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Cognitive change affecting patients after anaesthesia and surgery has been recognised for more than 100 yr. Research into cognitive change after anaesthesia and surgery accelerated in the 1980s when multiple studies utilised detailed neuropsychological testing for assessment of cognitive change after cardiac surgery. This body of work consistently documented decline in cognitive function in elderly patients after anaesthesia and surgery, and cognitive changes have been identified up to 7.5 yr afterwards. Importantly, other studies have identified that the incidence of cognitive change is similar after non-cardiac surgery. Other than the inclusion of non-surgical control groups to calculate postoperative cognitive dysfunction, research into these cognitive changes in the perioperative period has been undertaken in isolation from cognitive studies in the general population. The aim of this work is to develop similar terminology to that used in cognitive classifications of the general population for use in investigations of cognitive changes after anaesthesia and surgery. A multispecialty working group followed a modified Delphi procedure with no prespecified number of rounds comprised of three face-to-face meetings followed by online editing of draft versions.Two major classification guidelines [Diagnostic and Statistical Manual for Mental Disorders, fifth edition (DSM-5) and National Institute for Aging and the Alzheimer Association (NIA-AA)] are used outside of anaesthesia and surgery, and may be useful for inclusion of biomarkers in research. For clinical purposes, it is recommended to use the DSM-5 nomenclature. The working group recommends that 'perioperative neurocognitive disorders' be used as an overarching term for cognitive impairment identified in the preoperative or postoperative period. This includes cognitive decline diagnosed before operation (described as neurocognitive disorder); any form of acute event (postoperative delirium) and cognitive decline diagnosed up to 30 days after the procedure (delayed neurocognitive recovery) and up to 12 months (postoperative neurocognitive disorder). [ABSTRACT FROM AUTHOR]

Cognitive change affecting patients after anaesthesia and surgery has been recognised for more than 100 yr. Research into cognitive change after anaesthesia and surgery accelerated in the 1980s when multiple studies utilised detailed neuropsychological testing for assessment of cognitive change after cardiac surgery. This body of work consistently documented decline in cognitive function in elderly patients after anaesthesia and surgery, and cognitive changes have been identified up to 7.5 yr afterwards. Importantly, other studies have identified that the incidence of cognitive change is similar after non-cardiac surgery. Other than the inclusion of non-surgical control groups to calculate postoperative cognitive dysfunction, research into these cognitive changes in the perioperative period has been undertaken in isolation from cognitive studies in the general population. The aim of this work is to develop similar terminology to that used in cognitive classifications of the general population for use in investigations of cognitive changes after anaesthesia and surgery. A multispecialty working group followed a modified Delphi procedure with no prespecified number of rounds comprised of three face-to-face meetings followed by online editing of draft versions.Two major classification guidelines [Diagnostic and Statistical Manual for Mental Disorders, fifth edition (DSM-5) and National Institute for Aging and the Alzheimer Association (NIA-AA)] are used outside of anaesthesia and surgery, and may be useful for inclusion of biomarkers in research. For clinical purposes, it is recommended to use the DSM-5 nomenclature. The working group recommends that 'perioperative neurocognitive disorders' be used as an overarching term for cognitive impairment identified in the preoperative or postoperative period. This includes cognitive decline diagnosed before operation (described as neurocognitive disorder); any form of acute event (postoperative delirium) and cognitive decline diagnosed up to 30 days after the procedure (delayed neurocognitive recovery) and up to 12 months (postoperative neurocognitive disorder).

Anesthesia and surgery have been reported to produce long-term cognitive problems, and to accelerate neurodegenerative disorders in the elderly. In previous work, we found that inhaled anesthetics enhance fibril formation and cytotoxicity of amyloid beta peptide. In this work we show that the inhaled anesthetics halothane (2-bromo-2-chloro-1,1,1-trifluoroethane) and isoflurane (1-chloro-2,2,2-trifluoroethyl difluoromethyl ether) also favor intermediate oligomers of amyloid beta(1-40), and reduce solubility of amyloid beta(1-40) monomer. Size-exclusion chromatography, analytical ultracentrifugation and photo-induced cross-linking experiments indicate halothane enhancement of oligomeric species having molecular weight approximately 44-100 kDa. Bis-ANS fluorescence experiments revealed that halothane stabilizes a population of diffusible oligomers relative to the monomer or the mature fibril. These data show that inhaled anesthetics lower the amyloid beta(1-40) concentration necessary to initiate oligomer formation, probably by preferential binding to intermediate oligomers en route to fibril formation.

