Novel Insights on MRGPRX2-Mediated Hypersensitivity to Neuromuscular Blocking Agents And Fluoroquinolones.

Neuromuscular blocking agents (NMBAs) like atracurium and rocuronium as well as fluoroquinolones (FQs) cause mast cell-mediated anaphylaxis by activating Mas-related G protein-coupled receptor X2 (MRGPRX2), but many questions remain unanswered. Here, we address three of them, namely whether primary human mast cells show similar activation by these drugs as murine mast cells and mast cell lines, how sugammadex protects from atracurium-induced MRGPRX2-mediated mast cell activation, and why some but not all patients treated with rocuronium develop anaphylaxis. We used peripheral blood-derived cultured mast cells from healthy donors and patients, assessed mast cell activation and degranulation by quantifying intracellular calcium and CD63 expression, respectively, and made use of MRGPRX2-silencing, via electroporation with Dicer-substrate small interfering RNAs, and single cell flow cytometric analyses. Atracurium, ciprofloxacin, and levofloxacin activated and degranulated primary human mast cells, but only MRGPRX2-positive and not MRGPRX2-negative or -silenced mast cells. Sugammadex attenuated the atracurium-induced and MRGPRX2-mediated activation and degranulation of human mast cells by reducing free atracurium levels. The mast cells of patients with IgE-independent anaphylaxis to rocuronium were similar, in their MRGPRX2 expression and function, to those of patients with IgE-mediated anaphylaxis. These findings further improve our understanding of the role and relevance of MRGPRX2-driven mast cell activation in anaphylactic reactions to NMBAs and FQs and may help to improve their prediction, prevention, and treatment.

High gender -specific susceptibility to curare- a neuromuscular blocking agent.

Curare, a selective skeletal muscle relaxant, has been used clinically to reduce shivering and as an anesthetic auxiliary in abdominal surgery. It is also widely used in animal experiments to block neuromuscular junction activity. Effective doses of curare diminish muscle contraction without affecting brain function, but at higher doses it is known to be lethal. However, the exact dose of curare initiating muscle relaxation vs. lethal effect has not been fully characterized in mice. In this study we carefully examined the dose-response for achieving muscle inactivity over lethality in both male and female mice (C57BL6/J). The most striking finding of this study is that female mice were highly susceptible to curare; both the ED50 and LD50 were at least 3-fold lower than male littermates. This study shows that gender-specific differences can be an important factor when administering skeletal muscle relaxants, particularly curare or other analogous agents targeted to the neuromuscular junction.

High gender -specific susceptibility to curare- a neuromuscular blocking agent

Curare, a selective skeletal muscle relaxant, has been used clinically to reduce shivering and as an anesthetic auxiliary in abdominal surgery. It is also widely used in animal experiments to block neuromuscular junction activity. Effective doses of curare diminish muscle contraction without affecting brain function, but at higher doses it is known to be lethal. However, the exact dose of curare initiating muscle relaxation vs. lethal effect has not been fully characterized in mice. In this study we carefully examined the dose-response for achieving muscle inactivity over lethality in both male and female mice (C57BL6/J). The most striking finding of this study is that female mice were highly susceptible to curare; both the EDm and LDm were at least 3-fold lower than male littermates. This study shows that gender-specific differences can be an important factor when administering skeletal muscle relaxants, particularly curare or other analogous agents targeted to the neuromuscular junction.

An investigation of non-depolarizing muscle relaxants on embryonic development in cultured rat embryos.

BACKGROUND AND OBJECTIVE: We have investigated the toxic and teratogenic effects of certain non-depolarizing muscle relaxants on embryonic development in cultured rat embryos. METHODS: Rat embryos of 9.5 days were explanted and cultured in vitro for 48 h in rat serum. Whole rat serum was used as a culture medium for the control group while different concentrations of atracurium, cis-atracurium, rocuronium and mivacurium were added to rat serum for the experimental groups. Dose-dependent effects of these agents on embryonic developmental parameters were compared using morphological and biochemical methods. Each embryo was evaluated for the presence of any malformations. RESULTS: When compared to the control embryos, the muscle relaxants significantly decreased all growth and developmental parameters dose dependently with an increase in overall dismorphology. Among these malformations, maxillary deformity was most frequently observed. These effects were observed in much lower doses with atracurium and cis-atracurium compared to those with rocuronium and mivacurium. CONCLUSIONS: Our results suggest that non-depolarizing muscle relaxants cause dose-dependent toxicity on rat embryos at concentrations much greater than those in clinical practice. Although, these agents seems to have a low potential for causing developmental toxicity during organogenesis, because of the lower toxic effects observed with rocuronium and mivacurium, these agents may be preferred when recurrent administrations are necessary for parturients.

