Mode of action of sulfoxaflor on α-bungarotoxin-insensitive nAChR1 and nAChR2 subtypes: Inhibitory effect of imidacloprid.

Cockroach neurosecretory cells, dorsal unpaired median (DUM) neurons, express two distinct α-bungarotoxin-insensitive nicotinic acetylcholine receptor subtypes, nAChR1 and nAChR2 which are differently sensitive to the neonicotinoid insecticides and intracellular calcium pathways. The aim of this study is to determine whether sulfoxaflor acts as an agonist of nAChR1 and nAChR2 subtypes. We demonstrated that 1 mM sulfoxaflor induced high current amplitudes, compared to acetylcholine, suggesting that it was a full agonist of DUM neuron nAChR subtypes. Sulfoxaflor evoked currents were not inhibited by the nicotinic acetylcholine receptor antagonist d-tubocurarine (dTC) which reduced nAChR1. But, sulfoxaflor evoked currents were reduced in the presence of 5 µM mecamylamine which is known to reduce nAChR2 subtype. Interestingly, when 1 µM imidacloprid was added in the extracellular solution, sulfoxaflor-induced currents were significantly suppressed. Moreover, when extracellular calcium concentration was increased, bath application of 1 µM imidacloprid partially reduced sulfoxaflor activated currents when nAChR1 was inhibited with 20 µM dTC and completely suppressed sulfoxaflor currents when nAChR2 was inhibited with 5 µM mecamylamine. Our data demonstrated therefore that sulfoxaflor activates both nAChR1 and nAChR2 subtypes.

Where fMRI and electrophysiology agree to disagree: corticothalamic and striatal activity patterns in the WAG/Rij rat.

The relationship between neuronal activity and hemodynamic changes plays a central role in functional neuroimaging. Under normal conditions and in neurological disorders such as epilepsy, it is commonly assumed that increased functional magnetic resonance imaging (fMRI) signals reflect increased neuronal activity and that fMRI decreases represent neuronal activity decreases. Recent work suggests that these assumptions usually hold true in the cerebral cortex. However, less is known about the basis of fMRI signals from subcortical structures such as the thalamus and basal ganglia. We used WAG/Rij rats (Wistar albino Glaxo rats of Rijswijk), an established animal model of human absence epilepsy, to perform fMRI studies with blood oxygen level-dependent and cerebral blood volume (CBV) contrasts at 9.4 tesla, as well as laser Doppler cerebral blood flow (CBF), local field potential (LFP), and multiunit activity (MUA) recordings. We found that, during spike-wave discharges, the somatosensory cortex and thalamus showed increased fMRI, CBV, CBF, LFP, and MUA signals. However, the caudate-putamen showed fMRI, CBV, and CBF decreases despite increases in LFP and MUA signals. Similarly, during normal whisker stimulation, the cortex and thalamus showed increases in CBF and MUA, whereas the caudate-putamen showed decreased CBF with increased MUA. These findings suggest that neuroimaging-related signals and electrophysiology tend to agree in the cortex and thalamus but disagree in the caudate-putamen. These opposite changes in vascular and electrical activity indicate that caution should be applied when interpreting fMRI signals in both health and disease from the caudate-putamen, as well as possibly from other subcortical structures.

Telithromycin blocks neuromuscular transmission and inhibits nAChR currents in vitro.

Telithromycin, a ketolide antibiotic, is reported to exacerbate myasthenia gravis, potentially leading to respiratory failure and death. However, telithromycin is not associated with neuromuscular effects in animal toxicity studies. The objective of this study was to examine the effect of telithromycin on the neuromuscular junction in the isolated rat phrenic nerve-diaphragm preparation and to investigate its postsynaptic effects on the muscle-like nicotinic acetylcholine (ACh) receptors expressed on human TE671 cells. Telithromycin decreased the twitch contraction force of the rat diaphragm muscle in response to phrenic nerve stimulation in a concentration-dependent manner with an IC(50) of 22.3 microM and a maximal inhibition of approximately 70%. The trans-membrane current from the ACh receptors expressed in the TE671 neuromedulloblastoma cells was recorded in the whole-cell patch-clamp configuration. When applied to the TE671 cells, telithromycin caused a dose-dependent inhibition of the nicotinic ACh current with an IC(50) of 3.5 microM and maximal inhibition of nearly 100%. These results indicate that telithromycin inhibits postsynaptic nicotinic ACh receptors in vitro and partially blocks neuromuscular transmission in the isolated rat phrenic nerve-diaphragm preparation. Based on these findings, we propose that exacerbation of myasthenia gravis reported in some patients taking telithromycin results in part from postsynaptic neuromuscular transmission block.

