Endurance training increases skeletal muscle lactate transport

Lactate accumulation in skeletal muscle is reduced after a period of endurance training. Explanations for this phenomena include the increased oxidative capacity of the muscle, a reduction in lactate production, and increased lactate clearance. Muscle membrane transport of lactate can be seen to be a fundamental aspect of such clearance, and transmembrane lactate flux may well be an important aspect of the training response in skeletal muscle. Therefore, the lactate transport capacity in skeletal muscle sarcolemmal membranes in endurance-trained and sedentary rats was investigated. Training consisted of 6 weeks of progressively increased treadmill exercise. Twenty-four hours before being killed, both the trained and sedentary animals completed a brief exercise bout. Studies of lactate transport (zero-trans) were conducted using highly purified sarcolemmal vesicles. When low concentrations of L-lactate (1 mm) were used a 59.4% increase in lactate transport was observed (P < 0.05). However, when a high concentration of lactate (50 mm) was used no change in lactate transport was found (P > 0.05). Several interpretations are possible for these observations: (1) that there is an alteration in the K_m but not the V_max of the lactate transport system in skeletal muscle membranes; and (2) that specific changes occur in selected isoforms of the lactate transport protein which may co-exist in muscle.     

Current aspects of lactate exchange: lactate/H+ transport in human skeletal muscle

Skeletal muscle is capable of producing and releasing large amounts of lactate and at the same time taking up lactate and using it as a respiratory fuel. The release and uptake of lactate both involve transmembrane transport, which is mediated mainly by a membrane protein called the monocarboxylate transporter (MCT). MCTs mediate membrane transport with an obligatory 1:1 coupling between lactate and H⁺ fluxes, and is therefore of great importance for pH regulation, especially during intense muscle activity. The total lactate and H⁺ transport capacity is higher in membranes from oxidative fibers than in membranes from more glycolytic fibers. There are two isoforms of MCT present in skeletal muscle, MCT1 and MCT4. In human muscle samples, there is a positive correlation between the proportion of type I fibers and MCT1 density. In contrast, the MCT4 density in human muscle is independent of fiber type and displays a large interindividual variation. Although the two isoforms have identical transport kinetics (K _m), they may have different roles in muscle. MCT1 and MCT4 respond differently to a high-intensity training session, which suggests that these two isoforms are regulated differently. Electronic Publication     

Exercise-induced regulation of phospholemman (FXYD1) in rat skeletal muscle: implications for Na+/K+-ATPase activity

Background: Na⁺/K⁺‐ATPase activity is upregulated during muscle exercise to maintain ionic homeostasis. One mechanism may involve movement of α‐subunits to the outer membrane (translocation). Aim: We investigated the existence of exercise‐induced translocation and phosphorylation of phospholemman (PLM, FXYD1) protein in rat skeletal muscle and exercise‐induced changes in V_max and K_m for Na⁺ of the Na⁺/K⁺‐ATPase. Methods: Two membrane fractionation methods and immunoprecipitation were used. Results: Both fractionation methods revealed a 200–350% increase in PLM in the sarcolemma after 30 min of treadmill running, while the phosphorylation of Ser‐68 of PLM appeared to be unchanged. Exercise did not change V_max or K_m for Na⁺ of the Na⁺/K⁺‐ATPase in muscle homogenate, but induced a 67% increase in V_max in the sarcolemmal giant vesicle preparation; K_m for Na⁺ remained constant. The main part of the increase in V_max is related to a 36–53% increase in the level of α‐subunits; the remainder may be related to increased PLM content. Similar results were obtained with another membrane purification method. In resting muscle, 29% and 32% of α₁‐ and α₂‐subunits, respectively, were co‐immunoprecipitated by PLM antibodies. In muscle homogenate prepared after exercise, immunoprecipitation of α₁‐subunits was increased to 227%, whereas the fraction of precipitated α₂ remained constant. Conclusion: Exercise translocates PLM to the muscle outer membrane and increases its association with mainly the α₁‐subunit, which may contribute to the increased V_max of the Na⁺/K⁺‐ATPase.     

