Leniz F. Nurullin

NMDA receptors at the endplate of rat skeletal muscles: Precise postsynaptic localization

In this study we demonstrate expression of the N‐methyl‐D‐aspartate receptor NR1 subunit in the rat neuromuscular junction of skeletal muscles of different functional types (extensor digitorum longus, soleus, and diaphragm muscles) using fluorescence immunocytochemistry. Electron microscopic immunocytochemistry has shown that the NR1 subunit is localized solely on the sarcolemma in the depths of the postsynaptic folds. These findings suggest participation of the glutamatergic signaling system in functioning of the adult mammalian neuromuscular junction. Muscle Nerve, 2011

Non‐quantal release of acetylcholine from parasympathetic nerve terminals in the right atrium of rats

Acetylcholinesterase (AChE) inhibitors provoke typical cholinergic effects in the isolated right atrium of the rat due to the accumulation of acetylcholine (ACh). Our study was designed to show that in the absence of vagal impulse activity, ACh is released from the parasympathetic nerve fibres by means of non‐quantal secretion. The conventional microelectrode technique was used to study changes in action potential (AP) configuration in the right atrium preparation of rats during application of AChE inhibitors. Staining with the lipophilic fluorescent dye FM1‐43 was used to demonstrate the presence of endocytosis in cholinergic endings. The AChE inhibitors armin (10−7–10−5 m) and neostigmine (10−7 to 5 × 10−6 m) caused a reduction of AP duration and prolonged the cycle length. These effects were abolished by atropine and were therefore mediated by ACh accumulated in the myocardium during AChE inhibition. Putative block of impulse activity of the postganglionic neurons by tetrodotoxin (5 × 10−7 m) and blockade of ganglionic transmission by hexomethonium (2 × 10−4 m), as well as blockade of all forms of quantal release with Clostridium botulinum type A toxin (50 U ml−1), did not alter the effects of armin. Experiments with FM1‐43 dye confirmed the effective block of exocytosis by botulinum toxin. Selective inhibition of the choline uptake system using hemicholinium III (10−5 m), which blocks non‐quantal release at the neuromuscular junction, suppressed the effects of AChE inhibitors. Thus, accumulation of ACh is likely to be caused by non‐quantal release from cholinergic terminals. We propose that non‐quantal release of ACh, shown previously at the neuromuscular junction, is present in cholinergic postganglionic fibres of the rat heart in addition to quantal release.

Metabotropic GABAB receptors mediate GABA inhibition of acetylcholine release in the rat neuromuscular junction.

Gamma‐aminobutyric acid (GABA) is an amino acid which acts as a neurotransmitter in the central nervous system. Here, we studied the effects of GABA on non‐quantal, spontaneous, and evoked quantal acetylcholine (ACh) release from motor nerve endings. We found that while the application of 10 μM of GABA had no effect on spontaneous quantal ACh release, as detected by the frequency of miniature endplate potentials, GABA reduced the non‐quantal ACh release by 57%, as determined by the H‐effect value. Finally, the evoked quantal ACh release, estimated by calculating the quantal content of full‐sized endplate potentials (EPPs), was reduced by 34%. GABAs inhibitory effect remained unchanged after pre‐incubation with picrotoxin, an ionotropic GABAA receptor blocker, but was attenuated following application of the GABAB receptor blocker CGP 55845, which itself had no effect on ACh release. An inhibitor of phospholipase C, U73122, completely prevented the GABA‐induced decrease in ACh release. Immunofluorescence demonstrated the presence of both subunits of the GABAB receptor (GABABR1 and GABABR2) in the neuromuscular junction. These findings suggest that metabotropic GABAB receptors are expressed in the mammalian neuromuscular synapse and their activation results in a phospholipase C‐mediated reduction in the intensity of non‐quantal and evoked quantal ACh release.

