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W R Lieb

Publications and source records attributed to W R Lieb.

At least 37 records · Page 2Linked to original sources

Temperature dependence of the potency of volatile general anesthetics: implications for in vitro experiments.

BACKGROUND: When performing experiments at room temperature with volatile general anesthetics and in vitro mammalian preparations (such as isolated neurons), the question arises as to which concentrations of anesthetics are "clinically relevant." Different choices can lead to different interpretations of the anesthetic sensitivities of putative target sites. METHODS: Published data on the temperature dependence of minimum alveolar concentration were analyzed. RESULTS: Although gas-phase potencies of volatile anesthetics increase markedly with decreasing temperature, the corresponding aqueous-phase potencies are relatively constant. Changes in minimum alveolar concentration with temperature can be accounted for, on physical grounds, in terms of the temperature dependencies of anesthetics binding to their central nervous system target sites. CONCLUSION: When performing room-temperature in vitro experiments on simple mammalian preparations with a volatile anesthetic, the aqueous-phase (but not the gas- phase) minimum alveolar concentration calculated at normal body temperature is, to a first approximation, the appropriate choice for a clinically relevant anesthetic concentration. Recommended aqueous-phase minimum alveolar concentration values (in MM) for desflurane, enflurane, halothane, isoflurane, and sevoflurane have have been calculated.

Anesthetics, Inhalation↗

Actions of general anaesthetics on 5-HT3 receptors in N1E-115 neuroblastoma cells.

1. NIE-115 mouse neuroblastoma cells were studied under voltage clamp in the whole-cell patch-clamp configuration. Peak currents induced by bath application of 5-hydroxytryptamine (5-HT) were inwardly rectifying, reversed at 0.4 +/- 0.2 mV (mean +/- s.e.mean), and were approximately half-inhibited (at 1 microM 5-HT) by 2 nM of the 5-HT3 selective antagonist MDL-72222 (3-tropanyl-3,5-dichlorobenzoate). 2. Peak inward currents activated by a low concentration of 5-HT at a holding potential of -50 mV were potentiated by volatile general anaesthetics. At their human minimum alveolar concentrations (MACs), the degree of potentiation increased in the order isoflurane < halothane < enflurane < methoxyflurane. Potentiation by methoxyflurane was independent of membrane potential in the range -70 mV to +40 mV. The reversal potential was the same in the presence and absence of methoxyflurane. 3. Methoxyflurane shifted the 5-HT dose-response curve to lower 5-HT concentrations, without significantly changing the Hill coefficient or maximum response. The EC50 concentration for 5-HT decreased from 1.86 +/- 0.02 microM to 1.07 +/- 0.11 microM (means +/- s.e.mean) due to the presence of 1 MAC (270 microM) methoxyflurane. 4. In contrast to the volatile anaesthetics, the barbiturate anaesthetic, thiopentone, inhibited the 5-HT3 receptor. Hill analysis of thiopentone dose-response data gave an average IC50 = 117 +/- 8 microM thiopentone and Hill coefficient = 1.6 +/- 0.2 (means +/- s.e.mean). These parameters were not significantly different for data obtained at 5-HT concentrations above and below the control EC50 concentration for 5-HT, consistent with non-competitive inhibition. 5. The n-alcohols occupied an intermediate position between the volatile and barbiturate anaesthetics. The lower alcohols (butanol and hexanol) potentiated 5-HT responses at low alcohol concentrations but inhibited them at high concentrations. In contrast, the higher alcohols (octanol, decanol, dodecanol, tridecanol, tetradecanol and pentadecanol) produced no potentiation, but only inhibition, at all alcohol concentrations. 6. Inhibition of the 5-HT3 receptor by the n-alcohols exhibited a cutoff in potency similar to those previously found for tadpoles, luciferase enzymes and a neuronal nicotinic acetylcholine receptor channel.

Alcohols↗

Effects of inhalational general anaesthetics on native glycine receptors in rat medullary neurones and recombinant glycine receptors in Xenopus oocytes.

