Action Of Volatile Anesthetics Research Articles

Mechanosensing and anesthesia of single internodal cells of Chara

ABSTRACT The giant (2–3 × 10−2 m long) internodal cells of the aquatic plant, Chara, exhibit a rapid (>100 × 10−6 m s−1) cyclic cytoplasmic streaming which stops in response to mechanical stimuli. Since the streaming – and the stopping of streaming upon stimulation – is easily visible with a stereomicroscope, these single cells are ideal tools to investigate mechanosensing in plant cells, as well as the potential for these cells to be anesthetized. We found that dropping a steel ball (0.88 × 10−3 kg, 6 × 10−3 m in diameter) through a 4.6 cm long tube (delivering ca. 4 × 10−4 J) reliably induced mechanically-stimulated cessation of cytoplasmic streaming. To determine whether mechanically-induced cessation of cytoplasmic streaming in Chara was sensitive to anesthesia, we treated Chara internodal cells to volatilized chloroform in a 9.8 × 10−3 m3 chamber for 2 minutes. We found that low doses (15,000–25,000 ppm) of chloroform did not always anesthetize cells, whereas large doses (46,000 and higher) proved lethal. However, 31,000 ppm chloroform completely, and reversibly, anesthetized these cells in that they did not stop cytoplasmic streaming upon mechanostimulation, but after 24 hours the cells recovered their sensitivity to mechanostimulation. We believe this single-cell model will prove useful for elucidating the still obscure mode of action of volatile anesthetics.

Volatile anesthetics inhibit presynaptic cGMP signaling to depress presynaptic excitability in rat hippocampal neurons

Volatile anesthetics alter presynaptic function through effects on Ca2+ influx and neurotransmitter release. These actions are proposed to play important roles in their pleiotropic neurophysiological effects including immobility, unconsciousness and amnesia. Nitric oxide and cyclic guanosine monophosphate (NO/cGMP) signaling has been implicated in presynaptic mechanisms, and disruption of NO/cGMP signaling has been shown to alter sensitivity to volatile anesthetics in vivo. We investigated volatile anesthetic actions NO/cGMP signaling in relation to presynaptic function in cultured rat hippocampal neurons using pharmacological tools and genetically encoded biosensors and sequestering probes of cGMP levels. Using the fluorescent cGMP biosensor cGull, we found that electrical stimulation-evoked NMDA-type glutamate receptor-independent presynaptic cGMP transients were inhibited 33.2% by isoflurane (0.51 mM) and 26.4% by sevoflurane (0.57 mM) (p < 0.0001) compared to control stimulation without anesthetic. Stimulation-evoked cGMP transients were blocked by the nonselective inhibitor of nitric oxide synthase N-ω-nitro-l-arginine, but not by the selective neuronal nitric oxide synthase inhibitor N5-(1-imino-3-butenyl)-l-ornithine. Isoflurane and sevoflurane inhibition of stimulation-evoked increases in presynaptic Ca2+ concentration, measured with synaptophysin-GCaMP6f, and of synaptic vesicle exocytosis, measured with synaptophysin-pHlourin, was attenuated in neurons expressing the cGMP scavenger protein sponge (inhibition of exocytosis reduced by 54% for isoflurane and by 53% for sevoflurane). The anesthetic-induced reduction in presynaptic excitability was partially occluded by inhibition of HCN channels, a cGMP-modulated excitatory ion channel that can facilitate glutamate release. We propose that volatile anesthetics depress presynaptic cGMP signaling and downstream effectors like HCN channels that are essential to presynaptic function and excitability. These findings identify novel mechanisms by which volatile anesthetics depress synaptic transmission via second messenger signaling involving the NO/cGMP pathway in hippocampal neurons.

