[Effect of nicotine on electrogenic contribution of the Na,K-ATPase isoforms and contractile characteristics of the rat skeletal muscle].

Nicotine in concentration 100 nM which corresponds to concentration of nicotine circulating in tobacco smokes induced hyperpolarization by approximately 4 mV of muscle fibres of the rat isolated diaphragm, as well as an increase in amplitude and acceleration of action potentials. Similar hyperpolarization was induced by nicotine and acetylcholine in the rat soleus muscle. In this muscle, the hyperpolarization developed more slowly than in diaphragm revealing initial slight depolarization. Non-competitive blocker of open channel of nicotinic acetylcholine receptor, proadifen, abolished nicotine- or acetylcholine-induced depolarization but not the hyperpolarization. In the diaphragm, the hyperpolarization was blocked by specific inhibitors of the Na,K-ATPase, ouabain (50 nM) or marinobufagenin (2 nM) suggesting an involvement of the Na,K-ATPase. Estimation of elecrogenic contributions of isoforms of the Na,K-ATPase showed that the hyperpolarization was due to an increase in electrogenic contribution of alpha 2 isoform without change in contribution of alpha 1 isoform. Nicotine did not affect parameters of muscle contractions in response to direct stimulation.

[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.

[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.

[Analysis of the interaction between nicotinic acetylcholine receptor and Na+,K(+)-ATPase in the rat skeletal muscle and the Torpedo electric organ membrane preparation].

The interaction between the nicotinic acetylcholine receptor and Na+,K(+)-ATPase described previously was further studied in isolated rat diaphragm and in a membrane preparation of Torpedo californica electric organ. Three specific agonists of the nicotinic receptor: acetylcholine, nicotine and carbamylcholine (100 nmol/L each), all hyperpolarized the non-synaptic membranes of muscle fibers by up to 4 mV. Competitive antagonists of nicotinic acetylcholine receptor, d-tubocurarine (2 mcmol/L) or alpha-bungarotoxin (5 nmol/L) completely blocked the acetylcholine-induced hyperpolarization indicating that the effect requires binding of the agonists to their specific sites. The noncompetitive antagonist, proadifen (5 mcmol/L), exerted no effect on the amplitude of hyperpolarized but decreased K0.5 for this effect from 28.3 +/- 3.6 nmol/L to 7.1 +/- 2.3 nmol/L. Involvement of the Na+,K(+)-ATPase was suggested by data demonstrating that three specific Na+,K(+)-ATPase inhibitors: ouabain, digoxin or marinobufagenin (100 nmol/L each), all inhibit the hyperpolarizing effect of acetylcholine. Acetylcholine did not affectation either the catalytic activity of the Na+,K(+)-ATPase purified from sheep kidney or the transport activity of the Na+,K(+)-ATPase in the rat erythrocytes, i. e. in preparations not containing acetylcholine receptors. Hence, acetylcholine does not directly affect the Na+,K(+)-ATPase. In a Torpedo membrane preparation, ouabain (< or = 100 nmol/L) increased the binding of the fluorescent ligand: Dansyl-C6-choline (DCC). No ouabain effect was observed either when the agonist binding sites of the receptor were occupied by 2 mmol/L carbamylcholine, or in the absence Mg2+, when the binding of ouabain to the Na+,K(+)-ATPase is negligible. These results indicate that ouabain only affects specific DCC binding and only when bound to the Na+,K(+)-ATPase. The data obtained suggest that, in two different systems, the interaction between the nicotinic acetylcholine receptor and the Na+,K(+)-ATPase specifically involve the ligand binding sites of these two proteins.

[Effects of marinobufagenin on electrophysiological and contractile characteristics of the rat diaphragm].

Effects of Na+,K(+)-ATPase inhibitor: marinobufagenin, on contractile and electric characteristics of isolated rat diaphragm were studied for the first time. Marinobufagenin induced dose-dependent (EC50 = 0.3 +/- 0.1 nM) increase in the contraction force (positive inotropic effect). At 1-2 nM, it slowed down the fatigue induced by continuous direct stimulation (2/s) of the muscle. Marinobufagenin at the same concentrations did not affect resting membrane potential or parameters of action potentials of muscle fibers, while at 10 and 20 nM it induced hyperpolarization by approximately 2 mV. Marinobufagenin blocked dose-dependently (IC50 = 2.9 +/- 2.0 nM) hyperpolarizing effect of acetylcholine (100 nM) mediated by increase in electrogenic contribution of alpha2 isoform of the Na+,K(+)-ATPase. This result suggests a capability of marinobufagenin to inhibit this isoform of the Na+,K(+)-ATPase. Possible mechanisms of marinobufagenin effects in skeletal muscle are discussed.

[Ouabain-induced blockade of alpha2 isoform of the Na,K-ATPase on electrophysiological and contractile characteristics of the rat diaphragm].