Transepithelial dipeptide (glycylsarcosine) transport across epithelial monolayers of human Caco-2 cells is rheogenic

Net transepithelial transport (and cellular accumulation) of the dipeptide glycylsarcosine (Gly-Sar), across the apical membrane of human intestinal Caco-2 epithelia, is driven by a proton gradient (Na⁺-free conditions) and displays saturation kinetics (K_m 17.4±5.1 mM, V_max of 92.8±15.6 nmol.cm^–2.h^–1). Net Gly-Sar transport is associated with the stimulation of an inward short-circuit current (I_sc). This dipeptide-stimulated I_sc is observed in both Na⁺-containing and Na⁺-free conditions, is stimulated by apical acidity, and displays saturation kinetics (in Na⁺-free media at apical pH 6.0, K_m of 13.6±4.5 mM and a V_max of 284.1±39.3 nmol.cm^–2.h^–1). The maximal capacities of Gly-Sar transport and I_sc suggest a dipeptide/proton stoichiometry greater than unity (13).     

Effect of starvation on neutral amino acid transport in isolated small-intestinal cells from guinea pigs

The effects of starvation on neutral amino acid transport were examined in isolated enterocytes. Starvation stimulated L-alanine transport by the Na⁺-dependent system A and the Na⁺-independent system L without producing any changes in either the Na⁺-dependent systems ASC or the passive non-mediated uptake. Starvation produces a twofold increase in V _max of system A without any change in K _t. Starvation produces an increase in V _max of system L of 1.7 times without any change in K _t. Activation of systems A and L by starvation was reversible with subsequent refeeding. The effects of a series of amino acids on systems A and L were evaluated. A different inhibition pattern was found in starved animals as compared to controls. Starvation increases Na⁺-dependent L-alanine uptake and Na⁺-independent cycloleucine uptake by small-intestinal brushborder membrane vesicles. These results suggest that starvation stimulates amino acid transport across the apical plasma membrane of the enterocytes by inducing specific carrier units.     

High salt alone does not influence the kinetics of the Na+-H+antiporter

During a high-salt diet, tubular sodium reabsorption is decreased. This study concerns the effect of a high-salt diet on the proximal tubular (PT) Na⁺influx pathways. Brush-border membrane vesicles (BBMV) were prepared from rats on normal-salt (NS) and rats on high-salt (HS) diets. The initial uptake rates of Na⁺were the same in NS and HS rats, both in the absence and the presence of 1 mm amiloride. V_max and K_m for the amiloride-sensitive Na⁺/H⁺antiporter were also the same in the NS (V_max 3.69 ± 0.31 nmol mg prot^-110 s^-1, K_m 6.13 ± 0.58 mm ) and HS groups (V_max 3.54 ± 0.28 nmol mg prot^-110 s^-1, K_m 6.18 ± 0.64 mm ). There was no difference in the initial uptake rates of the Na⁺-glucose and the Na⁺-alanine symporters in NS and HS. V_max and K_m for the l -dopa-Na⁺symporter were also the same in NS (V_max 72 ± 2.5 pmol mg prot^-120 s^-1, K_m 98 ± 14 μm ) and HS groups (V_max 78 ± 6.0 pmol mg prot^-120 s^-1, K_m 106 ± 4 μm ). In summary, HS diet does not change the kinetics of the Na⁺transporters in the brush-border membrane of PT cells.     

