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Coastal taipan ( Oxyuranus scutellatus ) envenoming causes life-threatening neuromuscular paralysis in humans. We studied the time period during which antivenom remains effective in preventing and arresting in vitro neuromuscular block caused by taipan venom and taipoxin. Venom showed predominant pre-synaptic neurotoxicity at 3 µg/mL and post-synaptic neurotoxicity at 10 µg/mL. Pre-synaptic neurotoxicity was prevented by addition of Australian polyvalent antivenom before the venom and taipoxin and, reversed when antivenom was added 5 min after venom and taipoxin. Antivenom only partially reversed the neurotoxicity when added 15 min after venom and had no significant effect when added 30 min after venom. In contrast, post-synaptic activity was fully reversed when antivenom was added 30 min after venom. The effect of antivenom on pre-synaptic neuromuscular block was reproduced by washing the bath at similar time intervals for 3 µg/mL, but not for 10 µg/mL. We found an approximate 10-15 min time window in which antivenom can prevent pre-synaptic neuromuscular block. This time window is likely to be longer in envenomed patients due to the delay in venom absorption. Similar effectiveness of antivenom and washing with 3 µg/mL venom suggests that antivenom most likely acts by neutralizing pre-synaptic toxins before they interfere with neurotransmission inside the motor nerve terminals.

What We Already Know About This Topic: WHAT THIS ARTICLE TELLS US THAT IS NEW: BACKGROUND:: Structure-activity studies were performed to identify a new neuromuscular blocking agent retaining the ultra-short acting characteristics of gantacurium, including degradation and reversal by L-cysteine, but lacking its histaminoid properties in man. CW 1759-50 has emerged from this program., Methods: Adduction of CW 1759-50 with L-cysteine was studied by high-performance liquid chromatography and mass spectrometry. Institutional Animal Care and Use Committee-approved comparisons of CW 1759-50 to gantacurium were performed in rhesus monkeys. ED95 for neuromuscular blockade was established. Spontaneous recovery was compared to reversal by L-cysteine in paired studies of boluses or infusions. In addition, changes in mean arterial pressure and heart rate after very large doses of 15 to 60 × ED95 were compared., Results: The half-time of adduction of L-cysteine to CW 1759-50 in vitro was 2.3 min. The ED95 of CW 1759-50 was 0.069 ± 0.02 mg/kg; ED95 of gantacurium was 0.081 ± 0.05 mg/kg (P = 0.006). Duration of action (recovery to 95% twitch height after 98 to 99% blockade) was as follows: CW 1759-50, 8.2 ± 1.5 min; and gantacurium, 7.4 ± 1.9 min; (n = 8 and 9, P = 0.355). Administration of L-cysteine (30 mg/kg) shortened recovery (i.e., induced reversal) from CW 1759-50 after boluses or infusions (P always less than 0.0001). Recovery intervals (5 to 95% twitch) ranged from 6.1 to 6.7 min (and did not differ significantly) after boluses of 0.10 to 0.50 mg/kg, as well as control infusions (P = 0.426 by analysis of variance). Dose ratios comparing changes of 30% in mean arterial pressure or heart rate to ED95 for neuromuscular blockade (ED 30% Δ [mean arterial pressure or heart rate]/ED95) were higher for CW 1759-50 than for gantacurium., Conclusions: CW 1759-50, similar to gantacurium, is an ultra-short acting neuromuscular blocking agent, antagonized by L-cysteine, in the monkey. The circulatory effects, however, are much reduced in comparison with gantacurium, suggesting a trial in humans.

While some US populations of the Mohave rattlesnake (Crotalus scutulatus scutulatus) are infamous for being potently neurotoxic, the Mexican subspecies C. s. salvini (Huamantlan rattlesnake) has been largely unstudied beyond crude lethality testing upon mice. In this study we show that at least some populations of this snake are as potently neurotoxic as its northern cousin. Testing of the Mexican antivenom Antivipmyn showed a complete lack of neutralisation for the neurotoxic effects of C. s. salvini venom, while the neurotoxic effects of the US subspecies C. s. scutulatus were time-delayed but ultimately not eliminated. These results document unrecognised potent neurological effects of a Mexican snake and highlight the medical importance of this subspecies, a finding augmented by the ineffectiveness of the Antivipmyn antivenom. These results also influence our understanding of the venom evolution of Crotalus scutulatus, suggesting that neurotoxicity is the ancestral feature of this species, with the US populations which lack neurotoxicity being derived states., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Anuran toxins released from the skin glands are involved in defence against predators and microorganisms. Secretion from parotoid macroglands of bufonid toads is a rich source of bioactive compounds with the cytotoxic, cardiotoxic and hemolytic activity. Bufadienolides are considered the most toxic components of the toad poison, whereas the protein properties are largely unknown. In the present work, we analysed the cardio-, myo-, and neurotropic activity of extract and the selected proteins from Bufo bufo parotoids in in vitro physiological bioassays carried out on two standard model organisms: beetles and frogs. Our results demonstrate a strong cardioactivity of B. bufo gland extract. The toad poison stimulates (by 16%) the contractility of the insect heart and displays the cardioinhibitory effect on the frog heartbeat frequency (a 27% decrease), coupled with an irreversible cardiac arrest. The gland extract also exhibits significant myotropic properties (a 10% decrease in the muscle contraction force), whereas its neuroactivity remains low (a 4% decrease in the nerve conduction velocity). Among identified peptides present in the B. bufo parotoid extract are serine proteases, muscle creatine kinase, phospholipid hydroperoxide glutathione peroxidase, cytotoxic T-lymphocyte protein, etc. Some proteins contribute to the cardioinhibitory effect. Certain compounds display the paralytic (myo- and neurotropic) properties. As the toad gland extract exhibits a strong cardiotoxic activity, we conclude that the poison is a potent agent capable of slaying a predator. Our results also provide the guides for the use of toad poison-peptides in therapeutics and new drug development., (Copyright © 2018 Elsevier Inc. All rights reserved.)

A significant number of commonly administered medications in anesthesia show wide clinical interpatient variability. Some of these include neuromuscular blockers, opioids, local anesthetics, and inhalation anesthetics. Individual genetic makeup may account for and predict cardiovascular outcomes after cardiac surgery. These interactions can manifest at any point in the perioperative period and may also only affect a specific system. A better understanding of pharmacogenomics will allow for more individually tailored anesthetics and may ultimately lead to better outcomes, decreased hospital stays, and improved patient satisfaction., (Copyright © 2017 Elsevier Inc. All rights reserved.)

