Potency Of Halothane Research Articles

Impact of Gene Copy Number Variation on Anesthesia in Drosophila melanogaster

Chromosomal deletions and duplications, which result in halving or doubling of copy number in a block of genes, are an important source of variation between individuals. Phenotypic effects of copy number variation are commonly observed, but effects on sensitivity to volatile anesthetics have not been assessed in any organism. The potency with which halothane depresses the righting reflex of fruit flies was measured in congenic Drosophila strains, each of which was heterozygous for a deletion of average size 400 kb. Over 200 strains were examined, thereby scanning approximately half of the fly genome. Although the vast majority of deletion heterozygotes were indistinguishable from the control, eight had significantly altered sensitivity to halothane. Genetic tests supported the hypothesis that the change in anesthetic sensitivity was the result of reduction in copy number and not adventitious mutations in the strains. Among the eight outliers, the difference in halothane potency ranged from a 25% increase to a 15% decrease. Changes of similar magnitude but distinctive patterns were found when these lines were tested with enflurane, isoflurane, and sevoflurane. Variation in gene copy number has a significant impact on anesthetic sensitivity in Drosophila melanogaster. The level of transcription of a few genes must thus be limiting for a normal response to volatiles. Coupling between gene copy and gene expression is universal, and the components of the fly's nervous system are highly conserved; therefore, this work provides a rationale for investigating the clinical impact of copy number variation.

Kinetics and potency of halothane, isoflurane, and desflurane in the Northern Leopard frog Rana pipiens

Equilibration between delivered and effect site anesthetic partial pressure is slow in frogs. The use of less soluble agents or overpressure delivery may speed equilibration. Ten Northern leopard frogs were exposed to 3-4 constant concentrations of halothane, isoflurane or desflurane and their motor response to noxious electrical stimulation of the forelimb evaluated every 30 minutes until a stable proportion of frogs were immobile. Each frog received each anesthetic and concentration in random order and allowed at least 14 hours to recover between anesthetic exposures. An overpressure technique based upon the kinetics in the first study was then tested with 4 concentrations of desflurane. For halothane, isoflurane and desflurane respectively; the proportion of frogs immobile in response to stimulus became stable after 510, 480 and 180 minutes, and ED50 values were 1.36, 1.67 and 6.78 % atm. Desflurane ED50 delivered by overpressure was not significantly different at 6.85 % atm. Halothane, isoflurane and desflurane are effective general anesthetics in frogs with potencies similar to those reported in mammals. The time required for anesthetic equilibration is fastest with desflurane and can be hastened further by initial delivery of higher partial pressures.

Reduced inhibition of cortical glutamate and GABA release by halothane in mice lacking the K+ channel, TREK‐1

Deletion of TREK-1, a two-pore domain K(+) channel (K(2P)) activated by volatile anaesthetics, reduces volatile anaesthetic potency in mice, consistent with a role for TREK-1 as an anaesthetic target. We used TREK-1 knockout mice to examine the presynaptic function of TREK-1 in transmitter release and its role in the selective inhibition of glutamate vs GABA release by volatile anaesthetics. The effects of halothane on 4-aminopyridine-evoked and basal [(3)H]glutamate and [(14)C]GABA release from cerebrocortical nerve terminals isolated from TREK-1 knockout (KO) and littermate wild-type (WT) mice were compared. TREK-1 was quantified by immunoblotting of nerve terminal preparations. Deletion of TREK-1 significantly reduced the potency of halothane inhibition of 4-aminopyridine-evoked release of both glutamate and GABA without affecting control evoked release or the selective inhibition of glutamate vs GABA release. TREK-1 deletion also reduced halothane inhibition of basal glutamate release, but did not affect basal GABA release. The reduced sensitivity of glutamate and GABA release to inhibition by halothane in TREK-1 KO nerve terminals correlates with the reduced anaesthetic potency of halothane in TREK-1 KO mice observed in vivo. A presynaptic role for TREK-1 was supported by the enrichment of TREK-1 in isolated nerve terminals determined by immunoblotting. This study represents the first evidence for a link between an anaesthetic-sensitive 2-pore domain K(+) channel and presynaptic function, and provides further support for presynaptic mechanisms in determining volatile anaesthetic action.

Blockage of one class of potassium channel alters the effectiveness of halothane in a brain circuit of Drosophila.

