Low Ouabain Doses and AMP-Activated Protein Kinase as Factors Supporting Electrogenesis in Skeletal Muscle.

Many motor disorders are associated with depolarization of the membrane of skeletal muscle fibers due to the impaired functioning of Na,K-ATPase. Here, we studied the role of ouabain (specific Na,K-ATPase ligand) and AMP-activated protein kinase (key regulator of muscle metabolism) in the maintenance of muscle electrogenesis; the levels of these endogenous factors are directly related to the motor activity. After 4-day intraperitoneal administration of ouabain (1 µg/kg daily), a hyperpolarization of sarcolemma was registered in isolated rat diaphragm muscles due to an increase in the electrogenic activity of Na,K-ATPase. In acute experiments, addition of nanomolar ouabain concentrations to the bathing solution resulted in the muscle membrane hyperpolarization within 15 min. The effect of ouabain reversed to membrane depolarization with the increase in the external potassium concentration. It is possible that Na,K-ATPase activation by ouabain may be regulated by such factors as specific subcellular location, interaction with molecular partners, and changes in the ionic balance. Preventive administration of the AMP-activated protein kinase activator AICAR (5-aminoimidazole-4-carboxamide-1-ß-D-ribofuranoside; 400 mg/kg body weight daily for 7 days) in chronic experiments resulted in the stabilization of the endplate structure and abolishment of depolarization of the rat soleus muscle membrane caused by the motor activity cessation. The obtained data can be useful for creating approaches for correction of muscle dysfunction, especially at the early stages, prior to the development of muscle atrophy.

Correction to: Abnormal Membrane Localization of α2 Isoform of Na,K-ATPase in m. soleus of Dysferlin-Deficient Mice.

The third author's name should read: V. V. Matchkov.

Abnormal Membrane Localization of α2 Isoform of Na,K-ATPase in m. soleus of Dysferlin-Deficient Mice.

Dysferlin protein plays a key role in the multimolecular complex responsible for the maintenance of sarcolemma integrity and skeletal muscle cell functioning. We studied the membrane distribution of nicotinic acetylcholine receptors and α2 isoform of Na,K-ATPase in motor endplates of m. soleus in dysferlin-deficient Bla/J mice (a dysferlinopathy model). Endplates of Bla/J mice were characterized by increased area (without changes in fragmentation degree) and reduced density of the membrane distribution of nicotinic acetylcholine receptors in comparison with the corresponding parameters in control С57Bl/6 mice. The density of the membrane distribution of α2 isoform of Na,K-ATPase was also reduced, but the level of the corresponding mRNA remained unchanged. It can be hypothesized that abnormal membrane localization of α2 isoform of Na,K-ATPase results from adaptive skeletal muscle remodeling under conditions of chronic motor dysfunction.

Role of cholesterol in the maintenance of endplate electrogenesis in rat diaphragm.

Methyl-ß-cyclodextrin (0.1 mM) reduced resting potential of muscle fibers and abolished local endplate membrane hyperpolarization in rat diaphragm. This effect was associated with selective reduction of electrogenic activity of α2-isoform of Na,K-ATPase without changes in the level of intracellular acetylcholine. Experiments with cholesterol marker filipin showed that methyl-ß-cyclodextrin in this dose induced cholesterol translocation from lipid rafts to liquid phase of the membrane without its release into extracellular space. This modification of lipid rafts by methyl-ß-cyclodextrin presumably impaired the mechanism maintaining electrogenesis in endplates mediated by modulation of Na,K-ATPase by non-quantum acetylcholine. Cholesterol can serve as a molecular component of this mechanism.

[Functional interactions of Na,K-ATPase with molecular environment].

