[Disturbances in the function of cardiomyocyte ryanodine receptors of spontaneously hypertensive rats revealed by 4-chloro-m-cresol].

The rates of Ca2+ release from sarcoplasmic reticulum in response to the activation of ryanodine receptors by 4-chloro-m-cresol in cardiomyocytes of three rat lines: spontaneously hypertensive (SHR) and normotensive WKY, and Wistar rats during five weeks of their growth and development have been studied to reveal differences in the function of ryanodine receptors at different stages of hypertension. Whereas the efflux of Ca2+ from sarcoplasmic reticulum in Wistar and WKY rat cardiomyocytes decreased in response to 4-chloro-m-cresol, an abrupt rise in the rate of [Ca2+]i increase was observed in myocytes of spontaneously hypertensive rats after 17 days of development. A correlation between this phenomenon and the occurrence of genetic defect of ryanodine receptors in SHR seems improbable because we did not register any differences in the rates of Ca2+ release from sarcoplasmic reticulum by the action of 4-chloro-m-cresol in concentrations 0.5-2.0 mM in the cardiomyocytes of newborn WKY and spontaneously hypertensive rats. On the other hand, pathological changes in the function of ryanodine receptors may become apparent later during ontogenesis. The connection of this phenomenon with an increase in the role of ryanodine receptors during the excitation-contraction coupling in muscle cell and an increase in the calpain expression by the age of 3 weeks in spontaneously hypertensive, but not in WKY rats, is discussed. It is supposed that the disintegration of the subunit of ryanodine receptors by calpain notably intensifies the Ca2+ release from sarcoplasmic reticulum after the activation of ryanodine receptors without affecting the characteristics of receptor binding.

[A comparative analysis of contractile responses induced by acetylcholine and choline in twich and tonic frog skeletal muscle fibres].

A comparative analysis of contractile responses evoked by acetylcholine and external sodium removal in twitch and tonic frog skeletal muscle fibers has been performed. To elucidate the possible differences in the participation of various mechanisms of Ca2+ homeostasis in the regulation of these contractile reactions, selective pharmacological agents: dantrolene, 4-m-chlorocresol, tetrodotoxin, and d-tubocurarine were used. It was found that, although Ca2+ release from sarcoplasmic reticulum is a major source in the acetylcholine contracture for both types of muscle fibers, there are definite differences in the molecular structures of the alpha-isoform and/or the ratio of alpha- and beta-isoforms of ryanodine-sensitive receptor/channels for twitch and tonic fibers. The substitution of Na+ by choline in both twitch and tonic fibers evoked significant long-lasting contractile responses, which were sensitive to the inhibitory action of d-tubocurarine or dantrolene. The application of acetylcholine against the background of choline induced contracture with an amplitude closely related to that in normal solution. These results suggest that choline activates acetylcholine receptor/channel insignificantly, but this type of ionic channels is the main mechanism of its penetration through the surface membrane.

Changes in the functioning of the electromechanical connection during tetanic contraction.

The functioning of the electromechanical connection during tetanic contraction in frog skeletal muscle was studied. Analysis using caffeine, calcium-free medium, the ryanodine receptor blocker dantrolene, and the Ca-ATPase inhibitor thapsigargin showed that the initial increase in tetanus, as in twitch contractions, did not require the presence of calcium ions in the surrounding medium, which is in agreement with published data. Contraction was accompanied by activation of potential-dependent release of calcium from the sarcoplasmic reticulum. In contrast, the secondary rise phase and/or the duration of the tetanus plateau were critically dependent on the present of Ca2+ in the surrounding medium. Given that contraction in this situation was inhibited by dantrolene, activation of prolonged contraction was also mediated by calcium released from the sarcoplasmic reticulum, though ryanodine receptors were now activated not by changes in the membrane potential but by the influx of external calcium. Thus, external calcium plays a significant role in the formation of prolonged contractile responses, providing for longer-lasting maintenance of power in contracted muscles.

[Effects of agonists and antagonists of rhyanodine receptors on potassium contractures in twitch and tonic frog skeletal muscle fibers].

