W. R. Lieb

How does xenon produce anaesthesia

Since the discovery that the gas xenon can produce general anaesthesia without causing undesirable side effects, we have remained surprisingly ignorant of the molecular mechanisms underlying this clinical activity of an ‘inert’ gas. Although most general anaesthetics enhance the activity of inhibitory GABAA (γ-aminobutyric acid type-A) receptors,, we find that the effects of xenon on these receptors are negligible. Instead, xenon potently inhibits the excitatory NMDA (N-methyl-D-aspartate) receptor channels, which may account for many of xenons attractive pharmacological properties.

Differential sensitivities of mammalian neuronal and muscle nicotinic acetylcholine receptors to general anesthetics.

Background: Nicotinic acetylcholine receptors (nAChRs) are members of a superfamily of fast neurotransmitter‐gated receptor channels that includes the gamma‐aminobutyric acidA (GA‐BAA), glycine and serotonin type 3 (5‐HT3) receptors. Most previous work on the interactions of general anesthetics with nAChRs has involved the muscle‐type receptor. The authors investigate the effects of general anesthetics on defined mammalian neuronal and muscle nAChRs expressed in Xenopus oocytes. Methods: Complementary deoxyribonucleic acid (cDNA) or messenger ribonucleic acid (mRNA) encoding for various neuronal or muscle nAChR subunits was injected into Xenopus oocytes, and the resulting ACh‐activated currents were studied using the two‐electrode voltage‐clamp technique. The effects of halothane, isoflurane, sevoflurane, and propofol on the peak acetylcholine‐induced currents were investigated, and concentration‐response curves were constructed. Results: The neuronal nAChRs were found to be much more sensitive to general anesthetics than were the muscle nAChRs, with IC50 concentrations being 10‐ to 35‐fold less for the neuronal receptors. For the inhalational general anesthetics, the IC50 concentrations were considerably less than the free aqueous concentrations that cause general anesthesia in mammals. In addition, qualitative (dependence on acetylcholine concentration) and quantitative (steepness of concentration‐response curves) differences in the anesthetic interactions between the neuronal and muscle nAChRs suggest that different mechanisms of inhibition may be involved. Conclusions: Although there is considerable uncertainty about the physiologic roles that neuronal nAChRs play in the central nervous system, their extraordinary sensitivity to general anesthetics, particularly the inhalational agents, suggests they may mediate some of the effects of general anesthetics at surgical, or even subanesthetic, concentrations.

Contrasting Synaptic Actions of the Inhalational General Anesthetics Isoflurane and Xenon

Background: The mechanisms by which the inhalational general anesthetics isoflurane and xenon exert their effects are unknown. Moreover, there have been surprisingly few quantitative studies of the effects of these agents on central synapses, with virtually no information available regarding the actions of xenon. Methods: The actions of isoflurane and xenon on &ggr;-aminobutyric acid–mediated (GABAergic) and glutamatergic synapses were investigated using voltage-clamp techniques on autaptic cultures of rat hippocampal neurons, a preparation that avoids the confounding effects of complex neuronal networks. Results: Isoflurane exerts its greatest effects on GABAergic synapses, causing a marked increase in total charge transfer (by approximately 70% at minimum alveolar concentration) through the inhibitory postsynaptic current. This effect is entirely mediated by an increase in the slow component of the inhibitory postsynaptic current. At glutamatergic synapses, isoflurane has smaller effects, but it nonetheless significantly reduces the total charge transfer (by approximately 30% at minimum alveolar concentration) through the excitatory postsynaptic current, with the N-methyl-D-aspartate (NMDA) and &agr;-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate receptor–mediated components being roughly equally sensitive. Xenon has no measurable effect on GABAergic inhibitory postsynaptic currents or on currents evoked by exogenous application of GABA, but it substantially inhibits total charge transfer (by approximately 60% at minimum alveolar concentration) through the excitatory postsynaptic current. Xenon selectively inhibits the NMDA receptor–mediated component of the current but has little effect on the AMPA/kainate receptor–mediated component. Conclusions: For both isoflurane and xenon, the most important targets appear to be postsynaptic. The authors’ results show that isoflurane and xenon have very different effects on GABAergic and glutamatergic synaptic transmission, and this may account for their differing pharmacologic profiles.

