Binding of volatile anesthetics to serum albumin: measurements of enthalpy and solvent contributions

This study directly examines the enthalpic contributions to binding in aqueous solution of closely related anesthetic haloethers (desflurane, isoflurane, enflurane, and sevoflurane), a haloalkane (halothane), and an intravenous anesthetic (propofol) to bovine and human serum albumin (BSA and HSA) using isothermal titration calorimetry. Binding to serum albumin is exothermic, yielding enthalpies (DeltaH(obs)) of -3 to -6 kcal/mol for BSA with a rank order of apparent equilibrium association constants (K(a) values): desflurane > isoflurane approximately enflurane > halothane >or= sevoflurane, with the differences being largely ascribed to entropic contributions. Competition experiments indicate that volatile anesthetics, at low concentrations, share the same sites in albumin previously identified in crystallographic and photo-cross-linking studies. The magnitude of the observed DeltaH increased linearly with increased reaction temperature, reflecting negative changes in heat capacities (DeltaC(p)). These -DeltaC(p) values significantly exceed those calculated for burial of each anesthetic in a hydrophobic pocket. The enhanced stabilities of the albumin/anesthetic complexes and -DeltaC(p) are consistent with favorable solvent rearrangements that promote binding. This idea is supported by substitution of D(2)O for H(2)O that significantly reduces the favorable binding enthalpy observed for desflurane and isoflurane, with an opposing increase of DeltaS(obs). From these results, we infer that solvent restructuring, resulting from release of water weakly bound to anesthetic and anesthetic-binding sites, is a dominant and favorable contributor to the enthalpy and entropy of binding to proteins.     

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.     

Molecular genetic analysis of volatile-anesthetic action.

The mechanism(s) and site(s) of action of volatile inhaled anesthetics are unknown in spite of the clinical use of these agents for more than 150 years. In the present study, the model eukaryote Saccharomyces cerevisiae was used to investigate the action of anesthetic agents because of its powerful molecular genetics. It was found that growth of yeast cells is inhibited by the five common volatile anesthetics tested (isoflurane, halothane, enflurane, sevoflurane, and methoxyflurane). Growth inhibition by the agents is relatively rapid and reversible. The potency of these compounds as yeast growth inhibitors directly correlates with their lipophilicity as is predicted by the Meyer-Overton relationship, which directly correlates anesthetic potency of agents and their lipophilicity. The effects of isoflurane on yeast cells were characterized in the most detail. Yeast cells survive at least 48 h in a concentration of isoflurane that inhibits colony formation. Mutants resistant to the growth-inhibitory effects of isoflurane are readily selected. The gene identified by one of these mutations, zzz4-1, has been cloned and characterized. The predicted ZZZ4 gene product has extensive homology to phospholipase A2-activating protein, a GO effector protein of mice. Both zzz4-1 and a deletion of ZZZ4 confer resistance to all five of the agents tested, suggesting that signal transduction may be involved in the response of these cells to volatile anesthetics.     

Carbon monoxide production from five volatile anesthetics in dry sodalime in a patient model: halothane and sevoflurane do produce carbon monoxide; temperature is a poor predictor of carbon monoxide production

BACKGROUND: Desflurane and enflurane have been reported to produce substantial amounts of carbon monoxide (CO) in desiccated sodalime. Isoflurane is said to produce less CO and sevoflurane and halothane should produce no CO at all.The purpose of this study is to measure the maximum amounts of CO production for all modern volatile anesthetics, with completely dry sodalime. We also tried to establish a relationship between CO production and temperature increase inside the sodalime. METHODS: A patient model was simulated using a circle anesthesia system connected to an artificial lung. Completely desiccated sodalime (950 grams) was used in this system. A low flow anesthesia (500 ml/min) was maintained using nitrous oxide with desflurane, enflurane, isoflurane, halothane or sevoflurane. For immediate quantification of CO production a portable gas chromatograph was used. Temperature was measured within the sodalime container. RESULTS: Peak concentrations of CO were very high with desflurane and enflurane (14262 and 10654 ppm respectively). It was lower with isoflurane (2512 ppm). We also measured small concentrations of CO for sevoflurane and halothane. No significant temperature increases were detected with high CO productions. CONCLUSION: All modern volatile anesthetics produce CO in desiccated sodalime. Sodalime temperature increase is a poor predictor of CO production.     

