Low Concentrations Of Halothane Research Articles

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.

Reduced Threshold for Luminal Ca2+ Activation of RyR1 Underlies a Causal Mechanism of Porcine Malignant Hyperthermia

Naturally occurring mutations in the skeletal muscle Ca(2+) release channel/ryanodine receptor RyR1 are linked to malignant hyperthermia (MH), a life-threatening complication of general anesthesia. Although it has long been recognized that MH results from uncontrolled or spontaneous Ca(2+) release from the sarcoplasmic reticulum, how MH RyR1 mutations render the sarcoplasmic reticulum susceptible to volatile anesthetic-induced spontaneous Ca(2+) release is unclear. Here we investigated the impact of the porcine MH mutation, R615C, the human equivalent of which also causes MH, on the intrinsic properties of the RyR1 channel and the propensity for spontaneous Ca(2+) release during store Ca(2+) overload, a process we refer to as store overload-induced Ca(2+) release (SOICR). Single channel analyses revealed that the R615C mutation markedly enhanced the luminal Ca(2+) activation of RyR1. Moreover, HEK293 cells expressing the R615C mutant displayed a reduced threshold for SOICR compared with cells expressing wild type RyR1. Furthermore, the MH-triggering agent, halothane, potentiated the response of RyR1 to luminal Ca(2+) and SOICR. Conversely, dantrolene, an effective treatment for MH, suppressed SOICR in HEK293 cells expressing the R615C mutant, but not in cells expressing an RyR2 mutant. These data suggest that the R615C mutation confers MH susceptibility by reducing the threshold for luminal Ca(2+) activation and SOICR, whereas volatile anesthetics trigger MH by further reducing the threshold, and dantrolene suppresses MH by increasing the SOICR threshold. Together, our data support a view in which altered luminal Ca(2+) regulation of RyR1 represents a primary causal mechanism of MH.

Interaction between phospholipid monolayer and volatile anesthetics using quartz crystal oscillator methods

Abstract The interaction between phospholipid monolayer (dihexadecyl phosphate: DHP) and volatile anesthetics (halothane) has been studied using quartz crystal microbalance (QCM) method and quartz crystal impedance (QCI) method. The quartz crystal oscillator was attached horizontally on the DHP monolayer that was formed on the water surface. At quite low halothane concentrations (≤4 mM), no change of frequency for QCM and resistance for QCI was observed. At low concentrations (6–13 mM), the frequency decreased and the resistance increased concomitant with the increased concentration. Both frequency and resistance approached saturation values asymptotically. The concentration at which the frequency changed (6 mM) differed from that at which the resistance changed (9 mM). At high concentrations (>14 mM), both frequency and resistance showed a sudden and discontinuous change (a frequency decrease and a resistance increase). These results indicate the following action mechanism of halothane on DHP monolayer: at low halothane concentrations, the halothane hydrates adsorb into the DHP monolayer/water interface. Subsequently, DHP monolayer/water interface reconstruction occurs because of the interaction and aggregation between adsorbed halothane hydrates with an increase in the hydrate on the interface, i.e., an increase in the DHP monolayer viscosity. At high concentrations, multilayer formation of halothane hydrates and/or promotion of the aggregation of halothane hydrates occur on the DHP monolayer/water interface. The viscosity of the interfacial layer (DHP monolayer and interfacial structured water) changes according to the action of halothane hydrates.

