Amino acid transport and intracellular Na+ and K+ content of chicken erythrocytes genetically selected for high and low leucine transport activity

1. Amino acid transport and intracellular Na+ and K+ content have been studied in two lines of chickens, one high and the other low uptake, selected for their ability to transport leucine into erythrocytes. 2. Low line birds were less effective in absorbing glycine into erythrocytes than were high line birds, the difference in transport being due to a difference in maximal flux (Vmax), but not in apparent affinity for transport sites (Kt). 3. In contrast to glycine uptake, the greater ability of the high line to absorb lysine was found to be due to a difference in both Vmax and Kt. 4. High line erythrocytes were also observed to contain slightly more K+ (about 5%) and about 20% less Na+ than low line erythrocytes. 5. These results are discussed in terms of the ion dependency of amino acid transport.     

Sodium-dependent nucleoside transport in mouse leukemia L1210 cells

Nucleoside permeation in L1210/AM cells is mediated by (a) equilibrative (facilitated diffusion) transporters of two types and by (b) a concentrative Na(+)-dependent transport system of low sensitivity to nitrobenzylthioinosine and dipyridamole, classical inhibitors of equilibrative nucleoside transport. In medium containing 10 microM dipyridamole and 20 microM adenosine, the equilibrative nucleoside transport systems of L1210/AM cells were substantially inhibited and the unimpaired activity of the Na(+)-dependent nucleoside transport system resulted in the cellular accumulation of free adenosine to 86 microM in 5 min, a concentration three times greater than the steady-state levels of adenosine achieved without dipyridamole. Uphill adenosine transport was not observed when extracellular Na+ was replaced by Li+, K+, Cs+, or N-methyl-D-glucammonium ions, or after treatment of the cells with nystatin, a Na+ ionophore. These findings show that concentrative nucleoside transport activity in L1210/AM cells required an inward transmembrane Na+ gradient. Treatment of cells in sodium medium with 2 mM furosemide in the absence or presence of 2 mM ouabain inhibited Na(+)-dependent adenosine transport by 50 and 75%, respectively. However, because treatment of cells with either agent in Na(+)-free medium decreased adenosine transport by only 25%, part of this inhibition may be secondary to the effects of furosemide and ouabain on the ionic content of the cells. Substitution of extracellular Cl- by SO4(-2) or SCN- had no effect on the concentrative influx of adenosine.     

Electrophysiological study of L-lysine transport across Triturus proximal tubule: evidence for Na(+)-independent entry and Na(+)-dependent exit.

Cell membrane depolarization induced by intraluminal injection of lysine was entirely independent of the presence of Na+ in Triturus proximal tubule, confirming our previous observation. The amplitude of the depolarization conformed to Michaelis-Menten kinetics regardless of the presence or absence of Na+ in the perfusion solutions. pH of the intraluminal solution had no effect on the electrical response in its range from 5.5 to 8.5. In a Na(+)-free medium, particularly in a Tris-substituted medium, the depolarization induced by a constant concentration of lysine gradually decreased in its size when injection followed by washout of lysine was repetitively tested. The addition of Na+ to the peritubular side after extinction of the responsiveness resulted in a significant restoration of the voltage response to intraluminal lysine. In addition, influx of Na+ from the peritubular fluid into the cells was significantly greater in lysine-loaded tubules than in nonloaded tubules as indicated by a greater rate of increase in intracellular Na+ activity in the presence of ouabain. The data strongly suggest that lysine enters the cells via an electrogenic uniport mechanism and leaves the cells via Na+:amino acid exchange transport mechanism.     

Cell membrane amino acid transport processes in the domestic fowl (Gallus domesticus)

