Involvement of dihydropyridine-sensitive calcium channels in high asynchrony of transmitter release in neuromuscular synapses of newborn rats.

Experiments on neuromuscular synapses of rats at different stages of ontogenesis have been performed. It has been found that one of the reasons of higher asynchrony of the release of single quanta of acetylcholine in the synapses of newborn animals is the activity of the presynaptic dihydropyridine-sensitive calcium channels of the L-type.

FLUORESCENT MICROSCOPY IDENTIFICATION OF THE EXPRESSION OF a2 AND b1 NA +/K+-ATPase, a1S (L-TYPE), ÑÀ2+-CHANNEL, SERCA 1/2/3 CA2+-ATPase SUBUNITS AT FAST AND SLOW MUSCLES IN THE HYPOGRAVITY MODELING EXPERIMENTS IN RATS

The experiments of modeling hypogravity using fluorescent microscopy have shown a decrease of expression of b1 of Na +/K+-ATPase and Ca2+-ATPase subunits and the increase in the insensitivity of synthesis of a1S subunit of the L-type Ca2+-channel of the plasmatic membrane, whereas the synthesis of a2 subunit of Na+/K+-ATPase does not change. In «fast¼ muscle only observed similar for «slow¼ muscle decrease in the expression of b1 subunit without changing other parameters were studied. However, the decrease in fluorescence b1 subunit due to spread of data was not statistically significant. Thus hypogravity adversely affects the functioning primarily skeletal muscles, providing static load.

Immunohistochemical evidence of the presence of metabotropic receptors for γ-aminobutyric acid at the rat neuromuscular junctions.

in the synapses of the "fast" (m. EDL) and "slow" (m. soleus) skeletal muscles of the rat GABABR1 and GABABR2 subunits of metabotropic receptors for γ-aminobutyric acid (GABA), located primarily on the motor nerve ending membrane were detected by immunohistochemistry and fluorescence microscopy methods.

The role of chloride ions in the maintenance of resting membrane potential in rat fast and slow muscles during hypogravity modeling.

Antiorthostatic hindlimb suspension reduces resting membrane potential of rat fast and slow muscles within 7 days. Changes in Na+/K+ pump activity and shifts of the equilibrium potential for chloride ions are the main mechanism of the changes in the resting potential of muscle fibers. The latter is presumably associated with increased intracellular ion current due to activation of the second active Na+, K+, 2Cl- symport. Reduction of the membrane potential is related to muscle denervation. However, membrane depolarization of muscle fibers during antiorthostatic suspension cannot be explained solely by changes in the mechanisms of neurotrophic control from motor neurons.

[Influence of modeling of gravitational unloading on the postsynaptic acetylcholine receptor organization and acetylcholinesterase activity in neuromuscular synapses of rat fast and slow muscles].

Using immunofluorescent techniques, we have revealed that, after 35 days of rats hindlimb unloading, neuromuscular synapses of fast and slow muscles show enhanced fluorescence intensity and decreased area of fluorescent staining of acetylcholine receptors; increased fluorescent intensity and area of fluorescent staining for acetylcholinesterase. The ratio of the number of postsynaptic acetylcholine receptors and the amount of acetylcholinesterase changed as well as their spatial position in relation to each other. These rearrangements correspond to electrophysiological data on the reduction of the amplitude of the miniature endplate currents in both muscles. Identified synapses restructuring accompanied by a decrease in the volume of muscle fibers. Hindlimb unloading (simulation of hypogravity) leads to an increase in functional activity of acetylcholinesterase on the background of reduced postsynaptic membrane area occupied by acetylcholine receptors. This leads to a decrease in the amplitude of excitatory postsynaptic potentials thereby reducing the nerve-muscle excitation transmission safety factor.

[Myelination disorders in mechanism of hypogravity motor syndrome development].

