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ANESTHETIC PHARMACOLOGY

A Neurosteroid Anesthetic, Alphaxalone, Inhibits Nicotinic Acetylcholine Receptors in Cultured Bovine Adrenal Chromaffin Cells

Munehiro Shiraishi, MD^*, Izumi Shibuya, PhD, Kouichiro Minami, MD PhD^*, Yasuhito Uezono, MD PhD, Takashi Okamoto, MD^*, Nobuyuki Yanagihara, PhD, Susumu Ueno, MD PhD, Yoichi Ueta, MD PhD, and Akio Shigematsu, MD PhD^*

Departments of *Anesthesiology, Physiology, and Pharmacology, University of Occupational and Environmental Health, School of Medicine, Kitakyushu; and Department of Second Pharmacology, Nagasaki University, School of Medicine, Nagasaki, Japan

Address correspondence and reprint requests to Kouichiro Minami, MD, PhD, Department of Anesthesiology, University of Occupational and Environmental Health, School of Medicine, 1-1 Iseigaoka, Yahatanishiku, Kitakyushu 807-8555, Japan. Address e-mail to kminami{at}med.uoeh-u.ac.jp

	Abstract

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Several lines of evidence suggest that nicotinic acetylcholine receptors (nAChRs) are a target of general anesthetics. Alphaxalone (5-pregnan-3-ol-11, 20-dion) is a neurosteroid, which was used clinically for anesthesia, but its effects on the function of nAChRs have not been well investigated. We examined the effects of alphaxalone on nAChRs in cultured bovine adrenal chromaffin cells. We studied the effects of alphaxalone on nicotine-induced increases in the cytosolic Ca²⁺ concentration ([Ca²⁺]_i) and on membrane currents using Ca²⁺-imaging and whole-cell patch-clamp techniques, respectively, in these cells. We also examined the effects of alphaxalone on -aminobutyric acid A receptors in the same cells and compared them with the effects on nAChRs. Alphaxalone (0.1–100 µM) inhibited nicotine-induced [Ca²⁺]_i increases in a concentration-dependent manner. Alphaxalone inhibited high K⁺-induced [Ca²⁺]_i increases, but the inhibition was observed only at 100 µM. In voltage-clamp experiments using negative holding potentials, alphaxalone (0.1–100 µM) itself induced inward currents, which were abolished by the -aminobutyric acid A receptor antagonist picrotoxin. Alphaxalone also inhibited nicotine-induced inward currents, and the inhibition was unaffected by picrotoxin. We conclude that alphaxalone, at anesthetic concentrations, inhibits nAChRs in adrenal chromaffin cells. Alphaxalone may affect the sympathetic and other nervous systems via inhibition of nAChRs.

IMPLICATIONS: Alphaxalone inhibits the function of nAChRs at clinically relevant concentrations in adrenal chromaffin cells. Thus, the present findings may provide some information for understanding the anesthetic mechanism of alphaxalone.

	Introduction

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Alphaxalone (5-pregnan-3-ol-11, 20-dion) is a neurosteroid anesthetic and was used for both the induction and maintenance of general anesthesia (1). Enhancement of -aminobutyric acid A (GABA_A) receptor function by alphaxalone may be a mechanism of its anesthetic action. The potent and stereoselective potential of GABA_A receptor-mediated responses by alphaxalone was first shown in extracellular recordings performed on rat brain slices (2). This was subsequently confirmed in voltage-clamp experiments using dissociated neurons (3,4) and adrenal medullary chromaffin cells (5).

Volatile and IV general anesthetics have inhibitory effects on nicotinic acetylcholine receptors (nAChRs) (6). Isoflurane inhibited the human nAChRs, 2ß4, 3ß4, and 4ß2 expressed in Xenopus oocytes (7). Halothane suppressed nAChR subtypes and 4ß2 currents (8). Although much attention has been paid to the effects of anesthetics on nAChRs, there has been little information about the effects of alphaxalone on the nAChR function.

