Na+–K+ pump location and translocation during muscle contraction in rat skeletal muscle

Muscle contraction may up-regulate the number of Na(+)-K(+) pumps in the plasma membrane by translocation of subunits. Since there is still controversy about where this translocation takes place from and if it takes place at all, the present study used different techniques to characterize the translocation. Electrical stimulation and biotin labeling of rat muscle revealed a 40% and 18% increase in the amounts of the Na(+)-K(+) pump alpha(2) subunit and caveolin-3 (Cav-3), respectively, in the sarcolemma. Exercise induced a 36% and 19% increase in the relative amounts of the alpha(2) subunit and Cav-3, respectively, in an outer-membrane-enriched fraction and a 41% and 17% increase, respectively, in sarcolemma giant vesicles. The Na(+)-K(+) pump activity measured with the 3-O-MFPase assay was increased by 37% in giant vesicles from exercised rats. Immunoprecipitation with Cav-3 antibody showed that 17%, 11% and 14% of the alpha(1) subunits were associated with Cav-3 in soleus, extensor digitorum longus, and mixed muscles, respectively. For the alpha(2), the corresponding values were 17%, 5% and 16%. In conclusion; muscle contraction induces translocation of the alpha subunits, which is suggested to be caused partly by structural changes in caveolae and partly by translocation from an intracellular pool.     

Contraction-induced changes in TNFα and Akt-mediated signalling are associated with increased myofibrillar protein in rat skeletal muscle

Resistance training results in skeletal muscle hypertrophy, but the molecular signalling mechanisms responsible for this altered phenotype are incompletely understood. We used a resistance training (RT) protocol consisting of three sessions [day 1 (d1), day 3 (d3), day 5 (d5)] separated by 48 h recovery (squat exercise, 4 sets × 10 repetitions, 3 min recovery) to determine early signalling responses to RT in rodent skeletal muscle. Six animals per group were killed 3 h after each resistance training session and 24 and 48 h after the last training session (d5). There was a robust increase in TNFα protein expression, and IKK^Ser180/181 and p38MAPK^{Thr180/Tyr182} phosphorylation on d1 (P < 0.05), which abated with subsequent RT, returning to control levels by d5 for TNFα and IKK^Ser180/181. There was a trend for a decrease in MuRF-1 protein expression, 48 h following d5 of training (P = 0.08). Notably, muscle myofibrillar protein concentration was elevated compared to control 24 and 48 h following RT (P < 0.05). Akt^Ser473 and mTOR^Ser2448 phosphorylation were unchanged throughout RT. Phosphorylation of p70S6k^Thr389 increased 3 h post-exercise on d1, d3 and d5 (P < 0.05), whilst phosphorylation of S6^Ser235/236 increased on d1 and d3 (P < 0.05). Our results show a rapid attenuation of inflammatory signalling with repeated bouts of resistance exercise, concomitant with summation in translation initiation signalling in skeletal muscle. Indeed, the cumulative effect of these signalling events was associated with myofibrillar protein accretion, which likely contributes to the early adaptations in response to resistance training overload in the skeletal muscle.     

Changes in the concentrations of Na+, K+ and Cl− in secretion from the skin during progressive increase in exercise intensity

Summary A simple method for sampling skin secretion in 1-min periods was developed for investigating the effects of progressive increases in exercise intensity on Na⁺, K⁺ and CI^– secretions from the skin of the forearm. Ten healthy male subjects performed exercise consisting of eight stepwise increases in intensity from 50 to 225 W, with a 25-W increase at each step. Exercise at each step was for 3 min followed by a 1-min recovery period. Samples of blood and skin secretion were taken during the recovery period. Significant positive correlations were found between the mean concentrations of Na⁺ and Cl^– and between those of K⁺ and Cl^– in the skin secretion. The concentrations of electrolytes in the skin secretion also showed significant correlations with the blood lactate concentrations. The inflection points for secretions of Na⁺, K⁺ and Cl^– were 4.04, 3.61 and 3.83 mmol · l^– of blood lactate; 64.42, 61.96 and 62.14% of maximal oxygen consumption ( ); and exercise intensities of 123.01, 117.65 and 125.07 W, respectively. No significant differences were observed between the value of 67.27% of or 134.00W at the onset of blood lactate accumulation (OBLA) and the inflection points. From these results we concluded that changes in electrolyte concentrations in skin secretion during incremental exercise according to this protocol were closely related with the change in the blood lactate concentration, and that the inflection points for electrolytes may have been near the exercise intensity at OBLA.     

