[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.

[The role of stacking interactions in the mechanisms of clonidine binding].

The stacking interactions of the clonidine aromatic ring with the aromatic rings of Phe or Tyr of alpha2-adrenoreceptor and Tyr aromatic ring of the pore of the tetradotoxin-resistant channel have been investigated. Ab initio quantum chemical calculations for a model system of two parallel aromatic rings were performed by GAMESS software with 6-31G** basis set in the framework of the Moller-Plesset second-order perturbation theory with full geometry optimization without any symmetry. It was shown that the parallel shifted conformation of two aromatic rings is energetically most favorable. The 2,6-chlorination of one of the benzene rings leads to the amplification of the stacking interaction, an increase in the relative shift of the rings and possible growth of both the hypotensive and analgetic functions of clonidine due to the increase in the binding energy. The 4-fluoridization of the clonidine benzene ring can amplify its analgetic function but practically excludes its hypotensive action.

[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.

Effect of clonidine on potential-dependent sodium currents in sensory neurons.

In experiments on rat pups we studied the effect of clonidine on potential-dependent Na+ currents in dorsal root ganglia by the voltage clamp method. Clonidine decreased the amplitude of tetrodotoxin-sensitive and tetrodotoxin-resistant sodium currents. The range of acting concentrations and the absence of modulatory effect of norepinephrine on the efficiency of clonidine-induced blockade of sodium currents suggest that this blockade results from a direct interaction of clonidine with sodium 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.

[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.

Ethanol modulates the ionic permeability of sodium channels in rat sensory neurons.

The effects of ethanol on tetrodotoxin-sensitive (TTXs) and tetrodotoxin-resistant (TTXr) sodium channels in rat spinal ganglia were studied using a patch-clamp method. Application of ethanol (10 and 100 mM) to both sides of membranes resulted in decreases in the reversion potentials of both types of sodium channels. In the case of TTXr channels, ethanol decreased their selectivity in relation to Na ions and altered the sequence of ion selectivity of these channels for different cations from row X to row XI of the Eisenman selectivity classification. It is suggested that this change in ion selectivity is associated with ethanol-induced disruption of hydrogen bonds which stabilize the spatial structure of ion channel macromolecules, which may lead to changes in the steric parameters of the pores formed by these channels.

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.

[Ethanol modifies the ion selectivity of sodium channels in the rat sensory neurons]. / Etanol modifitsiruet ionnuiu izbiratel'nost' natrievykh kanalov sensornykh neironov krysy.

Ethanol was shown to decrease the reversal potential of tetrodotoxin-resistant (TTXr) and TTX-sensitive channels in short-term culture of the dorsal root ganglion cells. The ethanol led to alterations in ionic selectivity of the TTXr channels (its shifting from the X-th Eisenmann selectivity sequence to the XI-th one). The data obtained suggest that the findings were due to selectivity filter modification because of disturbed hydrogen bounds in the channel macromolecule.

[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.

[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.

Voltage-insensitive Na channels of different selectivity in human leukemic cells.

Patch clamp method was used to search for, and characterize ion channel activity which may participate in cation influx in human myeloid K562 cells. In cell-attached, outside-out and whole-cell experiments two types of voltage-insensitive Na-permeable channels were identified with different selectivities for monovalent cations, referred to as channels of high (HS) and low (LS) selectivity. The unitary conductance was similar for both channel types being 12 pS (145 mmol/l Na, 23 degrees C). The relative permeability PNa/PK estimated from the extrapolated reversal potential values were 10 and 3 for HS and LS channels, respectively. Both HS and LS channels were found to be impermeable to bivalent cations (Ca2+ or Ba2+). The activity of HS and LS channels displayed a tendency to increase with depolarization. Both channel types were not blocked by tetrodotoxin and were insensitive to amiloride in the concentration range of up to 100 mumol/l. At higher concentrations (0.1-2 mmol/l), amiloride reversibly inhibited HS channels only. The results obtained lead us to conclude that, under physiological conditions, both types of Na-permeable channels may provide sodium influx in leukemic cells. Our data imply the existence of a novel family of Na channels in blood cells.

Beta-adrenergic regulation of voltage-dependent calcium currents in cultured skeletal myocytes of the frog Rana temporaria.

Effects of beta-agonists isoproterenol (Isp) and adrenaline (Adr) and beta-adrenoblocker obsidan (Obs) on the voltage-dependent calcium currents in cultured embryonic skeletal myocytes were studied at various stages of development ranging from day 2 to 10, using the whole-cell patch-clamp technique at 19-21 degrees C. Adr (or Isp) in concentrations 0.1-10 mumol/l increases the amplitude of both the slow dihydropyridine(DHP)-sensitive calcium current (ICa) and the fast-activated DHP-insensitive ICa. From day 2 to 6 after myoblast plating, Adr and Isp did not change the amplitude of ICa at all or slightly increased it. Obvious strong positive effects (an approximately twofold amplitude increase) on the calcium channels have been observed in 7-10-day-old myocytes only. beta-adrenoblocker obsidan known to abolish the positive beta-agonist effect, had a positive effect on membrane calcium currents. It may have been a result of the immaturity of the beta-adrenergic regulatory system of the myocytes. It is concluded that the beta-adrenergic regulatory complex can stimulate the activity of the fast and the slow voltage-dependent calcium channels of the frog skeletal myocytes, and that there is a distinct developmental stage at which a functioning beta-adrenergic regulatory complex appears in the membrane of skeletal myocytes.

Pharmacological analysis of voltage-dependent potassium currents in cultured skeletal myocytes of the frog Rana temporaria.

Previously, the existence of nine types of outward potassium current (IK) was shown. The whole family of IK may be divided into two groups: fast transient currents (f) with time to peak less than 70 ms (at test potential near 0 mV), and slow (s) components (Lukyanenko et al. 1993). The latter were completely blocked by 4-aminopyridine (4-AP) and the former were more sensitive to TEA than slow IK. In the present study we analyzed the effects of calcium blockers on different types of IK using the whole-cell patch-clamp technique. One to seven-day-old myocytes without slow calcium current and without contact with nerve cells were examined. Extracellullar application of 40-80 mumol/l dihydropyridine (DHP) antagonist nifedipine did not change maximal conductance of K-channels, but induced a parallel shift by 5-10 mV of chord conductance curve along the voltage axis in the direction of more negative potentials. Quinidine in concentrations 30-200 mumol/l caused a reversible block of the fast and the slow IK (C0.5 = 75 mumol/l), and enhanced the current decay (2-3-fold at 150 mumol/l). Verapamil (VP) in concentrations 100-700 mumol/l reduced IK with dose-dependent effect (C0.5 = 200 mumol/l) and changed its kinetic properties. VP 100 mumol/l caused a complete irreversible block of the slow IK. VP reduced the time inactivation constant of fast IK with a dose-dependent effect (8-10-fold at 300 mumol/l), and this effect was stronger during depolarizing pulses. The latter points to the possibility that the fast K-channels preferentially bind VP in open state. An analysis of the effects suggests that K-channels of the frog myocytes could be divided into 2 groups: 1) K-channels which irreversibly blocked by VP and 4-AP (slow), and 2) those reversibly inhibited by VP and 4-AP (fast potassium channels).