Sevoflurane or halothane with target-controlled sufentanil infusions for coronary artery bypass surgery

Objectives Comparison of hemodynamics, circulatory efficiency, myocardial ischemia and recall during and after sevoflurane or halothane (0.6 MAC {Minimum Alveolar Concentration} inspired) combined with a target-controlled sufentanil infusion (2 ng.ml 1 ) for coronary artery bypass grafting. Methods Prospective randomised non-blinded study in a university teaching hospital of 45 patients undergoing on-pump surgery. Inhalation anesthetic agent was delivered before, during and after cardiopulmonary bypass. Results Intraoperative hemodynamics were maintained within predetermined limits using vasoactive and cardioactive drugs. Heart rate was unchanged from baseline, however the sevoflurane-sufentanil group required more glycopyrrollate, indicating a tendency towards a slower heart rate. In both groups, similar low incidences of myocardial ischemia were detected. After induction of anesthesia, as well as intraoperatively, oxygen consumption decreased by up to 36.6% compared with the awake values in both groups. Oxygen delivery remained unchanged during all measuring periods. Mixed venous oxygen tensions rose after induction, even in patients with cardiac indices below 2.2 L.min 1 .m 2 , indicating maintenance of an effective circulation when utilising these techniques. Twelve hours postoperatively, oxygen consumption exceeded awake values by 31%. No explicit recall was reported by any patient on enquiry on the third postoperative day. Conclusions Low concentrations of sevoflurane or halothane, supplementing target-controlled sufentanil infusions, were both suitable for providing anesthesia for coronary bypass surgery.

Mg2+ dependence of halothane-induced Ca2+ release from the sarcoplasmic reticulum in rat skeletal muscle.

The effect of cytosolic Mg2+ on halothane-induced Ca2+ release from the sarcoplasmic reticulum (SR) was investigated in mechanically skinned fibres from the rat extensor digitorum longus (EDL) muscle. Preparations were perfused with solutions mimicking the intracellular milieu and changes in [Ca2+] were detected using Fura-2 fluorescence. In the presence of 1 mM Mg2+, brief (500 ms) applications of 40 mM halothane failed to induce Ca2+ release from the SR. However, Ca2+ release became detectable when [Mg2+] was reduced to 0.4 mM, and the amplitude of the response increased progressively as [Mg2+] was further reduced to 0.2 and 0.1 mM. Lower halothane concentrations within the range found during anaesthesia or induction (0.1-1.2 mM) failed to induce SR Ca2+ release at 0.2 or 0.4 mM Mg2+. However, in further experiments, preparations were exposed to 1 mM halothane for 2-3 min under conditions where the volume of solution surrounding the preparation was restricted by stopping the flow. In the absence of perfusion, 1 mM halothane induced Ca2+ release from the SR at 0.4 mM Mg2+ in two out of six preparations, and release was observed consistently at 0.2 and 0.1 mM Mg2+. Responses to 1 mM halothane induced in the presence of 0.4 and 0.2 mM Mg2+ were typically delayed in onset and involved a localised release of Ca2+ that propagated along the fibre. These results suggest that halothane-induced Ca2+ release is strongly inhibited at normal physiological levels of Mg2+. However, when Mg2+-induced inhibition of the ryanodine receptor (RYR) is reduced, levels of halothane within the range found during anaesthesia can induce a marked efflux of Ca2+ from the SR. This may be of relevance to the condition of malignant hyperthermia, where the inhibition of RYRs by Mg2+ is reportedly reduced.

Halothane-induced intracellular calcium release in cholinergic cells

Low concentrations of halothane and isoflurane can release acetylcholine in an extracellular Ca 2+-independent manner. In the present study, a cholinergic cell line (SN56) was used to examine whether release of calcium from intracellular stores occurs in the presence of halothane. Changes in intracellular calcium concentration ([Ca 2+] i) were measured using fluo-3, a fluorescent calcium-sensitive dye and laser scanning confocal microscopy. Halothane, at sub-anesthetic concentrations (14, 28, 40 and 56 μM), increased [Ca 2+] i in SN56 cells. This effect remained even when the cells were perfused with medium lacking extracellular calcium, suggesting the involvement of intracellular Ca 2+ sources. SN56 cells responded to ryanodine by increasing [Ca 2+] i and this effect was blocked by dantrolene, an inhibitor of Ca 2+-release from ryanodine-sensitive stores. The effect of halothane was attenuated after the increase in [Ca 2+] i induced by ryanodine and it was suppressed by dantrolene, suggesting the participation of ryanodine-sensitive stores. Using cyclopiazonic acid, a Ca 2+-ATPase inhibitor, we investigated whether the depletion of intracellular Ca 2+ stores interfered with the effect of halothane. Cyclopiazonic acid significantly decreased the increase in [Ca 2+] i induced by the volatile anesthetic. It is suggested that sub-anesthetic concentrations of halothane may increase [Ca 2+] i by releasing Ca 2+ from intracellular stores in cholinergic cells.

