Presence Of Halothane Research Articles

Anesthetic Drugs and Onset of Malignant Hyperthermia

The time between the beginning of anesthetic administration and recognition of the first sign of malignant hyperthermia (MH) (MH onset time) could differ among anesthetic drugs. We examined the time of the first signs of suspected MH, anesthetic drugs administered, subject age, and year of event in Adverse Metabolic/Musculoskeletal Reaction to Anesthesia reports in the North American Malignant Hyperthermia Registry. Inclusion criteria were judgment by the reporting clinician that the event was possible or fulminant MH, documentation of the time when anesthetic administration began, and the time when the first MH sign was noted. Descriptive statistics, Kruskal-Wallis analysis, and nonparametric correlation were used to assess the difference in MH onset times under different conditions. Four hundred seventy-seven cases met inclusion criteria; 58.5% were possible MH and 41.5% fulminant MH. Inhaled anesthetic and succinylcholine were given in 53.9% of cases, inhaled anesthetic only in 41.7%, and succinylcholine without inhaled anesthetics in 2.9%. No causative anesthetic drugs were reported in 7 MH cases. In 394 patients exposed to only 1 of the 4 inhaled anesthetics, without regard for subject age, MH onset time was shorter in the presence of halothane than any of the other anesthetics and shorter after succinylcholine in all anesthetics. If succinylcholine was not given, MH onset was shorter during sevoflurane anesthesia than during desflurane or isoflurane. In 322 cases, 1 rather than multiple first signs of MH were reported with masseter spasm as the earliest MH sign. In 339 cases in which masseter spasm was not reported, there was no difference in MH onset time with or without succinylcholine. In 146 cases in which masseter spasm was not reported and succinylcholine was not given, MH onset was shorter during halothane anesthesia, than during exposure to desflurane, or isoflurane. MH onset time during sevoflurane was shorter than during desflurane or isoflurane. MH was reported later in the course of anesthesia after 1998, when halothane and succinylcholine were less often reported. MH occurred after succinylcholine administration in the absence of inhaled anesthetics. We could not separate an effect of age from that of other variables. The onset of MH has been observed later during desflurane and isoflurane anesthesia than during exposure to sevoflurane. Since 1998, MH signs have more often appeared later, in the second or third hour of anesthesia, than they did before 1998.

NMR resolved multiple anesthetic binding sites in the TM domains of the α4β2 nAChR

The α4β2 nicotinic acetylcholine receptor (nAChR) has significant roles in nervous system function and disease. It is also a molecular target of general anesthetics. Anesthetics inhibit the α4β2 nAChR at clinically relevant concentrations, but their binding sites in α4β2 remain unclear. The recently determined NMR structures of the α4β2 nAChR transmembrane (TM) domains provide valuable frameworks for identifying the binding sites. In this study, we performed solution NMR experiments on the α4β2 TM domains in the absence and presence of halothane and ketamine. Both anesthetics were found in an intra-subunit cavity near the extracellular end of the β2 transmembrane helices, homologous to a common anesthetic binding site observed in X-ray structures of anesthetic-bound GLIC (Nury et al., [32]). Halothane, but not ketamine, was also found in cavities adjacent to the common anesthetic site at the interface of α4 and β2. In addition, both anesthetics bound to cavities near the ion selectivity filter at the intracellular end of the TM domains. Anesthetic binding induced profound changes in protein conformational exchanges. A number of residues, close to or remote from the binding sites, showed resonance signal splitting from single to double peaks, signifying that anesthetics decreased conformation exchange rates. It was also evident that anesthetics shifted population of two conformations. Altogether, the study comprehensively resolved anesthetic binding sites in the α4β2 nAChR. Furthermore, the study provided compelling experimental evidence of anesthetic-induced changes in protein dynamics, especially near regions of the hydrophobic gate and ion selectivity filter that directly regulate channel functions.

