Anesth Analg 2003;96:746-749
© 2003 International Anesthesia Research Society
ANESTHETIC PHARMACOLOGY
Gamma-Aminobutyric Acid Neuropharmacological Investigations on Narcosis Produced by Nitrogen, Argon, or Nitrous Oxide
Jacques H. Abraini*,
Badreddine Kriem
,
Norbert Balon
,
Jean-Claude Rostain, and
Jean-Jacques Risso,
*UMR CNRS 6551 Mort Neuronale, Neuroprotection, Neurotransmission, Université de Caen, Centre Cyceron;
EMI INSERM 0014, Université Henri Poincaré Nancy 1;
UPRES EA3280, Université de la Méditerranée, Marseille; and
Institut de Médecine Navale du Service de Santé des Armées (IMNSSA), Toulon, France
Address correspondence and reprint requests to Dr. Jacques H. Abraini, Université de Caen, UMR CNRS 6551, Centre Cyceron, BP 5229, Boulevard Henri Becquerel, 14074 Caen Cedex, France. Address e-mail to abraini{at}neuro.unicaen.fr
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Abstract
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Inhaled anesthetics, including the gaseous anesthetics nitrous oxide and xenon, are thought to act by interacting directly with ion-channel receptors. In contrast, little is known about the mechanism of action of inert gases that show only narcotic potency at high pressures, such as nitrogen or argon. In the present study, we investigated the effects of selective 
-aminobutyric acid (GABA) receptor antagonists on narcosis produced by nitrogen, argon, and nitrous oxide. Pretreatment with the competitive GABAA receptor antagonist gabazine (0.2 nmol) but not the GABAB receptor antagonist 2-hydroxysaclofen (10 nmol) increased the nitrogen and argon threshold pressure for loss-of-righting-reflex (P < 0.005) but had no effect on nitrous oxide narcosis. Pretreatment with the GABAA benzodiazepine receptor antagonist flumazenil (5 nmol) also increased the narcosis threshold pressure of argon (P < 0.025). Given that neither 2-hydroxysaclofen, gabazine, nor flumazenil at the doses used induced hyperexcitability, our results support a selective antagonism by gabazine and flumazenil of the narcotic action of nitrogen and argon. Some mechanisms of nitrogen and argon narcotic action might be similar to those of clinical inhaled anesthetics.
IMPLICATIONS: We studied the effects in the rat of -aminobutyric acid (GABA) receptor antagonists on narcosis induced by nitrogen and argon that act only at high pressures. Our results show that the GABA A receptor may play a significant role, suggesting that some mechanisms might be similar to those of clinical inhaled anesthetics.
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Introduction
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Inhaled anesthetics (1–3), including the gaseous anesthetics nitrous oxide and xenon (4,5), interact directly at one or more superfamilies of ion-channel receptors such as the -aminobutyric acid type A (GABAA) receptor or the N-methyl-D-aspartate (NMDA) glutamate receptors. In contrast, only a few studies have investigated the mechanism of action of inert gases that only show narcotic potency at high pressures. As a consequence, because of the Meyer-Overton rule of a high correlation between hydrophobicity and narcotic potency, the traditional view is that inert gases that show only narcotic potency at high pressures dissolve in the cellular membrane, occupying or expanding its volume (6), and thereby indirectly disrupting the functioning of synaptic transmission.
To investigate the contribution of GABA transmission as a possible mechanism by which inert gases that only show narcotic potency at high pressures may act, we compared the effects of selective GABA receptor antagonists gabazine, flumazenil, and 2-hydroxysaclofen on narcosis induced by nitrogen and argon at increased pressure and that produced by nitrous oxide, which acts mainly through the NMDA glutamate receptor (4) in freely moving rats. Because narcosis by nitrogen or argon only occur at increased pressures, we further examined the effect of these GABA receptor antagonists at high helium pressures to discriminate whether these drugs might have favored the well known high helium pressure-induced hyperexcitability and thereby produced indirect antagonism of narcosis. Our data suggest that nitrogen and argon, but not nitrous oxide, may act directly by potentiating GABA neurotransmission at the GABAA receptor.
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Materials and Methods
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All animal use procedures were in accordance with The Declaration of Helsinki and the European Communities Council Directive No. 86/609/EEC. Male adult Sprague-Dawley rats (Iffa Credo, France) weighing 250–300 g were used. They were housed socially by groups of 6 at 21°C ± 0.5°C in Perspex home cages with light maintained on a 12-h light:dark cycle (light on from 7 AM to 7 PM) and free access to food and water for at least 3 days before surgery.