Intestinal absorption of amino acids in the chicken occurs by way of processes which are concentrative, Na+-dependent and dependent upon metabolic energy in the form of ATP. Intestinal transport is carrier-mediated, subject to exchange transport (trans-membrane effects) and is inhibitable by sugars, reagents which inactivate sulfhydryl groups, potassium ion, and by deoxpyridoxine, an anti-vitamin B6 agent. It is stimulated by phlorizin, a potent inhibitor of sugar transport, and in Na+-leached tissue by modifiers of tissue cyclic AMP levels, e.g. theophylline, histamine, carbachol and secretin. Separate transport sites with broad, overlapping specificities function in the intestinal absorption of the various classes of common amino acids. A simple model for these sites includes one for leucine and other neutral amino acids, one for proline, beta-alanine and related imino and amino acids, one for basic amino acids, and one for acidic amino acids. Absorption of amino acids appears to be widespread in occurrence in the digestive tract of the domestic fowl; transport has been reported to be present in the crop, gizzard, proventriculus, small intestine and in the colon. By the end of the first week of life post-hatch, the caecum loses its ability to transport. Similarly, the yolk sac loses its ability by the second day post-hatch. Intestinal transport was noted before hatch and was found to be maximal immediately post-hatch. A requirement for Ca2+ appears to be lost after the first week of life post-hatch. The cationic amino acids appear to be reabsorbed by a common mechanism in the kidney. Transport rates of leucine measured in the intestine or in the erythrocyte were found to cluster about discrete values when many individual chickens were surveyed; such patterns may be an expression of gene differences between individuals. Two lines of chickens have been developed, one high and the other low uptake, through selective breeding based on the ability of individual birds to absorb leucine in erythrocytes. High leucine absorbing chickens were found to be more effective in absorbing lysine and glycine, were more effectively stimulated by Na+, had greater erythrocyte Na+, K+-ATPase activity, and their erythrocytes contained about 20% less Na+ than low line erythrocytes. The underlying genetic difference between these lines may reside at the level of the Na+, K+-ATPase and (or) with a regulatory gene determining carrier copies. Amino acid transport in erythrocytes was noted to be highest in pre-hatch chicks and to diminish during post-hatch development.(ABSTRACT TRUNCATED AT 400 WORDS)     

Increase of Na+ gradient-dependent L-glutamate and L-aspartate transport in high K+ dog erythrocytes associated with high activity of (Na+, K+)-ATPase

As reported previously, some dogs possess red cells characterized by low Na+, high K+ concentrations, and high activity of (Na+, K+)-ATPase, although normal dog red cells contain low K+, high Na+, and lack (Na+, K+)-ATPase. Furthermore, these red cells show increased activities of L-glutamate and L-aspartate transport, resulting in high accumulations of such amino acids in their cells. The present study demonstrated: (i) Na+ gradient-dependent L-glutamate and L-aspartate transport in the high K+ and low K+ red cells were dominated by a saturable component obeying Michaelis-Menten kinetics. Although no difference of the Km values was observed between the high K+ and low K+ cells, the Vmax values for both amino acids' transport in the high K+ cells were about three times those of low ones. (ii) L- and D-aspartate, but not D-glutamate, competitively inhibited L-glutamate transport in both types of the cells. (iii) Ouabain decreased the uptake of the amino acids in the high K+ dog red cells, whereas it was not effective on those in the low K+ cells. (iv) The ATP-treated high K+ cells [(K+]i not equal to [K+]o, [Na+]i greater than [Na+]o) showed a marked decrease of both amino acids' uptake rate, which was almost the same as that of the low K+ cells. (v) Valinomycin stimulated the amino acids' transport in both of the high K+ and the ATP-treated low K+ cells [( K+]i greater than [K+]o, [Na+]o), suggesting that the transport system of L-glutamate and L-aspartate in both types of the cells might be electrogenic. These results indicate that the increased transport activity in the high K+ dog red cells was a secondary consequence of the Na+ concentration gradient created by (Na+, K+)-ATPase.     

An electronic mechanism in the actions of drugs and other cardinal adsorbents. I. Effects of ouabain on the relative affinities of the cell surface beta- and gamma-carboxyl groups for K+, Na+, glycine and other ions

The effects of ouabain on the effectiveness of glycine, Li+, Na+, K+, Rb+, and Cs+ in the external medium in reducing the rate of entry of labeled Cs+ into frog sartorius muscles were studied. The results showed that in the absence of ouabain the effectiveness of glycine and alkali-metal ions in inhibiting labeled Cs+ entry follows the rank order: K+ greater than Cs+, Rb+ greater than Na+, Li+ greater than glycine. Exposure to ouabain in essence reverses this order which then becomes: glycine greater than Li+, Na+ greater than K+, Rb+, greater than Cs+. These results confirm the prediction of the basic electronic interpretation of drug action according to the association-induction hypothesis. In addition, it shows that the action of ouabain on the surface beta- and gamma-carboxyl groups of frog muscle mediating Cs+ entry is quite similar to its action on the cytoplasmic beta- and gamma-carboxyl groups that are the seats of K+ accumulation in the bulk phase cytoplasm as well as to its action on the cell surface beta- and gamma-carboxyl groups responsible for the generation of the resting potential. In all these cases, ouabain acts as an electron-donating cardinal adsorbent (EDC). Finally the marked increase of the binding strength of glycine on the surface beta- and gamma-carboxyl groups was used to explain the primary pharmacodynamic effect of cardiac glycosides in combating heart failure.     

