Search

We report here experiments addressing the contribution of L-type Ca2+ channels to evoked acetylcholine secretion from frog and mouse motor nerve endings with active and inactivated voltage-gated K+ channels. These studies evaluated the effects of the specific L-type Ca2+ channel blocker nitrendipine on the quantum composition of endplate currents and the time course of the secretion of acetylcholine quanta in intact preparations and after preliminary blockade of voltage-gated K+ channels with 4-aminopyridine (4-AP) in medium with depressed and physiological Ca2+ levels. A fluorescence method was used to measure calcium transients reflecting the integral influx of Ca2+ into nerve endings; computer modeling was applied to the processes underlying exocytosis in the presence of the two types of Ca2+ channel (N and L) and with different durations of nerve ending action potentials. In frog synapses, L-type Ca2+ channels were found to contribute to evoked acetylcholine secretion in the presence of active K+ channels, but only in the presence of a depressed Ca2+ level in the medium; on inactivation of voltage-gated K+ channels, the contribution of L-type channels to the secretory process became less significant. At a physiological Ca2+ level, the involvement of L-type channels in evoked acetylcholine secretion was apparent, as in mouse synapses, only in conditions of blockade of voltage-gated K+ channels.

Experiments on frog neuromuscular preparations were performed to study the characteristics of the calcium response and the quantum secretion of acetylcholine in different pats of extended nerve terminals in different conditions of calcium influx. A calcium-sensitive fluorescent dye was used to analyze Ca2+ influx (Ca2+ transients) into the proximal and distal parts of nerve endings in conditions of increased K+ ion content, in response to blockers of N- and L-type calcium channels, and on blockade of calcium-activated potassium channels. These studies showed that at a uniform distribution density of voltage-gated calcium channels along nerve endings, the proximal-to-distal decrement in calcium transients and quantum secretion intensity persisted in conditions of additional opening of voltage-gated calcium channels by potassium depolarization, on “thinning” of these channels using specific blockers, but changed on blockade of calcium-activated potassium channels.

A long history of studies of the effect of catecholamines on various physiological processes, a multidirectional and ambiguous interpretation of these effects, and a widespread use of adrenergic drugs in clinical practice raises a question of the mechanisms of action of these compounds on various functionally important elements of a living organism. The neuromuscular synapse plays a leading role in ensuring locomotor and respiratory functions, as well as in the posture maintenance. There is a number of conflicting reports on the multidirectional effects of adrenergic agonists on the muscle contraction, the release of acetylcholine from the motor nerve endings, and the state of the postsynaptic membrane of muscle fibers. The purpose of this review is to systematize the information concerning the effects of adrenergic compounds on different stages of the process of excitation propagation in peripheral synapses and to highlight recently revealed opportunities of the application of adrenergic compounds for the treatment of various diseases associated with neuromuscular pathology.

In contrast to frog neuromuscular synapses, where noradrenaline (norepinephrine) and its analogues caused synchronization of the acetylcholine release process, in mouse diaphragm endplates noradrenaline increased the degree of asynchrony of neurosecretion. The effect of noradrenaline on release timing persisted at different levels of external calcium ions (0.25–2.0 mM) and was abolished in presence of both α- and β‑adrenoblockers phentolamine and propranolol. The computer reconstruction of multiquantal endplate currents accounting for experimentally observed modification of release kinetics under noradrenaline showed that the rise time of postsynaptic response changes to a greater extent than the amplitude and falling phase of the multiquantal responses. We conclude that there exists a principal difference in the action of noradrenaline in the cholinergic neuromuscular synapses of warm-blooded and cold-blooded animals that can be accounted for by the differences in the type of adrenoreceptors involved in the modulation of synaptic transmission and/or in the involvement of distinct intracellular pathways triggered by receptor activation.

At neuromuscular junctions, ATP inhibits both the evoked and spontaneous acetylcholine release and inward calcium current operating via presynaptic P2Y receptors. It was shown in the experiments with the frog neuromuscular synapse using specific calcium-sensitive dye Oregon Green Bapta 1 that exogenous ATP reduces the amplitude of calcium transient, which reflects the changes in the entry of calcium ions in response to the nerve pulse. The depressing effect of ATP on the transient was prevented by suramin, the blocker of P2 receptors. Nitrendipine, a specific blocker of L-type calcium channels, per se decreased the calcium transient amplitude and significantly attenuated the effect of ATP on the calcium signal. Contrariwise, the preliminary application of ATP to the neuromuscular junction completely eliminated the depressing effect of nitrendipine on the calcium response. The obtained data suggest that an essential component in the inhibitory action of ATP on the calcium transient amplitude is provided by reduction of the entry of calcium ions into a frog nerve ending via L-type voltage-gated calcium channels.