Structural Basis for the Inhibition of Firefly Luciferase by a General Anesthetic

The firefly luciferase enzyme from Photinus pyralis is probably the best-characterized model system for studying anesthetic-protein interactions. It binds a diverse range of general anesthetics over a large potency range, displays a sensitivity to anesthetics that is very similar to that found in animals, and has an anesthetic sensitivity that can be modulated by one of its substrates (ATP). In this paper we describe the properties of bromoform acting as a general anesthetic (in Rana temporaria tadpoles) and as an inhibitor of the firefly luciferase enzyme at high and low ATP concentrations. In addition, we describe the crystal structure of the low-ATP form of the luciferase enzyme in the presence of bromoform at 2.2-A resolution. These results provide a structural basis for understanding the anesthetic inhibition of the enzyme, as well as an explanation for the ATP modulation of its anesthetic sensitivity.

Stereoselective Effects of Etomidate Optical Isomers on Gamma-aminobutyric Acid Type A Receptors and Animals

Background The intravenous anesthetic etomidate is optically active and exists in two mirror‐image enantiomeric forms. However, although the R(+) isomer is used as a clinical anesthetic, quantitative information on the relative potencies of the R(+) and S(‐) isomers is lacking. These data could be used to test the relevance of putative molecular targets. Methods The anesthetic concentrations for a half‐maximal effect (EC50) needed to induce a loss of righting reflex in tadpoles (Rana temporaria) were determined for both etomidate enantiomers. The effects of the isomers on gamma‐aminobutyric acid (GABA)‐induced currents in stably transfected mouse fibroblast cells was also investigated using the patch‐clamp technique. In addition, the effects of the isomers on a lipid chain‐melting phase transition were determined. Results The EC50 concentrations for general anesthesia for the R(+) and S(‐) isomers were 3.4 +/‐ 0.1 micro Meter and 57 +/‐ 1 micro Meter, with slopes of n = 1.9 +/‐ 0.1 and n = 2.9 +/‐ 0.2, respectively. The R(+) isomer was also much more effective than the S(‐) isomer at potentiating GABA‐induced currents, although the degree of stereoselectivity varied with anesthetic concentration. R(+) etomidate potentiated the GABA‐induced currents by increasing the apparent affinity of GABA for its receptor. Both isomers were equally effective at disrupting lipid bilayers. Conclusions These data are consistent with the idea that the GABA sub A receptor plays a central role in the actions of etomidate. Etomidate exerts its effects on the receptor by binding directly to a specific site or sites on the protein and allosterically enhancing the apparent affinity of GABA for its receptor.

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 μm 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 μm 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 60–220%) 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 ≅ sevoflurane < halothane ≅ isoflurane ≅ 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 μm to 84 ± 7 μm glycine at 610 μm isoflurane, while that for the medullary neurones decreased from a control value of 30 ± 1 μm to 18 ± 2 μm 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 μm 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.

Which molecular targets are most relevant to general anaesthesia

1. In view of the large number of possible molecular targets of general anaesthetics, it is necessary to have some criteria for judging which targets are important for producing general anaesthesia and which are probably not. 2. We consider in detail two criteria: sensitivity to clinically relevant concentrations of anaesthetics and stereoselectivity to anaesthetic optical isomers. 3. The targets which currently emerge as most important belong to an anaesthetic-sensitive superfamily of genetically related fast neurotransmitter-gated receptor channels present at central synapses.

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 γ‐aminobutyric acid (GABA) were measured (using symmetrical Cl− solutions) at both low (2 μm) non‐desensitizing and high (300 μm) desensitizing concentrations of GABA. 2 At 2 μm 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 μm 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 μm GABA were stereoselective at moderate and high anaesthetic concentrations. The maximum stereoselectivity, about two fold, occurred at the EC50 concentration for general anaesthesia (310 μm 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 μm isoflurane). 4 The sustained residual current remaining after exposure of neurones to a desensitizing concentration of GABA (300 μm) 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 μm 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.

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

1 N1E‐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 μm 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 μm to 1.07 ± 0.11 μm (means ± s.e.mean) due to the presence of 1 MAC (270 μm) 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 μm 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.

ANAESTHETICS SET THEIR SITES ON ION CHANNELS

The effects of alcohol and general anaesthetics are obvious to everybody, but where in the brain — and how — do these agents act? By making hybrid GABAA- and glycine-receptor channels, one group has now identified two amino-acid residues that seem to be crucial for this process. Amino-acid substitutions at these positions remove the effects of anaesthetics, but its not yet possible to tell whether these amino acids represent a direct binding site, or whether they are involved more indirectly.