Skeletal muscle Na+/H+ exchange in rats: pH dependency and the effect of training

Skeletal muscle Na⁺/H⁺ exchange was studied using giant sarcolemmal vesicles obtained from rat hind limb muscle. Experiments with either the ²²Na tracer technique or with the Na⁺ sensitive fluorescent probe SBFI were conducted to determine the activity of the Na⁺/H⁺ exchanger, which was quantified from the amiloride or amiloride derivative 5-N-ethyl-N-propylamiloride (EIPA) sensitive Na⁺ influx. At a constant external pH of 7.4 the exchange system was close to half-activation at an internal pH of 7.2. A further activation was observed at lower internal pH values. The activity of the muscle Na⁺/H⁺ exchanger was elevated after 6 weeks of high-intensity treadmill training. In contrast, the activity of the system was unaffected by endurance training. The enhanced initial rate of amiloride-sensitive Na⁺/H⁺ exchange appears to be involved in the elevated in vivo (dynamical) buffer capacity reported for trained rats and human subjects, indicating that adaptive changes in the exchange system are of importance for pH regulation in association with high-intensity exercise.     

Lactate/proton co-transport in skeletal muscle: regulation and importance for pH homeostasis

In skeletal muscle, intracellular pH is more alkaline than would be predicted if H⁺ were passively distributed across the sarcolemma. Therefore, the passive influx of H⁺ must be counteracted by transport processes mediating H⁺ efflux. In resting skeletal muscle, these transport processes are Na⁺/H⁺ exchange and bicarbonate-dependent systems. During periods of high energy demand, skeletal muscle produces large amounts of lactic acid. The internal accumulation of lactic acid reduces pH, which may cause fatigue. It is therefore important for muscle cells to be able to regulate pH during and after activity. A part of the accumulated lactate and H⁺ is metabolized, but a considerable fraction is released from the cell. The efflux of H⁺ and lactate might be mediated by the lactate/proton co-transport system found in almost all cell types in the body. The role of lactate/proton co-transport in pH regulation has been studied both with intact cells and with sarcolemmal vesicles. In intact cells, inhibitors of lactate/proton transport have been shown to accelerate the development of fatigue, and to delay the recovery after activity. A comparison with vesicles has demonstrated that, at low pH, and with a high lactate concentration, the capacity for H⁺ removal is higher via the lactate/proton co-transport system than via the sum of the Na⁺/H⁺ exchange and bicarbonate-dependent exchange systems. Therefore, the carrier-mediated lactate/proton efflux is of major importance for pH regulation in connection with muscle activity. The lactate/proton transport system has been shown to undergo long-term changes depending on the level of physical activity. The capacity of the system was enhanced after intense training or chronic stimulation, and reduced after denervation. It is concluded that the lactate/proton transport system is of major importance for pH regulation in skeletal muscle, and that changes in the amount of transporters are one of the many adaptations to physical activity.     

Lactate/H+ transport kinetics in rat skeletal muscle related to fibre type and changes in transport capacity

 Lactate/H⁺ transport kinetics were determined by means of the pH-sensitive probe BCECF in sarcolemmal giant vesicles, obtained from rat skeletal muscle, and related to variations in lactate/H⁺ transport capacity. Vesicle preparations were made from red and white muscles, mixed muscles, denervated muscles, muscles of old rats and rats that had been subjected to high-intensity training, endurance training, repeated exposure to hypoxia, and hypothyroid or hyperthyroid treatments. The lactate/H⁺ transport capacity of red muscles was greater than that of white muscles, and this difference was associated with a higher maximal transport rate (V _max) in red muscles, whereas the K _m was similar in the two muscle types. High-intensity training and hyperthyroidism increased the lactate/H⁺ transport capacity by enhancing V _max without affecting K _m. Similarly, a reduced transport capacity with old age and hypothyroidism was due to a decrease in V _max. The denervation-induced decline in lactate/H⁺ transport capacity resulted from both an increased K _m and a reduced V _max. The present data show that muscle type differences and most changes in the lactate/H⁺ transport capacity are mediated by modifications in V _max, which is expected to represent the number of membrane transporter molecules. K _m is unaffected by most treatments and appears to be independent of fibre type. Received: 10 February 1998 / Received after revision: 21 April 1998 / Accepted: 24 April 1998     