Pathways of phosphate transport in chick jejunum

The effect of vitamin D₃ on intestinal phosphate (P_i) absorption was studied in everted sacs prepared from jejunum of either vitamin D-deficient (–D) or vitamin D-replete (+D) chicks. Vitamin D₃ stimulates the maximal velocity (V _max) of a mucosal active P_i transport mechanism from 125 to 314 nmol·min^–1·g^–1 tissue.K _m of this process remains virtually unchanged (–D: 0.15 mmol·l^–1; + D: 0.18 mmol·l^–1).Active P_i entry into the epithelium depends on extracellular Na⁺. Reduction of buffer Na⁺ reducesV _max in the + D group to 182 nmol·min^–1·g^–1 tissue but has no significant effect in the –D animals (V _max=105 nmol·min^–1·g^–1 tissue). In this group, the predominant effect of Na⁺ substitution is a shift ofK _m to 1.13 mmol·l^–1, whileK _m in the +D group is changed only to 0.53 mmol·l^–1.Transeptithelial P_i transport in the + D group involves the mucosal phosphate pump and hence an intracellular pathway, proceeding at a rate of 48 nmol·min^–1·g^–1 tissue. This is in contrast to –D P_i transfer (8 nmol·l^–1·g^–1 tissue) which is by a diffusional, Na⁺-insensitive, and presumably paracellular pathway.Transepithelial calcium transport (–D: 3.3 nmol·min^–1·g^–1; + D: 7.6 nmol·min^–1·g^–1 tissue) does not require the presence of extracellular Na⁺ and apparently involves pathways different from those of the P_i absorptive system.Presented in part at the Annual Meeting of the Austrian Biochemical Society, Salzburg, September 1978     

Muscle pH regulation: role of training

In most cell types, including resting skeletal muscle fibers, internal pH (pH_i) is kept constant at a relatively alkaline level. The high pH_i is obtained in spite of a chronic acid load resulting from cellular metabolism and passive influx of protons driven by electrochemical forces. Regulation of pH_i depends on continuous activity of membrane transport systems that mediate an outflux of H⁺ (or bicarbonate influx), whereby the acid load is counterbalanced. The transporters involved in muscle pH regulation at rest are the Na⁺/H⁺ exchange system as well as the Na⁺-dependent and Na⁺-independent Cl^–bicarbonate transport systems. The Na⁺/H⁺ exchanger seems to be active at resting pH_i levels in skeletal muscle. Therefore, pH homeostasis in skeletal muscle most likely involves an equilibrium between counter-directed H⁺ fluxes. A minor fraction of H⁺ release during intense exercise is mediated by the Na⁺/H⁺ exchanger. The capacity of this system is increased with training and hypoxia in rat skeletal muscle. The dominant acid extruding system associated with intense exercise is the lactate/H⁺ co-transporter. It has been demonstrated that the capacity of the lactate/H⁺ co-transporter of rat skeletal muscle is upregulated with training and chronic electrical stimulation, and that it is reduced upon denervation and hindlimb unweighting. Moreover, athletes can have an elevated lactate/H⁺ co-transport capacity, whereas the thigh muscle of spinal cord-injured individuals has a lower transport capacity than the one of healthy untrained subjects. Thus, it appears that the capacity of the lactate/H⁺ transporter is affected by the level of muscle activity in both rats and humans. In addition, the rate of H⁺ release from muscle may also be influenced by capillarization and local blood flow. Finally the resulting pH displacement during acid accumulation is determined by the cellular buffer capacity, which may also undergo adaptive changes.     

Proton transport mechanism in the cell membrane of Xenopus laevis oocytes

Mechanisms of H⁺ transport across the plasma cell membrane of prophase-arrested oocytes of Xenopus laevis were investigated by testing the effect of ion substitutions and inhibitors on cytoplasmic pH (pH_i), membrane potential (V _m) and membrane resistance (R _m). During superfusion with control solution of pH=7.4, pH_i was 7.49±0.12 (n=15), V _m was –61.9±7.8 mV (n=34) (cytoplasm negative), and R _m was 2.9±1.5 M (n=19). These data confirm that H⁺ ions are not distributed at electrochemical equilibrium. By following pH_i during recovery of the oocytes from an acid load (20 mmol/l NH₄Cl) in the presence and absence of extracellular Na⁺ or amiloride (1 mmol/l), a Na/H exchanger was identified. On the basis of the known Na⁺ gradient across the cell membrane, this transporter could suffice to generate the observed H⁺ disequilibrium distribution. Utilizing blockers or ion-concentration-step experiments no evidence was obtained for an ATP-driven H⁺ pump or for passive acid/base transporters such as H⁺ conductances or Na⁺ (HCO ₃ ^– )₃ cotransport. The membrane depolarization observed in response to extracellular acidification appeared to result from a pH-dependent, Ba²⁺-inhibitable K⁺ conductance.     

