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.

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.

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.

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.

[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.

[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.

[Characteristics of surface-active substances effect on K+-dependent phosphatase activity of brain tissue]. / Kharakteristika vliianiia poverkhnostno-aktivnykh veshchestv na K+zavisimuiu fosfataznuiu aktivnost' tkani mozga.

The K+-acetylphosphatase and K+-p-nitrophenylphosphatase activities in the fraction of brain microsomes were studied as affected by anionic (sodium desoxycholate and sodium dodecyl sulphate) and nonionic (triton X-100 and digitonin) surface-active substances. The most activating concentrations of these substances are determined and their similarity with those for Na+, K+-ATPase is marked. According to the character of the effect on the K+-phosphatase and Na+, K+-ATPase activities, the studied surface-active substances are grouped on the basis of the molecule configurations, rather than ionogenic factor. Their activating effect is supposed to result from an increase in the number of functioning catalytic centres rather than the molecular activity of the enzyme. It is shown that the digitonin high concentrations may completely inhibit the Na+, K+-ATPase activity and to some extent retain the K+-acetylphosphatase activity.

[Thermal sensitivity of Na+,K+-ATPase isoenzymes in the brain and kidney]. / Termochuvstvitel'nost' izofermentov Na+,K+-azy mozga i pochek.

Thermal stabilities of Na+, K(+)-ATP-ase preparations with different isozyme content from brain and kidneys of different animal species have been compared. The greater thermal lability of the alpha(+)-isoform of a catalytic subunit of Na+, K(+)-ATP-ase is established. The method of specific thermal inactivation of in brain preparations was used in comparative study of ouabain sensitivity of Na+, K(+)-ATP-ase isozymes from rat, cow and rabbit. It is concluded that the existing structural and functional model of the receptor site of Na+, K(+)-ATP-ase catalytic subunit is not sufficient for the complete explanation of the species heterogeneity of Na+, K(+)-ATP-ase affinity to heart glycosides.

[Receptor function stability of cerebral Na+,K+-ATPase under conditions of modification of their enzymatic activity]. / Stabil'nost' retseptornoi funktsii izofermentov Na+,K+-ATPazy mozga v usloviiakh modifikatsii ikh fermentativnoi aktivnosti.

Rat and bovine Na+, K(+)-ATP-ase and isozymes have been studied for their sensitivity to ouabain to evaluate the conformational stability of the receptor for cardioactive steroid and its interconnection with intramembrane organization of catalytic polypeptide under modification of native functional conformation of membrane-bound enzyme by treatment with agents known to affect the membrane integrity (such as SDS, heat, phospholipase A2). The higher sensitivity of alpha-isoform of Na, K-ATP-ase activity to detergent as compared to alpha + under their irreversible inactivation that is due to its preferential sollubilization from the membrane is discovered. The existence of differences between isozymes in the hydrophobic protein-lipid interaction has been supposed. The greater sensitivity of alpha-isozyme to disorganization of the enzyme native phospholipid environment with phospholipase A2 has been also established. It is shown that Na+, K(+)-ATP-ase receptor function of ouabain-sensitive and ouabain-resistant enzyme types is much more conservative in comparison with hydrolytic and resistant to possible disorders of protein-lipid interactions.

[Activity of Na+, K+-ATP-ase isoenzymes in the cerebral cortex and their sensitivity to sodium dodecyl sulfate in rat postnatal ontogenesis]. / Aktivnost' izofermentov Na+, K+-ATP-azy kory golovnogo mozga i ikh chuvstvitel'nost' k dodetsisul'fatu natriia v postnatal'nom ontogeneze krys.

The comparative study of activities of Na+, K(+)-ATPase alpha (+)- and alpha-isozymes in rat brain cortex in the postnatal period is conducted. It is shown, that the rates of isozymes expression are not different and levels of their activities reach maximum to the 30th day after birth. Na(+), K(+)-ATPase resistance to SDS inactivation increases with the age, that reflects possible changes in the peculiarities in structural organization of isozymes protein-lipid complex. Results are discussed from the view point of the adaptive changes in the process of structural-metabolic organization of biomembrane function occurring in ontogenesis.

[Thermostability of brain Na+, K+ -ATPase in the presence of surface-active agents]. / Termostabil'nost' Na+, K+ -ATPazy mozga v prisutstvii poverkhnostno-aktivnykh veshchestv.

