Human neuronal nicotinic acetylcholine receptors expressed in Xenopus oocytes predict efficacy of halogenated compounds that disobey the Meyer-Overton rule.

BACKGROUND: According to the Meyer-Overton rule, anesthetic potency of a substance can be predicted by its lipid solubility, but a group of halogenated volatile compounds predicted to induce anesthesia does not obey this rule. Thus, these compounds are useful tools for studies of molecular targets of anesthetics. Human neuronal nicotinic acetylcholine receptor (hnAChR) subunits have been recently cloned, which allowed the authors to assess whether these receptors could differentiate among volatile anesthetic and nonimmobilizer compounds. This study provides the first data regarding anesthetic sensitivity of hnAChRs. METHODS: alpha2beta4, alpha3beta4, and alphaabeta2 hnAChRs were expressed in Xenopus oocytes, and effects of volatile anesthetics isoflurane and F3 (1-chioro-1,2,2-triflurocyclobutane, 1A) and nonimmobilizers F6 (1,2-dichlorohexafluorocyclobutane, 2N) and F8 (2,3-dichlorooctafluorobutane) on the peak acetylcholine-gated currents were studied using the two-electrode voltage-clamp technique. RESULTS: Isoflurane and F3 inhibited all the hnAChRs tested in a concentration-dependent manner. Isoflurane at a concentration corresponding to 1 minimum alveolar concentration (MAC) inhibited 83, 69, and 71% of ACh-induced currents in alpha2beta4, alpha3beta4, and alpha4beta2 hnAChRs, respectively, and 1 MAC of F3 inhibited 64, 44, and 61% of currents gated in those receptors. F6 (8-34 microM) did not cause any changes in currents gated by any of the receptors tested. F8 (4-18 microM) did not alter the currents gated in either alpha3beta4 or alpha4beta2 receptors, but caused a small potentiation of alpha2beta4 hnAChRs without a concentration-response relation. CONCLUSION: The in vivo potency and effectiveness of volatile anesthetic and nonimmobilizer compounds were consistent with their actions on hnAChRs expressed in a recombinant expression system, suggesting a potential participation of these receptors in the mechanisms of anesthesia.     

Human Neuronal Nicotinic Acetylcholine Receptors Expressed in Xenopus Oocytes Predict Efficacy of Halogenated Compounds That Disobey the Meyer-Overton Rule

Background: According to the Meyer-Overton rule, anesthetic potency of a substance can be predicted by its lipid solubility, but a group of halogenated volatile compounds predicted to induce anesthesia does not obey this rule. Thus, these compounds are useful tools for studies of molecular targets of anesthetics. Human neuronal nicotinic acetylcholine receptor (hnAChR) subunits have been recently cloned, which allowed the authors to assess whether these receptors could differentiate among volatile anesthetic and nonimmobilizer compounds. This study provides the first data regarding anesthetic sensitivity of hnAChRs.

Methods: [alpha]2[beta]4, [alpha]3[beta]4, and [alpha]4[beta]2 hnAChRs were expressed in Xenopus oocytes, and effects of volatile anesthetics isoflurane and F3 (1-chloro-1,2,2-triflurocyclobutane, 1A) and nonimmobilizers F6 (1,2-dichlorohexafluorocyclobutane, 2N) and F8 (2,3-dichlorooctafluorobutane) on the peak acetylcholine-gated currents were studied using the two-electrode voltage-clamp technique.

Results: Isoflurane and F3 inhibited all the hnAChRs tested in a concentration-dependent manner. Isoflurane at a concentration corresponding to 1 minimum alveolar concentration (MAC) inhibited 83, 69, and 71% of ACh-induced currents in [alpha]2[beta]4, [alpha]3[beta]4, and [alpha]4[beta]2 hnAChRs, respectively, and 1 MAC of F3 inhibited 64, 44, and 61% of currents gated in those receptors. F6 (8-34[mu]M) did not cause any changes in currents gated by any of the receptors tested. F8 (4-18[mu]M) did not alter the currents gated in either [alpha]3[beta]4 or [alpha]4[beta]2 receptors, but caused a small potentiation of [alpha]2[beta]4 hnAChRs without a concentration-response relation.     