Lipid peroxidation and changes in cytochrome c oxidase and xanthine oxidase activity in organophosphorus anticholinesterase induced myopathy.

A possible role of radical oxygen species (ROS) initiated lipid peroxidation in diisopropylphosphorofluoridate (DFP)-induced muscle necrosis was investigated by quantifying muscle changes in F2-isoprostanes, novel and extremely accurate markers of lipid peroxidation in vivo. A significant increase in F2-isoprostanes of 56% was found in the diaphragm of rats 60 min after DFP-induced fasciculations. As possible source of ROS initiating lipid peroxidation, the cytocrome-c oxidase (Cyt-ox) and xanthine dehydrogenase-xanthine oxidase (XD-XO) systems were investigated. Within 30 min of onset of fasciculations Cyt-ox activity was reduced by 50% from 0.526 to 0.263 mumol/mg prot/min and XO activity increased from 0.242 to 0.541 mumol/mg prot/min. Total XD-XO activity was unchanged, indicating a conversion from XD into XO. In rats pretreatment with the neuromuscular blocking agent d-tubocurarine, prevented DFP-induced fasciculations, increases in F2-isoprostanes and changes in Cyt-ox or XD-XO. The decrease in Cyt-ox and increase in XO suggest that ROS are produced during DFP induced muscle fasciculations initiating lipid peroxidation and subsequent myopathy.

Comparative evaluation of mechanism of neuromuscular (n-m) blockade due to enhydrotoxin-A (EsNTx-a) and D-tubocurarine (d-TC).

Effects of major E. schistosa neurotoxin (NTx) EsNTx-a are compared with plant alkaloid d-TC for its onset (LP) and duration of action as the time required for 50% n-m blockade (DT1/2) in normal Ca2+ and modified Ca2+ Krebs Heinsleit (K-H) at two different (low and high) concentrations (conc.). Toxin-EsNTx-a in low Ca2+ K-H and d-TC in both normal and modified Ca2+ K-H did not show LP, whereas LP is increased significantly in 2 Ca2+ K-H in high conc. of EsNTx-a. DT1/2 is increased significantly in high conc. of EsNTx-a and in low conc. of d-TC in 2 Ca2+ K-H. N-m blocking actions are further compared with alpha-bungarotoxin (alpha-BuTx), beta-bungarotoxin (beta-BuTx), and botulinum toxin (B-Tx) for their effects on tetanus, Wednesky inhibition (W.I.), and post tetanic potentiation (PTP).

Prolonged d-tubocurarine infusion and/or immobilization cause upregulation of acetylcholine receptors and hyperkalemia to succinylcholine in rats.

