Does general anesthetic-induced desensitization of the Torpedo acetylcholine receptor correlate with lipid disordering?

We have tested the hypothesis that general anesthetics stabilize the desensitized state of the nicotinic acetylcholine receptor by disordering its surrounding lipids. Acetylcholine receptor-rich postsynaptic membranes from the electroplaques of Torpedo were used in this study to obtain the highest possible receptor specific activity in native membranes. We examined 18 general anesthetics, including six inhalation agents, eight 1-alcohols, the enantiomers of 2-octanol, and two intravenous general anesthetics (pentobarbital and ethylcarbamate). The degree of desensitization after preincubation with the general anesthetics was determined by brief exposure to [3H]acetylcholine, making use of the facts that desensitized receptors have much higher affinity than do those in the resting state and that interconversion between the states is slow. All of the general anesthetics desensitized the receptor within minutes, exhibiting steep concentration-response curves with Hill coefficients generally within the range of 2-4. At the highest general anesthetic concentrations, almost all receptors were desensitized. The concentrations that desensitized half of the resting state receptors varied by > 3000-fold. The 2-octanol enantiomers were without stereoselectivity. Membrane order was examined in parallel by using spin-labeled fatty acids doped into the native membranes. The spin label 5-doxylpalmitate reported from the most ordered part of the bilayer near the aqueous interface, whereas 12-doxylstearate reported from the less ordered region nearer the center of the bilayer. The spin label deeper in the membranes was 3 times more sensitive to a given anesthetic than was the other probe. At both depths in the membrane general anesthetics decreased lipid order linearly with increasing concentration. The range of disordering potencies (change in order parameter induced by a unit concentration of general anesthetic in the aqueous phase) was 5333 for 5-doxylpalmitate and 7143 for 12-doxylstearate, but the range of disordering compared at equally desensitizing concentrations was reduced by 875- and 1430-fold, respectively. The average degrees of disordering at concentrations that desensitized half of the resting state receptors were 1.5% and 4.4%, respectively. It is unlikely that changes in membrane order parameter per se cause desensitization, because the associated changes in order parameter can be reproduced by changes in cholesterol content or temperature that do not cause desensitization. We conclude that, although there is a strong association between anesthetic-induced membrane disordering and desensitization, more detailed tests of a mechanistic nature will be necessary to elucidate the mechanisms underlying the Meyer-Overton-type behavior we have observed.

Stereoselectivity of channel inhibition by secondary alkanol enantiomers at nicotinic acetylcholine receptors.

BACKGROUND: At the nicotinic acetylcholine receptor, long chain alkanols reduce, whereas short chain alkanols augment endplate currents. Using the enantiomers of five members of a homologous series of secondary alkanols (2-butanol through 2-octanol), we tested the hypothesis that these actions occur at a single hydrophobic site in the lumen of the channel. Small alkanols would bind to this site without blocking the channel, stabilizing the open state and enhancing the apparent affinity of the agonist for channel opening. Long chain alkanols would bind the same site and simply inhibit without affecting the agonist's apparent affinity. METHODS: Agonist-stimulated 86Rb+ efflux from acetylcholine receptor-rich vesicles from Torpedo nobiliana was studied by adding agonist and allowing efflux to proceed for 10 s before termination by filtration. RESULTS: All of the 2-alkanols inhibited 86Rb+ efflux elicited by a maximally stimulating concentration of agonist. Inhibitory potency increased logarithmically with the number of carbon atoms in the hydrocarbon chain of the alkanol. The inhibitory potency of the enantiomers of 2-butanol differed twofold, but the other enantiomers exhibited no stereoselectivity. The enantiomers of 2-octanol caused a concentration-dependent depression of carbamylcholine-stimulated 86Rb+ efflux without significantly altering the agonist's apparent dissociation constant. In contrast, the enantiomers of 2-butanol caused: (1) a nonstereoselective decrease in carbachol's apparent dissociation constant and (2) the expected stereoselective decrease in maximal carbamylcholine-stimulated 86Rb+ efflux. CONCLUSIONS: The alkanol site that modulates the apparent agonist affinity for channel opening is distinct from the site that results in inhibition of cation flux through the channel.

Continuous infusion of mivacurium in children.