Anesthetic Binding in a Pentameric Ligand-Gated Ion Channel: GLIC

Cys-loop receptors are molecular targets of general anesthetics, but the knowledge of anesthetic binding to these proteins remains limited. Here we investigate anesthetic binding to the bacterial Gloeobacter violaceus pentameric ligand-gated ion channel (GLIC), a structural homolog of cys-loop receptors, using an experimental and computational hybrid approach. Tryptophan fluorescence quenching experiments showed halothane and thiopental binding at three tryptophan-associated sites in the extracellular (EC) domain, transmembrane (TM) domain, and EC-TM interface of GLIC. An additional binding site at the EC-TM interface was predicted by docking analysis and validated by quenching experiments on the N200W GLIC mutant. The binding affinities (KD) of 2.3 ± 0.1 mM and 0.10 ± 0.01 mM were derived from the fluorescence quenching data of halothane and thiopental, respectively. Docking these anesthetics to the original GLIC crystal structure and the structures relaxed by molecular dynamics simulations revealed intrasubunit sites for most halothane binding and intersubunit sites for thiopental binding. Tryptophans were within reach of both intra- and intersubunit binding sites. Multiple molecular dynamics simulations on GLIC in the presence of halothane at different sites suggested that anesthetic binding at the EC-TM interface disrupted the critical interactions for channel gating, altered motion of the TM23 linker, and destabilized the open-channel conformation that can lead to inhibition of GLIC channel current. The study has not only provided insights into anesthetic binding in GLIC, but also demonstrated a successful fusion of experiments and computations for understanding anesthetic actions in complex proteins.

Unresponsive Correlated Motion in α7 nAChR to Halothane Binding Explains Its Functional Insensitivity to Volatile Anesthetics

Neuronal nicotinic acetylcholine receptors (nAChRs) have been implicated as targets for general anesthetics, but the functional responses to anesthetic modulation vary considerably among different subtypes of nAChRs. Inhaled general anesthetics, such as halothane, could effectively inhibit the channel activity of the alpha4beta2 nAChR but not the homologous alpha7 nAChR. To understand why alpha7 is insensitive to inhaled general anesthetics, we performed multiple sets of 20 ns molecular dynamics (MD) simulations on the closed- and open-channel alpha7 in the absence and presence of halothane and critically compared the results with those from our studies on the alpha4beta2 nAChR (Liu et al. J. Phys. Chem. B 2009, 113, 12581 and Liu et al. J. Phys. Chem. B 2010, 114, 626). Several halothane binding sites with fairly high binding affinities were identified in both closed- and open-channel alpha7, suggesting that a lack of sensitive functional responses of the alpha7 nAChR to halothane in the previous experiments was unlikely due to a lack of halothane interaction with alpha7. The binding affinities of halothane in alpha7 seemed to be protein conformation-dependent. Overall, halothane affinity was higher in the closed-channel alpha7. Halothane binding to alpha7 did not induce profound changes in alpha7 structure and dynamics that could be related to the channel function. In contrast, correlated motion of the open-channel alpha4beta2 was reduced substantially in the presence of halothane, primarily due to the more susceptible nature of beta2 to anesthetic modulation. The amphiphilic extracellular and transmembrane domain interface of the beta2 subunit is attractive to halothane and is susceptible to halothane perturbation, which may be why alpha4beta2 is functionally more sensitive to halothane than alpha7.

The Genetics of Malignant Hyperthermia and Related Muscular Syndromes

To the Editor: The review by Capacchione and Muldoon1 discusses relationships between malignant hyperthermia (MH) and other muscular diseases presenting with some of the clinical features of MH. The task is made more difficult because of the uncertain relationships between a positive in vitro contracture test for halothane and caffeine, specific expression of the triggering mechanism of MH, and the presence of a genetic defect in the muscular ryanodine receptor type 1 (RYR1) gene. Capacchione and Muldoon state that “a negative RYR1 screen with a positive contracture test does not necessarily mean that the patient is not MH susceptible as there are likely yet unidentified non-RYR1 mutations that confer MHS (MH susceptibility).” Indeed, in the 20% to 30% of individuals lacking an RYR1 mutation, genetic alterations of other proteins involved in calcium release or its regulation could be present.2 Monnier et al.3 described such a mutation in the α-1 subunit of the human dihydropyridine receptor responsible for MH in a French family. Protasi et al.2 and Dainese et al.,4 studying knockout mice lacking skeletal muscle calsequestrin, a calcium-binding and buffering protein involved in the modulation of calcium release from RYR1, suggested that this mutation could be involved in MH and environmental heat stroke. On the basis of analogies between MH and catecholaminergic polymorphic ventricular tachycardia, a cardiac disorder caused by mutation in the cardiac RYR2 gene, MacLennan and Chen5 suggested that both diseases may have a common mechanism. In fact, a mutation in RyR can decrease the threshold for calcium release from sarcoplasmic reticulum, and a mutation in calsequestrin can increase the free calcium concentration over the same threshold, giving rise, in the presence of halothane and catecholamines, to contracture (MH) or arrhythmias (catecholaminergic polymorphic ventricular tachycardia), respectively. It is also possible that a mutation in different genes involved in the production and regulation of different calcium handling proteins could explain not only MH and similar muscular diseases but also some cardiac-related diseases sharing the same genetic anomaly. Luca Siracusano, MD Viviana Girasole, MD Department of Neuroscience, Psychiatric and Anesthesiological Sciences University of Messina Messina, Italy [email protected] or [email protected]