The inhaled anesthetics were officially introduced to American medicine more than 160 years ago and rank among the most important medical advances in our time. These drugs are used to render patients insensible over twenty million times each year and are the most dangerous of all drugs that physicians currently use. An entire medical specialty, anesthesiology, has arisen out of the need for the special training to administer them safely. Nevertheless, side effects, toxicity, and long-term cognitive problems continue to plague their use, especially in the very sick or aged. Hence, it is essential that we develop an understanding of their molecular pharmacology so that safer alternatives can be developed.

Halothane, an inhaled anesthetic, destabilizes the folded structure of myoglobin. To determine whether this is due to preferential interactions with less stable folded conformers of myoglobin versus the completely unfolded state, we used photoaffinity labeling, hydrogen exchange, fluorescence spectroscopy, and circular dichroism spectroscopy. Apomyoglobin was used as a model of a less stable conformer of myoglobin. Halothane destabilizes myoglobin and binds with low affinity and stoichiometry but stabilizes and binds with higher affinity to apomyoglobin. The same halothane concentration has no effect on cytochrome c stability. The apomyoglobin/halothane complex is favored at pH 6.5 as compared to pH 4.5 or pH 2.5. Halothane photoincorporates into several sites in apomyoglobin, some allosteric to the heme pocket. Guanidinium unfolding of myoglobin, monitored by CD spectroscopy, shows destabilization at less than 1.3 M Gdm but stabilization at greater than 1.3 M Gdm, consistent with the hypothesis that less stable conformers of myoglobin bind halothane preferentially. We suggest the structural feature underlying preferential binding to less stable conformers is an enlarged cavity volume distribution, since myoglobin has several intermediate-sized cavities, while cytochrome c is more well packed and has no cavities detected by GRASP. Specific binding to less stable intermediates may underlie anesthetic potentiation of protein activity.

Hydrophobicity, a term used to describe a fundamental physicochemical property of local anesthetics, was in the past obtained by octanol/buffer partitioning. It has been suggested that the octanol method, despite its obvious advantages, also has some drawbacks. HPLC has become an attractive alternative for the measurement of hydrophobicity and has been applied to local anesthetics recently. However, the methods in current use for measuring the hydrophobicity of local anesthetics suffer from a number of limitations and remain obscure. This study introduces a new HPLC method for measuring the hydrophobicity of eight local anesthetics in current clinical use. Using a C(18) derivatized polystyrene-divinylbenzene stationary phase HPLC column, the log k'(w) values of local anesthetics were determined by measuring the capacity factor k'(i) in the process of chromatographic separation using a hydrophobic stationary phase and a hydrophilic mobile phase. A rapid reversed-phase HPLC method was developed to directly measure log k'(w) of eight local anesthetics. A high correlation between log k'(w) and hydrophobicity (log P(oct)) from the traditional shake-flask method was obtained for the local anesthetics, demonstrating the reliability of the method. The results reveal an improved method for measuring the hydrophobicity of the local anesthetic agents in the unionized form. This simple, sensitive and reproducible approach may serve as a valuable tool for describing the physicochemical properties of novel local anesthetics.

Xenon and dichloromethane are inhalational anesthetic agents whose binding to myoglobin has been demonstrated by X-ray crystallography. We explore the thermodynamic significance of such binding using differential scanning calorimetry, circular dichroism spectroscopy, and hydrogen-tritium exchange measurements to study the effect of these agents on myoglobin folding stability. Though specific binding of these anesthetics might be expected to stabilize myoglobin against unfolding, dichloromethane actually destabilized myoglobin at all examined concentrations of this anesthetic (15, 40, and 200 mM). On the other hand, xenon (1 atm) stabilized myoglobin. Thus, dichloromethane and xenon have opposite effects on myoglobin stability despite localization in comparably folded X-ray crystallographic structures. These results suggest a need for solution measurements to complement crystallography if the consequences of weak binding to proteins are to be appreciated.

Firefly luciferase is considered a reasonable model of in vivo anesthetic targets despite being destabilized by anesthetics, as reflected by differential scanning calorimetry (DSC). We examined the interaction between two inhaled anesthetics, ATP, luciferase, and temperature, using amide hydrogen exchange, tryptophan fluorescence, and photolabeling in an attempt to examine this apparent discrepancy. In the absence of ATP/Mg2+, halothane and bromoform cause destabilization, as measured by hydrogen exchange, suggesting nonspecific interactions. In the presence of ATP/Mg2+ and at room temperature, the anesthetics produce considerable stabilization with a negative DeltaH, indicating population of a conformer with a specific anesthetic binding site. Stabilizing interactions are lost, however, at unfolding temperatures. We suggest that preferential binding to aggregated forms of luciferase explain the higher temperature destabilization detected with DSC. Our results demonstrate a cooperative binding equilibrium between native ligands and anesthetics, suggesting that similar interactions could underlie actions at biologically relevant targets., (Copyright 2001 Wiley-Liss, Inc.)