Isoflurane inhibition of endocytosis is an anesthetic mechanism of action

The mechanisms of volatile anesthetic action remain among the most perplexing mysteries of medicine. Across phylogeny, volatile anesthetics selectively inhibit mitochondrial complex I, and they also depress presynaptic excitatory signaling. To explore how these effects are linked, we studied isoflurane effects on presynaptic vesicle cycling and ATP levels in hippocampal cultured neurons from wild-type and complex Imutant (Ndufs4(KO)) mice. To bypass complex I, we measured isoflurane effects on anesthetic sensitivity in mice expressing NADH dehydrogenase (NDi1). Endocytosis in physiologic concentrations of glucose was delayed by effective behavioral concentrations of isoflurane in both wild-type (τ [unexposed] 44.8± 24.2 s;τ [exposed] 116.1± 28.1 s; p<0.01) and Ndufs4(KO) cultures (τ [unexposed] 67.6± 16.0 s;τ [exposed] 128.4± 42.9 s; p= 0.028). Increasing glucose, to enhance glycolysis and increase ATP production, led to maintenance of both ATP levels and endocytosis (τ [unexposed] 28.0± 14.4;τ [exposed] 38.2± 5.7; reducing glucose worsened ATP levels and depressed endocytosis (τ [unexposed] 85.4± 69.3; τ [exposed]>1,000; p<0.001). The block in recycling occurred at the level of reuptake of synaptic vesicles into the presynaptic cell. Expression of NDi1 in wild-type mice caused behavioral resistance to isoflurane for tail clamp response (EC50 Ndi1(-) 1.27%± 0.14%; Ndi1(+) 1.55%± 0.13%) and halothane (EC50 Ndi1(-) 1.20%± 0.11%; Ndi1(+) 1.46%± 0.10%); expression of NDi1 in neurons improved hippocampal function, alleviated inhibition of presynaptic recycling, and increased ATP levels during isoflurane exposure. The clear alignment of cell culture data to invivo phenotypes of both isoflurane-sensitive and -resistant mice indicates that inhibition of mitochondrial complex I is a primary mechanism of action of volatile anesthetics.

Metabolomics and Whole-Exome Sequencing in Patients with Differential Sensitivity to Sevoflurane: A Protocol for a Prospective Observational Trial.

Introduction: Different sensitivity to volatile anesthetics in Drosophila, nematodes and mice is related to mutation of energy metabolism genes. In clinical practice, we find that the end-tidal sevoflurane concentration (ETsevo) differs among patients at the same depth of anesthesia, indicating that the sensitivity to sevoflurane varies among patients. However, the underlying mechanism remains unclear. The sensitivity of an anesthetic is associated with the postoperative outcomes of patients and the mechanism of action of volatile anesthetics. We therefore propose this protocol to determine whether differences in metabolite profile and genetic variations contribute to patients’ sensitivity to volatile anesthetics. Methods and Analysis: This is a single-centre, prospective observational study. 720 patients undergoing abdominal surgery were included. General anesthesia was induced with inhaled sevoflurane, a bolus of sufentanil (0.2–0.4 μg/kg) and cis-atracurium (0.2–0.3 mg/kg). The end-tidal sevoflurane concentration (ETsevo) was adjusted to maintain a BIS (bispectral index) value between 40–60. The mean ETsevo from 20 min after endotracheal intubation to 2 h after the beginning of surgery (steady state) was calculated for each patient. Patients were further divided into a high-sensitivity group (mean ETsevo – SD) and a low-sensitivity group (mean ETsevo + SD) to investigate the sensitivity to sevoflurane. Cases were paired from the high-sensitivity group (group H) and low-sensitivity group (group L) according to age, sex, body mass index (BMI), ASA physical status classification, vital signs, BIS, ephedrine use, sufentanildose, and cis-atracurium dose at anesthesia induction and steady state. Differences in metabolite levels, single nucleotide polymorphisms (SNPs) and protein-coding gene sequence variations between group H and group L will be determined through plasma metabolomics, whole-exome sequencing (WES), genome-wide association study (GWAS), and bioinformatics analyses. These results will be analysed to determine the reasons for the differential sensitivity to sevoflurane in humans. Ethics and Dissemination: This prospective observational study protocol has received ethical approval from the Ethical Committee of West China Hospital of Sichuan University on May 19, 2017 (Approval No. 78). Informed consent will be obtained before patient enrolment. The results will be submitted to international peer-review journals. Trial Registration Number: ChiCTR1800014327.

Gas-Induced Drying of Nanopores.

