Introduction

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Inhalational anesthetics induce a wide spectrum of clinical effects such as unconsciousness, amnesia, analgesia, muscle relaxation, attenuation of protective reflex, and hemodynamic suppression (Kissin, 1993; Stanski, 1994). These diverse effects could reflect an integration of separate pharmacological actions of anesthetics (Kissin, 1993). Ever since anesthetic potency was shown to correlate with the lipophilicity (Meyer, 1899), perturbation of the membrane lipid has been hypothesized as a cause of general anesthesia. In the past decade, however, there has been increasing evidence for the membrane proteins to be a target of general anesthetics (Franks and Lieb, 1994). Inhalational anesthetics have been demonstrated to modulate GABA_A, glycine, 5-hydroxytryptamine₃, and nicotinic acetylcholine receptors (nAChRs) (Franks and Lieb, 1994).

The inhibitory GABA_A receptor has been considered a potential target site for anesthetics because clinically relevant concentrations of inhalational anesthetics potentiate its response (Nakahiro et al., 1989; Yeh et al., 1991). Inhibition of neuronal nAChRs (nnAChRs) by inhalational anesthetics was also found in molluscan neurons (McKenzie et al., 1995). Recently, nnAChRs expressed in Xenopus laevis oocytes have been shown to be potently blocked by inhalational anesthetics (Violet et al., 1997; Flood et al., 1997; Cardoso et al., 1999). The rat 42 or 32 subunits of nnAChRs expressed in X. laevis oocytes are 20 to 30 times more sensitive to halothane and isoflurane than the muscle nAChRs (Violet et al., 1997). Isoflurane inhibited the 42 subunits with an IC₅₀ value of 85 µM but did not inhibit 7 subunits of nnAChRs expressed in X. laevis oocytes (Flood et al., 1997). Although inhalational anesthetics inhibited nnAChRs expressed in X. laevis oocytes, the structurally related nonimmobilizing compound, F6 or 1,2-dichlorohexafluorocyclobutane, did not (Cardoso et al., 1999). However, it remains to be seen whether anesthetics inhibit the nnAChRs in mammalian native neurons in the central nervous system. It should be pointed out that the pharmacological responses of recombinant receptors expressed in various systems are not necessarily the same as those of native neurons (Cooper and Millar, 1997; Lewis et al., 1997; Sivilotti et al., 1997).

nAChRs consist of pentameric oligomers and an ion channel that is ACh-gated and cation-selective. Neuronal nAChRs differ from skeletal muscle nAChRs (which consist of 11/) and Torpedo californica electric organ nAChRs in subunit composition, pharmacology, and biophysical profile (McGehee and Role, 1995; Lindstrom, 1996) and consist of various combinations of subunits (2-9, 2-4) (McGehee and Role, 1995; Colquhoun and Patrick, 1997b). 7, 8, and 9 subunits form functional and homo-oligomeric receptors, whereas other  subunits form functional receptors only when combined with  subunits (McGehee and Role, 1995; Colquhoun and Patrick, 1997b).

nnAChRs are found in the presynaptic, preterminal, and postsynaptic locations; those at the first two locations render nnAChRs the ability to modulate the release of a variety of neurotransmitters including dopamine, norepinephrine, GABA, glutamate, and ACh itself (Role and Berg, 1996; Alkondon et al., 1997, 1999; Wonnacott, 1997). In addition, ACh is one of the important neurotransmitters released from the brain stem, hypothalamus, basal forebrain, and cerebral cortex that participate in cognition, memory, alertness, learning, and antinociception (Lindstrom, 1997; McCormick and Bal, 1997; Changeux et al., 1998; Marubio et al., 1999). Therefore, the high sensitivity of nnAChRs to inhalational anesthetics suggests that they mediate anesthetic actions such as unconsciousness, drowsiness, amnesia, and cognitive and psychomotor impairment through modulation of the release of various neurotransmitters.

We now report the effects of halothane on nnAChRs in rat cortical neurons in long-term primary culture using the whole-cell, patch-clamp technique. Cortical neurons in culture exhibit two distinct types of ACh-induced currents, -bungarotoxin (-BuTX)-sensitive currents and -BuTX-insensitive currents (Aistrup et al., 1999; Marszalec et al., 1999). The predominant subunits of -BuTX-sensitive and -BuTX-insensitive receptors are generally thought to be composed of 7 and 42 subunits, respectively (Albuquerque et al., 1997; Changeux et al., 1998). The present study shows that halothane inhibits both -BuTX-sensitive, 7-type and -BuTX-insensitive, 42-type currents at clinically relevant concentrations. However, 42-type currents were more sensitive to halothane than 7-type currents. Therefore, the mechanisms that underlie the halothane inhibition of 42-type nnAChRs were investigated in detail.

