Allosteric inhibition of muscle-type nicotinic acetylcholine receptors by a neuromuscular blocking agent pancuronium.

Muscle relaxants are indispensable for surgical anesthesia. Early studies suggested that a classical non-depolarizing muscle relaxant pancuronium competitively binds to the ligand binding site to block nicotinic acetylcholine receptors (nAChR). Our group recently showed that nAChR which has two distinct subunit combinations are expressed in zebrafish muscles, αßδε and αßδ, for which potencies of pancuronium are different. Taking advantage of the distinct potencies, we generated chimeras between two types of nAChRs and found that the extracellular ACh binding site is not associated with the pancuronium sensitivity. Furthermore, application of either 2 µM or 100 µM ACh in native αßδε or αßδ subunits yielded similar IC50 of pancuronium. These data suggest that pancuronium allosterically inhibits the activity of zebrafish nAChRs.

Allosteric interaction of the neuromuscular blockers vecuronium and pancuronium with recombinant human muscarinic M2 receptors.

Neuromuscular blocking drugs produce muscle weakness by interaction with nicotinic-acetylcholine receptors. Cardiovascular side effects have been reported. In this study the neuromuscular blocking drug vecuronium and the controls gallamine and pancuronium slowed the rate of atropine induced [(3)H]N-methylscopolamine dissociation from Chinese hamster ovary cells expressing recombinant human muscarinic M2 receptors K(off) values min(-1); vecuronium (125 nM), atropine 0.45+/-0.07+blocker 0.04+/-0.02; gallamine (21 nM), atropine 0.42+/-0.05+blocker 0.15+/-0.04; pancuronium(21 nM), atropine 0.36+/-0.03+blocker 0.03+/-0.01). These data indicate that vecuronium, gallamine and pancuronium interact with an allosteric site on the muscarinic M2 receptor (located on the heart) and this may explain some of their cardiac side effects.

Competitive antagonists bridge the alpha-gamma subunit interface of the acetylcholine receptor through quaternary ammonium-aromatic interactions.

We recently demonstrated that conserved tyrosines Tyr198 of the alpha subunit and Tyr117 of the gamma subunit of the acetylcholine receptor stabilize binding of the curariform antagonist dimethyl-d-tubocurarine (DMT). To test the hypothesis that DMT interacts directly with these tyrosines, and therefore bridges the alpha-gamma subunit interface, we introduced point mutations into these key positions and expressed one or both mutant subunits in alpha 2 beta gamma 2 acetylcholine receptors in 293 HEK cells. Binding of DMT, measured by competition against the initial rate of 125I-alpha-bungarotoxin binding, shows high affinity for aromatic mutations, reduced affinity for polar mutations, and lowest affinity for arginine mutations. Similar side chain dependences were observed for both Tyr alpha 198 and Tyr gamma 117, indicating interaction of these residues with two symmetrical chemical groups in DMT. Two more bisquaternary antagonists, pancuronium and gallamine, show side chain dependences similar to that of DMT, indicating that the primary stabilizing interactions are aromatic-quaternary in both subunits. For the rigid ligands DMT and pancuronium, co-expressing mutant alpha and gamma subunits revealed independent contributions by each determinant, but strict independence was not observed for the flexible ligand gallamine. The free energy contributed by each aromatic-quaternary interaction was estimated to be 2-4 kcal/mol, as determined from the free energy difference between aromatic and alkyl hydroxyl mutations. Our results suggest that bis-quaternary competitive antagonists bridge the alpha-gamma subunit interface by fitting into a pocket bounded by tyrosines at positions 198 of the alpha subunit and 117 of the gamma subunit.

Selective enhancement of the interaction of curare with the nicotinic acetylcholine receptor.

