Nitric oxide and Na,K-ATPase activity in rat skeletal muscle.

AIM: It has been suggested that nitric oxide (NO) stimulates the Na,K-ATPase in cardiac myocytes. Therefore, the aims of this study were to investigate whether NO increases Na,K-ATPase activity in skeletal muscle and, if that is the case, to identify the underlying mechanism. METHOD: The study used isolated rat muscle, muscle homogenates and purified membranes as model systems. Na,K-ATPase activity was quantified from phosphate release due to ATP hydrolysis. RESULTS: Exposure to the NO donor spermine NONOate (10 µm) increased the maximal Na,K-ATPase activity by 27% in isolated glycolytic muscles, but had no effect in oxidative muscles. Spermine NONOate increased the maximal Na,K-ATPase activity by 58% (P < 0.05) in homogenates from glycolytic muscle, but had no effect in oxidative muscle. The stimulatory effect of NONOate was not related to one specific Na,K-ATPase α-isoform. Incubation with cGMP (1 mm) increased the maximal Na,K-ATPase activity in homogenates from glycolytic muscle by 16% (P < 0.05), but had no effect on homogenates from oxidative muscle. cGMP had no effect on phospholemman phosphorylation at serine 68. Spermine NONOate had no effect in muscle membranes in which the ATPase activity was depressed by oxidized glutathione. CONCLUSION: NO and cGMP stimulate the Na,K-ATPase in glycolytic skeletal muscle. Direct S-nitrosylation and interference with S-glutathionylation seem to be excluded. In addition, phosphorylation of phospholemman at serine 68 is not involved. Most likely, the NO/cGMP/protein kinase G signalling pathway is involved.

ß2-adrenergic stimulation enhances Ca2+ release and contractile properties of skeletal muscles, and counteracts exercise-induced reductions in Na+-K+-ATPase Vmax in trained men.

The aim of the present study was to examine the effect of ß2-adrenergic stimulation on skeletal muscle contractile properties, sarcoplasmic reticulum (SR) rates of Ca(2+) release and uptake, and Na(+)-K(+)-ATPase activity before and after fatiguing exercise in trained men. The study consisted of two experiments (EXP1, n = 10 males, EXP2, n = 20 males), where ß2-adrenoceptor agonist (terbutaline) or placebo was randomly administered in double-blinded crossover designs. In EXP1, maximal voluntary isometric contraction (MVC) of m. quadriceps was measured, followed by exercise to fatigue at 120% of maximal oxygen uptake (V̇O2, max ). A muscle biopsy was taken after MVC (non-fatigue) and at time of fatigue. In EXP2, contractile properties of m. quadriceps were measured with electrical stimulations before (non-fatigue) and after two fatiguing 45 s sprints. Non-fatigued MVCs were 6 ± 3 and 6 ± 2% higher (P < 0.05) with terbutaline than placebo in EXP1 and EXP2, respectively. Furthermore, peak twitch force was 11 ± 7% higher (P < 0.01) with terbutaline than placebo at non-fatigue. After sprints, MVC declined (P < 0.05) to the same levels with terbutaline as placebo, whereas peak twitch force was lower (P < 0.05) and half-relaxation time was prolonged (P < 0.05) with terbutaline. Rates of SR Ca(2+) release and uptake at 400 nm [Ca(2+)] were 15 ± 5 and 14 ± 5% (P < 0.05) higher, respectively, with terbutaline than placebo at non-fatigue, but declined (P < 0.05) to similar levels at time of fatigue. Na(+)-K(+)-ATPase activity was unaffected by terbutaline compared with placebo at non-fatigue, but terbutaline counteracted exercise-induced reductions in maximum rate of activity (Vmax) at time of fatigue. In conclusion, increased contractile force induced by ß2-adrenergic stimulation is associated with enhanced rate of Ca(2+) release in humans. While ß2-adrenergic stimulation elicits positive inotropic and lusitropic effects on non-fatigued m. quadriceps, these effects are blunted when muscles fatigue.

Potassium-transporting proteins in skeletal muscle: cellular location and fibre-type differences.

