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.

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.

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.

Muscle lactate transport studied in sarcolemmal giant vesicles.

Lactate transport was studied in giant (median diameter 6.3 microns) sarcolemmal vesicles obtained by collagenase treatment of rat skeletal muscle. The lactate transport displayed stereospecificity, had a high temperature coefficient, and could be inhibited up to 90% with known transport inhibitors (PCMBS and cinnamate). In equilibrium exchange experiments, the L-lactate flux demonstrated saturation kinetics with Km = 23.7 mM and Vmax = 108 pmol cm-2 s-1. With lactate present on only one side of the membrane, (zero trans conditions), Vmax was reduced to 48 pmol cm-2 s-1. The flux rate displayed transacceleration. The lactate flux was coupled to a parallel H+ flux. Under equilibrium exchange conditions, the carrier-mediated lactate flux was not pH-dependent. In the zero trans experiments, H+ on the trans side acted as an inhibitor. The loaded form of the carrier reorients faster than the unloaded form, and the protonated form with no lactate bound reorients slowly or is immobile. When compared to intact muscles, the giant sarcolemmal vesicles retain their transport characteristics both qualitatively and quantitatively.

Regulation of cellular pH in skeletal muscle fiber types, studied with sarcolemmal giant vesicles obtained from rat muscles.

Sarcolemmal giant vesicles obtained from rat hindlimb muscles were used as a model for the study of pH regulation in skeletal muscle. The transport systems involved in the recovery from 40 mM lactate and pHi 6.5 were quantified from both flux measurements of the co-transported ions and counter-ions, and from measurements of the rate of the internal pH change. The diffusion of lactic acid plus the carrier-mediated co-transport of lactate and H+ had the highest capacity to transport protons (240 nmol H+/mg protein per min). These systems are therefore responsible for a large part of the H+ efflux in periods with a high lactate production. The capacity of the HCO(3)- - dependent systems was 47 nmol/mg per min, and the capacity of the Na+/H+ exchange system was 33 nmol/mg per min in vesicles from mixed muscles. The capacity to remove H+ by the lactate/H+ co-transport system and by the bicarbonate-dependent systems was significantly higher in vesicles from predominantly red fibers than in vesicles from white fibers, whereas the distribution of the Na+/H+ exchange system was independent of fiber type. These observations demonstrate that the pH regulation during muscle activity in red muscles is more effective than in white muscles.

Symmetry and pH dependency of the lactate/proton carrier in skeletal muscle studied with rat sarcolemmal giant vesicles.

Muscle lactate transport was studied in rat sarcolemmal giant vesicles, in which the membrane orientation was similar to that of intact cells. The lactate concentration dependency was studied using influx experiments under equilibrium exchange. zero-trans. and infinite-cis conditions. At constant external and internal pH the carrier behaved symmetrically with regard to lactate transport from the two sides of the membrane. Lactate at the trans side stimulated the lactate tracer flux (trans-acceleration). Both in zero-trans influx and efflux experiments the lactate flux was stimulated in a symmetric way by H+ at the cis side, consistent with protons being the substrate for the carrier. In contrast, the lactate flux was symmetrically inhibited by H+ at the trans side (trans-inhibition), the underlying mechanism is likely that the protonated carrier with no lactate bound is unable to reorient in the membrane. These findings have implications both for the kinetic model and for understanding the physiological function of the lactate/proton transporter.

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.

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.

Enkephalin increases the efficacy of dopaminergic transmission in Helix pomatia.

A dopaminergic synaptic connection in Helix pomatia was studied. Transmitter mobilization was measured during incubation with met-enkephalin. Enkephalin decreased the time constant for transmitter mobilization. The effect was blocked by naloxone. It is postulated that the presynaptic cell possesses opiate receptors and that these receptors mediate the enkephalin-induced increase in efficacy of dopaminergic transmission.

Kinetics of lactate transport in sarcolemmal giant vesicles obtained from human skeletal muscle.