Here, we investigate the role of a dilute hydrophobic gas on the phase behavior of water confined in hydrophobic nanopores. Molecular dynamics showed that gas atoms are hydrophobically attracted within the pores, where even a single particle is able to induce spontaneous drying of the whole pore. The drying process is rationalized in terms of its free-energy landscape, revealing that the penetration of a gas atom is able to suppress the drying free-energy barriers of hydrophobic pores. Results provide insights into the role of gases on the wettability of nanopores and evidence of a possibile physical mechanism for the action of volatile anesthetics on some kinds of ion channels. Results also indicate a novel, bioinspired strategy for controlling bubble formation in nanopores for sensing and energy applications.

PD-1 Regulates GABAergic Neurotransmission and GABA-Mediated Analgesia and Anesthesia.

SummaryThe immune checkpoint inhibitor programmed cell death protein 1 (PD-1) plays a critical role in immune regulation. Recent studies have demonstrated functional PD-1 expression in peripheral sensory neurons, which contributes to neuronal excitability, pain, and opioid analgesia. Here we report neuronal expression and function of PD-1 in the central nervous system (CNS), including the spinal cord, thalamus, and cerebral cortex. Notably, GABA-induced currents in spinal dorsal horn neurons, thalamic neurons, and cortical neurons are suppressed by the PD-1-neutralizing immunotherapeutic Nivolumab in spinal cord slices, brain slices, and dissociated cortical neurons. Reductions in GABA-mediated currents in CNS neurons were also observed in Pd1−/− mice without changes in GABA receptor expression. Mechanistically, Nivolumab binds spinal cord neurons and elicits ERK phosphorylation to suppress GABA currents. Finally, both GABA-mediated analgesia and anesthesia are impaired by Pd1 deficiency. Our findings reveal PD-1 as a CNS-neuronal inhibitor that regulates GABAergic signaling and GABA-mediated behaviors.

Development of a simple method for differential delivery of volatile anesthetics to the spinal cord of the rabbit.

Emulsified volatile anesthetic can be directly injected into the circulation and eliminated from blood through lungs. Taking advantage of the unique pharmacokinetics of the emulsified volatile anesthetics, we aimed to develop a less traumatic method to differentially deliver them to the spinal cord of rabbit. Sixteen New Zealand White rabbits were randomly assigned to the isoflurane or sevoflurane group. A catheter was placed into the descending aorta, and emulsified isoflurane (8mg/kg/h) or sevoflurane (12mg/kg/h) was given respectively. The concentration and partial pressure of the anesthetics in the jugular and femoral vein were measured. Our results showed that the partial pressure for isoflurane was 3.91±1.11 mmHg and 12.61±1.60 mmHg (1.0MAC), and for sevoflurane was 3.89±1.00 mmHg and 19.92±1.84mmHg (1.0MAC), in the jugular vein and femoral vein, respectively. There was significant difference between jugular and femoral vein partial pressure for both isoflurane and sevoflurane groups (both P < 0.001). In conclusion, a simple and minimally invasive method has been successfully developed to selectively deliver isoflurane and sevoflurane to the spinal cord in the rabbit. Before the anesthetics taking action on the brain, 69% of isoflurane and 81% of sevoflurane were removed through lungs. This method can be used to investigate sites and mechanisms of volatile anesthetic action.

The actions of volatile anesthetics: a new perspective.

This article reviews recent work in applying neutron and X-ray scattering towards the elucidation of the molecular mechanisms of volatile anesthetics. Experimental results on domain mixing in ternary lipid mixtures, and the influence of volatile anesthetics and hydrostatic pressure are placed in the contexts of ion-channel function and receptor trafficking at the postsynaptic density.

Breakdown of Neural Function under Isoflurane Anesthesia: In Vivo, Multineuronal Imaging in Caenorhabditis elegans.