The present review covers the analysis of research in the field of intermolecular interactions of Na,K-ATPase which underlie novel cellular regulatory mechanisms. The capability of Na,K-ATPase to form multimolecular regulatory complexes and take part as a scaffolding protein in formation of cell functional and signaling microcompartments is considered. Particular attention is directed to membrane lipid microdomains (rafts, caveolae). Mechanisms of signaling and neuroprotective functions of Na,K-ATPase as well as the involvement of endogenous cardiotonic steroids in their realization are discussed. This review demonstrates novel data on functional interactions of Na,K-ATPase with neurotransmitter receptors and other proteins involved in regulation of synaptic transmission.

[Regulatory function of the Na,K-ATPase alpha2 isoform].

A present review is devoted to the analysis of literature data and results of our own research in the field of the Na,K-ATPase molecular diversity. Abundant evidence shows that the Na,K-ATPase alpha2 isoform is not only involved in various specific cell functions but also affected by different regulatory factors as compared to the alpha1 isoform which carries the main pump function. Data gathered suggest that these features of alpha2 isoform are determined by its functional and molecular environment, localization in specific cellular microdomains and also by less stable integration into the cell membrane as compared to other isoforms of the Na,K-ATPase alpha subunit.

[Effects of chronic nicotine exposure on electrogenic activity of the Na+, K(+)-ATPase and contractility in the rat diaphragm muscle].

Rats were chronically treated with nicotine via subcutaneous injections up to a dose 6 mg/kg/day during 2-3 weeks. After this period, resting membrane potential and action potentials of muscle fibres as well as isometric twitch and tetanic (20 s(-1) and 50(-1)) contractions of isolated rat diaphragm were studied. To estimate electrogenic contribution of the alpha2 isoform of the Na+, K(+)-ATPase ouabain in concentration 1 microM was used. Chronic nicotine exposure induced depolarization of resting membrane potential of 2.2 +/- 0.6 mV (p < 0.01). In rats chronically exposed to nicotine, electrogenic contribution of the Na+, K(+)-ATPase alpha2 isoform was twofold lesser than in control animals (3.7 +/- 0.6 mV and 6.4 +/- 0.6 mV, respectively, p < 0.01). Chronic nicotine exposure did not affect force of twitch and tetanic contractions in response to direct or indirect stimulation. A decrease in the twitch contraction time as well as in the rise time of tetanic contractions was observed. Fatigue dynamics was unchanged. The results suggest that chronic nicotine exposure leads to decrease of the Na+, K(+)-ATPase alpha2 isoform electrogenic activity, and as a consequence to damage of the rat diaphragm muscle electogenesis.

[Effect of functional unloading and dystrophin deficit on the local hyperpolarization of the postsynaptic membrane of a skeletal muscle fiber].

The data have been obtained that confirm the identity of the electrogenic mechanism of hyperpolarization by nanomolar concentrations of cholinergic ligands in the extrasynaptic region and endogenous nonquantal acetylcholine in the synaptic region of a skeletal muscle fiber. In both cases, this mechanism is realized through the involvement of the alpha2 isoform of Na, K-ATPase and operates in the absence of Na+ entry through membrane channels. At the same time, there are peculiar properties which take place under functional disorders. Thus, the effectiveness of this mechanism in the synaptic region selectively increases under rat hindlimb unloading and decreases in case of dystrophin deficit in mdx mice. The last fact suggests that dystrophin is a molecular component that is essential for the functioning of the electrogenic mechanism of local hyperpolarization of the end-plate membrane.

[Electrogenic activity of Na-K-ATPase and calcium ions in m. soleus fibers of rats and Mongolian gerbil during simulation of gravitational unloading].

Some of the electrophysiological parameters of m. soleus of rat and Mongolian gerbil, and Ca ions content in fiber myoplasm were compared in different periods of gravitational unloading simulated by tail-suspension. No difference was found between the control animals as for membrane potential at rest, electrogenic activities of Na-K-ATPase and its isoforms, and input resistance of m. soleus fibers. At the same time, unlike rats, gerbils exhibited a substantial Ca decrease in myoplasm. From day one to 14 of gravitational unloading the pace of electrophysiological changes in gerbil's m. soleus was noticeably slower than of rat's, whereas Ca ions depositing in myoplasm was observed in both species already at the beginning ofsuspension. Analysis of the results suggests that adaptive changes in m. soleus of Mongolian gerbil and rat during simulated gravitational unloading are fundamentally different due to, probably, peculiar water-electrolyte metabolism, type of locomotion, and other factors which are still unclear.