The effect of low concentrations of halothane on the cerebrovascular circulation in young children.

To determine the effect of halothane on cerebral blood flow velocity measured by transcranial Doppler, 23 healthy young children were studied during surgery. Anaesthesia was induced with thiopental, fentanyl and vecuronium, and maintained with halothane in 70% nitrous oxide in oxygen. A continuous epidural anaesthesia with 0. 25% bupivacaine was performed. End-tidal carbon dioxide pressure, temperature, heart rate and systolic blood pressure were kept constant. Three minimal alveolar concentrations (MAC; 0.5, 1.0 and 1. 5) of halothane were administered in stepwise increases. The cerebral blood flow velocity increased significantly at 1.0 (p < 0. 01) and 1.5 MAC (p < 0.001) compared with the value at 0.5 MAC. No further change in cerebral blood flow velocity was seen between 1.0 and 1.5 MAC. These data show that maximal changes in cerebral blood flow velocity are obtained at 1.0 MAC and that further increases in halothane concentration do not modify the cerebral circulation. It is suggested that young children differ from adults in that the maximal effect of halothane occurs at lower concentrations.

Utilização de alfentanil, sufentanil e fentanil em cães anestesiados com halotano

Os analgésicos opióides promovem analgesia intensa, possibilitando o emprego de concentrações reduzidas dos anestésicos inalatórios, minimizando assim a depressão cardiovascular que ocorre durante a anestesia. Vários opióides podem ser empregados na anestesia, sendo que dentre os mais potentes, o fentanil, sufentanil e alfentanil são os mais freqüentemente administrados no transoperatório. No presente estudo, comparou-se a ação no sistema cardiovascular desses três agentes durante a anestesia inalatória em cães, submetidos a procedimentos ortopédicos, bem como, avaliou-se a possibilidade de se manter a anestesia com baixas concentrações de halotano. Foram usados três grupos, de dez animais cada, que receberam, após estabilização da anestesia, doses equipotentes de um dos três agentes analgésicos - grupo I - 5µg/kg de fentanil IV, grupo II - 1µg/kg de sufentanil IV e grupo III - 25µg/kg de alfentanil IV. Os parâmetros cardiovasculares e respiratórios foram avaliados em diferentes tempos de observação. Os resultados obtidos foram analisados através de análise estatística (ANOVA seguida de Dunnett e Bonferroni). Os três agentes promoveram bradicardia importante durante o decorrer do estudo, sendo que apenas o alfentanil promoveu hipotensão significativa após sua administração. Apenas um animal tratado com fentanil apresentou aumento da pressão arterial e freqüência cardíaca durante a anestesia Na recuperação da anestesia, um número maior de animais tratados com alfentanil apresentou sinais de excitação. Através dos resultados obtidos, pode-se concluir que a administração de analgésicos opióides possibilita de fato o emprego de menores concentrações de halotano, promovendo anestesia estável com a manutenção da pressão arterial dentro dos valores normais para a espécie. Dos agentes empregados, o alfentanil é o que promove efeitos cardiovasculares mais pronunciados.

Halothane, an inhalational anesthetic agent, increases folding stability of serum albumin

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°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 Δ H for unfolding of 170 kcal/mol, the increase from 62 to 71°C corresponds to an additional Gibbs energy of stabilization (ΔΔ G) due to halothane of more than 4 kcal/mol. Analysis of the dependence of ΔΔ G 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.

