Changes in acetylcholine receptor number in muscle from critically ill patients receiving muscle relaxants: an investigation of the molecular mechanism of prolonged paralysis.

OBJECTIVE: Previous reports have described prolonged paralysis after the administration of muscle relaxants in critically ill patients. The purpose of this study was to examine possible pathophysiologic causes for this paralysis by measuring muscle-type, nicotinic acetylcholine receptor number in necropsy muscle specimens from patients who had received muscle relaxants to facilitate mechanical ventilation before death. DESIGN: Prospective laboratory study of human muscle collected at autopsy. SETTING: Medical and surgical intensive care units (ICUs) at a university hospital and a research laboratory. PATIENTS: Fourteen critically ill patients, with a variety of diagnoses, all of whom required mechanical ventilatory support before their deaths in the ICU and who underwent post mortem examination. Patients were arbitrarily divided into three groups, according to their total vecuronium dose and number of days mechanically ventilated before death. Three patients were in the control group (defined as dying within 72 hrs of initiation of ventilatory support and receiving a total dose of < 5 mg of vecuronium). Six patients were in the low-dose group (defined as requiring ventilatory support for > 3 days before death and receiving a total vecuronium dose of < or = 200 mg). Five patients were in the high-dose group (defined as requiring ventilatory support for > 3 days before death and receiving a total vecuronium dose of > 200 mg). INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Nicotinic acetylcholine receptor numbers as measured by specific 125I-alpha-bungarotoxin binding to human rectus abdominis muscle obtained at autopsy were determined. In general, receptor number reflected the clinical requirements for the muscle relaxants of each patient. Patients who had increasing requirements for muscle relaxants before death had increases in receptor number, as compared with control values. CONCLUSIONS: The increase in nicotinic acetylcholine receptor number in muscle from patients with an increasing requirement for muscle relaxants before death suggests that nicotinic acetylcholine receptor up-regulation may underlie the increased requirements for muscle relaxants seen in some patients. Furthermore, these findings suggest that muscle relaxant-induced, denervation-like changes may at least be partially responsible for prolonged muscle paralysis after the long-term administration of muscle relaxants. This study may provide the first information into the molecular mechanisms underlying prolonged paralysis.

Actions of pentobarbital enantiomers on nicotinic cholinergic receptors.

The enantiomers of pentobarbital had four different actions on the nicotinic receptor-rich membranes from Torpedo electroplaques. (i) Both inhibited cholinergically stimulated cation flux through the receptor's channel, with IC50 values of approximately 25 microM and extremely weak stereoselectivity. (ii) (R)-(+)-[14C]Pentobarbital bound to a saturable site with an apparent dissociation constant of 100 microM, a Hill coefficient of 1.2, and a stoichiometry of 1:1 with the acetylcholine binding sites. (S)-(-)-Pentobarbital also displaced (+)-[14C]pentobarbital but its IC50 was 4-fold higher than that of the (+)-enantiomer under the same conditions. (iii) Both enantiomers caused a stereoselective allosteric inhibition of [3H]acetylcholine binding, which occurred over the same concentration range and with the same stereoselectivity as barbiturate binding. (iv) Above 1 mM, pentobarbital caused an unexpected and sudden increase in [3H]acetylcholine binding, which lacked significant stereoselectivity. These results are consistent with a model where low concentrations of pentobarbital act on the receptor by binding to allosteric sites that have higher affinity but lower stereoselectivity for the open channel conformation than for the resting conformation, whereas the highest concentrations of pentobarbital act by nonspecific mechanisms mediated by general membrane perturbations.

Barbiturates bind to an allosteric regulatory site on nicotinic acetylcholine receptor-rich membranes.

The ability of barbiturates to bind to acetylcholine receptor-rich membranes purified from the electroplaques of Torpedo nobiliana was examined by centrifugation assay. [14C]Amobarbital both partitioned into the membrane and bound displaceably to a site with an equilibrium dissociation constant of 12 microM. This low affinity made the stoichiometry difficult to obtain despite the high specific activity of acetylcholine receptors in this membrane preparation. However, the data are not inconsistent with a stoichiometry of one barbiturate-binding site per acetylcholine-binding site. Displaceable [14C]amobarbital binding was completely inhibited by barbiturates (IC50: amobarbital, 28 microM; secobarbital, 110 microM; pentobarbital, 400 microM; phenobarbital, 690 microM; butabarbital, 690 microM; and barbital, 5.1 mM. alpha-Bungarotoxin had no effect, but cholinergic ligands that convert the acetylcholine receptor to the desensitized state (acetylcholine, carbamylcholine, and, to a lesser extent, d-tubocurarine) partially inhibited displaceable [14C]amobarbital binding. This cholinergic inhibition was prevented by preincubation with alpha-bungarotoxin, implying an allosteric mediation through the classical cholinergic site. This negative interaction between the cholinergic and the barbiturate sites was mutual with barbiturates partially decreasing equilibrium [3H]acetylcholine binding in a saturable fashion with relative affinities that parallel those for inhibiting [14C]amobarbital binding (IC50). These data establish a mutual negative heterotropic interaction between barbiturate-binding sites and cholinergic binding sites on the nicotinic acetylcholine receptor from Torpedo.