Previously, we found that muscarine downregulates the acetylcholine release at the frog neuromuscular junction acting via M3 muscarinic receptors. Here, the molecular mechanisms underlying the inhibitory effect of muscarine on the quantal secretion of acetylcholine were studied. Inhibition of phospholipase C (with U-73122) prevented the reduction of evoked neurotransmitter release induced by muscarine. Interruption of synthesis of phosphatidylinositol 3-phosphate by the inhibitor of phosphoinositide-3-kinase (wortmannin) did not affect the depressant action of muscarine but precluded the restoration of secretion after removal of muscarine from the bathing solution. The effect of muscarine was not significantly modified by the blockade of endocannabinoid receptors (with AM 281), but it was abolished by the inhibitor of nitric oxide synthase (L-NAME) as well as extracellular nitric oxide (NO) chelator (hemoglobin). Moreover, muscarine increased NO-sensitive dye fluorescence in junctional region, which was prevented by the M3 receptor antagonist 4-DAMP. The data obtained indicate that the attenuation of acetylcholine release mediated by muscarine is associated with a change in the activity of both lipid-metabolizing enzymes and NO synthases.

Olesoxime is a cholesterol-like neuroprotective compound that targets to mitochondrial voltage dependent anion channels (VDACs). VDACs were also found in the plasma membrane and highly expressed in the presynaptic compartment. Here, we studied the effects of olesoxime and VDAC inhibitors on neurotransmission in the mouse neuromuscular junction. Electrophysiological analysis revealed that olesoxime suppressed selectively evoked neurotransmitter release in response to a single stimulus and 20 Hz activity. Also olesoxime decreased the rate of FM1–43 dye loss (an indicator of synaptic vesicle exocytosis) at low frequency stimulation and 20 Hz. Furthermore, an increase in extracellular Cl− enhanced the action of olesoxime on the exocytosis and olesoxime increased intracellular Cl− levels. The effects of olesoxime on the evoked synaptic vesicle exocytosis and [Cl−]i were blocked by membrane-permeable and impermeable VDAC inhibitors. Immunofluorescent labeling pointed on the presence of VDACs on the synaptic membranes. Rotenone-induced mitochondrial dysfunction perturbed the exocytotic release of FM1–43 and cell-permeable VDAC inhibitor (but not olesoxime or impermeable VDAC inhibitor) partially mitigated the rotenone-driven alterations in the FM1–43 unloading and mitochondrial superoxide production. Thus, olesoxime restrains neurotransmission by acting on plasmalemmal VDACs whose activation can limit synaptic vesicle exocytosis probably via increasing anion flux into the nerve terminals.

Septins (Sept) are highly conserved Guanosine-5'-triphosphate (GTP)-binding cytoskeletal proteins involved in neuronal signaling in the central nervous system but their involvement in signal transmission in peripheral synapses remains unclear. Sept5 and Sept9 proteins were detected in mouse peripheral neuromuscular junctions by immunofluorescence with a greater degree of co-localization with presynaptic than postsynaptic membranes. Preincubation of neuromuscular junction preparations with the inhibitor of Sept dynamics, forchlorfenuron (FCF), decreased co-localization of Sept with presynaptic membranes. FCF introduced ex vivo or in vivo had no effect on the amplitude of the spontaneous endplate currents (EPCs), indicating the absence of postsynaptic effects of FCF. However, FCF decreased acetylcholine (ACh) quantal release in response to nerve stimulation, reduced the amplitude of evoked quantal currents and decreased the number of quanta with long synaptic delays, demonstrating the presynaptic action of FCF. Nevertheless, FCF had no effect on the amplitude of calcium transient in nerve terminals, as detected by calcium-sensitive dye, and slightly decreased the ratio of the second response amplitude to the first one in paired-pulse experiments. These results suggest that FCF-induced decrease in ACh quantal secretion is not due to a decrease in Ca2+ influx but is likely related to the impairment of later stages occurring after Ca2+ entry, such as trafficking, docking or membrane fusion of synaptic vesicles. Therefore, Sept9 and Sept5 are abundantly expressed in presynaptic membranes, and disruption of Sept dynamics suppresses the evoked synchronous and delayed asynchronous quantal release of ACh, strongly suggesting an important role of Sept in the regulation of neurotransmission in peripheral synapses.

Calcium entry into the nerve endings through voltage-dependent calcium channels triggers a chain of events leading to exocytosis of neurotransmitter, providing the transmission of excitation through the synapse. In this regard, a significant role of calcium ions and presynaptic calcium channels in the modulation of secretion is evident. However, the question of the contribution of different types of voltage-dependent calcium channels in the calcium regulation parameters of the quantal secretion still remains unclear. The secretion kinetics characterizes a degree of synchrony of the neurotransmitter release. In recent decades it is regarded as one of the important factors maintaining the effectiveness of the synaptic transmission. Since neuromuscular synapses of frogs and mice are classical objects of physiological and pharmacological studies, the results of which are summarized and extrapolated to other synapses, it is interesting to compare changes of the acetylcholine secretion in these synapses under different conditions of calcium entry into the nerve endings. In this review we discuss the data on the neuromuscular synapses of frogs and mice and analyze some aspects of calcium regulation and involvement of different types of voltage-dependent calcium channels in the modulation of the acetylcholine secretion kinetics.