Molecular Dynamics Investigation of Anesthetic Halothane Interactions with the Proton-Gated Ion Channel GLIC

Experimental studies found that general anesthetics inhibited the function of GLIC. To investigate the underlying inhibition mechanism, we studied the interaction of halothane with GLIC and determined the potential of mean force (PMF) for transporting a sodium ion across the GLIC channel in the presence and absence of halothane. Multiple halothane binding sites were identified through docking and multi-nanosecond molecular dynamics (MD) simulations; three important ones are: A) inter-subunit site near TM2-3 loop; B) inter-subunit site near D86; and C) intra-subunit site near W160. MD simulations demonstrated that halothane altered GLIC dynamics and electrostatic interactions between critical residues. Halothane near W160 reduced the stability of salt-bridges between D32 and R192, whose homologous electrostatic interaction was suggested to be important for channel gating in the ligand-gated ion channels. To assess potential impact of halothane binding to channel conductance, we calculated PMF using adaptive biased force method. Protonation state of E222 is a determinant for the PMF. PMF profile with five deprotonated E222 residues in the pentamer showed a significantly deeper energy well than that with three deprotonated E222, suggesting that five deprotonated E222 may trap an ion and that partial protonation of E222 is necessary for ion leaving the trap. While halothane near sites A and B introduced comparatively small effects on PMF, a profound PMF change near E222 was observed in the presence of halothane at W160. This change may likely affect the single channel conductance. Taken together, halothane may modulate GLIC function through altering salt bridges crucial for gating, coupled with a breaking of the fivefold symmetry of the pore-lining helices, leading to a change in the energy profile for ion passage through the channel. Supported by NIH (R01GM66358 and R01GM56257) and NCSA through PSC.

The role of structured water in mediating general anesthetic action on alpha4beta2 nAChR.

Water is an essential component for many biological processes. Pauling proposed that water might play a critical role in general anesthesia by forming water clathrates around anesthetic molecules. To examine potential involvement of water in general anesthesia, we analyzed water within alpha4beta2 nAChR, a putative protein target hypersensitive to volatile anesthetics. Experimental structure-derived closed- and open-channel nAChR systems in a fully hydrated lipid bilayer were examined using all-atom molecular dynamics simulations. At the majority of binding sites in alpha4beta2 nAChR, halothane replaced the slow-exchanging water molecules and caused a regional water population decrease. Only two binding sites had an increased quantity of water in the presence of halothane, where water arrangements resemble clathrate-like structures. The small number of such clathrate-like water clusters suggests that the formation of water clathrates is unlikely to be a primary cause for anesthesia. Despite the decrease in water population at most of the halothane binding sites, the number of sites that can be occupied transiently by water is actually increased in the presence of halothane. Many of these water sites were located between two subunits or in regions containing agonist binding sites or critical structural elements for transducing agonist binding to channel gating. Changes in water sites in the presence of halothane affected water-mediated protein-protein interactions and the protein dynamics, which can have direct impact on protein function. Collectively, water contributes to the action of anesthetics in proteins by mediating interactions between protein subunits and altering protein dynamics, instead of forming water clathrates around anesthetics.

Why Is α4β2 Nachr More Sensitive to Volatile Anesthetics Than α7 Nachr?