Inhibitory effects of isoflurane and nonimmobilizing halogenated compounds on neuronal nicotinic acetylcholine receptors

BACKGROUND: Neuronal nicotinic acetylcholine receptors (nAchRs) are inhibited by low concentrations of volatile anesthetics. However, it is not clear whether this phenomenon contributes to the anesthetic effects of volatile anesthetics. Effects of a volatile anesthetic (isoflurane) and structurally related nonimmobilizers (F6: 1,2-dichlorohexafluorocyclobutane, F8: 2,3-dichlorooctafluorobutane) on the current mediated through neuronal nAchRs were studied. METHOD: This study investigated neuronal nAchRs in PC12 cells and acutely dissociated rat medial habenula (MHb) neurons. Whole cell currents elicited by 30 microm nicotine were recorded in the absence and presence of the halogenated agents. The minimum alveolar concentrations (MACs) for F6 and F8 were predicted from Meyer-Overton correlation. RESULTS: All halogenated compounds inhibited the nicotine-induced current in a concentration-dependent manner in PC12 cells. In MHb neurons, while isoflurane and F6 significantly inhibited the nicotine-induced peak current, F8 failed to inhibit it. The peak currents in the presence of isoflurane at 1.7 MAC, of F6 at 2.4 MAC, and of F8 at 2.2 MAC were 12, 31, and 97% of control, respectively. CONCLUSIONS: Isoflurane, F6, and F8 inhibited ganglion-type nAchRs in PC12 cells independent from their abilities to produce the anesthetic state. In MHb neurons, isoflurane and F6, which lack the immobilizing effect but has the amnesic effect, inhibited nAchRs. Native brain nicotinic receptors in MHb neurons were almost insensitive to F8, which lacks both the immobilizing and the amnesic effect. These results are consistent with the hypothesis that inhibition of nAchRs in MHb neurons is not important for the anesthetic effect but may contribute to the amnesic effect of these agents.     

Nitrous oxide and xenon inhibit the human (alpha 7)5 nicotinic acetylcholine receptor expressed in Xenopus oocyte

The neuronal nicotinic acetylcholine (nACh) receptor is one of the ligand-gated ion channels that regulate the synaptic release of neurotransmitters in the central nervous system. Recently, neuronal nACh receptors have received attention as a potential target for general anesthetics because many general anesthetics inhibit their functions at clinical concentrations. Several general anesthetics are known to inhibit the homomeric (alpha(7))(5) nACh receptor, a subtype of neuronal nACh receptors, but the effects of two gaseous anesthetics, nitrous oxide (N(2)O) and xenon (Xe), remain unknown. Using the two-electrode voltage-clamping technique, we investigated the effects of N(2)O and Xe at the human (alpha(7))(5) nACh receptor expressed in Xenopus oocytes. At clinically relevant concentrations, N(2)O and Xe reversibly inhibited the ACh-induced currents of the (alpha(7))(5) nACh receptor in a concentration-dependent manner. The inhibitory actions of both anesthetics at the (alpha(7))(5) nACh receptor were noncompetitive and voltage-independent. Our results suggest that inhibition of the (alpha(7))(5) nACh receptor by N(2)O and Xe may play a role in their anesthetic effects.     

Nonhalogenated alkane anesthetics fail to potentiate agonist actions on two ligand-gated ion channels

BACKGROUND: Although ether, alcohol, and halogenated alkane anesthetics potentiate agonist actions or increase the apparent agonist affinity of ligand-gated ion channels at clinically relevant concentrations, the effects of nonhalogenated alkane anesthetics on ligand-gated ion channels have not been studied. The current study assessed the abilities of two representative nonhalogenated alkane anesthetics (cyclopropane and butane) to potentiate agonist actions or increase the apparent agonist affinity of two representative ligand-gated ion channels: the nicotinic acetylcholine receptor and y-aminobutyric acid type A (GABA(A)) receptor. METHODS: Nicotinic acetylcholine receptors were obtained from the electroplax organ of Torpedo nobiliana, and human GABA(A) receptors (alpha1beta2gamma2L) were expressed in human embryonic kidney 293 cells. The Torpedo nicotinic acetylcholine receptors apparent agonist affinity in the presence and absence of anesthetic was assessed by measuring the apparent rates of desensitization induced by a range of acetylcholine concentrations. The GABA(A) receptor's apparent agonist affinity in the presence and absence of anesthetic was assessed by measuring the peak currents induced by a range of GABA concentrations. RESULTS: Neither cyclopropane nor butane potentiated agonist actions or increased the apparent agonist affinity (reduced the apparent agonist dissociation constant) of the Torpedo nicotinic acetylcholine receptor or GABA(A) receptor. At clinically relevant concentrations, cyclopropane and butane reduced the apparent rate of Torpedo nicotinic acetylcholine receptor desensitization induced by low concentrations of agonist. CONCLUSIONS: Our results suggest that the in vivo central nervous system depressant effects of nonhalogenated alkane anesthetics do not result from their abilities to potentiate agonist actions on ligand-gated ion channels. Other targets or mechanisms more likely account for the anesthetic activities of nonhalogenated alkane anesthetics.     

