Kubasov 1 and M.G.Dobretsov 2

1- I.M. Sechenov Institute of Evolutionary Physiology and Biochemistry, Saint-Petersburg, Russia

2- Department of Anesthesiology, University of Arkansas for Medical Sciences, Little Rock, AR USA

It is known that skeletal muscle fibers have high Cl^- conductance (GCl), which constitutes up to 80-85% of overall ionic membrane conductance of these cells at rest. Remaining 15-20% of resting muscle fiber membrane per- meability can be attributed to the potassium conductance GK (1). High chloride conductance plays a critical role in the setting and maintaining of the resting membrane potential of muscle fibers and in determining the rate of repolarizing phase of the muscle fiber action potentials. Application of selective Cl^- channel blockers (anthracene-9-carboxylic acid (9-AC) results in generation of repeti- tive after-discharges of stimulated muscle fibers and in a substantial slowing of relaxation of contractile responses of the skeletal muscle (2, 3). Similar features of evoked electrical and contractile muscle responses are being observed in studies of animal models of myotonia and human subjects carrying CLCN1 gene mutation resulting in expression of CIC-1 chloride channels with sup- pressed conductivity (4). Despite that CIC-1 represents the major class of Cl^- channels expressed by skeletal muscles of vertebrates, the question about com- partmentalization of these channels within the muscle fiber plasma membrane remains unsettled. Immunohistochemical studies suggest that CIC-1 channels are present on sarcolemma, but almost lacking on the membrane of t-tubules of the skeletal muscle fibers (5). This view however, is difficult to reconcile with electrophysiological data suggesting that nearly 80% of the skeletal muscle fiber Cl^- conductance should be attributed to the channels of its t-system (6, 7, 8). Activity of ion channels and transporters localized within the t-system has a critical significance for contractility of skeletal muscle fibers; therefore the question above is of importance for our understanding of skeletal muscle physiology and pathophysiology. Our previous studies indicated that the analy- sis of characteristics of the third phase of propagating muscle fiber action po-

tentials recorded extracellularly using narrow-tipped pipettes may provide a valuable information regarding currents generated within t-tubules located just under the rim of the pipette and, therefore, regarding the role of ion- transporting mechanisms of the t-system of skeletal fibers (9). Considering these data and the question above, the major aim of this work was the study of the effects of specific CIC-1 chloride channels blocker, 9-AC on characteristics of bi- and tri-phasic (sarcolemma and sarcolemma + t-tubular membrane, re- spectively) extracellularly recorded AP of fibers of the frog (Rana temporaria) sartorius muscle. As in our previous experiments, either bi-phasic (type 1, T1) or tri-phasic (type 2, T2) APs were recorded in muscle fibers with the pipettes containing control solution. Respective temporal and amplitude characteristics of first and second phases of T1 and T2 APs did not differ. The presence or absence of the late positive (third) phase was, therefore, the only characteristic distinguishing these two types of the signals.

Fig. 1. T1 and T2 APs (labels) recorded immediately (a) and on 20^th minute (b) after establishing the loose seal contact between the muscle fiber and pipette loaded with 100 µM of 9-AC. Horizontal axes – time (ms), vertical axes – voltage (mV).

As under control conditions, either T1 or T2 responses were recorded during first minutes after the muscle fiber was approached with the pipette loaded with 9-AC (100 µM) (Fig. 1a). The difference of behavior of these two types of responses became obvious with time of recording. On the 10-15^th min- utes of continuous recording with 9-AC filled pipette from the fiber generating T1 response, a significant (p<0.05) increase in the duration of half-decay time (by 69 18 %) of the second phase of T1AP observed over the same time (27 fibers, 4 muscles; compare Fig.1a and b). Similar changes in temporal charac- teristics of T1 APs were also observed in experiments, in which 100 µM 9-AC was added to the bath solution. Unlike described above for the T1 type of re- sponses, amplitude and temporal characteristics of either of phases of T2 APs did not change significantly during over than 20 minutes of continuous re- cording with 100 µM 9-AC loaded pipettes (38 fibers, 4 muscles; compare Fig.

1a and b). However with 9-AC present in the extracellular solution, the second phase of T2 APs was observed to split into multiple (2 to 4) peaks (Fig. 2).

