Equilibration of Halothane with Brain Tissue In Vitro: Comparison to Brain Concentrations During Anesthesia

A method was devised for reproducing anesthetic concentrations of halothane in slice and membrane preparations of rat brain in vitro. Rats were anesthetized with varying concentrations of halothane, responsiveness was tested, and brain halothane content was determined by heptane extraction and gas chromatography. The inspired concentration of halothane at which half of all animals were unresponsive was 1.05%. At 1.25% halothane, all animals were unresponsive and brain halothane was determined to be 41 +/- 1.3 nmol/mg lipid. No significant differences in halothane concentration between whole brain and a variety of brain regions were detected. To obtain similar concentrations in vitro, membranes or slices of cerebral cortex were incubated in Krebs-Ringer bicarbonate buffer (KRB) that had been preequilibrated with anesthetic. Halothane equilibrated rapidly with the buffer and the tissues. The partition coefficient between gas and KRB was found to be 0.78, and between brain slices and KRB approximately 12. Slightly lower gas concentrations were necessary in vitro than in vivo to obtain the same tissue levels of anesthetic. Using this method, it was shown that there was no effect of anesthetic concentrations of halothane on the uptake of [3H]norepinephrine or [3H]choline into slices of rat cerebral cortex.     

Lack of effect of flurothyl, a non-anesthetic fluorinated ether, on rat brain synaptic plasma membrane calcium-ATPase

Plasma membrane Ca2+-ATPase (PMCA), a regulator of intracellular calcium, is inhibited by volatile anesthetics and by xenon and nitrous oxide. Response of a cellular system to anesthetics, particularly to volatile agents, raises the question of non-specific, even toxic, side effects unrelated to anesthetic action. Compounds with chemical and physical properties similar to halogenated anesthetics, but which lack anesthetic effect, have been used to address this question. We have compared the effects of halothane and flurothyl, a non-anesthetic fluorinated ether, on PMCA Ca2+ transport across isolated brain synaptic plasma membranes (SPM). Flurothyl, at concentrations predicted by the Meyer-Overton curve to range from 0.4 to 2.6 MAC (minimum alveolar concentration), had no significant on PMCA activity. In contrast halothane, 1.3 MAC, reduced Ca2+ transport 30 to 40%. These findings provide further evidence for a specific effect of inhalation anesthetics on neuronal plasma membrane Ca2+-ATPase.     

Chronic exposure to inhaled anesthetics increases cholesterol content in Acholeplasma laidlawii

Acholeplasma laidlawii cells were grown in cholesterol-enriched medium and exposed continuously to either air (control), 4.0 vol.% halothane in air at 1 atm pressure (4% atm halothane), or 80% cyclopropane in oxygen for 24 h at 37°C. Cells grown in the presence of 4% atm halothane or 80% cyclopropane had approximately twice as much membrane cholesterol content/mg protein as the control cells. Cells grown in an anesthetic environment also tended to have a higher membrane cholesterol/phospholipid molar ratio compared to control cells. Membranes isolated from halothane-exposed cells grown in a cholesterol-enriched medium were more ordered at 37°C (measurements were made with no anesthetic present) than membranes from control cells grown in an identically enriched medium. This difference in membrane physical state between control and anesthetic-exposed cells decreased as the temperature decreased, and disappeared at approx. 23°C. Continuous exposure of A. laidlawii to 4% atm halothane or 80% cyclopropane for 24 h did not markedly affect membrane fatty acid composition, either in cells grown on an unsupplemented medium or in cells grown in a medium enriched in myristic, palmitic or stearic acids. These results further support the hypothesis that an increased membrane cholesterol content may play a role in the tolerance or dependence that develops after chronic exposure to anesthetic agents.     

Nitrous oxide and xenon enhance phospholipid-N-methylation in rat brain synaptic plasma membranes

Halothane and isoflurane increase the rate of phospholipid methylation (PLM) in rat brain synaptosomal membranes, a process linked to the coupling of neuronal excitation to neurotransmitter release. In contrast, synaptic plasma membrane (SPM) Ca²⁺ ATPase (PMCA) pumping is reduced by exposure to halothane, isoflurane, xenon and nitrous oxide (N₂O). To examine further the relationship between PLM, PMCA and anesthetic action, we investigated the effect of clinically relevant concentrations of two less potent anesthetic gases, N₂O and xenon, on PLM in SPM. Biochemical assays were performed on SPM exposed to 1.3 MAC of N₂O (2 atm), 1.3 MAC of xenon (1.23 atm) or an equivalent pressure of helium for control. N₂O or xenon exposure increased PLM to 115% or 113%, respectively, of helium control (p < 0.02). Similar exposures to N₂O or xenon depressed PMCA activity to 78% and 85% of control (p < 0.05). Observations that PLM and PMCA are both altered by a wide variety of inhalation anesthetic agents at clinically relevant partial pressures lend support to a possible involvement and interaction of these processes in anesthetic action.     

