Recovery of residual curarization after red blood cell transfusion.

BACKGROUND: The muscle-relaxing effects of succinylcholine are terminated via hydrolysis by plasma cholinesterase. There are multiple genetic variants of this enzyme and clinical circumstances that might influence the activity of plasma cholinesterase and eventually lead to prolonged neuromuscular blockade following succinylcholine application. CASE REPORT: Here, we report a parturient woman with atonic bleeding who suffered significant blood loss (hemoglobin 6.0 g•dL-¹). For surgical curettage, general anesthesia was performed by using short-acting succinylcholine. By the end of the 105-minute procedure, the patient's trachea was extubated. After extubation she showed signs of the prolonged neuromuscular blocking action of succinylcholine. At this time, the patient received an AB0-compatible red blood cell transfusion and recovered instantly from neuromuscular blockade. The plasma cholinesterase (3.200 U•L-¹) was below the normal range (4.900-12.000 U•L-¹). Patient's blood DNA analysis revealed heterozygously the genetic K variant of plasma cholinesterase. After red blood cell transfusion, serum potassium was elevated (5.7 mmol•L-¹; 4.4 mmol•L-¹ prior to transfusion). CONCLUSIONS: Pregnancy, blood loss and genetic variation contributed to impairment of plasma cholinesterase. Due to high-speed red blood cell transfusion, hemolytic release of erythrocyte cholinesterase might have terminated the neuromuscular blocking succinylcholine effect.

Structural mechanism of muscle nicotinic receptor desensitization and block by curare.

Binding of the neurotransmitter acetylcholine to its receptors on muscle fibers depolarizes the membrane and thereby triggers muscle contraction. We sought to understand at the level of three-dimensional structure how agonists and antagonists alter nicotinic acetylcholine receptor conformation. We used the muscle-type receptor from the Torpedo ray to first define the structure of the receptor in a resting, activatable state. We then determined the receptor structure bound to the agonist carbachol, which stabilizes an asymmetric, closed channel desensitized state. We find conformational changes in a peripheral membrane helix are tied to recovery from desensitization. To probe mechanisms of antagonism, we obtained receptor structures with the active component of curare, a poison arrow toxin and precursor to modern muscle relaxants. d-Tubocurarine stabilizes the receptor in a desensitized-like state in the presence and absence of agonist. These findings define the transitions between resting and desensitized states and reveal divergent means by which antagonists block channel activity of the muscle-type nicotinic receptor.

An hypothesis concerning the molecular structure of the nicotinic acetylcholine receptor.

An hypothesis is presented concerning the molecular structure of the nicotinic acetylcholine receptor at the neuromuscular junction based on the actual amino acid sequence of the N-terminal segment of the alpha-subunit and the Chou and Fasman prediction of secondary structure from the primary sequence. This is mainly in the form of two alpha-helices cross-linked by four ionically bound complementary amino acids (arg/lys to glu). This structure (R) is complementary to a wide range of ACh agonists and to the antagonist beta-erythroidine. If the ionic cross-links are disrupted the two segments can separate by 2-3 A. This new conformation (R1) is now complementary to antagonists of the type of histrionicotoxin. A further separation (approximately 8 A) gives a conformation complementary to antagonist of the type of decamethonium. Experiments to test the hypothesis are suggested.

Evaluation of methods for estimating population pharmacokinetic parameters. II. Biexponential model and experimental pharmacokinetic data.

Individual pharmacokinetic parameters quantify the pharmacokinetics of an individual, while population pharmacokinetic parameters quantify population mean-kinetics, interindividual variability, and residual variability, including intraindividual variability and measurement error. Individual pharmacokinetics are estimated by fitting individual data to a pharmacokinetic model. Population pharmacokinetic parameters have been estimated either by fitting all individuals' data together as though there were no individual kinetic difference, the naive pooled data (NPD) approach, or by fitting each individuals' data separately and then combining the individual parameter estimates, the two stage (TS) approach. A third approach, NONMEM, takes a middle course between these. This study provides further evidence of NONMEM's validity by comparing, using simulation, the three approaches on three types of data sets corresponding to three typical types of pharmacokinetic studies. The estimates of population parameters provided by the NPD method are poorer than those provided by either of the other methods. The estimates provided by the TS method are adequate for mean values and for residual variability, but not for interindividual kinetic variability. NONMEM's estimates are as good as those of the TS method for mean parameters and for residual variability, and considerably better for interindividual variability. The latter estimates are still not acceptable in an absolute sense. This is probably due, not to an intrinsic fault of the method (as it is in the case of the TS approach), but to an insufficient number of individuals being studied.

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.

Localization of putative nicotinic cholinoreceptors in the early development of Paracentrotus lividus.