Fig. 2. The figure panels show four consecutive recordings of T2 APs, which were collected at from the same muscle fiber of the muscle stimulated at 0.1 Hz and in- cubated with 100 µM 9-AC for 20 minutes. Horizontal axes – time (ms), vertical axes – voltage (mV).

We did not observe multiple second phase responses generated in ei- ther of 27 recordings of T1 APs. Therefore, it is likely that these repetitive responses were generated in the t-tubules opened on the surface of the fiber under the rim of recording pipette and providing the outward current underly- ing generation of the third phase of this type of the signals.

Whether this suggestion is correct and whether these multiple peaks represent repetitive generation of inward spikes of Na current within a t- tubule or de-synchronization of Na currents generated in several t-tubules opened under the pipette remains to be determined. Whatever is the answer to the question above, we did not observe this type of responses in experi- ments with the Cl channel blocker included in the pipette solution. Thus the reason for putative repetitive or de-synchronized activity of Na channels of the t-tubular membrane should be attributed to some changes in electrogene- sis of the sarcolemma of the muscle fiber, which could be associated with the fiber depolarization and prolongation of spreading on its surface AP, evi- denced in prolongation of the second phase of T1 APs. Further experiments are needed to clarify these issues.

Conclusions

1. T1 and T2 APs, the action potentials presumably generated by currents originating in the skeletal muscle sarcolemma or both sarcolemma and t- system, respectively, respond differently to the selective block of C1C chloride channels with 9-AC.

2. Evaluation of the role of prolongation of T1 type of responses in genera- tion of multiple peaks of the second phase (Na current) of T2 skeletal muscle APs requires further studies.

This work was supported by grant 02.740.11.5135 by Federal Program of the Ministry of Science and Education of the Russian federation and in part by the COM UAMS pilot grant program .

References

1. Bretag A.H. Muscle chloride channels. // Physiol. Rev. 1987; 67: 618–724.

2. Bryant S.H, Morales-Aguilera A. Chloride conductance in normal and myotonic muscle fibres and the action of monocarboxylic aromatic acids. // J. Physiol. 1971;

219: 367–383.

3. Conte Camerino D., Tortorella V., Bettoni G. et al. A stereospecific binding site regulates the Cl^– ion channel in rat skeletal muscle. // Pharmacol. Res. Commun.

1988; 20: 1077–1078

4. Rudel R., Ricker K., Lehmann-Horn F. Transient weakness and altered membrane characteristic in recessive generalized myotonia (Becker). // Muscle Nerve 1988;

11: 202–211.

5. Lueck J.D., Rossi A.E.,Thornton C.A., et al. Sarcolemmal-restricted localization of functional ClC-1 channels in mouse skeletal muscle. // J. Gen. Physiol.

2010;136:597–613.

6. Dulhunty A.F. Distribution of potassium and chloride permeability over the sur- face and T-tubule membranes of mammalian skeletal muscle. // J. Membr. Biol.

1979; 45:293–310.

7. Palade P.T, Barchi R.L. Characteristics of the chloride conductance in muscle fi- bers of the rat diaphragm. // J. Gen. Physiol. 1977; 69: 325–342.

8. Dulhunty A.F. The dependence of membrane potential on extracellular chloride concentration in mammalian skeletal muscle fibres. J. Physiol. 1978; 276: 67–82 9. Kubasov I.V., Dobretsov M.G. Characterisnics of spreading action potentials re-

corded in various sites of skeletal muscle fibers of the frog Rana temporaria. // J.

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THE DIRECTION OF STEM CELLS MOVEMENT INTO THE BRAIN DEPENDS ON THE AREAS OF THEIR INJECTION

INTO PERIPHERAL PARTS OF THE NERVOUS SYSTEM V.A. Kulchitsky¹, Y.G. Shanko², P.G. Molchanov³, S.N. Cherehkevich³,

M.O. Chotianovich¹, A.A. Denisov^1,3, S.G. Pashkevich¹, I.V. Strizhak¹, M.V. Andrievskaya³, A.V. Rodich², T.N. Pitlik³, P.M. Bulay³

1Institute of Physiology, National Academy of Sciences of Belarus, 28 Akademicheskaya Street, 220072, Minsk, Belarus