Mivacurium is a new short-acting competitive neuromuscular blocking agent. Infusion requirements for the maintenance of a stable 90-99% muscle twitch depression were determined in 28 children anaesthetized with nitrous oxide and 1% halothane (inspired) in oxygen or nitrous oxide in oxygen and opioid. Neuromuscular block was assessed by monitoring the force of contraction of the adductor of the thumb during train-of-four (TOF) stimulation at 0.1 Hz. Infusion rate and twitch depression were analysed from 15 to 75 min and from 75 to 135 min after the start of the infusion. In the first period of evaluation, the mean infusion requirement was 10.4 (SEM 0.92) micrograms kg-1 min-1 during the halothane anaesthesia and 13 (1.4) micrograms kg-1 min-1 during the opiod anaesthesia (P less than 0.05). This difference was present also during the second 60-min period. There was no significant correlation between infusion rates required to maintain greater than 90% depression of the first twitch (T1) of the TOF and plasma cholinesterase concentrations. Regardless of the anaesthetic regimen, children recovered rapidly after discontinuing the infusion. The recovery index (25-75% recovery of T1) for all patients was 5.4 (0.57) min with no significant differences between the groups.

Neuromuscular and cardiovascular effects of doxacurium in children anaesthetized with halothane.

The neuromuscular and cardiovascular effects of doxacurium chloride (BW A938U) were evaluated in 27 children (2-12 yr) anaesthetized with 1% halothane and nitrous oxide in oxygen. In nine children the incremental technique was used to establish a cumulative dose-response curve by train-of-four stimulation. The remaining children received either 30 or 50 micrograms kg-1 of the drug as a single bolus. The median ED50 and ED95 of doxacurium in children were 19 and 32 micrograms kg-1, respectively. No clinically significant change in heart rate or arterial pressure occurred. Following doxacurium 30 micrograms kg-1 and 50 micrograms kg-1, recovery to 25% of control occurred in 25 (SEM 6) and 44 (3) min, respectively. The recovery index (25-75% of control) was 27 (2) min. The duration of action of doxacurium is similar to that of tubocurarine and dimethyl-tubocurarine in children. Compared with adults, children seem to require more doxacurium (microgram kg-1) to achieve a comparable degree of neuromuscular depression, and they recover more rapidly.

Neuromuscular and cardiovascular effects of mivacurium in children.

The neuromuscular and cardiovascular effects of mivacurium chloride (BW B1090U) were evaluated in 90 children (2-12 yr) during N2O:O2 halothane or N2O:O2 narcotic anesthesia. Neuromuscular response was evaluated by recording the force of contraction of the adductor of the thumb during train-of-four stimulation at 0.1 Hz. The children were divided into two groups. Patients in group A (n = 45) were anesthetized with N2O:O2 and halothane (1% inspired) and patients in group B (n = 45) were anesthetized with N2O:O2 and fentanyl or morphine. Each group was further divided into five subgroups of nine children. Children in the first three sets of subgroups (A1-A3, B1-B3) received an initial dose of 0.02, 0.04, 0.05, 0.06 or 0.07 mg/kg mivacurium to determine dose response relationships under the different anesthetic regimens. The ED50 and ED95 neuromuscular blocking doses calculated from this single dose technique were 0.051 mg/kg and 0.095 mg/kg, respectively, in children anesthetized with halothane N2O:O2, and 0.059 mg/kg and 0.11 mg/kg in children anesthetized with N2O:O2 narcotic. The fourth subset of each group (A4 and B4) received 0.09 mg/kg and 0.11 mg/kg mivacurium, the estimated ED95 for each respectively. The last subsets (A5 and B5) received 0.2 mg/kg. This dose induced 100% depression of the twitch response in all 18 patients in 1.8 +/- 0.1 min, with recovery to 5%, 25%, and 95% of control occurring in 8.4 +/- 0.5, 11.2 +/- 0.6 and 18.4 +/- 1.6 min, respectively. The recovery indices for all patients were 4.6 +/- 0.6 min for 25-75% recovery and 9.7 +/- 1.3 min for 5-95% recovery.(ABSTRACT TRUNCATED AT 250 WORDS)

Nonanesthetic alcohols dissolve in synaptic membranes without perturbing their lipids.

