Krivoi

St. Petersburg State University, St. Petersburg, Russia The Na,K-ATPase is a P-type ATPase which catalyzes the active transport of K⁺ into and Na⁺ out of the cell, thereby maintaining the steep Na⁺ and K⁺ gradients that underlie the resting membrane potential and electrical excitability of cells. A Na⁺ gradient ensures the work of coupled transporters of Ca²⁺ and H⁺, glucose, amino acids, neurotransmitters, vit- amins and reabsorption of Na⁺. Thus Na,K-ATPase plays the main role in regulation of water-salt exchange, presents an important factor resisting hypoxia and fatigue.

The Na,K-ATPase consists of alpha-catalytic and beta-glyco- protein subunits as well as a FXYD subunit which modulates enzyme activity. Presently mammals are known to have four isoforms of alpha subunit, three isoforms of beta subunit of Na,K-ATPase, and also seven proteins of the FXYD family. It is generally accepted that the ubiquitous alpha1 isoform plays the main “house-keeping” role while the other isoforms are expressed in a cell- and tissue-specific manner and possess additional regulatory functions which are still poorly understood.

Owing to specific structural domains the Na,K-ATPase may form multimolecular complexes with membrane, intracellular and cytoskeletal proteins and take part in formation of functional microcompartments of the cells and in intercellular interactions. An important role in compartmentali- zation and regulation of Na,K-ATPase is played also by membrane lipids, in particular cholesterol, which participates in control of fluidity and curva- ture of cell membrane. Cholesterol also is an essential component of mem- brane mobile heterogeneous domains – lipid rafts, participating in for- mation of functional cell microcompartments. In addition, Na,K-ATPase

forms multimolecular complexes in specialized microdomains of the mem- brane called caveolae. They are the invaginations of the plasma membrane characterized by specific lipid composition, in which some proteins are localized performing a signal function in the cell.

It is shown that in neurons, astrocytes and myocytes cardiotonic steroids (CTS) cause Ca²⁺ oscillations only in special microcompartments where Na,K-ATPase coordinates with Na⁺,Ca²⁺ exchanger (NCX), vari- ous Ca²⁺ channels and receptors of the plasma membrane in proximity to sarco(endo)plasmic reticulum. In accordance with this «PLasmERosome»

model inhibition of a part of Na,K-ATPase leads to Na⁺ accumulation in these narrow near-membrane spaces. This is accompanied by accumula- tion of Ca²⁺ as a result of a decrease in its extrusion by NCX. It is estab- lished that the Na,K-ATPase α2/α3 isoforms have specific structural do- mains that are responsible for their ability to clusterize in these microdomains. It is supposed that CTS-evoked slow Ca²⁺ oscillations af- fect not only muscle contractility but also genome and expression of pro- teins, proliferation, differentiation, etc. In this local Ca²⁺ handling there also involved SERCA, ryanodine and IP3-receptors of sar- co(endo)plasmic reticulum. It was shown that CTS may liberate Ca²⁺

from depot as a result of conformations changes of the Na,K-ATPase alpha subunit N-end interacting with the IP3-receptor of sarcoplasmic reticulum. In the formation of such Ca²⁺ microdomains an important role is played by various proteins, including ankyrin.

Many proofs has been shown for additional function of Na,K- ATPase as a signal molecule. This function is realized due to functional and direct molecular interactions of Na,K-ATPase with different neighbor- ing proteins. Major of such proteins appears to be Src kinase, which forms a functional complex with Na,K-ATPase, activating upon binding CTS a series of signal intracellular cascades. It is supposed that signal function is actualized by Na,K-ATPase in caveolae of the plasma membrane («Signalosome» model). According to this model, Na,K-ATPase alpha subunit is involved in a signal complex with Src kinase. These complexes also include NCX, Ca²⁺ channels, phospholipase C, IP3 receptor, caveolin etc. Due to such molecular organization of signal transmission even ul- tralow concentrations of CTS, without inhibition of Na,K-ATPase activity, are capable of regulating most diverse cellular functions: protein synthesis, proliferation and differentiation, processes of apoptosis, contractile proper- ties and others, influence cell survivability and death, exert anticancer and neuroprotector action. It is supposed that physiological stimuli triggering such a signal complex a presented by endogenous CTS.