2Republican Research and Clinical Center of Neurology and Neurosurgery, 24 F. Skorina Street, 220114, Minsk, Belarus

3Belarusian State University, 4 Nezavisimosti Avenue, 220030, Minsk, Belarus

Motility is an inherent property of all living cells. Because of the mo- tility various tissues and organs, including cell populations of different origin (which are topographically divided at different stages of ontogenesis) are

formed in the process of ontogenesis. After the formation of tissues, organs, systems and the whole organism the motility function is important for organi- zation of behavioral and defensive reactions, delivery of regulatory sub- stances and nutrients, providing interoperability between tissues, organs and cells, and realization of intracellular and intranuclear processes. In the current study the attention was not focused on the mechanisms of motility that is on those issues which are professionally elaborated by the organizers of the sci- entific forum. It is about the methods of stem cells delivery into various parts of the brain. The dominant methods are: systemic (intravenous) way of appli- cation or, conversely, the local way of stem cells placing into the damaged part of the brain after the preliminarily performed trepanation. According to the first way there are contradictory opinions based, on the one hand, on the relative simplicity and accessibility of the systemic approach, and, on the other hand, on the problems of blood-brain barrier overcoming and a certain diffuseness of stem cells allocation in the bloodstream and body tissues. The local way of stem cells delivery, as it was mentioned above, requires supple- mentary measures, for example, additional surgical intervention, that is not always possible in a number of situations [1].

There are some other ways; one of them is based on the well-known ability of stem cells to move via the perineural spaces. In this case, the injec- tion of stem cells into the peripheral parts of cranial nerves must be accom- panied by the migration of cells due to their inherent motility into the central parts of particular system of cranial nerves. As the stem cells have a certain tropism to regulatory substances (which are accumulated in damaged tissues) while moving, this factor was also taken into account when choosing targets for stem cells reparative potential use.

At the early stages the procedure was worked out and the series of ex- periments on stem cells injection to white rats conducted. The stem cells were previously obtained from the rats’ adipose tissue and cultured (at least two passages) in a CO2-incubator. FITC was used as a fluorescent label. Applica- tion of stem cells was performed into peripheral parts of the two cranial nerves. In the first series of experiments (n=8) cells were injected under the mucous membrane of the nasal cavity (i.e., into peripheral parts of the olfac- tory nerve). In the second series of experiments cells were injected into the Meckel’s cave (i.e., into the trigeminal ganglion region) (n=5).

Such a choice of cranial nerves was due to the central representation of each nerve chosen. Information firstly goes by the olfactory nerve to the ros- tral brain regions (frontal lobes of the brain and surrounding areas of nervous tissue). The signaling to the brainstem goes by the trigeminal nerve.

The histological material capture was carried out in 10 minutes, 2 and 24 hours after the injection. The preparations for fluorescent microscopy were made with the “squashed drop” method. In 10 minutes FITC-labeled stem cells were found in the place of application and in olfactory filaments after the injec- tion under the nasal mucous membrane, and in the trigeminal ganglion after the injection into the Meckel’s cave, but were not found in the brain. In 2 and 24

hours after the application under the nasal mucous membrane FITC-labeled stem cells were found in the olfactory filaments, olfactory bulbs, basal ganglia, the optic nerve, tuber cinereous of the diencephalons, on the basis of frontal lobes). In 2 and 24 hours the fluorescence was seen in the spinal nucleus of the trigeminal nerve and reticular formation of the brainstem.

Thus, in the experiments carried out a peculiar topographic feature of stem cells was noted. It depends on the location of their injection into the peripheral parts of the cranial nerves. The injection of cells in the vicinity of the olfactory nerve is accompanied by their movement and distribution to the rostral brain areas. The injection of stem cells into the trigeminal nerve sys- tem is accompanied by their migration to the caudal parts of the brain. Such pattern of stem cells motility from the periphery to the brain may be taken into account on trying to enhance the reparative potential of neural tissue after injuries or strokes namely in those brain areas, that were affected after the pathological process development [2].

Acknowledgements: We thank Victor Boksha (Neurosyntek Model- ing and Manufacturing Inc., Los Altos, CA.) for helpful discussions.