WHAT THIS ARTICLE TELLS US THAT IS NEW: BACKGROUND:: Previous work on the action of volatile anesthetics has focused at either the molecular level or bulk neuronal measurement such as electroencephalography or functional magnetic resonance imaging. There is a distinct gulf in resolution at the level of cellular signaling within neuronal systems. The authors hypothesize that anesthesia is caused by induced dyssynchrony in cellular signaling rather than suppression of individual neuron activity. Employing confocal microscopy and Caenorhabditis elegans expressing the calcium-sensitive fluorophore GCaMP6s in specific command neurons, the authors measure neuronal activity noninvasively and in parallel within the behavioral circuit controlling forward and reverse crawling. The authors compare neuronal dynamics and coordination in a total of 31 animals under atmospheres of 0, 4, and 8% isoflurane. When not anesthetized, the interneurons controlling forward or reverse crawling occupy two possible states, with the activity of the "reversal" neurons AVA, AVD, AVE, and RIM strongly intercorrelated, and the "forward" neuron AVB anticorrelated. With exposure to 4% isoflurane and onset of physical quiescence, neuron activity wanders rapidly and erratically through indeterminate states. Neuron dynamics shift toward higher frequencies, and neuron pair correlations within the system are reduced. At 8% isoflurane, physical quiescence continues as neuronal signals show diminished amplitude with little correlation between neurons. Neuronal activity was further studied using statistical tools from information theory to quantify the type of disruption caused by isoflurane. Neuronal signals become noisier and more disordered, as measured by an increase in the randomness of their activity (Shannon entropy). The coordination of the system, measured by whether information exhibited in one neuron is also exhibited in other neurons (multiinformation), decreases significantly at 4% isoflurane (P = 0.00015) and 8% isoflurane (P = 0.0028). The onset of anesthesia corresponds with high-frequency randomization of individual neuron activity coupled with induced dyssynchrony and loss of coordination between neurons that disrupts functional signaling.

Isoflurane disrupts excitatory neurotransmitter dynamics via inhibition of mitochondrial complex I

BackgroundThe mechanisms of action of volatile anaesthetics are unclear. Volatile anaesthetics selectively inhibit complex I in the mitochondrial respiratory chain. Mice in which the mitochondrial complex I subunit NDUFS4 is knocked out [Ndufs4(KO)] either globally or in glutamatergic neurons are hypersensitive to volatile anaesthetics. The volatile anaesthetic isoflurane selectively decreases the frequency of spontaneous excitatory events in hippocampal slices from Ndufs4(KO) mice. MethodsComplex I inhibition by isoflurane was assessed with a Clark electrode. Synaptic function was measured by stimulating Schaffer collateral fibres and recording field potentials in the hippocampus CA1 region. ResultsIsoflurane specifically inhibits complex I dependent respiration at lower concentrations in mitochondria from Ndufs4(KO) than from wild-type mice. In hippocampal slices, after high frequency stimulation to increase energetic demand, short-term synaptic potentiation is less in KO compared with wild-type mice. After high frequency stimulation, both Ndufs4(KO) and wild-type hippocampal slices exhibit striking synaptic depression in isoflurane at twice the 50% effective concentrations (EC50). The pattern of synaptic depression by isoflurane indicates a failure in synaptic vesicle recycling. Application of a selective A1 adenosine receptor antagonist partially eliminates isoflurane-induced short-term depression in both wild-type and Ndufs4(KO) slices, implicating an additional mitochondria-dependent effect on exocytosis. When mitochondria are the sole energy source, isoflurane completely eliminates synaptic output in both mutant and wild-type mice at twice the (EC50) for anaesthesia. ConclusionsVolatile anaesthetics directly inhibit mitochondrial complex I as a primary target, limiting synaptic ATP production, and excitatory vesicle endocytosis and exocytosis.

Identification of binding sites contributing to volatile anesthetic effects on GABA type A receptors.

Most general anesthetics enhance GABA type A (GABAA) receptor activity at clinically relevant concentrations. Sites of action of volatile anesthetics on the GABAA receptor remain unknown, whereas sites of action of many intravenous anesthetics have been identified in GABAA receptors by using photolabeling. Here, we used photoactivatable analogs of isoflurane (AziISO) and sevoflurane (AziSEVO) to locate their sites on α1β3γ2L and α1β3 GABAA receptors. As with isoflurane and sevoflurane, AziISO and AziSEVO enhanced the currents elicited by GABA. AziISO and AziSEVO each labeled 10 residues in α1β3 receptors and 9 and 8 residues, respectively, in α1β3γ2L receptors. Photolabeled residues were concentrated in transmembrane domains and located in either subunit interfaces or in the interface between the extracellular domain and the transmembrane domain. The majority of these transmembrane residues were protected from photolabeling with the addition of excess parent anesthetic, which indicated specificity. Binding sites were primarily located within α+/β- and β+/α- subunit interfaces, but residues in the α+/γ- interface were also identified, which provided a basis for differential receptor subtype sensitivity. Isoflurane and sevoflurane did not always share binding sites, which suggests an unexpected degree of selectivity.-Woll, K. A., Zhou, X., Bhanu, N. V., Garcia, B. A., Covarrubias, M., Miller, K. W., Eckenhoff, R. G. Identification of binding sites contributing to volatile anesthetic effects on GABA type A receptors.