Previous evidence suggests multiple anesthetic binding sites on human serum albumin, but to date, we have only identified Trp-214 in an interdomain cleft as contributing to a binding site. We used a combination of site-directed mutagenesis, photoaffinity labeling, amide hydrogen exchange, and tryptophan fluorescence spectroscopy to evaluate the importance to binding of a large domain III cavity and compare it to binding character of the 214 interdomain cleft. The data show anesthetic binding in this domain III cavity of similar character to the interdomain cleft, but selectivity for different classes of anesthetics exists. Occupancy of these sites stabilizes the native conformation of human serum albumin. The features necessary for binding in the cleft appear to be fairly degenerate, but in addition to hydrophobicity, there is evidence for the importance of polarity. Finally, myristate isosterically competes with anesthetic binding in the domain III cavity and allosterically enhances anesthetic binding in the interdomain cleft.

General anesthetics have been reported to alter the functions of G protein coupled receptor (GPCR) signaling systems. To determine whether these effects might be mediated by direct binding interactions with the GPCR or its associated G protein, we studied the binding character of halothane on mammalian rhodopsin, structurally the best understood GPCR, by using direct photoaffinity labeling with [(14)C]halothane. In the bleached bovine rod disk membranes (RDM), opsin and membrane lipids were dominantly photolabeled with [(14)C]halothane, but none of the three G protein subunits were labeled. In opsin itself, halothane labeling was inhibited by unlabeled halothane with an IC(50) of 0.9 mM and a Hill coefficient of -0.8. The stoichiometry was 1.1:1.0 (halothane:opsin molar ratio). The IC(50) values of isoflurane and 1-chloro-1,2, 2-trifluorocyclobutane were 5.0 and 15 mM, respectively. Ethanol had no effect on opsin labeling by halothane. A nonimmobilizer, 1, 2-dichlorohexafluorocyclobutane, inhibited halothane labeling by 50% at 0.05 mM. The present results demonstrate that halothane binds specifically and selectively to GPCRs in the RDM. The absence of halothane binding to any of the G protein subunits strongly suggests that the functional effects of halothane on GPCR signaling systems are mediated by direct interactions with receptor proteins.

Quantitative autoradiography of 14C-halothane direct photolabeled rat cerebellum sections was performed in the presence of increasing concentrations of gamma-aminobutyric acid (GABA) or glutamate to test the hypothesis that a coupled binding equilibrium between the anesthetic and neurotransmitter exists. The results show that halothane binding was enhanced in the presence of GABA by approximately 50% in the molecular layer and to a lesser extent in the granular layer, with no change in the myelin layer. Glutamate, however, did not enhance halothane binding in any layer. These data confirm the presence of coupling, and thus suggest a direct interaction of halothane with a GABA binding protein.

There is a distinct possibility that general anesthetics exert their action on the postsynaptic receptor channels. The structural requirements for anesthetic binding in transmembrane channels, however, are largely unknown. High-resolution (1)H nuclear magnetic resonance and direct photoaffinity labeling were used in this study to characterize the volatile anesthetic binding sites in gramicidin A (gA) incorporated into sodium dodecyl sulfate (SDS) micelles and into dimyristoylphosphatidylcholine (DMPC) bilayers, respectively. To confirm that the structural arrangement of the peptide side chains can affect anesthetic binding, gA in nonchannel forms in methanol was also analyzed. The addition of volatile anesthetic halothane to gA in SDS with a channel conformation caused a concentration-dependent change in resonant frequencies of the indole amide protons of W9, W11, W13, and W15, with the most profound changes in W9. These frequency changes were observed only for gA carefully prepared to ensure a channel conformation and were absent for gA in methanol. For gA in DMPC bilayers, direct [(14)C]halothane photolabeling and microsequencing demonstrated dominant labeling of W9, less labeling of W11 and W13, and no significant labeling of W15. In methanol, gA showed much less labeling of any residues. Inspection of the 3-D structure of gA suggests that the spatial arrangements of the tryptophan residues in the channel form of gA, combined with the amphiphilic regions of lipid, create a favorable anesthetic binding motif.