	Materials and Methods

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Cell Preparations. Rat cortical neurons were isolated and cultured by a procedure slightly modified from that described elsewhere (Marszalec and Narahashi, 1993). In brief, fetuses were removed from a 17-day pregnant Sprague-Dawley rat under methoxyflurane anesthesia. Small wedges of frontal cortex were excised and subsequently incubated in phosphate-buffered saline solution containing 0.25% (w/v) trypsin (Type XI; Sigma-Aldrich, St. Louis, MO) for 20 min at 37°C. The digested tissue was then mechanically triturated by repeated passages through a Pasteur pipette, and the dissociated cells were suspended in neurobasal medium with B-27 supplement (Life Technologies, Gaithersburg, MD) and 2 mM glutamine. The cells were added to 35-mm culture wells at a concentration of 100,000 cells/ml. Each well contained five 12-mm coverslips (previously coated with poly-L-lysine) overlaid with confluent glia that had been plated 2 to 4 weeks earlier. The cortical neuron/glia coculture was maintained in a humidified atmosphere of 90% air/10% CO₂ at 37°C. Cells cultured for 2 to 9 weeks were used for electrophysiological experiments.

Solutions for Current Recording. The external solution contained 150 mM NaCl; 5 mM KCl; 2.5 mM CaCl₂; 1 mM MgCl₂; 5.5 mM HEPES acid; 4.5 mM HEPES sodium; and 10 mM D-glucose. Tetrodotoxin (0.1 µM) was added to eliminate sodium channel currents, and atropine sulfate (20 nM) was added to block muscarinic ACh responses. The pH was adjusted to 7.3 and the osmolality was adjusted to 300 mOsmol by D-glucose. The internal solution contained 140 mM potassium-gluconate; 2 mM MgCl₂; 1 mM CaCl₂; 10 mM HEPES acid; 10 mM EGTA; 2 mM ATP-Mg²⁺; and 0.2 mM GTP-Na⁺. The pH was adjusted to 7.3 with KOH and the osmolality was adjusted to 300 mOsmol by adding D-glucose.

Current Recordings. The whole-cell, patch-clamp technique (Hamill et al., 1981) was used to record ionic currents induced by ACh application through a U-tube system. Recording pipettes were pulled in two stages on a vertical pipette puller (PP-83; Narishige, Tokyo, Japan). The pipettes with a relatively large tip diameter (electric resistance 2-3 M when filled with pipette solution) were used to facilitate the diffusion of pipette solution into the cell. Recording was started about 5 to 10 min after rupture of the membrane under the pipette tip to adequately equilibrate the cell interior with pipette solution. The currents were recorded with a patch-clamp amplifier (Axopatch-1B; Axon Instruments, Foster City, CA) and the membrane potential was held at 70 mV. The experiments were performed at room temperature (22 ± 2°C).

Drug Application. The speed of the U-tube system (Marszalec and Narahashi, 1993) had a rise time of 60 ms as measured by a change in junctional potential with a patch electrode and the solution exchange near the cell surface was complete within 200 ms, as assessed by the method of Liu and Dilger (1991). A computer-operated magnetic valve controlled this system. In the present study, the term "coapplication" is referred to as the simultaneous application of halothane with ACh through the U-tube only, whereas the term "preperfusion" is referred to as the application of halothane through the external bathing solution. Specific protocols for drug application are given in the respective Results subsections.

Analyses. Current records were initially analyzed via the ClampFit module of the PClamp6 to assess whole-cell current amplitude and decay kinetics. ACh concentration-response data and anesthetic inhibition data were fitted to the sigmoidal logistic equation (Hill equation) using the SigmaPlot (SPSS Science, Chicago, IL). Data were expressed as mean ± S.D. unless otherwise stated. Analyses of variance and/or Student's t tests were performed to assess significance of differences, if applicable. P values less than 0.05 were considered statistically significant.

