Anesthetic and ethanol effects on spontaneously opening glycine receptor channels

Strychnine-sensitive glycine receptors mediate inhibitory neurotransmission occurring in the brain stem and spinal cord. Alcohols, volatile anesthetics and inhaled drugs of abuse are positive allosteric modulators of glycine receptor function, normally enhancing function only in the presence of glycine. A complication in studying allosteric actions on ligand-gated ion channels is in the dissection of their effects on neurotransmitter binding from their effects on channel opening. Mutation of an aspartate residue at position 97 to arginine in the glycine receptor alpha1 subunit simulated the effects of glycine binding, producing receptors that exhibited tonic channel opening in the absence of neurotransmitter; i.e. these receptors demonstrated a dissociation of channel opening from neurotransmitter binding. In these receptors, ethanol, enflurane, chloroform, halothane, 1,1,1-trichloroethane and toluene elicited inward currents in the absence of glycine. We previously identified mutations on ligand-gated ion channels that eliminate ethanol, anesthetic and inhalant actions (such as S267I on alpha1 glycine receptors). The double mutant (D97R and S267I) receptors were both constitutively active and resistant to the enhancing effects of ethanol and enflurane. These data demonstrate that ethanol and volatile anesthetics can affect glycine receptor channel opening independently of their effects on enhancing neurotransmitter binding.     

Amino acid volume and hydropathy of a transmembrane site determine glycine and anesthetic sensitivity of glycine receptors.

Two specific amino acid residues in transmembrane segments (TM) 2 and 3 are critical for the enhancement of glycine receptor (GlyR) function by volatile anesthetics. To determine which physicochemical characteristics of these sites determine their roles in anesthetic actions, an extensive series of single amino acid mutations at amino acid residue 288 (Ala-288) in TM3 of the alpha1 GlyR subunit was tested for modulation by volatile anesthetics. The mutations changed the apparent affinities of receptors for glycine; replacements with larger volumes and less hydropathy exhibited higher affinities for glycine. Potentiation by anesthetics was reduced by specific mutations at Ala-288. The molecular volume of the substituents was negatively correlated with the extent of potentiation by isoflurane, enflurane, and 1-chloro-1,2,2-trifluorocyclobutane, whereas there was no correlation between anesthetic enhancement and polarity, hydropathy, or hydrophilicity of substituents. In contrast to anesthetics, no correlation was found between the effects of the nonanesthetics 1,2-dichlorohexafluorocyclobutane or 2, 3-dichlorooctafluorobutane and any physicochemical property of the substituent. These results suggest that the molecular volume and hydropathy of the amino acid at position 288 in TM3 regulate glycine and anesthetic sensitivity of the GlyR and that this residue might represent one determinant of an anesthetic binding site.     

General anesthetics modulate GABA receptor channel complex in rat dorsal root ganglion neurons

The effects of halothane, isoflurane, and enflurane on ionic currents induced by bath application of gamma-amino-butyric acid (GABA) were studied with the rat dorsal root ganglion neurons maintained in primary culture. The whole-cell patch clamp technique was used to record the current. In normal neurons before exposure to anesthetics, GABA at low concentrations (1-3 x 10(-6) M) induced a small sustained inward current. At higher concentrations (3 x 10(-5) M-1 x 10(-3) M), GABA induced a large inward current, which decayed to a steady-state level (desensitization). Halothane (0.86 mM), isoflurane (0.96 mM), and enflurane (1.89 mM), each equivalent to the respective 2 minimum alveolar concentration (MAC) units, augmented the sustained current evoked by 3 x 10(-6) M GABA to 330-350% of control and the peak current evoked by 3 x 10(-5) M of GABA to 136-145% of control. The decay phase of the current was accelerated by the anesthetics, the time for the current to decline to 70% of the peak being reduced to 23-39% of control. In contrast, the densitized steady-state current evoked by high concentrations of GABA was decreased by anesthetics. In conclusion, general anesthetics exert a dual effect on the GABA receptor channel complex: to potentiate the nondesensitized (both peak and sustained) current and to suppress the desensitized steady-state current. The potentiation of the GABA receptor channel response may be a primary action of anesthetics leading to surgical anesthesia.     