Elevated ornithine decarboxylase activity in the rat liver following exposure to halothane,enflurane and isoflurane

Exposure of rats to the volatile anesthetics, halothane, enflurane and isoflurane and low FIO₂ (0.8%) for two hours results in a transient induction of ODC appearing maximally four hours after exposure. Without the low oxygen accompanying the anesthetic or the low oxygen alone, no significant induction of ODC occurred. The concentration of anesthetic used to produce the ODC induction were 0.5% halothane, 1.5% enflurane and 1.4% isoflurane. Except for halothane, reducing the anesthetic concentration only slightly reduced the effect on ODC levels to control values. Reduction of halothane concentrations to 0.1% was required to reduce the values to control levels. Pretreatment of the animals with either cycloheximide or actinomycin D delayed the onset of ODC induction. The data support the fact that liver damage can occur in the absence of metabolism of the drug.     

Effects of halothane, enflurane, and isoflurane on measurements of Ca(2+) by calcium electrode and aequorin luminescence

We assessed the possible effects of the volatile halogenated anesthetics halothane, enflurane, and isoflurane on Ca(2+) electrode measurements and on the Ca(2+) sensitivity of the bioluminescent protein aequorin. In Ca(2+)-EGTA buffers of different pCa values (7. 870, 6.726, 6.033, 4.974, 4.038, and 2.995) and in serial Ca(2+) dilutions (10(-4), 10(-3), and 10(-2) M), halothane, enflurane, and isoflurane each caused a concentration-dependent and reversible increase in the absolute value of the negative electrode potential. Isoflurane and enflurane had larger effects than halothane. Neither of these anesthetics changed aequorin luminescence at any pCa tested in the range 2-8. There was no potentiation or inactivation of aequorin luminescence over a period of up to 2 h. These results suggest that (1) halothane, enflurane, and isoflurane interfere with Ca(2+) electrode measurements, most likely by changing the physicochemical properties of the membrane; (2) these anesthetics do not inactivate or otherwise modify the characteristics of the reaction of Ca(2+) with aequorin; and (3) these anesthetics do not change the apparent affinity of EGTA for Ca(2+).     

The inhibition of calcium efflux from rat liver mitochondria by halogenated anesthetics

The halogenated anesthetics halothane, enflurane and isoflurane inhibit the calcium efflux induced by Ruthenium Red in isolated rat liver mitochondria. The extent of the inhibition is higher for enflurane (approximately 50%) than for either isoflurane (approximately 35%) or halothane (approximately 15%), and does not increase significantly between 0.1 and 0.6-1.0 mM anesthetic. Both the mitochondrial respiratory rate and transmembrane electrical potential are unaffected by the halogenated anesthetics concentrations capable to inhibit the efflux of calcium.     

Volatile anesthetics selectively alter [3H]ryanodine binding to skeletal and cardiac ryanodine receptors.

The effect of clinical concentrations of volatile anesthetics on ryanodine receptors of cardiac and skeletal muscle sarcoplasmic reticulum was evaluated using [3H]ryanodine binding. At 2 volume percent, halothane and enflurane stimulated binding to cardiac SR by 238% and 204%, respectively, while isoflurane had no effect. In contrast, halothane and enflurane had no effect on [3H]ryanodine binding to skeletal ryanodine receptors, while isoflurane produced a significant stimulation. These results suggest that volatile anesthetics interact in a site-specific manner with ryanodine receptors of cardiac or skeletal muscle to effect Ca2+ release-channel gating.     

Estimation of minimum alveolar concentration (MAC) for halothane, enflurane and isoflurane in spontaneously breathing guinea pigs

MAC for halothane, enflurane and isoflurane was determined in guinea pigs (Cavia porcellus) exposed to constant anesthetic concentrations (2.5 hours each) in a flow-through glass chamber. The following values were obtained (N = 8 for each anesthetic): 1.01 +/- 0.03 vol% for halothane, 2.17 +/- 0.04 vol% for enflurane, and 1.15 +/- 0.05 vol% for isoflurane. In guinea pigs, MAC for halothane and enflurane are similar to those reported for other rodents, while MAC for isoflurane is lower. The data indicate that guinea pigs possibly are more susceptible to isoflurane's anesthetic actions than other rodents.     