Abstract Potassium (K(+)) displacement in skeletal muscle may be an important factor in the development of muscle fatigue during intense exercise. It has been shown in vitro that an increase in the extracellular K(+) concentration ([K(+)](e)) to values higher than approx. 10 mm significantly reduce force development in unfatigued skeletal muscle. Several in vivo studies have shown that [K(+)](e) increases progressively with increasing work intensity, reaching values higher than 10 mm. This increase in [K(+)](e) is expected to be even higher in the transverse (T)-tubules than the concentration reached in the interstitium. Besides the voltage-sensitive K(+) (K(v)) channels that generate the action potential (AP) it is suggested that the big-conductance Ca(2+)-dependent K(+) (K(Ca)1.1) channel contributes significantly to the K(+) release into the T-tubules. Also the ATP-dependent K(+) (K(ATP)) channel participates, but is suggested primarily to participate in K(+) release to the interstitium. Because there is restricted diffusion of K(+) to the interstitium, K(+) released to the T-tubules during AP propagation will be removed primarily by reuptake mediated by transport proteins located in the T-tubule membrane. The most important protein that mediates K(+) reuptake in the T-tubules is the Na(+),K(+)-ATPase alpha(2) dimers, but a significant contribution of the strong inward rectifier K(+) (Kir2.1) channel is also suggested. The Na(+), K(+), 2Cl(-) 1 (NKCC1) cotransporter also participates in K(+) reuptake but probably mainly from the interstitium. The relative content of the different K(+)-transporting proteins differs in oxidative and glycolytic muscles, and might explain the different [K(+)](e) tolerance observed.

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 alpha-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 alpha-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 alpha(1)- and alpha(2)-subunits, respectively, were co-immunoprecipitated by PLM antibodies. In muscle homogenate prepared after exercise, immunoprecipitation of alpha(1)-subunits was increased to 227%, whereas the fraction of precipitated alpha(2) remained constant. CONCLUSION: Exercise translocates PLM to the muscle outer membrane and increases its association with mainly the alpha(1)-subunit, which may contribute to the increased V(max) of the Na(+)/K(+)-ATPase.

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.

Effects of prolonged recombinant human erythropoietin administration on muscle membrane transport systems and metabolic marker enzymes.

Adaptations to chronic hypoxia involve changes in membrane transport proteins. The underlying mechanism of this response may be related to concomitant occurring changes in erythropoietin (Epo) levels. We therefore tested the direct effects of recombinant human erythropoietin (rHuEpo) treatment on the expression of muscle membrane transport proteins. Likewise, improvements in performance may involve upregulation of metabolic enzymes. Since Epo is known to augment performance we tested the effect of rHuEpo on some marker enzymes that are related to aerobic capacity. For these purposes eight subjects received 5,000 IU rHuEpo every second day for 14 days, and subsequently a single dose of 5,000 IU weekly for 12 weeks. Muscle biopsies were obtained before and after 14 weeks of rHuEpo treatment. The treatment increased hematocrit (from 44.7 to 48.8%), maximal oxygen uptake by 8.1%, and submaximal performance by approximately 54%. Membrane transport systems and carbonic anhydrases involved in pH regulation remained unchanged. Of the Na(+), K(+)-pump isoforms only the density of the alpha2 subunit was decreased (by 22%) after treatment. The marker enzymes cytochrom c and hexokinase remained unchanged with the treatment. In conclusion, changes in muscle membrane transport proteins and selected muscle enzymes do not contribute to the Epo-induced improvement in performance.

Prolonged administration of recombinant human erythropoietin increases submaximal performance more than maximal aerobic capacity.

The effects of recombinant human erythropoietin (rHuEpo) treatment on aerobic power (VO2max) are well documented, but little is known about the effects of rHuEpo on submaximal exercise performance. The present study investigated the effect on performance (ergometer cycling, 20-30 min at 80% of maximal attainable workload), and for this purpose eight subjects received either 5,000 IU rHuEpo or placebo every second day for 14 days, and subsequently a single dose of 5,000 IU/placebo weekly/10 weeks. Exercise performance was evaluated before treatment and after 4 and 11 weeks of treatment. With rHuEpo treatment VO2max increased (P<0.05) by 12.6 and 11.6% in week 4 and 11, respectively, and time-to-exhaustion (80% VO2max) was increased by 54.0 and 54.3% (P<0.05) after 4 and 11 weeks of treatment, respectively. However, when normalizing the workload to the same relative intensity (only done at time point week 11), TTE was decreased by 26.8% as compared to pre rHuEpo administration. In conclusion, in healthy non-athlete subjects rHuEpo administration prolongs submaximal exercise performance by about 54% independently of the approximately 12% increase in VO2max.

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) O(2) 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 O(2) 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 micromol min(-1)) of Ba(2+), 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.

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.