While many theories of general anesthesia postulate a lipid site of action, there has been no adequate explanation for the lack of anesthetic potency of the highly hydrophobic primary alkanols with more than 12 carbons (the cut-off). Some work suggests that these nonanesthetic alcohols do not dissolve in membranes. Other work contradicts this and suggests that an anesthetic site on a protein provides a better explanation. Here we show that both the anesthetic dodecanol and the nonanesthetic tetradecanol are taken up equally well into the tissues of animals and into isolated postsynaptic membranes. When a group of Rana pipiens tadpoles were treated with dodecanol, half were anesthetized by 4.7 microM (free aqueos concentration), and the corresponding concentration in the tissues was found to be 0.4 mmol per kg wet weight. Prolonged exposure (92 hr) to tetradecanol produced even higher tissue concentrations (0.7 mmol per kg wet weight), yet no anesthetic effects were observed. Furthermore, general anesthetics are thought to act on postsynaptic membranes but both alkanols partitioned into postsynaptic membranes from Torpedo electroplaques. The spin label, 12-doxyl stearate, was incorporated into these membranes. The lipid order parameter it reported was decreased by the anesthetic alcohols (octanol, decanol, and dodecanol), whereas the nonanesthetic alcohols either did not change it significantly (tetradecanol) or actually increased it (hexadecanol and octadecanol). Thus, although lipid solubility is unable to account for the pharmacology of the cut-off in potency of the long-chain alcohols, lipid perturbations provide an accurate description.

Anaesthetic potencies of primary alkanols: implications for the molecular dimensions of the anaesthetic site.

1. We have redetermined the anaesthetic potencies (EC50S) for a series of primary alkanols, to resolve uncertainties about the molecular dimensions of the anaesthetic site resulting from the use of data from different laboratories. 2. For each alkanol, concentration-response relationships for loss of righting reflex (LRR) were plotted for over one hundred tadpoles, and the median effective concentrations determined. Aqueous concentrations present during potency assays were determined independently, and for alkanols with chain length greater than nonanol, correction was made for depletion from the aqueous phase. 3. The EC50S were found to decrease logarithmically with increasing number of carbon atoms in the hydrocarbon chain of the alkanol (CN), such that, on average, each additional methylene group was associated with an approximately four fold increase in potency. 4. The relationship between log EC50 and CN was best described by the quadratic equation, log EC50 = 0.022 (+/- 0.0038) CN2 + 0.76 (+/- 0.051) CN + 3.7 (+/- 0.14) (r2 = 0.9951). 5. A previously described correlation between the apparent changes in the free energy of binding of an additional methylene group both to luciferase and to the sites for LRR in tadpoles was not confirmed. 6. A cut-off in potency beyond dodecanol was established in experiments where tadpoles were maintained in supersaturated solutions of tridecanol for 20 h without demonstrable LRR. 7. These findings indicate that the soluble enzyme firefly luciferase does not adequately model the anaesthetic site. Specifically, there are discrepancies in the position of cut-off, and the apparent changes in the free energy of binding, per methylene group, of an alkanol to luciferase do not parallel that for tadpoles.

Open versus closed chest cardiac massage in non-traumatic cardiac arrest.

Since the rediscovery and popularization of closed-chest cardiopulmonary resuscitation (CPR) in the early 1960s, this technique has largely replaced open-chest cardiac massage. However, in the ensuing quarter of a century, a large amount of data has been accumulated that seems to indicate that open-chest CPR is the physiologically superior method of cardiopulmonary resuscitation. This paper reviews that data comparing these two CPR modalities. The first part discusses data obtained from animal studies, while the second considers the limited amount of information that has been obtained from human investigations. The authors concludes that a randomized clinical trial of open-chest and closed-chest CPR is needed to fully evaluate the efficacy of these two resuscitative techniques as well as to define the most appropriate circumstances for the use of internal cardiac massage.

Anesthetic potencies of secondary alcohol enantiomers.

The Meyer-Overton rule has been interpreted to mean that general anesthetics act at a nonpolar site, either in a lipid bilayer or a protein. Optical isomers, also called enantiomers, are pairs of compounds with the same molecular formula and functional groups, but which differ in the arrangement of the groups around an "asymmetric" carbon atom and in the direction they rotate plane-polarized light. By definition, enantiomers that are anesthetics can distinguish between stereoselective and nonselective sites of anesthetic action. We used such enantiomers to determine whether anesthetics are stereoselective in their actions on animals by measuring the potencies of a homologous series of secondary aliphatic alcohols from 2-butanol through 2-octanol in tadpoles, using reversible loss of righting reflex as the endpoint. None of the isomeric pairs exhibited significant differences in potency. Anesthetic potency increased logarithmically with the number of carbon atoms in the hydrocarbon chain of the alcohol. The ED50 +/- SE (mM) for the (+) and (-) forms of the alcohols, respectively, were as follows: 2-butanol 17 +/- 1.2, 17 +/- 1.1; 2-pentanol 4.7 +/- 0.28, 4.8 +/- 0.27; 2-hexanol 1.33 +/- 0.068, 1.42 +/- 0.079; 2-heptanol 0.32 +/- 0.011, 0.33 +/- 0.020; and 2-octanol 0.063 +/- 0.0042, 0.061 +/- 0.0032. These data demonstrate a lack of stereoselectivity in the interactions between the anesthetic secondary alcohols and their site of action in animals.