Partitioning of anesthetics into a lipid bilayer and their interaction with membrane-bound peptide bundles

Molecular dynamics simulations have been performed to investigate the partitioning of the volatile anesthetic halothane from an aqueous phase into a coexisting hydrated bilayer, composed of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) lipids, with embedded alpha-helical peptide bundles based on the membrane-bound portions of the alpha- and delta-subunits, respectively, of nicotinic acetylcholine receptor. In the molecular dynamics simulations halothane molecules spontaneously partitioned into the DOPC bilayer and then preferentially occupied regions close to lipid headgroups. A single halothane molecule was observed to bind to tyrosine (Tyr-277) residue in the alpha-subunit, an experimentally identified specific binding site. The binding of halothane attenuated the local loop dynamics of alpha-subunit and significantly influenced global concerted motions suggesting anesthetic action in modulating protein function. Steered molecular dynamics calculations on a single halothane molecule partitioned into a DOPC lipid bilayer were performed to probe the free energy profile of halothane across the lipid-water interface and rationalize the observed spontaneous partitioning. Partitioned halothane molecules affect the hydrocarbon chains of the DOPC lipid, by lowering of the hydrocarbon tilt angles. The anesthetic molecules also caused a decrease in the number of peptide-lipid contacts. The observed local and global effects of anesthetic binding on protein motions demonstrated in this study may underlie the mechanism of action of anesthetics at a molecular level.     

Effects of isoflurane, halothane, and enflurane on myocardial flow and energy stores in the perfused rat heart

The effect of three volatile anesthetics (halothane, enflurane, and isoflurane) on coronary flow and metabolic state of isolated rat hearts was studied. These anesthetics are coronary dilators and their effects are dose dependent. At 2 MAC (minimum alveolar concentration), isoflurane, enflurane, and halothane increase coronary flow by 114 +/- 5.9, 93 +/- 6.1, and 77 +/- 6.4%, respectively (p less than 0.001). At these concentrations, they also have a modest but significant metabolic effect causing a 30% reduction in myocardial ATP and phosphocreatine levels, with no significant modification in ADP and AMP concentrations. Energy charge and lactate/pyruvate ratio were also unaffected by these anesthetics. The vascular and metabolic effects were reversible within 2 and 30 min, respectively. Perfusion of the hearts with a Krebs-Henseleit solution without Pi did not interfere with the vascular and the metabolic effect of the anesthetics; however, in this case, ATP and phosphocreatine concentration did not return to control levels after their discontinuation despite full recovery of the vascular effect. These data suggest that the volatile anesthetics have direct coronary vascular and myocardial metabolic effects and that these effects occur independently.     

Volatile anesthetics stimulate the phorbol ester evoked neurotransmitter release from PC12 cells through an increase of the cytoplasmic Ca2+ ion concentration

PC12 cells preloaded with [3H]norepinephrine release this neurotransmitter at a slow rate (basal release). This rate is increased by the addition of phorbol myristate acetate (PMA), but not by a biologically inactive phorbol ester. This effect most likely is mediated by protein kinase C, since desensitization of this kinase abolished the stimulation of the neurotransmitter release by PMA. Unexpectedly, clinical concentrations of the volatile anesthetics halothane, enflurane, isoflurane and methoxyflurane stimulated the PMA evoked neurotransmitter release in good correlation with their anesthetic potency. Since the volatile anesthetics increased the cytoplasmic Ca2+ concentration of the PC12 cells in a dose dependent manner it seems very likely that the effect of the anesthetics on the PMA-evoked neurotransmitter release is mediated by this rise in Ca2+ concentration.     