At concentrations comparable to those used in the clinic, halothane has profound effects on a neuronal pathway devoted to the escape reflex of the fruit fly, Drosophila melanogaster. We studied the influence of the potassium channel that is encoded by the Shaker gene on the halothane sensitivity of this circuit. Shaker channels were specifically inactivated either by genetic means, using strains with two different severe Shaker mutations, or by pharmacologic means, using ingestion of millimolar concentrations of 4-aminopyridine. In all cases, halothane potency decreased substantially. To ensure that the genetic alteration was specific, both mutations were studied as stocks that had been repeatedly backcrossed to a control strain. The specificity of the pharmacologic inhibition was demonstrated by the fact that 4-aminopyridine had no effect on halothane potency in a Shaker mutant. Quantitative differences in the effects of channel inhibition between males and females suggested a sexual dimorphism in the functional brain anatomy of the reflex circuit.

Blockage of One Class of Potassium Channel Alters the Effectiveness of Halothane in a Brain Circuit of Drosophila

At concentrations comparable to those used in the clinic, halothane has profound effects on a neuronal pathway devoted to the escape reflex of the fruit fly, Drosophila melanogaster. We studied the influence of the potassium channel that is encoded by the Shaker gene on the halothane sensitivity of this circuit. Shaker channels were specifically inactivated either by genetic means, using strains with two different severe Shaker mutations, or by pharmacologic means, using ingestion of millimolar concentrations of 4-aminopyridine. In all cases, halothane potency decreased substantially. To ensure that the genetic alteration was specific, both mutations were studied as stocks that had been repeatedly backcrossed to a control strain. The specificity of the pharmacologic inhibition was demonstrated by the fact that 4-aminopyridine had no effect on halothane potency in a Shaker mutant. Quantitative differences in the effects of channel inhibition between males and females suggested a sexual dimorphism in the functional brain anatomy of the reflex circuit.

Sensitization of the cardiac Na channel to alpha1-adrenergic stimulation by inhalation anesthetics: evidence for distinct modulatory pathways.

Alpha1-adrenergic receptor stimulation has been shown to inhibit cardiac Na+ current (INa). Furthermore, some form of synergistic interaction of alpha1-adrenergic effects on INa in combination with volatile anesthetics has been reported. In this study, the authors investigated the possible role of G proteins and protein kinase C in the effects of halothane and isoflurane in the absence and presence of alpha1-adrenergic stimulation on the cardiac INa. The standard whole-cell configuration of the patch-clamp technique was used. INa was elicited by depolarizing test pulses from a holding potential of -80 mV in reduced Na+ solution (10 mM). The experiments were conducted on ventricular myocytes enzymatically isolated from adult guinea pig hearts. The inhibitory effect of halothane (1.2 mM) and isoflurane (1 mM) on peak INa was significantly diminished in the presence of guanosine 5'-O-[2-thiodiphosphate (GDPbetaS). In myocytes pretreated with pertussis toxin (PTX), the potency of halothane was significantly enhanced, but the isoflurane effect was unchanged. In the presence of the protein kinase C (PKC) inhibitor bisindolylmaleimide (BIS), the effect of halothane was unchanged. In contrast, the effect of isoflurane on INa in the presence of BIS was significantly enhanced. The positive interaction between methoxamine and halothane was evident in the presence of G protein and PKC inhibitors. In contrast, the effect of methoxamine with isoflurane was additive in the presence of GDPbetaS or BIS. Different second messenger systems are involved in the regulation of cardiac Na+ current by volatile anesthetics. The effect of halothane involves a complex interaction with G proteins but is independent of regulation by PKC. In contrast, PKC is involved in the modulation of cardiac INa by isoflurane. In addition, non-PTX-sensitive G proteins may contribute to the effects of isoflurane. The positive interaction between methoxamine and anesthetics are independent of G proteins and PKC for halothane. In the case of isoflurane, the positive interaction with methoxamine is coupled to PTX-insensitive G proteins and PKC.

Behavioral effects of volatile anesthetics in Caenorhabditis elegans.