Two subtypes of neuronal nicotinic acetylcholine receptors (nAChRs) show different functional sensitivities to volatile anesthetics: the α4β2 nAChR is hypersensitive while the α7 nAChR is insensitive. To understand why these homologous proteins have different functional responses to volatile anesthetics, we performed multiple sets of 20-ns molecular dynamics (MD) simulations on the closed- and open-channel α7 in the absence and presence of halothane, and compared the results with those from a similar study on the α4β2 nAChR (Liu, et al. 2009). The details about construction of receptor structural models were published (Haddadian, et al. 2008). Initial halothane docking and subsequent MD simulations revealed several halothane-binding sites in α7. Consistent with observations from α4β2, free energy perturbation calculations showed that halothane had higher binding affinity in the closed- than the open-channel; α7 and α4β2 had a comparable number of high affinity sites. GNM analysis showed that halothane induced profound changes in correlated domain motion of the open-channel α4β2, especially between the Cys-loop and the TM2-TM3 linker, but had a negligible impact to the motion of the α7. Salt bridges between these loops in the β2 subunit may be responsible for the aforementioned observation. Flexibility of several key loops in the open-channel α4β2 changed considerably in the presence of halothane, but the same loops in the open-channel α7 showed little change. Taken together, our results suggest that halothane binding in nAChRs may be necessary, but not sufficient to produce essential dynamical changes that alter protein functions. Although α7 and α4β2 are homologous, specific residues in key loops may make α4β2 more susceptible to volatile anesthetics while α7 unaffected. Supported by NIH (R01GM66358, R01GM56257, and T32GM075770) and NCSA through the PSC.

Higher Susceptibility to Halothane Modulation in Open- Than in Closed-Channel α4β2 nAChR Revealed by Molecular Dynamics Simulations

The neuronal alpha4beta2 nicotinic acetylcholine receptor (nAChR) is a potential molecular target for general anesthetics. It is unclear, however, whether anesthetic action produces the same effect on the open and closed channels. Computations parallel to our previous open channel study (J. Phys. Chem. B 2009, 113, 12581) were performed on the closed-channel alpha4beta2 nAChR to investigate the conformation-dependent anesthetic effects on channel structures and dynamics. Flexible ligand docking and over 20 ns molecular dynamics simulations revealed similar halothane-binding sites in the closed and open channels. The sites with relatively high binding affinities (approximately -6.0 kcal/mol) were identified at the interface of extracellular (EC) and transmembrane (TM) domains or at the interface between alpha4 and beta2 subunits. Despite similar sites for halothane binding, the closed-channel conformation showed much less sensitivity than the open channel to the structural and dynamical perturbations from halothane. Compared to the systems without anesthetics, the amount of water inside the pore decreased by 22% in the presence of halothane in the open channel but only by 6% in the closed channel. Comparison of the nonbonded interactions at the EC/TM interfaces suggested that the beta2 subunits were more prone than the alpha4 subunits to halothane binding. In addition, our data support the notion that halothane exerts its effect by disturbing the quaternary structure and dynamics of the channel. The study concludes that sensitivity and global dynamics responsiveness of alpha4beta2 nAChR to halothane are conformation dependent. The effect of halothane on the global dynamics of the open-channel conformation might also account for the action of other inhaled general anesthetics.

Anesthetic effects on the structure and dynamics of the second transmembrane domains of nAChR α4β2

Channel functions of the neuronal α4β2 nicotinic acetylcholine receptor (nAChR), one of the most widely expressed subtypes in the brain, can be inhibited by volatile anesthetics. Our Na + flux experiments confirmed that the second transmembrane domains (TM2) of α4 and β2 in 2:3 stoichiometry, (α4) 2(β2) 3, could form pentameric channels, whereas the α4 TM2 alone could not. The structure, topology, and dynamics of the α4 TM2 and (α4) 2(β2) 3 TM2 in magnetically aligned phospholipid bicelles were investigated using solid-state NMR spectroscopy in the absence and presence of halothane and isoflurane, two clinically used volatile anesthetics. 2H NMR demonstrated that anesthetics increased lipid conformational heterogeneity. Such anesthetic effects on lipids became more profound in the presence of transmembrane proteins. PISEMA experiments on the selectively 15N-labeled α4 TM2 showed that the TM2 formed transmembrane helices with tilt angles of 12° ± 1° and 16° ± 1° relative to the bicelle normal for the α4 and (α4) 2(β2) 3 samples, respectively. Anesthetics changed the tilt angle of the α4 TM2 from 12° ± 1° to 14° ± 1°, but had only a subtle effect on the tilt angle of the (α4) 2(β2) 3 TM2. A small degree of wobbling motion of the helix axis occurred in the (α4) 2(β2) 3 TM2. In addition, a subset of the (α4) 2(β2) 3 TM2 exhibited counterclockwise rotational motion around the helix axis on a time scale slower than 10 – 4 s in the presence of anesthetics. Both helical tilting and rotational motions have been identified computationally as critical elements for ion channel functions. This study suggested that anesthetics could alter these motions to modulate channel functions.