The structural features of volatile anesthetic binding sites on proteins are being examined with the use of a defined model system consisting of a four-alpha-helix bundle scaffold with a hydrophobic core. Previous work has suggested that introducing a cavity into the hydrophobic core improves anesthetic binding affinity. The more polarizable methionine side chain was substituted for a leucine, in an attempt to enhance the dispersion forces between the ligand and the protein. The resulting bundle variant has an improved affinity (K(d) = 0.20 +/- 0.01 mM) for halothane binding, compared with the leucine-containing bundle (K(d) = 0.69 +/- 0.06 mM). Photoaffinity labeling with (14)C-halothane reveals preferential labeling of the W15 residue in both peptides, supporting the view that fluorescence quenching by bound anesthetic reports both the binding energetics and the location of the ligand in the hydrophobic core. The rates of amide hydrogen exchange were similar for the two bundles, suggesting that differences in binding affinity were not due to changes in protein stability. Binding of halothane to both four-alpha-helix bundle proteins stabilized the native folded conformations. Molecular dynamics simulations of the bundles illustrate the existence of the hydrophobic core, containing both W15 residues. These results suggest that in addition to packing defects, enhanced dispersion forces may be important in providing higher affinity anesthetic binding sites. Alternatively, the effect of the methionine substitution on halothane binding energetics may reflect either improved access to the binding site or allosteric optimization of the dimensions of the binding pocket. Finally, preferential stabilization of folded protein conformations may represent a fundamental mechanism of inhaled anesthetic action.

A loss of potency as one ascends a homologous series of compounds (cutoff effect) is often used to map the dimensions of binding sites on a protein target. The implicit assumption of steric hindrance is rarely confirmed with direct binding measurements, yet other mechanisms for cutoff exist. We studied the binding and effect of a series of n-alkanols up to hexadecanol (C16) on two model proteins, BSA and myoglobin (MGB), using hydrogen-tritium exchange and light scattering. BSA binds the n-alkanols specifically and, at 1 mM total concentration, is stabilized with increasing potency up to decanol (C10), where a loss in stabilizing potency occurs. Cutoff in stabilizing potency is concentration-dependent and occurs at progressively longer n-alkanols at progressively lower total n-alkanol concentrations. Light scattering measurements of n-alkanol/BSA solutions show a smooth decline in binding stoichiometry with increasing chain length until C14-16, where it levels off at approximately 2:1 (alkanol:BSA). MGB does not bind the n-alkanols specifically and is destabilized by them with increasing potency until C10, where a loss in destabilizing potency occurs. Like BSA, MGB demonstrates a concentration-dependent cutoff point for the n-alkanols. Derivation of the number of methylenes bound at K(D) and the free energy contribution per bound methylene showed that no discontinuity existed to explain cutoff, rendering steric hindrance unlikely. The data also allow an energetic explanation for the variance of the cutoff point in various reductionist systems. Finally, these results render cutoff an untenable approach for mapping binding site sterics in the absence of complementary binding measurements, and a poor discriminator of target relevance to general anesthesia.

Inhalational anesthetic agents are known to alter protein function, but the nature of the interactions underlying these effects remains poorly understood. We have used differential scanning calorimetry to study the effects of the anesthetic agent halothane on the thermally induced unfolding transition of bovine serum albumin. We find that halothane (0.6-10 mM) stabilizes the folded state of this protein, increasing its transition midpoint temperature from 62 to 71 degrees C. Binding of halothane to the native state of serum albumin thus outweighs any non-specific interactions between the thermally unfolded state of serum albumin and halothane in this concentration range. Based on the average enthalpy change DeltaH for unfolding of 170 kcal/mol, the increase from 62 to 71 degrees C corresponds to an additional Gibbs energy of stabilization (DeltaDeltaG) due to halothane of more than 4 kcal/mol. Analysis of the dependence of DeltaDeltaG on halothane concentration shows that thermal unfolding of a bovine serum albumin molecule is linked to the dissociation of about one halothane molecule at lower halothane concentrations and about six at higher halothane concentrations. Serum albumin is the first protein that has been shown to be stabilized by an inhalational anesthetic.

1. We have used differential scanning calorimetry to measure the halothane induced change in stability of five lipid-free proteins in aqueous solution. 2. The temperature at peak heat capacity (Tm) as the sample is heated provides a measure of stability. 3. Addition of halothane increases Tm for bovine and human serum albumin, but decreases Tm for hen egg white lysozyme, bovine pancreatic ribonuclease A, and horse skeletal muscle myoglobin. 4. A shift of Tm in either direction may model the action of inhaled anesthetics on relevant proteins in the central nervous system.

To determine whether specific or nonspecific interactions between inhaled anesthetics and proteins are more likely to underlie anesthetic actions, analysis of hydrogen/tritium exchange was used to measure effects on the stability of two model proteins that had been previously shown to bind anesthetics specifically (bovine serum albumin) or only nonspecifically (myoglobin). The data indicated that stabilization of albumin correlated with the potencies of a wide range of anesthetic compounds significantly better than did destabilization of myoglobin. In addition, sensitivity to nonanesthetics, isoflurane stereoselectivity, and temperature and pressure effects all influenced the stabilization of bovine serum albumin, but not the destabilization of myoglobin, in a manner strongly supporting the premise that specific binding interactions with protein targets underlie anesthetic action. These observations significantly increase the likelihood that such interactions can be found and optimized.