Simulation. The kinetic simulation was carried out with a C++ program for numerical solution for the conducting channel according to simplified schemes for halothane to modulate dose-response relationships and desensitization induced by ACh, and open channel block by chlorisondamine.

	Results

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Morphology and conditions of rat cortical neurons in long term culture were described elsewhere (Aistrup et al., 1999). Briefly, after 2 weeks in culture, neurons projected their neurites and established complex network. This was evidenced by spontaneous activity recorded as excitatory and/or inhibitory postsynaptic currents, primarily mediated by N-methyl-D-aspartate and GABA receptors, respectively (Marszalec et al., 1998). After about 3 weeks, neurons began to express nnAChRs to generate ACh-induced currents. The properties of ACh-induced currents observed in rat cortical neurons were examined and described in our previous article (Aistrup et al., 1999). ACh induced two distinct types of currents differing in pharmacology and decay kinetics. One is an -BuTX-sensitive current that exhibits fast desensitization and another is an -BuTX-insensitive current that exhibits slow desensitization. These two types of current are generally thought to be mediated by 7-type and 42-type nnAChRs, respectively. In some cells, a mixture of 7-type and 42-type currents was observed.

The effects of halothane on ACh-induced currents were examined using the following protocol unless otherwise stated. ACh and halothane were coapplied through a U-tube, and halothane was perfused through the bath starting 2 min before the coapplication. Two-minute preperfusion was long enough to exchange the whole bath solution and to allow halothane to exhibit maximal effect.

Halothane Inhibition of Mixed 7-Type and 42-Type Currents. The effect of halothane on neurons exhibiting both 7-type and 42-type currents (Fig. 1A) was studied first. Halothane at 250 µM inhibited the mixed type current (Fig. 1B). After washout of halothane, 25 nM -BuTX perfused in the bath blocked the 7-type, fast component of current without affecting 42-type, slow component of current (Fig. 1C). Halothane at 250 µM inhibited the 42-type currents (Fig. 1D). However, the 7-type currents estimated by subtracting current of C from A and D from B were only slightly inhibited by halothane (Fig. 1, E and F). This experiment clearly showed that halothane inhibited 42-type currents more potently than 7-type currents. In the following experiments, the effects of halothane on 7-type and 42-type currents were examined separately.

View larger version (19K): [in this window] [in a new window]   Fig. 1.   Differential sensitivity of 7-type and 42-type currents to halothane. To induce currents, ACh (300 µM) was applied through a U-tube for 3 s (thick horizontal bar). Halothane 250 µM was preperfused 2 min before the test ACh pulse and was also coapplied with ACh (thin horizontal bar). A, A mixture of 7-type and 42-type currents. B, halothane inhibited the mixed current. C, -BuTX blocked 7-type current without affecting 42-type current. D, halothane inhibited 42-type current. E and F, 7-Type currents were separated by subtracting C from A (E) as the control, and D from B (F) as the current in halothane. The 7-type current was only slightly inhibited by halothane. This clearly shows that halothane inhibits 42-type current more potently than 7-type current.

Halothane Inhibition of 7-Type Currents. To record 7-type currents, we tried to find cells that exhibited no 42-type currents. The concentration-dependent effect of halothane was examined at an ACh concentration of 300 µM, which is near the EC₅₀ value for 7-type currents. Halothane inhibited 7-type currents reversibly in a concentration-dependent manner (Fig. 2A). The IC₅₀ and Hill coefficient obtained from the halothane inhibition curve were 552 µM and 2.45, respectively (Fig. 2B). This concentration of halothane is clinically relevant but is twice as high as the MAC.

View larger version (20K): [in this window] [in a new window]   Fig. 2.   Halothane block of 7-type currents. A, halothane inhibited the currents induced by 300 µM ACh in a concentration-dependent manner. The inhibition was reversible after washout of halothane. B, concentration-response curve for halothane inhibition. The data points are the mean peak currents expressed as percentages of control current, and the error bars are standard deviations (n = 5). The line is unweighted least-squares fit of the data to a Hill equation. The IC50 and Hill coefficient obtained from halothane inhibition curve were 552 ± 52 µM and 2.45 ± 0.50, respectively.