1. Glycine responses were studied under voltage clamp in Xenopus oocytes injected with cDNA encoding mammalian glycine receptor subunits and in rat medullary neurones. Bath application of glycine gave strychnine-sensitive currents which reversed close to the expected equilibrium potentials for chloride ions. The peak currents for the receptors expressed in oocytes fitted a Hill equation with EC50 = 215 +/- 5 microM and Hill coefficient nH = 1.70 +/- 0.05 (means +/- s.e. means). The peak currents from the receptors in medullary neurones fitted a Hill equation with EC50 = 30 +/- 1 microM and Hill coefficient nH = 1.76 +/- 0.08. The current-voltage relationship for the receptors expressed in oocytes showed strong outward rectification (with Vrev = -21 +/- 2 mV), while that for the glycine responses from the medullary neurones in symmetrical Cl- was linear (with Vrev = 3.2 +/- 0.6 mV). 2. Inhalational general anaesthetics, at concentrations close to their human minimum alveolar concentrations (MACs), potentiated responses to low concentrations of glycine. The potentiation observed with the recombinant receptors (between 60-22%) was approximately twice that found with the medullary neurones (between 40-80%). For both the recombinant receptors and the receptors in medullary neurones, the degree of potentiation increased in the order of methoxyflurane approximately sevoflurane < halothane approximately isoflurane approximately enflurane. There was no significant difference between the potentiations observed for the two optical isomers of isoflurane. 3. For both the recombinant and native receptors, isoflurane potentiated the currents in a dose-dependent manner at low concentrations of glycine, although at high glycine concentrations the anaesthetic had no significant effect on the glycine-activated responses. The major effect of isoflurane was to cause a parallel leftward shift in the glycine concentration-response curves. The glycine EC50 concentration for the recombinant receptors decreased from a control value of 215 +/- 5 microM to 84 +/- 7 microM glycine at 610 microM isoflurane, while that for the medullary neurones decreased from a control value of 30 +/- 1 microM to 18 +/- 2 microM glycine at the same concentration of isoflurane. The potentiation was independent of membrane potential. 4. Isoflurane also potentiated responses to taurine, a partial agonist at the glycine receptor. This was observed for receptors expressed in oocytes at both low and saturating concentrations of taurine. The EC50 concentration decreased from a control value of 1.6 +/- 0.2 to 0.9 +/- 0.1 mM taurine in the presence of 305 microM isoflurane, while the maximum response to taurine increased from 47 +/- 2 to 59 +/- 2% of the maximum response to glycine. 5. Glycine receptors, like other members of the fast ligand-gated receptor superfamily, are sensitive to clinically relevant concentrations of inhalational general anaesthetics. Effects at these receptors may, therefore, play some role in the maintenance of the anaesthetic state.

Administration, Inhalation↗

Actions of general anaesthetics on a neuronal nicotinic acetylcholine receptor in isolated identified neurones of Lymnaea stagnalis.

1. Completely isolated identified neurones from the right parietal ganglion of the pond snail Lymnaea stagnalis were studied under two-electrode voltage-clamp. Neuronal nicotinic acetylcholine receptor currents were studied at low acetylcholine (ACh) concentrations (< or = 200 nM). At these levels, control currents were non-desensitizing and proportional to the square of the ACh concentration. 2. IC50 concentrations were determined for the steady-state inhibition of the ACh-activated current by 31 general anaesthetics plus the non-anaesthetic alcohol n-tridecanol. The general anaesthetics included inhalational agents, n-alcohols, n-alkane-(alpha,omega)-diols, cycloalcohols and an n-alkane. 3. Anaesthetic inhibition was independent of voltage and consistent with two anaesthetic-binding sites on the receptor. 4. IC50 concentrations for inhibiting the neuronal nicotinic ACh receptor correlated well (r = 0.97) with EC50 concentrations for general anaesthesia. The maximum deviation from the line of identity was less than fourfold. The inhalational agents tended to be more potent as inhibitors of the ACh receptor than as general anaesthetics, while the alcohols and diols were less potent. 5. The inhibition of the ACh-induced current by the homologous series of n-alcohols exhibited a cutoff at the same position (just after dodecanol) as found for the induction of general anaesthesia in tadpoles. 6. Polarity profile maps of the anaesthetic-binding sites on the neuronal nicotinic ACh receptor were calculated from IC50 concentrations for the homologous series of n-alcohols and n-alkane-(alpha,omega)-diols. They reveal amphiphilic sites with apolar regions capable of accommodating the hydrocarbon chains of n-alcohols as large as decanol. A striking resemblance was found to profiles previously calculated from data for tadpole general anaesthesia.