Luminal Na+/H+ exchange in the proximal tubule

The proximal tubule is critical for whole-organism volume and acid-base homeostasis by reabsorbing filtered water, NaCl, bicarbonate, and citrate, as well as by excreting acid in the form of hydrogen and ammonium ions and producing new bicarbonate in the process. Filtered organic solutes such as amino acids, oligopeptides, and proteins are also retrieved by the proximal tubule. Luminal membrane Na(+)/H(+) exchangers either directly mediate or indirectly contribute to each of these processes. Na(+)/H(+) exchangers are a family of secondary active transporters with diverse tissue and subcellular distributions. Two isoforms, NHE3 and NHE8, are expressed at the luminal membrane of the proximal tubule. NHE3 is the prevalent isoform in adults, is the most extensively studied, and is tightly regulated by a large number of agonists and physiological conditions acting via partially defined molecular mechanisms. Comparatively little is known about NHE8, which is highly expressed at the lumen of the neonatal proximal tubule and is mostly intracellular in adults. This article discusses the physiology of proximal Na(+)/H(+) exchange, the multiple mechanisms of NHE3 regulation, and the reciprocal relationship between NHE3 and NHE8 at the lumen of the proximal tubule.     

Expression of the Na+/H+ exchanger isoform NHE1 in rat skeletal muscle and effect of training

The expression of the Na⁺/H⁺ exchanger isoform NHE1 was quantified in homogenates of various rat skeletal muscles by means of immunoblotting, and the effect of 3 weeks of treadmill training on NHE1 expression was determined in a red (oxidative) as well as a white (glycolytic)‐muscle preparation. The NHE1 antibodies recognized a glycosylated protein at 101–111 kDa. There was a positive correlation between the NHE1 expression in the muscle and percent type IIB fibres and percent type IID/X fibres, whereas the NHE1 expressions were negatively correlated to percent type I fibres and percent type I + IIA fibres. Thus the highest NHE1 expression was evident in the most glycolytic fibres. Treadmill training increased (P < 0.05) the NHE1 content by 29 and 36% in oxidative and glycolytic fibres, respectively, suggesting that training enhanced the NHE1 content of all muscle‐fibre types. Therefore training may improve the capacity for pH regulation in skeletal muscle.     

Regulation of pH in human skeletal muscle: adaptations to physical activity

Regulation of pH in skeletal muscle is the sum of mechanisms involved in maintaining intracellular pH within the normal range. Aspects of pH regulation in human skeletal muscle have been studied with various techniques from analysis of membrane proteins, microdialysis, and the nuclear magnetic resonance technique to exercise experiments including blood sampling and muscle biopsies. The present review characterizes the cellular buffering system as well as the most important membrane transport systems involved (Na(+)/H(+) exchange, Na-bicarbonate co-transport and lactate/H(+) co-transport) and describes the contribution of each transport system in pH regulation at rest and during muscle activity. It is reported that the mechanisms involved in pH regulation can undergo adaptational changes in association with physical activity and that these changes are of functional importance.     

Buffer capacity and lactate accumulation in skeletal muscle of trained and untrained men

Buffer capacity (β) of skeletal muscle has been determined in trained (n=7) and in sedentary subjects (n=8). The trained subjects were active in ball games where a high degree of anaerobic energy utilization is required. Percentage fibre type occurrence in the thigh muscle was not significantly different in the two groups. However, there was a tendency towards a higher proportion of type I (slow-twitch) fibres (61.5±11.6% vs. 50.2±12.5%) and a lower proportion of type IIB fibres (2.1±3.5% vs 14.1±16.3%) in the trained subjects. The proportion of the cross-sectional area of the muscle biopsies that was made up of type I or type II fibres was not different in the two groups. All subjects performed an isometric contraction of the knee extensors to fatigue at 61% of their maximal voluntary contraction force. Muscle biopsies were taken from the quadriceps femoris muscle at rest and immediately after contraction. The buffer capacity of muscle was calculated from: β= (Muscle lactate (work)-Muscle lactate (rest))/(Muscle pH (rest) -Muscle pH (work)). A higher buffer capacity (p<0.05) was observed in the trained subjects (β=194±30 mmolxpH^-1xkg^-1 dry wt.) compared to the sedentary group (β=164±20) (mean±SD). An unexpected finding was that muscle lactate after contraction to fatigue was lower (30%, p<0.01) and muscle pH was higher (6.80±0.06 vs. 6.61±0.12, p<0.01) in the trained subjects than in the sedentary controls. Creatine phosphate stores were almost completely depleted in both groups. Post-exercise lactate values were related to the proportion of type II fibres in the muscle (p<0.01). There was, however, no statistical correlation betwe β and fibre type occurrence (p>0.05). In summary, the present results indicate that skeletal muscle buffer capacity can be changed by training in man. Furthermore, it is concluded that the lower lactate accumulation and pH decline after an isometric contraction to fatigue that was observed in the trained compared to the sedentary subjects is related to the training per se. However, the tendency towards a lower type I (slowtwitch) fibre percentage in the trained subjects is likely to have contributed to the observed differences.     