Ischemia/reperfusion-induced changes in intracellular free Ca2+ levels in rat skeletal muscle fibers – an in vivo study

Accumulation of intracellular free calcium (Ca2+i) may play an essential role in the ischemia/reperfusion injury of skeletal muscle. Although it has been shown that Ca2+i levels significantly increase during ischemia/reperfusion, it is still a matter of debate whether Ca2+i increases during ischemia alone. It was the aim of this study to monitor the in vivo Ca2+i levels in the rat spinotrapezius muscle during ischemia of varying duration and reperfusion, using a ratiometric fluorescence technique, and to investigate the relationship between the postischemic flow patterns and Ca2+i, if any. The muscle was loaded with Indo-1/AM and imaged by a cooled digital camera. Pre- and postischemic tissue perfusion was assessed by means of an analogue camera. Our results show that short-term ischemia (5, 15 and 30 min) and subsequent reperfusion (60 min) does not alter Ca2+i homeostasis and that tissue perfusion promptly recovers after the insult. One or two hours of ischemia resulted in changes in Ca2+i levels, varying from preparation to preparation; increases in some and no changes in others. In these preparations three distinct flow patterns - normal, compromised and no-reflow - could be distinguished during the 60-min reperfusion. Our main conclusion is that in skeletal muscle Ca2+i levels may increase, the increase probably depending on the muscle fiber type exposed.     

Investigation of changes in skeletal muscle α-actin expression in normal and pathological human and mouse hearts

We have developed a quantitative antibody-based assay to measure the content of skeletal muscle α-actin relative to cardiac α-actin. We found 21 ± 2% skeletal muscle α-actin content in normal heart muscle of adult man and mouse. In end stage failing heart 53 ± 5% of striated actin was skeletal muscle α-actin and in samples of inter-ventricular septum from patients with hypertrophic obstructive cardiomyopathy (HOCM) skeletal muscle α-actin was 72 ± 2% of sarcomeric actin. Thin filaments containing actin isolated from normal and HOCM heart muscle were functionally indistinguishable when studied by quantitative in vitro motility assay. We also found elevated skeletal muscle α-actin (60 ± 7%) in a mouse model of dilated cardiomyopathy.     

Cell-volume-dependent vascular smooth muscle contraction: role of Na+, K+, 2Cl− cotransport, intracellular Cl− and L-type Ca2+ channels

This study elucidates the role of cell volume in contractions of endothelium-denuded vascular smooth muscle rings (VSMR) from the rat aorta. We observed that hyposmotic swelling as well as hyper- and isosmotic shrinkage led to VSMR contractions. Swelling-induced contractions were accompanied by activation of Ca²⁺ influx and were abolished by nifedipine and verapamil. In contrast, contractions of shrunken cells were insensitive to the presence of L-type channel inhibitors and occurred in the absence of Ca²⁺_o. Thirty minutes preincubation with bumetanide, a potent Na⁺,K⁺,Cl^– cotransport (NKCC) inhibitor, decreased Cl^–_i content, nifedipine-sensitive ⁴⁵Ca uptake and contractions triggered by modest depolarization ([K⁺]_o=36 mM). Elevation of [K⁺]_o to 66 mM completely abolished the effect of bumetanide on these parameters. Bumetanide almost completely abrogated phenylephrine-induced contraction, partially suppressed contractions triggered by hyperosmotic shrinkage, but potentiated contractions of isosmotically shrunken VSMR. Our results suggest that bumetanide suppresses contraction of modestly depolarized cells via NKCC inhibition and Cl^–_i-mediated membrane hyperpolarization, whereas augmented contraction of isosmotically shrunken VSMR by bumetanide is a consequence of suppression of NKCC-mediated regulatory volume increase. The mechanism of bumetanide inhibition of contraction of phenylephrine-treated and hyperosmotically shrunken VSMR should be examined further.     