The nematode Caenorhabditis elegans offers many advantages as a model organism for studying volatile anesthetic actions. It has a simple, well-understood nervous system; it allows the researcher to do forward genetics; and its genome will soon be completely sequenced. C. elegans is immobilized by volatile anesthetics only at high concentrations and with an unusually slow time course. Here other behavioral dysfunctions are considered as anesthetic endpoints in C. elegans. The potency of halothane for disrupting eight different behaviors was determined by logistic regression of concentration and response data. Other volatile anesthetics were also tested for some behaviors. Established protocols were used for behavioral endpoints that, except for pharyngeal pumping, were set as complete disruption of the behavior. Time courses were measured for rapid behaviors. Recovery from exposure to 1 or 4 vol% halothane was determined for mating, chemotaxis, and gross movement. All experiments were performed at 20 to 22 degrees C. The median effective concentration values for halothane inhibition of mating (0.30 vol%-0.21 mM), chemotaxis (0.34 vol%-0.24 mM), and coordinated movement (0.32 vol% - 0.23 mM) were similar to the human minimum alveolar concentration (MAC; 0.21 mM). In contrast, halothane produced immobility with a median effective concentration of 3.65 vol% (2.6 mM). Other behaviors had intermediate sensitivities. Halothane's effects reached steady-state in 10 min for all behaviors tested except immobility, which required 2 h. Recovery was complete after exposure to 1 vol% halothane but was significantly reduced after exposure to immobilizing concentrations. Volatile anesthetics selectively disrupt C. elegans behavior. The potency, time course, and recovery characteristics of halothane's effects on three behaviors are similar to its anesthetic properties in vertebrates. The affected nervous system molecules may express structural motifs similar to those on vertebrate anesthetic targets.

The inhibition of serotonin evoked bovine coronary artery contraction by halothane, isoflurane and sevoflurane is endothelium-independent.

The present study was designed to determine the direct effects of halothane, isoflurane and sevoflurane on the bovine epicardial coronary artery as well as their mode of action. We chose serotonin as the vasoconstrictor because it also causes endothelium-dependent relaxation of coronary arteries. Isolated spiral strips of bovine epicardial coronary artery with and without endothelium were suspended for isotonic contraction recordings in Tyrode's solution. KCl (80.4 mM) solution induced maximal contraction, regarded as the reference value (100%). The muscle strips were then exposed to increasing concentrations of serotonin from 10(-8) to 10(-4) M in the presence and absence of 1.5 MAC halothane, isoflurane or sevoflurane. All three drugs attenuated serotonin-evoked contraction in the coronary artery strips both those strips with and without endothelium (P < 0.05-0.001). However, there were no significant differences in the attenuation of serotonin-induced contraction of the strips, both with and without endothelium, in each drug group. The attenuation potency of halothane was more than that of isoflurane and sevoflurane. The results demonstrate that halothane, isoflurane and sevoflurane attenuate contractile responses evoked by serotonin in bovine epicardial coronary artery both with and without endothelium.

The inhibition of serotonin evoked bovine coronary artery contraction by halothane, isoflurane and sevoflurane is endothelium-independent

The present study was designed to determine the direct effects of halothane, isoflurane and sevoflurane on the bovine epicardial coronary artery as well as their mode of action. We chose serotonin as the vasoconstrictor because it also causes endothelium-dependent relaxation of coronary arteries. Isolated spiral strips of bovine epicardial coronary artery with and without endothelium were suspended for isotonic contraction recordings in Tyrode's solution. KCl (80.4 mM) solution induced maximal contraction, regarded as the reference value (100%). The muscle strips were then exposed to increasing concentrations of serotonin from 10−8 to 10−4 M in the presence and absence of 1.5 MAC halothane, isoflurane or sevoflurane. All three drugs attenuated serotonin-evoked contraction in the coronary artery strips both those strips with and without endothelium (P<0.05−0.001). However, there were no significant differences in the attenuation of serotonin-induced contraction of the strips, both with and without endothelium, in each drug group. The attenuation potency of halothane was more than that of isoflurane and sevoflurane. The results demonstrate that halothane, isoflurane and sevoflurane attenuate contractile responses evoked by serotonin in bovine epicardial coronary artery both with and without endothelium.