View larger version (27K): [in this window] [in a new window]   Fig. 3.   Halothane block of 7-type currents as a function of ACh concentration. A, halothane 750 µM inhibited 7-type currents at all ACh concentrations tested. The inhibition was independent of ACh concentration. B, ACh concentration-response curves in the absence and presence of 750 µM halothane. The data points are the mean peak currents expressed as percentages of control current, and the error bars are standard deviations (n = 5). The lines are unweighted least-squares fit of the data to a Hill equation. The ACh EC50 and Hill coefficient were not changed by halothane (from 267 ± 51 µM to 259 ± 34 µM and from 1.18 ± 0.20 to 1.04 ± 0.12, respectively).

View larger version (28K): [in this window] [in a new window]   Fig. 4.   Halothane acceleration of the decay phase of 7-type currents. A, the peak of the currents in the absence and presence of 500 µM halothane was normalized to the same amplitude to compare the decay phase of 7-type currents. B, halothane at 250, 500, and 750 µM significantly and concentration dependently shortened the decay time constant which was obtained by fitting the current with a single exponential function (n = 5, p < 0.05). C, halothane significantly shortened the decay time constant at any concentrations of ACh tested (n = 5, p < 0.05).

Halothane Effect on 42-Type Currents. To isolate 42-type currents, -BuTX (25 nM) was applied in the bath to block 7-type currents. The concentration-dependent effect of halothane on 42-type currents was examined at an ACh concentration of 300 µM, which caused the maximum response. Halothane inhibited 42-type currents in a concentration-dependent manner at clinically relevant concentrations (Fig. 5A). The inhibition was reversible after washout of halothane. The IC₅₀ and Hill coefficient obtained from halothane inhibition curve were 105 µM and 1.1, respectively (Fig. 5B). This IC₅₀ value was less than half of the MAC. Therefore, 42-type currents were highly sensitive to halothane and much more sensitive than 7-type currents.

View larger version (19K): [in this window] [in a new window]   Fig. 5.   Halothane block of 42-type currents. A, halothane inhibited the currents induced by 300 µM ACh in a concentration-dependent manner. The inhibition was reversible after washout of halothane. B, concentration-response curve for halothane inhibition. The data points are the mean peak currents expressed as percentages of control current, and the error bars are standard deviations (n = 10). The line is unweighted least-squares fit of the data to a Hill equation. The IC50 and Hill coefficient obtained from halothane inhibition curve were 105 ± 7.0 µM and 1.10 ± 0.06, respectively.

View larger version (24K): [in this window] [in a new window]   Fig. 6.   Halothane block of 42-type currents as a function of ACh concentration. A, halothane at 100 µM reversibly inhibited 42-type currents at all ACh concentrations tested. The inhibition was slightly larger at high concentrations of ACh. B, ACh concentration-response curves in the absence and presence of 100 µM halothane. The data points are the mean peak currents expressed as percentages of control current, and the error bars are standard deviations (n = 10). The lines are unweighted least-squares fit of the data to a Hill equation. Halothane reduced the ACh EC50 value from 5.46 ± 2.40 µM to 1.53 ± 0.25 µM and increased the Hill coefficient from 0.61 ± 0.14 to 0.99 ± 0.14. Both changes are significant at a p value of < 0.05.

View larger version (20K): [in this window] [in a new window]   Fig. 7.   Halothane effect on the decay phase of 42-type currents. A, ACh 300 µM was applied for 25 s and the current was reversibly reduced in the presence of 75 µM halothane. The current in the presence of halothane was normalized to control peak and superimposed with control current. The decay could be well fit with a double exponential function. B, halothane increased both fast and slow decay time constants significantly (n = 5, p < 0.05).

Time Course of Halothane Block of the Activated and Resting 42-Type Receptors. Two protocols were used to monitor the time course of halothane block of ACh-induced currents of 42-type receptors. The protocol to monitor halothane block of the resting 42-type receptors is shown in Fig. 8. Two 1 mM ACh pulses were applied from Picospritzer II (General Valve Corporation, Fairfield, NJ) to generate test currents whereas halothane was applied for 4 s from a U-tube onto the cell. Halothane was washed out between each set of trials. The time to first test pulse from the beginning of halothane application was varied in each trial, and a second pulse was applied 2 s after the onset of halothane application. Plot of the amplitude of the first pulse current as a function of the period of halothane perfusion shows that halothane inhibition of the resting 42-type receptor is time dependent with a time constant of 218 ± 18 ms (Fig. 8B). Each  in Fig. 8B represents the amplitude of the second pulse current in each trial showing almost a constant amplitude, which represents a constant steady-state block. Halothane essentially reached the steady-state block of the resting receptors within 1 s.