The regulation of L-proline transport by insulin-like growth factor-I in human osteoblast-like SaOS-2 cells

The effect of insulin-like growth factor-I on amino acid transport was studied by measuring the uptake of tritiated L-proline in the cultured human osteoblast-like SaOS-2 cells. The uptake of L-proline was supported by both transport system A, ASC and Gly and by Na⁺-dependent amino acid transport system A, and by Na⁺-independent system L. The initial rate of total L-proline uptake as a function of concentration showed saturation and obeyed Michaelis-Menten kinetics with Michaelis constant (K _m) and maximum velocity (V _max) values of 1.87 mM and 8.89 nmol⋅(mg protein)⁻¹⋅(3 min)⁻¹, respectively. Na⁺-dependent L-proline uptake was significantly stimulated by insulin-like growth factor-I in a time- and concentration-dependent manner. Kinetic analysis showed that insulin-like growth factor-I enhanced transport activity by increasing the V _max of transport without significant changes in the affinity (K _m) of the carrier for the substrate. The increase in transport activity was significantly reduced by cycloheximide. The stimulated increment above basal L-proline uptake was completely inhibited by α-(methylamino) isobutyric acid, suggesting that only system A was affected by insulin-like growth factor-I. Na⁺-dependent L-proline uptake was also stimulated by insulin-like growth factor-II and insulin-like growth factor-I analogues. The insulin-like growth factor-I-stimulated L-proline uptake was inhibited by one of its binding protein, insulin-like growth factor binding protein-4, in a concentration-dependent manner. Received: 15 January 1996/Accepted 21 February 1996     

Selection of a lysine-resistant CHO-K1 mutant with reduced amino acid transport through multiple systems

High levels ofl-lysine were used to select for resistant variants of Chinese hamster ovary (CHO-K1) cells. Surviving colonies were screened for altered lysine transport and two with reduced uptake were picked. Clone CH-K^r, derived from the more severely affected colony, was analyzed in detail. In starved cells theV _max of lysine uptake in CH-K^r was half that of CHO whileK _m was unaltered. The intracellular pool of lysine, a substrate of cationic amino acid transport system y⁺, was significantly lower in CH-K^r. However, transport and pools of other amino acids, which are not substrates of y⁺, were also reduced in CH-K^r, as was the internal sodium concentration, while hexose import was increased. It appears that the mutation in CH-K^r is pleiotropic, affecting some general aspects of amino acid transport.     

Effect of repetitive stimulation on cell volume and its relationship to membrane potential in amphibian skeletal muscle

The effect of electrical stimulation on cell volume, V _c, and its relationship to membrane potential, E _m, was investigated in Rana temporaria striated muscle. Confocal microscope xz-plane scanning and histology of plastic sections independently demonstrated significant and reversible increases in V _c of 19.8±0.62% (n=3) and 27.1±8.62% (n=3), respectively, after a standard stimulation protocol. Microelectrode measurements demonstrated an accompanying membrane potential change, ΔE _m, of +23.6±0.98 mV (n=3). The extent to which this ΔE _m might contribute to the observed changes in V _c was explored in quiescent muscle exposed to variations in extracellular potassium concentration, [K⁺]_e. E _m and V _c varied linearly with log [K⁺]_e and [K⁺]_e, respectively, in the range 2.5–15 mM (R ²=0.99 and 0.96), and these results were used to reconstruct an approximately linear relationship between V _c and E _m (ΔV _c=0.85E _m+68.53; R ²=0.99) and hence derive the ΔV _c expected from the ΔE _m during stimulation. This demonstrated that both the time course and magnitude of the increase and recovery of V _c observed in active muscles could be reproduced by the corresponding [K⁺]_e-induced depolarisation in quiescent muscles, suggesting that the depolarisation associated with membrane activity makes a substantial contribution to the cell swelling during exercise. Furthermore, conditions of Cl⁻ deprivation abolished the relationship between E _m and V _c, supporting a mechanism in which the depolarisation of E _m drives a passive redistribution of Cl⁻ and hence cellular entry of Cl⁻ and K⁺ and an accompanying, osmotically driven, increase in V _c.     