Store overload‐induced Ca2+ release as a triggering mechanism for CPVT and MH episodes caused by mutations in RYR and CASQ genes

Store overload‐induced Ca²⁺ release as a triggering mechanism for CPVT and MH episodes caused by mutations in <i>RYR</i> and <i>CASQ</i> genes

Mechanism of Interaction between the General Anesthetic Halothane and a Model Ion Channel Protein, II: Fluorescence and Vibrational Spectroscopy Using a Cyanophenylalanine Probe

We demonstrate that cyano-phenylalanine (PheCN) can be utilized to probe the binding of the inhalational anesthetic halothane to an anesthetic-binding, model ion channel protein hbAP-PheCN. The Trp to PheCN mutation alters neither the α-helical conformation nor the 4-helix bundle structure. The halothane binding properties of this PheCN mutant hbAP-PheCN, based on fluorescence quenching, are consistent with those of the prototype, hbAP1. The dependence of fluorescence lifetime as a function of halothane concentration implies that the diffusion of halothane in the nonpolar core of the protein bundle is one-dimensional. As a consequence, at low halothane concentrations, the quenching of the fluorescence is dynamic, whereas at high concentrations the quenching becomes static. The 4-helix bundle structure present in aqueous detergent solution and at the air-water interface, is preserved in multilayer films of hbAP-PheCN, enabling vibrational spectroscopy of both the protein and its nitrile label (-CN). The nitrile groups' stretching vibration band shifts to higher frequency in the presence of halothane, and this blue-shift is largely reversible. Due to the complexity of this amphiphilic 4-helix bundle model membrane protein, where four PheCN probes are present adjacent to the designed cavity forming the binding site within each bundle, all contributing to the infrared absorption, molecular dynamics (MD) simulation is required to interpret the infrared results. The MD simulations indicate that the blue-shift of -CN stretching vibration induced by halothane arises from an indirect effect, namely an induced change in the electrostatic protein environment averaged over the four probe oscillators, rather than a direct interaction with the oscillators. hbAP-PheCN therefore provides a successful template for extending these investigations of the interactions of halothane with the model membrane protein via vibrational spectroscopy, using cyano-alanine residues to form the anesthetic binding cavity.

Anesthetic Modulation of Protein Dynamics: Insight from an NMR Study

Mistic (membrane integrating sequence for translation of integral membrane protein constructs) comprises the four-alpha-helix bundle scaffold found in the transmembrane domains of the Cys-loop receptors that are plausible targets for general anesthetics. Nuclear magnetic resonance (NMR) studies of anesthetic halothane interaction with Mistic in dodecyl phosphocholine (DPC) micelles provide an experimental basis for understanding molecular mechanisms of general anesthesia. Halothane was found to interact directly with Mistic, mostly in the interfacial loop regions. Although the presence of halothane had little effect on Mistic structure, (15)N NMR relaxation dispersion measurements revealed that halothane affected Mistic's motion on the microsecond-millisecond time scale. Halothane shifted the equilibrium of chemical exchange in some residues and made the exchange faster or slower in comparison to the original state in the absence of halothane. The motion on the microsecond-millisecond time scale in several residues disappeared in response to the addition of halothane. Most of the residues experiencing halothane-induced dynamics changes also exhibited profound halothane-induced changes in chemical shift, suggesting that dynamics modification of these residues might result from their direct interaction with halothane molecules. Allosteric modulation by halothane also contributed to dynamics changes, as reflected in residues I52 and Y82 where halothane introduction brought about dynamics changes but not chemical shift changes. The study suggests that inhaled general anesthetics could act on proteins via altering protein motion on the microsecond-millisecond time scale, especially motion in the flexible loops that link different alpha helices. The validation of anesthetic effect on protein dynamics that are potentially correlated with protein functions is a critical step in unraveling the mechanisms of anesthetic action on proteins.