Acetylcholine↗

The effects of temperature on the interactions between volatile general anaesthetics and a neuronal nicotinic acetylcholine receptor.

1. Completely isolated identified neurones from the right parietal ganglion of the pond snail Lymnaea stagnalis were investigated under two-electrode voltage clamp. Neuronal nicotinic acetylcholine receptor (AChR) currents were studied at low acetylcholine concentrations (< or = 200 nM). 2. Inhibition of the ACh-induced currents by three volatile general anaesthetics (halothane, isoflurane and methoxyflurane) and the specific inhibitor (+)-tubocurarine was studied as a function of temperature (over the range 4-25 degrees C). 3. The inhibition by the volatile anaesthetics increased (inhibition constants decreased) with decreasing temperature while the inhibition by (+)-tubocurarine did not change significantly near room temperature, but decreased at lower temperatures. The (+)-tubocurarine inhibition appeared to be competitive in nature and showed no significant voltage-dependence. 4. The van't Hoff plots (logarithms of the dissociation constants against reciprocal absolute temperature) were linear for the anaesthetics, but markedly non-linear for (+)-tubocurarine. From these plots, values for the changes in the standard Gibbs free energy delta G degrees water-->AChR, enthalpy delta H degree water-->AChR, entropy delta S degree water-->AChR and heat capacity delta Cp degree water-->AChR were determined. Tubocurarine was found to bind very much tighter to the receptor than the volatile anaesthetics due, entirely, to a favourable increase in entropy on binding. 5. A comparison between the temperature-dependence of the anaesthetic inhibition of the ACh receptor and that of general anaesthetic potencies in animals indicates that the temperature-dependence of animal potencies might be simply accounted for in terms of changes in anaesthetic/receptor binding.

Acetylcholine↗

Molecular and cellular mechanisms of general anaesthesia.

General anaesthetics are much more selective than is usually appreciated and may act by binding to only a small number of targets in the central nervous system. At surgical concentrations their principal effects are on ligand-gated (rather than voltage-gated) ion channels, with potentiation of postsynaptic inhibitory channel activity best fitting the pharmacological profile observed in general anaesthesia. Although the role of second messengers remains uncertain, it is now clear that anaesthetics act directly on proteins rather than on lipids.

Anesthesia, General↗

Can the stereoselective effects of the anesthetic isoflurane be accounted for by lipid solubility?

Isoflurane is an inhalational general anesthetic widely used in surgical operations as a racemic mixture of its two optical isomers. The recent availability of pure enantiomers of isoflurane has encouraged their use in experimental studies, and stereoselective effects have now been observed on anesthetic-sensitive neuronal ion channels. Although it has been assumed that such chiral effects demonstrate direct interactions with proteins, it is possible that they could be due to stereoselective interactions with chiral membrane lipids. We have determined the partition coefficients of the two optical isomers of isoflurane between lipid bilayers and water, using racemic isoflurane and gas chromatography with a chiral column. For lipid bilayers of phosphatidylcholine (PC) and 4 mol% phosphatidic acid (PA), both with and without cholesterol (CHOL), we found equal partitioning of the isoflurane optical isomers. The ratios of the S(+) to R(-) isoflurane partition coefficients were (mean +/- SEM): 1.018 +/- 0.010 for bilayers of PC/CHOL/PA (mole ratios 56:40:4) and 1.011 +/- 0.002 for bilayers of PC/PA (mole ratio 96:4). Molar partition coefficients for racemic isoflurane were 49 +/- 4 and 165 +/- 10, respectively. These findings support the view that the stereoselective effects on ion channels observed with isoflurane are due to direct actions on proteins rather than lipids.