Electrophysiology of L-lysine entry across the brush-border membrane of Necturus intestine

Microelectrode measurements of apical membrane potentials (Va) in absorptive cells of isolated Necturus intestine showed that, in the presence or absence of external Na+, 10 mM lysine added to the mucosal medium caused rapid depolarization followed by slower repolarization of Va. In Na+-free media the effects of 10 mM lysine on Va were abolished by 10 mM leucine which alone had no effect on Va under these conditions. This indicates that uncoupled electrodiffusion of lysine plays little or no role in lysine entry across the brush-border membrane. When external Na+ was greater than 10 mM the maximum depolarization of Va (delta Va') induced by [Lys] ranging from 5 to 30 mM was a simple saturable function of [Lys]. In Na+-free media, the relationship between delta Va' and [Lys] was biphasic. At first, delta Va' increased with increasing [Lys] reaching a maximum at 10 mM lysine. When [Lys] was further increased, delta Va' declined progressively to reach zero or near zero values. A single transport pathway model is proposed to account for rheogenic lysine entry across the brush-border membrane in the presence and absence of Na+. This postulates an amino acid transporter in the membrane with two binding sites. One is an amino acid site specific for the alpha-amino-alpha-carboxyl group. The other is a Na+ site. Neutral amino acids (e.g. leucine) compete with lysine for the amino acid site. The Na+ site has some affinity for the epsilon-amino group of lysine. When external Na+ is high the Na+ site is essentially 'saturated' with Na+ and formation of a mobile complex between an amino acid and the transporter depends in a saturable fashion on amino acid concentration. In Na+-free media or in media containing low [Na+]; at low external [Lys] the epsilon-amino group of a lysine molecule (simultaneously attached to the amino acid site) interacts with the Na+ site to form a mobile complex, as external [Lys] is increased, attachment of different lysine molecules to each site of an increasing number of transporters to form nontransported or poorly transported complexes results in substrate inhibition of the rheogenic lysine transport process.     

Cation and harmaline interactions with Na(+)-independent dibasic amino acid transport system y+ in human erythrocytes and in erythrocytes from a primitive vertebrate the pacific hagfish (Eptatretus stouti).

Transport systems y+, asc and ASC exhibit dual interactions with dibasic and neutral amino acids. For conventional Na(+)-dependent neutral amino acid system ASC, side chain amino and guanido groups bind to the Na+ site on the transporter. The topographically equivalent recognition site on related system asc binds harmaline (a Na(+)-site inhibitor) with the same affinity as asc (apparent Ki range 1-4 mM), but exhibits no detectable affinity for Ha. Although also classified as Na(+)-independent, dibasic amino acid transport system y+ accepts neutral amino acids when Na+ or another acceptable cation is also present. This latter observation implies that the y+ translocation site binds Na+ and suggests possible functional and structural similarities with ASC/asc. In the present series of experiments with human erythrocytes, system y(+)-mediated lysine uptake (5 microM, 20 degrees C) was found to be 3-fold higher in isotonic sucrose medium than in normal 150 mM NaCl medium. This difference was not a secondary consequence of changes in membrane potential, but resulted from Na+ functioning as a competitive inhibitor of transport. Apparent Km and Vmax values for lysine transport at 20 degrees C were 15.2 microM and 183 mumol/l cells per h, respectively, in sucrose medium and 59.4 microM and 228 mumol/l cells per h in Na+ medium. Similar results were obtained with y+ in erythrocytes of a primitive vertebrate, the Pacific hagfish (Eptatretus stouti), indicating that Na(+)-inhibition is a general property of this class of amino acid transporter. At a permeant concentration of 5 microM, the IC50 value for Na(+)-inhibition of lysine uptake by human erythrocytes was 27 mM. Other inorganic and organic cations, including K+ and guanidinium+, also inhibited transport. In parallel with its actions on ASC/asc harmaline competitively inhibited lysine uptake by human cells in sucrose medium. As predicted from mutually competitive binding to the y+ translocation site, the presence of 150 mM Na+ increased the harmaline inhibition constant (Ki) from 0.23 mM in sucrose medium to 0.75 mM in NaCl medium. We interpret these observations as further evidence that y+, asc and ASC represent a family of closely related transporters with a common evolutionary origin.     