Rats were anesthetized with intraperitoneal pentobarbital 30 mg/kg and intraperitoneal ketamine 100 mg/kg and mounted on a stereotaxic apparatus with the incisor bar set 3.9 mm below the horizontal zero. They were implanted with a stainless steel guide cannula (23-gauge) for intracerebroventricular (icv) drug infusion in the right lateral ventricle (A: 0.92, L: 1.4, V: 3.2 from Bregma (7)). The guide cannula was anchored to the skull with two stainless steel screws and dental cement, and a stainless steel wire stylet was inserted into the guide cannula to prevent occlusion. After surgery, the rats were housed individually in Perspex home cages with free access to food and water and allowed to recover for at least 5 days before being submitted to any experiment.
Drugs were purchased from Research Biochemicals Inc (RBI, Natick, MA) and delivered icv 10 min before compression (see below) in 5 µL of phosphate-buffered saline solution at an injection rate of 2.5 µL/min using an injection needle inserted through the cannula and a microsyringe and perfusion apparatus (PHD 2000, Harvard Apparatus, Holliston, MA). Drugs were injected icv to avoid systemic side effects. The following drugs selective for the GABAA or the GABAB receptor were used: the GABAA receptor antagonist gabazine that acts at the neurotransmitter recognition site, the GABAA receptor antagonist flumazenil that acts at the GABAA benzodiazepine site, and the GABAB receptor antagonist 2-hydroxysaclofen.
Because GABA receptor antagonists may cause hyperexcitability that could have affected the present investigations on narcosis, the effects of gabazine, 2-hydroxysaclofen, and flumazenil on basal locomotor activity were investigated during preliminary experiments. The rats were placed inside the pressure chamber (see below) maintained at normal pressure. The chamber was equipped with 2 individual Perspex activity cages, 0.25 m in diameter, in which piezoelectrical sensors were fixed under the floor to allow automatic recording of the rat’s locomotor activity (8). Recordings were continued for 1 h after the drug infusion.
The experiments were conducted in a 50-L hyperbaric chamber fitted with 3 viewing portholes (maximum pressure: 20 MPa 
200 atm). Oxygen of medical grade (Air Liquide, Paris, France) was controlled and maintained at a partial pressure of 0.030 MPa inside the pressure chamber, whereas carbon dioxide was kept at a value less than 300 ppm by continuously circulating chamber gases through a soda lime canister. A powerful fan ensured oxygen mixing with the added gases to produce narcosis or pressure-induced hyperexcitability.
Narcosis was induced using either nitrogen, argon, or nitrous oxide of medical grade (Air Liquide) at a dose just sufficient to induce total loss-of-righting-reflex, a reliable criterion of narcosis. Nitrogen and argon were admitted in the pressure chamber at a compression rate of 0.1 MPa/min, whereas nitrous oxide was admitted at a compression rate of 0.016 MPa/min. The chamber was equipped with a Perspex cylinder of 0.3 m in diameter in which 4 freely moving rats could be rotated individually at 1 m/min. To avoid interactions between temperature and anesthesia, the temperature inside the chamber was adjusted to maintain rectal temperature at 37°C ± 1°C in one additional restrained rat.
Because narcosis produced by nitrogen or argon only occur at increased pressures, the effect of gabazine, 2-hydroxysaclofen, and flumazenil were investigated at high helium pressures. Hyperbaric helium produces hyperexcitability that may affect both the sensory and motor aspects of a reflex. The rats were exposed to helium up to 4 MPa (the approximative pressure required to induce loss-of-righting-reflex with nitrogen) at a rate of 0.1 MPa/min and recorded as described above (8) only for locomotor activity; as a consequence, the sensory component was not studied. The temperature inside the pressure chamber was progressively increased from ambient temperature to 33°C to prevent hypothermia because of the significant specific heat of helium compared with air and to maintain the comfort of the rats.
Data are expressed as mean ± SEM. The effects of gabazine, flumazenil, and 2-hydroxysaclofen on nitrogen, argon, and nitrous oxide narcosis were expressed as percentage changes and analyzed using the nonparametric Mann-Whitney U-test. The level of significance was set at P < 0.05 (9).
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Results
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Because GABA receptor antagonists may lead to hyperexcitability that could have affected the present investigations on narcosis, the effects of gabazine, 2-hydroxysaclofen, and flumazenil on basal locomotor activity were investigated during preliminary experiments. As shown in Table 1, neither flumazenil 0.1, 1, or 5 nmol nor 2-hydroxysaclofen 0.1, 1, or 10 nmol produced arousal or hyperexcitability; in contrast, administration of gabazine 0.4 and 0.5 nmol, but not 0.1 and 0.2 nmol, resulted in an increase in locomotor activity and the additional development of clonic or clonic-tonic seizures. Given these data, the following doses were chosen for subsequent experiments on gas narcosis: 0.2 nmol of gabazine, 5 nmol of flumazenil, and 10 nmol of 2-hydroxysaclofen.