[Comparative analysis of structural and functional characteristics of soleus muscle in rats and Mongolian gerbils during gravitational unloading of various duration].

A comparative investigation of the dynamics of contractile properties of the whole soleus muscle and its fibers during 3- and 12-day-long hind limb suspension of Wistar rats and Mongolian gerbils (Meriones unguiculatus) has been performed. The data obtained indicate that the structural and functional changes caused by hypogravity in gerbils are slowed down compared with rats. A very intensive drop in water containment in gerbils was found, which can cause shifts in the ionic strength of the intracellular space of the muscle fiber. As a result, the photolytic activity of different enzymes may change, which can induce a less pronounced reduction in Z-disc and M-line stiffness and contractile capabilities in gerbils compared to rats.

[Effect of nicotine on electrogenic contribution of the Na,K-ATPase isoforms and contractile characteristics of the rat skeletal muscle].

Nicotine in concentration 100 nM which corresponds to concentration of nicotine circulating in tobacco smokes induced hyperpolarization by approximately 4 mV of muscle fibres of the rat isolated diaphragm, as well as an increase in amplitude and acceleration of action potentials. Similar hyperpolarization was induced by nicotine and acetylcholine in the rat soleus muscle. In this muscle, the hyperpolarization developed more slowly than in diaphragm revealing initial slight depolarization. Non-competitive blocker of open channel of nicotinic acetylcholine receptor, proadifen, abolished nicotine- or acetylcholine-induced depolarization but not the hyperpolarization. In the diaphragm, the hyperpolarization was blocked by specific inhibitors of the Na,K-ATPase, ouabain (50 nM) or marinobufagenin (2 nM) suggesting an involvement of the Na,K-ATPase. Estimation of elecrogenic contributions of isoforms of the Na,K-ATPase showed that the hyperpolarization was due to an increase in electrogenic contribution of alpha 2 isoform without change in contribution of alpha 1 isoform. Nicotine did not affect parameters of muscle contractions in response to direct stimulation.

[Decrease in the electrogenic contribution of Na,K-ATPase and resting membrane potential as a possible mechanism of calcium ion accumulation in filaments of the rat musculus soleus subjected to the short-term gravity unloading].

After three days of hind limb unloading, the depolarization of muscle fibers from -71.0 +/- 0.5 mV to -66.8 +/- 0.7 mV as well as a decrease in muscle excitability and a trend to fatigue acceleration were observed. After hind limb unloading, the electrogenic contribution of the ouabain-sensitive alpha2 isoform of Na,K-ATPase, tested as depolarization due to the administration of 1 microM ouabain, decreased from 6.2 +/- 0.6 to 0.5 +/- 0.8 mV. The contribution of the ouabain-resistant alpha1 isoform, estimated as additional depolarization after the administration of 500 microM ouabain, decreased from 4.6 +/- 0.6 to 2.6 +/- 0.6 mV. After hind limb unloading, the fluorescence intensity of single muscle fibers loaded with Fluo-4-AM increased more than four times, indicating an increase in intracellular Ca2+ concentration. The effect was prevented by local delivery of nifedipine, which blocks L-type Ca2+ channels. These data suggest the existence of a selective mechanism of suppression of the alpha2-pump electrogenic contribution, which led to the depolarization of soleus muscle fibers after 3 days of hind limb unloading. The depolarization in turn may activate L-type Ca2+ channels, resulting in intracellular Ca2+ accumulation.

[Contractile properties of the isolated rat musculus soleus and single skinned soleus fibers at the early stage of gravitational unloading: facts and hypotheses].