View larger version (19K): [in this window] [in a new window]   Fig. 8.   Time course of halothane block of the resting 42-type receptor. A, ACh 1 mM was applied from a Picospritzer as test pulse and 250 µM halothane was applied from a U-tube onto the cell. Halothane was washed out between trials. To evaluate the resting channel block, the time to the first test pulse from the beginning of halothane application was varied in each trial. A second pulse was applied 2 s after the onset of halothane application to monitor the stability of the response. B, plot of the currents () as a function of the period of halothane perfusion shows that halothane inhibition of the resting 42-type receptor developed with a time constant of 218 ±18 ms (n = 4). Second pulse currents () and their constant amplitude suggests halothane block is complete within 2 s.

View larger version (25K): [in this window] [in a new window]   Fig. 9.   Time course of halothane block of the activated 42-type receptor. Three currents induced by 300 µM ACh recorded in the same cell are superimposed. Control current was recorded in the absence of halothane. Current 1 was recorded when 250 µM halothane was coapplied with 300 µM ACh. Current 2 was recorded when 250 µM halothane was preperfused and coapplied with 300 µM ACh. When halothane was coapplied with ACh, the current exhibited time-dependent decay reaching the same level as that achieved by a combination of preperfusion and coapplication. This suggests that halothane has the same affinity for the resting state of receptor as well as for the activated receptor. The decay time constant estimated from current 1 was 295 ± 25 ms (n = 6).

Comparison of Time Course of Recovery from Halothane Block with Desensitized 42-Type Receptors. To assess whether halothane inhibition of 42-type currents was caused by receptor desensitization, the recovery of current from halothane block was compared with the recovery from the ACh-induced desensitization. The time course of recovery from halothane block was examined by two protocols. One is to assess the recovery in the resting state of receptors as shown in Fig. 10. ACh (3 mM) was applied from Picospritzer II as test pulse while halothane was applied in the bath. To observe recovery from resting block, halothane was washed out by halothane-free solution from a U-tube. The time to the first test pulse from the beginning of washout was varied in each set of trial. The second pulse and the third pulse were applied 2 and 3 s after the onset of washout, respectively. Plot of the peak amplitude of the first current as a function of the period of halothane washout shows that halothane recovered from inhibition of the resting receptor with a time constant of 499 ± 68 ms (Fig. 10).

View larger version (20K): [in this window] [in a new window]   Fig. 10.   The time course of recovery from halothane block in the resting 42-type receptors. ACh 3 mM was applied from a Picospritzer and halothane 250 µM was applied in the bath. To observe recovery of the resting receptor, halothane was washed out by halothane-free solution from a U-tube. The time to the first test pulse from the beginning of washout was varied in each set of trial. Second and third pulses were applied 2 and 3 s after the onset of washout, respectively. The current recovered almost completely from halothane block in 2 s. Plot of the first pulse current as a function of the period of halothane washout shows that the recovery from halothane inhibition of the resting 42-type receptor is time dependent (B) with a time constant of 499 ± 68 ms (n = 4).

View larger version (25K): [in this window] [in a new window]   Fig. 11.   Comparison of the recovery from halothane block in the 42-type receptor during receptor activation with the recovery from receptor desensitization. A, three currents evoked by 300 µM ACh are superimposed. Control current was recorded in the absence of halothane. Current 1 was recorded when halothane was only preperfused 2 min before ACh test pulse which did not contain halothane. Current 2 was recorded when halothane was preperfused and also coapplied during test pulse. Preperfused halothane inhibited initial peak current. This inhibition was removed by halothane-free ACh solution exhibiting the time-dependent rise to the control level with a time constant of 285 ± 51 ms (n = 7). B, ACh-induced desensitization of the 42-type receptor. A high concentration of ACh (1 mM) was applied for 10 s through a U-tube to induce receptor desensitization. The recovery of the 42-type receptor from desensitization was monitored by applying ACh test pulses (1 mM). The time constant of the recovery of the 42-type receptor from desensitization was 5.65 ± 0.29 s (n = 6) which was much longer than that of recovery from the halothane block.

Effects of Unstirred Layers on Rates of Halothane Blocking Action. The similarity between the onset of block of the resting receptor and the activated receptor made us to suspect that the onset of action might be rate-limited by solution exchange because of an unstirred layer surrounding the cell.