Time- and Dose-Dependent Effects of Desflurane in Sensitized Airways

Although the bronchodilatory actions of volatile anesthetics, such as halothane, isoflurane, and sevoflurane, have been well documented in previous studies, the properties of desflurane remain controversial. The aim of this study was to investigate the effects of desflurane at different concentrations and durations in an ovalbumin-sensitized guinea pig model of airway hyper-responsiveness. Ovalbumin-sensitized animals (n = 176) were randomly assigned to 5 groups according to the minimum alveolar concentration (MAC) of desflurane they received: 0.0, 0.5, 1.0, 1.5, and 2.0 MAC. Total lung resistance in vivo, airway smooth muscle tension in vitro, and intracellular cyclic adenosine monophosphate (AMP) levels were measured to evaluate the effects of desflurane. In 5 sensitized groups, total lung resistance increased from baseline to peak at approximately 8 minutes and then decreased slowly until about 17 minutes with extended administration of desflurane. Desflurane dose-dependently increased total lung resistance with or without incremental doses of acetylcholine and reduced muscle tension with increasing concentrations of carbacholine. Cyclic AMP levels were increased by desflurane: at the 60-minute time point, cyclic AMP concentrations (means ± SD) with 0.5 MAC (1.96 ± 0.40) and 1.0 MAC (2.11 ± 0.50) desflurane were higher than those at the 8-minute time point (1.11 ± 0.23 and 1.32 ± 0.32). Desflurane exerted time- and dose-dependent effects and could be used at 0.5 and 1.0 MAC concentrations without significant bronchoconstriction in ovalbumin-sensitized guinea pigs. Cyclic AMP-mediated airway smooth muscle relaxation might be one mechanism by which desflurane induces bronchodilation.

HCN1 Channels Contribute to the Effects of Amnesia and Hypnosis but not Immobility of Volatile Anesthetics.

Hyperpolarization-activated, cyclic nucleotide-gated (HCN) subtype 1 (HCN1) channels have been identified as targets of ketamine to produce hypnosis. Volatile anesthetics also inhibit HCN1 channels. However, the effects of HCN1 channels on volatile anesthetics in vivo are still elusive. This study uses global and conditional HCN1 knockout mice to evaluate how HCN1 channels affect the actions of volatile anesthetics. Minimum alveolar concentrations (MACs) of isoflurane and sevoflurane that induced immobility (MAC of immobility) and/or hypnosis (MAC of hypnosis) were determined in wild-type mice, global HCN1 knockout (HCN1) mice, HCN1 channel gene with 2 lox-P sites flanking a region of the fourth exon of HCN1 (HCN1) mice, and forebrain-selective HCN1 knockout (HCN1: cre) mice. Immobility of mice was defined as no purposeful reactions to tail-clamping stimulus, and hypnosis was defined as loss of righting reflex. The amnestic effects of isoflurane and sevoflurane were evaluated by fear-potentiated startle in these 4 strains of mice. All MAC values were expressed as mean ± SEM. For MAC of immobility of isoflurane, no significant difference was found among wild-type, HCN1, HCN1, and HCN1: cre mice (all ~1.24%-1.29% isoflurane). For both HCN1 and HCN1: cre mice, the MAC of hypnosis for isoflurane (each ~1.05% isoflurane) was significantly increased over their nonknockout controls: HCN1 versus wild-type (0.86% ± 0.03%, P < 0.001) and HCN1: cre versus HCN1 mice (0.84% ± 0.03%, P < 0.001); no significant difference was found between HCN1 and HCN1: cre mice. For MAC of immobility of sevoflurane, no significant difference was found among wild-type, HCN1, HCN1, and HCN1: cre mice (all ~2.6%-2.7% sevoflurane). For both HCN1 and HCN1: cre mice, the MAC of hypnosis for sevoflurane (each ~1.90% sevoflurane) was significantly increased over their nonknockout controls: HCN1 versus wild-type (1.58% ± 0.05%, P < 0.001) and HCN1: cre versus HCN1 mice (1.56% ± 0.05%, P < 0.001). No significant difference was found between HCN1 and HCN1: cre mice. By fear-potentiated startle experiments, amnestic effects of isoflurane and sevoflurane were significantly attenuated in HCN1 and HCN1: cre mice (both P < 0.002 versus wild-type or HCN1 mice). No significant difference was found between HCN1 and HCN1: cre mice. Forebrain HCN1 channels contribute to hypnotic and amnestic effects of volatile anesthetics, but HCN1 channels are not involved in the immobilizing actions of volatile anesthetics.