Measurement of Resting Cytosolic Ca2+ Concentrations and Ca2+ Store Size in HEK-293 Cells Transfected with Malignant Hyperthermia or Central Core Disease Mutant Ca2+ Release Channels

Malignant hyperthermia (MH) and central core disease (CCD) mutations were introduced into full-length rabbit Ca2+ release channel (RYR1) cDNA, which was then expressed transiently in HEK-293 cells. Resting Ca2+ concentrations were higher in HEK-293 cells expressing homotetrameric CCD mutant RyR1 than in cells expressing homotetrameric MH mutant RyR1. Cells expressing homotetrameric CCD or MH mutant RyR1 exhibited lower maximal peak amplitudes of caffeine-induced Ca2+ release than cells expressing wild type RyR1, suggesting that MH and CCD mutants might be "leaky." In cells expressing homotetrameric wild type or mutant RyR1, the amplitude of 10 mM caffeine-induced Ca2+ release was correlated significantly with the amplitude of carbachol- or thapsigargin-induced Ca2+ release, indicating that maximal drug-induced Ca2+ release depends on the size of the endoplasmic reticulum Ca2+ store. The content of endogenous sarco(endo)plasmic reticulum Ca2+-ATPase isoform 2b (SERCA2b), measured by enzyme-linked immunosorbent assay, 45Ca2+ uptake, and confocal microscopy, was increased in HEK-293 cells expressing wild type or mutant RyR1, supporting the view that endoplasmic reticulum Ca2+ storage capacity is increased as a compensatory response to an enhanced Ca2+ leak. When heterotetrameric (1:1) combinations of MH/CCD mutant and wild type RyR1 were expressed together with SERCA1 to enhance Ca2+ reuptake, the amplitude of Ca2+ release in response to low concentrations of caffeine and halothane was higher than that observed in cells expressing wild type RyR1 and SERCA1. In Ca2+-free medium, MH/CCD mutants were more sensitive to caffeine than wild type RyR1, indicating that caffeine hypersensitivity observed with a variety of MH/CCD mutant RyR1 proteins is not dependent on extracellular Ca2+ concentration.

Actions of general anaesthetics and arachidonic acid pathway inhibitors on K+ currents activated by volatile anaesthetics and FMRFamide in molluscan neurones

1. K+ currents activated by volatile general anaesthetics (IK(An)) and by the neuropeptide FMRFamide (IK(FMRFa)) were studied under voltage clamp in isolated identified neurones from the pond snail Lymnaea stagnalis. 2. IK(An) was activated by all the volatile anaesthetics studied. The maximal responses varied from agent to agent, with halothane sevoflurane > isoflurane > enflurane approximately chloroform. 3. IK(An) was inhibited rather than activated by the n-alcohols from hexanol to dodecanol and by the 6- and 8-carbon cycloalcohols. The n-alcohols exhibited a cutoff effect, with dodecanol being unable to half-inhibit IK(An). 4. Unlike IK(An) which did not desensitize at reasonable halothane concentrations, IK(FMRFa) desensitized at most FMRFamide concentrations studied. This desensitization could be substantially removed by halothane. Nonetheless, both IK(An) and IK(FMRFa) had similar sensitivities to the potassium channel blockers tetraethylammonium and 4-aminopyridine, consistent with both currents flowing through the same channels. Responses to low concentrations of halothane and FMRFamide showed synergy. 5. The phospholipase A2 inhibitor aristolochic acid inhibited IK(An), consistent with a role for arachidonic acid (AA). The lipoxygenase and cyclooxygenase inhibitor nordihydroguaiaretic acid blocked IK(FMRFa) but did not affect IK(An). IK(An) and IK(FMRFa) were little affected by the cyclooxygenase inhibitor indomethacin. These findings suggest that neither lipoxygenase nor cyclooxygenase pathways of AA metabolism are involved in the anaesthetic activation of IK(An. 6. Inhibitors of a third, cytochrome P450-mediated, pathway of AA metabolism (clotrimazole and econazole) potently blocked IK(An), suggesting possible roles for certain cytochrome P450 isoforms in the activation of IK(An).