View larger version (10K): [in this window] [in a new window]   Fig. 12.   Rate of solution exchange via the U-tube perfusion. A, the junction potential change in response to U-tube application of 15 mM Na+ solution to a 150 mM Na+ bathing solution was detected by a patch electrode with a rise time of 66.6 ± 3.30 ms (n = 5). B, ACh currents induced by the U-tube application of 0.3 mM ACh in 150 mM Na+ solution rose with a rise time of 65.4 ± 2.20 ms (n = 6). When 0.3 mM ACh was applied via the U-tube containing 75 mM Na+ solution to the cell bathing in 150 mM Na+ containing solution (hybrid), ACh current rose to the peak before settling to a steady-state level, which was about 50% of the current recorded when both the bathing solution and the U-tube solution contained 150 mM Na+. The settling times of 215 ± 30.0 ms (n = 5) arose from the effect of unstirred layers.

Interaction of Halothane with Chlorisondamine. To get insight into the nature of halothane block of the 42-type receptors, we studied the interaction of halothane with chlorisondamine. Chlorisondamine (Tocris Cookson Inc., Ballwin, MO) is an irreversible open channel blocker of nAChRs of frog muscle (Neely and Lingle, 1986), sympathetic ganglia (Rogers et al., 1997), and nnAChRs expressed in X. laevis oocytes (Colquhoun and Patrick, 1997a). When 3 µM chlorisondamine was coapplied with 300 µM ACh for 3 s, the decay of ACh-induced current was greatly accelerated indicating open channel block (Fig. 13A). After 3 to 5 min of washout of chlorisondamine, the current was significantly reduced, indicating that chlorisondamine block was almost irreversible. In the presence of 250 µM and 750 µM halothane, however, the current after washout of halothane and chlorisondamine was larger than the control group (Fig. 13, B and C versus A). The current amplitude after washout was normalized to the control and is plotted as a function of halothane concentration (Fig. 13D). The fraction of receptor not blocked by chlorisondamine was significantly increased with an increase in halothane concentration. The halothane protection could be overcome by increasing chlorisondamine concentration. The protection by 750 µM halothane was drastically reduced when chlorisondamine was increased from 3 to 10 µM. These results suggest that halothane competitively protects the 42-type receptor from chlorisondamine block.

View larger version (14K): [in this window] [in a new window]   Fig. 13.   Interaction of halothane with the open channel blocker chlorisondamine. A, currents were induced by 300 µM ACh for 3 s. After control recording, 3 µM chlorisondamine was coapplied with ACh. The decay of ACh-induced current was greatly accelerated suggesting open channel block. After 3 to 5 min of washout of chlorisondamine, the current was significantly reduced indicating that chlorisondamine block was almost irreversible. B and C, in the presence of halothane, the current after washout of halothane and chlorisondamine was larger than the control group in A. D, the current amplitude after washout was normalized to the control and is plotted as a function of halothane concentration. The fraction of unblocked receptor was significantly increased with an increase in halothane concentration (n = 8-12). The protection by 750 µM halothane was decreased with an increase in chlorisondamine concentration.

View larger version (21K): [in this window] [in a new window]   Fig. 14.   Halothane 250 µM slows the current decay accelerated by 3 µM chlorisondamine. A, to facilitate the comparison of the decay phases, the peak current in the presence of 250 µM halothane was normalized to the control. Chlorisondamine-induced acceleration of the decay was slowed by halothane. B, the decay phase was fit with a single exponential function to calculate the decay time constant which was significantly increased in the presence of halothane (n = 9-12) (p < 0.05).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

The present study demonstrated that halothane reversibly inhibited both 7-type and 42-type currents of nnAChRs in a concentration-dependent manner at clinically relevant concentrations. Furthermore, halothane block of 7-type currents was independent of ACh concentration, whereas the block of 42-type currents became less with decreasing ACh concentration.

The decay phases of 42-type and 7-type currents were differentially affected by halothane. Halothane accelerated the decay phase of 7-type currents while slowing the decay of 42-type currents (Figs. 4 and 7). This acceleration of the decay phase of the 7-type currents suggest that halothane accelerates desensitization or causes open channel block. However, the fact that the halothane block of 7-type currents is independent of ACh concentration is not consistent with open channel block. On the other hand, halothane block of 42-type currents is dependent on ACh concentration, suggesting open channel block. The ACh concentration-dependent block of 42-type currents will be addressed later under Discussion.