Effect of halothane on the natural-abundance 13C NMR spectra of excised rat brain

Halothane increases the intensity of the 30.5- and 129-ppm resonances in 13C nuclear magnetic resonance spectra of excised rat brain, of phospholipid vesicles prepared from chloroform-methanol extract of rat brain, and of brain excised from rats anesthetized with halothane. The 13C spin-lattice relaxation times of the 30.5- and 129-ppm resonances are increased in excised brain, or phospholipid vesicles, upon addition of halothane, and they are also increased in brain excised from rats anesthetized with halothane. Excised brain and its membrane-rich subcellular fractions interact with [14C]halothane reversibly. The interaction is virtually abolished when the phospholipids are extracted from the brain. The [14C]halothane content of the brain membranes is correlated with the halothane-induced increase in the integral of the 129-ppm resonance. From this correlation and from the phospholipid content of the membranes, a halothane concentration of 3.34 mM and a partition of 0.057 mol of halothane/mol of phospholipid may be calculated in the brain of anesthetized rats.     

Halothane enhances the phosphorylation of H1 histone and rat brain cytoplasmic proteins by protein kinase C

The effect of halothane, a typical volatile anesthetic, on the calcium- and phospholipid-dependent protein kinase (PKC), which is one of the key enzymes of membrane signal transduction, was examined. PKC was partially purified from the cerebral tissue of male Wistar rats. Halothane increased PKC-mediated phosphorylation of calf thymus H1 histone in the presence or absence of phorbol ester or diolein, and also increased phosphorylation of the rat brain cytosolic proteins (47 kDa and 80 kDa). A similar but slight increase in H1 histone phosphorylation was observed with isoflurane and enflurane, less lipid soluble volatile anesthetics. These findings suggest that halothane may increase PKC-mediated phosphorylation by the modification of phospholipid membrane and affect membrane signal transduction of the nerve cell under the anesthetic state.     

Halothane accumulation in rat brain and liver

Halothane concentration (g/g wet weight) was measured in rat brain and liver following exposure to various concentrations of halothane in air. Because of the difficulty of determining the amount of a volatile compound in brain, we analyzed tissue fixed by two different methods. The apparent concentration of halothane in brain was higher following direct decapitation into liquid nitrogen, than after decapitation, removal of fresh tissue, and then freezing. However, the relative effects of altering the inspired concentration were essentially the same in each case. Thus, absolute quantitative accuracy remains a point for discussion; however, we can reach several conclusions regarding the relative accumulation of halothane in brain tissue following various conditions of exposure. Resultant tissue concentrations of halothane were not linearly related to ambient concentrations. Above an inspired concentration of 1.0%, an increase to 1.5% inspired concentration caused little further increase in the halothane concentration in brain, although the liver concentration increased in proportion to the dose increase. Below an inspired concentration of 0.5%, tissue concentrations were less than expected, probably as a result of metabolic degradation occurring at a rate that becomes more noticeable at lower inspired concentrations. Body size was shown to be an important variable affecting the time required for each tissue to reach equilibrium at a given inspired concentration. These data indicate that tissue concentrations at low exposure levels may be less than proportional to dose and that concentrations in small laboratory animals may be expected to exceed values in humans under equivalent conditions of exposure.     