Influence of alkali metal cations on the transport of calcium by phosphatidate in multi-phase systems

The effects of alkali metal cations on the rates at which Ca2+ and phosphatidic acid were cotransported from aqueous to hydrocarbon medium were examined. The alkali metal cations remained in the aqueous phase yet specifically influenced the transport of Ca2+ into the hydrocarbon solvent. For the physiological cations, Na+ and K+, there were critical concentration ranges in which small changes in concentration effected sharp changes in transport rates. The maximal rate observed with Na+ was an order of magnitude greater than that with K+; however, unlike Na+, K+ promoted low levels of transport below the critical concentration range. Li+ effected only low levels of transport even at high concentrations, whereas Rb+ and Cs+ induced transport at rates proportional to their concentrations. These results are discussed in terms of a classical ionophore model for the complex composed of a neutral phosphatidic acid dimer bridged by Ca2+.     

Amino acid transport by resealed ghosts from pigeon erythrocytes.

Resealed ghosts from pigeon erythrocytes were shown to haemolyse during incubation in isotonic media with pH values greater than about 7 and high concentrations of Na+ inside the ghosts seemed to enhance this effect. At lower pH values the ghosts were stable but still highly permeable to Na+ and K+, and moderately permeable to sucrose. Under the latter conditions the ghosts transported amino acids in a way qualitatively but not quantitatively similar to intact erythrocytes. The Na+-dependent transport of serine and alanine by the ghosts consisted essentially of an exchange of extracellular for intracellular amino acids, with no significant net flux. In contrast, net fluxes of glycine in the direction of the Na+-concentration gradient across the ghost membrane were demonstrated. However, under one condition a small net influx of glycine occurred against the prevailing Na+-concentration gradient. Unlike Na+-dependent glycine uptake, the uptake of six other amino acids by intact pigeon erythrocytes was not influenced by the nature of the anion present. The significance of these findings in relation to previous work on the Na+-gradient hypothesis of membrane transport is discussed.     

Na- and Cl-dependent glycine transport in human red blood cells and ghosts. A study of the binding of substrates to the outward-facing carrier

Na- and Cl-dependent glycine transport was investigated in human red blood cells. The effects of the carrier substrates (Na, Cl, and glycine) on the glycine transport kinetics were studied with the goal of learning more about the mechanism of transport. The K1/2-gly was 100 microM and the Vmax-gly was 109 mumol/kg Hb.h. When cis Na was lowered (50 mM) the K1/2-gly increased and the Vmax-gly decreased, which was consistent with a preferred order of rapid equilibrium loading of glycine before Na. Na-dependent glycine influx as a function of Na concentration was sigmoidal, and direct measurement of glycine and Na uptake indicated a stoichiometry of 2 Na:1 glycine transported. The sigmoidal response of glycine influx to Na concentration was best fit by a model with ordered binding of Na, the first Na with a high K1/2 (greater than 250 mM), and the second Na with a low K1/2 (less than 10.3 mM). In the presence of low Cl (cis and trans 5 mM), the K1/2-gly increased and the Vmax-gly increased. The Cl dependence displayed Michaelis-Menten kinetics with a K1/2-Cl of 9.5 mM. At low Cl (5 mM Cl balanced with NO3), the glycine influx as a function of Na showed the same stoichiometry and Vmax-Na but a decreased affinity of the carrier for Na. These data suggested that Cl binds to the carrier before Na. Experiments comparing influx and efflux rates of transport using red blood cell ghosts indicated a functional asymmetry of the transporter. Under the same gradient conditions, Na- and Cl-dependent glycine transport functioned in both directions across the membrane but rates of efflux were 50% greater than rates of influx. In addition, the presence of trans substrates modified influx and efflux differently. Trans glycine largely inhibited glycine efflux in the absence or presence of trans Na; trans Na largely inhibited glycine influx and this inhibition was partially reversed when trans glycine was also present. A model for the binding of these substrates to the outward-facing carrier is presented.     

alpha-aminoisobutyrate transport into cells from R3230AC mammary adenocarcinoma. Evidence for sodium ion-dependent and -independent carrier-mediated entry and effects of diabetes.