Evidence has been obtained in support of the notion that general anesthetics directly act on ion channel proteins (Dilger et al., 1994; Forman et al., 1995; Eckenhoff, 1996). The M2 domain of 42-type nnAChRs could be the site of halothane binding as suggested by Forman et al. (1995), who showed in mutagenesis experiments with muscle type nAChRs that anesthetics act on a specific amino acid in the M2 hydrophobic region that forms the pore lining. It is interesting to note that the N-terminal domain of 7 receptor outside the membrane was shown to be important for the inhibitory action of inhalational anesthetics using chimeric receptors (Zhang et al., 1997).

We have also found that halothane caused a shift in the ACh dose-response curve of 42-type currents and protected the receptor from another open channel blocker, chlorisondamine. The experimental protocol failed to show unequivocally that halothane acts on the resting 42-type nnAChRs as well as the activated receptors as was seen in muscle type nAChRs (Dilger et al., 1993, 1994; Wachtel, 1995; Scheller et al., 1997), because halothane blocked and unblocked the activated and resting receptors at the rates similar to that of solution exchanges in the unstirred layer. The time constants of the isoflurane binding to and unbinding from the muscle type nAChRs were estimated to be around 500 µs using a rapid perfusion technique (Dilger et al., 1993). Direct modulation of GABA_A receptors by halothane has recently been reported to occur within milliseconds (Li and Pearce, 2000). Thus, it is entirely possible that the resting block observed here is caused by a fast open channel block as seen with the muscle type nAChRs (Dilger et al., 1993).

Simulation of Halothane Effects on 42-Type nnAChRs. In the present study with the 42-type nnAChRs, halothane caused a shift in the ACh dose-response curve, slowed desensitization and protected the receptor from another open channel blocker, chlorisondamine. All of these halothane effects can be simulated by a simple model in which halothane rapidly blocks nnAChRs in the open state. In addition, the halothane-blocked receptor undergoes little or no desensitization. In the following simulation, we use a conventional scheme for the activation of nnAChRs. To simplify the simulation, we assume that the activated receptor undergoes desensitization and the halothane-bound and chlorisondamine-bound ones do not.

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Effects of Halothane on Activation and Desensitization. Halothane modified ACh activation by shifting the ACh dose-response relationship in the direction of lower ACh concentration and by increasing the Hill coefficient. Such changes in activation could be largely accounted for by simulation of halothane block of the open channel (Fig. 15A). At high ACh concentrations, almost all receptors enter the open state, and the steady-state block by halothane is governed by the blocking and unblocking rate constants. At lower ACh concentrations (e.g., 10 µM), about 50% of the receptors that have entered the open state can be blocked rapidly by halothane. Because of this redistribution of the open state into conducting and blocked states, the transiently depleted conducting pool is replenished by converting more R to R2A*. The percentage of the blocked state is no longer determined solely by the blocking and unblocking rate constants. Consequently, less block is seen at lower ACh concentrations displaying a left shift in the ACh dose-response curve.

View larger version (16K): [in this window] [in a new window]   Fig. 15.   Simulation of the effects of halothane on ACh concentration-response relathionship, desensitization, and chlorisondamine block. Numerical solution of the kinetic Scheme 1 was carried out with C++ program. The following rate constants were used: kon = 1 × 107 M1 s1, koff =100 s1,  = 3000 s1,  = 500 s1, 1 = 1.4 s1, 1 = 1.4 s1, 2 = 0.28 s1, 2 = 0.084 s1, k+1 = 1 × 107 M1 s1, k1 = 1 × 103 s1 for governing halothane block, k+2 = 5 × 106 M1 s1, k2 = 0.01 s1 for governing chlorisondamine block. A, symbols are simulated data fitted with the Hill equation. Halothane at 100 µM decreased the EC50 value for ACh from 6.08 µM to 3.95 µM and increased the Hill coefficient from 1.44 to 1.54. B, ACh current peaked rapidly and was followed by two phases of decay during a prolonged application of 300 µM ACh. In the presence of 75 µM halothane, the peak was reduced and current decay was slowed. The fast time constant was increased from 0.4 to 0.8 s and the slow time constant was increased from 5 to more than100 s. C, halothane protects the receptor from chlorisondamine block. In the presence of 3 µM chlorisondamine, the ACh current induced by 300 µM ACh reached the peak rapidly and then decayed with a time constant of 515 ms. The decay phase was slowed by 250 µM halothane with a decay time constant of 1.28 s. At the end of 3 s, 3 µM blocked 80% of the receptors in the control and 60% of the receptors in the presence of 250 µM halothane as seen experimentally (Fig. 13D).