Human skeletal muscle and erythrocyte proteins involved in acid-base homeostasis: adaptations to chronic hypoxia.

Chronic hypoxia is accompanied by changes in blood and skeletal muscle acid-base control. We hypothesized that the underlying mechanisms include altered protein expression of transport systems and the enzymes involved in lactate, HCO3- and H+ fluxes in skeletal muscle and erythrocytes. Immunoblotting was used to quantify densities of the transport systems and enzymes. Muscle and erythrocyte samples were obtained from eight Danish lowlanders at sea level and after 2 and 8 weeks at 4100 m (Bolivia). For comparison, samples were obtained from eight Bolivian natives. In muscle membranes there were no changes in fibre-type distribution, lactate dehydrogenase isoforms, Na+,K+-pump subunits or in the lactate-H+ co-transporters MCT1 and MCT4. The Na+-H+ exchanger protein NHE1 was elevated by 39 % in natives compared to lowlanders. The Na+-HCO3- co-transporter density in muscle was elevated by 47-69 % after 2 and 8 weeks at altitude. The membrane-bound carbonic anhydrase (CA) IV in muscle increased in the lowlanders by 39 %, whereas CA XIV decreased by 23-47 %. Levels of cytosolic CA II and III in muscle and CA I and II in erythrocytes were unchanged. The erythrocyte lactate-H+ co-transporter MCT1 increased by 230-405 % in lowlanders and was 324 % higher in natives. The erythrocyte inorganic anion exchanger (Cl--HCO3- exchanger AE1) was increased by 149-228 %. In conclusion, chronic hypoxia induces dramatic changes in erythrocyte proteins, but only moderate changes in muscle proteins involved in acid-base control. Together, these changes suggest a hypoxia-induced increase in the capacity for lactate, HCO3- and H+ fluxes from muscle to blood and from blood to erythrocytes.

Molecular characterization of Danish Cryptosporidium parvum isolates.

The genetic polymorphism among 271 Danish Cryptosporidium isolates of human and animal origin was studied by partial amplification and sequencing of the Cryptosporidium oocyst wall protein (COWP) gene, the 1 8S rDNA, and a microsatellite locus. Furthermore, the microsatellite locus was studied directly using fragment analysis. A comparative analysis of DNA sequences showed the presence of 3 different subgenotypes (Cl, C2 and C3) in C. parvum isolates from Danish cattle, with prevalences of 16.7, 17.2 and 73.1% including 13 (7.0%) mixed infections. Subgenotype Cl was significantly more prevalent (P < 0.001) in the southern part of Denmark. In Cryptosporidium isolates of human origin the anthroponotic subgenotype H1 was identified, in addition to the zoonotic subgenotypes C1, C2, and C3. Of 44 human samples, 56.8% were anthroponotic, whereas 40.9% were zoonotic genotypes. One human isolate was characterized as C. meleagridis. The porcine Cryptosporidium isolates (N = 4) revealed a pattern which was genetically distinct from human and bovine isolates. Cryptosporidium in a hedgehog (Erinaceus europaeus L.) was identified for the first time. By microsatellite sequencing the hedgehog isolate showed a subgenotype distinct from the previously detected types. The assignment to subgenotype by microsatellite sequencing and fragment typing was 100% identical in samples where results were achieved by both methods. In addition, the fragment analysis proved more sensitive, easier, faster, and less expensive compared to sequencing.

Interstitial pH in human skeletal muscle during and after dynamic graded exercise.

1. In this study a new method has been used to measure interstitial pH continuously in human muscle during graded exercise. Human subjects performed 5 min of one-legged knee-extensor exercise at power outputs of 30, 50 and 70 W. Muscle interstitial pH was measured continuously in microdialysis dialysate using the pH-sensitive fluorescent dye 2',7'-bis-(2-carboxyethyl)-5-(and -6)-carboxyfluorescein (BCECF). 2. The mean interstitial pH at rest was 7.38 +/- 0.02. Interstitial pH gradually reduced during exercise in a nearly linear manner. The mean value (range) of the lowest interstitial pH at 30, 50 and 70 W exercise was 7.27 (7.18-7.34), 7.16 (7.05-7.24) and 7.04 (6.93-7.12), respectively. 3. The lowest pH was obtained 1 min after exercise, irrespectively of the workload, after which interstitial pH recovered in a nearly exponential manner. The mean half-time for recovery was 5.2 min (range 4.1-6.1 min). The changes in interstitial pH exceeded the changes in venous blood pH. 4. The present study showed that interstitial pH decreased during exercise in relation to intensity. These pH changes could have implications for blood flow regulation as well as for modulations of membrane transport systems.