A comparative analysis of the effects of the concentrations of Ca2+ in external medium and the inhibitor (dantrolene) and activator (4-chloro-m-cresol) of rhyanodine-sensitive Ca2+ channels of carcoplasmic reticulum on the characteristics of potassium contracture in frog twitch and tonic skeletal muscles has been performed. It was shown that the duration of contracture in tonic muscles is not restricted by the presence of Ca2+, as distinct from twitch muscles. Dandrolene does not practically affect the contractile responses of tonic fibres, and the concentration of cresol eliciting the contracture for tonic fibres is substantially higher (1 mM) than for twitch fibers (0.25 mM). In twitch fibers, the potassium contracture activated in the presence of cresol is comparable in amplitude and dynamics with the contracture under control conditions, and in tonic fibers a summing of responses without relaxation after the washing of excessive potassium is observed. This suggests that, in twitch fibers, the influx of Ca2+ can directly create the concentration sufficient for the maintenance of contraction, and in tonic fibers its involvement is mediated through the Ca(2+)-dependent activation of the beta-isoform of rhyanodine-sensitive channels.

Inhibitory effect of clonidine on cholinergic transmission.

Equilibrium geometry and electronic structure of three local anesthetic molecules and clonidine were computed. It was shown that clonidine molecule could incorporate into local anesthetic binding centers in potential-operated Na+ channels and in ionic channels coupled to nicotinic cholinergic receptors. The modulatory effect of clonidine on contractile responses of skeletal muscle showed that blockade of Na+ channels and nicotinic cholinergic receptors is a part of its analgesic action.

[Changes in the electromechanical activity in the course of tetanic contraction].

The functioning of excitation-contraction coupling during tetanic contraction was investigated on frog skeletal muscle. The effect of the calcium release blocker dantrolene was tested on electrically evoked twitches and tetanic contractions. It was shown that the first: developmental stage of tetanus is inhibited by dantrolene as well as a twitch contraction, and does not influenced by calcium-free medium. This substantiates it as based on "voltage dependent Ca-release" (VDCR) mechanism of activation, when depolarization directly opens the rhyanodin receptor calcium channels. The next stage: the long lasting plateau of tetanic contraction, is directly dependent on external Ca2+ entry and also inhibited by dantrolene, and therefore may be described as "calcium-induced Ca-release" (CICR) activation mechanism. It is proposed that such change in ECC mechanism taking place during tetanic contraction, can occur also in conditions of natural muscle activity, because of its rhythmical nature.

[Functioning of the electromechanical connection in the course of the contracture contraction]. / O funktsionirovanii élektromekhanicheskoi sviazi na raznykh stadiiakh kontrakturnogo sokrashcheniia.

The effects of calcium release blocker dantrolene was tested on electrically evoked twitches and on contractures induced by potassium depolarization, by acetylcholine or caffeine. It was shown that the first: developmental, stage of potassium or acetylcholine contracture is inhibited by dantrolene and is not influenced by calcium free medium, therefore we may interpret it as based on a "voltage-dependent Ca release" (VDCR) mechanism of activation, whereas depolarization directly opens the rhyanodin receptor calcium channels. On the contrary, the next stage: the long-lasting plateau of contracture, is directly dependent on external Ca2+ and inhibited by dantrolene, and therefore can be described as "calcium induced Ca-release" (CICR) activation mechanism. In this case stored calcium is also released by rhyanodine receptors, although by means of entering the extracellular Ca2+. Finally, the last stage of low amplitude is not influenced by dantrolene nor by calcium-free medium. Therefore the activation of contraction on this stage is not based on the Ca2+ release through the rhyanodin receptor calcium channels.

[The role of electro-mechanical coupling in development of experimental atrophy of skeletal muscles]. / O roli élektromekhanicheskoi sviazi v razvitii éksperimental'noi atrofii skeletnykh myshts.

The available data of functioning of excitation-contraction coupling in atrophied skeletal muscles after denervation, immobilization, or hindlimb unloading, were summed up and analyzed. The analogy and differences between activation of contraction under these experimental conditions were elucidated in order to understand possible mechanisms of adaptation of the excitation-contraction coupling to different modes of atrophy. It was supposed that denervation and immobilization are the pathogenic factors for triggering the excitation-contraction coupling, whereas unloading is the factor which evokes active adaptation of this system.