Functional specialization and peculiarities of the Na,K-ATPase al- pha2 regulation isoform are studied more precise. The alpha2 Na,K- ATPase of astrocytes is involved in clearance of the intercellular space from accumulated potassium ions. In addition, alpha2 isoform partici- pates in glutamate level control due to functional and molecular interac- tion with the glutamate transporter, which is considered to be an im- portant factor in the pathophysiology of migraine.

In a vertebrates body Na,K-ATPase predominantly located in muscle tissues where the alpha1 and alpha2 isoforms of alpha subunit are ex- pressed. In skeletal muscle the Na,K-ATPase is critically important for ex- citability, electrogenesis and contractility. It was shown that skeletal mus- cle use and disuse differently regulates the alpha1 and alpha2 isoforms however mechanisms of this regulation remain to be elucidated. Numerous evidences suggest that the Na,K-ATPase alpha2 isoform play key role as regulator of calcium balance and contractile properties of cardiac and smooth muscles due to functional interaction with NCX and co-localization with sarcoplasmic reticulum. It is established that it is the alpha2 isoform that plays the main role in realization of positive inotropic action of cardiac glycosides in cardiac, smooth and presumably skeletal muscles.

In sum, data obtained from different cells and tissues indicates that the Na,K-ATPase alpha2 isozyme is the more regulated subunit com- pared to alpha1. Regulation of alpha2 Na,K-ATPase is determined by its functional and molecular environment, localization in specific cellular microdomains. These peculiarities of the alpha2 Na,K-ATPase are ac- companied with its less stable integration into the cell membrane com- pared to other Na,K-ATPase alpha isoforms. Instability of the alpha2 iso- form is explained by its solely inherent peculiarities of transmembrane domains М8–М10, responsible for interaction with phospholipids, and also by weaker association with subunit FXYD1.

Supported by RFBR #13-04-00973а and St. Petersburg State Uni- versity research grant #1.38.231.2014.

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EVOLUTION OF INDIVIDUAL CONTRACTILE EVENTS DURING TETANIC STIMULATION OF FAST- AND SLOW-

TWITCH RAT MUSCLES

I.V. Kubasov, R.S.Arutyunyan, E.V. Matrosova

Sechenov Institute of Evolutionary Physiology and Biochemistry RAS, 194223, 44, Thorez av., St-Petersburg, Russia

The purpose of this study was to evaluate behavior of individual contractile components (ICC) of muscle response to a tetanic stimulation.

To achieve this, we analyzed characteristics (peak amplitude and half- relaxation time) of the last ICC (force transient) in the tetanic constraction evoked by train of 5, 10, 20, 30, 40, 50 or 100 stimuli deliv- ered at 20 or 50 Hz rate (m. Soleus and m. EDL, respectively). In each set of stimulating conditions, N^th ICC was isolated by point-by-point subtrac- tion of di-

Fig. 1. Effect of duration of tetanic stimulation on global and last ICC mus- cle contractile responses . A, Representative record of global and last ICC responses of m. Soleus to a trains of 10 -50 stimuli (delivered at 20 Hz in- tra-train rate). B, Mean amplitude of last ICC of m. Soleus (circles, n=7) and m. EDL (triangles, n=6).

gitally recorded profiles of tetanic contractions evoked by stimulation with N and N-1 pulses-long trains. The results of this analysis were used to estimate the effect of duration of tetanic stimulation on characteristics of individual twitch as an elementary unit of overall tetanic contractile response of a muscle. Our data demonstrate a significant effect of pro- longed stimulation on both amplitude and half- relaxation of ICC. With an increase in duration of tetanic stimulation, ICC of m. EDL showed progressive potentiation, while changes in ICC of m. Soleus were transi- ent. The amplitude of ICC elicited by first 5 to10 pulses in the train was decreased by 30-40% but this effect was attenuated and eventually re- placed with potentiation of ICC with prolongation of the tetanus. Fig.1A shows the representative example of global (solid lines) and last stimulus ICC (dotted lines) of m. Soleus’s contractile response to a 5 - 50 pulses- long tetanic stimulation. Unlike that for slow, m. Soleus (fig. 1A and B, circles), ICC of fast, EDL muscle show potentiation with the increase in duration of tetanic stimulation (fig. 1B, triangles).