Extracellular formation and uptake of adenosine during skeletal muscle contraction in the rat: role of adenosine transporters.

1. The existence of adenosine transporters in plasma membrane giant vesicles from rat skeletal muscles and in primary skeletal muscle cell cultures was investigated. In addition, the contribution of intracellularly or extracellularly formed adenosine to the overall extracellular adenosine concentration during muscle contraction was determined in primary skeletal muscle cell cultures. 2. In plasma membrane giant vesicles, the carrier-mediated adenosine transport demonstrated saturation kinetics with Km = 177 +/- 36 microM and Vmax = 1.9 +/- 0.2 nmol x ml(-1) x s(-1) (0.7 nmol (mg protein)(-1) x s(-1)). The existence of an adenosine transporter was further evidenced by the inhibition of the carrier-mediated adenosine transport in the presence of NBMPR (nitrobenzylthioinosine; 72% inhibition) or dipyridamol (64% inhibition; P < 0.05). 3. In primary skeletal muscle cells, the rate of extracellular adenosine accumulation was 5-fold greater (P < 0.05) with electrical stimulation than without electrical stimulation. Addition of the adenosine transporter inhibitor NBMPR led to a 57% larger (P < 0.05) rate of extracellular adenosine accumulation in the electro-stimulated muscle cells compared with control cells, demonstrating that adenosine is taken up by the skeletal muscle cells during contractions. 4. Inhibition of ecto-5'-nucleotidase with AOPCP in electro-stimulated cells resulted in a 70% lower (P < 0.05) rate of extracellular adenosine accumulation compared with control cells, indicating that adenosine to a large extent is formed in the extracellular space during contraction. 5. The present study provides evidence for the existence of an NBMPR-sensitive adenosine transporter in rat skeletal muscle. Our data furthermore demonstrate that the increase in extracellular adenosine observed during electro-stimulation of skeletal muscle is due to production of adenosine in the extracellular space of skeletal muscle and that adenosine is taken up rather than released by the skeletal muscle cells during contraction.

Reversibility of exercise-induced translocation of Na+-K+ pump subunits to the plasma membrane in rat skeletal muscle.

Exercise-induced translocation of Na+-K+ pump subunits to the sarcolemmal membrane was studied using sarcolemmal giant vesicles as a membrane purification procedure. The subunit content was quantified by Western blotting or by ouabain labeling. Low-intensity treadmill running increased (P<0.01) the alpha1, alpha2, beta1, and beta2 subunit contents by 19-32% in membranes from oxidative muscle fibers and the alpha1, alpha2, and beta2 contents increased by 13-25% in membranes from glycolytic muscle fibers. Ouabain labeling of membranes from mixed fibers was increased by 29% after exercise. A similar increase in subunit content could be induced by 5 min of fatiguing, high-intensity electrical stimulation of isolated soleus muscles. An increased subunit content was just detectable in vesicles produced 30 min after exercise, and the content was completely back to control levels 3 h after exercise. It is concluded that both low-intensity long-lasting running and short-lasting high-intensity contractions are able to induce a translocation of pump subunits to the sarcolemmal membrane. The post-exercise disappearance of the extra subunits (half-time approximately 20 min) from the membrane demonstrates the reversible nature of the translocation process.

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 (Km), 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.

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.

Exercise-induced translocation of Na(+)-K(+) pump subunits to the plasma membrane in human skeletal muscle.

Six human subjects performed one-legged knee extensor exercise (90 +/- 4 W) until fatigue (exercise time 4.6 +/- 0.8 min). Needle biopsies were obtained from vastus lateralis muscle before and immediately after exercise. Production of giant sarcolemmal vesicles from the biopsy material was used as a membrane purification procedure, and Na(+)-K(+) pump alpha- and beta-subunits were quantified by Western blotting. Exercise significantly increased (P < 0.05) the vesicular membrane content of the alpha(2)-, total alpha-, and beta(1)-subunits by 70 +/- 29, 35 +/- 10, and 26 +/- 5%, respectively. The membrane content of alpha(1) was not changed by exercise, and the densities of subunits in muscle homogenates were unchanged. The ratio of vesicular to crude muscle homogenate content of the alpha(2)-, total alpha-, and beta(1)-subunits was elevated during exercise by 67 +/- 33 (P < 0.05), 23 +/- 6 (P < 0.05), and 40 +/- 14% (P = 0.06), respectively. It is concluded that translocation of subunits is an important mechanism involved in the short time upregulation of the Na(+)-K(+) pumps in association with human muscle activity.