Interaction of fluorinated ether anesthetics with artificial membranes.

Fluorine-19 nuclear magnetic resonance spectroscopy is applied to the study of the environment of dipalmitoyl phosphatidylcholine-bound fluorinated ether anesthetics (enflurane, fluoroxene and methoxyflurane) both below and above the lipid gel to liquid crystal phase transition temperature. Line widths and spin-lattice relaxation time (T1) measurements are consistent with substantial immobilization of the lipid-bound anesethetic molecules. Heating anesthetic/lipid mixtures above the lipid transition temperature leads to narrowing of the lipid-bound anesthetic fluorine resonances accompanied by little or no change in anesthetic fluorine-19 chemical shifts, suggesting that although the mobility of the bound anesthetic increases at the higher temperature, the nature of the anesthetic-lipid interaction changes little as a result of this phase change. Differential scanning calorimetric studies of the effects of these anesthetics on the phase transition behavior of the phospholipid indicate that the regions of the bilayer in which volatile anesthetics partition at lower concentrations are different from the regions in which they partition at higher concentrations.     

Interaction of fluorinated ether anesthetics with artificial membranes

Fluorine-19 nuclear magnetic resonance spectroscopy is applied to the study of the environment of dipalmitoyl phosphatidylcholine-bound fluorinated ether anesthetics (enflurane, fluoroxene and methoxyflurane) both below and above the lipid gel to liquid crystal phase transition temperature. Line widths and spin-lattice relaxation time (T₁) measurements are consistent with substantial immobilization of the lipid-bound anesthetic molecules. Heating anesthetic/lipid mixtures above the lipid transition temperature leads to narrowing of the lipid-bound anesthetic fluorine resonances accompanied by little or no change in anesthetic fluorine-19 chemical shifts, suggesting that although the mobility of the bound anesthetic increases at the higher temperature, the nature of the anesthetic-lipid interaction changes little as a result of this phase change. Differential scanning calorimetric studies of the effects of these anesthetics on the phase transition behavior of the phospholipid indicate that the regions of the bilayer in which volatile anesthetics partition at lower concentrations are different from the regions in which they partition at higher concentrations.     

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.     

Changes in voltage-dependent ion currents during meiosis and first mitosis in eggs of an ascidian.

Different patterns of voltage-dependent ion currents are present in mature eggs and in early embryos of the ascidian Boltenia villosa, as if each ion current is regulated in a different manner between fertilization and the early cleavages of embryogenesis. The ion currents appear and/or disappear with precise timing suggesting that they play important roles at specific times during early development. We investigated changes in three voltage-dependent ion currents (an inwardly rectifying chloride current, a calcium current, and a sodium current) and membrane surface area over time between the resumption of meiosis (with fertilization or activation) and the first mitotic cleavage. Using time-lapse video recordings made during whole-cell patch-clamp experiments, we were able to correlate electrophysiological changes with morphological changes and cell cycle related events. Between fertilization and first cleavage, INa was lost exponentially, the density of ICa remained relatively constant, and the amplitudes of both ICl and membrane surface area fluctuated in time with the cell cycle. ICl and surface area increased whenever the cell began dividing--with the polar body extrusions and the formation of the first cleavage furrow. This suggested that the values of ICl and surface area were largest during interphase and smallest during M-phase of each cell cycle. This hypothesis was supported by an experiment in which entry into M-phase was blocked in fertilized eggs by inhibiting protein synthesis. This prevented the decreases of ICl and surface area but allowed the increases to occur normally. Patterns of change in ion currents are current specific and, as is the case with ICl, are tightly correlated with developmental events.     

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.     