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Table 1. Effect of Intracerebroventricular Infusion of the GABAA Receptor Antagonist Gabazine, the GABAB Receptor Antagonist 2-Hydroxysaclofen, and the GABAA Benzodiazepine Receptor Antagonist Flumazenil on Basal Locomotor Activity
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Measurement of the anesthetic action yielded a threshold pressure for loss-of-righting-reflex of 4.16 ± 0.09 MPa for nitrogen, 2.1 ± 0.04 MPa for argon, and 0.15 ± 0.006 MPa for nitrous oxide (Table 2). Both these absolute and relative anesthetic potencies are in agreement with previous data in rodents (10,11) and the Meyer-Overton rule of a high correlation between hydrophobicity and anesthetic potency (10). Pretreatment with gabazine resulted in a highly significant increase of the nitrogen threshold pressure and argon threshold pressure for the onset of loss-of-righting-reflex (P < 0.005), which were increased, respectively, by 13% and 15% compared with the control rats injected with saline alone but had no significant effect on narcosis induced by nitrous oxide. Pretreatment with flumazenil resulted in a modest, but significant, increase of the threshold pressure of argon, which was increased by 7% (P < 0.025) compared with control rats injected with saline but had no significant effect on narcosis produced by nitrogen or nitrous oxide (14 < U < 18; not significant). Pretreatment with 2-hydroxysaclofen showed no significant effect on narcosis produced by either nitrogen, argon, or nitrous oxide.
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Table 2. Effect of Intracerebroventricular Administration of the GABAA Receptor Antagonist Gabazine (0.2 nmol), the GABAB Receptor Antagonist 2-Hydroxysaclofen (10 nmol), and of the GABAA Benzodiazepine Receptor Antagonist Flumazenil (5 nmol) on the Threshold Pressure of Nitrogen, Argon, or Nitrous Oxide for Loss-of-Righting-Reflex
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Because narcosis produced by nitrogen or argon only occur at increased pressures, the effects of gabazine, 2-hydroxysaclofen, and flumazenil were investigated at equivalent helium pressures to discriminate whether these drugs, at the doses that induced no arousal at normobaric pressure, might have produced hyperexcitability at increased pressures. Compared with control rats, helium at a pressure up to 4 MPa (the pressure approximatively required to induce loss-of-righting-reflex with nitrogen as a phase) produced a significant increase in locomotor activity (P < 0.02) that is part of the high-pressure neurological syndrome (12). Pretreatment with gabazine, 2-hydroxysaclofen, or flumazenil produced no additional hyperexcitability (Table 3).
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Table 3. Effect of Intracerebroventricular Administration of the GABAA Receptor Antagonist Gabazine (0.2 nmol), the Benzodiazepine Receptor Antagonist Flumazenil (5 nmol), and of the GABAB Receptor Antagonist 2-Hydroxysaclofen (10 nmol) on Helium Pressure-Induced Hyperlocomotor Activity
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Discussion
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Pretreatment with the GABAA receptor antagonist gabazine, but not the GABAB receptor antagonist 2-hydroxysaclofen, led to a significant increase of the nitrogen and argon threshold pressure for the onset of loss-of-righting-reflex but did not change the threshold pressure of nitrous oxide. In addition, pretreatment with flumazenil, an antagonist of the GABAA receptor at the benzodiazepine site, increased the threshold pressure of argon for loss-of-righting-reflex but had no effect on the threshold pressures of nitrogen or nitrous oxide. Alternatively, pretreatment with gabazine, 2-hydroxysaclofen, or flumazenil at the doses used to modulate narcosis produced no hyperexcitability at normal or hyperbaric pressures up to 4 MPa (the approximative pressure required to induce loss-of-righting-reflex with nitrogen). Our flumazenil data agree with a previous report (13). Neither 2-hydroxysaclofen nor gabazine have been investigated at high helium pressures. Our data support a pharmacologic, rather than a physiologic, antagonistic action of gabazine and flumazenil on narcosis induced by argon and nitrogen at increased pressures.
These findings may be consistent with either a direct or indirect mechanism of action on GABAA receptors. Indeed, it may still be plausible to interpret our data as identifying one of the synaptic functions that nitrogen and argon disrupt by dissolving in the cell membrane. However, the fact that neither gabazine nor flumazenil had a significant effect on narcosis induced by nitrous oxide, which acts via the NMDA receptor, further suggests that nitrogen and argon may interact directly with the GABAA receptor to produce loss-of-righting-reflex. In addition, our results also suggest that argon, but not nitrogen, may produce its narcotic action by interacting partly, but not exclusively, with the benzodiazepine site of the GABAA receptor; this suggests that argon may bind at multiple discrete sites on the GABAA receptor. The fact that the competitive GABAA receptor antagonist gabazine increased the narcotic threshold pressure of nitrogen and argon, whereas flumazenil, which acts at the benzodiazepine site of the GABAA receptor, only enhanced the narcotic threshold pressure of argon, may indicate that nitrogen and argon do not bind, at least partly, at the same discrete sites on the GABAA receptor. Although plausible, this hypothesis remains to be proven. Our results extend recent reports suggesting that nitrogen may induce its sedative subanesthetic action on locomotor activity by interacting with the GABAA, but not the GABAB, receptor (14,15) and further shows that the GABAA receptor is poorly affected by nitrous oxide, which mainly produces its narcotic effect through the NMDA glutamate receptor (4,5).