BACKGROUND: Hyperkalemic cardiac arrest after the administration of succinylcholine (SCh) to critically ill intensive care patients has been attributed to changes in the acetylcholine receptors (AChRs) at the muscle membrane. The current study attempts to characterize the contributory roles of chronic administration of nondepolarizing muscle relaxants typified by d-tubocurarine (dTC) and/or of immobilization on AChR upregulation and the relationship of these AChR changes to SCh-induced hyperkalemia. METHODS: Rats received chronic subparalytic infusion of saline or dTC for 28 days via subcutaneous osmotic pumps inserted while they were under anesthesia. Approximately half of the saline- or dTC-treated rats underwent bilateral hind-limb immobilization with plaster casts for the same duration as the infusion. After 4 weeks, the osmotic pumps were removed, and 24-48 h later, the blood potassium concentrations were measured at baseline and at 1, 3, 5, 7, and 10 min after SCh (3 mg/kg). At the end of this period, the gastrocnemius muscle was excised for quantitation of AChR number using (125)I-alpha-bungarotoxin. RESULTS: At 28 days, the weight gain in mobile animals receiving saline or dTC infusion did not differ, nor did that in immobilized animals receiving saline or dTC infusion, confirming that infusion of dTC did not unduly affect the ability of the animals to feed. The maximal potassium change after SCh occurred at 5 min. Potassium responses to SCh changed (mean +/- SE): (1) from 3.9 +/- 0.04 to 4.5 +/- 0.1 mEq/1 in the mobile saline- treated control group, where the AChR concentration was 18.4 +/- 2 fmol/mg protein; (2) from 3.9 +/- 0.03 to 5.1 +/- 0.1 in the mobile dTC-infused group (AChRs = 48.6 +/- 7); (3) from 3.8 +/- 0.1 to 5.5 +/- 0.3 in the immobilized saline- treated group (AChRs = 107.4 +/- 14); and (4) from 3.8 +/- 0.1 to 6.3 +/- 0.2 in the immobilized-dTC-treated group (AChRs = 183.5 +/- 23). There was a significant positive correlation between maximal change in blood potassium concentration and the respective AChR concentration in the gastrocnemius of the same animal (r = 0.81, P<0.01). CONCLUSIONS: Subtherapeutic (subparalytic) doses of chronic infusion of dTC (with no immobilization) or immobilization alone (with no dTC) independently increased number of AChRs. The infusion of dTC with immobilization caused the greatest upregulation of AChRs. The magnitude of the increase in blood potassium to SCh was directly dependent on AChR number. This study shows direct evidence and confirms previous speculation that AChR number plays an important role in the magnitude of the hyperkalemic response to SCh. Presuming this represents an appropriate model for patients who are immobilized and/or receiving nondepolarizing muscle relaxants for prolonged periods, exaggerated blood potassium responses to SCh are possible when either or both of these perturbations are present in patients.

Interaction of lipopolysaccharide endotoxin produced from Escherichia coli with D-tubocurarine at the nicotinic2 receptor and adenosine 3':5' cyclic monophosphate during physiological contraction in skeletal muscle.

In this report the murine model of endotoxicosis was used to evaluate hyposensitivity to the neuromuscular relaxant D-tubocurarine (dTC). This hyposensitivity was expressed in terms of a decreased potency to dTC. A rightward shift of the dose-response curve due to endotoxin was observed. Mice were subjected to cumulative intraperitoneal doses of Escherichia coli endotoxin over a 2-wk period. The interaction between endotoxin and dTC was examined during an acute (1 wk) and chronic (2 wk) period of endotoxicosis. Muscle twitch analyses were performed and samples of gastrocnemius muscle were assayed for adenosine 3':5' cyclic monophosphate (cAMP) by [125I]radioimmunoassay. A parallel shift in the dose-response curve occurred in the endotoxin group subjected to doses corresponding to one-third the dose evoking 50% lethality for 2 wk. Both skeletal muscle tension and cAMP levels decreased as cumulative endotoxin doses increased. A relationship between decreasing cAMP levels and increasing dTC and effective dose required to achieve 50% muscle paralysis values was thought to be evoked by the agonistic activity of E. coli endotoxin leading to desensitizing of adenylate cyclase. The perturbations of the classical second messenger cAMP system by endotoxin may be responsible for skeletal muscle dysfunction observed in immunocompromised patients.

Activation of brain acetylcholine receptors by neuromuscular blocking drugs. A possible mechanism of neurotoxicity.