Inhibitory Effects of Isoflurane and Nonimmobilizing Halogenated Compounds on Neuronal Nicotinic Acetylcholine Receptors

Background: Neuronal nicotinic acetylcholine receptors (nAchRs) are inhibited by low concentrations of volatile anesthetics. However, it is not clear whether this phenomenon contributes to the anesthetic effects of volatile anesthetics. Effects of a volatile anesthetic (isoflurane) and structurally related nonimmobilizers (F6: 1,2-dichlorohexafluorocyclobutane, F8: 2,3-dichlorooctafluorobutane) on the current mediated through neuronal nAchRs were studied.

Method: This study investigated neuronal nAchRs in PC12 cells and acutely dissociated rat medial habenula (MHb) neurons. Whole cell currents elicited by 30 [mu]m nicotine were recorded in the absence and presence of the halogenated agents. The minimum alveolar concentrations (MACs) for F6 and F8 were predicted from Meyer-Overton correlation.

Results: All halogenated compounds inhibited the nicotine-induced current in a concentration-dependent manner in PC12 cells. In MHb neurons, while isoflurane and F6 significantly inhibited the nicotine-induced peak current, F8 failed to inhibit it. The peak currents in the presence of isoflurane at 1.7 MAC, of F6 at 2.4 MAC, and of F8 at 2.2 MAC were 12, 31, and 97% of control, respectively.     

Nonhalogenated Alkane Anesthetics Fail to Potentiate Agonist Actions on Two Ligand-gated Ion Channels

Background: Although ether, alcohol, and halogenated alkane anesthetics potentiate agonist actions or increase the apparent agonist affinity of ligand-gated ion channels at clinically relevant concentrations, the effects of nonhalogenated alkane anesthetics on ligand-gated ion channels have not been studied. The current study assessed the abilities of two representative nonhalogenated alkane anesthetics (cyclopropane and butane) to potentiate agonist actions or increase the apparent agonist affinity of two representative ligand-gated ion channels: the nicotinic acetylcholine receptor and [gamma]-aminobutyric acid type A (GABAA) receptor.

Methods: Nicotinic acetylcholine receptors were obtained from the electroplax organ of Torpedo nobiliana, and human GABAA receptors ([alpha]1[beta]2[gamma]2L) were expressed in human embryonic kidney 293 cells. The Torpedo nicotinic acetylcholine receptors apparent agonist affinity in the presence and absence of anesthetic was assessed by measuring the apparent rates of desensitization induced by a range of acetylcholine concentrations. The GABAA receptor's apparent agonist affinity in the presence and absence of anesthetic was assessed by measuring the peak currents induced by a range of GABA concentrations.

Results: Neither cyclopropane nor butane potentiated agonist actions or increased the apparent agonist affinity (reduced the apparent agonist dissociation constant) of the Torpedo nicotinic acetylcholine receptor or GABAA receptor. At clinically relevant concentrations, cyclopropane and butane reduced the apparent rate of Torpedo nicotinic acetylcholine receptor desensitization induced by low concentrations of agonist.     

Modulation of GABA(A) receptor function by nonhalogenated alkane anesthetics: the effects on agonist enhancement,direct activation,and inhibition

At clinically relevant concentrations, ethers, alcohols, and halogenated alkanes enhance agonist action on the gamma-aminobutyric acid(A) (GABA(A)) receptor, whereas nonhalogenated alkanes do not. Many anesthetics also directly activate and/or inhibit GABA(A) receptors, actions that may produce important behavioral effects; although, the effects of nonhalogenated alkane anesthetics on GABA(A) receptor direct activation and inhibition have not been studied. In this study, we assessed the abilities of two representative nonhalogenated alkanes, cyclopropane and butane, to enhance agonist action, directly activate, and inhibit currents mediated by expressed alpha(1)beta(2)gamma(2L) GABA(A) receptors using electrophysiological techniques. Our studies reveal that cyclopro- pane and butane enhance agonist action on the GABA(A) receptor at concentrations that exceed those required to produce anesthesia. Neither nonhalogenated alkane directly activated nor inhibited GABA(A) receptors, even at concentrations that approach their aqueous saturated solubilities. These results strongly suggest that the behavioral actions of nonhalogenated alkane anesthetics do not result from their abilities to enhance agonist actions, directly activate, or inhibit alpha(1)beta(2)gamma(2L) GABA(A) receptors and are consistent with the hypothesis that electrostatic interactions between anesthetics and their protein binding sites modulate GABA(A) receptor potency. IMPLICATIONS: When normalized to either their in vivo anesthetic potencies or hydrophobicities, cyclopropane and butane are 1-1.5 orders of magnitude less potent enhancers of agonist action on alpha(1beta2gamma2L) GABA(A) receptors than isoflurane. Additionally, cyclopropane and butane fail to directly activate or inhibit receptors, even at near aqueous saturating concentrations. Thus, it is unlikely that either enhancement or inhibition of the most common GABA(A) receptor subtype in the brain accounts for the behavioral activities of cyclopropane and butane.     