To understand further the weak molecular interactions between inhaled anesthetics and proteins, we studied the character and dynamic consequences of halothane binding to bovine serum albumin (BSA) and myoglobin using photoaffinity labeling and hydrogen-tritium exchange (HX). We find that halothane binds saturably and with submillimolar affinity to BSA, but either nonspecifically or with considerably lower affinity to myoglobin. Titration of halothane binding with guanidine hydrochloride suggested more protection of binding sites from solvent in BSA as compared with myoglobin. Protection factors for slowly exchanging albumin hydrogens are increased in a concentration-dependent manner by up to 27-fold with 10 mM halothane, whereas more rapidly exchanging groups of albumin hydrogens have either unaltered or decreased protection factors. Protection factors for slowly exchanging hydrogens in myoglobin are decreased by halothane, suggesting destabilization through binding to an intermediate or completely unfolded conformer. These results demonstrate the conformation dependence of halothane binding and clear dynamic consequences that correlate with the character of binding in these model proteins. Preferential binding and stabilization of different conformational states may underlie anesthetic-induced protein dysfunction, as well as provide an explanation for heterogeneity of action.

14C-halothane direct photoaffinity labeling was used to characterize the distribution of halothane binding in rat brain to test the hypothesis that anesthetics bind preferentially to a specific, heterogeneously distributed, receptor or channel. Slide-mounted sagittal rat brain sections were placed in gas-tight quartz cuvettes with 100 microM 14C-halothane in phosphate buffered saline with 0 to 7.5 mM unlabeled halothane, or unlabeled chloroform and isoflurane at 10 times the clinical EC50, and then exposed to UV light for 60 to 100 sec. Autoradiograms of nine brain regions (cortex, corpus callosum, hippocampal molecular and pyramidal layers, dentate molecular and granule cell layers, and cerebellar molecular, granular and white matter layers) were prepared and quantitated using Image 1.44. Total label incorporation was widespread, but exhibited subtle heterogeneity. There was significantly more total labeling in regions of high synaptic density than in regions containing primarily cell bodies or white matter. Most labeling (approximately 80%) was displaced by unlabeled halothane and can therefore be considered specific. Significantly more specific labeling was found in regions of high synaptic density. Isoflurane did not inhibit halothane photolabeling significantly, but chloroform inhibited it by approximately 50%. In conclusion, halothane photolabeling distribution in the mammalian brain is widespread, saturable and selective, but does not mimic the distribution of any individual receptor or channel. Brain regions with high synaptic density displayed the greatest degree of specific binding, consistent with transmission being an important functional target of volatile anesthetics. These results suggest a remarkably widespread individual target, or more likely, similar binding sites in multiple targets, and are consistent with the notion that anesthesia is the result of action at multiple sites.

The fundamental interactions of inhalational anesthetics with proteins have been considered in some detail, using specific examples where appropriate to illustrate these interactions and demonstrate progress. It is now clear that these low-affinity volatile molecules with rapid kinetics can specifically bind to discrete sites in some proteins at reasonable pharmacological concentrations, and some general features of these sites are beginning to emerge. The structural or dynamic consequences of anesthetic binding, however, are still vague at best. The remaining challenge is to define which interactions produce anesthetic binding to relevant targets and what the features of this relevant anesthetic binding site are. Finally, and most importantly, how does the occupancy of these pockets, patches, or cavities result in the subtle alterations in protein conformation and dynamics that confound their function and ultimately produce the behavioral response that we term "anesthesia"?

The activity of various Ca2+-ATPases is affected by volatile anesthetics, such as halothane, commonly used in clinical practice. The effect on the enzyme in skeletal muscle sarcoplasmic reticulum (SR) is biphasic, including stimulation at clinical anesthetic concentrations and subsequent inhibition at higher concentrations. We have previously proposed that the action of a volatile anesthetic on Ca2+-ATPases results from its binding in the interior of the enzyme molecule [Lopez, M.M. and Kosk-Kosicka, D. (1995) J. Biol. Chem. 270, 28239-28245]. Presently, we investigated whether the anesthetic interacts directly with the skeletal muscle SR Ca2+-ATPase (SERCA1) as evidenced by binding. Photoaffinity labeling with [14C]halothane demonstrated that the anesthetic binds saturably to SR membranes, and that approximately 80% of the binding is specific, with a KI of 0.6 mM. The KI value agrees well with the concentration at which halothane half-maximally activates SERCA1. SDS gel electrophoresis of labeled membranes indicates that 38-56% of [14C]halothane incorporates into SERCA1, and 38-53% in lipids. Distribution of label among the three fragments produced by controlled tryptic digestion of SERCA1 suggests heterogeneous halothane binding presumably in discrete sites in the enzyme. The results provide the first direct evidence that halothane binds to SERCA1. Potentially this binding could be related to anesthetic effect on enzyme's function.