Interfacial preference of anesthetic action upon the phase transition of phospholipid bilayers and partition equilibrium of inhalation anesthetics between membrane and deuterium oxide

The half-height linewidth (v 1/2) of the 1H-NMR spectra of dipalmitoylphosphatidylcholine vesicles changes abruptly at the phase transition temperature. In the absence of inhalation anesthetics, proton signals from the choline head group (hydrophilic interface) and acyl-chain tails (lipid core) change at the same temperature of 39.6 degrees C. The present study compared the effect of four inhalation anesthetics, i.e., methoxyflurane, chloroform, halothane and enflurane, upon the ligand-induced phase transition of phosphatidylcholine vesicle membranes at 37 degrees C. The anesthetics showed differential action upon the phase transition of the phospholipid vesicle membranes between the lipid core and the hydrophilic interface. The concentrations of anesthetics which induced the phase transition of the lipid core were about 2-fold greater than those required for the phase transition of the interfacial choline head groups. From the area under the proton signals of inhalation anesthetics in the NMR spectra, the maximum solubilities of methoxyflurane, chloroform and halothane in 2H2O at 37 degrees C were determined to be 0.671 . 10(-4), 2.637 . 10(-4) and 1.398 . 10(-4) (expressed as mole fractions), or 3.35, 13.17 and 6.98 mmol/1000 g 2H2O, respectively. The solubilities of the anesthetic vapor in 2H2O expressed as mole fractions according to Henry's law ere 9.586 . 10(-4), 6.432 . 10(-4) and 2.311 10(-4)/atm (1.013 . 10(5) Pa) partial pressure, respectively. The presence of phospholipid vesicles in 2H2O increased the solubility of the inhalation anesthetics. From difference between solubility in 2H2O and a dipalmitoylphosphatidylcholine vesicle suspension, the partition coefficients of methoxyflurane, chloroform and halothane between the phospholipid vesicle membranes and 2H2O were estimated. These values, calculated from the mole fractions, were 3364, 1660 and 3850, respectively at 37 degrees C.     

In vivo 19F-NMR study of isoflurane elimination from brain

The time course of isoflurane elimination from rabbit brain was studied in vivo with 19F-NMR spectroscopy. Two exponential decay functions with different time constants were observed and assigned to two distinct brain compartments. Isoflurane has a 26 min time constant for one compartment (similar to a value of 25 min with halothane) but 174 min in the second one, compared with 320 min for halothane. The shorter half-life for isoflurane may be due to lower solubility of this agent in brain tissue. Comparison of isoflurane 19F chemical shifts in solvents in isolated brain lipids and in whole brain tissue indicates that the anesthetic present in the brain exists in a single environment (on the NMR time scale), which is a weighted average of both hydrophilic and hydrophobic environments.     

Concentration effects of volatile anesthetics on the properties of model membranes: a coarse-grain approach

To gain insights into the molecular level mechanism of drug action at the membrane site, we have carried out extensive molecular dynamics simulations of a model membrane in the presence of a volatile anesthetic using a coarse-grain model. Six different anesthetic (halothane)/lipid (dimyristoylphosphatidylcholine) ratios have been investigated, going beyond the low doses typical of medical applications. The volatile anesthetics were introduced into a preassembled fully hydrated 512-molecule lipid bilayer and each of the molecular dynamics simulations were carried out at ambient conditions, using the NPT ensemble. The area per lipid increases monotonically with the halothane concentration and the lamellar spacing decreases, whereas the lipid bilayer thickness shows no appreciable differences and only a slight increase upon addition of halothane. The density profiles of the anesthetic molecules display a bimodal distribution along the membrane normal with maxima located close to the lipid-water interface region. We have studied how halothane molecules fluctuate between the two maxima of the bimodal distribution and we observed a different mechanism at low and high anesthetic concentrations. Through the investigation of the reorientational motions of the lipid tails, we found that the anesthetic molecules increase the segmental order of the lipids close to the membrane surface.     

The fluorinated anesthetic halothane as a potential NMR biologic probe

Fluorinated anesthetics such as halothane preferentially partition into hydrophobic environments such as cell membranes. The 19F-NMR spectrum of halothane in a rat adenocarcinoma (with known altered lipid metabolism and membrane composition) shows an altered chemical shift pattern compared to the anesthetic in normal tissue. In eight tumor samples examined, the 19F-NMR spectra exhibit two distinct resonances, compared to a single resonance observed in normal tissues. This is explained by an enhanced or altered hydrophobic component in the tumor tissue giving rise to two discrete halothane environments. Another fluorinated anesthetic, isoflurane, shows similar behavior in distinguishing normal from diseased tissue. Given the large chemical shift range of fluorine and the inherent sensitivity of this nucleus, 19F-NMR spectra of fluorinated anesthetics can also be used to follow anesthetic degradation by the liver. The ability of fluorinated anesthetics to discriminate tissues and to monitor metabolic processes is potentially useful for in vivo 19F-NMR surface coil and imaging studies.     