Nonanesthetic Volatile Drugs Obey the Meyer-Overton Correlation in Two Molecular Protein Site Models

Background: Nonanesthetic volatile compounds fail to inhibit movement in response to noxious stimulation at concentrations predicted to induce anesthesia from their oil-water partitioning. Thus they represent tools to determine whether molecular models behave like the targets that mediate in vivo anesthetic actions. The effects of volatile anesthetics and non-anesthetics were examined in two experimental models in which anesthetics interact directly with proteins: the pore of the nicotinic acetylcholine receptor and human serum albumin.

Methods: Wild-type mouse muscle nicotinic receptors and receptors containing pore mutations ([Greek small leter alpha] S252I + [Greek small letter beta] T263I) were studied electrophysiologically in membrane patches from Xenopus oocytes. Patch currents evoked by brief pulses of acetylcholine were measured in the presence of enflurane and two nonanesthetics, 1,2-dichlorohexafluorocyclobutane and 2,3-dichlorooctafluorobutane. Nonanesthetic interactions with human serum album were assessed by quenching of intrinsic protein fluorescence.

Results: Both anesthetic and nonanesthetic volatile compounds inhibited wild-type and [Greek small letter alpha] S252I + [Greek small letter beta] T263I mutant nicotinic channels but displayed different selectivity for open versus resting receptor states. Median inhibitory concentrations (IC50 s) in wild-type nicotinic receptors were 870 +/- 20 [micro sign]M for enflurane, 37 +/- 3 [micro sign]M for 1,2-dichlorohexafluorocylcobutane, and 11.3 +/- 5.6 [micro sign]M for 2,3-dichlorooctafluorobutane. For all three drugs, ratios of wild-type IC50 s to mutant IC50mut ranged from 7-10, and ratios of wild-type IC50 s to predicted anesthetic median effective concentrations (EC50 s) ranged from 1.8-2.3. 1,2-Dichlorohexafluorocyclobutane quenched human serum albumin with an apparent dissociation constant (Kd) of 160 +/- 11 [micro sign]M. The ratios of dissociation constants to predicted EC50 s for the nonanesthetics were within a factor of two of the dissociation constant:EC (50) ratios calculated for halothane and chloroform from previous published results.     

Nonhalogenated alkanes cyclopropane and butane affect neurotransmitter-gated ion channel and G-protein-coupled receptors: differential actions on GABAA and glycine receptors

BACKGROUND: Anesthetic mechanisms of nonhalogenated alkanes cyclopropane and butane are not understood. This study was designed to look at which neurotransmitter receptors are possible targets for these anesthetics. METHODS: Effects of cyclopropane and butane on eight recombinant receptors expressed in Xenopus oocytes were examined electrophysiologically. To address molecular mechanisms of interaction with glycine and gamma-aminobutyric acid type A (GABA(A)) receptors, cyclopropane was further tested on alpha1(S267C) glycine receptor and alpha2(S270X)beta1 GABA(A) receptors that were mutated to amino acids with larger side chains. RESULTS: Cyclopropane (1, 2, and 5 minimum alveolar concentration [MAC]) potentiated glycine responses by 39, 62, and 161%, respectively, and butane (1 MAC) potentiated by 64% with an increase in apparent affinity for glycine, but yielded barely detectable potentiation of GABA(A) receptors. The efficacy of cyclopropane for glycine receptors was less than isoflurane and halothane. The potentiation by cyclopropane was eliminated for the alpha1(S267C) glycine receptor. Mutant GABA(A) receptors in which the corresponding amino acid was substituted with larger amino acids did not produce significant potentiation. Cyclopropane and butane inhibited nicotinic acetylcholine and N-methyl-D-aspartate receptors, potentiated G-protein-coupled inwardly rectifying potassium channels, and did not change 5-hydroxytryptamine(3A) or muscarinic(1) receptor function. Only cyclopropane markedly inhibited alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors. CONCLUSIONS: Glycine, nicotinic acetylcholine, and N-methyl-D-aspartate receptors are sensitive to nonhalogenated alkanes, and the authors propose that glycine and N-methyl-D-aspartate receptors are good candidates for anesthetic immobility. The authors also suggest that the distinct effects on glycine and GABA(A) receptors are not due to the small volumes of these anesthetics.     