Comparison of open-chest cardiac massage techniques in dogs.

Manual compression of the heart during open-chest cardiac massage (OCPR) has been shown to be superior to closed-chest compression. Our study sought to determine, in a canine model, the optimal hand position for manual compression of the heart. Twelve dogs were anesthetized with ketamine, an orotracheal tube was placed, and anesthesia was maintained with halothane and nitrous oxide. Cannulae were placed to monitor diastolic (DBP) and systolic (SBP) blood pressures, intracranial pressure (ICP), and common carotid blood flow (CCBF). Control values were obtained under light general anesthesia, and ventricular fibrillation was induced. External CPR (ECPR) was performed with a mechanical compressor before opening the chest and pericardium through the left fifth interspace. The following sequence of three hand positions was used for OCPR: technique A, one-handed technique with thumb on left ventricle, fingers over the right ventricle, and apex in palm; technique B, two-handed technique with right ventricle cupped in left hand and fingers of right hand over left ventricle; and technique C, one-handed technique with fingers of right hand over left ventricle and heart against sternum. Each was done at a rate of 60 compressions per minute with the operator blind to results during performance. All three techniques produced significantly (P less than .05) greater DBPs and CCBFs when compared with ECPR. All three also produced significantly lower (P less than .05) ICPs when compared with ECPR. DBPs, SBPs, CCBFs, and cerebral perfusion pressures were similar for techniques B and C, and all were significantly greater (P less than .05) than those achieved with technique A.(ABSTRACT TRUNCATED AT 250 WORDS)

Enflurane, halothane and isoflurane do not inhibit angiotensin converting enzyme activity.

We studied the effect of halothane, enflurane and isoflurane on angiotensin converting enzyme (ACE) activity using [3H]-benzoyl-phenylalanyl-alanyl-proline (BPAP) as a substrate. Isolated rabbit lungs were perfused in a recirculating system in vitro with BPAP in Krebs-Ringer solution. The rate of metabolism and per cent metabolism were determined before and after treatment for 30 minutes with four MAC multiples of enflurane, halothane or isoflurane. The effects of the anaesthetics on ACE activity were determined by calculating per cent inhibition of metabolism of BPAP using data from the control and test period for each lung. The average metabolism of BPAP at 15 minutes during the control period was 76.5 per cent (+/- 1.92 SEM). No anaesthetic significantly inhibited metabolism of BPAP. Likewise there was no effect on BPAP first order kinetics. Although potent inhalation anaesthetics may alter the renin-angiotensin-aldosterone axis, they do not affect this crucial step.

Kinetics and stoichiometry of Na-dependent Li transport in human red blood cells.

This paper describes the kinetics and stoichiometry of a tightly coupled Na-Li exchange transport system in human red cells. The system is inhibited by phloretin and furosemide but not by ouabain. Li influx by this system increases and saturates with increasing concentrations of external Li and internal Na and is inhibited competitively by external Na. Comparable functions relate Li efflux and Na efflux to internal and external Li and Na concentrations. Analysis of these relations yields the following values for the ion concentrations required to half-maximally activate the transport system: internal Na and Li 9.0 and 0.5 mM, respectively, external Na and Li 25 and 1.5 mM, respectively. The system performs a 1:1 exchange of Na and Li moving in opposite directions across the red cell membrane. We found no evidence for a simultaneous transport of more than one Na and Li by the system. The maximum transport rate of Na-dependent Li transport varied between 0.1 and 0.37 mmol/(liter of cells X h) in the red cells of the five normal male subjects studied. No significant variations between individual subjects were observed for bicarbonate-stimulated Li transport and for the residual Li fluxes which occur in the absence of bicarbonate and in the presence of ouabain plus phloretin.