β2-adrenoceptor agonist-evoked reactive oxygen species generation in mouse atria: implication in delayed inotropic effect

Fenoterol, a β2-adrenoceptor agonist, has anti-apoptotic action in cardiomyocytes and induces a specific pattern of downstream signaling. We have previously reported that exposure to fenoterol (5 μM) results in a delayed positive inotropic effect which is related to changes in both Ca2+ transient and NO. Here, the changes in reactive oxygen species (ROS) production in response to the fenoterol administration and the involvement of ROS in effect of this agonist on contractility were investigated in mouse isolated atria. Stimulation of β2-adrenoceptor increases a level of extracellular ROS, while intracellular ROS level rises only after removal of fenoterol from the bath. NADPH-oxidase inhibitor (apocynin) prevents the increase in ROS production and the Nox2 isoform is immunofluorescently colocalized with β2-adrenoceptor at the atrial myocytes. Treatments with antioxidants (N-acetyl-L-cysteine, NADPH inhibitors, exogenous catalases) significantly inhibit the fenoterol induced increase in the contraction amplitude, probably by attenuating Ca2+ transient and up-regulating NO production. ROS generated in a β2-adrenoceptor-dependent manner can potentiate the activity of some Ca2+ channels. Indeed, inhibition of ryanodine receptors, TRPV-or L-type Ca2+- channels shows a similar efficacy in reduction of positive inotropic effect of both fenoterol and H2O2. In addition, detection of mitochondrial ROS indicates that fenoterol triggers a slow increase in ROS which is prevented by rotenone, but rotenone has no impact on the inotropic effect of fenoterol. We suggest that stimulation of β2-adrenoceptor with fenoterol causes the activation of NADPH-oxidase and after the agonist removal extracellularly generated ROS penetrates into the cell, increasing the atrial contractions probably via Ca2+ channels.

Kinetics of neurotransmitter release in neuromuscular synapses of newborn and adult rats.

The kinetics of the phasic synchronous and delayed asynchronous release of acetylcholine quanta was studied at the neuromuscular junctions of aging rats from infant to mature animals at various frequencies of rhythmic stimulation of the motor nerve. We found that in infants 6 (P6) and 10 (P10) days after birth a strongly asynchronous phase of quantal release was observed, along with a reduced number of quanta compared to the synapses of adults. The rise time and decay of uni‐quantal end‐plate currents were significantly longer in infant synapses. The presynaptic immunostaining revealed that the area of the synapses in infants was significantly (up to six times) smaller than in mature junctions. The intensity of delayed asynchronous release in infants increased with the frequency of stimulation more than in adults. A blockade of the ryanodine receptors, which can contribute to the formation of delayed asynchronous release, had no effect on the kinetics of delayed secretion in the infants unlike synapses of adults. Therefore, high degree of asynchrony of quantal release in infants is not associated with the activity of ryanodine receptors and with the liberation of calcium ions from intracellular calcium stores.

Mechanisms of carbacholine and GABA action on resting membrane potential and Na+/K+-ATPase of Lumbricus terrestris body wall muscles

This work was aimed to identify the action of several ion channel and pump inhibitors as well as nicotinic, GABAergic, purinergic and serotoninergic drugs on the resting membrane potential (RMP) and assess the role of cholinergic and GABAergic sensitivity in earthworm muscle electrogenesis. The nicotinic agonists acetylcholine (ACh), carbacholine (CCh) and nicotine depolarize the RMP at concentrations of 5 μM and higher. The nicotinic antagonists (+)tubocurarine, α-bungarotoxin, muscarinic antagonists atropine and hexamethonium do not remove or prevent the CCh-induced depolarization. Verapamil, tetrodotoxin, removal of Cl(-) and Ca(2+) from the solution also cannot prevent the depolarization by CCh. In a Na(+)-free medium, however, CCh lost this depolarization ability and this indicates that the drug opens the sodium permeable pathway. Serotonin, glutamate, glycine, adenosine triphosphate (ATP) and cis-4-aminocrotonic acid (GABA(C) receptor antagonist) had no effect on the RMP. On the other hand, isoguvacin, γ-aminobutyric acid (GABA) and baclofen (GABA(B) receptor agonist) hyperpolarized the RMP. Ouabain, bicucullin (GABA(A) antagonist) and phaclofen (GABA(B) antagonist), as well as the removal of Cl(-), suppressed the effect of GABA and baclofen. CCh did not enhance the depolarization generated by ouabain but, on the other hand, hindered the hyperpolarizing activity of baclofen both in the absence and presence of atropine and (+)tubocurarine. The long-term application of CCh depolarizes the RMP primarily by inhibiting the Na(+)/K(+)-ATPase. The muscle membrane also contains A and B type GABA binding sites, the activation of which increases the RMP at the expense of increasing the action of ouabain- and Cl(-) -sensitive electrogenic pumps.