Acetylcholine receptors and thresholds for convulsions from flurothyl and 1,2-dichlorohexafluorocyclobutane

There are acetylcholine receptors throughout the central nervous system, and they may mediate some forms and aspects of convulsive activity. Most high-affinity binding sites on nicotinic acetylcholine receptors for nicotine, cytisine, and epibatidine in the brain contain the beta2 subunit of the receptor. Transitional inhaled compounds (compounds less potent than predicted from their lipophilicity and the Meyer-Overton hypothesis) and nonimmobilizers (compounds that do not produce immobility despite a lipophilicity that suggests anesthetic qualities as predicted from the Meyer-Overton hypothesis) can produce convulsions. The nonimmobilizer flurothyl [di-(2,2,2,-trifluoroethyl)ether] blocks the action of gamma-aminobutyric acid on gamma-aminobutyric acid(A) receptors, whereas the nonimmobilizer 1,2-dichlorohexafluorocyclobutane (2N, also called F6) does not. 2N can block the action of acetylcholine on nicotinic acetylcholine receptors. We examined the relative capacities of these compounds to cause convulsions in mice having and lacking the beta2 subunit of the acetylcholine receptor. The partial pressure causing convulsions in half the mice (the 50% effective concentration [EC(50)]) was the same as in control mice. For the knockout mice, the EC(50) for flurothyl was 0.00170 +/- 0.00030 atm (mean +/- SD), and for 2N, it was 0.0345 +/- 0.0041 atm. For the control mice, the respective values were 0.00172 +/- 0.00057 atm and 0.0341 +/- 0.0048 atm. The ratio of the 2N to flurothyl EC(50) values was 20.8 +/- 3.5 for the knockout mice and 21.7 +/- 7.0 for the control mice. These results do not support the notion that acetylcholine receptors are important mediators of the capacity of 2N or flurothyl to cause convulsions. However, we also found that both nonimmobilizers inhibit rat alpha4beta2 neuronal nicotinic acetylcholine receptors at EC(50) partial pressures (0.00091 atm and 0.062 atm for flurothyl and 2N, respectively) that approximate those that produce convulsions (0.0015 atm and 0.04 atm). IMPLICATIONS: The results from the present study provide conflicting data concerning the notion that acetylcholine receptors mediate the capacity of nonimmobilizers to produce convulsions.     

Effects of gaseous anesthetics nitrous oxide and xenon on ligand-gated ion channels. Comparison with isoflurane and ethanol

BACKGROUND: Ligand-gated ion channels are considered to be potential general anesthetic targets. Although most general anesthetics potentiate the function of gamma-aminobutyric acid receptor type A (GABAA), the gaseous anesthetics nitrous oxide and xenon are reported to have little effect on GABAA receptors but inhibit N-methyl-d-aspartate (NMDA) receptors. To define the spectrum of effects of nitrous oxide and xenon on receptors thought to be important in anesthesia, the authors tested these anesthetics on a variety of recombinant brain receptors. METHODS: The glycine, GABAA, GABA receptor type C (GABAC), NMDA, alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA), kainate, 5-hydroxytryptamine3 (5-HT3), and nicotinic acetylcholine (nACh) receptors were expressed in Xenopus oocytes and effects of nitrous oxide and xenon, and as equipotent concentrations of isoflurane and ethanol, were studied using the two-electrode voltage clamp. RESULTS: Nitrous oxide (0.58 atmosphere [atm]) and xenon (0.46 atm) exhibited similar effects on various receptors. Glycine and GABAA receptors were potentiated by gaseous anesthetics much less than by isoflurane, whereas nitrous oxide inhibited GABAC receptors. Glutamate receptors were inhibited by gaseous anesthetics more markedly than by isoflurane, but less than by ethanol. NMDA receptors were the most sensitive among glutamate receptors and were inhibited by nitrous oxide by 31%. 5-HT3 receptors were slightly inhibited by nitrous oxide. The nACh receptors were inhibited by gaseous and volatile anesthetics, but ethanol potentiated them. The sensitivity was different between alpha4beta2 and alpha4beta4 nACh receptors; alpha4beta2 receptors were inhibited by nitrous oxide by 39%, whereas alpha4beta4 receptors were inhibited by 7%. The inhibition of NMDA and nACh receptors by nitrous oxide was noncompetitive and was slightly different depending on membrane potentials for NMDA receptors, but not for nACh receptors. CONCLUSIONS: Nitrous oxide and xenon displayed a similar spectrum of receptor actions, but this spectrum is distinct from that of isoflurane or ethanol. These results suggest that NMDA receptors and nACh receptors composed of beta2 subunits are likely targets for nitrous oxide and xenon.     

Anesthetic and Nonanesthetic Halogenated Volatile Compounds Have Dissimilar Activities on Nicotinic Acetylcholine Receptor Desensitization Kinetics

Background: The Meyer-Overton rule predicts that an anesthetic's potency will correlate with its oil solubility. A group of halogenated volatile compounds that disobey this rule has been characterized. These compounds do not induce anesthesia in rats at partial pressures exceeding those predicted by the Meyer-Overton rule to be anesthetic. The observation that potentiation of GABAA receptor responses by anesthetic and nonanesthetic halogenated volatile compounds correlates with their abilities to induce general anesthesia suggests that this receptor is involved in the mechanism of general anesthesia. However, the GABAA receptor is only one member of a superfamily of structurally similar ligand-gated ion channels. This study compares the actions of both anesthetic and nonanesthetic halogenated volatile compounds on another member of this superfamily of receptors, the nicotinic acetylcholine receptor (nAcChoR).