Saturable binding of various inhaled anesthetics to serum albumin has been shown with a variety of approaches. In order to determine the location of halothane binding sites in serum albumin, both human and bovine serum albumins (HSA and BSA) were photolabeled with [14C]halothane, and subjected to proteolysis and microsequencing. BSA was found to have a higher affinity for halothane than HSA, and it contained two specifically labeled sites. One site was characterized by diffuse labeling from Trp212-Leu217, and the other by a more discrete and higher affinity labeling at Trp134-Gly135. HSA contained only a single labeled site, and although lower affinity, was determined to be analogous to BSA Trp212. The position 130-140 region of HSA, having a leucine instead of tryptophan at position 134, was not labeled. These results demonstrate specific and discrete binding of an inhaled anesthetic to a mammalian-soluble protein, and further suggest the importance of aromatic residues as one feature of inhaled anesthetic binding sites.

The present study makes use of direct photoaffinity labeling and fluorescence and circular dichroism spectroscopy to examine the interaction of the inhalational anesthetic halothane with the uncharged alpha-helical form of poly(L-lysine) over a range of chain lengths. Halothane bound specifically to long chain homopolymers (190 to 1060 residues), reaching a stable stoichiometry of 1 halothane to 160 lysine residues in polymers longer than 300 residues. Halothane bound only non-specifically to an alpha-helical 30 residue polymer and to all of the polymers in their charged, random coil form. The data suggest that halothane binding is a function of supersecondary structure whereby intramolecular helix-helix clusters form in the longer polymers, resulting in the creation of confined hydrophobic domains. Circular dichroism spectroscopy cannot demonstrate changes in poly(L-lysine) secondary structure at any chain length with up to 12 mM halothane, suggesting that extensive hydrogen bond disruption by the anesthetic does not occur.

To determine inhalational anesthetic binding domains on a ligand-gated ion channel, I used halothane direct photoaffinity labeling of the nicotinic acetylcholine receptor (nAChR) in native Torpedo membranes. [14C]Halothane photoaffinity labeling of both the native Torpedo membranes and the isolated nAChR was saturable, with Kd values within the clinically relevant range. All phospholipids were labeled, with greater than 95% of the label in the acyl chain region. Electrophoresis of labeled nAChR demonstrated no significant subunit selectivity for halothane incorporation. Within the alpha-subunit, greater than 90% of label was found in the endoprotease Glu-C digestion fragments which contain the four transmembrane regions, and the pattern was different from that reported for photoactivatable phospholipid binding to the nAChR. Unlabeled halothane reduced labeling more than did isoflurane, suggesting differences in the binding domains for inhalational anesthetics in the nAChR. These data suggest multiple similar binding domains for halothane in the transmembrane region of the nAChR.

Because hydrostatic pressure can antagonize the behavioral effects of anesthetics in many organisms, we examined whether ethanol-induced immobility of the nematode Caenorhabditis elegans was antagonized by 100 ATA pressure. Nematode mobility was examined at pressures ranging from 1 ATA to 132 ATA, with and without increasing concentrations of ethanol (0-1 M). Pressure > 100 ATA alone inhibited movement of the nematode (EP50 = 111.5 +/- 0.6 ATA), as did increasing concentrations of ethanol (EC50 = 487 +/- 44 mM). Pressure and ethanol appeared to be additive, and not antagonistic. Because of previous results implicating glycine receptor antagonism as a mechanism of pressure reversal, and our current inability to observe significant behavioral effects of strychnine or glycine inhibition of high affinity strychnine binding, we suggest that an absence of glycine receptors in these organisms is the basis for a lack of pressure antagonism of ethanol immobility.

We have shown previously that inhalation anaesthetics inhibit dopamine transport in rat synaptosomes. In order to determine if this inhibition is associated with occupancy of the cocaine site, we examined binding of [3H] (2 beta-carbomethoxy-3 beta-(4-fluorophenyl)-tropane) (3H-CFT) in the presence of halothane or isoflurane 0.01-5 mmol litre-1 in rat brain synaptosomes. Both anaesthetics inhibited 3H-CFT binding (mean Ki 0.61 (SEM 0.12) and 0.75 (0.21) mmol litre-1, respectively), by increasing Kd (13.8 (0.6) and 29.8 (12.8) nmol litre-1, respectively) compared with control (8.02 (0.5) nmol litre-1) (P < 0.01). Halothane did not change Bmax, but isoflurane increased it significantly. Cocaine protected CFT sites from N-ethylmaleimide alkylation, but neither anaesthetic did. Photoaffinity labelling with halothane significantly inhibited 3H-CFT binding compared with UV-exposed controls. We conclude that clinically relevant concentrations of both anaesthetics inhibit high-affinity CFT binding, and the data suggest overlapping sites for halothane and CFT.