Effects of concentric and repeated eccentric exercise on muscle damage and calpain–calpastatin gene expression in human skeletal muscle

The purpose of this study was to compare the responsiveness of changes in Ca²⁺-content and calpain–calpastatin gene expression to concentric and eccentric single-bout and repeated exercise. An exercise group (n = 14) performed two bouts of bench-stepping exercise with 8 weeks between exercise bouts, and was compared to a control-group (n = 6). Muscle strength and soreness and plasma creatine kinase and myoglobin were measured before and during 7 days following exercise bouts. Muscle biopsies were collected from m. vastus lateralis of both legs prior to and at 3, 24 h and 7 days after exercise and quantified for muscle Ca²⁺-content and mRNA levels for calpain isoforms and calpastatin. Exercise reduced muscle strength and increased muscle soreness predominantly in the eccentric leg (P < 0.05). These responses as well as plasma levels of creatine kinase and myoglobin were all attenuated after the repeated eccentric exercise bout (P < 0.05). Total muscle Ca²⁺-content did not differ between interventions. mRNA levels for calpain 2 and calpastatin were upregulated exclusively by eccentric exercise 24 h post-exercise (P < 0.05), with no alteration in expression between bouts. Calpain 1 and calpain 3 mRNA did not change at any specific time point post-exercise for either intervention. Our mRNA results suggest a regulation on the calpain–calpastatin expression response to muscle damaging eccentric exercise, but not concentric exercise. Although a repeated bout effect was demonstrated in terms of muscle function, no immediate support was provided to suggest that regulation of expression of specific system components is involved in the repeated bout adaptation. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

The role of AMPK in regulation of Na+,K+-ATPase in skeletal muscle: does the gauge always plug the sink?

Journal of Muscle Research and Cell Motility - AMP-activated protein kinase (AMPK) is a cellular energy gauge and a major regulator of cellular energy homeostasis. Once activated, AMPK stimulates...     

Chaperone activity of α-crystallin in relation to the sarcoplasmic reticulum Ca2+ pump of skeletal muscles in stress and adaptation

α-Crystallin, an endogenous low-molecular-weight protein with chaperone activity, exerted protective effects on membrane systems of Ca²⁺ transport into the sarcoplasmic reticulum of skeletal muscles. Protective action of α-crystallin depended on the body state. This effect was not observed in the control and after adaptation to stress, while after stress, especially against the background of adaptation, α-crystallin increased the rate of Ca²⁺ transport into the sarcoplasmic reticulum and thermal resistance of Ca²⁺ pump. The mechanisms of α-crystallin activation during stress are discussed. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 128, No. 9, pp. 279–282, September, 1999     

Long-term depolarization regulates the α1s subunit of skeletal muscle Ca2+ channels and the amplitude of L-type Ca2+ currents

The effects of long-term depolarization on the level of alpha1s and on L-type Ca2+ currents of skeletal muscle were investigated. Long-term depolarization (14 h) caused a 50% decrease of alpha1s, revealed with the Western blot technique. This decline was prevented by preincubation with the Ca2+ channel blocker nifedipine. Electrophysiological experiments using the voltage-clamp technique were performed to measure the actions of long-term depolarization on Ca2+ currents and charge movement. A progressive decline in the amplitude of the Ca2+ currents by depolarizations lasting 0.5-14 h was observed. Similar to Western blot results, the fall in current amplitude was prevented by nifedipine, and it depended on external Ca2+. The nonlinear charge mobilized by step pulses was also significantly reduced (50%) by long-term depolarization. It is suggested that alpha1s subunit is down-regulated by long-term depolarization by a very stringent mechanism and that, in this process, Ca2+ ions permeating through L-type channels play a key role. A new role for the L-type Ca2+ current in skeletal muscle fibers in which the channels are self-regulated is proposed.     

Na/K pump α subunit expression in rabbit ventricle and regional variations of intracellular sodium regulation

We examined the isoform distribution and expression of the alpha subunit of the Na/K-ATPase in the left ventricular muscle of rabbit heart in order to determine whether previously reported regional differences in intracellular sodium regulation derive from differences in pump expression. Immunohistochemical techniques show that only the alpha1 isoform is present in rabbit ventricle; therefore, regional variation in isoform distribution is not possible. Western blots of samples taken from subendocardial and sub-epicardial regions confirm the absence of alpha2 and alpha3 isoforms but also show that levels of the alpha1 isoform do not differ significantly in the two regions. The ratio of densitometric readings from blot bands was 1.18+/-0.17 (epicardial:endocardial; mean +/-SEM). Measurements of fully activated pump current in voltage-clamped cells were achieved by dialysing the cell via the patch pipette with 50 mM Na and applying 1 mM dihydroouabain. The current measured was 0.16+/-0.02 pA/pF in epicardial cells and 0.17+/-0.02 pA/pF in endocardial cells. These results indicate that the capacity of cells from the two regions to generate sodium efflux is identical. Regional differences in intracellular sodium regulation, therefore, are more likely to arise from differences in influx of sodium.     