Alteration of the ligand-binding domain of the nicotinic acetylcholine receptor through site-directed mutagenesis offers a powerful approach to the elucidation of structure-function relations in the receptor. Several conserved tyrosine residues in the large extracellular amino terminus of the alpha subunit of the receptor have been implicated by both chemical labeling and mutagenesis studies as playing an important role in the interaction of acetylcholine with the receptor. We and others have previously shown that substitution of phenylalanine for tyrosine at position 198 of the alpha subunit (alpha Y198F) leads to a rightward shift in the dose-response curve for acetylcholine-elicited currents. We have further investigated this particular mutation by examining the interaction of the competitive antagonist d-tubocurarine (curare) with the receptor. In contrast to the effect on the interaction of agonists with the receptor, this mutation leads to a marked increase in the affinity of the receptor for curare. Furthermore, this enhancement in affinity is selective for curare and is not seen with other competitive antagonists (pancuronium, beta-erythroidine, and gallamine). Examination of the structures of these competitive antagonists leads to the proposal that this enhancement is due to the formation of an aromatic-aromatic interaction between the phenylalanine ring at position alpha 198 in the mutant and one of the aromatic rings of curare and that this can provide information about the spatial arrangement of this residue in the binding site.

Inhibition of ionotropic neurotransmitter receptors by antagonists: strategy to estimate the association and the dissociation rate constant of antagonists with very strong affinity to the receptors.

Since binding of an agonist to an ionotropic neurotransmitter receptor causes not only channel opening, but also desensitization of the receptor, inhibition of the receptor by the antagonist sometimes becomes very complicated. The transient state kinetics of ligand association and dissociation, and desensitization of the receptor were considered on the basis of the minimal model proposed by Hess' group, and the following possibilities were proposed. 1) When an agonist is simultaneously applied to the receptor with an antagonist whose affinity to the receptor is extremely strong and different from that of the agonist, it is usually impossible to estimate the real inhibition constant exactly from the responses because desensitization of the receptor proceeds before the equilibrium of the ligand binding. Simultaneous addition of the antagonist with strong affinity to the receptor may apparently accelerate inactivation (desensitization) of the receptor. The association rate constant of the antagonist can be estimated by analyses of the rate of the inactivation in the presence and the absence of the antagonist. 2) A preincubated antagonist with a slow dissociation rate constant, i.e., a very effective inhibitor, may cause apparent noncompetitive inhibition of the receptor, since the receptor is desensitized by an agonist as soon as the antagonist dissociates from the receptor and the dissociation of the antagonist from the receptor becomes the rate-determining step. A nicotinic acetylcholine receptor (nAChR) was expressed in Xenopus oocytes by injecting mRNA prepared from Electrophorus electricus electroplax and used for the experiments on inhibition by an antagonist.(ABSTRACT TRUNCATED AT 250 WORDS)

[Determinations of steroidal muscle relaxants and their metabolites].

Pancuronium, vecuronium and pipecuronium are quaternary ammonium steroidal neuromuscular blocking agents. These drugs are typical nondepolarizing muscle relaxants. The steroidal compounds and/or their metabolites have been determined using mass spectrometry or gas chromatography. In this review, these analytical methods and application of the methods to pharmacokinetics are introduced.

Relationship of train-of-four ratio to twitch depression during pancuronium-induced neuromuscular blockade.

The relationship of train-of-four (TOF) ratio to the depression of the first twitch (TD) was investigated in 16 patients given pancuronium in doses insufficient to eliminate the fourth response in the train. In ten patients the block was allowed to spontaneously recover (Group 1) and in six it was reversed with neostigmine (Group 2) once maximum depression of TOF ratio had occurred. Maximum depression of the first response occurred in 6.5 +/- 4.3 min (mean +/- SD). Maximum depression of the TOF ratio was not present until 28.0 +/- 11.0 min. TOF ratio was usually still decreasing when recovery of TD had begun. However, once the TOF ratio began to recover, the recovery rates for TD and TOF ratio were similar in both the spontaneously recovering and the reversed groups. TOF ratio at complete TD recovery was 74.9 +/- 15.7 and 86.2 +/- 15.4% for the two groups. A kinetic analysis yielded significantly different elimination rate constants for the two effects from the "effect compartment." These findings suggest that these two effects of pancuronium are exerted at different sites.