The synthesis, biological evaluation and molecular modeling study of 6,7-dihydro-[1,3,4] thiadiazolo[3,2-a][1,3]diazepine analogues as new class of neuromuscular blocking agents are described. The new compounds act via competitive mechanism with ACh which could be reversed by the anticholinesterase - Physostigmine. Compounds GS-53 (30) and AAH1 (33) induced dose-dependent neuromuscular blockade with onset time of 3 and 10 min, ED 50 0.15 and 0.36 mmol/kg i.p., respectively, in rats. Compound 30 proved to be as twice as potent as 33 with rapid onset and shorter duration (P < 0.05). Docking profile of 30 and 33 closely resembles HIE-124 (3), in α7β2 nAChR receptor. Molecular modeling analysis indicated that hydrogen bonding to Thr120 and Thr124 beside hydrophobic interactions play effective role incorporating the active ligands to nAChR. The obtained model could be useful for further development of new skeletal muscle relaxants., (Copyright © 2016 Elsevier Masson SAS. All rights reserved.)

Neuromuscular blocking agents are widely used in perioperative medicine to aid in endotracheal intubation, facilitate surgery, and in critical care/emergency medicine settings. Muscle relaxants have profound clinical uses in current surgical and intensive care and emergency medical therapy. This article reviews cyclodextrins, development of selective binding agents, clinical development, and future directions of sugammadex., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Snakes of the opisthoglyphous genus Philodryas are widespread in South America and cause most bites by colubrids in this region. In this study, we examined the neurotoxic and myotoxic effects of venom from Philodryas patagoniensis in biventer cervicis and phrenic nerve-diaphragm preparations and we compared the biochemical activities of venoms from P. patagoniensis and Philodryas olfersii. Philodryas patagoniensis venom (40 microg/mL) had no effect on mouse phrenic nerve-diaphragm preparations but caused time-dependent neuromuscular blockade of chick biventer cervicis preparations. This blockade was not reversed by washing. The highest concentration of venom tested (40 microg/mL) significantly reduced (p<0.05) the contractures to exogenous acetylcholine (55 microM and 110 microM) and K(+) (13.4 mM) after 120 min; lower concentrations of venom had no consistent or significant effect on these responses. Venom caused a concentration- and time-dependent release of creatine kinase (CK) from biventer cervicis preparations. Histological analysis showed contracted muscle fibers at low venom concentrations and myonecrosis at high concentrations. Philodryas venoms had low esterase and phospholipase A(2) but high proteolytic activities compared to the pitviper Bothrops jararaca. SDS-PAGE showed that the Philodryas venoms had similar electrophoretic profiles, with most proteins having a molecular mass of 25-80 kDa. Both of the Philodryas venoms cross-reacted with bothropic antivenom in ELISA, indicating the presence of proteins immunologically related to Bothrops venoms. RP-HPLC of P. patagoniensis venom yielded four major peaks, each of which contained several proteins, as shown by SDS-PAGE. These results indicate that P. patagoniensis venom has neurotoxic and myotoxic components that may contribute to the effects of envenoming by this species.

Objectives: Therapeutic hypothermia has been shown to decrease neurologic damage in patients experiencing out-of-hospital cardiac arrest. In addition to being treated with hypothermia, critically ill patients are treated with an extensive pharmacotherapeutic regimen. The effects of hypothermia on drug disposition increase the probability for unanticipated toxicity, which could limit its putative benefit. This review examines the effects of therapeutic hypothermia on the disposition, metabolism, and response of drugs commonly used in the intensive care unit, with a focus on the cytochrome P450 enzyme system., Data Sources and Study Selection: A MEDLINE/PubMed search from 1965 to June 2006 was conducted using the search terms hypothermia, drug metabolism, P450, critical care, cardiac arrest, traumatic brain injury, and pharmacokinetics., Data Extraction and Synthesis: Twenty-one studies were included in this review. The effects of therapeutic hypothermia on drug disposition include both the effects during cooling and the effects after rewarming on drug metabolism and response. The studies cited in this review demonstrate that the addition of mild to moderate hypothermia decreases the systemic clearance of cytochrome P450 metabolized drugs between approximately 7% and 22% per degree Celsius below 37degreesC during cooling. The addition of hypothermia decreases the potency and efficacy of certain drugs., Conclusions: This review provides evidence that the therapeutic index of drugs is narrowed during hypothermia. The magnitude of these alterations indicates that intensivists must be aware of these alterations in order to maximize the therapeutic efficacy of this modality. In addition to increased clinical attention, future research efforts are essential to delineate precise dosing guidelines and mechanisms of the effect of hypothermia on drug disposition and response.

Neuroendocrine chromaffin cells were used to study the mechanism of the snake phospholipase A2 (PLA2) neurotoxin enhancement of exocytosis. Notexin, beta-bungarotoxin, taipoxin or textilotoxin enhanced the fast release of catecholamines elicited by flash photolysis of cytosolic caged calcium. Such an increase correlates with the capacity of these neurotoxins to cause fragmentation of the F-actin cortical barrier with subsequent accumulation of vesicles in the proximity of the plasma membrane. These PLA2 neurotoxins do not act via protein kinase C activation, which is known to promote F-actin fragmentation. Lithium, RO31-8220 and SB216763, three inhibitors of the glycogen synthase kinase 3, prevent both the alteration of the F-actin peripheral cortex and the enhancement of fast release elicited by these neurotoxins. In addition, glycogen synthase kinase 3 has been detected by immunolocalization in a membranous compartment of the chromaffin cell endoplasmic reticulum (ER). These results suggest that the activation of this enzyme plays a major role in the enhancement of exocytosis of the readily releasable granules caused by PLA2 neurotoxins in neuroendocrine chromaffin cells.