When modeling effects of hypogravitation by the method of hindlimb unloading in rats the area of cross-section in lumbar part of a spinal cord was found to reduce. The analysis of spinal cord slides showed that these changes are associated with a decrease in the area of white substance of a spinal cord. Data obtained are consistent with our previous observation of a decrease in expression of the genes encoding myelin proteins. Results of our researches give the good reasons to believe that miyelinization failure in CNS is one of the factors that underlie the development of hypogravitational motor syndrome.

[Neuromuscular synaptic transmission at different stages of postnatal development in rats].

On the nerve-muscle preparation of rats diaphragm muscle on different stages of postnatal development, the comparison of morphological features and functions of synaptic apparatus, including induced secretion time parameters was carried out. It was found that, along with the reduced, compared to the adult animals, area of nerve endings in the newborn the speed of the motor nerve excitation was slower, intensity of spontaneous and induced secretion of quantum fluctuations was reduced and real synaptic delays in the end plate were intense. Severe degree of acetylcholine quanta asynchronous secretion with longer open state of the ion channel in newborns synapses can compensate reduction in reliability of synaptic transmission due to a decrease of the quantal content of the postsynaptic response.

Voltage-dependent P/Q-type calcium channels at the frog neuromuscular junction.

It is well known that antagonists of N-type voltage-gated calcium channels inhibit the evoked quantal release of acetylcholine in amphibian neuromuscular synapses. This, however, does not exclude the functional expression of other types of voltage-gated calcium channels in these nerve terminals. Using immunocytochemistry, we detected the expression of the alpha1A subunit of P/Q-type calcium channels (that is otherwise typical of mammalian motor nerve endings) in the frog neuromuscular junction. In addition, we demonstrated that the P/Q-type channel blocker omega-agatoxin IVA (20 nM) reduced the action potential-induced calcium transient and significantly decreased both spontaneous and evoked mediator release. Our data indicates the functional expression of P/Q-type calcium channels in the frog motor nerve ending which participate in acetylcholine release.

Miniature excitatory synaptic ion currents in the earthworm Lumbricus terrestris body wall muscles.

The miniature excitatory postsynaptic currents (MEPCs) of the muscle cells of the earthworm Lumbricus terrestris were recorded by glass microelectrodes. In a single synaptic zone, three types of MEPC were recorded: a fast single-exponential type that decayed with tau =0.9 ms, a slow single-exponential with tau = 9.2 ms and a two-exponential MEPC with tau = 1.3 and 8.5 ms, respectively. The muscle cells of earthworms contain populations of yet-unidentified ionic channels that might be different from the common nicotinic and muscarinic groups of acetylcholine receptors, since these MEPCs are not sensitive to d-tubocurarine, atropine, benzohexonium or proserine. Alternatively, besides ACh receptors, the membrane may contain receptors for another yet-unidentified excitatory transmitter.

Effect of GABAergic and adrenergic agents on activity of Na+/K+ pump and Cl(-)-cotransport in somatic muscle cells of earthworm Lumbricus Terrestris.

GABA, baclofen, epinephrine, and norepinephrine hyperpolarized the membrane of earthworm somatic cells. This effect was prevented by furosemide, removal of Cl- from the medium, or activation of Na+/K+ pump by 3-fold increase external potassium concentration. It was hypothesized that GABA, baclofen, epinephrine, and norepinephrine stimulate Na+/K+ transport via specific receptor inputs, but their effect on resting potential can be realized only under conditions of working Cl- symport.

Functional heterogeneity of the "transporter" of electrogenic ionic pump of the Lumbricus terrestris somatic myocyte membrane.

Potential created by electrogenic ionic pumps under conditions of maximum activation in a warm standard ionic medium with K(+) after preincubation in cold potassium-free solution has two components: a higher ouabaine-insensitive "stationary" component, and a lower "regulatory" component sensitive to ouabaine, furosemide, and removal of Cl(-) or Ca(2+) from the medium. Functional heterogeneity of electrogenic ionic pumps is hypothesized implying the existence of two components: "stationary" (not regulated extracellularly) and "regulatory" (controlled and directly related to active Cl(-) transfer).