Adrenal chromaffin cells are derived from the embryonic neural crest and are homologous with sympathetic postganglionic neurons. Cultured bovine adrenal chromaffin cells contain a variety of ion channels that are involved in catecholamine secretion; nAChR-associated cation channels and voltage-dependent Ca²⁺ channels (VDCC) are activated by membrane depolarization (9). Moreover, there are also GABA_A receptor-associated Cl^- channels in adrenal chromaffin cells (10), and activation of these channels causes catecholamine secretion because of membrane depolarization as a result of Cl^- efflux (11). The properties of VDCC in adrenal chromaffin cells have been well studied (12,13), and we have reported the effects of volatile and IV anesthetics on the nAChR-induced responses in adrenal medullary cells (14,15). It follows that adrenal chromaffin cells are a good tool for detailed analysis of actions of anesthetics on nAChRs, VDCC, and GABA_A receptors.

The purpose of the present study is to clarify the mechanism of action of alphaxalone. For this purpose, we studied the effects of alphaxalone on nAChRs using whole-cell patch-clamp and Ca²⁺ imaging techniques. In addition, we studied the effects of alphaxalone on responses mediated by VDCC and GABA_A receptors and compared them with the effects on nAChRs.

	Materials and Methods

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Eagle’s minimum essentials medium is from Nissui Pharmaceuticals (Tokyo, Japan). The calf serum and nicotine were from Nacalai Tesque, (Kyoto, Japan), and the collagenase is from Nitta Zerachine (Osaka, Japan). GABA and picrotoxin were from Tocris Cookstone Ltd (Bristol, UK). Alphaxalone, magnesium-adenosine triphosphate, and HEPES were from Sigma (St Louis, MO). Fura-2 acetoxymethyl (AM) was from Dojin Laboratory (Kumamoto, Japan).

Adrenal chromaffin cells were isolated by collagenase digestion of slices of the bovine adrenal medulla, as described elsewhere (14). The cells were plated at a density of 4 x 10⁶ cells/dish (35 mm in diameter) in Eagle’s minimum essential medium containing 10% calf serum and antibiotics (60 mg/mL of aminobenzylpenicillin, 100 mg/mL of streptomycin, 0.3 mg/mL of amphotericin B, and 3.0 mM of cytosine arabinoside). The cells were cultured in 5% CO₂/95% air in a culture chamber and used on the first to fifth day of culture.

The method used for electrophysiological experiments has been described elsewhere (16). Cells are plated on a glass cover slip (11 mm in diameter) and used after being maintained in culture for 2–4 days. Standard perfusion medium (HEPES-buffered solution [HBS]) contained NaCl 140 mM, KCl 5 mM, CaCl₂ 2 mM, MgCl₂ 1 mM, HEPES 10 mM, and glucose 10 mM (pH value of 7.4 adjusted with NaOH). The pipette solution used in the recording electrodes contained KCl 140 mM, MgCl₂ 1 mM, CaCl₂ 1 mM, EGTA 10 mM, HEPES 10 mM, and magnesium-adenosine triphosphate 2 mM (pH value of 7.2 adjusted with Tris base) unless otherwise noted. When current-voltage relation was measured, 140 mM of CsCl was used instead of 140 mM of KCl, and tetrodotoxin was added to the bath solution to suppress voltage-dependent K⁺ and Na⁺ currents, respectively. The electrodes were made with a pullar (P-97; Sutter Instrument Co, Novato, CA) from thick-walled borosilicate glass (GD-1.5; Narishige, Tokyo, Japan) and had a final resistance of 3 to 6 M when filled with the electrode solution. The volume of recording chamber was approximately 0.5 mL, and the flow rate of the perfusion medium was 1.5 mL/min. The solution level was kept constant by a low-pressure aspiration system. All electrophysiological recordings were performed at a room temperature of around 23°C. Membrane currents were recorded with a patch-clamp amplifier (AxoPatch 200A; Axon Instruments Inc, Union City, CA) and were digitized using AxoGraph software (version 4.6; Axon Instruments Inc) for subsequent off-line analysis. Data were analyzed also using the AxoGraph software. The sampling rate was 1–10 kHz. The drugs were added by changing the bath solution, which was introduced to the chamber with a peristaltic pump.

The method used for [Ca²⁺]_i measurements was described elsewhere in detail (17). Cultured cells plated on a cover glass were incubated in HBS with the addition of AM esters of fura-2 (fura-2 AM; 3 µM) at room temperature for 1 h. The cells were then washed with dye-free HBS and kept at room temperature until used.