Lactate transport in isolated mouse muscles studied with a tracer technique—kinetics,stereospecificity, pH dependency and maximal capacity

Lactate transport across the sarcolemma of isolated mouse muscles was studied with a ¹⁴C tracer technique. The cellular tracer uptake could be inhibited by unlabelled l -lactate (and pyruvate) and to a lesser extent by d-lactate. The stereospecific fraction had a K_m of 3.5 mm, and made up 50% of the total transport. The tracer uptake was unaffected by 0.05 mM DIDS and 0.2 mM amiloride, but was inhibited by cinnamate (K_i= 8 mm) and PCMBS (K_i= 0.8 mm). With high concentrations of the latter inhibitor compounds or with high concentrations of unlabelled l -lactate, the tracer uptake was inhibited 80%, which indicates that the main part of the transport involves facilitated diffusion. The remaining fraction (20%) was non-saturable, reduced at high pH, and could not be inhibited; it is probably mediated by diffusion of undissociated lactic acid. Lactate transport was pH-dependent, which is consistent with a lactate-H⁺ symport. The maximal transport capacity, as calculated from the pH changes measured with pH-sensitive micro-electrodes while the lactate gradient was 30 mM, was 11.8 mmol kg^-1 min^-1 (pH 6.2).     

Mechanical characteristics of chemically skinned guinea-pig taenia coli

Strips of intact and chemically skinned (Triton X-100) taenia coli were mounted for isometric and quick-release experiments at 23°C. Active force increased in repeated high-K⁺ induced contractures in the intact muscle. Stable maximal force was 313±24 mN/mm² (n=6). The skinned preparations activated by Ca²⁺, at 2 mM Mg²⁺, 3.2 mM MgATP and ionic strength 0.085 M, gave half maximal force atpCa=5.62±0.4 and a maximal force (63±8 mN/mm²) atpCa=4.5 (20–25 of the control K⁺-responses prior to skinning but about 60% of the first K⁺-response). Force-velocity relations were obtained from intact muscles and from the same muscles chemically skinned and activated at optimal Ca²⁺. Maximal shortening velocity (V _max) was unaltered in the skinned preparation compared to the intact muscle (0.138±0.011 vs 0.140±0.006 L/s) indicating similar kinetics of actomyosin interaction. In the intact muscle a decrease inV _max was found when the Ca²⁺ concentration was reduced. Calmodulin (1M) increased Ca²⁺ sensitivity (by about 0.6 log units) of the skinned preparation but at optimal Ca²⁺ caused no alteration in isometric force orV _max Apreliminary report of some of the results presented here was given at the Scandinavian Physiology Society Meeting in Århus, November 1981. Arner A, Hellstrand P (1982) Acta Physiol Scand (Abstract) 114: 38 A.     