BACKGROUND: Neuromuscular blocking drugs cause excitement and seizures when introduced into the central nervous system. We examined the possibility that these drugs produce paradoxical activation of acetylcholine or glutamate receptors, the chief types of brain receptors involved in excitatory neurotransmission. METHODS: Because activation of central glutamate or acetylcholine receptors causes calcium influx into postsynaptic neurons, we measured intracellular calcium concentration ([Ca2+]i) as an index of receptor activation. Changes in [Ca2+]i were compared in brain slices exposed to neuromuscular blocking drugs or acetylcholine and glutamate receptor agonists. [Ca2+]i was measured with the fluorescent dye fura-2. RESULTS: Pancuronium and vecuronium caused sustained increases in [Ca2+]i in approximately the same potency ratio as for seizure activity in vivo (concentrations at which the increase in [Ca2+]i was 95% of maximal: 100 and 400 microM, respectively). Atracurium and laudanosine did not increase [Ca2+]i in cortical slices. Increases in [Ca2+]i caused by both pancuronium and vecuronium were prevented by the non-subtype-specific nicotinic acetylcholine receptor antagonist D-tubocurarine and were reduced 44-73% by atropine. Blockade of glutamate receptors or voltage-gated calcium or sodium channels had no effect on calcium influx. CONCLUSIONS: The results suggest that the acute excitement and seizures caused by introduction of pancuronium and vecuronium into the central nervous system is due to accumulation of cytosolic calcium caused by sustained activation of acetylcholine receptor ion channels.

Regulation and activity-dependence of N-cadherin, NCAM isoforms, and polysialic acid on chick myotubes during development.

Muscle development in vivo involves a complex sequence of cell-cell interactions in which secondary myotubes first form in association with primary myotubes and subsequently separate from them. We show here that during this process N-cadherin and the different structural forms of NCAM are regulated in a pattern that involves both temporal changes in expression and localization to particular regions of the muscle cell surface. In particular, levels of N-cadherin on maturing myotubes are decreased, and the form of NCAM synthesized by the muscle changes from a transmembrane non-polysialylated to a lipid-linked polysialylated membrane protein. Moreover, while NCAM was distributed on all myotube surfaces, the polysialyated form of NCAM was restricted to regions of the myotube surface that had recently separated from neighboring cells. We previously found that blockade of nerve-induced activity by d-Tubocurarine perturbed muscle cell interactions, resulting in a failure of myotubes to separate. We now show that this activity blockade also alters adhesion molecule expression. First, N-cadherin was no longer down-regulated in maturing myotubes, and its persistence on the surfaces of mature myotubes may partly explain their failure to separate. Secondly, the developmental switch from transmembrane to lipid-linked NCAM did not occur, and polysialylated NCAM was no longer formed. As the unusual physical properties of PSA have been proposed to impede cell-cell interactions, this alteration would also be expected to compromise cell separation. Together, these results suggest that the regulated expression of both N-cadherin and NCAM isoforms including their polysialylation, is an essential mechanism for the normal separation of secondary myotubes from primary myotubes.

Developmental toxicity of nondepolarizing muscle relaxants in cultured rat embryos.

Evidence of developmental toxicity of clinically used nondepolarizing muscle relaxants was sought in rat embryos grown in culture. Embryos were explanted at 8 AM on day 9 of gestation (presomite stage, plug day = day 0), and were cultured in rotating bottles with medium containing various concentrations of d-tubocurarine, pancuronium, atracurium, and vecuronium. At 10 AM on day 11 of gestation (forelimb bud stage), culture was terminated and embryos were examined for general morphology. Treatment with tested agents resulted in dose-dependent developmental toxicity; namely, growth retardation seen as decreased crown-rump length, decreased number of somite pairs, and morphologic abnormalities. However, the concentrations that caused toxicity were at least 30-fold greater than serum concentrations clinically achieved in the mother. We conclude that these muscle relaxants have a low potential for causing developmental toxicity during organogenesis.

Potentiation of atracurium toxicity in isolated rat hepatocytes by inhibition of its hydrolytic degradation pathway.

This study tested the hypothesis that the esters of acrylic acid might be responsible for the previously observed cytotoxic effect of atracurium. Rats were pretreated with triorthotolyl phosphate (TOTP), an inhibitor of the hydrolytic degradation of atracurium. Because hydrolysis of acrylates is also inhibited by TOTP and because the hydrolysis represents a detoxification pathway for these esters, we postulated that the leak of lactic dehydrogenase (LDH) induced by atracurium would be enhanced in hepatocytes harvested from rats pretreated with TOTP. Hepatocytes isolated from rats previously treated with TOTP (25 or 50 mg/kg intraperitoneally, 20 hr before induced death) were incubated for 4 hr in the absence of muscle relaxants or in the presence of either atracurium (0.008-0.8 mM) or metocurine (0.015-0.85 mM). Atracurium produced a concentration-dependent leakage of LDH. The leakage out of cells obtained from TOTP-pretreated rats was greater than was the leakage out of hepatocytes harvested from animals pretreated only with corn oil (a vehicle for TOTP). Metocurine did not produce a leak of LDH. It is concluded that the LDH leakage was produced by ester-type products of atracurium degradation. Acrylates appear to be the toxic agent.