Methods: The actions of both anesthetic and nonanesthetic compounds on desensitization kinetics were characterized from the time-dependent binding of the fluorescent acetylcholine analogue, Dns-C6 -Cho, to the nAcChoR.

Results: At concentrations predicted by the Meyer-Overton rule to be equianesthetic, the anesthetics isoflurane and enflurane were significantly more effective than the nonanesthetics 1,2-dichlorohexafluorocyclobutane and 2,3-dichlorooctafluorobutane in enhancing the fraction of receptors preexisting in the slow desensitized state and increasing the apparent rates of agonist-induced fast and slow desensitization.     

Effects of Gaseous Anesthetics Nitrous Oxide and Xenon on Ligand-gated Ion Channels: Comparison with Isoflurane and Ethanol

Background: Ligand-gated ion channels are considered to be potential general anesthetic targets. Although most general anesthetics potentiate the function of [gamma]-aminobutyric acid receptor type A (GABAA), the gaseous anesthetics nitrous oxide and xenon are reported to have little effect on GABAA receptors but inhibit N-methyl-d-aspartate (NMDA) receptors. To define the spectrum of effects of nitrous oxide and xenon on receptors thought to be important in anesthesia, the authors tested these anesthetics on a variety of recombinant brain receptors.

Methods: The glycine, GABAA, GABA receptor type C (GABAC), NMDA, [alpha]-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA), kainate, 5-hydroxytryptamine3 (5-HT3), and nicotinic acetylcholine (nACh) receptors were expressed in Xenopus oocytes and effects of nitrous oxide and xenon, and as equipotent concentrations of isoflurane and ethanol, were studied using the two-electrode voltage clamp.

Results: Nitrous oxide (0.58 atmosphere [atm]) and xenon (0.46 atm) exhibited similar effects on various receptors. Glycine and GABAA receptors were potentiated by gaseous anesthetics much less than by isoflurane, whereas nitrous oxide inhibited GABAC receptors. Glutamate receptors were inhibited by gaseous anesthetics more markedly than by isoflurane, but less than by ethanol. NMDA receptors were the most sensitive among glutamate receptors and were inhibited by nitrous oxide by 31%. 5-HT3 receptors were slightly inhibited by nitrous oxide. The nACh receptors were inhibited by gaseous and volatile anesthetics, but ethanol potentiated them. The sensitivity was different between [alpha]4[beta]2 and [alpha]4[beta]4 nACh receptors; [alpha]4[beta]2 receptors were inhibited by nitrous oxide by 39%, whereas [alpha]4[beta]4 receptors were inhibited by 7%. The inhibition of NMDA and nACh receptors by nitrous oxide was noncompetitive and was slightly different depending on membrane potentials for NMDA receptors, but not for nACh receptors.     

Molecular properties important for inhaled anesthetic action on human 5-HT3A receptors

Although inhaled anesthetics have diverse effects on 5-hydroxytryptamine type 3 (5-HT3A) receptors, the mechanism accounting for this diversity is not understood. Studies have shown that modulation of 5-HT3A receptor currents by n-alcohols depends on molecular volume, suggesting that steric interactions between n-alcohols and their binding sites define their action on this receptor. Electrostatic interactions also play an important role in anesthetic action on other ligand-gated receptors. We aimed to determine the contribution of molecular volume and electrostatics in defining volatile anesthetic actions on 5-HT3A receptors. Human 5-HT3A receptors were expressed in, and recorded from, Xenopus oocytes using the two-electrode voltage-clamp technique. The effects of a range of volatile anesthetics, n-alcohols, and nonhalogenated alkanes on submaximal serotonin-evoked peak currents, and full serotonin concentration-response curves were defined. Volatile anesthetics and n-alcohols, but not alkanes, smaller than 0.120 nm3 enhanced submaximal serotonin-evoked peak currents whereas all larger agents reduced currents. Most compounds tested inhibited maximal serotonin-evoked peak currents to varying degrees. However, only agents smaller than 0.120 nm3 shifted the 5-HT3A receptor's serotonin concentration-response curve to the left, whereas larger anesthetics shifted them to the right. Modulation of human 5-HT3A-mediated currents by volatile anesthetics exhibits a dependence on molecular volume consistent with the n-alcohols, suggesting that both classes of agents may enhance 5-HT3A receptor function via the same mechanism. Furthermore, the enhancing but not inhibiting effects of anesthetic compounds on 5-HT3A receptor currents are modulated by electrostatic interactions.     