In addition to the effect on amplitude of ICC duration of tetanic stimulation had also affected the time characteristics of ICCs. Duration of half- relaxation of ICC decreased from 224 ± 8 ms to 115 ± 12 ms in m.

Soleus and from 31 ± 4 ms to 24 ± 3 ms in m. EDL (fig. 2). These effects of prolongation of tetanic stimulation could be interpreted as the result of activation of Ca²⁺-ATPase.

As it is shown in fig.3, for both types of muscles, latency of ICC increases with an increase in duration of tetanic train from nearly zero for single twitch response to 26 ± 9 ms in m. EDL (n=6) and 157 ± 12 ms in m. Soleus (n=7) at the end of 100 pulses-long tetanic stimulation. Proba- bly, this increase in latency of ICC reflects changes in the dynamics of

Fig. 2. Post-peak decline of single muscle twitch (solid trace) and ICC (dashed trace) at the end of tetanic train with 100 pulses. Averages of re- cordings collected in 7 and 6 independent experiments are shown in panels A (m. Soleus) and B (m EDL), respectively.

Fig. 3. Single muscle twitch response (solid trace) and ICC (dashed trace) at the end tetanic train with 100 pulses. Averages of recordings collected in 6 (m EDL, panel A) and 7 (m Soleus, panel B) independent experiments are shown. All recordings were normalized to the peak amplitude of respective muscle twitch response.

occupation by Ca²⁺ of troponin C binding sites situated close to points of Ca⁺² release. As to the difference between latencies of m. EDL and m.

Soleus ICCs, it could be attributed to a different sensitivity of fast and slow muscle fibers to Ca²⁺.

The potentiation of ICCs during tetanic train is not associated with any detectable changes in either amplitude or duration of muscle fiber action potentials (APs). Therefore, it seems reasonable to speculate that changes in operation of other mechanisms such as mechanisms of intra- cellular Ca²⁺ release, binding and/or uptake are responsible for this poten- tiation. To test the possibility of activation of Ca²⁺-indused Ca²⁺-release (CICR) we evaluated behavior of ICCs during tetanic stimulation of caf- feine-treated muscles. In experiments in m. EDL, application of 10 mM of caffeine in bath solution led to prolongation and potentiation of single twitch and tetanic muscle responses but it decreased amplitude of ICCs to about half of that of the first twitch in the tetanus (fig. 4).

The same picture was observed in experiments with caffeine-treated m. Soleus (data not shown). Thus initialy activated by caffeine CICR does

Fig. 4. Single twitch response (solid trace) and 10th, 50th and 100th ICCs (dash traces) during tetanic stimulation at 50.

not support subsequent ponentiation of ICCs during tetanic stimulation. In tetanus decrease in amplitude of ICCs may be associated with depletion in intracellular Ca²⁺ stores in sarcoplasmic reticulum. Potentiation of tetanic force is likely conditioned by initialy prolongated by caffeine of ICCs de- cay phases only. Part of our finding are similar to that has been shown by B.R. MacIntosh et al. in surgically isolated in situ gastrocnemius muscle for train stimulation consisned of 1-5 pulses [MacIntosh et al., 2006]. Po- tentiation contractile events during tetanic stimulation and its elimination by caffeine can be evidence of increasing of Ca²⁺-release during tetanuses development. But in experiments with fluorscent indicators was shown decline of the amount released in repeated action potentials. The fifth depo- larization in high-frequency train (50 Hz) of action potentials released only the amount of calcium corresponding to one-fifth of calcium released by the first action potential in slow and one-six in fast fiber (Baylor, Hollingworth, 2003). In our experiments we observed only short-lasting decreasing in transient responses (by 30-40 %) in slow but not fast mus- cles. Reasons of this disparitet is not clear and need in further study.