Background and Purpose: Nicotinic acetylcholine receptors (AChRs) are valuable therapeutic targets. To exploit them fully requires rapid assays for the evaluation of potentially therapeutic ligands and improved understanding of the interaction of such ligands with their receptor binding sites., Experimental Approach: A variety of neuromuscular blocking agents (NMBAs) were tested for their ability to inhibit the binding of [(125)I]alpha-bungarotoxin to TE671 cells expressing human muscle AChRs. Association and dissociation rate constants for vecuronium inhibition of functional agonist responses were then estimated by electrophysiological studies on mouse muscle AChRs expressed in Xenopus oocytes containing either wild type or mutant alpha1 subunits., Key Results: The TE671 inhibition binding assay allowed for the rapid detection of competitive nicotinic AChR ligands and the relative IC(50) results obtained for NMBAs agreed well with clinical data. Electrophysiological studies revealed that acetylcholine EC(50) values of muscle AChRs were not substantially altered by non-conservative mutagenesis of phenylalanine at alpha1:189 and proline at alpha1:194 to serine. However the alpha1:Phe189Ser mutation did result in a 3-4 fold increase in the rate of dissociation of vecuronium from mouse muscle AChRs., Conclusions and Implications: The TE671 binding assay is a useful tool for the evaluation of potential therapeutic agents. The alpha1:Phe189Ser substitution, but not alpha1:Pro194Ser, significantly increases the rate of dissociation of vecuronium from mouse muscle AChRs. In contrast, these non-conservative mutations had little effect on EC(50) values. This suggests that the AChR agonist binding site has a robust functional architecture, possibly as a result of evolutionary 'reinforcement'.

Predicting a patient's response to a particular drug has long been a goal of clinicians. Rapid advances in molecular biology have enabled researchers to identify associations between an individual's genetic profile and drug response. Pharmacogenetics is the study of the molecular mechanisms that underlie individual differences in drug metabolism, efficacy, and side effects. The pharmacogenetics of commonly used anesthetic and analgesic agents are reviewed.

Although numerous reversal agents for neuromuscular block (NMB) have been known for some time, investigations on new approaches were initiated only recently. The different approaches used in an attempt to avoid the muscarinic side effects associated with the antagonists of NMB that are currently available are reviewed.

Laudanosine is a metabolite of the neuromuscular-blocking drugs atracurium and cisatracurium with potentially toxic systemic effects. It crosses the blood-brain barrier and may cause excitement and seizure activity. Its interest in recent years has increased because of the recognized interaction with gamma-aminobutyric acid, opioid and nicotinic acetylcholine receptors. It has been shown to produce analgesia in animals. In the cardiovascular system, high plasma concentrations produce hypotension and bradycardia. In hepatic failure, its elimination half-life is prolonged but only moderate accumulation occurs in adults, whereas in infants and children plasma concentration are greater. In patients undergoing liver transplantation, laudanosine concentrations are increased during preanhepatic, anhepatic and postanhepatic stages. Patients with renal failure have higher plasma concentrations and a longer mean elimination half-life. In pregnancy, laudanosine crosses the placental barrier. The mean transplacental transfer is 14% of maternal blood concentrations. Except for prolonged administration of atracurium in intensive care units, laudanosine accumulation and related toxicity seem unlikely to be achieved in clinical practice. When cisatracurium is used, plasma concentrations of laudanosine are lower. Further studies are needed, especially around the interactions with gamma-aminobutyric acid, opioid and nicotinic acetylcholine receptors.

The historical development, pharmacology, pharmacodynamics, pharmacokinetics, clinical applications, pharmacologic basis for selection, adverse effects, and cost of neuromuscular blockers (NMBs) are discussed. The first NMB to be used was tubocurarine. During neurotransmission, acetylcholine is synthesized, stored in vesicles at the neuromuscular junction, released into the synapse, and bound to nicotinic receptors in the muscle end plate. For muscle contraction to occur, the impulse generated in a neuron's cell body must create an action potential that is chemically transmitted across the synapse. The postsynaptic nicotinic receptor at the neuromuscular junction is the major site of action of depolarizing and nondepolarizing NMBs. All NMBs have the potential for cross-reactivity at other nicotinic and muscarinic sites. Drug interactions most commonly occur between NMBs and inhalation anesthetics, certain antimicrobials, calcium-channel blockers, and anticholinesterases. When selecting an NMB, an agent's onset and duration of action must be considered. NMBs can be used on a short-term or long-term basis. Apart from cost, the choice of an NMB is made on the basis of its adverse-reaction profile, pharmacokinetics, and indications for use. Monitoring tools, their use, the rationale for their use, and the interpretation of the results they provide are unique. The patterns of peripheral nerve stimulation vary and elicit different characteristics of nondepolarizing neuromuscular blockade. The effectiveness of reversal agents is proportional to the degree of blockade. The mechanism of action of anticholinesterases involves inhibition of acetylcholinesterase. The expensive NMBs should be conserved for use in surgery, while the cheaper, long-acting [corrected] agents should be used in the intensive care unit. An understanding of the pharmacology, pharmacodynamics, and pharmacokinetics of NMBs will help health care providers gain expertise in the selection and use of these agents.

Botulinum toxin is a zinc-dependent endoprotease that acts on vulnerable cells to cleave polypeptides that are essential for exocytosis. To exert this poisoning effect, the toxin must proceed through a complex sequence of events that involves binding, productive internalization, and intracellular expression of catalytic activity. Results presented in this study show that soluble chelators rapidly strip Zn(2+) from its binding site in botulinum toxin, and this stripping of cation results in the loss of catalytic activity in cell-free or broken cell preparations. Stripped toxin is still active against intact neuromuscular junctions, presumably because internalized toxin binds cytosolic Zn(2+). In contrast to soluble chelators, immobilized chelators have no effect on bound Zn(2+), nor do they alter toxin activity. The latter finding is because of the fact that the spontaneous loss of Zn(2+) from its coordination site in botulinum toxin is relatively slow. When exogenous Zn(2+) is added to toxin that has been stripped by soluble chelators, the molecule rebinds cation and regains catalytic and neuromuscular blocking activity. Exogenous Zn(2+) can restore toxin activity either when the toxin is free in solution on the cell exterior or when it has been internalized and is in the cytosol. The fact that stripped toxin can reach the cytosol means that the loss of bound Zn(2+) does not produce conformational changes that block internalization. Similarly, the fact that stripped toxin in the cytosol can be reactivated by ambient Zn(2+) or exogenous Zn(2+) means that productive internalization does not produce conformational changes that block rebinding of cation.