Thermogenesis Inhibition in Brown Adipocytes Is a Specific Property of Volatile Anesthetics

Background: This investigation examined the possibility that the inhibitory effect of halothane on nonshivering thermogenesis (heat production) in brown adipocytes is not a universal effect of all anesthetic agents but related to the type of anesthetic.

Methods: Brown adipocytes from hamster were isolated with a collagenase digestion method and incubated with anesthetic agents. The rate of oxygen consumption was measured with an oxygen electrode. The effect of clinically relevant (and higher) doses of anesthetics of different classes on basal and norepinephrine-induced thermogenesis (oxygen consumption) was tested.

Results: Two distinct groups of anesthetics could be distinguished: thermogenesis inhibitors and noninhibitors. Thermogenesis inhibitors include volatile anesthetics such as halothane (IC50, 1.1 mm), ether (IC50, 20 mm), and chloroform (IC50, 2.2 mm) (nominal concentrations), but also tribromoethanol (IC50, 0.6 mm), all inducing inhibition of norepinephrine-induced thermogenesis without affecting the EC50 for norepinephrine. Thermogenesis noninhibitors include the nonvolatile anesthetics pentobarbital, propofol, ketamine, and urethane, the inhalation anesthetic nitrous oxide, and, notably, also the volatile nonanesthetics (nonimmobilizers) 1,2-dichlorohexafluorocyclobutane and 2,3-dichlorooctafluorobutane; none of these compounds had any effect on norepinephrine-induced thermogenesis at any concentration tested.     

Different effects of volatile anesthetics and polyhalogenated alkanes on depolarization-evoked glutamate release in rat cortical brain slices.

Anesthetics cause a reduction in excitatory neurotransmission that may be important in the mechanisms of in vivo anesthetic action. Because glutamate is the major excitatory neurotransmitter in mammalian brain, evaluation of anesthetic effects on induced glutamate release is relevant for studying this potential mechanism of anesthetic action. In the present study, we compared the effects of anesthetics and nonanesthetics (halogenated alkanes that disobey the Meyer-Overton hypothesis) on depolarization-evoked glutamate release. Glutamate released from rat cortical brain slices after chemically induced depolarization (50 mM KCl) was measured continuously using an enzymatic fluorescence assay. The effects of the volatile anesthetics isoflurane and enflurane were compared with the effects of the transitional compound 1,1,2-trichlorotrifluoroethane, the nonanesthetic compound 1,2-dichlorohexafluorocyclobutane, and other polyhalogenated alkanes. Tested concentrations included effective anesthetic concentrations for the anesthetics and transitional compounds, and concentrations predicted to be anesthetic based on lipid solubility for the nonanesthetics. Isoflurane dose-dependently reduced depolarization-evoked glutamate release in cortical brain slices. Isoflurane and enflurane at concentrations equivalent to 1 minimum alveolar anesthetic concentration (MAC) reduced the KCl-evoked release to 20% and 17% of control, respectively. The transitional compound 1,1,2-trichlorotrifluoroethane at 210 microM (approximately 1.2 MAC) reduced glutamate release to 47%, and the nonanesthetic 1,2-dichlorohexafluorocyclobutane increased glutamate release at 70 microM (approximately 3 MAC). These findings support the hypothesis that the modulation of excitatory neurotransmission might be responsible, in part, for in vivo anesthetic action. IMPLICATIONS: The volatile anesthetics isoflurane and enflurane reduce depolarization-evoked glutamate release in rat brain slices. The transitional compound 1,1,2-trichlorotrifluoroethane reduces glutamate release to a much lesser extent, and the nonanesthetic 1,2-dichlorohexafluorocyclobutane does not reduce glutamate release. These findings support the hypothesis that the modulation of excitatory neurotransmission might be responsible, in part, for in vivo anesthetic action.     

Subunit-dependent inhibition of human neuronal nicotinic acetylcholine receptors and other ligand-gated ion channels by dissociative anesthetics ketamine and dizocilpine