Simulation of Apparent Slowing in Desensitization. Halothane slowed current decay during a prolonged application of 300 µM ACh, which might be construed as indicative of slowing of receptor desensitization. In the simulation of the effect of halothane in slowing the ACh current decay, we have to assume that the halothane-blocked receptors undergo little or no desensitization. In the simulation shown in Fig. 15B, the halothane-bound receptor was assumed not to undergo desensitization. After the application of 300 µM ACh without halothane, the receptors enter the conducting state rapidly, and then decayed slowly. The kinetics of current decay are indicative of receptor desensitization and are determined by forward and backward rate constants governing the transition into two desensitized states. The slowing of current decay in the presence of 75 µM halothane could be simulated by the above kinetic scheme on the assumption that about 50% of the receptors are rapidly blocked by halothane without undergoing desensitization whereas the rest undergo normal desensitization. The halothane-blocked state initially serves as current sink and later becomes current source. Under this condition, the current decay is no longer determined solely by the rate constants governing the desensitization step and is influenced by the late arrival of conducting state from the blocked state.

Halothane Protects Receptors from Chlorisondamine Block. Halothane decreased the chlorisondamine block of 42-type receptors in a concentration-dependent manner. This suggests that halothane somehow prevents chlorisondamine from binding to the open receptor channel. In the simulation of competitive block of open channels by halothane and chlorisondamine, we added a chlorisondamine-blocked state, B2, with forward and backward rate constants governing its block. As shown in Fig. 15C, chlorisondamine at 3 µM blocked the receptor with a time constant of 600 ms to 20% of the control. In the presence of 250 µM halothane, block by 3 µM chlorisondamine became slower and its time constant was increased to 1.87 s from the control of 0.60 s. Mechanistically, the slowing in the current decay is caused by the late arrival of the conducting state from the halothane-bound state. In addition, only 40% of the receptors are blocked by chlorisondamine because fewer receptors are available due to competitive block by halothane.

Comparison with Previous Results. The results of the present experiments with the nnAChRs of rat cortical neurons may be compared with those with the chicken receptors expressed in X. laevis oocytes (Flood et al., 1997). Halothane and isoflurane both potently inhibit the 42-type current with similar IC₅₀ values (105 µM or 0.4 MAC for halothane and 85 µM or 0.3 MAC for isoflurane). However, their block differs in the ACh concentration dependence. Halothane block increases with ACh concentration, where isoflurane block decreases. The 7-type receptor is much less sensitive to the anesthetics than the 42-type receptor. Halothane at 552 µM (2.2 MACs) inhibits the 7-type current 50% whereas isoflurane at 640 µM (2 MACs) has no effect. These differences may be attributed to different expression systems (native cortical neurons versus X. laevis oocytes) and/or different animal species (rat versus chicken).

Clinical Implication of Potent Effect of Halothane on nnAChRs. There is general agreement that nnAChRs located in presynaptic and preterminal sites modulate the release of various neurotransmitters such as norepinephrine, dopamine, GABA, glutamate, serotonin, and ACh itself (Role and Berg, 1996; Alkondon et al., 1997, 1999; Wonnacott, 1997). Along this line, several studies demonstrated that the modulation of neurotransmitter release mediated by nnAChRs was affected by inhalational anesthetics. At clinically relevant concentrations, inhalational anesthetics suppressed nicotine-induced dopamine release in rat striatum (Salord et al., 1997), nicotine-induced catecholamine release in chromaffin cells (Sumikawa et al., 1982; Pocock and Richards, 1988), and ACh release in rat striatum and cerebral cortex (Shichino et al., 1998). Thus, the inhibition of nnAChRs by halothane observed in the present study could potently modulate the neurotransmitter release in the central nervous system. Our preliminary experiments using cortical neurons in long-term culture showed that the increase in the frequency of miniature excitatory postsynaptic currents by exogenously administered ACh in the presence of tetrodotoxin was inhibited by halothane without change in the amplitude.