Increased Risk of Intraoperative Awareness in Patients with a History of Awareness

Patients with a history of intraoperative awareness with explicit recall (AWR) are hypothesized to be at higher risk for AWR than the general surgical population. In this study, the authors assessed whether patients with a history of AWR (1) are actually at higher risk for AWR; (2) receive different anesthetic management; and (3) are relatively resistant to the hypnotic actions of volatile anesthetics. Patients with a history of AWR and matched controls from three randomized clinical trials investigating prevention of AWR were compared for relative risk of AWR. Anesthetic management was compared with the use of the Hotelling's T statistic. A linear mixed model, including previously identified covariates, assessed the effects of a history of AWR on the relationship between end-tidal anesthetic concentration and bispectral index. The incidence of AWR was 1.7% (4 of 241) in patients with a history of AWR and 0.3% (4 of 1,205) in control patients (relative risk = 5.0; 95% CI, 1.3-19.9). Anesthetic management did not differ between cohorts, but there was a significant effect of a history of AWR on the end-tidal anesthetic concentration versus bispectral index relationship. Surgical patients with a history of AWR are five times more likely to experience AWR than similar patients without a history of AWR. Further consideration should be given to modifying perioperative care and postoperative evaluation of patients with a history of AWR.

The effects of volatile anesthetics on the extracellular accumulation of [(3)H]GABA in rat brain cortical slices.

GABA is an inhibitory neurotransmitter that appears to be associated with the action of volatile anesthetics. These anesthetics potentiate GABA-induced postsynaptic currents by synaptic GABAA receptors, although recent evidence suggests that these agents also significantly affect extrasynaptic GABA receptors. However, the effect of volatile anesthetics on the extracellular concentration of GABA in the central nervous system has not been fully established. In the present study, rat brain cortical slices loaded with [(3)H]GABA were used to investigate the effect of halothane and sevoflurane on the extracellular accumulation of this neurotransmitter. The accumulation of [(3)H]GABA was significantly increased by sevoflurane (0.058, 0.11, 0.23, 0.46, and 0.93mM) and halothane (0.006, 0.012, 0.024, 0.048, 0072, and 0.096mM) with an EC50 of 0.26mM and 35μM, respectively. TTX (blocker of voltage-dependent Na(+) channels), EGTA (an extracellular Ca(2+) chelator) and BAPTA-AM (an intracellular Ca(2+) chelator) did not interfere with the accumulation of [(3)H]GABA induced by 0.23mM sevoflurane and 0.048mM halothane. SKF 89976A, a GABA transporter type 1 (GAT-1) inhibitor, reduced the sevoflurane- and halothane-induced increase in the accumulation of GABA by 57 and 63%, respectively. Incubation of brain cortical slices at low temperature (17°C), a condition that inhibits GAT function and reduces GABA release through reverse transport, reduced the sevoflurane- and halothane-induced increase in the accumulation of [(3)H]GABA by 82 and 75%, respectively, relative to that at normal temperature (37°C). Ouabain, a Na(+)/K(+) ATPase pump inhibitor, which is known to induce GABA release through reverse transport, abolished the sevoflurane and halothane effects on the accumulation of [(3)H]GABA. The effect of sevoflurane and halothane did not involve glial transporters because β-alanine, a blocker of GAT-2 and GAT-3, did not inhibit the effect of the anesthetics. In conclusion, the present study suggests that sevoflurane and halothane increase the accumulation of GABA by inducing the reverse transport of this neurotransmitter. Therefore, volatile anesthetics could interfere with neuronal excitability by increasing the action of GABA on synaptic and extrasynaptic GABA receptors.