[Characteristics of functioning of electromechanical coupling in striated muscles of higher and lower vertebrates]. / Osobennosti funktsionirovaniia élektromekhanicheskogo sopriazheniia poperechno-polosatykh myshts u vysshikh i nizshikh pozvonochnykh.

A comparative pharmacological analysis of relative contributions of different signal transduction pathways in the activation of contraction (excitation-contraction coupling, ECC) in intact fast striated muscles of frog and lamprey was performed. It was found that the major mechanism responsible for the ECC in muscles of both animals is Ca2+ release from the sarcoplasmic reticulum through the ryanodine-sensitive channels. However, the ECC in lamprey muscle displays some important differences in the units of electromechanical coupling, which precede the calcium release from sarcoplasmic reticulum. The maximum contraction force in frog muscle develops during caffeine-induced contracture, which indicates that all Ca2+ stored in sarcoplasmic reticulum is released through ryanodine-sensitive channels. In contrast, in lamprey muscle, the maximum force develops not in response to high caffeine concentration, but in response to repetitive electrical stimulation. Hence, in addition to stores liberated by ryanodine-sensitive channels, some other sources of calcium ions should exist, which contribute to the contraction activation. A source of this additional Ca2+ ions can be external medium, because acetylcholine contracture is abolished in a calcium-free medium. In frog muscle, the acetylcholine contracture was abolished in a Na(+)-free solution. It was concluded that in frog muscle ECC can be triggered by changes in the transmembrane potential (depolarization-induced calcium release), while in lamprey muscle the entry of calcium ions into myoplasm as the trigger in ECC (calcium-induced calcium release). The lamprey muscle was found to be more resistant to tetrodotoxin and tetracaine, which is indicative of a role in the activation of contraction of tetrodotoxin-resistant Na+ and/or Ca2+ channels. It was concluded, that ECC mechanism in striated muscles of low vertebrates is not limited by the generally accepted scheme of depolarization-induced calcium release but can include some other schemes, which require the Ca2+ influx into the cell.

Development of muscle-specific features in cultured frog embryonic skeletal myocytes.

To study the development of muscle-specific features during myogenesis, we analysed the ultrastructure and voltage-dependent currents of frog embryonic skeletal myocytes maintained in culture for 10 days. The cells were maintained under culture conditions that prevented cell division, fusion and cell contacts with neuroblasts. The cell surface was estimated morphometrically and from cell capacity and the values obtained were used to calculate ion current densities. It was shown that the expression of all main types of voltage dependent ionic currents occurs during the first 3-5 days. Na+ maximum specific conductance at days 1-2 was low but by day 7 it showed a 20-fold increase. The magnitude of Na+ current densities increased 16-fold from day 1 (3.6 microA/cm) to the day 7 (58.1 microA/cm). The maximum specific K+ conductance increased almost 3-fold during the first 5 days. In contrast to the other types of currents, I(K) undergoes qualitative changes. Sodium action potentials, whose amplitude and time course depend on gNa/gK ratio, appeared from day 4 in culture, when myofibrils and the T-system also developed. The amplitude of DHP-sensitive slow I(Ca) increased in parallel with the development of the T-membrane. I(Ca,S) density per unit of T-membrane area reached an equilibrium of ca., 17 microA/cm2 on the day 4 and then remained stable until the end of the period of observation. These studies demonstrate that muscle-specific characteristics including morphology and excitatory properties begin to develop on the third day and resemble those of adult muscle cells by the sixth day in culture.

[Inward rectifier potassium current in the frog embryonic skeletal myocytes]. / Kalievyi tok vkhodiashchego vypriamleniia v émbrional'nykh skeletnykh miotsitakh liagushki.

Pharmacological and kinetic properties of the inward rectifier potassium current Iir the frog embryonic skeletal myocytes were found to be identical to those of adult frog skeletal muscle fibres. The data obtained suggest that the Iir plays the main role in maintaining the myocytes resting membrane potential (RMP) when chloride conductance is insignificant. Changes of the integral conductance Gir and the RMP values correlated with the T-system development. The inward rectifier K+ channels, from the early stages of the muscle seem to be located in the T-tubule membranes.