This work was supported by grant RFBR №13-04-00509А.

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Physiol. 2003. 551(1):125–138.

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Physiol. Rev. 2010. 91: 1447-1531.

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LOAD PARAMETERS TO PRESERVE THE POWER OF ENDURANCE IN THE PROCESS OF RESISTANCE TRAINING OF ASTRONAUTS IN WEIGHTLESSNESS

T.B. Kukoba, Е.V. Fomina

Institute of Biomedical Problems of the Russian Acad. Sci., Moscow, 123007, Russia

The Russian physical countermeasure system is a complex training with the use of both active and passive methods. Among the active meth-

ods is the NASA ARED that allows a vast variety of resistive exercises for the main groups of muscles. These are NASA coaches who develop and introduce the ARED training programs to cosmonauts. According to the US partners’ concept, astronauts perform a high-intensity resistive training, i.e. the “loading weight” amounts to 70 - 90% of a rep max and number of repetitions, 3 to 10. Russian instructors give preference to a less intensive regimen; they ease the force loading and increase the number of repetitions and attempts instead. For the ISS Russian crewmembers, resistive sessions are scheduled on days 2 and 4 of the training microcycle so that cosmo- nauts exercise with the ARED two or three times a week.

In order to evaluate effectiveness of the resistive training with dif- ferent levels of loading, the strength endurance of cosmonauts’ femoral muscles was tested pre and post flight.

The experiment involved 11 cosmonauts who were on 145 to 167-day long ISS missions. Depending on the ARED resistive loading, the cosmo- nauts were distributed into 2 groups. One group trained with heavy loading that reached about 60% of a rap max; each exercise was done in 4 attempts with 12 to 16 repetitions. The other group performed the same set of exercis- es but with a moderate loading that is the “weight” did not change, attempts reduced to 3 and the number of repetitions was no more than 12.

Evaluation was performed on pre-launch days 60 and 30, and on day 4 post recovery. Strength endurance of the femoral muscles was es- timated by the results of isokinetic testing. The subject fulfilled 22 cyclic maximal contractions (flexion/extension of the knee joint) at the angular velocity of 120°/s without break. The strength endurance of knee flexors and extensors before and after space flight was estimated.

The groups showed significant differences in the parameters of strength endurance of the knee flexors (P<0.04) and extensors (P<0.03).

Data analysis evidenced that the group of cosmonauts who applied greater loads for resistive training increased strength endurance of equally the knee flexors and extensors. Endurance of the flexors gained 10% to 50% and extensors, 11% to 59%. One cosmonaut made an exception in the group as his flexors lost 17% of the strength endurance.

In the group where cosmonauts trained with moderate loads the pa- rameters under study degraded in each one without exception, i.e. the flexor endurance dropped by 22% to 55% and extensors, 2% to 61%.

It was brought out that the resistive training with heavy loading in microgravity is favorable to maintenance of the strength endurance of cosmonauts.

The work was carried out with support of RFFI grant No. 13-04-02182.

POSSIBLE EFFECTS OF HYPOXIA INDUCED BY EXOGENIC NITRITE ON RAT HEART

AND SKELETAL MUSCLE PROTEINS N.V. Kuleva, D.S.Aleksejeva, T.E. Shumilova

Departments of Biochemistry and General Physiology, St.-Petersburg State University, Universitetskaya nab. 7-9,