Identification of sodium-dependent and sodium-independent dicarboxylate transport systems in rat liver basolateral membrane vesicles

The mechanisms involved in the hepatocellular uptake of Krebs-cycle intermediates were investigated in isolated basolateral (sinusoidal and lateral) rat liver plasma membrane (blLPM) vesicles. An inwardly directed Na⁺ gradient markedly stimulated uptake of 2-oxoglutarate and succinate into voltage- and pH-clamped blLPM vesicles. This Na⁺-dependent portion of the dicarboxylate uptake was characterized by (a) saturability with increasing substrate concentrations (K _m= 6.4–10 mM; V _max0.2 nmol min^–1 mg protein^–1), (b) cisinhibition by lithium (10 mM), other Krebs-cycle dicarboxylates (1 mM) and DIDS (4,4-diisothiocyanostilbene-2,2-disulfonic acid; 1 mM) but not by sulphate, monocarboxylates, oxalate, acidic amino acids, bile salts and probenecid, (c) stimulation by an intravesicular negative K⁺-diffusion potential indicating electrogenic [(Na⁺) _n>2-succinate] cotransport, and (d) a pH optimum for transport between 7.0 and 7.5. In the absence of Na⁺, an inside alkaline pH gradient also markedly stimulated 2-oxoglutarate uptake. This pH-gradient-driven 2-oxoglutarate uptake was insensitive to lithium, but could also be inhibited by DIDS and succinate. Furthermore, saturation kinetics demonstrated K _m ( 34 mM) and V _max ( 0.8 nmol min^–1 mg protein^–1) values that were clearly different from those of the Na⁺-dependent uptake system. These results indicate the occurrence of two separate dicarboxylate transport systems along the sinusoidal border of hepatocytes, one being a Na⁺-dicarboxylate symporter and the other representing an anion-exchange system.     

Glucose transport in Leishmania donovani promastigotes

Mid-log phase Leishmania donovani promastigotes accumulated 2-deoxy-d-glucose (2-DOG) via a carrier mediated transport system, maintaining an apparent K_m of 24.4 μM and a V_max of 3.12 nmol mg^?1 protein min^?1. d-Glucose but not l-glucose competitively inhibited the 2-DOG transport with an apparent K_i of 18.7 μM. Transport of 2-DOG was inhibited by 2,4-dinitrophenol and NaN₃. The parasites maintained a 2-DOG gradient of at least 79 fold across the surface membrane, demonstrating the active nature of the transport system.     

Mn2+ transport in the unicellular cyanobacterium Anacystis nidulans

Mn²⁺ uptake by wild type of Anacystis nidulans IU 625 follows Michaelis-Menten type kinetics with apparent saturation at 27 μmol/l of Mn²⁺. Lineweaver-Burk plot of the data reveals a Km of 25 μmol/l and V_max of 1.2 nmol/mg protein · min. The uptake values at different pH (pH 4.0 to 9.0) exhibit a characteristic profile with an optimum at pH 6.0. The Mn²⁺ uptake rate in the dark is found to be similar to that in the light suggesting that energy for transport may not be directly derived from light reactions. The system seems to be specific for Mn²⁺ transport since simultaneous addition of Mg²⁺ partially inhibited the uptake in a competitive manner. Further, the alterations in Km and V_max values of mutants with respect to that of wild type strains suggest a genetic control of the transport system.     

Muscle lactate transport studied in sarcolemmal giant vesicles: dependence on fibre type and age

Lactate transport was studied in sarcolemmal giant vesicles obtained from rat or rabbit skeletal muscle. With this technique it is possible to obtain quantitative information on sarcolemmal transport characteristics. In equilibrium exchange experiments with 10, 30 and 60 mm lactate, vesicles from ‘red’ rat muscles had a 50% higher lactate transport capacity than vesicles from ‘white’ muscles. Giant vesicles made from rabbit red muscles had a 39% higher lactate transport capacity than vesicles from white muscles. These differences probably reflect a different number of lactate transporters, whereas the lactate affinity in red and white muscles are identical. Lactate transport capacity decreased with age. Sarcolemmal giant vesicles made from 22-month-old rats had a 28% lower transport capacity than vesicles from 2-month old rats. In absolute terms, the initial exchange flux with 30 mm lactate was 92 and 127 pmol cm^-2 sec^-1 for old and young rats, respectively. In supplementary studies in which microelectrode measurements were made in single mouse muscle fibres, it was shown that the cellular acidification rate due to lactate incubation, was 38% lower in fibres from 18-month old mice than in fibres from 2-month old mice.     