References

1. Kalyunov V.N., Kulchitsky V.A., Chernov A.N. Stem cells: reality, perspectives, illusions and myths // News of Biomedical Sci. 2011. Vol.3, No1, P.120-136.

2. Cherenkevich T.N., et al. The effect of electrical stimulation on the stem cells pro- liferation and differentiation // News of Natl. Acad. Sci. 2011. No2. P. 110-121.

ARACHIDONIC ACID METABOLISM INHIBITORS MODULATE THE EFFECT OF DRUG MOLIXAN ON INTRACELLULAR Ca²⁺

CONCENTRATION IN MACROPHAGES

L.S. Kurilova, Z.I. Krutetskaya, O.E.Lebedev, N.I. Krutetskaya, V.G. Antonov, V.V.Lastochkin, K.O. Vojtcehovitch, A.A.Naumova

Saint-Petersburg State University, 7/9 University emb., Saint-Petersburg, Russia

A number of disulfide-containing drugs affecting redox-state and having prominent physiological effect have clinical value. Thus, a synthetic analogue of oxidized glutathione (GSSG) pharmacological drug glutoxim^® (GSSG diso- dium salt with platinum nanoaddition, PHARMA-VAM, Moscow, Russia), is applied as an immunomodulator and hemostimulator in therapy of bacterial and viral diseases, psoriasis , radio- and chemotherapy of oncological disorders.

Another disulfide-containing drug molixan^® (complex of glutoxim with inosine nucleoside) has the similar application. However, the cellular and molecular mechanisms underlying these drugs action are poorly understood.

Earlier we found that GSSG and glutoxim increase intracellular Ca²⁺- concentration, [Ca²⁺]i, inducing Ca²⁺-mobilization from thapsigargin- sensitive Ca²⁺-stores and subsequent Ca²⁺-entry in rat peritoneal macrophages [1]. Later we demonstrated that the same effect on [Ca²⁺]i is observed in macrophages treated with molixan [2]. In addition, it was found that tyrosine kinases, tyrosine phosphatases [1], phosphatidylinositol kinases [3], small G-

ptoteins from Ras family as well as phospholipase C and protein kinase C, the key molecules of phosphoinositol signaling pathway [4] and actin cy- toskeleton [5] are the critical components of the signaling cascade, triggered by GSSG and glutoxim and leading to [Ca²⁺]i increase in macrophages. Also it was found that the metabolites of cyclooxygenase pathway of arachidonic acid (AA) metabolism are involved in the regulation of glutoxim effect on [Ca²⁺]i in macrophages [6].

It is known that AA, released from membrane phospholipids, can be metabolized via both cyclooxygenase and lipoxygenase pathways, resulting in the formation of prostaglandins and leukotrienes in macrophages. AA itself has been shown to affect cell signaling elements in different types of cells. AA and its metabolites formed by the cyclooxygenase or lipoxygenase pathways play an important role in regulation of various physiological processes.

Therefore, the aim of the present work was to elucidate whether en- zymes and/or products of AA metabolism are involved in the effect of molixan on [Ca²⁺]i in rat peritoneal macrophages. Using Fura-2AM micro- fluorimetry, we have studied the influence of inhibitors of AA oxidation pathways on [Ca²⁺]i rise induced by molixan in rat peritoneal macrophages.

Evidence is obtained which indicates the involvement of AA metabolites, probably of the cyclooxygenase and lipoxygenase pathways, in the formation of the Ca²⁺-response to molixan in macrophages; in the presence of AA me- tabolism inhibitors molixan induces no [Ca²⁺]i increase.

In order to investigate the possible involvement of cyclooxygenase pathway of AA metabolism in the effect of molixan on [Ca²⁺]i in macro- phages we used two structurally different cyclooxygenase inhibitors aspirin (acetylsalicylic acid) and indomethacin. Control experiments, shown in Fig.

1a, demonstrate that preincubation of the cells with 100 g/ml molixan in Ca²⁺-free solution leads to a gradual increase of [Ca²⁺]i in macrophages due to the mobilization of Ca²⁺ from the intracellular Ca²⁺- stores. The addition of Ca²⁺ ions in the external medium causes the additional [Ca²⁺]i increase medi- ated by Ca²⁺-influx from the external medium.