Metabotropic and ionotropic glutamate receptors mediate the modulation of acetylcholine release at the frog neuromuscular junction

There is some evidence that glutamate (Glu) acts as a signaling molecule at vertebrate neuromuscular junctions where acetylcholine (ACh) serves as a neurotransmitter. In this study, performed on the cutaneous pectoris muscle of the frog Rana ridibunda, Glu receptor mechanisms that modulate ACh release processes were analyzed. Electrophysiological experiments showed that Glu reduces both spontaneous and evoked quantal secretion of ACh and synchronizes its release in response to electrical stimulation. Quisqualate, an agonist of ionotropic α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic receptors and metabotropic Group I mGlu receptors, also exerted Glu‐like inhibitory effects on the secretion of ACh but had no effect on the kinetics of quantal release. Quisqualates inhibitory effect did not occur when a blocker of Group I mGlu receptors (LY 367385) or an inhibitor of phospholipase C (U73122) was present. An increase in the degree of synchrony of ACh quantal release, such as that produced by Glu, was obtained after application of N‐methyl‐D‐aspartic acid (NMDA). The presence of Group I mGlu and NMDA receptors in the neuromuscular synapse was confirmed by immunocytochemistry. Thus, the data suggest that both metabotropic Group I mGlu receptors and ionotropic NMDA receptors are present at the neuromuscular synapse of amphibians, and that the activation of these receptors initiates different mechanisms for the regulation of ACh release from motor nerve terminals.

Quantal and non-quantal acetylcholine release at neuromuscular junctions of muscles of different types in a model of hypogravity.

4 In the space flight environment and in models of the effects of weightlessness on Earth, the functional unloading of skeletal muscles leads to the development of the so called hypogravity syndrome expressed, in particular, in a number of morphological and func tional changes in the neuromuscular system. The characteristic features of this syndrome include hypo tonia, displacement of the myosin profile of the skele tal muscle in the direction of the fast form of myosin, hypotrophy, and reduction of the maximum contrac tion force [1, 2].

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.

The effect of modelling of hypogravity on postsynaptic acetylcholine receptors and activity of acetylcholinesterase in neuromuscular synapses of fast and slow muscles of rat

Rats were subjected to antiorthostatic hindlimb unloading for 35 days. Using immunofluorescent techniques, we found increased intensity of fluorescence and decreased area of staining of acetylcholine receptors and the increased intensity and area of staining of acetylcholinesterase in neuromuscular synapses of “fast” and “slow” muscles. Changes in the ratio of the number of acetylcholine receptors on the postsynaptic membrane and acetylcholinesterase and the alterations in their spatial location relative to each other in neuromuscular synapses of “fast” and “slow” muscles were also observed. These modifications are in accordance with the electrophysiological data on the decreased amplitudes of miniature end plate currents in both muscles. They were accompanied by a decrease in the volume of muscle fibers. Antiorthostatic unloading, a model of hypogravity, resulted in increased functional activity of acetylcholinesterase associated with decreased area of the postsynaptic membrane occupied by acetylcholine receptors, which led to a decrease in the amplitude of postsynaptic excitatory potentials and thus, to the decreased reliability threshold of excitation transmission from a nerve to a muscle.