Experiments are described, showing the presence of putative nicotinic cholinoreceptors in the egg after fertilization. The experiments were carried out on gametes and early embryos of the sea urchin Paracentrotus lividus, by using nicotinic agonists and antagonists. 1 mM Acetylcholine (ACh), 100 microM nicotine, 100 nM alpha-bungarotoxin (alpha-BuTx) and 100 microM curare inhibit sperm motility and fertilization, while they have no effect on unfertilized eggs. The drugs added within 1 min. after the raising of the fertilization layer had stronger effects on cleavage and development; when added more than 15 min. after the raising of the fertilization layer, they had lesser effects on further development up to pluteus stage. In all the experiments, nicotine was the most effective drug. The binding of fluorescein-labelled alpha-BuTx did not point out any affinity sites on unfertilized eggs, while they were localized on the sperms and on the eggs fertilized by sperms, but not on the eggs activated artificially. The binding was prevented by pretreatment of sperms and activated eggs with 10 nM native alpha-BuTx and 10 microM curare. We conclude that, in the fertilized egg, putative nicotinic cholinoreceptors are present, which are able to bind alpha-BuTx and curare. Fertilization by sperms is needed to trigger the formation of alpha-BuTx receptors.

Characterization of curaremimetic neurotoxin binding sites on membrane fractions derived from the human medulloblastoma clonal line, TE671.

Studies were conducted on curaremimetic neurotoxin binding to the nicotinic acetylcholine receptor present on membrane fractions derived from the human medulloblastoma clonal line, TE671. High-affinity binding sites (KD = 2 nM for 1-h incubation at 20 degrees C) and low-affinity binding sites (KD = 40 nM) for 125I-labeled alpha-bungarotoxin are present in equal quantities (60 fmol/mg membrane protein). The kinetically determined dissociation constant for high-affinity binding of toxin is 0.56 nM (k1 = 6.3 X 10(-3) min-1 nM-1; k-1 = 3.5 X 10(-3) min-1) at 20 degrees C. Nicotine, d-tubocurarine, and acetylcholine are among the most effective inhibitors of high-affinity toxin binding. The quantity of toxin binding sites and their affinity for cholinergic agonists is sensitive to reduction, alkylation, and/or oxidation of membrane sulfhydryl residues. High-affinity toxin binding sites that have been subjected to reaction with the sulfhydryl reagent dithiothreitol are irreversibly blocked by the nicotinic receptor affinity reagent bromoacetylcholine. High-affinity toxin binding is inhibited in the presence of either of two polyclonal antisera or a monoclonal antibody raised against nicotinic acetylcholine receptors from fish electric tissue. Taken together, these results indicate that curaremimetic neurotoxin binding sites on membrane fractions of the TE671 cell line share some properties with nicotinic acetylcholine receptors of peripheral origin and with toxin binding sites on other neuronal tissues.

A model for the acetylcholine binding site of the nicotinic acetylcholine receptor.

A detailed model for the acetylcholine binding site on the nicotinic acetylcholine receptor is proposed. It is derived from assumptions based on existing biochemical, structural, and pharmacological data, combined with molecular modeling and principles of protein evolution and architecture. Acetylcholine is proposed to fit into a pocket on one face of an antiparallel beta-pleated sheet formed by residues 128-142 on the alpha-subunit. This sheet is flexible yet stable, in part because of a double cystine bridge at its end. Asp138, Thr133, and Gln140 provide a ring of negative charges around the quaternary ammonium group of acetylcholine, Ile131 and alkane segments of the other residues in the binding site provide hydrophobic interactions, and Gln140 provides a hydrogen bond for acetylcholine's carbonyl group; Glu129 would form part of the second anionic subsite for the bis-quaternary ammonium compounds and curares. The model is compatible with the available evidence pertaining to the binding site and with structure-activity relationship studies. It is precise and detailed, thereby making clear predictions, which are directly testable by affinity labeling and site-directed mutagenesis. It should prove useful in the design of such experiments.

Mimicry between receptors and antibodies. Identification of snake toxin determinants recognized by the acetylcholine receptor and an acetylcholine receptor-mimicking monoclonal antibody.

In several instances, a monoclonal antibody raised against a receptor ligand has been claimed to mimic the ligand receptor. Thus, a specific monoclonal antibody (Malpha2-3) raised against a short-chain toxin from snake was proposed to mimic the nicotinic acetylcholine receptor (AChR) (). Further confirming this mimicry, we show that (i) like AChR, Malpha2-3 elicits anti-AChR antibodies, which in turn elicit anti-toxin antibodies; and (ii) the region 106-122 of the alpha-chain of AChR shares 66% primary structure identity with complementarity-determining regions of Malpha2-3. Also, a mutational analysis of erabutoxin a reveals that the epitope recognized by Malpha2-3 consists of 10 residues, distributed within the three toxin loops. Eight of these residues also belong to the 10-residue epitope recognized by AChR, a result that offers an explanation as to the functional similarities between the receptor and the antibody. Strikingly, however, most of the residues common to the two epitopes contribute differentially to the energetic formation of the antibody-toxin and the receptor-toxin complexes. Together, the data suggest that the mimicry between AChR and Malpha2-3 is partial only.