In conclusion, the findings of the present study are consistent with the general consensus that has emerged during the past few decades that inhaled anesthetics produce their action by acting directly at one or more superfamilies of ligand ion-channels. That the sensitivity of ion-channel receptors, including the GABAA receptor, to inhaled anesthetics may be modulated by specific mutations in the channel receptor domains (16–18), with the sensitivity of the receptor to anesthetics being increased as hydrophobicity increases, together with our findings, lead us to believe that some mechanisms of action of inert gases that only show narcotic potency at high pressures might be shared with those of volatile and gaseous clinical anesthetics.
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Acknowledgments
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Supported, in part, by grant no. 98/10033 and grant no. 98/0809 and a postdoctoral fellowship to NB from PACA-COMEX PRO and DGA/DSP/SRAA/SC/EXP no. 0160084/A000, respectively.
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References
|
|---|
- Franks NP, Lieb WR. Molecular and cellular mechanisms of general anesthesia. Nature 1994; 367: 607–14.[Medline]
- Peoples RW, Li C, Weight F. Lipid vs protein theories of alcohol action in the nervous system. Annu Rev Pharmacol Toxicol 1996; 36: 185–201.[ISI][Medline]
- Franks NP, Lieb WR. Which molecular targets are most relevant to general anaesthesia? Toxicol Lett 1998; 100–101: 1–8.
- Franks NP, Dickinson R, de Sous SL, et al. How does xenon produce anaesthesia? Nature 1998; 396: 324.[Medline]
- Jevtovic-Todorovic V, Todorovic SM, Mennerick S, et al. Nitrous oxide (laughing gas) is an NMDA antagonist, neuroprotectant and neurotoxin. Nat Med 1998; 4: 460–3.[ISI][Medline]
- Bennett PB. Inert gas narcosis. In: Bennett PB, Elliott DH, eds. The physiology and medicine of diving. London: Saunders, 1993: 171–93.
- Paxinos G, Watson C. The rat brain. In: Stereotaxic coordinates. New York: Academic Press, 1998.
- Tomei C, Abraini JH, Rostain J-C. A new device for behavioral analysis on rats exposed to high pressure. Physiol Behav 1991; 49: 393–6.[Medline]
- Siegel S, Castellan NJ. Nonparametric statistics for the behavioral sciences: statistical series. New York: McGraw-Hill International Editions, 1988.
- Miller KW, Wilson MW, Smith RA. Pressure resolves two sites of action of inert gases. Mol Pharmacol 1978; 14: 950–9.[Abstract/Free Full Text]
- Smith RA, Smith M, Eger EI II, et al. Nonlinear antagonism of anesthesia in mice by pressure. Anesth Analg 1979; 58: 19–22.[Abstract/Free Full Text]
- Bennett PB, Rostain J-C. The high pressure nervous syndrome. In: Bennett PB, Elliott DH, eds. The physiology and medicine of diving. London: Saunders, 1993: 194–207.
- Bichard AR, Little HJ. The benzodiazepine antagonist, Ro 15–1788, prevents the effects of flurazepam on the high pressure neurological syndrome. Neuropharmacology 1982; 21: 877–80.[ISI][Medline]
- David HN, Abraini JH. Physiologic antagonism of the sedative action of nitrogen pressure by the GABA B receptor antagonist saclofen in the rat. Eur J Underwater Hyperbaric Med 2001; 2: 27–31.
- David HN, Balon N, Rostain J-C, Abraini JH. Nitrogen at raised pressure interacts with the GABA A receptor to produce its narcotic effect in the rat. Anesthesiology 2001; 95: 921–7.[ISI][Medline]
- Forman SA, Miller KW, Yellen G. A discrete site for general anesthetics on a post-synaptic receptor. Mol Pharmacol 1995; 48: 574–81.[Abstract]
- Mihic SJ, Ye Q, Wick MJ, et al. Sites of alcohol and volatile anaesthetic action on GABA(A) and glycine receptors. Nature 1997; 25: 385–9.
- Belelli D, Pistis M, Peters JA, Lambert JJ. The interaction of general anaesthetics and neurosteroids with GABA(A) and glycine receptors. Neurochem Int 1999; 34: 447–52.[ISI][Medline]
Accepted for publication November 19, 2002.
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Abstract
Introduction