Background The neuronal mechanisms responsible for dissociative anesthesia remain controversial. N-methyl-D-aspartate (NMDA) receptors are inhibited by ketamine and related drugs at concentrations lower than those required for anesthetic effects. Thus, the authors studied whether ligand-gated ion channels other than NMDA receptors might display a sensitivity to ketamine and dizocilpine that is consistent with concentrations required for anesthesia. METHODS: Heteromeric human neuronal nicotinic acetylcholine receptors (hnAChR channels alpha2beta2, alpha2beta4, alpha3beta2, alpha3beta4, alpha4beta2 and alpha4beta4), 5-hydroxytryptamine3 (5-HT3), alpha1beta2gamma2S gamma-aminobutyric acid type A (GABAA) and alpha1 glycine receptors were expressed in Xenopus oocytes, and effects of ketamine and dizocilpine were studied using the two-electrode voltage-clamp technique. RESULTS: Both ketamine and dizocilpine inhibited hnAChRs in a noncompetitive and voltage-dependent manner. Receptors containing beta1 subunits were more sensitive to ketamine and dizocilpine than those containing beta2 subunits. The inhibitor concentration for half-maximal response (IC50) values for ketamine of hnAChRs composed of beta4 subunits were 9.5-29 microM, whereas those of beta2 subunits were 50-92 microM. Conversely, 5-HT3 receptors were inhibited only by concentrations of ketamine and dizocilpine higher than the anesthetic concentrations. This inhibition was mixed (competitive/noncompetitive). GABAA and glycine receptors were very resistant to dissociative anesthetics. CONCLUSIONS: Human nAChRs are inhibited by ketamine and dizocilpine at concentrations possibly achieved in vivo during anesthesia in a subunit-dependent manner, with beta subunits being more critical than alpha subunits. Conversely, 5-HT3, GABAA, and glycine receptors were relatively insensitive to dissociative anesthetics.     

Depressant and convulsant barbiturates both inhibit neuronal nicotinic acetylcholine receptors.

Neuronal nicotinic acetylcholine receptors (neuronal nAchRs) are sensitive to many anesthetics, including barbiturates, which suggests that these receptors are potential sites for anesthetic action. Subtle changes in molecular structures of the anesthetic barbiturates can produce compounds with potent convulsant activity. Whereas R(-) isomer of 1-methyl-5-phenyl-5-propyl barbituric acid (MPPB) exerts anesthetic action, S(+)MPPB exhibits pure excitatory effects, including convulsion. 5-(2-cyclohexilidene-ethyl)-5-ethyl barbituric acid is another example of a convulsant barbiturate. We compared the effects of depressant and convulsant barbiturates on the neuronal nAchR-mediated current to determine whether inhibition of neuronal nAchRs contributes to the anesthetic action of barbiturates. Whole cell nicotine-induced currents were recorded in PC12 derived from rat pheochromocytoma, using the conventional whole cell patch clamp technique in the presence and absence of barbiturates. Both depressant and convulsant barbiturates inhibited the nicotine-induced inward current reversibly and in a dose-dependent manner when co-applied with nicotine. All barbiturates accelerated the current decay. There was no significant difference between the concentrations for 50% inhibition for MPPB isomers. There was no correlation between inhibition of ganglionic nAchRs and anesthetic effects of the barbiturates. These results strongly oppose the idea that inhibition of neuronal nAchRs contributes to the anesthetic action of barbiturates. IMPLICATIONS: We found that both convulsant and depressant barbiturates inhibit the current mediated through ganglionic nicotinic acetylcholine receptors in PC12 cells. This finding suggests that the inhibition of neuronal nicotinic acetylcholine receptors does not contribute to the anesthetic action of barbiturates.     

Isoflurane modulation of neuronal nicotinic acetylcholine receptors expressed in human embryonic kidney cells

BACKGROUND: It is well established that neuronal nicotinic acetylcholine receptors (nAChRs) are sensitive to inhalational anesthetics. The authors previously reported that halothane potently blocked alpha4beta2-type nAChRs of rat cortical neurons. However, the effect of isoflurane, which is widely used clinically, on nAChRs largely remains to be seen. The authors studied the effects of isoflurane as compared with sevoflurane and halothane on the human alpha4beta2 nAChRs expressed in human embryonic kidney cells. METHODS: The whole-cell and single-channel patch clamp techniques were used to record currents induced by acetylcholine. RESULTS: Isoflurane, sevoflurane, and halothane suppressed the acetylcholine-induced currents in a concentration-dependent manner with 50% inhibitory concentrations of 67.1, 183.3, and 39.8 microM, respectively, which correspond to 0.5 minimum alveolar concentration or less. When anesthetics were coapplied with acetylcholine, isoflurane and sevoflurane decreased the apparent affinity of receptor for acetylcholine, but halothane, in addition, decreased the maximum acetylcholine current. When isoflurane was preapplied and coapplied, its inhibitory action was independent of acetylcholine concentration. Isoflurane blocked the nAChR in both resting and activated states. Single-channel analyses revealed that isoflurane at 84 microM decreased the mean open time and burst duration without inducing "flickering" during channel openings. Isoflurane increased the mean closed time. As a result, the open probability of single channels was greatly reduced by isoflurane. CONCLUSIONS: Isoflurane, sevoflurane, and halothane potently blocked the alpha4beta2 nAChR. Isoflurane suppression of whole-cell acetylcholine currents was a result of decreases in the open time, burst duration, and open probability and an increase in the closed time of single channels. The high sensitivity of neuronal nAChRs to inhalational anesthetics is expected to play an important role in several stages of anesthesia.