Vecuronium induced neuromuscular blockade. The effect of increasing dose on speed of onset.

The speed of onset of neuromuscular blockade was measured for increasing doses of vecuronium. Onset times were found to be shorter with larger doses, up to a limit of ED95 X 3 (0.15 mg/kg). Thereafter, increasing the dose, even up to ED95 X 8 (0.4 mg/kg), did not appreciably shorten the time to full neuromuscular blockade. The clinical implications of, and pharmacokinetic basis for, these findings are discussed.

Comparative pharmacokinetics and dynamics of vecuronium and pancuronium in anesthetized patients.

Plasma concentrations and the degree of neuromuscular blockade after a 2-min infusion of 0.1 mg/kg of vecuronium bromide or pancuronium bromide (equipotent doses) were studied in 12 gynecologic patients. The plasma concentrations of both drugs declined in a triphasic manner. The difference between the intercepts and rate constants of the two drugs was not significant. Vecuronium was removed faster from the plasma than pancuronium; this was reflected in a significantly larger plasma clearance rate for vecuronium (4 ml X min-1 X kg-1 vs 1.1 ml X min-1 X kg-1 for pancuronium). The effective plasma concentrations at 50% recovery of the twitch height were 0.11 +/- 0.02 (vecuronium) and 0.2 +/- 0.03 microgram/ml (pancuronium). The disposition kinetics were adequately described by a three-compartment model. An effect compartment was added to the model to correlate the neuromuscular effects and plasma concentrations of both drugs. The ratio between concentrations of vecuronium and pancuronium in the effect compartment at 50% twitch height was 0.83. In spite of its greater potency, vecuronium has a shorter duration of action than pancuronium.

Disposition and urinary excretion of vecuronium bromide in anesthetized patients with normal renal function or renal failure.

The effect and plasma concentrations of vecuronium bromide were measured in normal patients after an intravenous dose of 50, 100, or 150 micrograms/kg and in patients with renal failure after 50 or 100 micrograms/kg. Urinary excretion of vecuronium was studied in normal patients after the 150 micrograms/kg dose. Pharmacokinetic parameters of patients with or without renal failure were similar. No metabolites of vecuronium were found in the plasma. Twenty percent of vecuronium was excreted unchanged in the urine; 5% as the 3-hydroxy derivative. No other metabolites of vecuronium were found in the urine. Increasing doses of vecuronium shortened the onset, but prolonged the duration of action and the recovery rate, to a similar extent in patients with or without renal failure. It was concluded that the disposition of vecuronium was best described by a three compartment model. Both the disposition and the effect of vecuronium are only marginally disturbed by renal failure.

Neuromuscular blockade for critical patients in the emergency department.

This retrospective study examines the indications and the effects of 119 doses of succinylcholine or pancuronium given in the emergency department during a 24-month period to patients considered to have immediately life-threatening emergencies. The most common indication for succinylcholine was to accomplish tracheal intubation (20 of 25 patients). Indications for pancuronium included computerized tomography of the head (60 of 94), control of agitation (40 of 94), facilitation of tracheal intubation (20 of 94), control of ventilation (12 of 94), and control of seizure unresponsive to anticonvulsants (4 of 94). Deterioration following succinylcholine occurred in three cases. These included two involving bradycardia and one involving ventricular tachycardia. Major complications following pancuronium included four incidences of ventricular arrhythmias. Intubation failure requiring surgical airway occurred in one patient given succinylcholine, two patients given pancuronium, and one patient who received both succinylcholine and pancuronium. Inadequate documentation of neurological examination prior to blockade was noted in six of 25 succinylcholine and nine of 94 pancuronium cases. Failure to sedate patients who might be aware of paralysis occurred in three of 25 succinylcholine and eight of 94 pancuronium uses. Neuromuscular blocking agents facilitate expeditious management of selected critical patients in the ED. Their prudent use requires anticipation of potential complications, preparation for surgical airway should intubation fail, documentation of physical examination before paralysis, and prior sedation when the patient responds to pain.