We developed a method that allows the measurement of muscle lactate transport in humans. The transport studies were carried out with giant (1.8- to 36-microns-diam) sarcolemmal vesicles obtained by collagenase treatment of needle biopsy material. Marker enzyme analyses demonstrated that the vesicular membrane is predominantly of sarcolemmal origin, contamination with sarcoplasmic reticulum membranes is very low, and mitochondrial membranes are not a major contaminant. The vesicles were loaded with labeled lactate, and the efflux was measured. The system displayed saturation kinetics and inhibitor sensitivity. In equilibrium exchange experiments (pH 7.4, 21 degrees C), the Michaelis-Menten constant (Km) for the carrier-mediated flux was 30 +/- 8 (SD) mM and maximal transport rate (Vmax) was 184 +/- 24 pmol.cm-2.s-1 (142 nmol.mg protein-1.min-1). In zero-trans efflux experiments, Km was 24 +/- 8 mM and Vmax was 81 +/- 11 pmol.cm-2.s-1 (63 nmol.mg protein-1.min-1). In infinite-cis experiments with a variable lactate concentration on the outside of the vesicles, Km was 8 +/- 4 mM and Vmax was 136 +/- 9 pmol.cm-2.s-1 (105 nmol.mg protein-1.min-1). Thus, the system displayed transacceleration. Low pH (6.4) had no significant effect on equilibrium exchange experiments, whereas in zero-trans experiments low pH at the trans side inhibited the flux by 50%. We concluded that lactate transport can be studied in giant vesicles obtained from a single human muscle biopsy. Our data provide evidence for the existence of a lactate carrier in human sarcolemma. This transport system must be taken into account in models of human lactate kinetics.

Lactate transport studied in sarcolemmal giant vesicles from human muscle biopsies: relation to training status.

The present study examined sarcolemmal lactate transport capacity in humans of widely different training status. Muscle biopsies were obtained from m. vastus lateralis in 39 subjects divided into untrained (n = 13), trained (n = 7), and athlete [sprint runners (n = 2), endurance runners (n = 5), triathletes (n = 3), and road (n = 6) and track (n = 3) bicyclists] groups. From the biopsy sample giant vesicles were produced with collagenase treatment to determine the sarcolemmal lactate transport capacity, and histochemical analyses were made. The athletes had a higher capacity to transport lactate than the untrained and trained subjects (P < 0.01). Within the group of athletes, the bicyclists had a higher lactate transport capacity than the runners (P < 0.05), whereas there was no difference among trained subjects, runners, and triathletes. The lactate transport capacity was related to the occurrence of type I muscle fibers (r = 0.48, P < 0.01). The present results suggest that the capacity to transport lactate is higher in athletes than in untrained and less trained subjects. It might indicate that lactate transport capacity in human skeletal muscle can be changed by a high volume of training including frequent high-intensity sessions. In addition, sarcolemmal lactate transport capacity appears to be related to the fiber type distribution of a muscle.

Limb skeletal muscle adaptation in athletes after training at altitude.

Morphological and biochemical characteristics of biopsies obtained from gastrocnemius (GAS) and triceps brachii muscle (TRI), as well as maximal O2 uptake (VO2 max) and O2 deficit, were determined in 10 well-trained cross-country skiers before and after a 2-wk stay (2,100 m above sea level) and training (2,700 m above sea level) at altitude. On return to sea level, VO2 max was the same as the prealtitude value, whereas an increase in O2 deficit (29%) and in short-term running performance (17%) was observed (P less than 0.05). GAS showed maintained capillary supply but a 10% decrease in mitochondrial enzyme activities (P less than 0.05), whereas an increase in capillary supply (P less than 0.05) but unchanged mitochondrial enzyme activities were observed in TRI. Buffer capacity was increased by 6% in both GAS and TRI (P less than 0.05). A positive correlation was found between the relative increase in buffer capacity of GAS and short-term running time (P less than 0.05). Thus the present study indicates no effect of 2 wk of altitude training on VO2 max but provides evidence to suggest an improvement in short-term exercise performance, which may be the result of an increase in muscle buffer capacity.

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.

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.

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.

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.

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.

The effect of beta 2-adrenoceptor activation on ion-shifts and fatigue in mouse soleus muscles stimulated in vitro.

The resting membrane potential (RMP), the intracellular free Na+ concentration ([Na+]i) and the intracellular free K+ concentration ([K+]i), were measured with double-barrelled ion-selective microelectrodes in mouse soleus muscles in vitro. In addition, the Na+ contents and K+ contents have been measured with the flame photometric technique. At rest the beta 2-selective adrenoceptor agonist terbutaline (10(-5) M) increased the membrane potential and [K+]i, and decreased [Na+]i when compared with control muscles. During a 5 min stimulation period the muscles, which were incubated with the beta 2-adrenoceptor agonist, showed a smaller depolarization, a smaller decrease in [K+]i and a smaller increase in [Na+]i than stimulated control muscles. This difference was probably associated with an increased rate of Na-K-pumping in the beta 2-adrenoceptor stimulated muscles. The beta 2-agonist treated muscles were more resistant to fatigue than control muscles. This effect was significant with 10(-6) M terbutaline (25 degrees C). A depolarization obtained by increasing [K+]o was shown to reduce the maximal tension. It is postulated, that the K+ shifts, which are responsible for the depolarization during muscle activity, are one of the mechanisms underlying muscle fatigue.