Tetanus neurotoxin (TeNT) is a powerful bacterial protein toxin that cleaves VAMP/synaptobrevin, an essential protein of the synaptic vesicle fusion machinery, and consequently blocks neurotransmission. The extreme neurospecificity of TeNT is determined by the binding of its C-terminal domain (fragment C or H(C)) to neuronal receptors. Whereas polysialogangliosides are known acceptors for the toxin, the existence of additional protein receptors has also been suggested. We have reported previously on a 15 kDa cell-surface glycoprotein that interacts with TeNT in neuronal cell lines and motoneurons [Herreros, Lalli, Montecucco and Schiavo (2000) J. Neurochem., in the press]. Here, on the basis of the structural information provided by the crystallization of fragment C of TeNT, we have expressed its C-and N-terminal halves as recombinant proteins and analysed their binding abilities to rat phaeochromocytoma (PC12) cells differentiated with nerve growth factor. We found that the C-terminal subdomain of the fragment C of TeNT is necessary and sufficient for cell binding and for the interaction with the 15 kDa putative receptor. In contrast, the N-terminal half showed a very poor interaction with the cell surface. These results restrict the binding domain of TeNT to the C-terminal half of the fragment C and highlight the importance of this domain for the neurospecific interaction of the toxin with the synapse. Furthermore, these findings support the use of this portion of TeNT as a neurospecific targeting device, pointing to an involvement of the N-terminal subdomain in later steps of the intoxication pathway.

Factors driving the development of neuromuscular blocking agents are discussed. The goal of recent development of neuromuscular blocking agents is to develop agents with fewer adverse effects than succinylcholine and greater control. Greater control can be achieved through a short duration of action and a fast onset, similar to that found with succinylcholine. Duration control can be achieved through rapid, reliable metabolism that is organ independent, as with cisatracurium, or that occurs in the liver, because this will fail only in patients with severe hepatic disease. Rapid onset can be achieved by giving higher doses of drugs that have a fast, reliable metabolism or a rapid distribution to tissue. This has been done with succinylcholine, cisatracurium, and mivacurium. It has been shown that a low-potency drug has a faster onset than a higher-potency drug, even at the same relative dose. Rocuronium was the first neuromuscular blocking agent marketed that was developed specifically as a low-potency drug for achieving a rapid onset. In a comparison with two other intermediate-duration neuromuscular blocking agents at equipotent doses, rocuronium's onset was about one minute, which was two minutes faster than that of either vecuronium or atracurium. Rapacuronium is an investigational intermediate-duration agent and is even less potent than rocuronium. Clinical studies have shown it to have a very rapid onset (about 50 seconds) and a short duration of action (15-25 minutes, depending on the dose). Its short duration of action will make it potentially useful for short surgical procedures and procedures in which nerve integrity must be monitored. At the same time, its onset time approaches that of succinylcholine. Recent advances have made the administration of neuromuscular blocking agents much easier because the newer nondepolarizing relaxants offer faster onset and greater control.

A cholinergically disrupted laboratory animal has been produced by administration of the cholinotoxin ethylcholine aziridinium mustard (AF64A), which produced a dysfunction in the cholinergic forebrain system. After AF64A treatment, a reduction of choline acetyl transferase (ChAT) activity was measured in the hippocampal regions. ChAT activity was preferentially reduced in tissue samples of the dorsal with respect to the ventral hippocampus, and concomitantly with this reduction, a compensatory increase in ChAT activity in the medial septum was found. Tissue gamma-aminobutyric acid (GABA) content in the hippocampal and septal brain areas was not affected by AF64A, indicating a specific effect on the cholinergic septohippocampal projection. The rate of GABA accumulation induced by aminooxyacetic acid administration was higher in the dorsal hippocampus and medial septum of AF64A-treated animals, but not in their ventral hippocampus and lateral septum, where significant changes occurred in ChAT activity. Concomitantly with the changes in GABA metabolism, a significant Bmax increase and Kd reduction of 3H-flunitrazepam binding in the hippocampus of AF64A-treated animals were associated with changes in the ChAT activity. This finding suggests an increase of GABA input on the cholinergic somas of the medial septum and an uncompensated GABAergic interneuron activity in the hippocampus. In this study, we present an adaptive mechanism of homotypic compensatory metabolism by cholinergic somas, and a heterotypic response of the GABAergic septohippocampal projection system, which was elicited by AF64A administration.

Objective: To review current data on butyrylcholinesterase., Data Sources: Search through Medline data bases of articles in French or English., Study Selection: Original articles and case reports were selected. Letters to editor were excluded., Data Extraction: The articles were analyzed in order to obtain current data on biochemical structure, action, major pathological variations, especially with regard to the recent informations obtained by molecular biology concerning the identification of genetic variants., Data Synthesis: Butyrylcholinesterase must be differentiated from acetylcholinesterase, which cannot hydrolyse succinylcholine. The physiological action of butyrylcholinesterase remains unknown, although it can hydrolyse many drugs. Excluding genetical mutations, several physiopathological situations alter butyryl-cholinesterase activity. Butyrylcholinesterase activity assessment does not allow the diagnosis of genetic variants. Whatever the origin, only deficits of more than 50% modify significantly the metabolism of succinylcholine or mivacurium. The diagnosis of a prolonged neuromuscular blockade is obtained with systematic monitoring of the neuromuscular function in case of administration of mivacurium or succinylcholine. Mivacurium should only be re-injected when one response at train of four is obtained. In case of prolonged neuromuscular blockade, the anticholinesterasic agent should not be administered when no response at train of four is obtained. The biochemical methods using inhibitors (dibucaine, fluoride) of the butyrylcholinesterase and a familial study lead to the diagnosis in most cases because the atypical and fluoride variants are the most frequent. When results are doubtful, genetic molecular methods with the use of PCR and restriction enzymes allow a rapid diagnosis.

Plasma cholinesterase is an enzyme which has importance to the anaesthetist primarily for its role in the metabolism of suxamethonium, although other anaesthetic related drugs that this enzyme metabolises are also increasingly important. In this article we review current thoughts on the function, profile and chemistry of plasma cholinesterase. Causes of variations in the activity of the enzyme are described and the basis of genetic variations is explained.

