: Effect of sodium on influx

Summary Marine mussels can accumulate amino acids from seawater into the epithelial cells of the gill against chemical gradients in excess of 5×10⁶ to 1. Uptake of both alanine and taurine into gill tissue isolated fromMytilus californianus was found to be dependent upon Na⁺ in the external solution. Uptake of these amino acids was described by Michaelis-Menten kinetics, and a reduction in external [Na⁺] (from 425 to 213mm) increased the apparent Michaelis constants (alanine, from 8 to 17 m; taurine, from 4 to 39 m) without a significant influence on theJ _max's of these processes. Fivemm harmaline, an inhibitor of Na-cotransport processes in many systems, reduced both alanine and taurine uptake by more than 95%; this inhibition appeared to be competitive in nature, with an apparentK _i of 43 m for the interaction with alanine uptake. Increasing the external [Na⁺] from 0 to 510mm produced a sigmoid activation of alanine and taurine uptake withK _Na's of approximately 325mm. The apparent Hill coefficients for this activation were 7.3 and 7.4 for alanine and taurine, respectively. These data are consistent with uptake mechanisms which require comparatively high concentrations of Na⁺ to activate transport, and which couple several Na⁺ ions to the transport of each amino acid. These characteristics, in conjunction with the previously demonstrated low passive permeability of the apical membrane to amino acids, result in systems capable of i) accumulating amino acids from seawater to help meet the nutritional needs of this animal, and ii) maintaining the high intracellular amino-acid concentrations associated with volume regulation in the gill.