Biomedical subjects

C Juel

Publications and source records attributed to C Juel.

At least 19 recordsLinked to original sources

PubMed2004

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.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMed2003

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.

Acid-Base Equilibrium↗

PubMed2001

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.

Acids↗

PubMed2001

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.

Adenosine↗

PubMed2001

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.

Biological Transport↗

PubMed2001

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.

Animals↗

PubMed2000

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.

Animals↗

PubMed2000

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.

Adult↗

PubMed2000

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.

Adult↗

PubMed1999

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.

Amino Acid Sequence↗

PubMed1999

Kinetics of lactate and pyruvate transport in cultured rat myotubes.

Skeletal muscle transport of lactate and pyruvate was studied in primary cultures of rat myotubes, applying the pH-sensitive fluorescent indicator 2', 7'-bis(carboxyethyl)-5(6)-carboxyfluorescein. The initial rate of decrease in intracellular pH (pHi) upon lactate or pyruvate incubation was used to determine total transport (carrier mediated and diffusion). Both lactate and pyruvate transport could be inhibited by a combination of 0.5 mM 4,4'-diisothiocyanostilbene-2, 2'-disulfonic acid, 5 mM mersalyl and 10 mM alpha-cyano-4-hydroxycinnamate. The kinetic parameters, Km and Vmax, for carrier-mediated transport of lactate were 9.9+/-1.1 mM and 0. 69+/-0.02 mmol l-1 s-1, respectively. For pyruvate, Km and Vmax were 4.4+/-1.3 mM and 0.30+/-0.05 mmol l-1 s-1, respectively. The diffusion component of the total transport was 0.0040+/-0.0005[S] (n=4) and 0.0048+/-0.0003[S] (n=4) for lactate and pyruvate, respectively. Furthermore, it was observed that the two monocarboxylate transporter isoforms present in mature skeletal muscles, MCT1 and MCT4 (formerly called MCT3 (M.C. Wilson, V.N. Jackson, C. Heddle, N.T. Price, H. Pilegaard, C. Juel, A. Bonen, I. Montgomery, O.F. Hutter, A.P. Halestrap, Lactic acid efflux from white skeletal muscle is catalyzed by the monocarboxylate transporter isoform MCT3, J. Biol. Chem. 273 (1998) 15920-15926)), were also expressed in primary culture of myotubes.

Animals↗

PubMed1999

Effect of high-intensity exercise training on lactate/H+ transport capacity in human skeletal muscle.

The present study examined the effect of high-intensity exercise training on muscle sarcolemmal lactate/H+ transport and the monocarboxylate transporters (MCT1 and MCT4) as well as lactate and H+ release during intense exercise in humans. One-legged knee-extensor exercise training was performed for 8 wk, and biopsies were obtained from untrained and trained vastus lateralis muscle. The rate of lactate/H+ transport determined in sarcolemmal giant vesicles was 12% higher (P < 0.05) in the trained than in untrained muscle (n = 7). The content of MCT1 and MCT4 protein was also higher (76 and 32%, respectively; n = 4) in trained muscle. Release of lactate and H+ from the quadriceps muscle at the end of intense exhaustive knee-extensor exercise was similar in the trained and untrained leg, although the estimated muscle intracellular-to-interstitial gradients of lactate and H+ were lower (P < 0.05) in the trained than in the untrained muscle. The present data show that intense exercise training can increase lactate/H+ transport capacity in human skeletal muscle as well as improve the ability of the muscle to release lactate and H+ during contractions.

Acid-Base Equilibrium↗

PubMed1999

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.

Adult↗

PubMed1999

Potassium fluxes in contracting human skeletal muscle and red blood cells.

The present study examined K+ fluxes in red blood cells and muscle during muscle contractions. Seven subjects performed two-legged submaximal knee-extensor exercise for 30 min. After 10 min of leg exercise (L1), intense arm exercise was also performed for 10 min (L2+A). Plasma epinephrine and norepinephrine concentrations were higher (P < 0.05) in L2+A compared with L1. Arterial plasma K+ at the end of L2+A was higher than in L1 (5.6 vs. 4.4 mM, P < 0.05) and returned to the L1 level on cessation of arm exercise. A net K+ release of 0.16 mmol/min from the active legs during L1 was turned to a net K+ uptake of 0.79 mmol/min during L2+A. Both arterial and venous red blood cell K+-to-hemoglobin ratios were constant during exercise. The present data suggest that contracting muscle can take up K+ probably by a combination of K+ and hormone activation of the Na+-K+ pump. Furthermore, changes in red blood cell K+ concentrations during muscle activity appear to be due to water movements and not transmembrane fluxes of K+.

Adult↗

PubMed1998

Lactic acid efflux from white skeletal muscle is catalyzed by the monocarboxylate transporter isoform MCT3.

The newly cloned proton-linked monocarboxylate transporter MCT3 was shown by Western blotting and immunofluorescence confocal microscopy to be expressed in all muscle fibers. In contrast, MCT1 is expressed most abundantly in oxidative fibers but is almost totally absent in fast-twitch glycolytic fibers. Thus MCT3 appears to be the major MCT isoform responsible for efflux of glycolytically derived lactic acid from white skeletal muscle. MCT3 is also expressed in several other tissues requiring rapid lactic acid efflux. The expression of both MCT3 and MCT1 was decreased by 40-60% 3 weeks after denervation of rat hind limb muscles, whereas chronic stimulation of the muscles for 7 days increased expression of MCT1 2-3-fold but had no effect on MCT3 expression. The kinetics and substrate and inhibitor specificities of monocarboxylate transport into cell lines expressing only MCT3 or MCT1 have been determined. Differences in the properties of MCT1 and MCT3 are relatively modest, suggesting that the significance of the two isoforms may be related to their regulation rather than their intrinsic properties.

Animals↗

PubMed1998

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.

Animals↗

PubMed1998

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.

Adaptation, Physiological↗

PubMed1998

Muscle pH regulation: role of training.

In most cell types, including resting skeletal muscle fibers, internal pH (pHi) is kept constant at a relatively alkaline level. The high pHi is obtained in spite of a chronic acid load resulting from cellular metabolism and passive influx of protons driven by electrochemical forces. Regulation of pHi 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 pHi 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.

Animals↗