Curare binding and the curare-induced subconductance state of the acetylcholine receptor channel.

The curare-induced subconductance state of the nicotinic acetylcholine receptor (AChR) of mouse skeletal muscle was examined using the patch-clamp technique. Two mechanisms for the generation of subconductance states were considered. One of these mechanisms entails allosteric induction of a distinct channel conformation through the binding of curare to the agonist binding site. The other mechanism entails the binding of curare to a different site on the protein. Occupation of this site would then limit the flow of ions through the channel. The voltage dependence and concentration dependence of subconductance state kinetics are consistent with curare binding to a site within the channel. The first order rate constant for binding is 1.2 X 10(6) M-1s-1 at 0 mV, and increases e-fold per 118 mV of membrane hyperpolarization. The rate of curare dissociation from this site is 1.9 X 10(2)s-1 at 0 mV, and decreases e-fold per 95 mV hyperpolarization. The equilibrium constant is 1.4 X 10(-4) M at 0 mV, and decreases e-fold per 55 mV hyperpolarization. This voltage dependence suggests that the fraction of the transmembrane potential traversed by curare in binding to this site is 0.46 or 0.23, depending on whether one assumes that one or both charges of curare sense the electric field. Successive reduction and alkylation of the AChR agonist binding sites with dithiothreitol (DTT) and N-ethyl maleimide (NEM), a treatment which results in the loss of responsiveness of the AChR to agonists, produced no change in curare-induced subconductance events, despite the fact that after this treatment most of the channel openings occurred spontaneously. Mixtures of high concentrations of carbamylcholine (CCh) with a low concentration of curare, which produce channel openings gated predominantly by CCH, resulted in subconductance state kinetics similar to those seen in curare alone at the same concentration. Thus displacement by CCh of curare from the agonist binding sites does not prevent curare from inducing subconductances. The results presented here support the hypothesis that curare induces subconductance states by binding to a site on the receptor other than the agonist binding sites, possibly within the channel pore. It is the occupation of this site by curare that limits the flow of ions through an otherwise fully opened channel.

Adrenaline inhibits muscarinic transmission in bullfrog sympathetic ganglia.

The effect of adrenaline (Ad) on muscarinic transmission was examined in B neurones of bullfrog sympathetic ganglia by using intracellular and voltage-clamp recording methods. Bath-application of Ad (5-500 microM) caused a depression of the slow excitatory postsynaptic potential (EPSP) elicited by repetitive stimulations of preganglionic nerve fibres in the presence of curare (30 microM). Ad also depressed the 'muscarinic' ACh potential induced by ionophoretic application of ACh directly to curarized sympathetic neurones in a concentration-dependent manner. Isoprenaline mimicked the effect of Ad in producing the inhibition of the 'muscarinic' ACh potential. Propranolol antagonized the inhibitory action of Ad. Dibutyryl adenosine 3',5'-monophosphate had no significant effect on the 'muscarinic' ACh potential. Under voltage-clamp conditions, Ad caused an inward current associated with inhibition of the M-current (Brown and Adams 1980). Ad depressed the amplitude of slow postsynaptic currents produced by applications of ACh and muscarinic. At a concentration of 100 microM, Ad produced a 68 +/- 8% (n = 12) depression of the amplitude of the muscarinic ACh current. The inhibition of muscarinic transmission induced by Ad is due to a direct suppression of the muscarinic current at the postsynaptic membrane in bullfrog sympathetic ganglia.

Electrophysiology: a method to investigate the functional properties of ligand-gated channels.

Ligand-gated channels (LGCs) play a fundamental role in the fast transmission of electrical activity from neuron to neuron and/or to effector cells. Studies of LGCs in isolation have become possible since the identification of genes coding for these membrane proteins together with the establishment of reconstitution techniques in host systems. Methods for electrophysiological investigations of LGCs reconstituted either in the Xenopus oocytes or stably tranfected in cell lines are discussed. Functional studies of reconstituted receptors enable fast determination of LGCs' pharmacological profiles and comparison of their physiological properties. Combination of molecular engineering with physiological measurements allows studies with unpreceeding resolution and it is now possible to examine at the amino-acid level the contribution of some residues in the formation of the ligand-binding site or the ionic channel domains.