Interstitial K(+) in human skeletal muscle during and after dynamic graded exercise determined by microdialysis.

Interstitial K(+) concentrations were measured during one-legged knee-extensor exercise by use of microdialysis with probes inserted in the vastus lateralis muscle of the subjects. K(+) in the dialysate was measured either by flame photometry or a K(+)-sensitive electrode placed in the perfusion outlet. The correction for fractional K(+) recovery was based on the assumption of identical fractional thallium loss. The interstitial K(+) was 4. 19 +/- 0.09 mM at rest and increased to 6.17 +/- 0.19, 7.48 +/- 1.18, and 9.04 +/- 0.74 mM at 10, 30, and 50 W exercise, respectively. The individual probes demonstrated large variations in interstitial K(+), and values >10 mM were obtained. The observed interstitial K(+) was markedly higher than previously found for venous K(+) concentrations at similar work intensities. The present data support a potential role for interstitial K(+) in regulation of blood flow and development of fatigue.

Lactate transport in skeletal muscle - role and regulation of the monocarboxylate transporter.

Skeletal muscle is the major producer of lactic acid in the body, but its oxidative fibres also use lactic acid as a respiratory fuel. The stereoselective transport of L-lactic acid across the plasma membrane of muscle fibres has been shown to involve a proton-linked monocarboxylate transporter (MCT) similar to that described in erythrocytes and other cells. This transporter plays an important role in the pH regulation of skeletal muscle. A family of eight MCTs has now been cloned and sequenced, and the tissue distribution of each isoform varies. Skeletal muscle contains both MCT1 (the only isoform found in erythrocytes but also present in most other cells) and MCT4. The latter is found in all fibre types, although least in more oxidative red muscles such as soleus, whereas expression of MCT1 is highest in the more oxidative muscles and very low in white muscles that are almost entirely glycolytic. The properties of MCT1 and MCT2 have been described in some detail and the latter shown to have a higher affinity for substrates. MCT4 has been less well characterized but has a lower affinity for L-lactate (i.e. a higher Km of 20 mM) than does MCT1 (Km of 5 mM). MCT1 expression is increased in response to chronic stimulation and either endurance or explosive exercise training in rats and humans, whereas denervation decreases expression of both MCT1 and MCT4. The mechanism of regulation is not established, but does not appear to be accompanied by changes in mRNA concentrations. However, in other cells MCT1 and MCT4 are intimately associated with an ancillary protein OX-47 (also known as CD147). This protein is a member of the immunoglobulin superfamily with a single transmembrane helix, whose expression is known to be increased in a range of cells when their metabolic activity is increased.

Distribution of the lactate/H+ transporter isoforms MCT1 and MCT4 in human skeletal muscle.

The profiles of the lactate/H+ transporter isoforms [monocarboxylate transporter isoforms (MCT)] MCT1 and MCT4 (formerly MCT3 of Price, N. T., V. N. Jackson, and A. P. Halestrap. Biochem. J. 329: 321-328, 1998) were studied in the soleus, triceps brachii, and vastus lateralis muscles of six male subjects. The fiber-type compositions of the muscles were evaluated from the occurrence of the myosin heavy chain isoforms, and the fibers were classified as type I, IIA, or IIX. The total content of MCT1 and MCT4 was determined in muscle homogenates by Western blotting, and MCT1 and MCT4 were visualized on cross-sectional muscle sections by immunofluorescence microscopy. The Western blotting revealed a positive, linear relationship between the MCT1 content and the occurrence of type I fibers in the muscle, but no significant relation was found between MCT4 content and fiber type. Moreover, the interindividual variation in MCT4 content was much larger than the interindividual variation in MCT1 content in homogenate samples. The immunofluorescence microscopy showed that within a given muscle section, the MCT4 isoform was clearly more abundant in type II fibers than in type I fibers, whereas only minor differences existed in the occurrence of the MCT1 isoform between type I and II fibers. Together the present results indicate that the content of MCT1 in a muscle varies between different muscles, whereas fiber-type differences in MCT1 content are minor within a given muscle section. In contrast, the content of MCT4 is clearly fiber-type specific but apparently quite similar in various muscles.