The contractile properties of the postural rat soleus muscle at the early stage of the gravitational unloading (3-day rat hindlimb suspension) have been studied using different modes of muscle contraction (twitch and tetanic contraction of the isolated muscle, Ca-induced contraction of isolated skinned fibers). A significant enhancement of the twitch maximal tension of unloaded muscles without changes in time-dependent characteristics was observed, although the half-relaxation time tended to increase. The fiber diameter did not change (42.37 +/- 0.76 vs 43.43 +/- 1.15 microm in controls). The Ca-induced maximal isometric tension in unloaded soleus was significantly decreased (32.1 +/- 1.05 vs 37.6 +/- 1.52 mg in controls, p < 0.05). The maximal specific tension was respectively decreased (23.14 +/- 0.77 vs 27.6 +/- 2.36 kN/m in controls). The pCa50 in unloaded muscle decreased from 6.05 +/- 0.02 in controls to 5.97 +/- 0.02 (p < 0.05), indicating the loss of myofibrillar calcium sensitivity. The analysis with the calcium probe Fluo-4AM demonstrated that the intracellular [Ca2+] was sufficiently increased after hindlimb suspension. At the same time, the relative content of titin and nebulin did not change.

[Role of the alpha2-isoform of Na+, K(+)-ATPase in the positive inotropic effect of ouabain and marinobufagenin in the rat diaphragm].

It was shown that the specific inhibitors of Na+, K(+)-ATPase ouabain and marinobufagenin increased the contraction of an isolated rat diaphragm (positive inotropic effect) by up to approximately 15% in a dose-dependent manner with EC50 = 1.2 +/- 0.3 and 0.3 +/- 0.1 nM, respectively. The results indicate the involvement of the ouabain-sensitive alpha 2 isoform of Na+, K(+)-ATPase. The analysis of ouabain-resting membrane potential dose-response relationships in the presence and absence of hyperpolarizing concentration of acetylcholine (100 nM) suggests the existence of two pools of alpha 2 Na+, K(+)-ATPase with different affinities for ouabain. The pool with a higher ouabain affinity is involved in the hyperpolarizing effect of acetylcholine and, presumably, in the positive inotropic effect of ouabain, which might be a mechanism of regulation of muscle efficiency by circulating endogenous inhibitors of Na+, K(+)-ATPase.

[Analysis of the interaction between nicotinic acetylcholine receptor and Na+,K(+)-ATPase in the rat skeletal muscle and the Torpedo electric organ membrane preparation].

The interaction between the nicotinic acetylcholine receptor and Na+,K(+)-ATPase described previously was further studied in isolated rat diaphragm and in a membrane preparation of Torpedo californica electric organ. Three specific agonists of the nicotinic receptor: acetylcholine, nicotine and carbamylcholine (100 nmol/L each), all hyperpolarized the non-synaptic membranes of muscle fibers by up to 4 mV. Competitive antagonists of nicotinic acetylcholine receptor, d-tubocurarine (2 mcmol/L) or alpha-bungarotoxin (5 nmol/L) completely blocked the acetylcholine-induced hyperpolarization indicating that the effect requires binding of the agonists to their specific sites. The noncompetitive antagonist, proadifen (5 mcmol/L), exerted no effect on the amplitude of hyperpolarized but decreased K0.5 for this effect from 28.3 +/- 3.6 nmol/L to 7.1 +/- 2.3 nmol/L. Involvement of the Na+,K(+)-ATPase was suggested by data demonstrating that three specific Na+,K(+)-ATPase inhibitors: ouabain, digoxin or marinobufagenin (100 nmol/L each), all inhibit the hyperpolarizing effect of acetylcholine. Acetylcholine did not affectation either the catalytic activity of the Na+,K(+)-ATPase purified from sheep kidney or the transport activity of the Na+,K(+)-ATPase in the rat erythrocytes, i. e. in preparations not containing acetylcholine receptors. Hence, acetylcholine does not directly affect the Na+,K(+)-ATPase. In a Torpedo membrane preparation, ouabain (< or = 100 nmol/L) increased the binding of the fluorescent ligand: Dansyl-C6-choline (DCC). No ouabain effect was observed either when the agonist binding sites of the receptor were occupied by 2 mmol/L carbamylcholine, or in the absence Mg2+, when the binding of ouabain to the Na+,K(+)-ATPase is negligible. These results indicate that ouabain only affects specific DCC binding and only when bound to the Na+,K(+)-ATPase. The data obtained suggest that, in two different systems, the interaction between the nicotinic acetylcholine receptor and the Na+,K(+)-ATPase specifically involve the ligand binding sites of these two proteins.