Animals↗

Insensitivity of P-type calcium channels to inhalational and intravenous general anesthetics.

BACKGROUND: Voltage-gated Ca2+ channels long have been considered plausible targets for general anesthetics. Previous anesthetic studies have focused on L-, T-, or N-type channels, but there have been no studies on channels identified as P-type. Since P-type channels may be the most important voltage-gated Ca2+ channels involved in synaptic transmission in mammalian brain, it is important to establish their sensitivity to clinically relevant concentrations of general anesthetics. METHODS: Acutely dissociated cerebellar Purkinje neurons were obtained from 7-14-day-old Sprague-Dawley rats. P-type currents were measured using the whole-cell version of the patch-clamp technique, with Ba2+ as the current carrier. General anesthetics were applied to the neurons in aqueous solution at room temperature (20-23 degrees C). RESULTS: P-type Ca2+ channels were found to be very insensitive to a variety of general anesthetics and ethanol. Inhibitions of less than 10% were produced by 0.35 mM halothane, 0.35 mM isoflurane, 32 microM thiopental, 50 microM pentobarbital, 2 microM propofol, and 200 mM ethanol. Substantial anesthetic inhibition was found only at free aqueous concentrations much greater than those that are clinically relevant. For halothane, the dose-response curve showed an IC50 concentration of 1.17 +/- 0.02 mM and a Hill coefficient of 2.02 +/- 0.04 (mean +/- SEM). CONCLUSIONS: The relatively small inhibitions of P-type Ca2+ channels produced by volatile and intravenous anesthetics at their free aqueous EC50 concentrations for general anesthesia in mammals suggest that these channels do not play a major role in the induction of general anesthesia.

Administration, Inhalation↗

Stereoselective and non-stereoselective actions of isoflurane on the GABAA receptor.

1. Acutely dissociated cerebellar Purkinje neurones from 8-14 day old rats were studied under voltage clamp in the whole-cell patch-clamp configuration. Cl- currents induced by bath application of gamma-aminobutyric acid (GABA) were measured (using symmetrical Cl- solutions) at both low (2 microM) non-desensitizing and high (300 microM) desensitizing concentrations of GABA. 2. At 2 microM GABA, the bicuculline-sensitive Cl- currents were potentiated by racemic isoflurane and both of its optical isomers. Isoflurane had no effect on membrane current in the absence of GABA. The dose-response data for potentiation by racemic isoflurane could be fitted with a Hill equation with an EC50 = 320 +/- 20 microM isoflurane and a Hill coefficient of h = 2.7 +/- 0.4 (means +/- s.e.mean). 3. The potentiations produced by the optical isomers of isoflurane at 2 microM GABA were stereoselective at moderate and high anaesthetic concentrations. The maximum stereoselectivity, about two fold, occurred at the EC50 concentration for general anaesthesia (310 microM isoflurane), with S(+)-isoflurane being more effective than R(-)-isoflurane. At sub-anaesthetic concentrations, the stereoselectivity was less marked and vanished at the lowest concentration used (77 microM isoflurane). 4. The sustained residual current remaining after exposure of neurons to a desensitizing concentration of GABA (300 microM) was inhibited non-stereoselectively, but only at high concentrations of isoflurane. The ratio of inhibitions by S(+)- and R(-)-isoflurane (mean +/- s.e.mean) was 1.14 +/- 0.21 at 770 microM isoflurane. At the EC50 concentration for general anaesthesia, however, the inhibition was barely significant. 5. The above results are discussed in relation to the possible role of the GABAA receptor channel in general anaesthesia.

