Konstantin A. Petrov

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.

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 10 000‐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, 3 d (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.

Unfolded and macrocyclic ammonium derivatives of diterpenoids steviol and isosteviol having choline moieties. Synthesis and inhibitory activities toward acetylcholine- and butyrylcholinesterases

A series of unfolded and macrocyclic ammonium derivatives of diterpenoids isosteviol 2 and steviol 17 possessing choline moieties have been synthesized and assayed for inhibitory activities toward AchE and BchE. Compounds 5–8, 13, 16, 20, and 21 showed moderate activity within the range of IC50 values 8.0 × 10−4 to 2.2 × 10−6 mol L−1. Isosteviol derivative 16 exhibited the best inhibition selectivity against acetylcholinesterase among the compounds tested. It inhibited AchE of human erythrocytes at IC50 = 80 μM, whereas inhibition of BchE occurred at IC50 > 1000 μM.

Different sensitivities of rat skeletal muscles and brain to novel anti‐cholinesterase agents, alkylammonium derivatives of 6‐methyluracil (ADEMS)

The rat respiratory muscle diaphragm has markedly lower sensitivity than the locomotor muscle extensor digitorum longus (EDL) to the new acetylcholinesterase (AChE) inhibitors, alkylammonium derivatives of 6‐methyluracil (ADEMS). This study evaluated several possible reasons for differing sensitivity between the diaphragm and limb muscles and between the muscles and the brain.

Luminescent silica nanoparticles for sensing acetylcholinesterase-catalyzed hydrolysis of acetylcholine

This work highlights the H-function of Tb(III)-doped silica nanoparticles in aqueous solutions of acetic acid as a route to sense acetylcholinesterase-catalyzed hydrolysis of acetylcholine (ACh). The H-function results from H(+)-induced quenching of Tb(III)-centered luminescence due to protonation of Tb(III) complexes located close to silica/water interface. The H-function can be turned on/switched off by the concentration of complexes within core or nanoparticle shell zones, by the silica surface decoration and adsorption of both organic and inorganic cations on silica surface. Results indicate the optimal synthetic procedure for making nanoparticles capable of sensing acetic acid produced by enzymatic hydrolysis of acetylcholine. The H-function of nanoparticles was determined at various concentrations of ACh and AChE. The measurements show experimental conditions for fitting the H-function to Michaelis-Menten kinetics. Results confirm that reliable fluorescent monitoring AChE-catalyzed hydrolysis of ACh is possible through the H-function properties of Tb(III)-doped silica nanoparticles.

Mechanisms of Cardiac Muscle Insensitivity to a Novel Acetylcholinesterase Inhibitor C-547

We compared the effects of the novel acetylcholinesterase (AChE) inhibitor C-547 on action potential configuration and sinus rhythm in the isolated right atrium preparation of rat with those of armin and neostigmine. Both armin (10−7, 10−6, and 10−5 M) and neostigmine (10−7, 10−6, and 5 × 10−6 M) produced a marked decrease in action potential duration and slowing of sinus rate. These effects were abolished by atropine and are attributable to the accumulation of acetylcholine in the myocardium. The novel selective AChE inhibitor C-547 (10−9 to 10−7 M), an alkylammonium derivative of 6-methyluracil, had no such effects. The inhibition constant of C-547 on cardiac AChE is 40-fold higher than that on extensor digitorum longus muscle AChE. These results suggest that C-547 might be employed to treat diseases such as myasthenia gravis or Alzheimer disease, without having unwanted effects on the heart.

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.

Selective Blockade of Locomotor Muscles by Uracil-Containing Tetraalkylammonium Acetylcholinesterase Inhibitors

The ratio between muscle-relaxing and lethal doses of onium cholinesterase inhibitors characterizes the selectivity of their effect on the locomotor muscles and the corresponding level of “pharmacological safety” ( LD 50 / ED 50 ). The greatest selectivity is characteristic of oxazyl (ambenonium), whereas the lowest selectivity is exhibited by the majority of cholinesterase inhibitors [1]. Taking into account the fact that the specific effect of cholinesterase inhibitors lasts insufficiently long, the search for ways of increasing the selectivity of their effect is a topical problem.

Nanoparticle-Delivered 2-PAM for Rat Brain Protection against Paraoxon Central Toxicity

Solid lipid nanoparticles (SLNs) are among the most promising nanocarriers to target the blood-brain barrier (BBB) for drug delivery to the central nervous system (CNS). Encapsulation of the acetylcholinesterase reactivator, pralidoxime chloride (2-PAM), in SLNs appears to be a suitable strategy for protection against poisoning by organophosphorus agents (OPs) and postexposure treatment. 2-PAM-loaded SLNs were developed for brain targeting and delivery via intravenous (iv) administration. 2-PAM-SLNs displayed a high 2-PAM encapsulation efficiency (∼90%) and loading capacity (maximum 30.8 ± 1%). Drug-loaded particles had a mean hydrodynamic diameter close to 100 nm and high negative zeta potential (-54 to -15 mV). These properties contribute to improve long-term stability of 2-PAM-SLNs when stored both at room temperature (22 °C) and at 4 °C, as well as to longer circulation time in the bloodstream compared to free 2-PAM. Paraoxon-poisoned rats (2 × LD50) were treated with 2-PAM-loaded SLNs at a dose of 2-PAM of 5 mg/kg. 2-PAM-SLNs reactivated 15% of brain AChE activity. Our results confirm the potential use of SLNs loaded with positively charged oximes as a medical countermeasure both for protection against OPs poisoning and for postexposure treatment.

Effect of tissue-specific acetylcholinesterase inhibitor C-547 on α3β4 and αβεδ acetylcholine receptors in COS cells

The C-547 is the most effective muscle and tissue-specific anticholinesterase among alkylammonium derivatives of 6-methyluracil (ADEMS) acting in nanomolar concentrations on locomotor muscles but not on respiratory muscles, smooth muscles and heart and brain acetylcholine esterases (AChE). When applied systematically it could influence peripheral acetylcholine receptors. The aim of the present study was to investigate the effect of C-547 on rat α3β4 (ganglionic type) and αβεδ (muscle type) nicotinic receptors expressed in COS cells. Currents evoked by rapid application of acetylcholine or nicotine were recorded in whole-cell mode by electrophysiological patch-clamp technique 2-4 days after cell transfection by plasmids coding the α3β4 or αβεδ combination of receptor subunits. In cells sensitive to acetylcholine, the application of C-547 evoked no responses. When acetylcholine was applied during an already running application of C-547, acetylcholine responses were only inhibited at concentrations higher than 10(-7)M. This inhibition is not voltage-dependent, but is accompanied by an increased rate of desensitization. Thus in both types of receptors, effective doses are approximately 100 times higher than those inhibiting AChE in leg muscles and similar to those inhibiting respiratory diaphragm muscles and external intercostal muscles. These observations show that C-547 can be considered for symptomatic treatment of myasthenia gravis and other congenital myasthenic syndromes as an inhibitor of AChE in leg muscles at concentrations much lower than those inhibiting muscle and ganglion types of acetylcholine receptors.