Analysis of exercise-induced Na+-K+ exchange in rat skeletal muscle in vivo

We aimed to quantify the Na(+)-K(+) exchange occurring during exercise in rat skeletal muscle in vivo. Intracellular Na(+) and K(+) content, Na(+) permeability ((22)Na(+) influx), Na(+)-K(+) pump activity (ouabain-sensitive (86)Rb(+) uptake) and Na(+)-K(+) pump alpha(2) subunit content ([(3)H]ouabain binding) were measured. Six-week-old rats rested (control animals) or performed intermittent running for 10-60 min and were then killed or were killed at 15 or 90 min following 60 min exercise. In the soleus muscle, intracellular Na(+) was 80% higher than in control rats after 60 min exercise, was still elevated (38%) after 15 min rest and returned to control levels after 90 min rest. Intracellular K(+) showed corresponding decreases after 15-60 min exercise, returning to control levels 90 min postexercise. Exercise induced little change in Na(+) and K(+) in the extensor digitorum longus muscle (EDL). In soleus, the exercise-induced rise in Na(+) and reduction in K(+) were augmented by pretreatment with ouabain or by reducing the content of muscular Na(+)-K(+) pumps by prior K(+) depletion of the animals. Fifteen minutes after 60 min exercise, ouabain-sensitive (86)Rb(+) uptake in the soleus was increased by 30% but was unchanged in EDL, and there was no effect of exercise on [(3)H]ouabain binding measured in vitro or in vivo in either muscle. In conclusion, in the soleus, in vivo exercise induces a rise in intracellular Na(+), which reflects the excitation-induced increase in Na(+) influx and leads to augmented Na(+)-K(+) pump activity without apparent change in Na(+)-K(+) pump capacity.     

Characterization of bumetanide-sensitive Na+ and K+ transport in rat skeletal muscle

In isolated rat soleus muscle an average of 23% of the total ²²Na influx was found to be suppressible by bumetanide (K_0.5=0.1 mm ) and furosemide (K_0.5=1 mm ), whereas the influx and efflux of ⁴²K were not affected. In extensor digitorum longus muscle, around 25% of the total ²²Na influx was suppressible by bumetanide (1 mm ). In the presence of ouabain, both diuretics decreased net intracellular accumulation of Na⁺, but caused no change in K⁺ content. In extensor digitorum longus (but not in soleus), bumetanide-suppressible ²²Na influx was stimulated by increasing extracellular osmolarity with the bumetanide having no effect on ⁴²K influx. Bumetanide-suppressible Na⁺ influx was almost abolished in Cl^--free buffer, but was unaffected by the omission of K⁺. In rat soleus, the inhibitory effects of bumetanide, amiloride and tetrodotoxin on ²²Na influx were found to be additive. The results indicate that a NaCl cotransport system is present in both fast- and slow-twitch skeletal muscles, and may participate in volume regulation. Due to the large pool of muscle cells, activation of NaKCl₂ cotransport is likely to entail the hazards of hypokalemia. The advantage of exerting volume control via NaCl cotransport is that this risk can be avoided.     

Evidence for Na/H exchange and Cl/HCO3 exchange in A10 vascular smooth muscle cells

In the present study we used the pH sensitive absorbance of 5(and6)-carboxy-4,5-dimethylfluorescein to investigate intracellular pH (pH_i) regulation in A10 vascular smooth muscle cells: (1) The steady state pH_i in A10 cells averaged 7.01±0.1 (mean±SEM,n=26) at an extracellular pH of 7.4 (28 mM HCO₃/5% CO₂). (2) Removal of extracellular sodium led to an intracellular acidification of 0.36±0.07 pH-units (mean±SEM,n=8). (3) pH_i-Recovery after an acute intracellular acid load (by means of NH₄Cl-prepulse) was reversibly blocked by 1 mM amiloride and was dependent on the presence of sodium. The velocity of pH_i recovery increased with increasing sodium concentrations with an apparentK _m for external sodium of about 30 mM and aV _max of about 0.35 pH units/min. These findings are compatible with a Na/H exchanger being responsible for pH_i recovery after an acid load. (4) Removal of extracellular chioride induced an intracellular alkalinization of 0.23±0.03 pH-units (mean±SEM,n=10). The alkalinization was dependent on the presence of extracellular bicarbonate (5) Removal of chloride during pH_i recovery from an alkaline load (imposed by acetate prepulse) stopped and reversed pH_i backregulation. Chloride removal had no effect in the absence of bicarbonate or in the presence of 10^–4 M DIDS, suggesting that the effects were mediated by a Cl/HCO₃ exchanger. In conclusion we have demonstrated evidence for a Na/H exchanger and a Cl/HCO₃ exchanger in A10 vascular smooth muscle cells.Abbreviations used CDMF 5(and6)-carboxy-4,5-dimethylfluorescein - DIDS 4,4-diisothiocyanostilbene-2,2-disulfonic acid - NMDG N-methyl-d-glucamine; pH_i, intracellular pH - pH_o extracellular pH - Mops 3-[N-Morpholino]propanesulfonic acid - Hepes 2-[4-(2-Hydroxyethyl)-1-piperazinyl]-ethanesulfonic acid - Tris Tris(hydroxymethyl)-aminomethane - EDTA ethylenediamine-tetraacetic acid - EGTA ethyleneglycol-bis-(-amino-ethylether)N,N-tetraacetic acid     