Accumulation and disappearance of enflurane from rat brain

Enflurane accumulation and loss from rat brain tissue was determined by direct analysis of tissue concentrations. Equilibration is rapid and concentrations resultant from administering different dosages are non-linear at very low subanesthetic levels and above the anesthetic range. Clearance from brain following termination of exposure is at least an order of magnitude faster than for halothane.     

Halothane-induced functional and structural modifications in sarcoplasmic reticulum membranes from pig skeletal muscle

We investigated the effect of halothane on lipid and protein components of sarcoplasmic reticulum membranes isolated from pig trapezius muscle. We studied the relationships between the (Ca2(+)-Mg2+)-ATPase activity and the interaction of the anesthetic with lipid and protein moieties by means of EPR and fluorescence spectroscopic techniques. Our results clearly show that below 5 mumol per mg protein, halothane interacts mainly with the lipid components of the membrane. This interaction is shown to be localized in the central core of the phospholipid bilayer and to induce an increase of the membrane calcium permeability. The interaction with protein components only occurs at higher halothane concentrations and affects its conformational and functional states. These results are discussed with respect to new insights into diethylether-SR membrane interaction and to malignant hyperthermia syndrome in the pig.     

The fluorinated anesthetic halothane as a potential NMR biologic probe

Fluorinated anesthetics such as halothane preferentially partition into hydrophobic environments such as cell membranes. The ¹⁹F-NMR spectrum of halothane in a rat adenocarcinoma (with known altered lipid metabolism and membrane composition) shows an altered chemical shift pattern compared to the anesthetic in normal tissue. In eight tumor samples examined, the ¹⁹F-NMR spectra exhibit two distinct resonances, compared to a single resonance observed in normal tissues. This is explained by an enhanced or altered hydrophobic component in the tumor tissue giving rise to two discrete halothane environments. Another fluorinated anesthetic, isoflurane, shows similar behavior in distinguishing normal from diseased tissue. Given the large chemical shift range of fluorine and the inherent sensitivity of this nucleus, ¹⁹F-NMR spectra of fluorinated anesthetics can also be used to follow anesthetic degradation by the liver. The ability of fluorinated anesthetics to discriminate tissues and to monitor metabolic processes is potentially useful for in vivo ¹⁹F-NMR surface coil and imaging studies.     

Correlation of 19F-NMR spectra of halothane in rat tumor and non-tumor tissues with membrane alterations

The membrane environments in normal and tumor rat tissue and the effect of hyperthermia thereon are studied with 19F-NMR spectroscopy of the general anesthetic halothane. Normal and tumor cell types are clearly differentiated by the halothane resonance. A hydrophobic environment prominent in tumor tissue is more sensitive to heat treatment than the corresponding environments of normal cells. Studies of extracted lipids suggest that this may be due in part to the considerable difference in lipid temperature response which exists between normal and kidney tumor cells.     

Modulation of the Benzodiazepine/γ-Aminobutyric Acid Receptor Chloride Channel Complex by Inhalation Anesthetics

Inhalation anesthetics, such as diethyl ether, halothane, and enflurane, increase 36Cl- uptake into rat cerebral cortical synaptoneurosomes in a concentration-dependent, picrotoxin-sensitive fashion. At concentrations consistent with those that stimulate 36Cl- uptake, inhalation anesthetics also inhibit the binding of t-[35S]butylbicyclophosphorothionate ([35S]TBPS) to well-washed cortical membranes. Scatchard analysis of [35S]TBPS binding indicates that these agents reduce the apparent affinity of this radioligand and have little effect on the Bmax. The ability of inhalation anesthetics to directly stimulate 36Cl- uptake and inhibit [35S]TBPS binding is a property shared by nonvolatile anesthetics. Nonetheless, there are differences between nonvolatile agents (such as barbiturates and alcohols) and inhalation anesthetics, because the former compounds augment muscimol (a GABAmimetic) stimulated 36Cl- uptake, whereas the latter group (such as ether and enflurane) inhibit this effect. These findings demonstrate that therapeutically relevant concentrations of inhalation anesthetics perturb the benzodiazepine/gamma-aminobutyric acid receptor chloride channel complex, and suggest this oligomeric protein may be a common mediator of some aspects of anesthetic action.     

Do inhalation general anesthetic drugs induce the neuronal release of endogenous opioid peptides?