Binding of Halothane to Serum Albumin: Relevance to Theories of Narcosis

Professor and Vice Chairman for Research.Professor, Department of Anesthesia, University of California, San Francisco, Box 0464, Science 455, 513 Parnassus Avenue, San Francisco, California 94143–0464.To the Editor:--The report by Johansson et al. provides further insight into the molecular site at which general anesthetics act. The investigators found that halothane quenches the tryptophan fluorescence of bovine serum albumin in a concentration-dependent manner with a dissociation constant of 1.8 mM. They also reported that diethyl ether competes with halothane with a 50% inhibition concentration of 39 mM.These concentrations surpass those required for anesthesia. At 1.8 mM, halothane equals a partial pressure at 37 degrees Celsius of 0.06 atm (1.8 mM =(1.8 x 10 sup -6 mol/ml)(2.5436 x 104ml/mol)/0.75, where 0.75 is the partition coefficient for halothane in Krebs' solution at 37 degrees Celsius. This exceeds the anesthetizing partial pressure of halothane in humans by a factor of 8. Similarly, the partial pressure of ether at 39 mM equals 0.76 atm, assuming a partition coefficient of 13. This exceeds the anesthetizing partial pressure of ether in humans by a factor of 40. .Although Johansson et al. performed their studies at 25 degrees Celsius, the above ratios (8 for halothane and 40 for ether) for 37 degrees Celsius will approximate ratios at 25 degrees Celsius because of the counterbalancing changes in solubility and potency of anesthetics with decreasing temperature. Furthermore, the dissociation constant of 1.8 mM for halothane quenching found by Johansson et al. is also an order of magnitude greater than the anesthetic potency of halothane measured in animals at lower temperatures: The righting reflex EC50of halothane in tadpoles is approximately 0.1 mM at 20 degrees Celsius. The calculations of partial pressure also assume that the solution used in the experiment was equivalent to an isotonic salt solution. If the albumin added appreciably to the solubility of halothane, this would lower the partial pressure calculated for halothane but not that for ether, whose solubility in blood scarcely differs from that in water. .Even allowing for these factors, it appears that the partial pressures applied exceed those that produce anesthesia. If so, can the results provide us with insights into mechanisms of anesthetic action? Does the five-fold difference in the ratios for ether and halothane (8 vs. 40) mean that the finding for halothane does not apply equally to all anesthetics, and thus that the tryptophan site is not representative of a relevant anesthetic site of action? Finally, do results obtained at 25 degrees Celsius apply at the higher temperatures sustained by homeotherms?Edmond I. Eger II, M.D., Professor and Vice Chairman for Research.Donald D. Koblin, Ph.D., M.D., Professor, Department of Anesthesia, University of California, San Francisco, Box 0464, Science 455, 513 Parnassus Avenue, San Francisco, California 94143–0464.

O-235 - Histaminergic modulation of anesthetic potency of halothane.

O-235 - Histaminergic modulation of anesthetic potency of halothane.

Halothane's effects on GABA-gated chloride flux in mice selectively bred for sensitivity or resistance to diazepam

The DS (diazepam-sensitive) and DR (diazepam-resistant) lines of mice, selected on the basis of their ataxic response to diazepam, also diverge in the physiologic response of their brain γ-aminobutyric acid A (GABA A) receptors to benzodiazepines, as indicated by augmentation of GABA-mediated chloride flux. Cross-sensitivity and -resistance to other sedatives known to interact with the GABA A-receptor have also been demonstrated in DS and DR mice. Based on the finding that these mice also show cross-sensitivity and -resistance to obtundation by halothane, we predicted that their GABA A-receptors would also exhibit a differential response to halothane as assayed by an in vitro 36Cl − influx assay using purified brain microvesicles. Consistent with this prediction, therapeutic concentrations of halothane enhanced 1 μmol/1 GABA-gated flux with significantly greater potency in DS than in DR mice (halothane EC 50 336±64 μmol/1 (S.E.M.) vs. 605±110 μmol/1, respectively, P = 0.03), but there was no difference in maximal flux enhancement between the two lines (DS 4.7±0.4 nmol·mg −1·3 −1, vs. DR 4.7±0.5nmol·mg −1·3s −). Halothane (500 μmol/1) also shifted the entire GABA concentration-flux relationship significantly to the left, decreasing the EC 50 for GABA in both the DS and DR lines. Importantly, the shift in the GABA concentration-flux response in the presence of halothane was more pronounced in the DS mice (GABA EC 50 1.8±0.4 μmol/1vs.14.7±0.9 μmol/1 without halothane) than in the DR mice (GABA EC 50 4.7±0.6 μmol/1vs.14.7±0.9 μmol/1 without halothane). This effect of halothane was highly significant, both when compared to control, and between the selected lines ( P < 0.001). The findings that halothane enhances GABA-gated flux and enhances GABA's channel gating potency support the hypothesis that differential enhancement of agonist-stimulated chloride permeability at GABA A receptors could be a mechanism underlying the differential obtunding potency of halothane in DS and DR mice. However, at high GABA concentrations halothane decreased maximal chloride flux, more in DS than in DR mice ( P < 0.001), which is not consistent with such a mechanism.

Effects of inhalational general anaesthetics on intact Daphnia magna (Cladocera: Crustacea)

1. The potency of general anaesthetics was studied in the water flea, Daphnia magna. 2. Anaesthesia was defined as a reversible lack of movement to stimulation with strong light. 3. ec 50 s for methoxyflurane, halothane, isoflurane and enflurane were 0.095, 1.005, 1.170 and 1.422 (% v v in 100% oxygen). 4. Nitrous oxide had no significant additive effect on the ec 50 s of the other agents, nor any effect on its own. 5. Oxygen concentrations in the carrier gas did not significantly affect the ec 50 of halothane. 6. Clonal differences in halothane potency were observed.