The effects of β-adrenergic stimulation and exercise on NR4A3 protein expression in rat skeletal muscle

β-Adrenergic stimulation and exercise up-regulate the mRNA expression of nuclear receptor NR4A3, which is involved in the regulation of glucose and fatty acid utilization genes in skeletal muscle. The objective of our study was to examine the effects of β-adrenergic stimulation and exercise on the expression of NR4A3 protein in rat skeletal muscle. A single subcutaneous injection of clenbuterol, which is a β2-adrenergic receptor (β2-AR) agonist, increased NR4A3 mRNA and protein expression in the fast-twitch glycolytic triceps muscle. On the other hand, an acute 3-h session of either treadmill running or swimming did not increase the NR4A3 protein level in the exercised muscle, although both treadmill running and swimming increased NR4A3 mRNA. Finally, loss of postural contractile activity because of hindlimb immobilization reduced NR4A3 mRNA and protein in the slow-twitch oxidative soleus muscle. These results suggest that: β-adrenergic stimulation up-regulates not only NR4A3 mRNA but also NR4A3 protein in fast-twitch glycolytic muscle; exercise may increase NR4A3 mRNA but not NR4A3 protein in skeletal muscle; and local postural contractile activity plays a crucial role in maintaining NR4A3 protein expression level in postural muscle.     

Effect of GABAergic and adrenergic agents on activity of Na+/K+ pump and Cl−-cotransport in somatic muscle cells of earthworm Lumbricus Terrestris

GABA, baclofen, epinephrine, and norepinephrine hyperpolarized the membrane of earthworm somatic cells. This effect was prevented by furosemide, removal of Cl⁻ from the medium, or activation of Na⁺/K⁺ pump by 3-fold increase external potassium concentration. It was hypothesized that GABA, baclofen, epinephrine, and norepinephrine stimulate Na⁺/K⁺ transport via specific receptor inputs, but their effect on resting potential can be realized only under conditions of working Cl⁻ symport. __________ Translated from Byulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 141, No. 5, pp. 572–574, May, 2006     

Physical activity and plasma interleukin-6 in humans – effect of intensity of exercise

The present study included data from three marathon races to investigate the hypothesis that a relationship exists between running intensity and elevated concentrations of interleukin (IL)-6 in plasma. The study included a total of 53 subjects whose mean age was 30.6 [95% confidence interval (CI) 1.4] years, mean body mass 77.7 (95%CI 2.0) kg, mean maximal oxygen uptake (V˙O_2max) 59.3 (95%CI 1.4) ml · min⁻¹ · kg⁻¹, and who had participated in the Copenhagen Marathons of 1996, 1997 or 1998, achieving a mean running time of 206 (95%CI 7) min. Running intensity was calculated as running speed divided by V˙O_2max. The concentration of IL-6 in plasma peaked immediately after the run. There was a negative correlation between peak IL-6 concentration and running time (r=−0.30, P < 0.05) and a positive correlation between peak IL-6 concentration and running intensity (r=0.32, P < 0.05). The IL-1 receptor antagonist (IL-1ra) plasma concentration peaked 1.5 h after the run and there was a positive correlation between the peak plasma concentrations of IL-6 and IL-1ra (r=0.39, P < 0.01). Creatine kinase (CK) plasma concentration peaked on the 1st day after the run, but no association was found between peak concentrations of IL-6 and CK. In conclusion, the results confirmed the hypothesized association between plasma IL-6 concentration and running intensity, but did not confirm the previous finding of a connection between IL-6 plasma concentration and muscle damage. Accepted: 6 August 2000     

β-Adrenoceptor-mediated depolarization of the resting membrane in guinea-pig papillary muscles: changes in intracellular Na+, K+ and Cl− activities