The arrangements for perfusing cells are the same as those used for electrophysiological measurements described above. Fluorescence was measured from fura-2-loaded cells in the perfusion chamber, which had a glass coverslip bottom and was positioned on the stage of an inverted microscope (IX-70) equipped with a Ca²⁺-imaging system (Quanticell/900, JOEL, Japan). Once cells were selected in the optical field under microscope, fluorescence intensities with excitation at 340 nm and 380 nm were alternatively recorded at an interval of 3–5 s with an intensified charge-coupled device (CCD) camera. [Ca²⁺]_i was calculated from the ratio (R) of the fluorescence measured with excitation at 340 nm to that at 380 nm using the following equation:

equation

where Kd is the dissociation constant of the fura-2 (224 nM), Rmax and Rmin are the ratios for the unbound and bound forms of the fura-2/Ca²⁺ complex, and ß is the ratio between the maximum and the minimum fluorescence intensities of fura-2 at 380 nm excitation. Rmax and Rmin were estimated with the fluorescence intensities of fura-2 solution (3 µM) containing 5 mM of CaCl₂ and 5 mM of EGTA, respectively. Autofluorescence in chromaffin cells was negligible compared with the fluorescence in the fura-2 loaded cells. The experiments were performed at room temperature of 23°C.

The electrophysiological and [Ca²⁺]_i results are expressed as percentages of two control responses recorded before the test and after the recovery to minimize the variability and run-down of the responses in the cells. All values are presented as the mean ± SEM. The n values refer to the number of cells studied. Statistical analyses were performed using a one-way analysis of variance. Estimation of the half-maximal inhibitory concentration and Hill coefficient values from the concentration-response curve fit was performed using GraphPad Prism Software (San Diego, CA).

	Results

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Effects of alphaxalone on basal [Ca²⁺]_i and nicotine-induced [Ca²⁺]_i increases were studied. Nicotine (10 µM) induced a rapid [Ca²⁺]_i increase in single bovine adrenal chromaffin cells (Fig. 1). Bath application of alphaxalone (0.1–100 µM), which was added 90 s before nicotine stimulation, caused concentration-dependent and transient increases in [Ca²⁺]_i (Fig. 1A–C, Fig. 2A, and Fig. 3A). Alphaxalone inhibited the [Ca²⁺]_i increase induced by nicotine (10 µM) (Fig. 1A). The inhibitory effects were reversible within a few minutes and also concentration-dependent over alphaxalone concentrations ranging from 0.1 to 100 µM. The mean ± SEM of the inhibition were 33% ± 2% and 78% ± 2% of the control at 100 µM and 10 µM, respectively (n = 16 for each concentration of alphaxalone) (Fig. 1C). Because alphaxalone had an agonistic effect on GABA_A receptors, picrotoxin (10 µM) was used to prevent the opening of Cl^- channels. In this situation, although the [Ca²⁺]_i increasing effects of alphaxalone were abolished, the inhibition of nicotine-induced [Ca²⁺]_i increases by alphaxalone persisted (Fig. 1, B and C). The magnitude of alphaxalone-induced inhibition of the nicotine responses obtained in the presence of picrotoxin was not significantly different from that obtained in the absence of picrotoxin.

View larger version (40K): [in this window] [in a new window]   Figure 1. Effects of alphaxalone on the basal [Ca2+]i and nicotine-induced [Ca2+]i increases in bovine adrenal chromaffin cells. (A) A representative time course of changes in [Ca2+]i induced by nicotine (10 µM) with or without various concentrations of alphaxalone (0.1, 1, 10, and 100 µM). Nicotine was added for 20 s after pretreatment (90 s) with alphaxalone. Alphaxalone itself caused increases in [Ca2+]I and the inhibition of nicotine-induced [Ca2+]i increases, both in a concentration-dependent manner. Open and closed bars mean the period for nicotine and alphaxalone applications, respectively. (B) A representative time course of changes in [Ca2+]i induced by nicotine (10 µM) with or without various concentrations of alphaxalone (0.1, 1, 10, and 100 µM) obtained in the presence of picrotoxin (10 µM). Alphaxalone alone did not induce any change in the basal [Ca2+]I yet inhibited nicotine-induced [Ca2+]i increases. Open, closed, and hatched bars mean the period for nicotine, alphaxalone, and picrotoxin applications, respectively. (C) Summary results for percent inhibition of nicotine-induced [Ca2+]i increases by alphaxalone and for alphaxalone-induced [Ca2+]i increases in the presence and absence of picrotoxin. The ordinate for the effects of alphaxalone on basal cytosolic Ca2+ concentrations in the absence (hatched bar; 50% effective concentration = 37.4 ± 9.3 µM, Hill coefficient = 0.44) or presence (cross hatched bar) of picrotoxin is shown on the right (n = 16 for each concentration of alphaxalone). *P < 0.05, **P < 0.01, and ***P < 0.001 using one-way analysis of variance (Bonferroni’s multiple comparison test). #P < 0.01 compared between the two columns using analysis of variance (Bonferroni’s multiple comparison test).