The antagonism of some effects of inhalation general anesthetic agents by naloxone suggests that there may be an opioid component to anesthetic action. There is evidence that this opioid action component is due to neuronal release of endogenous opioid peptides. The strongest evidence is provided by studies that monitor changes in the concentration of opioid peptides in the perfused brain following inhalation of the anesthetic. Indirect or circumstantial evidence also comes from studies of anesthetic effects on regional brain levels of opioid peptides, antagonism of selected anesthetic effects by antisera to opioid peptides and anesthetic-induced changes radioligand binding to opioid receptors. It is likely that some inhalation general anesthetics (e.g., nitrous oxide) can induce neuronal release of opioid peptides and that this may contribute to certain components of general anesthesia (e.g., analgesia). More definitive studies utilizing in vivo microdialysis or autoradiography in selected areas of the brain during induction and successive states of general anesthesia have yet to be conducted.     

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 degrees C. Binding of halothane to the native state of serum albumin thus outweighs any non-specific interactions between the thermally unfolded state of serum albumin and halothane in this concentration range. Based on the average enthalpy change DeltaH for unfolding of 170 kcal/mol, the increase from 62 to 71 degrees C corresponds to an additional Gibbs energy of stabilization (DeltaDeltaG) due to halothane of more than 4 kcal/mol. Analysis of the dependence of DeltaDeltaG on halothane concentration shows that thermal unfolding of a bovine serum albumin molecule is linked to the dissociation of about one halothane molecule at lower halothane concentrations and about six at higher halothane concentrations. Serum albumin is the first protein that has been shown to be stabilized by an inhalational anesthetic.     

Interaction modes of long-chain fatty acids in dipalmitoylphosphatidylcholine bilayer membrane: contrast to mode of inhalation anesthetics

The effects of long-chain fatty acids (four saturated and two unsaturated fatty acids, one derivative) on phase transitions of dipalmitoylphosphatidylcholine (DPPC) bilayer membranes were examined in the low concentration region, and the results were compared with those for an inhalation anesthetic. The effects of all fatty acids on the pre- and main-transition temperatures of the DPPC bilayer membrane appeared in the concentration range of μM order while that of the anesthetic appeared in the mM order. The appearance modes of these ligand actions were significantly different from one another. The three differential partition coefficients of the ligands between two phases of the DPPC bilayer membrane were evaluated by applying the thermodynamic equation to the variation of the phase-transition temperatures. The DPPC bilayer membranes showed the different receptivity for the ligands; the saturated fatty acids had an affinity for gel phase whereas unsaturated fatty acids and an anesthetic had an affinity for liquid-crystalline phase to the contrary. In particular, the receptivity for the ligands in the gel phase markedly changed depending on kinds of ligands. The interaction modes between the DPPC and fatty acid molecules in the gel phase were considered from the hexagonal lattice model. The disappearance compositions of the pretransition by the fatty acids coincided with the compositions at which the membrane is all covered by the units in each of which two fatty acids molecules are regularly distributed in the hexagonal lattice in a different way, and the distribution depended on the chain length and existence of a double bond for the fatty acids. The interpretation did not hold for the case of the anesthetic at all, which proved that a number of anesthetic molecules act the surface region of the bilayer membrane nonspecifically. The present study clearly implies that DPPC bilayer membranes have high ability to recognize kinds of ligand molecules and can discriminate among them with specific interaction by the membrane states.     

Inhibition of leukotriene formation in human leukocytes by halothane

The effects of an inhalation anesthetic, halothane (2-bromo-2-chloro-1,1,1-trifluoroethane) on the formation of 5-lipoxygenase metabolites such as leukotriene B4, 5(S)-hydroxyeicosatetraenoic acid (5-HETE), 6-trans-isomers of leukotriene B4 and leukotriene C4 were studied in human leukocytes stimulated with calcium ionophore A23187. Halothane inhibited the formation of all these metabolites dose dependently and the formation was restored by removal of the drug. The anesthetic also reversibly inhibited the release of [3H]arachidonic acid from neutrophils with a half-inhibition concentration of less than 0.19 mM. The formation of 5-lipoxygenase metabolites was not inhibited by the anesthetic when leukocytes were stimulated with the ionophore in the presence of exogenous arachidonic acid. These observations indicate that the inhibitory effect of halothane on the formation of 5-lipoxygenase metabolites in leukocytes is mainly due to the inhibition of arachidonic acid release.