In the presence of Na+, alpha-aminoisobutyrate was transported by saturable and non-saturable processes into R3230AC mammary tumour cells isolated by enzymic treatment. Eadie-Hofstee analysis for the saturable process gave a curvilinear plot, suggesting that transport occurred by more than one carrier. In the absence of Na+, alpha-aminoisobutyrate was also transported by both saturable and non-saturable processes. This Na+-independent saturable process gave a linear plot according to Eadie-Hofstee analysis: V, 708 +/- 105 pmol/min per 5 X 10(6) cells; Km, 0.36 +/- 0.33 mM (mean +/- S.E.M.). Subtracting alpha-aminoisobutyrate entry in the absence of Na+ from total alpha-aminoisobutyrate uptake (in the presence of Na+) showed the presence of another saturable process (Na+-dependent), accounting for 75% of total alpha-aminoisobutyrate uptake. This component gave a linear Eadie-Hofstee plot: V, 2086 +/- 213; Km, 1.75 +/- 0.16 alpha-(Methylamino)isobutyrate, a substrate specifically taken up by the A system, inhibited 80% of alpha-aminoisobutyrate entry. The presence of both alhpa-(methylamino)isobutyrate and phenylalanine inhibited alpha-aminoisobutyrate entry completely. 2-Aminobicyclo[2.2.1]heptane-2-carboxylate, an analogue specifically taken up by the Na+-independent system, inhibited completely the Na+-independent entry of alpha-aminoisobutyrate. In the presence of Na+, the distribution ratio, which is defined as the amino acid concentration in the intracellular space divided by that in the incubation medium for alpha-aminoisobutyrate, at 90 min was 19, and in the absence of Na+ at 60 min was 5. These concentrative processes were sensitive to the metabolic inhibitor pentachlorophenol. The Na+-dependent, but not the Na+-independent, alpha-aminoisobutyrate uptake was increased in cells from diabetic rats. This was primarily due to an increase in the V for the Na+-dependent component (164%) with no effect on the Km. We conclude, therefore, that alpha-aminoisobutyrate entry into cells from this mammary tumour is mediated by two transport systems, one Na+-dependent and another Na+-independent. Furthermore, the Na+-dependent component of alpha-aminoisobutyrate is sensitive to alterations of insulin in vivo.     

Characteristics and adaptive regulation of glycine transport in cultured glial cells.

The transport of glycine in C6 glioma cells takes place mainly in a heterogeneous Na+-dependent manner which can be resolved into different components. A Na+- and Cl(-)-dependent component with high affinity for glycine is pH-sensitive and inhibited by sarcosine, all these characteristics corresponding to System Gly. The low-affinity component of the transport of glycine can be discriminated as two components, namely System A and System ASC. The main proportion of glycine transport through the low-affinity system is carried out by the ASC System, which appears to be constitutively expressed by the cells. The adaptive response of the low-affinity Na+-dependent transport of glycine to amino acid deprivation was identified with System A on the basis of its ion-dependency, pH-sensitivity and by inhibition analysis. The possible physiological role of the high- and low-affinity components of the transport system for glycine in glial cells is discussed.     

Further studies on amino acid transport in murine P388 leukemia cells in vitro. Presence of system y+

The transport of glycine and L-lysine into murine P388 leukemia cells has been examined. Glycine transport appears to be shared by both systems A and ASC in P388 cells. Glycine transport is Na+-dependent and is effectively blocked by alpha-(methylamino)isobutyric acid, threonine and alanine but only a marginal reduction in transport is seen with 100-fold excess cold 2-aminobicyclo[2,2,1]heptane-2-carboxylic acid. System gly is not expressed in P388 cells. Lysine is largely transported by a Na+-independent, pH-insensitive system with a Km of 0.079 mM. Lysine transport is relatively unaffected by the addition of 100-fold excess cold alpha-(methylamino)isobutyric acid, 2-aminobicyclo[2,2,1]heptane-2-carboxylic acid and the anionic amino acids, L-glutamate and L-aspartate. A partial inhibition of lysine transport was observed with L-threonine and L-leucine while L-arginine and L-histidine radically decreased lysine transport. Lysine appears to be transported by a system similar to the system y+ seen in cultured human fibroblasts, Ehrlich ascites cells, and hepatoma cell lines.     