Anesthetic-induced alteration of Ca2+ homeostasis in neural cells: a temperature-sensitive process that is enhanced by blockade of plasma membrane Ca2+-ATPase isoforms.

Many inhalation anesthetics at clinically relevant concentrations inhibit plasma membrane Ca2+-adenosine triphosphatase (PMCA) ion pumping in brain synaptic membranes and in cultured cells of neural origin. In this study, the authors investigated the effect of inhalation anesthetics on cytosolic calcium homeostasis in cortical neurons maintained at physiologic and room temperatures and on cortical neurons and pheochromocytoma cells with antisense blockade of specific PMCA isoforms. Using Ca2+-specific confocal microfluorimetry, the anesthetic effects on Ca2+ dynamics were examined in mouse embryonic cortical neurons in association with ligand-stimulated Ca2+ influx. Studies were done at 21 degrees C and 37 degrees C. Mouse embryonic cortical neurons with oligodeoxyribonucleotide blockade of PMCA2 expression and transfected rat pheochromocytoma cells with blocked expression of PMCA1 were also examined. Baseline and poststimulation peak cytosolic calcium concentrations ([Ca2+]i) were increased, and Ca2+ clearance was delayed in cells exposed at 37 degrees C, but not at 21 degrees C, to concentrations < or = 1 minimum alveolar concentration (MAC)-equivalent of halothane, isoflurane, and sevoflurane. Neurons exposed to xenon solutions < or = 0.4, 0.6, and 0.8 MAC showed dose-related perturbations of cytosolic Ca2+. Calcium dynamics were altered in neural cells with blocked PMCA isoform production, but at much lower halothane concentrations: 0.5 MAC for cortical neurons and 0.1 MAC for pheochromocytoma cells. By extruding Ca2+ through the plasma membrane, PMCA maintains resting neuronal [Ca2+]i at low levels and clears physiologic loads of Ca2+ after influx through calcium channels. Inhalation anesthetics perturb this process and thus may interfere with neurotransmitter release, altering interneuronal signaling.

Caffeine and Halothane Sensitivity of Intracellular Ca2+ Release Is Altered by 15 Calcium Release Channel (Ryanodine Receptor) Mutations Associated with Malignant Hyperthermia and/or Central Core Disease

Malignant hyperthermia (MH) and central core disease (CCD) are autosomal dominant disorders of skeletal muscle in which a potentially fatal hypermetabolic crisis can be triggered by commonly used anesthetic agents. To date, 17 mutations in the human RYR1 gene encoding the Ca2+ release channel of skeletal muscle sarcoplasmic reticulum (the ryanodine receptor) have been associated with MH and/or CCD. Although many of these mutations have been linked to MH and/or CCD, with high lod (log of the odds favoring linkage versus nonlinkage) scores, others have been found in single, small families. Independent biochemical evidence for a causal role for these mutations in MH is available for only two mutants. Mutations corresponding to the human MH mutations were made in a full-length rabbit RYR1 cDNA, and wild type and mutant cDNAs were transfected into HEK-293 cells. After about 48 h, intact cells were loaded with the fluorescent Ca2+ indicator, fura-2, and intracellular Ca2+ release, induced by caffeine or halothane, was measured by photometry. Ca2+ release in cells expressing MH or CCD mutant ryanodine receptors was invariably significantly more sensitive to low concentrations of caffeine and halothane than Ca2+ release in cells expressing wild type receptors or receptors mutated in other regions of the molecule. Linear regression analysis showed that there is a strong correlation (r = 0.95, p < 0.001) between caffeine sensitivity of different RYR1 mutants measured by the cellular Ca2+ photometry assay and by the clinical in vitro caffeine halothane contracture test (IVCT). The correlation was weaker, however, for halothane (r = 0.49, p > 0.05). Abnormal sensitivity in the Ca2+ photometry assay provides supporting evidence for a causal role in MH for each of 15 single amino acid mutations in the ryanodine receptor. The study demonstrates the usefulness of the cellular Ca2+ photometry assay in the assessment of the sensitivity to caffeine and halothane of specific ryanodine receptor mutants.