PKC independent inhibition of voltage gated calcium channels by volatile anesthetics in freshly isolated vascular myocytes from the aorta

In this study we used barium currents through voltage gated L-type calcium channels (recorded in freshly isolated cells with a conventional patch-clamp technique) to elucidate the cellular action mechanism for volatile anesthetics. It was found that halothane and isoflurane inhibited (dose-dependently and voltage independently) Ba2+ currents through voltage gated Ca2+ channels. Half maximal inhibitions occurred at 0.64±0.07mM and 0.86±0.1mM. The Hill slope value was 2 for both volatile anesthetics, suggesting the presence of more than one interaction site. Current inhibition by volatile anesthetics was prominent over the whole voltage range without changes in the peak of the current voltage relationship. Intracellular infusion of the GDPβS (100μM) together with staurosporine (200nM) did not prevent the inhibitory effect of volatile anesthetics. Unlike pharmacological Ca2+ channel blockers, volatile anesthetics blocked Ca2+ channel currents at resting membrane potentials. In other words, halothane and isoflurane induced an ‘initial block’. After the first 4–7 control pulses, the cells were left unstimulated and anesthetics were applied. The first depolarization after the pause evoked a Ca2+ channel current whose amplitude was reduced to 41±3.4% and to 57±4.2% of control values. In an analysis of the steady-state inactivation curve for voltage dependence, volatile anesthetics induced a negative shift of the 50% inactivation of the calcium channels. By contrast, the steepness factor characterizing the voltage sensitivity of the channels was unaffected. Unitary L-type Ca2+ channels blockade occurred under cell-attached configuration, suggesting a possible action of volatile anesthetics from within the intracellular space or from the part of the channel inside the lipid bilayer.

Propofol Binding to the Resting State of the Gloeobacter violaceus Ligand-gated Ion Channel (GLIC) Induces Structural Changes in the Inter- and Intrasubunit Transmembrane Domain (TMD) Cavities

General anesthetics exert many of their CNS actions by binding to and modulating membrane-embedded pentameric ligand-gated ion channels (pLGICs). The structural mechanisms underlying how anesthetics modulate pLGIC function remain largely unknown. GLIC, a prokaryotic pLGIC homologue, is inhibited by general anesthetics, suggesting anesthetics stabilize a closed channel state, but in anesthetic-bound GLIC crystal structures the channel appears open. Here, using functional GLIC channels expressed in oocytes, we examined whether propofol induces structural rearrangements in the GLIC transmembrane domain (TMD). Residues in the GLIC TMD that frame intrasubunit and intersubunit water-accessible cavities were individually mutated to cysteine. We measured and compared the rates of modification of the introduced cysteines by sulfhydryl-reactive reagents in the absence and presence of propofol. Propofol slowed the rate of modification of L240C (intersubunit) and increased the rate of modification of T254C (intrasubunit), indicating that propofol binding induces structural rearrangements in these cavities that alter the local environment near these residues. Propofol acceleration of T254C modification suggests that in the resting state propofol does not bind in the TMD intrasubunit cavity as observed in the crystal structure of GLIC with bound propofol (Nury, H., Van Renterghem, C., Weng, Y., Tran, A., Baaden, M., Dufresne, V., Changeux, J. P., Sonner, J. M., Delarue, M., and Corringer, P. J. (2011) Nature 469, 428-431). In silico docking using a GLIC closed channel homology model suggests propofol binds to intersubunit sites in the TMD in the resting state. Propofol-induced motions in the intersubunit cavity were distinct from motions associated with channel activation, indicating propofol stabilizes a novel closed state.

Sevoflurane-induced loss of consciousness is paralleled by a prominent modification of neural activity during cortical down-states