Effects of cholinergic receptor agonists and antagonists on miniature stimulatory postsynaptic ionic currents in somatic muscle cells of lumbricus terrestris.

Miniature stimulating postsynaptic currents of Lumbricus terrestris somatic muscle cells were recorded. Atropine, d-tubocurarin, alpha-bungarotoxin, carbacholine, and proserin did not modify the amplitude and temporal parameters of miniature stimulatory postsynaptic currents, while carbacholine and nicotine depolarized the muscle membrane. Presumably, Lumbricus terrestris muscle cells contain acetylcholine-sensitive channel-receptor complexes not belonging to classical nicotinic or muscarinic acetylcholine receptors.

Effects of some transmitters on resting membrane potential of somatic cell in Lumbricus terrestris muscle wall.

Serotonin, glutamate, glycine, ATP, and muscarine had no effect on resting membrane potential of muscle cell in earthworm Lumbricus terrestris. Nicotine depolarizes and GABA hyperpolarizes the muscle membrane. Removal of K(+), Cl(-) and addition of ouabaine and strychnine to the solution abolished the effect of GABA. The authors conclude that the Lumbricus terrestris myocyte membrane contains nicotine receptors and GABAergic receptors sensitive to strychnine. Stimulation of these receptors activates sarcolemmal ionic pumps and causes membrane hyperpolarization.

Chloride cotransport in the membrane of earthworm body wall muscles.

The resting membrane potential (V(m)) of isolated somatic longitudinal muscles of the earthworm Lumbricus terrestris was studied by glass microelectrodes. The inhibition of chloride permeability by low pH did not affect V(m) of the muscle fibers in isolated somatic longitudinal muscles of the earthworm Lumbricus terrestris which was -48.7 mV (inside negative) at pH 7.3 and -49.1 at pH 5.6. On the other hand, bathing the muscles in Cl(-) and Na(+)-free solutions, or application of the chloride transporter inhibitor furosemide and Na(+)-K(+)-ATPase inhibitor ouabain depolarized the V(m) by 3-5 mV. The effects of a Cl(-) -free solution and ouabain were not additive. This demonstrates relatively small contribution of equilibrium potential for Cl(-) to the resting membrane potential and electrogenic effect of Na(+)K(+)-ATPase which is dependent on the supply of Na(+)(i) ions by furosemide-sensitive and Cl(-)(e)- and Na(+)(e)-dependent electroneutral transport (most probably Na(+)K(+)Cl(-) cotransport).

Two populations of miniature excitatory synaptic ionic currents in somatic muscle cells of Lumbricus terrestris earthworm body wall.

Three types of miniature excitatory synaptic currents were recorded in the same synaptic region of earthworm muscle cells: monoexponential (tau=1.2 msec) and biexponential (tau(1)=1.2 and tau(2)=8.0 msec). It was hypothesized that earthworm muscle cells contain at least two populations of acetylcholine-sensitive ionic channels, which do not belong to classical nicotinic and muscarinic acetylcholine receptors.

Calcium mechanism of norepinephrine activation of ionic pump in somatic cells of lumbricus terrestris earthworm muscle wall.

Blockade of norepinephrine-induced hyperpolarization in earthworm muscle cells was observed in a calcium-free medium, after substitution of Ca(2+)with Mn(2+), and in the presence of verapamil or chlorpromazine to the incubation saline. Changes in Ca(2+)concentration in the saline and addition of caffeine had no effect on the resting potential of muscle cells. It was hypothesized that signal transduction from norepinephrine-activated membrane adrenoceptors to the ionic pump in earthworm muscle cells depends on influx of extracellular Ca(2+)and subsequent involvement of Ca(2+)-accepting proteins similar to calmodulin in vertebrates.