Role of K+ channels in the regulation of electrical spontaneous activity of the mouse small intestine

The roles of K⁺ channels in the regulation of slow waves and pacemaker potentials recorded from mouse small intestine were investigated using intracellular recording techniques in the presence of nifedipine. Iberiotoxin (0.1 μM) and charybdotoxin (0.1 μM) had no effect on the generation of slow waves recorded from circular smooth muscle cells. Apamin (0.3 μM) depolarized the membrane and decreased the amplitude of early, rapid repolarization of slow waves, without altering the amplitude, frequency, duration, or maximum rate of rise of the initial upstroke phase (dV/dt _max). The early, rapid repolarization was enhanced by phenylephrine (15 μM). 4-Aminopyridine (4-AP, 5 mM) depolarized the membrane and increased the amplitude and dV/dt _max of slow waves. Both apamin and 4-AP depolarized the membrane and decreased the amplitude and dV/dt _max of pacemaker potentials recorded from interstitial cells of Cajal distributed in the myenteric region (ICC-MY). Membrane depolarization with a high-K⁺ solution decreased the amplitude and dV/dt _max of slow waves. These results suggest that apamin-sensitive K⁺ conductance and 4-AP-sensitive K⁺ conductance may contribute to the resting membrane potential of circular smooth muscle cells. The early, rapid repolarization of slow waves appears to result from the opening of apamin-sensitive K⁺ conductance. 4-AP-sensitive K⁺ conductance is likely to be activated in the initial upstroke component (primary component) of slow waves. In ICC-MY, membrane depolarization induced by apamin or 4-AP may result from electrotonic spread from smooth muscle cells.     

Effects of intra- and extracellular H+ and Na+ concentrations on Na+-H+ antiport activity in the lacrimal gland acinar cells

Kinetic properties of the Na⁺-H⁺ antiport in the acinar cells of the isolated, superfused mouse lacrimal gland were studied by measuring intracellular pH (pH_i) and Na⁺ activity (aNa_i) with the aid of double-barreled H⁺- and Na⁺-selective microelectrodes, respectively. Bicarbonate-free solutions were used throughout. Under untreated control conditions, pH_i was 7.12±0.01 and aNa_i was 6.7±0.6 mmol/l. The cells were acid-loaded by exposure to an NH ₄ ⁺ solution followed by an Na⁺-free N-methyl-d-glucamine (NMDG⁺) solution. Intracellular Na⁺ and H⁺ concentrations were manipulated by changing the duration of exposure to the above solutions. Subsequent addition of the standard Na⁺ solution rapidly increased pH_i. This Na⁺-induced increase in pH_i was almost completely inhibited by 0.5 mmol/l amiloride and was associated with a rapid, amiloride-sensitive increase in aNa_i. The rate of pH_i recovery induced by the standard Na⁺ solution increased in a saturable manner as pH_i decreased, and was negligible at pH_i 7.2–7.3, indicating an inactivation of the Na⁺-H⁺ antiport. The apparent K _m for intracellular H⁺ concentration was 105 nmol/l (pH 6.98). The rate of acid extrusion from the acid-loaded cells increased proportionally to the increase in extracellular pH. Depletion of aNa_i to less than 1 mmol/l by prolonged exposure to NMDG⁺ solution significantly increased the rate of Na⁺-dependent acid extrusion. The rate of acid extrusion increased as the extracellular Na⁺ concentration increased following Michaelis-Menten kinetics (V _max was 0.55 pH/min and the apparent K _m was 75 mmol/l at pH_i 6.88). The results clearly showed that the Na⁺-H⁺ antiport activity is dependent on the chemical potential gradient of both Na⁺ and H⁺ ions across the basolateral membrane, and that the antiporter is asymmetric with respect to the substrate affinity of the transport site. The data agree with the current model of activation and inactivation of the antiporter by an intracellular site through changes in the intracellular Na⁺ and H⁺ concentrations.