HALOTHANE MEDIATED INHIBITION OF CALMODULIN-NEUROPEPTIDE BINDING

Binding of volatile anesthetics by proteins has been proposed as a mechanism of anesthesia. There is no evidence, however, that anesthetics at clinically relevant concentrations can interact directly with lipid - free preparations of receptors or signal transducing proteins. Calmodulin is a key regulator of metabolism in neurons and muscle cells. This study describes the effects of halothane on the binding of the neuropeptide vasoactive intestinal peptide (VIP) by calmodulin. It was found that : (1) Saturable complexation of (tyr10-125 1)VIP by calmodulin was absolutely Ca2+ -dependent, requiring mM metal concentrations for optimal binding. (2) Halothane produced a dose - dependent, biphasic inhibition of VIP - calmodulin complexation . Relatively low concentrations of halothane (0.15-0.27 mM) inhibited the complexation strongly up to 67% inhibition. (3) The inhibitory concentrations of halothane did not influence binding of VIP by monoclonal antibody. It could be concluded that calmodulin may be the target molecule for halothane and that the mechanism of anesthesia by halothane involves direct interaction with cellular signal-transducing proteins .

Postoperative Tracheal Extubation

Postoperative Tracheal Extubation

Postoperative Tracheal Extubation

Postoperative Tracheal Extubation

Effects of halothane and diltiazem on L-type calcium currents in single smooth muscle cells from rabbit portal veins

We have studied the effects of halothane and diltiazem on L-type voltage-dependent calcium currents (ICa) in single smooth muscle cells from rabbit portal veins using a whole cell voltage clamp technique. The threshold of ICa was -30mV and the peak current was reached at 0mV. Halothane (0.25, 0.5, 1.0, 1.5 and 2.0%) decreased ICa in a concentration-dependent manner and shifted the ICa activation threshold to the depolarizing side. Halothane 2.0% abolished ICa. Diltiazem 10(-8)-10(-6) mol litre-1, a calcium channel antagonist, also depressed ICa in a concentration-dependent manner. Administration of both 0.5% halothane and diltiazem 10(-7) mol litre-1 (concentrations lower than the clinical therapeutic range) abolished ICa; however, halothane did not exhibit use-dependent inhibition of ICa whereas diltiazem showed partial use-dependency. We conclude that the decrease in ICa produced by halothane is associated with a direct vasodilator effect of this anaesthetic, but is not explained by block of Ca2+ channels similar to the action of diltiazem. Furthermore, administration of low concentrations of both halothane and diltiazem decreased ICa and may reduce the contractility of the vascular smooth muscle cells.

Halothane acts on many potassium channels, including a minimal potassium channel

There has been considerable controversy over whether general anesthetics act directly on membrane proteins, and if so, whether there are uniquely sensitive protein targets upon which they act. Here, we examine the actions of halothane on a diverse collection of voltage-gated potassium channels expressed in Xenopus oocytes, and find that they are all sensitive at clinically relevant concentrations. To investigate further the molecular basis of this commonality, human and rat minimal potassium (minK) channels, which have exceedingly short amino acid sequences, were examined. Current through these channels is reversibly reduced to 68% of control values by 0.5% (0.34 mM) halothane. A double deletion mutant of the 130-amino acid minK protein, in which 30 amino acids of the N-terminus, thought to be extracellular, and 37 amino acids of the putative intracellular C-terminus are deleted (resulting in a protein in which more than half of both the extracellular and intracellular domains have been removed) responds to low halothane concentrations similarly to the parent channel. While alternative explanations are possible, this result is consistent with a model whereby halothane interacts with the channel protein from within the lipid bilayer.