In experiments with isolated neuromuscular preparation of the rat diaphragm, selective blockade of alpha2 isoform of the Na,K-ATPase with ouabain (1 mcmol/L) induced steady depolarization of muscle fibers that reached a maximum of 4 mV, a decrease in amplitude of muscle fiber action potential, and prolonged raising and decline phases of the action potential. At the same time, the force, time to peak, and half relaxation time of the isometric muscle twitch were increased, as well as the area under the contraction curve. During continuous fatiguing stimulation (2/s), a more pronounced decline of contraction speed was observed in presence of ouabain; dynamics of the half-relaxation time remaining unchanged. It is suggested that blockade of alpha2 isoform of the Na,K-ATPase impairs excitation-contraction coupling resulting in a delay of Ca2+ release from sarcoplasmic reticulum. The increase in contraction force seems to result from a mechanism similar to that of positive inotropic effect of cardiac glycosides in heart muscle. Physiological significance of the skeletal muscle alpha2 isoform of the Na,K-ATPase in regulation of Ca2+ and Na+ concentrations near triadic junctions and in regulatory processes involving the Na,K-ATPase endogenous modulators or transmitter acetylcholine is discussed.

[From diversity of molecular forms to functional specialization of oligomeric proteins, nicotinic acetylcholine receptor, acetylcholinesterase and Na+, K+-ATPase]. / Ot raznoobraziia molekuliarnykh form k funktsional'noi spetsializatsii oligomernykh belkov, nikotinovyi kholinoretseptor, atsetilkholinésterasa i Na+, K+-ATFaza.

The review is devoted to the issue of diversity of molecular forms of oligomeric proteins using as examples members of the three protein classes: nicotinic acetylcholine receptor, acetylcholinesterase, and Na,K-ATPase. The data are presented on the molecular structure of proteins, subunit compositions, and isoforms of subunits, as well as on some features of gene expression. Particular emphasis has been made on the functional specialization of different molecular forms of one and the same oligomeric protein. The three above proteins, which serve seemingly quite different cellular processes, demonstrate many common principles of molecular mechanisms of physiological function.

[Functional interaction between nicotinic cholinergic receptors and Na, K-ATPase in the skeletal muscles]. / Funktsional'naia sviaz' nikotinovykh kholinoretseptorov i Na, K-ATFazy v skeletnoi myshtse.

Acetylcholine (ACh) hyperpolarized the rat diaphragm muscle fibers by 4.5 +/- 0.8 mV (K0.5 = = 36 +/- 6 nmol/l). The AC-induced hyperpolarization was blocked by d-tubocurarine and ouabain in nanomolar concentrations. This effect of ACh was not observed in cultured C2C12 muscle cells and in Xenopus oocytes with expressed embryonic mouse muscle nicotinic acetylcholine receptors (nAChR) or with neuronal alpha 4 beta 2 nAChR. In membrane preparations from the Torpedo californica electric organ, containing both nAChR and Na, K-ATPase, 10 nmol/l ouabain modulated the binding kinetics of the cholinergic ligand dansyl-C6-choline to the nAChR. These results suggest that in-sensitive alpha 2 isoform) and nAChR in a state with high affinity to Ach and d-tubocurarine may form a functional complex in which binding of ACh to nAchR is coupled to activation of the Na, K-ATPase.

The modulating effects of various muscle factors on the function of motor nerve terminals.

Studies reported here showed that the incubation medium used for frog muscle contains factors able to modulate the secretion of mediator from motor nerve terminals, with increases in release from low-efficiency synapses and decreases in release from high-efficiency synapses. Both the stimulatory and the inhibitory effects of the incubation medium are mediated by the corresponding changes in the number of available mediator quanta stored. Factors with stimulatory presynaptic actions were present mainly in the low-molecular-weight (<10 kDal) fraction of the incubation medium. The extent of the stimulatory action of this fraction on low-efficiency synapses correlated with the concentration of histidine-containing substances.

[Modulatory effects of various factors of muscular origin on the function of the motor nerve endings]. / O moduliruiushchem deistvii nekotorykh faktorov myshechnogo proiskhozhdeniia na funktsiiu motornogo nervnogo okonchaniia.

Muscle incubate was shown to contain factors capable to increase the transmitter release in low-effective synapses and decrease it in high-effective those, dueto activation and inhibition, respectively, of the processes governing formation of available store of the transmitter. Activatiory effect of the low-molecular fraction on the low-effective synapses was correlated with the concentration of histidine-containing substances in this fraction.

Synaptic feed-backs mediated by potassium ions.