Diverse range of fixed positional deformities and bone growth restraint provoked by flaccid paralysis in embryonic chicks.

Pancuronium bromide (PB) is used in neonates and pregnant women to induce limp, flaccid paralysis in order to allow mechanical ventilation during intensive care. Such non-depolarizing neuromuscular blocking drugs are administered to 0.1% of all human births in the UK. In this study, we examined PB effects on skeletal development in chick embryos. PB treatment produced skeletal deformities associated with significant reduction in longitudinal growth of all appendicular elements. This was associated with greater cartilage to bone ratios, indicating a preferential reduction in osteogenesis. PB also increased the incidence of knee joint flexion and tibiotarsal joint hyperextension. In addition to limb, spinal and craniofacial deformities, flaccid immobility appears to convert the normal geometric pattern of weight gain to a simple arithmetic accretion. This novel study highlights the potentially harmful effects of pharmacologically induced flaccid immobility on chick embryonic skeletal development. Whilst in ovo avian development clearly differs from human, our findings may have implications for the fetus, premature and term neonate receiving such non-depolarizing neuromuscular blocking drugs.

A mechanism for rapacuronium-induced bronchospasm: M2 muscarinic receptor antagonism.

BACKGROUND: A safe and effective ultra-short-acting nondepolarizing neuromuscular blocking agent is required to block nicotinic receptors to facilitate intubation. Rapacuronium, which sought to fulfill these criteria, was withdrawn from clinical use due to a high incidence of bronchospasm resulting in death. Understanding the mechanism by which rapacuronium induces fatal bronchospasm is imperative so that newly synthesized neuromuscular blocking agents that share this mechanism will not be introduced clinically. Selective inhibition of M2 muscarinic receptors by muscle relaxants during periods of parasympathetic nerve stimulation (e.g., intubation) can result in the massive release of acetylcholine to act on unopposed M3 muscarinic receptors in airway smooth muscle, thereby facilitating bronchoconstriction. METHODS: Competitive radioligand binding determined the binding affinities of rapacuronium, vecuronium, cisatracurium, methoctramine (selective M2 antagonist), and 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP; selective M3 antagonist) for M2 and M3 muscarinic receptors. RESULTS: Rapacuronium competitively displaced 3H-QNB from the M2 muscarinic receptors but not from the M3 muscarinic receptors within clinically relevant concentrations. Fifty percent inhibitory concentrations (mean +/- SE) for rapacuronium were as follows: M2 muscarinic receptor, 5.10 +/- 1.5 microm (n = 6); M3 muscarinic receptor, 77.9 +/- 11 microm (n = 8). Cisatracurium and vecuronium competitively displaced 3H-QNB from both M2 and M3 muscarinic receptors but had affinities at greater than clinically achieved concentrations for these relaxants. CONCLUSIONS: Rapacuronium in clinically significant doses has a higher affinity for M2 muscarinic receptors as compared with M3 muscarinic receptors. A potential mechanism by which rapacuronium may potentiate bronchoconstriction is by blockade of M2 muscarinic receptors on prejunctional parasympathetic nerves, leading to increased release of acetylcholine and thereby resulting in M3 muscarinic receptor-mediated airway smooth muscle constriction.

Weal and flare responses to intradermal rocuronium and cisatracurium in humans.

Thirty volunteers underwent intradermal skin testing with increasing concentrations of rocuronium and cisatracurium to evaluate weal and flare responses, and whether either agent would cause mast cell degranulation and sensitization upon re-exposure. We found that intradermal injection of rocuronium and cisatracurium at concentrations > 10(-4) M resulted in positive weal (>8 mm) responses, and positive flare responses at > 10(-4) and > 10(-5) M respectively. Only cisatracurium caused mild to moderate mast cell degranulation, and neither drug caused significant in vitro histamine release from whole blood collected from study subjects 4 weeks after skin testing. Skin testing with rocuronium and cisatracurium should be performed at concentrations < 10(-4) and < 10(-5) M respectively to avoid false-positive responses. The ability of these agents to produce positive weal and flare responses at relatively low concentrations may explain the high incidence of potential reactions reported.

[Synthesis and pharmacologic study of 1,5-dialkyl-1,5-benzodiazepine-2,4-dithiones]. / Synthèse et étude pharmacologique des 1,5-dialkyl-1,5-benzodiazépine-2,4-dithiones.