The primary virulence factors of many pathogenic bacteria are secreted protein toxins which bind to glycolipid receptors on host cell surfaces. The binding specificities of three such toxins for different glycolipids, mainly from the ganglioside series, were determined by surface plasmon resonance (SPR) using a liposome capture method. Unlike microtiter plate and thin layer chromatography overlay assays, the SPR/liposome methodology allows for real time analysis of toxin binding under conditions that mimic the natural cell surface venue of these interactions and without any requirement for labeling of toxin or receptor. Compared to conventional assays, the liposome technique showed more restricted oligosaccharide specificities for toxin binding. Cholera toxin demonstrated an absolute requirement for terminal galactose and internal sialic acid residues (as in GM1) with tolerance for substitution with a second internal sialic acid (as in GD1b). Escherichia coli heat-labile enterotoxin bound to GM1 and tolerated removal or extension of the internal sialic acid residue (as in asialo-GM1 and GD1b, respectively) but not substitution of the terminal galactose of GM1. Tetanus toxin showed a requirement for two internal sialic acid residues as in GD1b. Extension of terminal galactose with a single sialic acid was tolerated to some extent. The SPR analyses also yielded rate and affinity constants which are not attainable by conventional assays. Complex binding profiles were observed in that the association and dissociation rate constants varied with toxin:receptor ratios. The sub-nanomolar affinities of cholera toxin and heat-labile enterotoxin for liposome-anchored gangliosides were attributable largely to very slow dissociation rate constants. The SPR/liposome technology should have general applicability in the study of glycolipid-protein interactions and in the evaluation of reagents designed to interfere with these interactions.

Drugs administered to patients undergoing anaesthesia may complicate the use of the neuromuscular blockers that are given to provide good surgical conditions. The various sites of interaction include actions on motor nerve conduction and spinal reflexes, acetylcholine (ACh) synthesis, mobilisation and release, sensitivity of the motor end plate to ACh and the ease of propagation of the motor action potential. In addition, many drugs affect the pharmacokinetics of neuromuscular blockers, especially as most drugs depend to a greater or lesser extent upon renal excretion. The clinically significant interaction between nondepolarisers and depolarisers may be due to blockade of the pre-synaptic nicotinic receptors by the depolarisers, leading to decreased ACh mobilisation and release. Synergism between nondepolarisers probably results from post-synaptic receptor mechanisms. Volatile anaesthetic agents affect the sensitivity of the motor end-plate (post-synaptic receptor blockade) in addition to having effects on pre-synaptic nicotinic function. The effects of nondepolarisers are likely to be potentiated and their action prolonged by large doses of local anaesthetics due to depression of nerve conduction, depression of ACh formation, mobilisation and release, decreases in post-synaptic receptor channel opening times and reductions in muscular contraction. Most antibacterials have effects on pre-synaptic mechanisms. Procainamide and quinidine principally block nicotinic receptor channels. Magnesium has a marked inhibitory effect on ACh release. Calcium antagonists could theoretically interfere with neurotransmitter release and muscle contractility. Phenytoin and lithium decrease ACh release, whilst corticosteroids and furosemide (frusemide) tend to increase the release of the transmitter. Ecothiopate, tacrine, organophosphates, propanidid, metoclopramide and bambuterol depress cholinesterase activity and prolong the duration of the neuromuscular block. The probability of clinically significant interactions increases in patients receiving several drugs with possible effects on neuromuscular transmission and muscle contraction.

Many organelles are energized by proton pumps: mitochondria form an inside-negative membrane potential by means of the respiratory chain and endomembrane structures, such as lysosomes and synaptic vesicles, establish an internal acidic pH by means of a vacuolar-type H(+)-ATPase (V-ATPase). Various amphipathic drugs such as local anesthetics and neuron blockers are accumulated in acidic organelles upon energization by proton pumps. However, this process does not require any transporters specific for the drugs: these drugs penetrate through the lipid bilayer against a concentration gradient so as to accumulate inside the energized organelles. Essentially the same transport process takes place in liposomes that have been reconstituted with purified V- or F-ATPase. Various hydrophobic cations are also accumulated in mitochondria by a similar mechanism. The energy-dependent but transporter-independent accumulation does not belong to the known transport categories and seems to represent a new type of transport which may be important for understanding the mode of action of drugs and toxins.

[3H]Choline mustard aziridinium ion binds irreversibly to the sodium-coupled high-affinity choline transport protein in a sodium-dependent and hemicholinium-sensitive manner, and thus is a useful affinity ligand. In rat striatal synaptosomal membranes, it radiolabels two polypeptides with apparent molecular masses of 58 and 35 kDa. Based upon the use of two different experimental approaches, it appears that neither of these polypeptides is glycosylated.

Crotoxin and taipoxin are both neurotoxic phospholipases A2 capable of affecting the presynaptic activity to bring about ultimate blockade of synaptic transmission. The enzymatic activity has generally been considered to be necessary but not sufficient for the blockade. Since many phospholipases A2 with comparable or even higher enzymatic activity are not toxic, it has been postulated that the difference lies in the affinity of binding to the presynaptic membrane. In confirmation of this proposition, we and others have previously shown that iodinated crotoxin and taipoxin bind specifically with high affinity to the isolated synaptic membrane fraction from guinea-pig brain, whereas specific binding is not detected with the nontoxic pancreatic phospholipase A2. Experiments based on photoaffinity labeling and simple chemical cross-linking techniques have led to the identification of three polypeptides preferentially present in neuronal membranes as (subunits of) the binding protein(s) for crotoxin and/or taipoxin. Some, but not all, other toxic phospholipases A2 also appear to be ligands for the three polypeptides. We now report studies on partial purification of these polypeptides using affinity chromatography and other techniques. In order to learn the normal physiological roles played by the toxin-binding proteins, the phospholipase-independent effects of the toxins on the synaptosomes have been sought. We have found that under Ca(2+)-free condition, taipoxin or crotoxin inhibits with IC50 of 20-1000 nM the Na(+)-dependent uptake of norepinephrine, dopamine and serotonin by the synaptosomes. In contrast, choline uptake is not affected.(ABSTRACT TRUNCATED AT 250 WORDS)

We have identified, by affinity chromatography, a binding protein for the snake venom toxin taipoxin. The sequence of this 47 kDa protein is unique, is characteristic of a secreted protein, and has homology to the acute phase proteins serum amyloid P protein and C-reactive protein of the pentraxin family. We have named this protein neuronal pentraxin (NP), as Northern analysis and in situ hybridization demonstrate high message levels in neurons of cerebellum, hippocampus, and cerebral cortex. Because NP may be released synaptically and has homology to immune proteins potentially involved in uptake of lipidic, toxic, or other antigenic material, we suggest that NP may be involved in a general uptake of synaptic macromolecules.