Two pools of cholesterol in acetylcholine receptor-rich membranes from Torpedo.

The acetylcholine receptor (AChR)-containing electroplax membranes from Torpedo californica have a relatively high cholesterol content. Reconstitution studies suggest that this cholesterol may be important in preserving or modulating the function of the acetylcholine receptor-channel complex. We have manipulated cholesterol levels in intact Torpedo AChR-rich membrane fragments using small, unilamellar phosphatidylcholine liposomes. Conditions have been established that allow further subfractionation of sucrose gradient purified Torpedo electroplax membranes into AChR-rich and ATPase-rich populations and that, at the same time, achieve cholesterol depletion without phospholipid back exchange or fusion. The incubation of membranes with excess liposomes could only achieve about a 50% reduction in the molar ratio of cholesterol to phospholipid. In no case was the number of cholesterol molecules per AChR oligomer reduced below 36. The remaining cholesterol could not be depleted either by longer incubations or by multiple, sequential depletions. Cholesterol depletion was accompanied by a significant increase in bulk membrane fluidity as measured by electron spin resonance spectroscopy, but the equilibrium binding parameters of acetylcholine to its receptor were unaltered. This suggests strongly that there exist two pools of cholesterol in the AChR-rich Torpedo electroplax membrane: an easily depleted fraction that influences bulk fluidity, and a tightly-bound fraction perhaps surrounding the AChR oligomer.

Actions of general anesthetics on acetylcholine receptor-rich membranes from Torpedo californica.

The molecular mechanisms by which general anesthetics act on postsynaptic membranes can only be worked out in a highly purified, homogeneous system. The nicotinic acetylcholine receptor-rich membranes from the electric tissue of Torpedo californica are currently the only postsynaptic membranes that fulfill this condition. Is this peripheral synapse acted on with a pharmacologic specificity similar to that for general anesthesia, and how much less sensitive is it to anesthetic action than the unknown central site? To answer these questions, the authors studied the effects of 13 anesthetic compounds, including volatile general anesthetics, alkanols, and urethane, on the equilibrium binding of 3H-acetylcholine to these nicotinic receptors. As the anesthetic concentration was raised, all the agents first increased acetylcholine binding steeply and then, with few exceptions, decreased it again at higher concentrations. Anesthetics increased acetylcholine binding by decreasing acetylcholine's dissociation constant without changing the Hill coefficient or the number of sites. To a first approximation, the relative ability of these agents to increase 3H-acetylcholine binding parallels that of anesthesia in vivo as predicted by the Meyer-Overton lipid solubility rule. On average, they produced half maximal increases in acetylcholine binding (EC50) at about four times the concentration that causes loss of righting reflex in one-half of a group of animals (ED50). However, a few agents deviated from this relationship. They were the agents with greatest general anesthetic potency in both the volatile anesthetic series (thiomethoxyflurane) and the normal alcohol series (octanol), and required up to 17 times their ED50s to achieve a half effect on acetylcholine binding. Although the concentrations required were high, these effects were reversible.(ABSTRACT TRUNCATED AT 250 WORDS)

Pressure reversal of the action of octanol on postsynaptic membranes from Torpedo.

Octanol increases the binding of [3H]-acetylcholine to the desensitized state of the nicotinic receptor in postsynaptic membranes prepared from Torpedo californica. This increase in binding results from an increase in the affinity of [3H]-acetylcholine for its receptor without any change in the number of sites or the shape of the acetylcholine binding curve. High pressures of helium (300 atm) decrease [3H]-acetylcholine binding by a mechanism that changes only the affinity of acetylcholine binding. Helium pressure reverses the effect of octanol on the affinity of [3H]-acetylcholine for its receptor. This pressure reversal of the action of octanol at a postsynaptic membrane is consistent either with pressure counteracting an octanol-induced membrane expansion or with independent mechanisms for the actions of octanol and pressure. The data do not conform with a mechanism in which pressure displaces octanol from a binding site on the receptor protein.