NMR study of volatile anesthetic binding to nicotinic acetylcholine receptors

New lines of evidence suggest that volatile anesthetics interact specifically with proteins. Direct binding analysis, however, has been largely limited to soluble proteins. In this study, specific interaction was investigated between isoflurane, a clinically important volatile anesthetic, and membrane-bound nicotinic acetylcholine receptors (nAChRs) from Torpedo electroplax, using (19)F nuclear magnetic resonance spectroscopy and gas chromatography. The receptors were reconstituted into 1, 2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) lipid vesicles. After correcting for nonspecific partitioning into the lipid, the equilibrium dissociation constant, K(d), of isoflurane binding to nAChR at 15 degrees C was found to be 0.36 +/- 0.03 mM. This value is within the clinically relevant concentration range of the agent. Based on the receptor concentrations in the vesicle suspension assayed by the bicinchoninic acid method and the fraction of bound isoflurane, X(b), determined by gas chromatography, an estimate of an average of 9-10 specifically bound isoflurane molecules can be made for each receptor, or two for each subunit. Upon binding, the transverse relaxation time constant (T(2)) of (19)F resonance of isoflurane is decreased by nearly three orders of magnitude, indicating a dramatic reduction in the mobility of specifically bound isoflurane. Kinetic analysis reveals that the off rate of binding, k(-1), is 1.7 x 10(4) s(-1). The on rate, k(+1), can thus be calculated to be approximately 4.8 x 10(7) M(-1) s(-1), suggesting a nearly diffusion-limited association. This is in contrast to anesthetic binding to a soluble protein, bovine serum albumin (BSA), where k(+1) and k(-1) are at least an order of magnitude slower. It is concluded that the presence of lipids may be critical for the correct evaluation of binding kinetics between volatile anesthetics and neuronal receptors.     

Volatile anesthetics inhibit the ion flux through Ca2+-activated K+ channels of rat glioma C6 cells

Ca2+-activated K+ channels in rat glioma C6 cells were investigated using monolayers of these cells in petri dishes. The ion flux through the channels was studied with 86Rb+ after addition of a Ca2+-ionophore to the incubation medium. Both the influx and efflux of 86Rb+ through these Ca2+-activated K+ channels were inhibited by the general anesthetic halothane (at clinical concentrations). Other volatile anesthetics such as isoflurane, enflurane and methoxyflurane also inhibited the Ca2+-activated K+ channels at clinical concentrations. Inhibition of these channels by general anesthetics could have profound effects on signal transmission in the brain.     

Transient increases of intracellular Ca2+ induced by volatile anesthetics in rat hepatocytes

The affects of volatile anesthetics on mobilization of intracellular Ca2+ was monitored in primary cultures of rat hepatocytes using the fluorescent Ca2+ probe Fura-2. The use of Fura-2 was limited by several factors which complicated the quantitative analysis of the results, such as: (i) a high rate of dye leakage; (ii) changes in the redox state of the hepatocytes which interfered with the fluorescence produced by the dye at various excitation wavelengths; (iii) compartmentalization of the dye producing high local intracellular concentrations; and, of particular importance for this study, (iv) enhanced photobleaching of the dye in the presence of halothane. To aid in the interpretation of the Fura-2 data, the Ca2(+)-sensitive photoprotein aequorin was also used to monitor changes in [Ca2+]i. The aequorin and Fura-2 techniques qualitatively yielded the same result, that the volatile anesthetic agents halothane, enflurane, and isoflurane induce an immediate and transient increase of [Ca2+]i. The durations of these transients were approximately between 5 and 10 min and were not related to any evident acute cell toxicity. The [Ca2+]i increases induced by the volatile anesthetic agents were dose-dependent, with halothane the most potent. The exact mechanism governing these increases in [Ca2+]i induced by these anesthetics in rat hepatocytes is unknown, but is likely to involve effects on both the cell surface membrane and endoplasmic reticulum components of the signal transducing system.     

Effects of volatile anesthetics on bacteriorhodopsin in purple membrane, Halobacterium halobium cells and reconstituted vesicles.