Anesthesia, General↗

Synthesis and use of the n-bromododecane-1,12-diols as conformational probes for general anesthetic target sites.

The n-bromododecane-1,12-diols with bromine on carbons 2, 3, 5, and 6, respectively, were synthesized and found to be potent general anesthetics. They were also found to be potent inhibitors of firefly luciferase, a protein model for the primary target sites underlying general anesthesia. However, their effects on lipid bilayers were small, lowering the chain-melting phase transition temperature by less than 1 degree C at their EC50 concentrations for general anesthesia. A large dependence upon the position of the bromine atom was found for both n-hexadecane/water partition coefficients and inhibition constants for firefly luciferase; a much smaller positional dependence was found for induction of general anesthesia and for disrupting lipids. These results are consistent with the bulky bromine atom inhibiting the conformational flexibility of the diol hydrocarbon chain, making these bromo diols useful probes for ascertaining the shapes of apolar binding sites. In particular, our measurements suggest that these novel anesthetics produce general anesthesia by binding to long and relatively narrow apolar target sites in the central nervous system.

Anesthesia, General↗

Molecular organization of liquid n-octanol: an X-ray diffraction analysis.

We recorded X-ray diffraction patterns from n-octane, n-octanol, and fully hydrated n-octanol. These patterns were analyzed using a modified version of the Percus-Yevick hard-sphere theory for liquids. A model is presented for the molecular organization of n-octanol that accurately predicts the observed diffraction patterns. The octanol molecules are associated in aggregates, with their hydroxyl groups forming roughly spherical clusters and their hydrocarbon chains pointing outwards, almost fully extended. When n-octanol is hydrated, the overall organization does not change greatly, but water molecules coordinate the hydroxyl groups in the clusters. Estimates are made for the maximum number of octanol molecules in the aggregates.

1-Octanol↗

A simple method for recording single-channel activity from synaptic plasma membranes.

Due to the small size of most nerve terminals, the ion channels which underlie presynaptic currents are usually inaccessible to investigation by conventional electrophysiological techniques. Here we describe a simple method for obtaining single-channel recordings from synaptic plasma membranes that does not require exposure of the native membranes to exogenous lipids or fusogens. To illustrate the method, we have recorded single-channel activity from rat cerebrocortical synaptosomal membranes. Under conditions designed to isolate calcium-independent currents, we describe three channel types that are most commonly observed.

Action Potentials↗

Thermodynamics of anesthetic/protein interactions. Temperature studies on firefly luciferase.

Firefly luciferase is a soluble enzyme which is unusually sensitive to general anesthetics. The inhibition of the highly purified enzyme by three inhalational and three alcohol general anesthetics has been studied as a function of temperature, in the range from 5 to 20 degrees C. Inhibition constants Ki were determined at different temperatures, and van't Hoff plots of ln (Ki) versus reciprocal absolute temperature were found to be linear for all agents. Analysis of these plots gave values for the standard Gibbs free energy, enthalpy and entropy changes for transferring each anesthetic from water to the anesthetic-binding pocket on the protein. The most striking finding was that the enthalpy changes were much more negative for anesthetics binding to the protein than for binding to lipids or simple solvents. Furthermore, amongst the set of anesthetics studied, it was found that increasing potency correlated with favorable enthalpy rather than entropy changes. We discuss our results with respect to the molecular mechanisms underlying general anesthesia.

Anesthesia, General↗

Effects of temperature on the anaesthetic potency of halothane, enflurane and ethanol in Daphnia magna (Cladocera: Crustacea).

1. The effects of temperature on the anesthetic potencies of halothane, enflurane and ethanol have been studied in the water flea Daphnia magna. 2. In the absence of anaesthetics, decreasing temperature resulted in decreased activity by the daphnids. 3. Potencies in the gas phase decreased with increasing temperature for all of the anaesthetics, while aqueous potency decreased for halothane and enflurane but increased for ethanol. 4. Enthalpy calculations suggest that the observed potency changes for the inhalational anaesthetics cannot be accounted for in terms of changing solubility in lipid bilayers but most likely reflect more specific interactions with animal target sites.