Four- α-Helix Bundle with Designed Anesthetic Binding Pockets. Part II: Halothane Effects on Structure and Dynamics

As a model of the protein targets for volatile anesthetics, the dimeric four- α-helix bundle, (A α 2-L1M/L38M) 2, was designed to contain a long hydrophobic core, enclosed by four amphipathic α-helices, for specific anesthetic binding. The structural and dynamical analyses of (A α 2-L1M/L38M) 2 in the absence of anesthetics (another study) showed a highly dynamic antiparallel dimer with an asymmetric arrangement of the four helices and a lateral accessing pathway from the aqueous phase to the hydrophobic core. In this study, we determined the high-resolution NMR structure of (A α 2-L1M/L38M) 2 in the presence of halothane, a clinically used volatile anesthetic. The high-solution NMR structure, with a backbone root mean-square deviation of 1.72 Å (2JST), and the NMR binding measurements revealed that the primary halothane binding site is located between two side-chains of W15 from each monomer, different from the initially designed anesthetic binding sites. Hydrophobic interactions with residues A44 and L18 also contribute to stabilizing the bound halothane. Whereas halothane produces minor changes in the monomer structure, the quaternary arrangement of the dimer is shifted by about half a helical turn and twists relative to each other, which leads to the closure of the lateral access pathway to the hydrophobic core. Quantitative dynamics analyses, including Modelfree analysis of the relaxation data and the Carr-Purcell-Meiboom-Gill transverse relaxation dispersion measurements, suggest that the most profound anesthetic effect is the suppression of the conformational exchange both near and remote from the binding site. Our results revealed a novel mechanism of an induced fit between anesthetic molecule and its protein target, with the direct consequence of protein dynamics changing on a global rather than a local scale. This mechanism may be universal to anesthetic action on neuronal proteins.

The Effects of General Anesthesia on Whole Body and Regional Pharmacokinetics of Local Anesthetics at Toxic Doses

Local anesthetic toxicity is often studied experimentally in anesthetized subjects, but clinical toxicity usually occurs in conscious patients. In this study, we determined the influence of general anesthesia on the pharmacokinetics of six local anesthetics administered i.v. at approximately the highest recommended doses. Chronically instrumented ewes (approximately 45-50 kg, n = 18) were infused over 3 min with (base doses as HCl salts) bupivacaine (100 mg), levobupivacaine (125 mg), ropivacaine (150 mg), lidocaine (350 mg), mepivacaine (350 mg), or prilocaine (350 mg), on separate occasions when conscious and halothane anesthetized. Serial arterial, heart, and brain venous blood drug concentrations were measured by achiral/chiral high-performance liquid chromatography, as relevant. Whole body pharmacokinetics were assessed by noncompartmental analysis; heart and brain pharmacokinetics were assessed by mass balance. Drug blood binding, in the absence and presence of halothane, was assessed by equilibrium dialysis in vitro. Blood local anesthetic concentrations were doubled with anesthesia because of decreased whole body distribution and clearance (respectively, to 33% and 52% of values when conscious). Heart and brain net drug uptake were greater under anesthesia, reflecting slower efflux from both regions. Clearances of R-bupivacaine > S-bupivacaine and R-prilocaine > S-prilocaine, but, mepivacaine clearance was not enantioselective. Halothane did not influence blood binding of the local anesthetics. General anesthesia significantly changed whole body and regional pharmacokinetics of each local anesthetic as well as the systemic effects. General anesthesia is thus an important but frequently overlooked factor in studies of local anesthetic toxicity.

Halothane induces vesicular and carrier-mediated release of [ 3H]serotonin from rat brain cortical slices

The serotonergic system may play a role during general anesthesia but the effect of the volatile anesthetic halothane on the release of serotonin (5-HT) is not fully understood. Rat brain cortical slices were labeled with [ 3H]5-HT to investigate the effects of halothane on the release of this neurotransmitter from the central nervous system. Halothane induced an increase on the release of [ 3H]5-HT that was dependent on incubation time and anesthetic concentration (0.006, 0.012, 0.024, 0.036, 0.048 and 0.072 mM). This effect was independent of extracellular calcium and was not affected by tetrodotoxin (blocker of voltage dependent Na + channels). In contrast, the halothane-evoked [ 3H]5-HT release was reduced by BAPTA-AM, a membrane-permeable BAPTA analog that chelates intracellular Ca 2+. The anesthetic-induced [ 3H]5-HT release depends on the ryanodine-sensitive intracellular calcium store since it was blocked by dantrolene and azumolene (inhibitors of the calcium-release through ryanodine receptors) but was not affected by aminoethoxydiphenylborate (2-APB), an inhibitor of inositol 1,4,5-triphosphate receptor. The [ 3H]5-HT release induced by halothane comes mainly from the vesicular pool since it was reduced in about 70% by reserpine, a blocker of vesicular monoamine transporter. The halothane-evoked release of [ 3H]5-HT release is reduced by fluoxetine, an inhibitor of 5-HT uptake, and the volatile agent also decreased the uptake of [ 3H]5-HT into rat brain cortical slices. Moreover, a decrease on halothane-induced release of [ 3H]5-HT was also observed when the brain cortical slices were incubated at low temperature, which is known to interfere with the carrier-mediated release of the neurotransmitter. Ouabain, a Na +/K + ATPase pump inhibitor, which induces 5-HT release through reverse transport, also decreased [ 3H]5-HT release induced by halothane, confirming the involvement of a carrier-mediated release of the neurotransmitter in the presence of halothane. In conclusion, these data suggest that halothane induces vesicular and carrier-mediated release of [ 3H]5-HT in rat brain cortical slices.