In this study, we have investigated effects of volatile anesthetics on absorption spectra, proton pumping activity and decay of photointermediate M of bacteriorhodopsin (bR) in differently aggregated states. Anesthetics used in this study are ether-type general anesthetics; enflurane and sevoflurane. The observed effects on bR depend not only on variety or concentration of anesthetics but also strongly on the aggregation state of bR molecules in the membrane. In purple membrane (PM), bR having maximum light absorption at 567 nm (bR567) is formed in the presence of sevoflurane or a small amount of enflurane, while a species absorbing maximally at 480 nm (bR480) is formed upon the addition of large amounts of enflurane. X-ray diffraction studies show that the former species maintains crystallinity of PM, but the latter does not. In reconstituted vesicles where bR molecules exist as monomer, even sevoflurane forms bR480. Flash photolysis experiments show that bR567 contains a shorter-lived M intermediate absorbing maximally at 412 nm in the photoreaction cycle than bR does and that bR480 contains at least two long-lived M intermediates which seem to absorb maximally near and at lower than 380 nm. The measurements of light-induced pH changes of the whole cells and of the reconstituted vesicles in the presence of the anesthetics indicate that bR567 has a enhanced proton pumping efficiency, while bR480 has a quite low or no activity. No significant difference was observed in the anesthetic action between two inversely pumping vesicles. These observations suggest that on the formation of bR480, anesthetics enter into the membrane and affect the protein-lipid interaction.     

Simultaneous determination of fluorinated inhalation anesthetics in blood by gas chromatography–mass spectrometry combined with a headspace autosampler

Although the fluorinated inhalation anesthetics, including desflurane, sevoflurane, isoflurane, enflurane, and halothane are commonly used, fatal cases resulting from their abuse or misuse have been reported. To date, gas chromatography (GC) equipped with different kinds of detectors has been utilized to analyze inhalation anesthetics. However, none of them can detect desflurane reliably or analyze all five common anesthetics simultaneously. The purpose of the present work is to further modify the previously developed headspace (HS) GC–MS method for blood isoflurane determination to analyze and distinguish five common clinical inhalation anesthetics, simultaneously. The modified HS-GC–MS method adopts a 60 m×0.25 mm I.D., 0.25 μm film thickness DB-5 capillary column along with an adequate GC temperature program, which gives the five inhalation anesthetics, including isoflurane and its isomer, enflurane, a high resolution. The method also takes both the volatility and the influence of the top space on the obtained concentration into consideration and therefore keeps the sample loss acceptable even for analyzing the highly volatile desflurane. Within a certain concentration range of the calibration standard (about 20–300 μg/ml), this method shows a good linearity with correlation coefficients greater than 0.999. In addition, both within- and between-run precision and accuracy results meet the validation requirements as well as the tested results of practical blood samples of desflurane. In summary, this is a reliable analytical method to simultaneously determine the concentration of five common inhalation anesthetics in blood. Such a method is very practical for both clinical and occupational monitoring, as well as for analytical toxicology.     

The alpha-helix to beta-sheet transition in poly(L-lysine): effects of anesthetics and high pressure.

Poly(L-lysine) exists in a random-coil formation at a low pH, alpha-helix at a pH above 10.6, and transforms into beta-sheet when the alpha-helix polylysine is heated. Each conformation is clearly distinguishable in the amide-I band of the infrared spectrum. The thermotropic alpha-to-beta transition was studied by using differential scanning calorimetry. At pH 10.6, the transition temperature was 43.5 degrees C and the transition enthalpy was 170 cal/mol residue. At pH 11.85, the measurements were 36.7 degrees C and 910 cal/mol residue, respectively. Volatile anesthetics (chloroform, halothane, isoflurane and enflurane) partially transformed alpha-helix polylysine into beta-sheet. The transformation was reversed by the application of hydrostatic pressure in the range of 100-350 atm. Apparently, the alpha-to-beta transition was induced by anesthetics through partial dehydration of the peptide side-chains (beta-sheet surface is less hydrated than alpha-helix). High pressure reversed this process by re-hydrating the peptide. Because the membrane spanning domains of channel and receptor proteins are predominantly in the alpha-helix conformation, anesthetics may suppress the activity of excitable cells by transforming them into a less than optimal structure for electrogenic ion transport and neurotransmission. Proteins and lipid membranes maintain their structural integrity by interaction with water. That which attenuates the interaction will destabilize the structure. These data suggest that anesthetics alter macromolecular conformations essentially by a solvent effect, thereby destroying the solvation water shell surrounding macromolecules.