Animals↗

Mapping the polarity profiles of general anesthetic target sites using n-alkane-(alpha, omega)-diols.

The effects of the homologous series of n-alkane-(alpha, omega)-diols have been studied on the inhibition of the purified firefly luciferase enzyme from Photinus pyralis, the inhibition of the purified bacterial luciferase enzyme from Vibrio harveyi, and the induction of general anesthesia in Xenopus laevis tadpoles. All but one of the diols tested were found to be reversible general anesthetics. The diols inhibited firefly luciferase by competing with its normal substrate firefly luciferin, and they inhibited bacterial luciferase by competing with the substrate n-decanal. For all but the smallest agent (1,4-butanediol), only a single diol molecule was found to be involved in the inhibition of the enzymes. Inhibition constants Ki were determined for the enzymes, and general anesthetic EC50 concentrations were determined for tadpoles. These data were then used in conjunction with previously determined n-alkane and n-alcohol data to calculate, as a function of chain length, the incremental standard Gibbs free energies delta (delta G0) for adding apolar -CH2- groups and for converting apolar terminal -CH3 groups to polar -CH2OH groups. The resulting plots of delta (delta G0) versus chain length gave a consistent mapping of the polarity profiles of the anesthetic-binding pockets. They clearly reveal the existence of two substantial and distinct polar regions in the anesthetic-binding pocket of firefly luciferase but only one such region for bacterial luciferase and for the unknown target sites underlying general anesthesia. The polarities and geometric properties of these different binding sites for straight-chain anesthetics are discussed in terms of simple models.

Anesthetics↗

Stereospecific effects of inhalational general anesthetic optical isomers on nerve ion channels.

Although it is generally agreed that general anesthetics ultimately act on neuronal ion channels, there is considerable controversy over whether this occurs by direct binding to protein or secondarily by nonspecific perturbation of lipids. Very pure optical isomers of the inhalational general anesthetic isoflurane exhibited clear stereoselectivity in their effects on particularly sensitive ion channels in identified molluscan central nervous system neurons. At the human median effect dose (ED50) for general anesthesia, the (+)-isomer was about twofold more effective than the (-)-isomer both in eliciting the anesthetic-activated potassium current IK(An) and in inhibiting a current mediated by neuronal nicotinic acetylcholine receptors. For inhibiting the much less sensitive transient potassium current IA, the (-)-isomer was marginally more potent than the (+)-isomer. Both isomers were equally effective at disrupting lipid bilayers.

Anesthesia, Inhalation↗

Modulation of the general anesthetic sensitivity of a protein: a transition between two forms of firefly luciferase.

The activities of most proteins are relatively insensitive to general anesthetics. A notable exception is firefly luciferase, whose sensitivity to a wide range of anesthetic agents closely parallels that of whole animals. We have now found that this sensitivity can be controlled by ATP. The enzyme is insensitive at low (microM) concentrations of ATP and very sensitive at high (mM) concentrations. The differential sensitivity varies from anesthetic to anesthetic, being greatest (about a 100-fold difference) for molecules with large apolar segments. This suggests that anesthetic sensitivity is modulated by changes in the hydrophobicity of the anesthetic-binding pocket. Parallel changes in the binding of the substrate firefly luciferin, for which anesthetics compete, indicate that anesthetics bind at the same site as the luciferin substrate. These changes in the nature of the binding pocket modify not only the sensitivity to anesthetics but also the position of the "cutoff" in the homologous series of primary alcohol anesthetics; the cutoff position can vary from octanol to pentadecanol, depending upon the concentration of ATP. Our results suggest that particularly sensitive anesthetic target sites in the central nervous system may possess anesthetic-binding pockets whose polarities are regulated by neuromodulatory agents.

Adenosine Triphosphate↗