View larger version (35K): [in this window] [in a new window]   Figure 2. Effects of alphaxalone on high K+-induced [Ca2+]i increases in bovine adrenal chromaffin cells. (A) A representative time course of changes in [Ca2+]i induced by high K+ (50 mM) with or without various concentrations of alphaxalone (0.1, 1, 10, and 100 µM) in the absence of picrotoxin. The extracellular K+ concentration was increased from 5 to 50 mM for 20 s after pretreatment (90 s) with alphaxalone. Alphaxalone induced concentration-dependent increases in the basal [Ca2+]i. (B) A representative time course of changes in [Ca2+]i induced by high K+ (50 mM) with or without various concentrations of alphaxalone (0.1, 1, 10, and 100 µM) obtained in the presence of picrotoxin (10 µM). Alphaxalone alone did not induce any change in the basal [Ca2+]i, although it inhibited high K+-induced [Ca2+]i increases. Open bars mean the period for high K+ applications, and closed bars indicate the period for alphaxalone applications. (C) Summary results for percent inhibition of high-K+-induced [Ca2+]i increases by alphaxalone in the presence or absence of picrotoxin (n = 25 for each concentration of alphaxalone). ***P < 0.001 and #P < 0.001 compared between the two columns using one-way analysis of variance (Bonferroni’s multiple comparison test).

View larger version (24K): [in this window] [in a new window]   Figure 3. Effects of alphaxalone on -aminobutyric acid (GABA)-induced [Ca2+]i increases in bovine adrenal chromaffin cells. (A) A representative time course of changes in [Ca2+]i induced by GABA (10 µM) with or without various concentrations of alphaxalone (0.1, 1, 10, and 100 µM). GABA was added for 20 s after pretreatment (90 s) with alphaxalone. Alphaxalone alone induced concentration-dependent increases in basal [Ca2+]i. The combined applications of alphaxalone with GABA elicited potentiation except for alphaxalone at 100 µM. Open bars mean the period for GABA applications, and closed bars indicate the period for alphaxalone applications. (B) Summary results for the potentiation of GABA-evoked [Ca2+]i increases by alphaxalone (n = 20 for each concentration of alphaxalone). ***P < 0.001 using one-way analysis of variance (Bonferroni’s multiple comparison test).

We also studied effects of alphaxalone on GABA-induced [Ca²⁺]_i increases. GABA (10 µM) induced rapid [Ca²⁺]_i increases in a concentration-dependent manner (30 ± 9 nM, 256 ± 32 nM, and 571 ± 26 nM more than the baseline level in response to 1, 10, and 100 nM of GABA, respectively; n = 9), presumably as a result of membrane depolarization caused by Cl^- channel opening (Fig. 3). Alphaxalone administered at 0.1–10 µM enhanced the GABA-induced [Ca²⁺]_i increases in a reversible and concentration-dependent manner; however, 100 µM of alphaxalone did not enhance the GABA-induced [Ca²⁺]_i increases (Fig. 3, A and B; n = 20 for each concentration of alphaxalone).

In the next series of experiments, bovine adrenal chromaffin cells were voltage-clamped at -60 mV by the whole-cell patch-clamp technique to record membrane currents. Bath application of nicotine (10 µM) induced inward currents that desensitized slowly. After sufficient time for recovery from the desensitization, bath application of alphaxalone, which was added 90 s before nicotinic stimulation, suppressed the nicotine-induced currents (Fig. 4, A and B). The inhibitory effects of alphaxalone on nicotinic currents were reversible. Alphaxalone itself induced inward currents at negative holding potentials in a concentration-dependent manner (Fig. 4, A and C). The currents induced by alphaxalone (100 µM) showed a linear current-voltage relationship and had a reversal potential of around 0 mV when a ramp command from -80 to +20 mV was applied (Fig. 4A (inset)). The properties of the alphaxalone-induced currents showed close similarity to the currents induced by GABA (10 µM). Moreover, the alphaxalone-induced currents were abolished by picrotoxin (10 µM) (Fig. 4B), indicating that the currents were Cl^- currents mediated by GABA_A receptors. Significant changes in membrane currents were observed with alphaxalone at 0.1 µM and higher. Alphaxalone inhibited nicotine-induced inward currents in a concentration-dependent manner (0.1–100 µM), and the inhibition was reversible and repeatable (Fig. 4C). In the absence of picrotoxin (10 µM), alphaxalone administered at 1 and 10 µM inhibited nicotine-induced inward currents to 75% ± 4% (n = 7) and 61% ± 4% (n = 7) of control, respectively (Fig. 4C). In the presence of picrotoxin, alphaxalone inhibited the currents in a similar manner (Fig. 4, B and C). The half-maximal inhibitory concentration of alphaxalone to inhibit the nicotine-induced currents was estimated to be 30.7 ± 1.1 µM and 32.4 ± 1.2 µM in the absence and presence of picrotoxin, respectively.