Paralytic shellfish poison (PSP) has historically been a problem for the shellfish industry. In order to prevent the marketing of contaminated seafood products, governments have implemented monitoring programs where standards of toxins are necessary. The stability of these standard toxins is very important. In this paper we analysed the stability of saxitoxin (STX) and neosaxitoxin in acidic solution and lyophilized samples. Individual toxins were determined in each sample using a high-performance liquid chromatographic procedure employing post-column oxidation of the toxins to form fluorescent derivatives. Our results demonstrate that STX is very stable in solution samples and could be adopted as a reference standard. This toxin can be kept in dilute acidic solutions for 18 months without loss of potency. However, neosaxitoxin is unstable, possibly due to transformation to other toxins.

Since early time the bark and fruit of some species of plants belonging to family melia have been used as ascaris vermifuge in traditional Chinese medicine. In our previous studies it was shown that toosendanin, a triterpenoid derivative extracted from the bark of Meli toosendan Seib et Zucc was a neuromuscular blocker acting selectively and irreversibly on the release of acetylcholine from the nerve terminals. It was thus suggested that toosendanin binding sites should exist in presynaptic nerve terminals. The binding experiments of 3H-toosendanin to the pellet of rat cerebral cortex homogenate demonstrated that the binding site of 3H-toosendanin exists in the cortex homogenate. The equilibrium dissociation constant (KD) of toosendanin-binding site complex is about 2.0 x 10(-7) mol/L. The specific binding activity of the homogenate pellet estimated according to the binding data is about 2.7 x 10(-12) mol per mg protein. The Hill coefficient of this binding site is 0.97. Our data also indicated that the synaptosomes prepared by sucrose density gradient ultracentrifugation are enriched with components of toosendanin binding site.

Evoked hind limb digital extensor tension (hoof twitch) was maintained at 40% of baseline for 1 h by atracurium infusion in 7 horses anaesthetised with halothane. After 1 h, atracurium was discontinued and hoof twitch allowed to recover to 75%. Atracurium was again given by infusion to maintain 40% twitch for a second hour, then 2 mg gentamycin/kg bwt were given i.v. Atracurium infusion was continued for a third hour, and then hoof twitch was again allowed to recover spontaneously to 75%. Gentamycin reduced twitch strength from 40 +/- 1% (mean +/- sem) to 29 +/- 4% within 7.0 +/- 1.5 min (P = 0.02). Twitch gradually returned to pre-gentamycin strength over the course of the next hour. Recovery of hoof twitch from 50% to 75% took 7.7 +/- 0.7 min for atracurium alone and 11.5 +/- 2.7 min for atracurium plus gentamycin (P = 0.03). Recovery from 50% twitch to 75% fade recovery took 13.8 +/- 0.8 min for atracurium alone and 13.7 +/- 1.2 min for atracurium plus gentamycin. At 75% recovery of fade, hoof twitch was 87 +/- 3% for atracurium alone and 82 +/- 4% for atracurium plus gentamycin. Reversal of the block with edrophonium and subsequent recovery of the horses from anaesthesia were uneventful. It was concluded that, although gentamycin did augment the neuromuscular blockade of atracurium, the effect was minimal.

Toxin production of a Malaysian isolate of the toxic red tide dinoflagellate Pyrodinium bahamense var. compressum was investigated at various stages of the batch culture growth cycle and under growth conditions affected by temperature, salinity, and light intensity variations. In all the experiments conducted, only 5 toxins were ever detected. Neosaxitoxin (NEO) and gonyautoxin V (GTX5) made up 80 mole percent or more of the cellular toxin content and saxitoxin (STX), GTX6 and decarbamoylsaxitoxin (dcSTX) made up the remainder. No gonyautoxins I-IV or C toxins were ever detected. In nutrient-replete batch cultures, toxin content rapidly peaked during early exponential phase and just as rapidly declined prior to the onset of plateau phase. Temperature had a marked effect on toxin content, which increased 3-fold as the temperature decreased from the optimum of 28 degrees C to 22 degrees C. Toxin content was constant at salinities of 24% or higher, but increased 3-fold at 20%. Toxin content decreased 2-fold and chlorophyll content increased 3-fold when light intensity was reduced from 90 to 15 microE m-2 s-1. This accompanied a 30% decrease in growth rate. Toxin composition (mole % individual toxin cell-1) remained constant throughout the course of the nutrient-replete culture and during growth at various salinities, but varied significantly with temperature and light intensity changes. At 22 degrees C, GTX5 was 25 mole % and NEO was 65 mole %, while at 34 degrees C, GTX5 increased to 55 mole % and NEO decreased proportionally to 40 mole %. When light intensity was reduced from 90 to 15 microE m-2 s-1, NEO decreased from 55 to 38 mole %, while GTX5 increased from 40 to 58 mole %. These data suggest that low light and high temperature both somehow enhance sulfo-transferase activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Brevetoxin PbTx-3 and non-toxic derivative 4 were investigated for their abilities to bind to the specific brevetoxin receptor site on rat brain synaptosomes and to modulate the normal function of voltage-gated sodium channels as determined by patch clamping of cultured neurons. Compounds 4 and 5 are produced from PbTx-3 by opening of the A-ring lactone to the saturated and unsaturated diols using sodium borohydride in ethanol. Natural PbTx-3 exhibited tighter binding to rat brain synaptosomes by at least 3 orders of magnitude as determined by competitive radioligand binding experiments, and was also more effective at activating voltage-gated channels. Patch clamping revealed the 3 orders of magnitude greater potency of PbTx-3 toxin over 5, although each produced delayed sodium channel opening and a pronounced delay in inactivation. Conformational modeling of the Brevetoxin B backbone indicates that the two molecules are identical except for the region of the A-Ring lactone. Thus, we conclude that the brevetoxin PbTx-3 backbone requires electrophilic functionality in the region of the lactone in PbTx-3, and that opening of the ring in 5 is sufficient to substantially reduce both binding and activity.