Stability of Na+, K+-ATPase (EC 3.6.1.3) of the brain microsomal fraction towards thermoinactivation was studied under the effect of surfactants--alkyl sulphates with the C8-C15 chain length of a hydrocarbon radical, desoxycholate, triton X-100, twin-80 and digitonin. It is shown that NaCl treatment of the enzyme before thermoinactivation, and also in the presence of surfactants, increases its resistance to the effect of temperature (octyl and decyl sulphates are an exception, in their presence transport ATPase preincubated with NaCl is completely inactivated). In the presence of most surfactants used in the maximum activating concentrations the activity of Na+, K+-ATPase remained at a considerably high level (80-110% of the initial one) and did not change essentially under NaCl pretreatment of the enzyme. Besides octyl and decyl sulphate, desoxycholate, was also an exception, in the presence of NaCl it inhibited sharply the Na+, K+-ATPase activity. An interrelation is found between the ability of surfactants to decrease the activity of transport ATPase and to solubilize proteins and lipids from the microsomal fraction.

[Activation of brain K+-phosphatase and Mg2+, Na+, K+-AtPase by sodium alkyl sulfates]. / Aktivatsiia K+-fosfatazy i Mg2+,Na+,K+-ATP-azy mozga alkilsul'fatami natriia.

K+-dependent phosphatase and Mg2+, Na+, K+-ATPase were studied under the activating effect of surfactant homologs of the alkyl sulphate series with the hydrocarbon radical long chain C4-C15. The homologs are shown to activate the enzymes when they are in the molecular-disperse but not in micellar state. A clear regularity is observed in the effect of these surfactants on K+-phosphatase depending on the length of the hydrocarbon radical chain: the degree of the activating effect rises with the chain lengthening, reaching the maximum value when the number of carbon atoms is 12. The lower and upper bounds of the alkyl sulphate hydrocarbon radical chain length necessary for manifestation of the activating effect shift somewhat for K+-dependent phosphatase as compared with Mg2+, Na+, K+-ATPase. The data obtained evidence for a stronger stability of the phosphatase to a destructive effect of the surfactants as compared with transport ATPase.

[The role of magnesium ions in the regulation of the catalytic activity of kidney Na+,K+-ATPase]. / Rol' ionov magniia v reguliatsii kataliticheskoi aktivnosti Na+,K+-ATP-azy pochek.

Free Mg2+ is studied for its effect on the activation kinetics of pig kidney Na+, K+-ATPase by monovalent cations (nH and K0.5 for Na+ and K+ are determined). It is established that at the saturating concentration of complementary ion-activator an increase of free Mg2+ concentration up to 12 mM is accompanied by a rise of nH and K0.5 for Na+ and a fall of K0.5 for K+ without nH changes for this cation. The analysis of inhibition kinetics shows that free Mg2+ is a competitive inhibitor as to Na+ and noncompetitive as to K+. It is concluded that inhibition of Na+, K+-ATPase by free Mg2+ is a complex process including competition with Na+ at its binding sites and the "occluding" of enzyme at the stage, preceding dissociation of cation and also the weakening of subunit interactions in the enzyme.

[The thermally induced transformation of the protein-detergent complexes of the Na+,K(+)-ATPase catalytic subunits of the kidneys and brain in PAG electrophoresis]. / Termoindutsiruemaia transformatsiia belkovo-detergentnykh kompleksov kataliticheskikh sub''edinits Na+,K(+)-ATP-azy pochek i mozga pri élektroforeze v PAAG.

Electrophoretic properties of the protein bands of Na+, K(+)-ATPase catalytic subunits of the kidney and brain have been comparatively studied for some types of animals after heat treatment of the preparations in the presence of DS-Na. The diffuse protein zone with lower electrophoretic mobility after heat-induced conformational transformation of alpha appears on gels parallel with protein band alpha II which is typical of this process. The prolonged heat treatment causes more rapid electrophoretic degradation of the alpha-isoform of the Na+, K(+)-ATPase catalytic subunit of the brain in comparison with alpha +. The results of the investigations are discussed in the aspect of existence of heat-induced transformation of the protein-detergent complexes of catalytic subunits of the enzyme.

[Kinetics of thermoinactivation of kidney Na+-,K+-ATPase]. / Kinetika termoinaktivatsii Na+-,K+-Atpazy pochek.

The studies of Na+,K+-ATPase (the fraction of microsomes and highly active preparation) thermoinactivation in kidneys at 50, 55 and 60 degrees C under conditions of the different ionic composition of the medium has shown that 20 mM K+ protects the enzymic complex from the thermal denaturation more effectively than 182 mM Na+ does. An increase in the Mg2+ concentration in the medium from 1 to 5 mM decreases Na+,K+-ATPase resistance to thermoinactivation. The enzyme half-life becomes almost thrice as low in this case. The results obtained are discussed from the standpoint of specificity of ion-induced conformational states of Na+,K+-ATPase and regulatory role of cations in the process of its functioning.

[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.