View larger version (36K): [in this window] [in a new window]   Figure 4. Effects of alphaxalone on nicotine-induced inward currents recorded by the whole-cell patch-clamp technique in bovine adrenal chromaffin cells. (A) A representative current trace showing the effects of alphaxalone (1–100 µM) on 10 µM of nicotine-induced currents. Nicotine was applied for 20 s with or without 100-s pretreatment with 100, 10, or 1 µM of alphaxalone. The membrane potential was voltage-clamped to -60 mV. (Inset) An example for the current-voltage relationship of currents induced by alphaxalone (100 µM) or by -aminobutyric acid (GABA) (10 µM) obtained by applying a ramp voltage command from -80 to +20 mV lasting for 200 ms. CsCl (140 mM) was used instead of 140 mM of KCl for the electrode solution and tetrodotoxin (1 µM) was added to the bath solution to suppress voltage-dependent K+ and Na+ currents, respectively. Note that both currents had a similar linear current-voltage relationship and a similar reversal potential around 0 mV. (B) A representative current trace showing the effects of alphaxalone (1–100 µM) on 10 µM of nicotine-induced currents in the presence of picrotoxin (10 µM). Note that alphaxalone-induced currents were abolished, whereas the inhibition of nicotinic currents persisted. (C) Summary results for the concentration-response relationship of the effects of alphaxalone on the basal membrane currents and on the nicotine-induced currents. Open bars mean the effect of alphaxalone on nicotine-induced currents in the absence of picrotoxin (half-maximal inhibitory concentration = 30.7 ± 1.1; Hill coefficient was 0.34), and closed bars mean the effect of alphaxalone on nicotine-induced currents in the presence of picrotoxin (half-maximal inhibitory concentration = 32.4 ± 1.2; Hill coefficient was 0.39). The ordinate for the effects of alphaxalone on basal membrane currents in the absence (hatched bar; 50% effective dose = 20.7 ± 3.3 µM; Hill coefficient = 1.2) or presence (cross hatched bar) of picrotoxin is shown on the right. Data represent the mean ± SEM (n = 4, 7, 7, and 4 for 0.1, 1.0, 10, and 100 µM of alphaxalone, respectively). **P < 0.01 and ***P < 0.001 compared with the control response using analysis of variance (Dunnett’s multiple comparison test). #P < 0.01 compared between the two columns using analysis of variance (Bonferroni’s multiple comparison test).

View larger version (17K): [in this window] [in a new window]   Figure 5. Effects of alphaxalone on -aminobutyric acid (GABA)-induced inward currents in bovine adrenal chromaffin cells. (A) A representative current trace showing the effect of alphaxalone (10 and 100 µM) on 10 µM of GABA-induced currents. GABA was applied for 20 s with or without 100-s pretreatment with different concentrations of alphaxalone. Membrane potential was voltage-clamped at -60 mV. (B) Summary results for the effects of alphaxalone (0.1–100 µM) on 10 µM of GABA-induced inward currents (n = 4 for each concentration of alphaxalone). ***P < 0.001 using one-way analysis of variance (Dunnett’s multiple comparison test).