[Effects of marinobufagenin on electrophysiological and contractile characteristics of the rat diaphragm].

Effects of Na+,K(+)-ATPase inhibitor: marinobufagenin, on contractile and electric characteristics of isolated rat diaphragm were studied for the first time. Marinobufagenin induced dose-dependent (EC50 = 0.3 +/- 0.1 nM) increase in the contraction force (positive inotropic effect). At 1-2 nM, it slowed down the fatigue induced by continuous direct stimulation (2/s) of the muscle. Marinobufagenin at the same concentrations did not affect resting membrane potential or parameters of action potentials of muscle fibers, while at 10 and 20 nM it induced hyperpolarization by approximately 2 mV. Marinobufagenin blocked dose-dependently (IC50 = 2.9 +/- 2.0 nM) hyperpolarizing effect of acetylcholine (100 nM) mediated by increase in electrogenic contribution of alpha2 isoform of the Na+,K(+)-ATPase. This result suggests a capability of marinobufagenin to inhibit this isoform of the Na+,K(+)-ATPase. Possible mechanisms of marinobufagenin effects in skeletal muscle are discussed.

[Ouabain-induced blockade of alpha2 isoform of the Na,K-ATPase on electrophysiological and contractile characteristics of the rat diaphragm].

In experiments with isolated neuromuscular preparation of the rat diaphragm, selective blockade of alpha2 isoform of the Na,K-ATPase with ouabain (1 mcmol/L) induced steady depolarization of muscle fibers that reached a maximum of 4 mV, a decrease in amplitude of muscle fiber action potential, and prolonged raising and decline phases of the action potential. At the same time, the force, time to peak, and half relaxation time of the isometric muscle twitch were increased, as well as the area under the contraction curve. During continuous fatiguing stimulation (2/s), a more pronounced decline of contraction speed was observed in presence of ouabain; dynamics of the half-relaxation time remaining unchanged. It is suggested that blockade of alpha2 isoform of the Na,K-ATPase impairs excitation-contraction coupling resulting in a delay of Ca2+ release from sarcoplasmic reticulum. The increase in contraction force seems to result from a mechanism similar to that of positive inotropic effect of cardiac glycosides in heart muscle. Physiological significance of the skeletal muscle alpha2 isoform of the Na,K-ATPase in regulation of Ca2+ and Na+ concentrations near triadic junctions and in regulatory processes involving the Na,K-ATPase endogenous modulators or transmitter acetylcholine is discussed.

[From diversity of molecular forms to functional specialization of oligomeric proteins, nicotinic acetylcholine receptor, acetylcholinesterase and Na+, K+-ATPase]. / Ot raznoobraziia molekuliarnykh form k funktsional'noi spetsializatsii oligomernykh belkov, nikotinovyi kholinoretseptor, atsetilkholinésterasa i Na+, K+-ATFaza.

The review is devoted to the issue of diversity of molecular forms of oligomeric proteins using as examples members of the three protein classes: nicotinic acetylcholine receptor, acetylcholinesterase, and Na,K-ATPase. The data are presented on the molecular structure of proteins, subunit compositions, and isoforms of subunits, as well as on some features of gene expression. Particular emphasis has been made on the functional specialization of different molecular forms of one and the same oligomeric protein. The three above proteins, which serve seemingly quite different cellular processes, demonstrate many common principles of molecular mechanisms of physiological function.