Training-induced changes in membrane transport proteins of human skeletal muscle

Training improves human physical performance by inducing structural and cardiovascular changes, metabolic changes, and changes in the density of membrane transport proteins. This review focuses on the training-induced changes in proteins involved in sarcolemmal membrane transport. It is concluded that the same type of training affects many transport proteins, suggesting that all transport proteins increase with training, and that both sprint and endurance training in humans increase the density of most membrane transport proteins. There seems to be an upper limit for these changes: intense training for 6–8 weeks substantially increases the density of membrane proteins, whereas years of training (as performed by athletes) have no further effect. Studies suggest that training-induced changes at the protein level are important functionally. The underlying factors responsible for these changes in transport proteins might include changes in substrate concentration, but the existence of “exercise factors” mediating these responses is more likely. Exercise factors might include Ca²⁺, mitogen-activated protein kinases, adenosine monophosphate kinases, other kinases, or interleukin-6. Although the magnitudes of training-induced changes have been investigated at the protein level, the underlying signal mechanisms have not been fully described.     

Expression of Na+/HCO3− co‐transporter proteins (NBCs) in rat and human skeletal muscle

Aim: Sodium/bicarbonate co‐transport (NBC) has been suggested to have a role in muscle pH regulation. We investigated the presence of NBC proteins in rat and human muscle samples and the fibre type distribution of the identified NBCs. Methods and results: Western blotting of muscle homogenates and sarcolemmal membranes (sarcolemmal giant vesicles) were used to screen for the presence of NBCs. Immunohistochemistry was used for the subcellular localization. The functional test revealed that approximately half of the pH recovery in sarcolemmal vesicles produced from rat muscle is mediated by bicarbonate‐dependent transport. This indicates that the NBCs are preserved in the vesicles. The western blotting experiments demonstrated the existence of at least two NBC proteins in skeletal muscle. One NBC protein (approximately 150 kDa) seems to be related to the kidney/pancreas/heart isoform NBC1, whereas the other protein (approximately 200 kDa) is related to the NBC4 isoform. The two NBC proteins represent the electrogenic isoforms named NBCe1 and NBCe2. Membrane fractionation and immunofluorescence techniques confirmed that the two NBCs are located in the sarcolemmal membrane as well as in some internal membranes, probably the T‐tubules. The two NBCs localized in muscle have distinct fibre type distributions. Conclusions: Skeletal muscle possesses two variants of the sodium/bicarbonate co‐transporter (NBC) isoforms, which have been called NBCe1 and NBCe2.     

Polarized expression of Na+/H+ exchange activities in clonal LLC-PK1 cells (Clone4 and PKE20)