Experiments were conducted on mouse hemidiaphragm preparations using five phospholipase A2 neurotoxins of differing chain structures and antigenicities [notexin (one chain); crotoxin (two chains not covalently bound), beta-bungarotoxin (two chains covalently bound); taipoxin (three chains), and textilotoxin (five chains; one copy each of three chains and two copies of a fourth chain)]. Three clostridial neurotoxins (botulinum neurotoxin types A and B, and tetanus toxin) were used in comparison experiments. Phospholipase A2 neurotoxins produced concentration-dependent blockade of neuromuscular transmission. There was no obvious relationship between chain structure and potency, but there was an indication of a relationship between chain structure and binding. The binding of notexin was substantially reversible, the binding of crotoxin was slightly reversible, and the binding of beta-bungarotoxin, taipoxin and textilotoxin was poorly reversible. Experiments with neutralizing antibodies indicated that phospholipase A2 neurotoxins became associated with binding sites on or near the cell surface. This binding did not produce neuromuscular blockade. When exposed to physiological temperatures and nerve stimulation, bound toxin disappeared from accessibility to neutralizing antibody. This finding suggests that there was some form of molecular rearrangement. The two most likely possibilities are: (1) there was a change in the conformation of the toxin molecule, or (2) there was a change in the relationship between the toxin and the membrane. The molecular rearrangement step did not produce neuromuscular blockade. At a later time there was onset of paralysis; the amount of time necessary for onset of blockade was a function of toxin concentration. Phospholipase A2 neurotoxins were not antagonized by drugs that inhibit receptor-mediated endocytosis. In addition, phospholipase A2 neurotoxins did not display the pH-induced conformational changes that are typical of other endocytosed proteins, such as clostridial neurotoxins. However, phospholipase A2 neurotoxins were antagonized by strontium, and this antagonism was expressed against toxins that were free in solution and toxins that were bound to the cell surface. Limited antagonism was expressed after toxins had undergone molecular rearrangement, and no antagonism was expressed after toxin-induced neuromuscular blockade. The cumulative data suggest that phospholipase A2 neurotoxins are not internalized to produce their poisoning effects. These toxins appear to act on the plasma membrane, and this is the site at which they initiate the events that culminate in neuromuscular blockade.

Neuromuscular blocking agents are frequently used in the Intensive Care Unit to facilitate tracheal intubation and the application of continuous paralysis. This review will focus on various conditions of the critically ill patient such as multi-organ dysfunction, acid-base and electrolyte imbalance, prolonged immobility, multiple drug interactions and specific disease/injury processes that may affect the pharmacokinetic and pharmacodynamic behaviour of muscle relaxants. As such, due to the complex nature of the critically ill patients, the effects of neuromuscular blocking agents are unpredictable. Therefore, guidelines regarding their administration and the methodology and requirement for continuous bedside monitoring of neuromuscular function will be presented.

Long-term phenytoin therapy induces resistance to the neuromuscular blocking effects of metocurine. The hypothesis that this is attributable to increased plasma protein binding of the drug (decreased free fraction) related to increased concentrations of alpha 1-acid glycoprotein (AAG) or attributable to the proliferation of acetylcholine receptors (AChR) at the muscle membrane was tested in the rat. After 14 days of phenytoin 40 mg.kg-1, administered intraperitoneally twice daily (n = 12), the neuromuscular pharmacodynamics were evaluated and compared with those of time-matched controls (n = 10). Protein binding was measured by equilibrium dialysis, AAG concentrations by radial immunodiffusion assay, and AChR by 125I-alpha-bungarotoxin binding. The effective dose for 50% inhibition of baseline twitch height (ED50) was significantly greater in the phenytoin group than in the control group (15.03 +/- 1.65 micrograms.kg-1 vs. 9.98 +/- 0.69 micrograms.kg-1, respectively). The concentrations of AAG increased gradually from 133.8 +/- 7.8 micrograms.ml-1 at day 0, to 343.1 +/- 58.0 micrograms.ml-1 at day 7, to 1,729.5 +/- 422.3 micrograms.ml-1 at day 14 in the phenytoin group. The induction of AAG concentrations in plasma was dependent on plasma phenytoin concentrations and was most prominent after 14 days of phenytoin (r = 0.77; P less than 0.01; n = 22). The free fraction of metocurine was significantly decreased in the phenytoin group compared to the control group (67.2 +/- 0.18% vs. 74.5 +/- 2.5%). There was a significant negative correlation between increased AAG concentrations and decreased free fraction (r = 0.65).(ABSTRACT TRUNCATED AT 250 WORDS)

The pharmacokinetics of pipecuronium bromide (Arduan) studied in animals (rat, dog, cat) and in humans can be described by a two-compartment open model. The elimination half-life in animals was found to be around 40 min, in humans between 44-137 min. The main route of elimination is the excretion of the unchanged molecule in urine. In kidney patients the elimination becomes longer. The metabolism in the elimination is less important, in rats 3-hydroxy-, 17-hydroxy and 3,17-dihydroxy metabolites have been identified.

The non-toxic binding fragment of tetanus toxin (fragment C) binds avidly to neural tissue and has a growing number of neurobiological uses. Its current utility is limited by both its high commercial cost and the complex procedure for its preparation requiring highly purified tetanus toxin. We have developed a short procedure which prepares fragments of tetanus toxin from crude C. tetani extracts. The resultant proteins are atoxic with molecular sizes and immunological properties closely resembling fragment C. These proteins undergo retrograde axonal and apparent transneuronal transport in a fashion similar to fragment C.

Neosaxitoxin (neoSTX) differs structurally from saxitoxin (STX) in that the hydrogen on N-1 is replaced by a hydroxyl group. On single frog skeletal muscle fibers in the vaseline-gap voltage clamp, the concentrations for reducing the maximum sodium current by 50% (ED50) at pH's 6.50, 7.25, and 8.25 are, respectively, 4.9, 5.1, and 8.9 nM for STX and 1.6, 2.7, and 17.2 nM for neoSTX. The relative potencies of STX at the different pH's closely parallel the relative abundance of the protonated form of the 7,8,9 guanidinium function, but the relative potencies of neoSTX at the same pH's vary with the relative abundance of the deprotonated N-1 group. In constant-ratio mixtures of the two toxins, the observed ED50's are consistent with the notion that the two toxins compete for the same site. At pH's 6.50 and 7.25, the best agreement between observed and computed values is obtained when the efficacy term (epsilon) for either toxin is 1. At pH 8.25 the best agreement is obtained if the efficacy is 1 for STX but 0.75 for neo-STX. The marked pH dependence of the actions of neoSTX probably reflects the presence of a site in the receptor that interacts with the N-1 -OH, in addition to those interacting with the 7,8,9 guanidinium and the C-12 hydroxyl groups. Considering the three-dimensional structure of the STX and neoSTX molecules, the various site points are probably located in a fold or a crevice of the channel protein, where the extracellular orifice of the sodium channel is located.