A number of different types of presynaptic receptors was revealed in central and peripheral chemical synapses activated both by main mediator and co-mediators released simultaneously. Physiological significance and mechanisms of functioning of these receptors are not clear yet. They are assumed to provide negative or positive feedback decreasing or increasing the number of neurotransmitter quanta released in response to nerve impulse and thus regulating synaptic transmission. At the same time, there is one more way of secretion process modulation associated with the changes of timing of transmitter release. This mechanism was shown to contribute to the efficiency of synaptic transmission. The role of presynaptic receptors in regulation of the kinetics of quanta release is one of the interesting questions of modern neurophysiology. This paper overviews the results obtained by the authors that demonstrate the contribution of presynaptic receptors of different types into the regulation of temporal parameters of quantal secretion at the vertebrates neuromuscular junction. It was shown that activation of the cholinergic nicotinic receptors leads to a decrease of the amplitude of postsynaptic response not only due to reduction of the quantity of released quanta but also due to increased the level of asynchronous release. On the contrary, the facilitating effect of catecholamines on the neuromuscular synapse is the result of activation of presynaptic β1-adrenoreceptors which leads to greater synchronization of release process and, consequently, to the increase of the amplitude of the postsynaptic response. Presynaptic purine receptors, involved in the modulation the intensity of secretion, are also capable of alteration of the time course of secretion. Activation of ryanodine receptors results in the increase of the number of quanta released with prolonged latencies leading to appearance of the phase of delayed asynchronous neurotransmitter release.

Reactive oxygen species (ROS) are potent regulators of transmitter release in chemical synapses, but the mechanism of this action remains almost unknown. Presynaptic modulation can change either the release probability or the time course of quantal release, which was recently recognized as an efficient mechanism determining synaptic efficiency. The nonuniform structure and a big size of the frog neuromuscular junction make it a useful model to study the action of ROS in compartments different in release probability and in time course of transmitter release. The time course (or kinetics) of quantal release could be estimated by measuring the dispersion of the synaptic delays for evoked uniquantal endplate currents (EPCs) under low release probability. Using two-electrode recording technique, the action of ROS on kinetics and release probabilities were studied at the proximal and distal parts within the same neuromuscular junction. The stable ROS hydrogen peroxide (H2O2) increased the dispersion of synaptic delays of EPCs (i.e. desynchronized quantal release) within the distal part but decreased delay dispersion (synchronized quantal release) within the proximal part of the same synapse. Unlike the opposite modulation of kinetics, H2O2 reduced release probability in both distal and proximal parts. Since ATP is released from motor nerve terminals together with acetylcholine and can be involved in ROS signaling, we tested the presynaptic action of ATP. In the presence of the pro-oxidant Fe2+, extracellular ATP, similarly to H2O2, induced significant desynchronization of release in the distal regions. The antioxidant N-acetyl-cysteine attenuated the inhibitory action of ATP on release probability and abolished the action of H2O2 and ATP in the presence of Fe2+, on release kinetics. Our data suggest that ROS induced during muscle activity could change the time course of transmitter release along the motor nerve terminal to provide fine tuning of synaptic efficacy.

A relationship between energy exchange and ion transport is the basis for the functional response of cells to stress factors. The aim of the present work was to estimate the energy expenditure for response reactions and adaptation that are dependent on the permeability of the plasma membrane of the excised root cells. We studied the dynamics of changes in the (i) energy flow as measured by the oxygen uptake rate and the heat production rate, (ii) the electrical membrane potential (MP) and (iii) the loss of potassium ions of the cells on prolonged (5 h) treatment with membrane active compounds, namely the specific K + -ionophore valinomycin (Val) and chlorpromazine (CPZ), an antagonist of calmodulin but with a wide spectrum of other action. It was shown that the early (2 h) response of the cells exposed to these compounds was an increase in the loss of K + ions and a decrease in the MP that were more pronounced in the presence of CPZ. The rates of oxygen uptake and heat production by the cells in the presence of Val increased with time and were coupled with the restoration of ion homeostasis as measured by the reduced loss of K + ions. It is supposed that in the presence of Val the energy dependent recovery of ion homeostasis occurred through the compensatory activation of a plasma membrane H + -ATPase and the increase of K + /H + -exchange. Compared to Val, CPZ had larger effect on the alteration of the membrane characteristics and energy expenditure even during the relatively short time of treatment. It is supposed that this happened because CPZ damaged the membranes of the cells as a result of its partition with membrane lipids. Therefore, more energy is needed to restore the cellular homeostasis in the presence of CPZ compared to Val.