	Discussion

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The question arises as to how alphaxalone inhibits nAChR-mediated responses. Previously, Kataoka et al. (20) reported that a GABA receptor antagonist, bicuculline, facilitates the release of catecholamines from chromaffin cells induced by nicotinic receptor stimulation. Moreover, the authors identified immuno-staining of the enzymes involved in GABA synthesis, glutamic acid decarboxylase, and GABA aminotransferase in adrenal chromaffin cells. These results suggest that endogenous GABA released from chromaffin cells and acting on GABA_A receptors on these cells significantly affects the secretory response to nicotinic stimulation. Alphaxalone has an agonistic effect on GABA_A receptors (2–5). In fact, our results indicated that alphaxalone itself stimulat-ed the GABA_A receptors and activated membrane currents in adrenal chromaffin cells. However, the present results also revealed that alphaxalone inhibited the nicotine-induced inward currents and [Ca²⁺]_i increases. Furthermore, the inhibition was observed both in the presence and absence of the GABA_A receptors antagonist picrotoxin. These results clearly indicate that the inhibitory effects of alphaxalone on nAChRs are independent of GABA_A receptors and suggest that the effect could be direct on nAChRs. A study using chimera and point-mutated rat nAChRs expressed in Xenopus oocytes identified that single amino acid residues 4-Val(254) and ß2-Val(253) near the middle of the second transmembrane segment alter the channel gating and determine volatile anesthetic, isoflurane, and pentobarbital sensitivity of nAChRs (21). In fact, several steroids inhibit nicotinic responses in HEK293 cells expressing 4ß2 subunits (22). However, adrenal chromaffin cells express 3, 5, and 7 together with ß4 subunits (23), and there is no evidence for the presence of 4 in these cells. It may be that alphaxalone possesses broad inhibitory actions on several different types of nAChRs. To provide further information concerning the molecular mechanism of alphaxalone-induced inhibition on nAChRs, the effects of alphaxalone on point-mutated nAChRs of different subclasses with special reference to amino acids in the second transmembrane segment region should be determined.

In the present study, alphaxalone inhibited high K⁺-induced [Ca²⁺]_i increases, but the inhibition was observed only at the largest concentration of alphaxalone tested, which is much larger than clinically relevant concentrations. These results indicate that alphaxalone can inhibit VDCC, but the inhibition requires much larger concentrations of alphaxalone than the inhibition of nAChRs or the activation and enhancement of GABA_A receptors. Previously, we reported that several general anesthetics, such as ketamine (24) and isoflurane (14), inhibit the nAChR-ion channels at much smaller concentrations than VDCC in adrenal medullary cells. It has been proposed that ligand-gated ion channels in postsynaptic membranes are most sensitive to general anesthetics (25–27). Taken together, our results suggest that alphaxalone affects the two ligand-gated ion channels, GABA_A receptor/Cl^- channels and nAChRs, much more potently than VDCC, and these properties are similar to those of other general anesthetics.

The result that the inhibition of high-K⁺-induced responses by alphaxalone persisted in the presence of picrotoxin suggests that the inhibition of voltage-gated Ca²⁺ channels by alphaxalone is not mediated mainly by GABA_A receptors and could be a direct effect on the channels. The significant reduction in the magnitudes of inhibition by picrotoxin could be because of activation of GABA_A receptor/Cl^- channel complex, which would keep the membrane potential near the Cl^- equilibrium potential and thereby make the cell less sensitive to a change in the extracellular K⁺ concentration and counteract the depolarizing effect of the high K⁺ challenge. A similar example of inhibition of high-K⁺-induced responses by GABA_A receptor activation has been reported (28).

In conclusion, alphaxalone inhibits the function of nAChRs at clinically relevant concentrations in adrenal chromaffin cells. Because adrenal chromaffin cells share various properties, such as ion channels and receptors with neuronal cells, alphaxalone may exert similar inhibitory effects on neuronal cells in the central and peripheral nervous systems. Although alphaxalone is not currently used in clinical situations, it seems to be important and required to study the mechanisms of neurosteroid anesthetics because of their potential roles as a therapeutic drug, and this study may provide some information for the anesthetic action in clinical situations in the future.

	Acknowledgments

 
Supported, in part, by Grants-in-Aid (Nos. 11671532, 13671626, 12770851, 11770878, 12671515, 12671516, 12770849 and 12470013) from the Ministry of Education, Science and Culture of Japan, by a UOEH Research Grant for Promotion of Occupational Health, by a Grant-in Aid for the Promotion of Occupational Health for the Post Graduate Student of UOEH, by the Japan Research Foundation for Clinical Pharmacology, Uehara Memorial Foundation, and by the Kanehara-Ichiro Memorial Medical Foundation.

	References

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