Acidic extracellular pH-activated outwardly rectifying chloride current in mammalian cardiac myocytes

Extracellular acidic pH was found to induce an outwardly rectifying Cl- current (I(Cl,acid)) in mouse ventricular cells, with a half-maximal activation at pH 5.9. The current showed the permeability sequence for anions to be SCN- > Br- > I- > Cl- > F- > aspartate, while it exhibited a time-dependent activation at large positive potentials. Similar currents were also observed in mouse atrial cells and in atrial and ventricular cells from guinea pig. Some Cl- channel blockers (DIDS, niflumic acid, and glibenclamide) inhibited ICl,acid, whereas tamoxifen had little effect on it. Unlike volume-regulated Cl- current (ICl,vol) and CFTR Cl- current (ICl,CFTR), ICl,acid was independent of the presence of intracellular ATP. Activation of ICl,acid appeared to be also independent of intracellular Ca2+ and G protein. ICl,acid and ICl,vol could develop in an additive fashion in acidic hypotonic solutions. Isoprenaline-induced ICl,CFTR was inhibited by acidification in a pH-dependent manner in guinea pig ventricular cells. Our results support the view that ICl,acid and ICl,vol stem from two distinct populations of anion channels and that the ICl,acid channels are present in cardiac cells. ICl,acid may play a role in the control of action potential duration or cell volume under pathological conditions, such as ischemia-related cardiac acidosis.     

Interaction between artificial membranes and enflurane,a general volatile anesthetic: DPPC-enflurane interaction

The structural modifications of the dipalmitoylphosphatidylcholine (DPPC) organization induced by increasing concentration of the volatile anesthetic enflurane have been studied by differential scanning calorimetry, small-angle, and wide-angle x-ray scattering. The interaction of enflurane with DPPC depends on at least two factors: the enflurane-to-lipid concentration ratio and the initial organization of the lipids. At 25 degrees C (gel state), the penetration of enflurane within the lipids induces the apparition of two different mixed lipid phases. At low anesthetic-to-lipid molar ratio, the smectic distance increases whereas the direction of the chain tilt changes from a tilt toward next-neighbors to a tilt between next-neighbors creating a new gel phase called L(beta')(2NNN). At high ratio, the smectic distance is much smaller than for the pure L(beta') DPPC phase, i.e., 50 A compared to 65 A, the aliphatic chains are perpendicular to the membrane and the fusion temperature of the phase is 33 degrees C. The electron profile of this phase that has been called L(beta)(i), indicates that the lipids are fully interdigitated. At 45 degrees C (fluid state), a new melted phase, called L(alpha)(2), was found, in which the smectic distance decreased compared to the initial pure L(alpha)(1) DPPC phase. The thermotropic behavior of the mixed phases has also been characterized by simultaneous x-ray scattering and differential scanning calorimetry measurements using the Microcalix calorimeter of our own. Finally, titration curves of enflurane effect in the mixed lipidic phase has been obtained by using the fluorescent lipid probe Laurdan. Measurements as a function of temperature or at constant temperature, i.e., 25 degrees C and 45 degrees C give, for the maximal effect, an enflurane-to-lipid ratio (M/M), within the membrane, of 1 and 2 for the L(alpha)(2) and the L(beta)(i) lamellar phase respectively. All the results taken together allowed to draw a pseudo-binary phase diagram of enflurane-dipalmitoylphosphatidylcholine in excess water.     

Antagonism of inhalant and volatile anesthetic enhancement of glycine receptor function

Recent studies suggest that alcohols, volatile anesthetics, and inhaled drugs of abuse, which enhance gamma-aminobutyric acid, type A, and glycine receptor-activated ion channel function, may share common or overlapping molecular sites of action on these receptors. To investigate this possibility, these compounds were applied singly and in combination to wild-type glycine alpha(1) receptors expressed in Xenopus laevis oocytes. Data obtained from concentration-response curves of the volatile anesthetic enflurane constructed in the presence and absence of ethanol, chloroform, or toluene were consistent with competition for a common binding pocket on these receptors. A mutant glycine receptor, insensitive to the enhancing effects of ethanol but not anesthetics or inhalants, demonstrated antagonism of anesthetic and inhalant effects on this receptor. Although ethanol (25-200 mm) had no effect on its own in this receptor, it was able to inhibit reversibly the enhancing effect of enflurane, toluene, and chloroform in a concentration-dependent manner. These data suggest the existence of overlapping molecular sites of action for ethanol, inhalants, and volatile anesthetics on glycine receptors and illustrate the feasibility of pharmacological antagonism of the effects of volatile anesthetics.     