Sodium-dependent, concentrative nucleoside transport in Walker 256 rat carcinosarcoma cells

Nucleoside transport in Walker 256 cells was reexamined using formycin B, a nonmetabolized analog of inosine. In the presence of dipyridamole to inhibit the equilibrative (facilitated diffusion) transporter previously described in these cells, the initial rate of uptake of 1 microM formycin B was 10-fold greater in Na(+)-containing medium than in Na(+)-free medium. In the presence of Na+ and dipyridamole the intracellular concentration of formycin B exceeded that in the medium within one min and was 6-fold greater than that of the medium by 5 min. Na(+)-dependent transport of formycin B was inhibited by low concentrations of inosine, but not thymidine. Furthermore, Na(+)-dependent transport of uridine, but not thymidine, was apparent in the presence of dipyridamole. These data indicate that Walker 256 cells have, in addition to the previously described equilibrative transporter, a concentrative nucleoside transporter. The specificity of this transporter appears to correspond to one of the two Na(+)-dependent transporters previously described in mouse intestinal epithelial cells.     

Regulation of K+ transport in tomato roots by the TSS1 locus. Implications in salt tolerance

The tss1 tomato (Lycopersicon esculentum) mutant exhibited reduced growth in low K+ and hypersensitivity to Na+ and Li+. Increased Ca2+ in the culture medium suppressed the Na+ hypersensitivity and the growth defect on low K+ medium of tss1 seedlings. Interestingly, removing NH4+ from the growth medium suppressed all growth defects of tss1, suggesting a defective NH4(+)-insensitive component of K+ transport. We performed electrophysiological studies to understand the contribution of the NH4(+)-sensitive and -insensitive components of K+ transport in wild-type and tss1 roots. Although at 1 mm Ca2+ we found no differences in affinity for K+ uptake between wild type and tss1 in the absence of NH4+, the maximum depolarization value was about one-half in tss1, suggesting that a set of K+ transporters is inactive in the mutant. However, these transporters became active by raising the external Ca2+ concentration. In the presence of NH4+, a reduced affinity for K+ was observed in both types of seedlings, but tss1 at 1 mm Ca2+ exhibited a 2-fold higher Km than wild type did. This defect was again corrected by raising the external concentration of Ca2+. Therefore, membrane potential measurements in root cells indicated that tss1 is affected in both NH4(+)-sensitive and -insensitive components of K+ transport at low Ca2+ concentrations and that this defective transport is rescued by increasing the concentration of Ca2+. Our results suggest that the TSS1 gene product is part of a crucial pathway mediating the beneficial effects of Ca2+ involved in K+ nutrition and salt tolerance.     

Reversed transport of amino acids in Ehrlich cells.

Gramicidin induces a marked Na+-dependent efflux of amino acids from Ehrlich cells. In absence of Na+, gramicidin does not alter the efflux. In presence gramicidin, glycine efflux is inhibited by methionine and less so by leucine. Glycine efflux caused by HgCl2 is neither Na+ dependent nor inhibitable by amino acids. Neither efflux of inositol which is transported by an Na+-dependent route, nor efflux of several other solutes which are transported by Na+-independent routes, is affected by gramicidin. The antibiotic appears to permit a reversal in the direction of of the operation of the Na+-dependent amino acid transport system. The increased efflux is partly, but not entirely, due to an increase in the cellular Na+ concentration and a reduction of the electrochemical potential difference for Na+.     

