E. E. Nikolsky

Effect of nitric oxide and NO synthase inhibition on nonquantal acetylcholine release in the rat diaphragm.

After anticholinesterase treatment, the postsynaptic muscle membrane is depolarized by about 5u2003mV due to nonquantal release of acetylcholine (ACh) from the motor nerve terminal. This can be demonstrated by the hyperpolarization produced by the addition of curare (H‐effect). The magnitude of the H‐effect was decreased significantly to 3u2003mV when the nitric oxide (NO) donors, sodium nitroprusside (SNP) and S‐nitroso‐N‐acetylpenicillamine (SNAP) were applied to the muscle, or when NO production was elevated by adding l‐arginine, but not d‐arginine, as a substrate. The H‐effect was increased to 8–9u2003mV by inhibition of NO synthase by l‐nitroarginine methylester (l‐NAME), or by guanylyl cyclase inhibition by methylene blue and 1H‐[1,2,4]oxidiazolo[4,3‐a]quinoxalin‐1‐one (ODQ). ODQ increased the H‐effect to 7.3u2003±u20030.2u2003mV and diminished the SNP‐induced decrease of the H‐effect when applied together with SNP. The effects of NO donors and l‐arginine were eliminated by adding reduced haemoglobin, an extracellular NO scavenger. The present results, together with earlier evidence for the presence of NO synthase in muscle fibres, indicate that nonquantal release of ACh is modulated by NO production in the postsynaptic cell.

The glutamate and carbachol effects on the early post-denervation depolarization in rat diaphragm are directed towards furosemide-sensitive chloride transport.

The membrane potentials of denervated muscle fibres of the rat diaphragm kept in a tissue culture medium are depolarized by about 8-10 mV (10-12%) within 3 h after denervation. This early post-denervation depolarization (EPD) is substantially reduced (2-3 mV) when muscle strips are bathed with 1 mM L-glutamate (GLU) which is found in motor nerve endings, or with 5 x 10(-8) M carbachol (CCh), which mimics the effect of nonquantally released acetylcholine (ACh). The hyperpolarizing effects of GLU and CCh on EPD are not influenced by ouabain, an active sodium transport inhibitor, but are absent when Cl- transport is augmented by increased osmolarity (500 mosmol/l) produced by addition of sucrose or NaCl. The EPD and the effect of hyperosmolarity are effectively prevented by the Cl- transport inhibitor furosemide (1 x 10(-4) M) or by a chloride-free bathing medium. It is suggested that the post-denervation cessation of nonquantal ACh release, and probably also GLU release, from nerve endings leads to the activation of the furosemide-sensitive Cl- transport in the sarcolemma, which is responsible for the early post-denervation depolarization.

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

Negative cross‐talk between presynaptic adenosine and acetylcholine receptors

Functional interactions between presynaptic adenosine and acetylcholine (ACh) autoreceptors were studied at the frog neuromuscular junction by recording miniature end‐plate potentials (MEPPs) during bath or local application of agonists. The frequency of MEPPs was reduced by adenosine acting on presynaptic adenosine A1 receptors (EC50u2003=u20031.1u2003µm) or by carbachol acting on muscarinic M2 receptors (EC50u2003=u20031.8u2003µm). However, carbachol did not produce the depressant effect when it was applied after the action of adenosine had reached its maximum. This phenomenon implied that the negative cross‐talk (occlusion) had occurred between A1 and M2 receptors. Moreover, the occlusion was receptor‐specific as ATP applied in the presence of adenosine continued to depress MEPP frequency. Muscarinic antagonists [atropine or 1‐[[2‐[(diethylamino)methyl)‐1‐piperidinyl]acetyl]‐5,11‐dihydro‐6H‐pyrido [2,3‐b][1,4]benzodiazepine‐6‐one) (AFDX‐116)] had no effect on the inhibitory action of adenosine and adenosine antagonists [8‐(p‐sulfophenyl)theophylline (8‐SPT) or 1,3‐dipropyl‐8‐cyclopentylxanthine (DPCPX)] had no effect on the action of carbachol. These data suggested that membrane–delimited interactions did not occur between A1 and M2 receptors. Both carbachol and adenosine similarly inhibited quantal release triggered by high potassium, ionomycin or sucrose. These results indicated a convergence of intracellular pathways activated by M2 and A1 receptors to a common presynaptic effector located downstream of Ca2+ influx. We propose that the negative cross‐talk between two major autoreceptors could take place during intense synaptic activity and thereby attenuate the presynaptic inhibitory effects of ACh and adenosine.