Partitioning of four modern volatile general anesthetics into solvents that model buried amino acid side-chains.

Partitioning of four modern inhalational anesthetics (halothane, isoflurane, enflurane, and sevoflurane) between the gas phase and nine organic solvents that model different amino acid side-chains and lipid membrane domains was performed in an effort to define which microenvironments present in proteins and lipid bilayers might be favored. Compared to a purely aliphatic environment (hexane), the presence of an aromatic-, alcohol-, thiol- or sulfide group on the solvent improved anesthetic partitioning, by factors of 1.3-5.2 for halothane, 1.7-5.6 for isoflurane, 1.7-7.6 for enflurane, and 1.5-7.3 for sevoflurane. The most favorable solvent for halothane partitioning was ethyl methyl sulfide, a model for methionine. Enflurane and isoflurane partitioned most extensively into methanol, a model for serine, and sevoflurane into ethanol, a model for threonine. Isoflurane also partitioned favorably into ethyl methyl sulfide. The results suggest that volatile general anesthetics interact better with partly polar groups, which are present on amino acids frequently found buried in the hydrophobic core of proteins, compared to purely aliphatic side-chains. Furthermore, if an anesthetic molecule was located in a saturated region of a phospholipid bilayer membrane, there would be an energetically favorable driving force for it to move into several higher dielectric microenvironments present on membrane proteins. The results provide evidence that proteins rather than lipids are the likely targets of volatile general anesthetics in biological membranes.     

A model for the secondary structure of beta-lactamases

The effects of the quaternary agent meproadifen on ACh-activated channel currents were studied on myoballs cultured from hind limb muscles of neonatal rats. Meproadifen (0.02-0.1 microM) combined with ACh (0.1-0.3 microM) in the patch pipette caused an increase, followed by a decrease, in the frequency of channel openings. At concentrations greater than 0.2 microM the initial phase was not detected and a rapid and marked reduction in the opening frequency was observed. Meproadifen (up to 2.5 microM) produced no change in the duration or conductance of the open state of ACh-activated channels. In addition, this agent induced the appearance of events with a marked increase in the 'noise' during the opening phase. The lack of effect under inside-out patch conditions suggested that meproadifen binds to a site located at the external portion of the nicotinic macromolecule and has no access to it through the cell membrane. This study indicated that non-competitive antagonists such as meproadifen can facilitate receptor activation and desensitization.     

Volatile anesthetics cause conformational changes of bacteriorhodopsin in purple membrane

We examined the effects of volatile anesthetics on the structure of the bacteriorhodopsin in the purple membrane by measurements of the absorption spectrum and the visible circular dichroism (CD) spectrum and assay of the retinal composition. As the concentrations of halothane, enflurane and methoxyflurane were increased, the absorption at 560 nm decreased but that at 480 nm increased with an isosbestic point around 510 nm. These anesthetic-induced spectroscopic changes were reversible. The CD spectrum showed the biphasic pattern with a positive and a negative band. As the concentration of halothane was increased from 4 mM to 8mM, the negative band reversibly diminished more drastically than the positive band, and at 8 mM of halothane the positive band shifted to around 480 nm. These results show that halothane disturbed the exciton coupling among bacteriorhodopsin molecules. The retinal isomer composition was analyzed using high performance liquid chromatography. The ratio of 13-cis- to all-trans-retinal was 47:53, 34:66 and 19:81 at control, 7.4 mM and 14.9 mM enflurane, respectively. After elimination of enflurane, the ratio returned to the control value. These findings indicate that volatile anesthetic directly affect a bacteriorhodopsin in the purple membrane and induce conformational changes in it.     

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.     

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.     