Stimulation of glucose transport in skeletal muscle by the sodium ionophore monensin

The tissue/medium distribution of the nonmetabolized glucose analog 3-O-methyl-D-glucose was measured in mouse diaphragm muscle and related to changes in 45Ca influx, Na+ content and Na+-pump activity. In the presence of external Ca2+ the sodium ionophore monensin greatly increased cellular Na+ content (and decreased K+ content) although 86Rb uptake, reflecting Na+-pump activity was increased. Concomitantly, 45Ca influx was stimulated, presumably through activation of Na+-Ca2+ exchange. In parallel to the rise in Ca2+ influx sugar transport was also increased. Sugar transport was also increased by monensin in the nominal absence of external Ca2+, when Ca2+ influx was minimal. To test if monensin releases Ca2+ from intracellular storage sites in the absence of external Ca2+, the ionophore was added to medium perfusing rat hind limb preparations and the total Ca content of muscle mitochondria was determined. When Ca2+ was present in the perfusate, monensin increased the mitochondrial Ca content. In the absence of Ca2+, the mitochondrial Ca content was lower and was further depressed by monensin, suggesting that elevation of internal Na+ by monensin may increase mitochondrial Ca2+ loss via activation of Na+-Ca2+ exchange across the mitochondrial membrane. The above results are consistent with the effect of monensin on sugar transport being due to alterations in Ca2+ distribution. They support the earlier conclusion that regulation of sugar transport in muscle is Ca2+ dependent.     

Sodium-dependent glucose transport by cultured proximal tubule cells

The cotransport of sodium ion and alpha-methyl glucose, a non-metabolized hexose, was studied in rabbit proximal tubule cells cultured in defined medium. The rate of uptake of alpha-methyl glucose shows saturation kinetics, in which Km, but not Vmax, is dependent upon the Na+ concentration in the medium. The transport system was found to be of the high-affinity type, characteristic of the straight portion of the proximal tubule. Analysis of the rates of initial uptake within the context of a generalized cotransport model, suggests that two Na+ ions are bound in the activation of the hexose transport. The steady-state level of accumulation of alpha-methyl glucose also depends upon sodium concentration, consistent with the initial rate findings. The uptake of alpha-methyl glucose is inhibited by other sugars with the relative potencies of D-glucose greater than alpha-methyl glucose greater than D-galactose = 3-O methylglucose. L-Glucose, D-fructose, and D-mannose show no inhibition. Phlorizin inhibits the alpha-methyl glucose uptake with a Ki of 9 X 10(-6) M. Ouabain (10(-3) M) decreases the steady-state alpha-methyl glucose accumulation by 60%. In the absence of sodium, the accumulation of alpha-methyl glucose is 7-fold less than at 142 mM Na+, reaching a level comparable to the sodium-independent accumulation of 3-O-methyl-D-glucose. These findings are similar to those observed in the proximal tubule of the intact kidney.     

The mechanism of insulin stimulation of (Na+,K+)-ATPase transport activity in muscle

Since the mechanism underlying the insulin stimulation of (Na+,K+)-ATPase transport activity observed in multiple tissues has remained undetermined, we have examined (Na+,K+)-ATPase transport activity (ouabain-sensitive 86Rb+ uptake) and Na+/H+ exchange transport (amiloride-sensitive 22Na+ influx) in differentiated BC3H-1 cultured myocytes as a model of insulin action in muscle. The active uptake of 86Rb+ was sensitive to physiological insulin concentrations (1 nM), yielding a maximum increase of 60% without any change in 86Rb+ permeability. In order to determine the mechanism of insulin stimulation of (Na+,K+)-ATPase activity, we demonstrated that insulin also stimulates passive 22Na+ influx by Na+/H+ exchange transport (maximal 200% increase) and an 80% increase in intracellular Na+ concentration with an identical time course and dose-response curve as insulin-stimulated (Na+,K+)-ATPase transport activity. Incubation of the cells with high [Na+] (195 mM) significantly potentiated insulin stimulation of ouabain-inhibitable 86Rb+ uptake. The ionophore monensin, which also promotes passive Na+ entry into BC3H-1 cells, mimics the insulin stimulation of ouabain-inhibitable 86Rb+ uptake. In contrast, incubation with amiloride or low [Na+] (10 mM), both of which inhibit Na+/H+ exchange transport, abolished the insulin stimulation of (Na+,K+)-ATPase transport activity. Furthermore, each of these insulin-stimulated transport activities displayed a similar sensitivity to amiloride. These results indicate that insulin stimulates a large increase in Na+/H+ exchange transport and that the resulting Na+ influx increases the intracellular Na+ concentration, thus activating the internal Na+ transport sites of the (Na+,K+)-ATPase. This Na+ influx is, therefore, the mediator of the insulin-induced stimulation of membrane (Na+,K+)-ATPase transport activity classically observed in muscle.