We have analysed the mechanisms of Na⁺-dependent pH_i recovery from an acid load in LLC-PK₁/ Clone₄ and LLC-PK₁/PKE₂₀ cells by using the intracellular pH indicator 2,7-bis(carboxyethyl)-5,6-carboxyfluorescein acetoxymethyl ester. By analysis using single-cell microspectrofluorometry, we obtained evidence for polarized expression of Na⁺/H⁺ exchange activities with different properties in apical and basolateral cell surfaces, respectively. In Clone₄ cells, Na⁺/H⁺ exchange activity is only visible on the basolateral cell surface; in PKE₂₀ cells, Na⁺/H⁺ exchange activities with equal capacities are present on both cell surfaces. In Clone₄ cells, the apparent K _m value for Na⁺ is around 10 mM; in PKE₂₀ cells it is around 20 mM and indistinguishable for the two cell poles. Ethylisopropylamiloride (EIPA) inhibition for all three activities measured in monolayer configuration is reduced by increasing Na⁺ concentration. Measured in the same cells, EIPA inhibition of transport of PKE₂₀ cells is weaker for apical Na⁺/H⁺ exchange as compared to basolateral activity. In Clone₄ and PKE₂₀ cells kept in suspension, Na⁺/H⁺ exchange activities with similar properties for the two cell lines are observed. However, Na⁺/H⁺ exchange activities in cells in suspension are different from either activity measured in monolayer configuration: affinity for Na⁺ is higher (PKE₂₀ cells) and inhibition by amiloride is weak and not influenced by increasing Na⁺ concentrations (PKE₂₀ and Clone₄ cells). It is concluded that PKE₂₀ cells contain different Na⁺/H⁺ exchange activities on the two cell surfaces; this cell line should be a useful model to study regulatory aspect of different Na⁺/H⁺ exchange functions (epithelial/housekeeping). Furthermore, measurements on cells in suspension do not reflect the properties of Na⁺/H⁺ exchange activities of cells in epithelial configuration.     

K+ as a vasodilator in resting human muscle: implications for exercise hyperaemia

Aim: Potassium (K⁺) released from contracting skeletal muscle is considered a vasodilatory agent. This concept is mainly based on experiments infusing non‐physiological doses of K⁺. The aim of the present study was to investigate the role of K⁺ in blood flow regulation. Methods: We measured leg blood flow (LBF) and arterio‐venous (A‐V) O₂ difference in 13 subjects while infusing K⁺ into the femoral artery at a rate of 0.2, 0.4, 0.6 and 0.8 mmol min^?1. Results: The lowest dose increased the calculated femoral artery plasma K⁺ concentration by approx.1 mmol L^?1. Graded K⁺ infusions increased LBF from 0.39 ± 0.06 to 0.56 ± 0.13, 0.58 ± 0.17, 0.61 ± 0.11 and 0.71 ± 0.17 L min^?1, respectively, whereas the leg A‐V O₂ difference decreased from 74 ± 9 to 60 ± 12, 52 ± 11, 53 ± 9 and 45 ± 7 mL L^?1, respectively (P < 0.05). Mean arterial pressure was unchanged, indicating that the increase in LBF was associated with vasodilatation. The effect of K⁺ was totally inhibited by infusion (27 μmol min^?1) of Ba²⁺, an inhibitor of Kir2.1 channels. Simultaneous infusion of ATP and K⁺ evoked an increase in LBF equalled to the sum of their effects. Conclusions: Physiological infusions of K⁺ induce significant increases in resting LBF, which are completely blunted by inhibition of the Kir2.1 channels. The present findings in resting skeletal muscle suggest that K⁺ released from contracting muscle might be involved in exercise hyperaemia. However, the magnitude of increase in LBF observed with K⁺ infusion suggests that K⁺ only accounts for a limited fraction of the hyperaemic response to exercise.     

Ion transport in human skeletal muscle cells: disturbances in myotonic dystrophy and Brody's disease

After excitation of skeletal muscle, the disturbed ion homeostasis is restored by Na⁺, K⁺ ATPase of the sarcolemma and Ca²⁺ ATPase of the sarcoplasmic reticulum (SR). Contrary to Na⁺, K⁺ ATPase, the concentration and isoenzyme distribution of SR Ca²⁺ ATPase in human skeletal muscle depend on fibre type and age. In cultured human muscle cells the concentration and activity of Na⁺, K⁺ ATPase and SR Ca²⁺ ATPase increase with maturation. In skeletal muscle and cultured muscle cells of patients suffering from myotonic dystrophy (MyD), the activity and the concentration of both Na⁺, K⁺ ATPase and SR Ca²⁺ ATPase are decreased by about 40%. In addition, we measured in cultured MyD muscle cells at rest an increased cytosolic Ca²⁺ concentration ([Ca²⁺]_i) caused by active voltage-operated Ca²⁺ channels, which are inactive in resting control cells. However, the restoration of a stimulus-induced Ca²⁺ transient is unaffected. A differentiation-related disturbance of membranes or a modulation defect of membrane proteins may play a role in MyD. In skeletal muscle and cultured muscle cells of patients suffering from Brody's disease, which is characterized by impaired muscle relaxation, the SR Ca²⁺ ATPase activity is reduced by about 50%, but the concentrations of total SR Ca²⁺ ATPase and the predominant SERCA1 isoform are normal. Diseased muscle cells show a delayed restoration of [Ca²⁺]_i after stimulation, which might be explained by structural modifications of SERCA1. Reduction of the Ca²⁺ release by drugs balances the excitation–relaxation cycle of the pathological cells.