Non-linear fitting program for biological data.

A simple microcomputer program written in Microsoft Basic estimates pharmacokinetic parameters using the coordinate search technique to minimize the sum of squared errors. The program developed for portable computers combines a plot of data and curve fitting so as to find rapidly the initial parameters with the subsequent optimization of the parameter estimate.

Paradoxical antagonism of neuromuscular block by vecuronium metabolites.

Vecuronium is hydrolyzed in the body to 3-deacetyl (ORG 7268), 17-deacetyl (ORG NC58), and 3, 17-bis-deacetyl (ORG 7402) derivatives. Interactions of vecuronium and these metabolites were studied in phrenic nerve-hemidiaphragm preparations of rats. As already reported, ORG 7268 had a potent neuromuscular blocking action, and ORG NC58 and ORG 7402 had a weak neuromuscular blocking action. As expected, ORG 7268 increased the degree of neuromuscular block by vecuronium. However, a low concentration (10 microM) of ORG NC58 and ORG 7402 reversed the block by vecuronium. At a high concentration (50 microM), ORG NC58 and ORG 7402 increased the degree of block by vecuronium. Although we do not have enough data to explain these paradoxical reversal of neuromuscular block at this moment, we postulate that these results reflect the interaction between "slow" and "fast" competitive antagonists. Regardless of the mechanism, it should be emphasized that the concentrations of ORG NC58 and ORG 7402 which are necessary to reverse the block are much lower than those which facilitate the block. It is conceivable that this paradoxical reversal of the block occurs in experimental and clinical situations. Therefore, in determining the neuromuscular blocking action of a compound, the "antagonistic" effect of its metabolites should also be considered.

[Atracurium: neuromuscular blockade in repeated administration]. / Atracurium: Die neuromuskuläre Blockade bei wiederholter Gabe.

The neuromuscular blocking action of repeated injections of atracurium and vecuronium was studied in 74 surgical patients during balanced anaesthesia (methohexitone or etomidate, intubation after suxamethonium, fentanyl, droperidol, N2O). The initial bolus dose (ID) of atracurium was 0.25 mg/kg and of vecuronium 0.05 mg/kg followed by repeated increments (RD) of atracurium 0.1 mg/kg and vecuronium 0.0125 mg/kg when neuromuscular function (EMG) had recovered to about 30% of pre-relaxant control. Dose-response relationships revealed atracurium to be about 1/5 as potent as vecuronium; the ED50 of atracurium was 0.13 +/- 0.03 mg/kg and of vecuronium 0.023 +/- 0.007 mg/kg. The ID of both relaxants produced a neuromuscular blockade of about 90% within 4 min. The duration from the time of injection to 30% recovery was slightly longer in atracurium 26 +/- 9 min. In all patients the RD produced within 3.5 min satisfactory muscle relaxation with a neuromuscular block of about 85%. The mean duration of atracurium (18 min) was 5-10 min longer than of vecuronium (12 min). To maintain good surgical relaxation (more than 70% blockade) atracurium 0.32 mg/kg X h and vecuronium 0.056 mg/kg X h were required. No cumulation could be measured after repeated injections. The recovery time of atracurium and vecuronium at the end of anaesthesia was 10-12 min. Neither cardiovascular side-effects nor signs of histamine release were observed after both relaxants in our particular dose range. It is concluded, that atracurium is a favourable blocker for anaesthetic practice: The time of onset is approximately the same compared with vecuronium. The duration of action, however, is slightly longer but still truly intermediate long.(ABSTRACT TRUNCATED AT 250 WORDS)