Effects of temperature on the anaesthetic potency of halothane, enflurane and ethanol in Daphnia magna (Cladocera: Crustacea)

1. The effects of temperature on the anaesthetic potencies of halothane, enflurane and ethanol have been studied in the water flea Daphnia magna. 2. In the absence of anaesthetics, decreasing temperature resulted in decreased activity by the daphnids. 3. Potencies in the gas phase decreased with increasing temperature for all of the anaesthetics, while aqueous potency decreased for halothane and enflurane but increased for ethanol. 4. Enthalpy calculations suggest that the observed potency changes for the inhalational anaesthetics cannot be accounted for in terms of changing solubility in lipid bilayers but most likely reflect more specific interactions with animal target sites.

Temperature-dependent effects of halothane and isoflurane on the isolated left atrium

The aim of the present study was to examine whether changes in temperature alter the effects of halothane and isoflurane on isolated left atria. Concentration-response curves for inotropic effects at different temperatures (30 degrees C, 37 degrees C, 40 degrees C) on electrically stimulated left atria of the rat were obtained. The change of temperature modified the maximal negative inotropic response to halothane. The maximal decrease induced by halothane was 12 +/- 2.3 per cent at 37 degrees C and 18 +/- 2.5 per cent at 30 degrees C. When the temperature increased up to 40 degrees C the maximal decrease of atrial inotropism was 46 +/- 2.1 per cent--significantly higher than obtained at 37 degrees C. However, the maximal effect obtained by isoflurane was not significantly affected by temperature (30 degrees C = 7 +/- 1.6 per cent; 37 degrees C = 8 +/- 1.8 per cent; 40 degrees C = 2 +/- 0.8 per cent). Furthermore the potency of halothane (expressed as the concentration which produced 50 per cent inhibition - IC 50 per cent), decreased significantly at 30 degrees C (IC 50 = 1.34 +/- 0.18) and increased at 40 degrees C (IC 50 = 0.44 +/- 0.17) when compared with its potency at 37 degrees C (IC 50 = 0.96 +/- 0.08). On the other hand changes in temperature did not significantly modify the IC 50 for isoflurane obtained at 37 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)

Fluroxene and Isolated Heart Muscle

The direct myocardial effects of fluroxene were examined in isometrically and isotonically contracting isolated rat heart muscle. MAC, the minimum anesthetic concentration needed to prevent movement in response to tail clamping, was found to be 5.0 vol per cent fluroxene in the rat. At 4.6 vol per cent fluroxene, peak developed isometric tension and maximum rate of tension development were decreased 29 and 24 per cent, respectively. At 11 vol per cent, the depressions were 39 and 33 per cent. At 26.4 vol per cent, the depressions were around 60 per cent. Vmax (the maximum shortening velocity of unloaded muscle) of the force-velocity relation was unaltered by fluroxene concentrations of 0.8, 4.6, and 11 vol per cent. Even at 26.4 vol per cent, the depression in Vmax was only 25 per cent. Po (the maximum force at zero velocity), work, and power were lowered much more, with reductions ranging from 15 to 27 per cent at 4.6 vol per cent, from 40 to 42 per cent at 11.0 vol per cent, and from 65 to 69 per cent at the 26.4 vol per cent. Series elastic extension was unchanged at 0.8 and 4.6 vol per cent fluroxene, but was decreased 16 per cent at 11.0 vol per cent and 46 per cent at 26.4 vol per cent fluroxene. The data indicate the fluroxene has a direct negative inotropic effect that is associated with increased series elastic stiffness, but does not involve Vmax of the force-velocity relation until quite high anesthestic concentrations are reached. Comparative studies were also carried out with halothane. MAC for halothane in the rat was 1.0 vol per cent. The relative potency of halothane compared with fluroxene in depression of Vmax was 13.2, and its relative potency in depression of Po, 4.0.

DEPENDENCE OF HALOTHANE POTENCY ON pH

SUMMARY The effect of halothane on the amplitude and conduction time of the evoked nerve action potential of a mammalian nerver preparation was determined in vitro. The action of 4 per cent v/v halothane on the action potential was measured at three different hydrogen ion concentrations in the Krebs perfusate, namely, pH 6.57, pH 7.40, pH 8.00. Halothane potency, assessed by the depression of the action potential amplitude, was inversely proportional to the extracellular pH and represented a mean 18.7 per cent decrease of halothane potency for a rise of one pH unit. The change in never conduction time induced by halothane was found to be less with a high extracellular pH than with a normal pH, and represented an 18.8 per cent decrease in halothane potency for a rise of one pH unit.