Networks of neocortical neurons display a bistable activity pattern characterised by phases of high frequency action potential firing, so called up-states, and episodes of low discharge activity (down-states). We hypothesised that during down-states neocortical neurons are vulnerable to anaesthetic agents. To tackle this issue, it is necessary to identify analytical methods, which are sufficiently sensitive for resolving anaesthetic effects during phases of scarce neuronal activity. The local field potential was recorded in organotypic cultures (OTC) from rat neocortex under control conditions and in the presence of increasing concentrations of sevoflurane by extracellular electrodes. Epochs from down-states were cut from the local field potential and analysed using power spectrum density as well as non-linear parameters approximate entropy (ApEn) and order recurrence rate (ORR). ApEn and ORR proved to be suitable tools for analysing the actions of volatile anaesthetics on cortical down-states. During these phases of low neuronal activity, sevoflurane caused prominent changes in the local field potential. Time series analysis using ApEn showed a reduction of signal predictability in the presence of sevoflurane. Furthermore, the ORR displayed an abrupt decrease at sevoflurane concentrations corresponding to loss of consciousness in vivo, indicating a drug-induced decrease in the signal to noise ratio. The actions of volatile anaesthetics on cortical down-states have been neglected so far, perhaps due to the lack of suitable analysis tools. In the current in vitro study the non-linear parameters ApEn and ORR are introduced to characterise volatile anaesthetics actions. Sevoflurane alters cortical down-states as indicated by non-linear parameter analysis of local field potential recording from cultured neuronal networks. ORR even displays an abrupt change, i.e., a step-like behaviour indicating an increased signal complexity at concentrations of sevoflurane corresponding to loss of consciousness in humans.

Myocardial Protection by Volatile Anesthetics in Patients Undergoing Cardiac Surgery: A Critical Review of the Laboratory and Clinical Evidence

JOHN BLAND AND EDWARD LOWENSTEIN first reported in 1976 that administration of halothane (0.75 minimum alveolar concentration [MAC]) significantly (p o 0.001) attenuates ST-segment elevation caused by brief coronary artery occlusion in 6 dogs anesthetized with chloraloseurethane concomitant with a reduction in the primary determinants of myocardial oxygen consumption. These experiments demonstrated, for the first time, that volatile anesthetics are capable of producing essential anti-ischemic effects. Since the publication of this landmark paper, several studies provided further evidence that volatile anesthetics protected myocardium against reversible and irreversible ischemic injury. For example, isoflurane and halothane enhanced the functional recovery of postischemic-reperfused (‘‘stunned’’) canine myocardium when either volatile anesthetic was administered before a 15-min coronary artery occlusion and reperfusion. This beneficial effect occurred in conjunction with the preservation of high-energy phosphates. Isoflurane also reduced the magnitude of myocardial necrosis and improved recovery of left ventricular (LV) function after global ischemia and reperfusion. Halothane was shown to maintain contractile function and ultrastructural integrity during reperfusion after normothermic cardioplegic arrest in isolated rat hearts. These results were especially notable because they implied that the protective action of volatile anesthetics occur independent of any favorable alteration in coronary perfusion or myocardial oxygen supply-demand relations. In 1997, three research groups simultaneously reported that the administration of halothane or isoflurane before prolonged coronary artery occlusion and reperfusion reduced myocardial infarct size in vivo. This beneficial effect was termed ‘‘anesthetic preconditioning’’ (APC), occurred independent of changes in systemic and coronary hemodynamics, and persisted despite discontinuation of the volatile anesthetic before coronary artery occlusion, a ‘‘memory’’ period similar to that observed during ischemic preconditioning (IPC). Subsequently, volatile anesthetics were shown to protect myocardium against ischemic

Binding of Isoflurane to a Bacterial Voltage-Gated Sodium Channel: Structure and Accessibility of Distinct Interaction Sites

The most likely targets for volatile anesthetics (VA) are ion channels. The mechanism of volatile anesthetic action is not completely understood. Identifying the molecular pathway for drug binding is crucial to understand the effect of VA on voltage gated sodium channels. We use Molecular Dynamics simulations to identify the binding sites for a hydrophobic general anesthetic isoflurane, on voltage gated bacterial sodium channel NaChBac. Apart from the voltage sensing domain (S1-S4), linker and the pore domain (S5-S6), bacterial sodium channels also have fenestrations, which provide a hydrophobic tunnel through the lipid-embedded portion of the channel to the central cavity, where the known local anesthetic site is located. Unbiased “flooding” simulations were performed on the activated open confirmation of NaChBac. We performed a cluster analysis to identify all the possible binding sites of isoflurane. The three most important ones among them are: a region near the selectivity filter, called the extracellular site, a region near the S4-S5 linker, called the linker site, and a region within the cavity, called the cavity site. The most important observation is that isoflurane enters the central cavity through the fenestrations. Free energy perturbation method was employed to calculate the binding affinities of isoflurane for each of these sites. We also studied the interactions between isoflurane and the amino acids in these three binding sites.