Alzheimer’s Disease: Halothane Induces Aβ Peptide to Oligomeric Form—Solution NMR Studies

Alzheimer's disease (AD) is a significant contributor to cognitive decline and is responsible for about half of the cases of dementia in later life. Although exact etiology of AD is not known, however, many risk factors for AD are identified. Anesthesia for elderly patients is considered as a risk factor in AD as they frequently experience deterioration in cognitive function with long exposure to anesthetics during surgery. Inhaled anesthetic agents remain the mainstay for patients undergoing major surgical operations. This study using multidimensional NMR spectroscopy provides the first direct evidence in vitro that inhaled anesthetic, halothane specifically interacts with Abeta40 and Abeta42 peptide. Halothane induces structural alternation of Abeta peptide from soluble monomeric alpha-helical form to oligomeric beta-sheet conformation, which may hasten the onset of AD. Abeta42 is more prone to oligomerization compared to Abeta40 in the presence of halothane. The molecular mechanism of halothane induced structural alternation of Abeta peptide is discussed.

Tandem-Pore K+ Channels Mediate Inhibition of Orexin Neurons by Glucose

Glucose-inhibited neurons orchestrate behavior and metabolism according to body energy levels, but how glucose inhibits these cells is unknown. We studied glucose inhibition of orexin/hypocretin neurons, which promote wakefulness (their loss causes narcolepsy) and also regulate metabolism and reward. Here we demonstrate that their inhibition by glucose is mediated by ion channels not previously implicated in central or peripheral glucose sensing: tandem-pore K(+) (K(2P)) channels. Importantly, we show that this electrical mechanism is sufficiently sensitive to encode variations in glucose levels reflecting those occurring physiologically between normal meals. Moreover, we provide evidence that glucose acts at an extracellular site on orexin neurons, and this information is transmitted to the channels by an intracellular intermediary that is not ATP, Ca(2+), or glucose itself. These results reveal an unexpected energy-sensing pathway in neurons that regulate states of consciousness and energy balance.

Effect of an inhalation anesthetic on the viscosity of aqueous bovine serum albumin solutions

The viscosities of aqueous bovine serum albumin (BSA) solutions were measured as a function of pH in the neutral and acidic region in the absence and presence of inhalation anesthetic halothane. The reduced viscosities of BSA solutions in the presence of halothane at any pH measured had smaller values than those in the absence of halothane. The finding suggests that the volume of a BSA molecule in the solution shrinks by adding halothane irrespective of its structure.

Anesthetic Interaction with Ketosteroid Isomerase: Insights from Molecular Dynamics Simulations

The nature and the sites of interactions between anesthetic halothane and homodimeric Δ 5-3-ketosteroid isomerase (KSI) are characterized by flexible ligand docking and confirmed by 1H- 15N NMR. The dynamics consequence of halothane interaction and the implication of the dynamic changes to KSI function are studied by multiple 5-ns molecular dynamics simulations in the presence and absence of halothane. Both docking and MD simulations show that halothane prefer the amphiphilic dimeric interface to the hydrophobic active site of KSI. Halothane occupancy at the dimer interface disrupted the intersubunit hydrogen bonding formed either directly through side chains of polar residues or indirectly through the mediation of the interfacial water molecules. Moreover, in the presence of halothane, the exchange rate of the bound waters with bulk water was increased. Halothane perturbation to the dimer interface affected the overall flexibility of the active site. This action is likely to contribute to the halothane-induced reduction of the KSI activity. The allosteric halothane modulation of the dynamics-function relationship of KSI without direct competition at the enzymatic active sites may be generalized to offer a unifying explanation of anesthetic action on a diverse range of multidomain neuronal proteins that are potentially relevant to clinical general anesthesia.