A [Na+]o-independent, pHo-dependent mechanism for reduction of intracellular [Ca2+] after influx through Ca2+ channels in mouse pituitary cells

The effect of extracellular pH (pHo) on the duration of calcium-dependent chloride currents (ICl(Ca] was studied in voltage clamped AtT-20 pituitary cells. ICl(Ca) was activated by Ca2+ influx through plasma membrane Ca2+ channels, which were opened by step depolarization to voltages between -20 and +60 mV. Increasing pHo from 7.3 to 8.0 reversibly prolonged ICl(Ca) tail currents in perforated patch recordings from cells bathed in both Na(+)-containing and Na(+)-free solutions. This prolongation was prevented in standard whole cell recordings when the pipette solution contained 0.5 mM EGTA. The effects of raised pHo were not due to alteration of intracellular pH, since tail current prolongation still occurred when intracellular pH was buffered at 7.3 with 80 mM HEPES. The prolongation of ICl(Ca) at pHo 8 could not be accounted for by a direct action on Ca2+ channels, since tail currents were prolonged when pHo was changed rapidly during the tail current, after all Ca2+ channels were closed. The effects of increasing pHo on ICl(Ca) also could not be explained by a direct action on Cl- channels, since changing to pHo 8 did not prolong Cl- tail currents when intracellular Ca2+ concentration [( Ca2+]i) was fixed by EGTA in whole cell recordings. Raising pHo did, however, prolong depolarization-evoked [Ca2+]i transients, measured directly with the Ca2+ indicator dye, fura-2. Taken together, these data demonstrate the presence of a Na(+)-independent, pHo-sensitive mechanism for reduction of [Ca2+]i after influx through Ca2+ channels. This mechanism is associated with the plasma membrane, and is active on a time scale that is relevant to the duration of single action potentials in these cells. We suggest that this mechanism is the plasma membrane Ca2+ ATPase.     

Diazepam action onγ-aminobutyric acid-activated chloride currents in internally perfused frog sensory neurons

The Cl- current (ICl) in gamma-aminobutyric acid (GABA)-sensitive frog sensory neuron was separated from other Na+, Ca2+, and K+ currents using a suction pipette technique which allows internal perfusion under a single-electrode voltage clamp. Diazepam (DZP) itself evoked no response but facilitated the dose- and time-dependently GABA-induced ICl without changing the GABA equilibrium potential (EGABA) at concentrations ranging widely, from 3 X 10(-9) to 10(-4) M. In the presence of DZP, the GABA dose-response curve shifted to the left without changing the maximum current, indicating that DZP modifies the interaction between GABA and its receptor rather than affecting directly the channel activation step. The enhancement of the GABA-induced ICl by DZP depended neither on the membrane voltage nor on the inward or outward direction of the ICl. DZP also potentiated the ICl elicited by GABA agonists such as beta-alanine, taurine, homotaurine, 5-aminovaleric acid, l-GABOB, d-GABOB, glycine, and muscimol. The GABA response enhanced by pentobarbital (PB) was further enhanced by adding DZP, indicating that DZP and PB do not act in the same way. Ro5-3663, a diazepam analogue, enhanced the GABA-induced ICl only in a narrow range of the concentrations but inhibited the current at concentrations higher than 2 X 10(-6) M.     

Interactions ofγ-aminobutyric acid (GABA), pentobarbital,and homopantothenic acid (HOPA) on internally perfused frog sensory neurons

Augmentatory actions among Cl- currents (ICl) induced by gamma-aminobutyric acid (GABA), pentobarbital (PB), and homopantothenic acid (HOPA) were investigated in isolated frog sensory neurons after suppression of Na+, K+, and Ca2+ currents using a suction pipette technique which combines internal perfusion with voltage clamp. GABA-sensitive neurons responded to both PB and HOPA, and the responses behaved as a simple Cl- electrode and reversed at the Cl- equilibrium potential (ECl). The dose-response curve for GABA-induced Cl- conductance was sigmoidal with the GABA concentration producing a half-maximum response (4.2 X 10(-5) M). Both GABA and HOPA dose-response curves shifted to the left in the presence of PB, though the facilitatory action of PB on GABA- and HOPA-induced ICl was more effective in the former. There was a significant facilitatory interaction between GABA- and HOPA-induced ICl. It is concluded that HOPA affects the GABA-GABA or PB-PB receptor interactions.