Leakage of mitochondrial and cytosolic GOT in isolated rat hepatocytes exposed to halothane: an immunohistochemical study.

The leakage of mitochondrial (m-) and cytosolic (s-) aspartate aminotransferases (glutamic-oxaloacetic transaminase: GOT) from isolated hepatocytes exposed to halothane was investigated immunohistochemically. In control isolated hepatocytes, a large number of cells were immunopositive (m-GOT, 85%; s-GOT, 98.5%) at various intensities. Reaction products of m-GOT-immunohistochemistry (m-GOT-I) were seen exclusively in mitochondria, while those of s-GOT (s-GOT-I) were seen in the cytoplasm. When treated with low concentrations of halothane (up to 0.75%), the number of strongly m-GOT-I-immunopositive cells was reduced to half, while that of s-GOT-I showed no noticeable change. The number of m-GOT-I-immunonegative cells showed a negligible increase, while that of s-GOT-I increased gradually. At higher concentrations of halothane (1% or more), strongly immunopositive cells in both m- and s-GOT-I almost disappeared, while immunonegative cell numbers predominantly increased. This study showed that the isolated hepatocyte system was not homogeneous with regard to the quantity of GOT isozymes, and that halothane could induce the leakage of these isozymes from hepatocytes, along with inducing ultrastructural abnormalities, even at the lowest concentration used (0.5%). Furthermore, the data appear to indicate that the sensitivity of isolated hepatocytes to halothane is dependent on the nature of the hepatocyte itself and on the nature of both types of GOT isozymes.

Comparison of Low Concentrations of Halothane and Isoflurane as Bronchodilators

Although high concentrations of all currently used inhalational anesthetics are thought to be good bronchodilators, studies using traditional measures of airway tone fail to show differences in airway responsiveness during halothane, enflurane, and isoflurane use. Using a more sensitive technique, the authors compared the ability of halothane and isoflurane to dilate histamine-constricted airways at equivalent MAC concentrations. Responses of histamine-constricted individual airways to increasing doses of halothane and isoflurane were directly measured using high-resolution computed tomography (HRCT). Fifteen studies were performed in five dogs. All dogs were initially anesthetized with thiopental 15 mg/kg followed by a 10-mg.kg-1 x h-1 maintenance dose. Following tracheal intubation, the lungs were mechanically ventilated (15 ml/kg, 15 bpm). The airways were constricted with intravenous histamine 200 micrograms/min. On alternate days, the dogs subsequently received increasing concentrations of either halothane or isoflurane (0.6, 1.1, and 1.7 MAC). On a separate day, the dogs received atropine 0.2 mg/kg after the histamine infusion and the study was repeated. Histamine decreased airway area 34 +/- 2.5% (mean +/- SEM). All preconstricted airways showed a significant dose-dependent dilation to halothane and isoflurane at concentrations of 0.6, 1.1, and 1.7 MAC. Halothane significantly dilated airways to a greater extent than isoflurane at 0.6 and 1.1 MAC (P < 0.001). This effect was most pronounced in airways less than 3 mm in diameter. At 1.7 MAC, there was no significant difference between the two agents (P = 0.42). Atropine (0.2 mg/kg) reversed the airway constriction elicited by intravenous histamine. The histamine-preconstricted airways area increased 370 +/- 34% (P < 0.0001) after atropine. Halothane and isoflurane dilate histamine-constricted airways in a dose-dependent manner. However, at low concentrations, halothane was a more effective bronchodilator than isoflurane at equivalent MAC doses.