St.Petersburg,199034, Russia

Nitrite anion is an oxidative breakdown product of nitric oxide (NO), a labile biological mediator from group of gasotransmitters. This gas was firstly discovered in the late 1700s and during two next centuries was consid- ered to be as toxic and dangerous for environment (Calvert J., Lefer D., 2009). The importance NO on the field of biology and medicine was not ful- ly appreciated until the 1980s when several independent research groups found that NO is generated in mammals including human, by nitric oxide syntases (NOSs) and plays a prominent role in controlling blood pressure via regulation of vascular tone (Ignarro L., 1999). The localization of NOS in the vascular epithelium (eNOS) is of particular importance for cardiovascular system , as eNOS maintains basal vascular tone trough its release of low lev- els of NO (Loscalso J.,Welch G.,1995). The synthesis of NO is significantly influenced by numerous cofactors such as tetrahydrobiopterin, flavin mono- nucleotide and flavin adenine dinucleotide, the presence of reduced thiols and endogenous NOS inhibitor asymmetric dimetylarginine , as well as sub- strate and oxygen availability. During ischemia the ability of eNOS to gener- ate NO is severely reduced because of inadequate delivery of oxygen and cofactors (Becker B.F. et al., 2000). But it is a hypoxic state when the main role in NO formation nitrite begins to play. Recently there has been a para- digm shift in nitrite biology with discovery that nitrite is a physiologically relevant storage reservoir of NO in the blood and tissues that can be readily reduced to NO under pathological conditions such as ischaemia and hypoxia (Zweier J.L. et al., 1995). Nitrite reductase activity in mammalian tissues has been linked to mitochondrial electron transport system, non-enzymatic acidic disproportionation, deoxyhemoglobin, xanthine oxidase, and more recently myoglobin (Gladwin M.T. et al., 2005).

So, nitrite injected to animals in hypoxic conditions is able to pro- duce NO that regulates vasodilatation and blood pressure. It means that nitrite represent a novel salvage pathway for NO equivalents that are bio- logically important and appear to be attractive targets for therapeutic pur- poses. The diseases include heart ischaemia-reperfusion injury (Gladwin M.T.et al., 2005), pulmonatory hypertension ( Zuckerbraun et al., 2011),

chronic ischaemic tissue disease (Patillo E.B. et al., 2011) and other.

However, several key questions remain: What signaling mechanisms does nitrite influence? How nitrite cellular uptake is regulated, and is it com- partmentalized upon entry into cell? What are the molecular and bio- chemical targets of nitrite- mediated protection in disease-models? Are there adverse consequences of low levels of nitrite besides carcinogenesis at high concentrations?

In this study we have made an attempt to answer the question about adverse consequences of low levels of exogenic nitrite for rat hearts and skeletal muscle and to look for oxidative modifications of water-soluble proteins at nitrite injection into rates in doses of 1-5 mg 100 gm of body weight. These injections resulted in hemic hypoxia induced by methemoglobinonemia. In our experiments the content of blood plasma methemoglobin was increased to mean value of 10 % of total hemoglobin content. Circulation parameters of nitrite hypoxia are characterized by fast and significant decrease of arterial pressure, bradycardia develop- ment, decreasing of heart output and heart stroke volume as well as total peripheral resistance decreasing. After 1-1 ,5 hours the indexes of sys- temic and peripheral hemodynamics became near to original values. At this moment there were taken blood samples and samples of skeletal and heart muscles. The tissues were homogenized in 10 volumes of 0,01 M phosphate buffer, pH 7,4, and supernatant formed at centrifugation (at 5000g , for 15 min) was used to study oxidative modifications in proteins.

As the hypoxic state there may be conditions for free radicals formation followed by oxidative modifications of cardiac and muscle proteins, so, we have considered a possibility of reversible modifications, transfor- mation sulfhydryl groups into disulfide, and non-reversible modifications, protein carbonylation. In the first case, for registration so called “diagonal electrophoresis” was used (Eaton J. et al., 2006), in the second one, thespectrophotometric registration of interaction of carbonylated proteins with 2,4-dinitrophenyl hydrazine was (Dalle-Donne I. at al., 2003).

The reversible oxidative modifications were found in proteins with molecular masses 38, 43 and 79 kDa of skeletal muscle and in those with 37, 48, 52, 95 kDa of heart, but significant differences between density of stained spots on electrophoregrams for normal and hypoxic animals were not found. The same situation of absence of significant differences was found in carbonylation of water-soluble proteins of heart and skeletal muscle of control animals and animals with nitrite hypoxia. This result may be considered as an evidence of absence of harmful action of nitrite