The objective of this study was to measure the uptake and metabolism of [3H]choline mustard aziridinium ion in rat brain synaptosomes. In previous investigations, we showed that this compound binds irreversibly to the choline carrier thereby inhibiting choline transport into nerve terminals; it also acts as both a substrate and inhibitor of the acetylcholine biosynthetic enzyme choline acetyltransferase. We now report that [3H]choline mustard aziridinium ion was transported into purified rat brain synaptosomes by a hemicholinium-sensitive mechanism, but at only a fraction of the rate of uptake of [3H]choline. Following 5 min incubation with the nerve terminal preparation, uptake of [3H]choline mustard aziridinium ion was 20% of that of [3H]choline transport, but this fell to 10% of [3H]choline accumulation at 30 min incubation. Apparent Michaelis constants derived from double reciprocal plots of velocity of transport versus substrate concentration revealed that the apparent affinity constants (Km) of the high-affinity choline carrier for [3H]choline mustard aziridinium ion and [3H]choline were not different (1.44 +/- 0.15 and 2.14 +/- 0.80 microM for choline and choline mustard aziridinium ion, respectively). Increasing the incubation time from 5 to 30 min, during which time a proportion of the high-affinity choline carriers were irreversibly inactivated by choline mustard aziridinium ion, did not alter the binding affinity for this compound. The maximum velocity of transport (Vmax) for the two compounds were significantly different with the maximum uptake of [3H]choline mustard aziridinium ion being 19.5% of that for choline at 5 min incubation, and falling to only 10.6% of the maximum rate of choline transport by 30 min incubation. [3H]Choline mustard aziridinium ion transported into synaptosomes on the high-affinity choline carrier was metabolized, with 27% being recovered as [3H]acetylcholine mustard aziridinium ion, 27% as [3H]phosphorylcholine mustard aziridinium ion, 7% as unmetabolized [3H]choline mustard aziridinium ion and 16% recovered as an unidentified metabolite. In parallel samples, [3H]choline taken up into synaptosomes was recovered as [3H]acetylcholine (71%) and unmetabolized [3H]choline (18%) with no net production of [3H]phosphorylcholine. Acetylation of [3H]choline mustard aziridinium ion amounted to only 7.6% of [3H]acetylcholine synthesized under the same conditions. These results show clearly that choline mustard aziridinium ion was accumulated into the cholinergic nerve terminals by the high-affinity choline carrier, but the amount was small relative to the uptake of choline and probably restricted by progressive inactivation of the transporters through covalent bond formation.

Neuromuscular blocking drugs have a high affinity for muscarinic acetylcholine receptors in the heart atria and ileal smooth muscle. In experiments on homogenates, alcuronium, gallamine, pancuronium, tercuronium and ritebronium inhibited the binding of the muscarinic antagonist (3H)quinuclidinyl benzilate (QNB) to rat heart atria with IC50 values of 0.15-0.53 mumol X 1(-1) and to ileal longitudinal muscles with IC50 values of 0.12-0.45 mumol X 1(-1). d-Tubocurarine and decamethonium inhibited (3H)QNB binding to these tissues with IC50 values of 6.2-8.5 mumol X 1(-1). For each neuromuscular blocking drug, the IC50 values were virtually identical for (3H)QNB displacement in the homogenates of the atria and of the ileal muscle. Alcuronium and gallamine differed from the other blocking agents in that they produced less steep (3H)QNB displacement curves both in the atria and the ileal muscle; Hill coefficients for the binding of alcuronium and gallamine to atrial and ileal homogenates were lower than unity. On isolated atria, gallamine, pancuronium, ritebronium and tercuronium antagonized the inhibition of tension development caused by the muscarinic agonist, methylfurmethide, with Kd values which were of the same order of magnitude as the IC50 values for the displacement of (3H)QNB binding to homogenates; the Kd of alcuronium was 12.5 times higher. d-Tubocurarine and decamethonium did not antagonize the effects of methylfurmethide at concentrations up to 100 mumol X 1(-1). On isolated ileal longitudinal muscle, gallamine and pancuronium antagonized the effects of methylfurmethide with Kd values that were 53 times and 100 times higher than their respective Kd values in the atria.(ABSTRACT TRUNCATED AT 250 WORDS)

Pharmacodynamics of vecuronium have been studied in children 1 to 10 years old. Doses from 0.015 to 0.1 mg/kg were given and the effects measured mechanically and by electromyography. For 0.1 mg/kg the pharmacokinetics were established. A smaller distribution volume and higher plasma clearance in children as compared to adults have been found, while the dose-effect ratio is the same. The action of vecuronium is shorter in children than in adults.

Neuromuscular blocking agents can be evaluated in anesthetized cats, dogs, or rhesus monkeys using dose-response relationships to assess the degree of separation between neuromuscular blocking activity and effects on cardiovascular and autonomic systems. Histamine release can be studied in anesthetized dogs by establishing whether any induced hypotension is reduced by pretreatment with H1 and H2 receptor antagonists. In anesthetized cats, the role of the liver in metabolism is investigated after administration of the drug by the hepatic portal vein and determining whether this "first pass" effect through the liver reduces potency. The involvement of the kidneys in the elimination of the drug can be assessed by ascertaining whether or not the potency and duration of action are enhanced after bilateral renal ligation. In anesthetized man, the simultaneous measurement of the tetanic (50 Hz) and single twitch contractions of the adductor pollicis muscle of the hand has proved a valuable technique for the quantitative assessment of skeletal muscle relaxants. Furthermore, the tetanic response overcomes the relative insensitivity of the single twitch, and tetanic fade provides a measure of the effects of these drugs at presynaptic sites. Cumulative effects may be evident as a prolonged recovery after repeated dosage or after an infusion. The effectiveness of neostigmine as an antagonist should be investigated both from partial and complete block, and recovery of respiration should be compared with that of peripheral neuromuscular blockade.