Comparative toxicity of atracurium and metocurine in isolated rat hepatocytes.

Primary cultures of liver cells isolated from seven rats were used to study the possible toxicity of atracurium and metocurine. The muscle relaxants were separately added to the culture medium and the cells then incubated for 4 hr. The amount of lactic dehydrogenase (LDH) that leaked into the culture medium was determined at the end of incubation. The customary assumption was made that the exudation of LDH reflects the toxic effects of the relaxants. In untreated dishes, approximately 11% of the total intracellular LDH leaked out during the incubation. The net leakage of LDH produced by the relaxants was obtained by subtracting this amount from the LDH activity determined in the media of dishes with the relaxants added. On this basis, metocurine, in concentrations of 12-850 X 10(-6)M, did not cause a net leak of LDH. On the other hand, atracurium, in similar molar concentrations, caused a statistically significant and concentration-dependent leak of LDH that, at its maximum, amounted to more than one half of the intracellular LDH. The results are interpreted in terms of damage to cellular membranes produced by atracurium or its metabolites. Although the exact biochemical process was not identified, we hypothesize that acrylates--produced by Hofmann elimination from atracurium--might be the likely toxic species.

A comparison of the antagonisms by neostigmine and diaminopyridine against the neuromuscular block caused by cobrotoxin and (+)-tubocurarine.

Cobrotoxin was about 11-fold more potent than (+)-tubocurarine on a weight basis in blocking neuromuscular transmission in mouse isolated phrenic nerve-diaphragm preparations. Neostigmine and diaminopyridine increased the concentrations of cobrotoxin for 70% inhibition of indirect contraction by 290 and 320%, and increased those of (+)-tubocurarine by 180 and 230%, respectively. More than additive increases were obtained when neostigmine and diaminopyridine were used simultaneously. Cobrotoxin, however, was only 6-fold more toxic than (+)-tubocurarine after intraperitoneal injection in mice. The lethal dose of (+)-tubocurarine was increased by 80% when both antidotes were used together, but only by 15-20% when used alone. In contrast, the lethality of cobrotoxin was not decreased by these drugs. Unexpectedly, the time to death after treatment with cobrotoxin was shortened when mice were pretreated with these antidotes.

Potentiation between neostigmine and 3,4-diaminopyridine in antagonizing the neuromuscular block induced by D-tubocurarine.

The antagonisms to the d-tubocurarine-induced neuromuscular blockade by neostigmine and 3,4-diaminopyridine (DAP) were studied quantitatively in the isolated phrenic nerve-diaphragm preparation of mice in vitro and in lethality of mice in vivo by assaying concentrations of d-tubocurarine needed to produce 70% block of indirect muscle contraction and LD50, respectively. The "antagonist efficacies", defined as the ratio of d-tubocurarine concentration (dose) after pretreatment with antagonistic agents over that of control, were 1.87, 2.24 and 14.7, respectively, for neostigmine, DAP and both agents combined when the stimulus pulses were at 0.1 Hz. Under 50 Hz train stimulation, the antagonist efficacies were lower, being 1.85, 1.64 and 5.33, respectively. For the lethality to d-tubocurarine, the values were still lower, being only 1.21, 1.33 and 1.84, respectively. The synergism between neostigmine and DAP, as evident from the marked increase of antagonist efficacy in vitro, is more than expected from the possible interaction of the major pharmacological actions of these two agents.

Cardiac vagolytic action of some neuromuscular blockers.