The sensitivity of cells to stress factors is associated with the function of electrogenic pumps and depends on their energy conditions. Regulation of cell metabolism and corresponding energy expense can be achieved by means of shifts in ion homeostasis via changes in the structural and functional properties of the plasma membrane. In the present work, we studied the changes in the rates of respiration and heat production along with the electrical membrane potential (MP) in cells of the excised roots of 5 day old wheat seedlings ( Triticum aestivum L.) while the ion conductivity of their plasma membranes was modulated by 20 μM valinomycin (Val) and 100 μM chlorpromazine (CPZ). It is shown that both of these compounds induced an enhancement of potassium ion loss. CPZ also reduces both respiration intensity and heat production after 2 h of exposure. The enhancement of K + loss caused by the specific K + ionophore, Val, had no disorganizing effect on plasma membranes, and was compensated by an increase in ATPase activity on the plasma membrane. The essential rise of plasma membrane ion conductivity induced by CPZ, the compound with a pronounced membrane effect, was followed by a considerable inhibition of root respiration and heat production. These results point to the lack of cell energy resources to eliminate the damaging effect of CPZ.

We investigated the effects of repetitive high-frequency (10 sec-1) nerve stimulation on the time course of evoked and miniature end-plate currents (EPC and mEPC, respectively) in the frog neuromuscular junction. The data obtained indicate that at a physiological Ca2+ level in the bath medium, 10 sec-1 stimulation results in prolongation of the growth phase of multiquantum EPC without any effect on the mEPC time course. It is concluded that timing of acetylcholine quantum secretion may be affected by high-frequency stimulation.

Calcium ions entering into the nerve terminal through voltage-dependent calcium channels during action potential initiate neurotransmitter release and thereby play an important role in the transmission of excitation. In addition to the amount of released neurotransmitter quanta (the quantal content of the endplate potential), another important characteristic of exocytosis is the kinetics of release of individual quanta that form total multiquantal response. Previously, we showed the dependence of the temporal parameters of neurotransmitter quantum release on the extracellular calcium ion concentration, but it remained unclear which calcium channel types are involved in the modulation of the quantal content and release kinetics. It is commonly accepted that, in contrast to the central nervous system, wherein the secretion take part various types of voltage-dependent calcium channels, exocytosis in the peripheral nervous system of adult animals mainly occurs owing to one type of calcium channels. This is the P/Q type (Ca V 2.1 in the latest classification) blocked with FTX toxin and ω -agatoxin IVA in mammalian neuromuscular junction. In frog synapses, this is the N type channels (Ca V 2.2), which are blocked by the ω -conotoxin GVIA. At the same time, the ability of FTX, a specifc P/Q channel blocker, to decrease the quantal content in frog synapses and effects of dihydropyridines that block L type (Ca V 1.2) channels in the developing Xenopus synapses, and L type (Ca V 1.2) channels activator effects in frog synapses, taken together, indicate the possibility of participation of other types of calcium channels in the exocytosis regulation in peripheral synapses. The possible role of changes in the activity of not only P/Q channels, but also other calcium channel types, in modulation of the temporal parameters of neurotransmitter release was shown in peripheral synapses of mouse and crayfish. In order to identify the type of voltage-dependent calcium channels modulating of the quanta secretion in frog and mouse synapses, the effects of specific blockers of various channel types on the endplate potentials quantal content and transmitter release kinetics were analyzed. On isolated Rana ridibunda neuromuscular preparation of m. cutaneus pectoris and on the mouse diaphragm the evoked uniquantal endplate currents were registered by the extracellular electrodes and the fluctuations of the real synaptic latency were estimated. Blocking of the P/Q-, N-, and L-channels in nerve-muscle junction of the frog by the appropriate specific blockers led to a decrease in the amount of released quanta, as well as in the synaptic latency variance, that indicate the synchronization of the process of synaptic vesicles exocytosis. Obtained results indicate that in low-calcium solution the kinetics of quanta secretion in the frog synapse may be regulated by N-, P/Q-, and L-calcium channels. In the mouse nerve muscle junctions at low calcium concentration and infrequent stimulation, when asynchrony of the secretion was more pronounced, the block of the P/Q-channels led to a decrease in the amount of released quanta without changing the kinetics of their secretion, while blocking the L-type channels only decreased variance of synaptic latency. The fact that the number of released quanta and the time course of their secretion may change independently when various types of voltage-dependent calcium channels are blocked witnesses that the role of N-, P/Q-, and L-channels in modulation of the process of synaptic vesicles exocytosis is different.