Regulation of acetylcholinesterase activity by nitric oxide in rat neuromuscular junction via N-methyl-D-aspartate receptor activation

Acetylcholinesterase (AChE) is an enzyme that hydrolyses the neurotransmitter acetylcholine, thereby limiting spillover and duration of action. This study demonstrates the existence of an endogenous mechanism for the regulation of synaptic AChE activity. At the rat extensor digitorum longus neuromuscular junction, activation of N‐methyl‐d‐aspartate (NMDA) receptors by combined application of glutamate and glycine led to enhancement of nitric oxide (NO) production, resulting in partial AChE inhibition. Partial AChE inhibition was measured using increases in miniature endplate current amplitude. AChE inhibition by paraoxon, inactivation of NO synthase by Nω‐nitro‐l‐arginine methyl ester, and NMDA receptor blockade by dl‐2‐amino‐5‐phosphopentanoic acid prevented the increase in miniature endplate current amplitude caused by amino acids. High‐frequency (10 Hz) motor nerve stimulation in a glycine‐containing bathing solution also resulted in an increase in the amplitude of miniature endplate currents recorded during the interstimulus intervals. Pretreatment with an NO synthase inhibitor and NMDA receptor blockade fully eliminated this effect. This suggests that endogenous glutamate, released into the synaptic cleft as a co‐mediator of acetylcholine, is capable of triggering the NMDA receptor/NO synthase‐mediated pathway that modulates synaptic AChE activity. Therefore, in addition to well‐established modes of synaptic plasticity (e.g. changes in the effectiveness of neurotransmitter release and/or the sensitivity of the postsynaptic membrane), another mechanism exists based on the prompt regulation of AChE activity.

The effect of glutamate and inhibitors of NMDA receptors on postdenervation decrease of membrane potential in rat diaphragm.

The early postdenervation depolarization of rat diaphragm muscle fibers (8-10 mV within 3 h in vitro) is substantially smaller (3 mV) when muscles are bathed with 1 x 10(-3) M L-glutamate (Glu) or 1 x 10(-3) M N-methyl-D-aspartate (NMDA). The effects of Glu and NMDA are inhibited in a dose-dependent manner by competitive inhibitor 2-amino-5-phosphonovaleric acid (APV) with Ki 6.3 x 10(-4) M, by 2 x 10(-7) M MK-801, which acts as an open channel inhibitor, by 2-3 x 10(-4) Zn2+, which reacts with surface-located sites of the NMDA subtype of the glutamate receptor, and also by glycine-free solutions and 7-Cl-kynurenic acid, which inhibits the glycine binding sites on NMDA receptors. It follows that the effect of glutamate on early post-denervation depolarization is mediated by the NMDA subtype of glutamate receptor with similar pharmacological properties to those found in neurons. The only exception found was the glutamate-like action of 1 x 10(-7) M MK-801, which partially prevented the early postdenervation depolarization when present in the muscle bath during the first 3 h after nerve section.

Opposite modulation of time course of quantal release in two parts of the same synapse by reactive oxygen species.

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.

6-Methyluracil Derivatives as Bifunctional Acetylcholinesterase Inhibitors for the Treatment of Alzheimer's Disease.

Novel 6‐methyluracil derivatives with ω‐(substituted benzylethylamino)alkyl chains at the nitrogen atoms of the pyrimidine ring were designed and synthesized. The numbers of methylene groups in the alkyl chains were varied along with the electron‐withdrawing substituents on the benzyl rings. The compounds are mixed‐type reversible inhibitors of cholinesterases, and some of them show remarkable selectivity for human acetylcholinesterase (hAChE), with inhibitory potency in the nanomolar range, more than 10u2009000‐fold higher than that for human butyrylcholinesterase (hBuChE). Molecular modeling studies indicate that these compounds are bifunctional AChE inhibitors, spanning the enzyme active site gorge and binding to its peripheral anionic site (PAS). In vivo experiments show that the 6‐methyluracil derivatives are able to penetrate the blood–brain barrier (BBB), inhibiting brain‐tissue AChE. The most potent AChE inhibitor, 3u2009d (1,3‐bis[5‐(o‐nitrobenzylethylamino)pentyl]‐6‐methyluracil), was found to improve working memory in scopolamine and transgenic APP/PS1 murine models of Alzheimers disease, and to significantly decrease the number and area of β‐amyloid peptide plaques in the brain.

Redox-sensitive synchronizing action of adenosine on transmitter release at the neuromuscular junction

The kinetics of neurotransmitter release was recognized recently as an important contributor to synaptic efficiency. Since adenosine is the ubiquitous modulator of presynaptic release in peripheral and central synapses, in the current project we studied the action of this purine on the timing of acetylcholine quantal release from motor nerve terminals in the skeletal muscle. Using extracellular recording from frog neuromuscular junction we tested the action of adenosine on the latencies of single quantal events in the pro-oxidant and antioxidant conditions. We found that adenosine, in addition to previously known inhibitory action on release probability, also synchronized release by removing quantal events with long latencies. This action of adenosine on release timing was abolished by oxidants whereas in the presence of the antioxidant the synchronizing action of adenosine was further enhanced. Interestingly, unlike the timing of release, the inhibitory action of adenosine on release probability was redox-independent. Modulation of release timing by adenosine was mediated by purinergic A1 receptors as it was eliminated by the specific A1 antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) and mimicked by the specific A1 agonist N(6)-cyclopentyl-adenosine. Consistent with data obtained from dispersion of single quantal events, adenosine also reduced the rise-time of multiquantal synaptic currents. The latter effect was reproduced in the model based on synchronizing effect of adenosine on release timing. Thus, adenosine which is generated at the neuromuscular junction from the breakdown of the co-transmitter ATP induces the synchronization of quantal events. The effect of adenosine on release timing should preserve the fidelity of synaptic transmission via cost-effective use of less transmitter quanta. Our findings also revealed important crosstalk between purinergic and redox modulation of synaptic processes which could take place in the elderly or in neuromuscular diseases associated with oxidative stress like lateral amyotrophic sclerosis.

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.