Background: Recently, halothane and isoflurane have been shown to bind in a saturable manner to serum albumin using NMR and gas chromatography methods. To validate a novel direct photoaffinity labeling method developed in our laboratory, the authors also determined the binding characteristics of halothane to serum albumin, and then extended this approach to other soluble proteins in an initial attempt to understand the interaction of volatile anesthetics and proteins., Methods: Serum albumin (BSA), bacterial luciferase (BL), poly-(L-lysine)(PLL), and poly-(L-glutamate)(PLG) were dissolved in 0.154 M NaCl containing 14C-halothane with or without other volatile anesthetics or ligands, and exposed to 254 nm UV light for 10 s. Covalently bound label was quantitated by scintillation counting after precipitation, filtration, and washing. Binding parameters were calculated by nonlinear least-squares fitting of rectangular hyperbolas or logistic equations., Results: Serum albumin bound halothane in a saturable manner at an apparent KD between 0.3 and 0.5 mM. Other volatile anesthetics inhibited binding (KI, in mM): halothane (0.36), chloroform (1.26), methoxyflurane (2.66), isoflurane (1.47), diethyl ether (45.5), and ethanol (1,040). Oleate and BSA conformational changes (low pH) also inhibited label incorporation. Binding to BL and PLL at pH 7 was nonsaturable and not displaced by unlabeled halothane or the BL substrate decanal. Conversion of PLL to an alpha-helical conformation (pH > 10) increased binding and created a saturable component with an apparent KD of 0.55 mM. Alkaline conditions decreased binding to PLG consistent with the loss of alpha-helical domains., Conclusions: Photoaffinity labeling produced results in close agreement with more conventional methods for studying halothane binding, and should be a useful tool for the study of volatile anesthetic binding sites. Halothane binding to soluble proteins depended on their type and conformation, and, in some cases, was saturable within the clinical concentration range, increasing the tenability of discrete proteinaceous sites of action for the inhalational anesthetics.

Background: Volatile anesthetics may depress transmission by altering synaptic concentrations of neurotransmitter. Microdialysis studies have found an increase in brain extracellular dopamine concentration on exposure to volatile anesthetics. We investigated the possibility that synaptosomal dopamine transport is reversibly inhibited by the halothane and isoflurane., Methods: Rat brain synaptosomes were incubated with 5 nM 3H-DA and increasing concentrations of anesthetic in Teflon-sealed microvials. Cocaine (100 microM) was used to quantify non-specific binding/uptake. Uptake was stopped by vacuum filtration and washing; label incorporation into synaptosomes was determined by liquid scintillation counting. 3H-DA release from preloaded synaptosomes also was studied in the presence of the anesthetic to allow distinction between uptake inhibition and release stimulation in the synaptosomes., Results: Both halothane and isoflurane inhibited the specific 3H-DA uptake in a concentration-dependent fashion with an IC50 of 0.72 +/- 0.14 mM for halothane and 2.24 +/- 0.85 mM for isoflurane. No stereoselectivity of isoflurane's action on Dopamine (DA) uptake was observed. The inhibition produced by halothane and isoflurane was kinetically characterized as noncompetitive, but full reversal was demonstrated after removal of the anesthetic from the incubation mixture. The anesthetics did not stimulate 3H-DA release from preloaded synaptosomes., Conclusions: These results demonstrate volatile anesthetic-induced inhibition of the dopamine transporter in this preparation of synaptosomes. The calculated IC50S suggest this inhibition occurs with clinically relevant concentrations of halothane but not with isoflurane. The results are consistent with and may explain the increase in extracellular dopamine concentrations demonstrated by microdialysis.

The hypothesis that volatile anesthetics act directly on or bind specifically to membrane proteins remains controversial. In earlier in situ electron probe microanalysis studies in cardiac muscle we showed preferential partitioning of halothane into mitochondria. To determine whether partitioning represents saturable binding or simple solubility, a photoaffinity labeling method was developed for halothane to examine binding in rat brain synaptosomes. Radioligand binding assays were then used to determine binding parameters for this important inhalational anesthetic. UV-light exposure of synaptosomes incubated with clinical concentrations of [14C]halothane resulted in sufficient labeling to allow characterization of binding sites. Analysis of saturation and competition curves showed that greater than 60% of [14C]halothane photolysis product binding to synaptosomes was specific, with low affinity (Kd = 0.49 +/- 0.16 mM) and high binding site concentration (Bmax = 1.87 +/- 0.75 nmol/mg of protein). Halothane photoaffinity labeling was partially inhibited by isoflurane (20%), chloroform (44%), 2-bromotrifluoroethane (20%), and dichlorotrifluoroethane (20%) but not by ethanol. The Kd measured with this photoaffinity approach is similar to the concentration of halothane required to produce anesthesia in rats.