Cardiac vagolytic effect of four commonly used neuromuscular blockers, (viz. D-tubocurarine, decamethonium, pancuronium and gallamine) was compared in midcollicular decerebrate rats. The intravenous doses of neuromuscular blockers used (d-tubocurarine: 0.1 mg/kg; decamethonium: 2 mg/kg; pancuronium: 0.1 mg/kg; gallamine: 20 mg/kg) were sufficient to produce the paralysis of respiratory muscles. Bradycardia was induced by electrical stimulation of the vagus or by injecting dimethyl-phenyl-piperazinium (DMPP; a ganglionic stimulant). It was observed that d-tubocurarine and decamethonium were devoid of cardiac vagolytic action. On the other hand, pancuronium and gallamine inhibited significantly the bradycardia induced by electrical stimulation of the vagus or injection of DMPP; gallamine was found to have greater vagolytic action. The pressor responses to DMPP were not attenuated by pancuronium and gallamine indicating that in the dose administered, these agents did not block the ganglia. Bradycardia induced by the administration of acetylcholine in the left atrium was also attenuated by pancuronium and gallamine suggesting that the drugs produce cardiac vagolytic action by acting on the post-synaptic cholinergic receptors of the heart.

[Muscle-relaxant effects of intravenously administered micronomicin].

Pharmacological effects, mainly on its muscle-relaxant of micronomicin (MCR) were studied. Intravenous infusion of MCR at a dose of 20 mg/kg/hour did not influence on both respiration rate and cardiovascular functions in anesthetized rabbits. When MCR was injected to anesthetized rabbit at an intravenous dose of 100 mg/kg, a respiratory arrest followed by a cardiac arrest was observed. This effect was antagonized by either treatment with CaCl2 or artificial respiration. In slant test, MCR at a dose of 100 mg/kg (i.v.) in mice induced muscle relaxation. This effect was weakened by slowing the injection speed of the drug. Intravenous injection of MCR potentiated the lethality induced by either d-tubocurarine or succinylcholine. Intravenous injection of MCR influenced on neither pentobarbital sodium-, ethyl ether-, nor halothane-induced anesthesia in mice. From these results, intravenous infusion of MCR at a dose of 20 mg/kg/hour that is considered as 5 approximately 10 times of clinical dose (120 approximately 240 mg/day) did not influence on both respiration and cardiovascular functions.

[Comparative study of the effect of verapamil and tubocurarine on acetylcholinesterase of skeletal muscle sarcolemma]. / Sravnitel'noe issledovanie deistviia verapamila i tubokurarina na atsetilkholinésterazy sarkolemmy skeletnykh myshts.

There are many similarities between verapamil and tubocurarine action on acetylcholinesterase of skeletal muscle sarcolemma: verapamil is equipotent with tubocurarine in its inhibitory effect; the kinetic analysis demonstrated the same (mixed) type of inhibition, the Hill coefficient is 0.7 and 0.6 for verapamil and tubocurarine suggested possible binding of verapamil and tubocurarine to the same sites.

Effects of corticosteroids on neuromuscular blocking actions of d-tubocurarine.

Dexamethasone (50 microgram/kg) significantly increased the LD50 of d-tubocurarine (d-TC) when administered i.p. simultaneously with d-TC. Choline (50 and 100 mg/kg) gave some protection against the lethal effects of d-TC and the cholinesterase inhibitors neostigmine (250 microgram/kg) and physostigmine (1000 microgram/kg) provided full protection against doses of d-TC twice the LD50. The blocking effect of d-TC (75 microgram/kg) on the sciatic nerve-tibialis anterior muscle preparation was antagonized by dexamethasone. Prednisolone delayed the occurrence of a complete neuromuscular block caused by d-TC in the phrenic nerve-diaphragm preparation, and antagonized the effect of d-TC on short tetanic contractions. d-TC (5 mumol/l) inhibited the [14C]choline uptake in the endplate-rich region of the rat diaphragm during stimulation. This inhibition was antagonized by dexamethasone as well as by physostigmine. The incorporation of radioactive choline into acetylcholine was inhibited in the presence of d-TC (15 mumol/l), and both dexamethasone and physostigmine counteracted this inhibition. It is concluded from these experiments that d-TC very probably has an effect on the choline carrier system. These experimental results support the hypothesis that glucocorticoids may improve reduced muscle performance by direct presynaptic effects at the neuromuscular junction.