The synthesis and psychotropic activity of 1,5-diakyl-1,5-benzodiazepine-2,4-dithiones (alkyl = methyl, ethyl and benzyl radicals) were studied. Alkylation reactions were performed in catalytic conditions by phase transfer. These reactions allowed us to isolate only one kind of product N-alkyl. Acute toxicity studies were conducted according to European protocols in two species of appropriate mammals in order to discover the lethal doses. The activity of the compounds on the CNS was then studied, using a battery of compartmental tests used in psychopharmacology. No toxicity was demonstrated at therapeutic doses. Each product had a sedative effect more or less pronounced and different from the reference substance clobazam (Urbanyl). They also had myorelaxant and anxiolytic effects, even lengthening the hypnotic effect of thiopental (synergic action).

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.

Tone burst-evoked otoacoustic emissions in cats with acoustic overstimulation and anoxia.

Transient evoked otoacoustic emissions (TEOAE) produced by a 2 kHz tone burst could be detected in 30 out of 37 ears (81% detectability) in 21 cats. The amplitude of tone burst-evoked TEOAE was saturated at a stimulus level between 45 and 50 dB SPL and the latency time of peak amplitude was 6.23 ms on average (5.53-7.28 ms). The effects of pure tone overstimulation and short-term anoxia on the tone burst-evoked TEOAE in cats were evaluated. A permanent detection threshold shift of the TEOAE was confirmed at 24 h and 1 week after the overstimulation at 125 dB SPL. In these cases, damaged first row outer hair cells and inner hair cells were observed over an average length of 3.3 mm (16% of the entire cochlear length) by scanning electron microscopy. These findings suggested that the TEOAE can detect localized cochlear hair cell damage. A temporary detection threshold shift of the TEOAE was observed after the overstimulation at 105 dB SPL, and the threshold shift recovered in 107.5 min on average. In the short-term anoxia trial, the TEOAE amplitude started to decrease 45-90 s after the anoxia and recovered completely when the duration of anoxia was under 1 min. However, the TEOAE amplitude did not recover pre-anoxia values (it remained below 80% of its initial value) after 5 min when the anoxia was over 2 min. These findings demonstrated that the detection threshold and amplitude of the TEOAE were also affected by metabolic changes of the cochlear hair cells. Tone burst-evoked TEOAE are useful for the evaluation of localized histological and functional damage of the cochlear hair cells.

How do snake curaremimetic toxins discriminate between nicotinic acetylcholine receptor subtypes.

Curaremimetic toxins from snake venoms form a large family of small proteins that adopt a similar fold and which bind to Torpedo nicotinic acetylcholine receptors with high affinity. Notwithstanding its apparent homogeneity, the toxin family is subdivided into short-chain (60-62 residues and four disulfide bonds) and long-chain toxins (66-74 residues and five disulfide bonds). In agreement with this structurally-based distinction we recently showed that only long-chain toxins bind with high affinity to the neuronal nicotinic acetylcholine alpha7 receptor. We suggested that a small loop cyclized by a disulfide bond and uniquely present in long-chain toxins may act as a major discriminative element. To assess the validity of this proposal we prepared various derivatives of a long-chain toxin, using stepwise solid-phase synthesis. We found that replacement of both half cystines of the small loop by a serine caused a 35-fold affinity decrease for the neuronal receptor and only a 6-fold affinity decrease for Torpedo receptor. In addition, insertion of this loop at a homologous position of a short-chain toxin caused a 20-fold affinity increase for the neuronal receptor whereas it did not modify its affinity for the Torpedo receptor. Our findings, therefore, reveal that a small structural deviation from a toxin fold can generate exquisite discriminative recognition for some receptor subtypes.

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.

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.

Doxacurium and mivacurium do not trigger malignant hyperthermia in susceptible swine.

The role of succinylcholine in the precipitation of malignant hyperthermia (MH) necessitates the testing of new neuromuscular relaxants for their ability to trigger MH in MH-susceptible swine before general human use. We tested doxacurium and mivacurium, two new nondepolarizing bis-benzylisoquinolinium neuromuscular relaxants, at ED95 and at four times ED95 doses in swine previously documented to be MH-susceptible. In none of the 16 animals was MH triggered after administration of these relaxants, whereas all animals developed fatal MH after administration of halothane or halothane plus succinylcholine. Muscle biopsy specimens taken before administration of the relaxant confirmed that all animals had increased sensitivity to halothane, caffeine, or both. Thus, we conclude that doxacurium and mivacurium are not triggering agents of malignant hyperthermia in MH-susceptible swine.