Effects of -adrenergc stimulation on the membrane potential and intracellular Na⁺, K⁺ and Cl^– activities were examined in isolated guinea-pig ventricular muscles using conventional and ion-selective elctrodes. Isoproterenol in concentrations of 30 nM–1 M produced a transient depolarization followed by a slight hyperpolarization in electrically stimulated or quiescent papillary muscles. The negative logarithm of the concentration producing 50% maximum effect (pD₂) for the membrane-depolarizing effect of isoproterenol was smaller than that for the positive inotropic effect, suggesting that a higher level of cAMP accumulation is required to produce the transient depolarization. Whereas the isoproterenol(1 M)-induced depolarization was not blocked by tetrodotoxin (10 M), nifedipine (10 M), Cs⁺ (5 mM), Ba²⁺ (0.3 mM), amiloride (1 mM) or ouabain (10 M), it was significantly attenuated by anthracene-9-carboxylic acid (1 mM), a Cl^–-channel blocker. Intracellular K⁺ activity increased, whereas intracellular Na⁺ activity slightly decreased during the transient depolarization. Intracellular Cl^– activity significantly decreased during the isoproterenol-induced depolarization of the resting membrane. These results suggest that an inward current resulting from outward Cl^– movement, rather than inward Na⁺ movement, may be involved in the -adrenoceptor-mediated membrane depolarization.     

Voltage‐gated Ca2+ influx through L‐type channels contributes to sarcoplasmic reticulum Ca2+ loading in skeletal muscle

Key points

• Muscle contraction is triggered by Ca²⁺ ions released from the sarcoplasmic reticulum (SR) in response to depolarization of skeletal muscle fibres. Muscle activation is also known to be associated with a voltage‐activated trans‐sarcolemmal Ca²⁺ influx.
• Because removal of external Ca²⁺ does not impede fibres from contracting, a negligible role has been initially attributed to this Ca²⁺ entry. Furthermore, it is not clearly established whether Ca²⁺ exclusively flows through L‐type channels.
• By monitoring the quenching of fura‐2 fluorescence resulting from Mn²⁺ influx in voltage‐controlled mouse and zebrafish muscle fibres, we show that the L‐type current is the only contributor to Ca²⁺ influx during long‐lasting depolarizations.
• Calibration of the Mn²⁺ quenching signal allowed us to estimate an Mn²⁺ current of 0.31 A F^–1 flowing during a train of action potentials.
• Measurements of SR Ca²⁺ changes with fluo‐5N in response to depolarization indicated that voltage‐activated Ca²⁺/Mn²⁺ influx contributes to SR Ca²⁺/Mn²⁺ loading.

Abstract

Muscle contraction is triggered by Ca²⁺ ions released from the sarcoplasmic reticulum (SR) in response to depolarization of skeletal muscle fibres. Muscle activation is also associated with a voltage‐activated trans‐sarcolemmal Ca²⁺ influx early identified as a current flowing through L‐type Ca²⁺ channels. Because removal of external Ca²⁺ does not impede fibres from contracting, a negligible role was given to this voltage‐activated Ca²⁺ entry, although the decline of Ca²⁺ release is more pronounced in the absence of Ca²⁺ during long‐lasting activation. Furthermore, it is not clearly established whether Ca²⁺ exclusively flows through L‐type channels or in addition through a parallel voltage‐activated pathway distinct from L‐type channels. Here, by monitoring the quenching of fura‐2 fluorescence resulting from Mn²⁺ influx in voltage‐controlled mouse and zebrafish isolated muscle fibres, we show that the L‐type current is the only contributor to Ca²⁺ influx during long‐lasting depolarizations in skeletal muscle. Calibration of the Mn²⁺ quenching signal allowed us to estimate a mean Mn²⁺ current of 0.31 ± 0.06 A F^–1 flowing through L‐type channels during a train of action potentials. Measurements of SR Ca²⁺ changes with fluo‐5N in response to depolarization revealed that an elevated voltage‐activated Ca²⁺ current potentiated SR Ca²⁺ loading and addition of external Mn²⁺ produced quenching of fluo‐5N in the SR, indicating that voltage‐activated Ca²⁺/Mn²⁺ influx contributes to SR Ca²⁺/Mn²⁺ loading.