Fig. 1b shows that the preincubation of macrophages with 40 M in- domethacin for 5 min before molixan addition almost completely suppressed Ca²⁺-response induced by molixan. Fig. 1c demonstrates that preincubation of the cells with 100 M aspirin for 5 min before molixan application has the same effect on Ca²⁺-responses induced by molixan. The results suggest the involvement of cyclooxygenase pathway of AA metabolism in the effect of molixan on [Ca²⁺]i in macrophages.

In order to investigate the possible involvement of enzymes and/or products of lipoxygenase pathway of AA metabolism in the effect of molixan on [Ca²⁺]i in macrophages we used a well known lypoxygenase inhibitor nordihydroguaiaretic acid (NDGA). Fig. 2b shows that preincubation of macrophages with 10 M NDGA for 5 min before 100 g/ml molixan addi- tion leads to the almost complete inhibition of Ca²⁺-responses induced by molixan in comparison with control experiments (Fig. 2a).

Fig 1. The effect of aspirin and indomethacin on Ca²⁺-responses induced by molixan in macrophages.

Here and in Fig. 2 the abscissa axis shows time, min; the ordinate axis, Ca²⁺- concentration in the cytosol, nM.

a - cells were incubated with 100 g/ml molixan in Ca²⁺-free medium for 25 min, then Ca²⁺-entry was induced by addition of 2 mM Ca²⁺ to the external medium. b, c – cells were preincubated with 40 M indomethacin (b) or 100 M aspirin (c) in Ca²⁺-free medium for 5 min, then 100 g/ml molixan was applied; 20 min later 2 mM Ca²⁺ was added to the external medium.

Fig. 2. The effect of nordihydroguaiaretic acid (NDGA) on Ca²⁺-responses induced by molixan in macrophages.

a - cells were incubated with 100 g/ml molixan in Ca²⁺-free medium for 20 min, then Ca²⁺-entry was induced by addition of 2 mM Ca²⁺ to the external medium. b – cells were preincubated with 10 M NDGA in Ca²⁺-free medium for 5 min, then 100 g/ml molixan was applied; 20 min later 2 mM Ca²⁺ was added to the external medium.

The results suggest that lipoxygenase pathway of AA metabolism is also involved in the effect of molixan on [Ca²⁺]i in macrophages.

Thus, the present study has shown that some interacting mechanisms between AA formation and molixan-induced Ca²⁺-responses are present in rat peritoneal macrophages. NDGA, an inhibitor of lipoxygenase pathway of AA metabolism inhibits the Ca²⁺-response to molixan. Cyclooxygenase inhibitors indomethacin and aspirin also completely eliminate the Ca²⁺-response in- duced by the drug. The data presented indicate that in rat peritoneal macro- phages AA metabolites, probably of cyclooxygenase and lipoxygenase path- ways, are involved in the generation of the Ca²⁺ responses to molixan.

References

1. Kurilova L.S., Krutetskaya Z.I., Lebedev O.E., Antonov V.G. The effect of oxidized glutathione and its pharmacological anologue glutoxim on intracellular Ca²⁺ concen- tration in macrophages. Cell and Tissue Biology. 2008. V. 2. P. 322-332.

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The role of the key enzymes of the phosphoinositide signaling pathway in the ef- fect of oxidized glutathione and glutoxim on intracellular Ca²⁺ concentration in macrophages. Doklady Biol. Sci. 2009. V. 428. P. 407-409.

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SYSTEMIC HAEMODYNAMIC CHANGES DURING ISOMETRIC RHYTHMIC CONTRACTIONS OF KNEE EXTENSORS WITH DIFFERENT PATTERNS OF FORCE DEVELOPMENT S.Y. Kuznetsov, V.A. Makarov, A.S. Borovik, O.L. Vinogradova

SSC RF Institute for Biomedical Problems RAS, Moscow, Russia 10 young healthy male volunteers (23.7±2.8 yrs; 177.5±4.0 cm;

75.5±8.8 kg) with maximal knee extension torque 238.9±34.6 N·m performed isometric rhythmic contractions of knee extensors in regime 20 s contraction – 20 s rest till exhaustion at Biodex dynamometer (USA). Muscle contrac-