Repetitive activity of the neuromuscular system and of neuronal centers leads to K+ efflux from excited cells and to its accumulation within extracellular spaces and synaptic clefts, especially during the generation of postsynaptic responses such as end-plate potentials or excitatory postsynaptic potentials. K+ ions accumulated within the synaptic cleft during activity modulate the transmitter secretion from motor nerve terminals. Depending on the concentration, K+ can either increase the transmitter release due to a specific presynaptic action or decrease it due to depolarization of the presynaptic membrane. The dual antidromic action of K+ can be the basis of functional self-regulation of the synapse. The significance of the positive presynaptic action of K+ can be assumed to enhance the reliability of the synaptic transmission at moderate activation rates. The negative presynaptic action of K+, which predominates at high-frequency activities or during neuromuscular fatigue, leads to randomized failures of transmissions at individual synapses, the overall pattern of activation of the entire system being reproduced. This might save the general capability of the system and protect its weakest elements. The positive antidromic action of K+ can be assumed to be essential to the mechanism of heterosynaptic facilitation and long-term potentiation at learning synapses of the brain.

The influence of skeletal muscle incubation medium on fatigue of neuromuscular preparation and on transmitter release at neuromuscular junctions in the frog.

The effect of frog skeletal muscle incubate on fatigue was studied in frog sciatic nerve, sartorius muscle preparation. Fatigue was produced by prolonged repetitive (1 s-1) stimulation of motor nerve or of curarized muscle. The incubate partially restored isometric contraction amplitudes of muscle fatigued by nerve stimulation. This effect of partial recovery from fatigue (PRF effect) was exerted mainly by a relatively low-molecular fraction (LMF; < 10 kDa) of the incubate. The incubate and its fractions failed to produce the PRF effect in experiments with directly stimulated muscle. The action of LMF on synaptic transmission in unfatigued cutaneous-pectoris muscle was examined using binomial analysis of quantal transmitter release. LMF produced an increase in the end-plate potential quantal content (m) at synapses with low initial m values. In contrast, it produced a decrease i n m at synapses with higher m values. Both effects were due to respective changes in binomial parameter n. It is assumed that the stimulatory presynaptic action of the incubate on synapses the effectiveness of which was lowered during fatigue, could account for the PRF effect. A possible contribution of low- and high-molecular components of the incubate is discussed.

[The effect of carnosine on synaptic transmission in the frog neuromuscular junction]. / Issledovanie vliianiia karnozina na sinapticheskuiu peredachu v nervno-myshechnom soedinenii liagushki.

The influence of carnosine on quantal content of the e.p.p. of the frog cutaneous pectoris muscle was studied. The e.p.p. quantal content decreased in presence of carnosine (8 mmol/l). Removal of carnosine from the incubation medium potentiated synaptic transmission as compared with its level prior to addition of carnosine to the medium (after-potentiation). Depressant presynaptic action of carnosine did not depend on external Ca+2 concentration or initial level of evoked quantum secretion whereas the after-potentiation only occurred in conditions of decreased Ca+2 concentration in the medium. In the blockade of cyclic nucleotides phosphodiesterase with theophylline, carnosine induced the same effects as in the experiments without theophylline. However, the presence of theophylline in the medium during removal of carnosine prevented the after-potentiation. Possible mechanisms of presynaptic effects of carnosine are discussed.

[Biochemical characteristics of a muscle perfusate and its effects on mediator release in the neuromuscular synapse]. / Nekotorye biokhimicheskie kharakteristiki myshechnogo perfuzata i ego vliianie na osvobozhdenie mediatora v nervno-myshechnom sinapse.

Histidine-containing compounds (HCC) were found, using diazoreaction and reaction with diethylpyrocarbonate, a specific histidine reagent, in the perfusate of the frog hindlimb vasculature. After gel filtration on sephadex G-25 about 80% of HCC were eluted with a fraction with molecular mass less than 5000 kD. The studies of the perfusate influence on the characteristics of quantum secretion of transmitter in the preparation of frog cutaneopectoral muscle have shown that the perfusate increased the quantum content of end-plate potentials (EPP) due to increasing binominal parameter and decreasing frequency of miniature EPP. The characteristics of presynaptic action of the perfusate were similar to those of exogenous histidine.

[Effect of a brain extract containing the postural asymmetry factor on the frog neuromuscular synapse]. / Vliianie ékstrakta mozga, soderzhashchego faktor poznoi asimmetrii, na nervno-myshechnyi sinaps liagushki.

The influence of the rat brain extract containing a postural asymmetry factor on the synaptic transmission in the right and left preparations of the frog cutaneus-pectoralis muscle was studied. The extract (2.10(-4) mg/ml) increased the quantal content of end-plate potentials (e.p.p.) in synapses of the right muscle and decreased it in synapses of the left muscle. In both right and left muscles relative changes in quantal content of e.p.p. correlated with relative changes in binomial parameter n. The results indicate an asymmetrical influence of the extract containing a postural asymmetry factor on the transmitter release in synapses of the right and left muscles.