Antagonistic postsynaptic and presynaptic actions of cyclohexanol on neuromuscular synaptic transmission and function

Abstract

Intentional ingestion of agricultural organophosphorus insecticides is a significant public health issue in rural Asia, causing thousands of deaths annually. Some survivors develop a severe, acute or delayed myasthenic syndrome. In animal models, similar myasthenia has been associated with increasing plasma concentration of one insecticide solvent metabolite, cyclohexanol. We investigated possible mechanisms using voltage and current recordings from mouse neuromuscular junctions (NMJs) and transfected human cell lines. Cyclohexanol (10–25 mM) reduced endplate potential (EPP) amplitudes by 10–40% and enhanced depression during repetitive (2–20 Hz) stimulation by up to 60%. EPP decay was prolonged more than twofold. Miniature EPPs were attenuated by more than 50%. Cyclohexanol inhibited whole‐cell currents recorded from CN21 cells expressing human postjunctional acetylcholine receptors (hnAChR) with an IC50 of 3.74 mM. Cyclohexanol (10–20 mM) also caused prolonged episodes of reduced‐current, multi‐channel bursting in outside‐out patch recordings from hnAChRs expressed in transfected HEK293T cells, reducing charge transfer by more than 50%. Molecular modelling indicated cyclohexanol binding (−6 kcal/mol) to a previously identified alcohol binding site on nicotinic AChR α‐subunits. Cyclohexanol also increased quantal content of evoked transmitter release by ∼50%. In perineurial recordings, cyclohexanol selectively inhibited presynaptic K+ currents. Modelling indicated cyclohexanol binding (−3.8 kcal/mol) to voltage‐sensitive K+ channels at the same site as tetraethylammonium (TEA). TEA (10 mM) blocked K+ channels more effectively than cyclohexanol but EPPs were more prolonged in 20 mM cyclohexanol. The results explain the pattern of neuromuscular dysfunction following ingestion of organophosphorus insecticides containing cyclohexanol precursors and suggest that cyclohexanol may facilitate investigation of mechanisms regulating synaptic strength at NMJs. Key points Intentional ingestion of agricultural organophosphorus insecticides is a significant public health issue in rural Asia, causing thousands of deaths annually. Survivors may develop a severe myasthenic syndrome or paralysis, associated with increased plasma levels of cyclohexanol, an insecticide solvent metabolite. Analysis of synaptic transmission at neuromuscular junctions in isolated mouse skeletal muscle, using isometric tension recording and microelectrode recording of endplate voltages and currents, showed that cyclohexanol reduced postsynaptic sensitivity to acetylcholine neurotransmitter (reduced quantal size) while simultaneously enhancing evoked transmitter release (increased quantal content). Patch recording from transfected cell lines, together with molecular modelling, indicated that cyclohexanol causes selective, allosteric antagonism of postsynaptic nicotinic acetylcholine receptors and block of presynaptic K+‐channel function. The data provide insight into the cellular and molecular mechanisms of neuromuscular weakness following int...

“Calcium bombs” as harbingers of synaptic pathology and their mitigation by magnesium at murine neuromuscular junctions

“…Thus, the findings reported in the present paper are based on rather few, sometimes individual, observations. Nevertheless, our optical recordings were consistent with more robust, but indirect, muscle force measurements ( Dissanayake et al, 2021a , b ).…”

“…Aftercontractions were not exclusive to muscles treated with omethoate. Similar responses were observed after muscles were exposed to other classes of anti-AChE, including the carbamate AChE antagonist neostigmine ( Hong and Chang, 1993 ; Dissanayake et al, 2021b ) and the piperidine anti-AChE donepezil (Redman et al, submitted).…”

“…Imaging muscles in unstained preparations during and after stimulation showed that both the tetanic responses and the aftercontractions induced by omethoate most likely arose from direct effects on motor endplates. In a subsequent study ( Dissanayake et al, 2021b ) we found that omethoate caused prolonged, summating endplate currents (EPCs) and endplate potentials (EPPs).…”

“…However, other potential mechanisms could explain the complex profiles of post-tetanic decay of endplate Ca 2+ . Inhibiting AChE results in prolonged EPCs and summation of EPCs during repetitive stimulation, causing prolonged endplate depolarization ( Dissanayake et al, 2021b ). Moreover, increasing extracellular [Mg 2+ ] reduces the conductance of the adult (ε-subunit) form of AChR at NMJs ( Grassi and Degasperi, 2000 ; Piccari et al, 2011 ; Deflorio et al, 2012 ).…”

“…For instance, inhibitors of acetylcholinesterase (anti-AChEs), which prolong cholinergic synaptic depolarization, have been implicated in the increased incidence of ALS in military personnel and veterans diagnosed with “Gulf War Syndrome” ( Haley, 2003 ; Kasarskis et al, 2009 ). NMJ dysfunction, paralysis and degeneration also occur following acute exposure to agricultural insecticides containing organophosphorus (OP) anti-AChE compounds dissolved in organic solvents, with possible links to ALS ( Eddleston et al, 2012 ; Pamphlett, 2012 ; Merwin et al, 2017 ; Dissanayake et al, 2021a , b ). Acute exposure of rodent muscles to OP anti-AChEs has a number of pathophysiological effects and consequences, including hypercontraction of muscle fibers in the region of their NMJs, and this has been linked to subsequent development of a focal Ca 2+ -dependent myopathy, and associated in some studies with degeneration of motor nerve terminals ( Leonard and Salpeter, 1979 ; Duxson and Vrbová, 1985 ; Meshul et al, 1985 ; Ferry and Cullen, 1991 ; Zhu et al, 2014 ).…”