[Structural differentiation of dissociated skeletal embryonal myocytes of frog under conditions of cell culture]. / Strukturnaia differentsirovka dissotsiirovannykh skeletnykh émbrional'nykh miotsitov liagushki v usloviiakh kletochnoi kultury.

The development of membrane structures, providing E-C coupling, and the contractile apparatus organization were investigated in frog skeletal myocytes cultured for 1 to 10 days in conditions preventing both myocyte division and fusion. Ruthenium red was used to determine the membranous structures being in contact with the extracellular environment. The marked membrane structures (vesicles and short tubules) appeared to be near the cell membrane on the first days of culturing. The increase in the ratio of the surface area of all internal membranous structures, marked by Ruthenium red, to the external membrane area with aging was proven by morphometric calculations, that means a progressive development. Contractile filaments were found near the cell membrane on the first days of development. Bundles of filaments with initial signs of sarcomere organization were observed on the 3rd-4th days, and myofibrils with highly organized sarcomeres occupied the main part of the sarcoplasm on the 6th day of culturing. The triads appeared also on the sixth day, being regularly inserted into the sarcomere structure. Degenerative signs in the myocytes (sarcomere disorganization and T-tubule swelling) were observed on the 8-10th days, but the area occupied by contractile elements was increased. These results show that the myocyte fusion into myotubules is not a necessary condition for either sarcomere formation, or the formation of all membranous structures providing the E-C coupling.

[The formation of electrical excitability in cultured frog embryonic myocytes]. / Formirovanie élektrovozbudimosti v kul'tiviruemykh émbrional'nykh miotsitakh liagushki.

Quantitative changes of sodium and potassium ionic currents were studied in the frog embryonic skeletal myocytes cultures under conditions preventing cell fusion and division. During 7 days of culturing the mean values of the sodium and potassium currents densities were 7- and 1.8-fold increased, resp. In currents clamp records the APs were observed only after 5 days of culturing, and in cells with proportion of sodium and potassium conductances above the critical level.

Contractile properties of rat skeletal muscles after hindlimb unloading and beta-GPA administration.

NASA: Soleus and EDL muscles of rats were examined following hindlimb unloading. Some of the rats were given beta-GPA, a creatine analog which depletes high-energy phosphates in muscle tissue, in their food. The contractile properties and fatigue resistance of these muscles were studied, with and without incubation in calcium solution. The increased fatigue resistance after beta-GPA feeding was less in calcium-free solution.^ieng

The effects of calcium channel modulators on contractions of tonic frog muscle fibres.

Effects of adrenaline (ADR) and the dihydropyridine Ca channel agonist CGP-IOS were studied on twitch and tetanic contractions of isolated tonic muscle fibres or small muscle fibre bundles containing tonic fibres of the frog Rana temporaria. Tetanization caused a gradual increase of tension between 10 and 70 Hz. CGP-IOS produced an increase in twitch amplitude. After CGP-IOS administration (2 x 10(-6) - 10(-5) mmol/l), the twitch amplitude increased and the high frequency tetanus developed more rapidly. However, during the phase of high tension level a breakdown of tetanic tension appeared and this occurred earlier at higher stimulation frequencies. After the end of tetanization the contraction curve did not return to the initial level, so that the remaining contraction (contracture) lasted 2 to 5 min. Sometimes just after replacement of CGP-IOS by Ringer, a large enhancement of tetanus amplitude was observed followed by long lasting contractures. ADR (10(-5) mmol/l) increased the tetanic tension without changing the shape of tetanus but did not affect twitch amplitude. Adrenergic modulation of tension requires extracellular Ca2+. Combined administration of ADR and CGP-IOS had an integrative effect, so that independent action of each of them can be assumed. It is to suggest that direct and indirect Ca channel modulators, DHP derivatives and ADR, have different targets. The sites responsible for adrenergic modulation of the contraction may be Ca channels not identical to DHP-sensitive channels, i.e. Ca-releasing channels in SR or the DHP-insensitive Ca channels in muscle membrane.