To further define the relationship between oxygen dependence of lung injury during ischemia and ischemia-reperfusion, we used the isolated, perfused, and ventilated rat lung model, so that oxygenation and perfusion could be separated. During ischemia, lungs were ventilated with various oxygen concentrations and then ventilated with 95% oxygen during the 60-min reperfusion period. Other lungs were ventilated with 0% oxygen (nitrogen) during ischemia, and the reperfusion phase oxygen concentration was varied. Tissue and perfusate lipid peroxidation products (thiobarbituric acid-reactive substances and conjugated dienes), dry-to-wet weight ratio, and lactate dehydrogenase were measured as indexes of lung damage. In addition, electron microscopy of some lungs was performed. Results demonstrate an oxygen dependence of lipid peroxidation in both the ischemic and reperfusion phases, but lipid peroxidation is severalfold greater in the reperfusion than in the ischemic phase. Products of lipid peroxidation closely correlate with indexes of lung injury (dry-to-wet weight ratio, lactate dehydrogenase, and electron microscopy).

The effect of alveolar oxygen tension on lung lipid peroxidation during lung ischemia was evaluated by using isolated rat lungs perfused with synthetic medium. After a 5-min equilibration period, global ischemia was produced by discontinuing perfusion while ventilation continued with gas mixtures containing 5% CO2 and a fixed oxygen concentration between 0 and 95%. Lipid peroxidation was assessed by measurement of tissue thiobarbituric acid-reactive products and conjugated dienes. Control studies (no ischemia) showed no change in parameters of lipid peroxidation during 1 h of perfusion and ventilation with 20% or 95% O2. With 60 min of ischemia, there was increased lipid peroxidation which varied with oxygen content of the ventilating gas and was markedly inhibited by ventilation with N2. Perfusion with 5-, 8-, 11-, 14-eicosatetraynoic acid indicated that generation of eicosanoids during ischemia accounted for approximately 40-50% of lung lipid peroxide production. Changes of CO2 content of the ventilating gas (to alter tissue pH) or of perfusate glucose concentration had no effect on lipid peroxidation during ischemia, but perfusion at 8% of the normal flow rate prevented lipid peroxidation. Lung dry/wet weight measured after 3 min of reperfusion showed good correlation between lung fluid accumulation and lipid peroxidation. These results indicate that reperfusion is not necessary for lipid peroxidation with ischemic insult of the lung and provide evidence that elevated PO2 during ischemia accelerates the rate of tissue injury.

We exposed 34 subjects to a 48-h, 6.25-msw dive, and administered ethanol (0.5-1.0 ml pure ethanol.kg-1 body weight) orally to 11 of them immediately after direct decompression. Doppler monitoring of both precordial and subclavian sites for 24 h postsurfacing revealed that all subjects from both groups had detectable bubbles, and that there was no difference in timing or magnitude between the 2 groups. These results do not support the recently suggested role for ethanol in the treatment of decompression sickness.

The dose-response relationship for decompression magnitude and venous gas emboli (VGE) formation in humans was examined. Pressure exposures of 138, 150, and 164 kPa (12, 16, and 20.5 ft of seawater gauge pressure) were conducted in an underwater habitat for 48 h. The 111 human male volunteer subjects then ascended directly to the surface in less than 5 min and were monitored for VGE with a continuous-wave Doppler ultrasound device over the precordium or the subclavian veins at regular intervals for a 24-h period. No signs or symptoms consistent with decompression sickness occurred. However, a large incidence of VGE detection was noted. These data were combined with those from our previously reported experiments at higher pressures, and the data were fit to a Hill dose-response equation with nonlinear least-squares or maximum likelihood routines. Highly significant fits of precordial VGE incidences were obtained with the Hill equation (saturation depth pressure at which there is a 50% probability of detectable VGE [D(VGE)50] = 150 +/- 1.2 kPa). Subclavian monitoring increased the sensitivity of VGE detection and resulted in a leftward shift [D(VGE)50 = 135 +/- 2 kPa] of the best-fit curve. We conclude that the reduction in pressure necessary to produce bubbles in humans is much less than was previously thought; 50% of humans can be expected to generate endogenous bubbles after decompression from a steady-state pressure exposure of only 135 kPa (11 ft of seawater). This may have significant implications for decompression schedule formulation and for altitude exposures that are currently considered benign. These results also imply that endogenous bubbles arise from preexisting gas collections.