Modulation of Neuronal Nicotinic Acetylcholine Receptors by Halothane in Rat Cortical Neurons

Materials and Methods

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 byd-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 addingd-glucose.

ACh (Sigma) was first dissolved in distilled water to make stock solution. Halothane (Fluothane) was obtained from Ayerst Laboratories (New York, NY). Saturated halothane solutions were made by stirring halothane in the external solution over 8 h in a sealed glass container with very little air space. Halothane test solutions were prepared immediately before experiments by dilution of the saturated solution and were kept in air-free, closed glass bottles to prevent evaporation of halothane. Using ¹⁹F-NMR spectroscopy (GE NMR Instruments), the saturated solution was found to contain 18.0 mM halothane, a value identical with that determined previously (Seto et al., 1992). Taking into account the solubility of halothane (Smith et al., 1981) and temperature (T) (Franks and Lieb, 1993), the aqueous effective concentration for 50% effect (EC₅₀) in rat neurons (Franks and Lieb, 1996) was estimated by the equation EC₅₀‖_T(°C) = exp[(−4.08 × (37 − T))/(273.15 + T)] × EC₅₀‖_37°C. The concentration of halothane in aqueous phase at 22°C (0.25 mM) is very close to 0.23 mM in equilibrium with 0.4% halothane in air, which corresponds to one minimum alveolar concentration (MAC) for rat (1.03% at 37°C). The concentrations of halothane used in the present experiments were 7.5 to 2500 μM.

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 MΩ 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).

ACh-induced currents were filtered at 5 kHz and digitized at 1 to 10 kHz via a Digidata 1200 analog-to-digital/digital-to-analog converter interfaced to a microcomputer under control of the ClampEx module of the PClamp6 software package (Axon Instruments).

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

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 α-BuTX-sensitive current that exhibits fast desensitization and another is an α-BuTX-insensitive current that exhibits slow desensitization. These two types of current are generally thought to be mediated by α7-type and α4β2-type nnAChRs, respectively. In some cells, a mixture of α7-type and α4β2-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 α7-Type and α4β2-Type Currents.

The effect of halothane on neurons exhibiting both α7-type and α4β2-type currents (Fig.1A) was studied first. Halothane at 250 μM inhibited the mixed type current (Fig. 1B). After washout of halothane, 25 nM α-BuTX perfused in the bath blocked the α7-type, fast component of current without affecting α4β2-type, slow component of current (Fig. 1C). Halothane at 250 μM inhibited the α4β2-type currents (Fig. 1D). However, the α7-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 α4β2-type currents more potently than α7-type currents. In the following experiments, the effects of halothane on α7-type and α4β2-type currents were examined separately.

Halothane Inhibition of α7-Type Currents.

To record α7-type currents, we tried to find cells that exhibited no α4β2-type currents. The concentration-dependent effect of halothane was examined at an ACh concentration of 300 μM, which is near the EC₅₀ value for α7-type currents. Halothane inhibited α7-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.

The effect of halothane on the ACh concentration-response curve of α7-type currents was tested at a concentration of 750 μM. Halothane inhibited α7-type currents at all ACh concentrations tested ranging from 100 μM to 3 mM (Fig. 3A). This inhibition was independent of ACh concentration. The ACh EC₅₀ values before and after halothane application were 267 to 259 μM, respectively, and the Hill coefficients were 1.18 and 1.04, respectively (Fig. 3B), indicating that halothane inhibition of α7-type currents is noncompetitive in nature.

The decay phase of α7-type current was accelerated by halothane. When the amplitudes of currents recorded in the absence and presence of halothane (500 μM) were normalized (Fig.4A), a slight acceleration of the decay phase by halothane was noted (Fig. 4A). The decay phase could be fitted by a single exponential function. Halothane at concentrations of 250, 500, and 750 μM reduced the decay time constant in a concentration-dependent manner and at ACh concentrations of 30 μM to 3 mM (Fig. 4, B and C). This suggests that halothane either accelerates the desensitization or blocks open channel.

Halothane Effect on α4β2-Type Currents.

To isolate α4β2-type currents, α-BuTX (25 nM) was applied in the bath to block α7-type currents. The concentration-dependent effect of halothane on α4β2-type currents was examined at an ACh concentration of 300 μM, which caused the maximum response. Halothane inhibited α4β2-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, α4β2-type currents were highly sensitive to halothane and much more sensitive than α7-type currents.

The effect of halothane on ACh concentration-response curve of α4β2-type currents was tested at a halothane concentration of 100 μM. Halothane inhibited the currents at all ACh concentrations tested ranging from 0.3 μM to 1 mM (Fig. 6A). The inhibition was ACh concentration-dependent, being larger at higher concentrations of ACh. Consequently, halothane reduced the ACh EC₅₀ value from 5.46 to 1.53 μM and increased the Hill coefficient from 0.61 to 0.99 (Fig. 6B). A shift in ACh dose-response curve toward the direction of lower ACh concentrations and an increase in the Hill coefficient by halothane are consistent with its open channel blocking action. A simulation from such a model will be given later under Discussion.

The effect of halothane on the decay phase of α4β2-type currents was also examined. Because α4β2-type currents exhibited slow desensitization, ACh was applied for as long as 25 s. In the presence of halothane at 75 μM, the current was reversibly reduced (Fig. 7A). To clarify the difference in the decay phase, the peak current recorded in the presence of halothane was normalized to control peak current (Fig. 7A). The decay phase could be well fit with double exponential functions, and halothane increased both fast and slow decay time constants significantly (Fig. 7B). Thus, the decay phase of α7-type and α4β2-type currents was differentially affected by halothane.

Time Course of Halothane Block of the Activated and Resting α4β2-Type Receptors.

Two protocols were used to monitor the time course of halothane block of ACh-induced currents of α4β2-type receptors. The protocol to monitor halothane block of the resting α4β2-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 α4β2-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.

The protocol to examine an interaction of halothane with the activated receptor is illustrated in Fig. 9. Three currents recorded from the same cell and induced by 300 μM ACh are superimposed. Control current was recorded in the absence of halothane. Current 1 was induced by coapplication of 250 μM halothane and ACh. Current 2 was induced by ACh when 250 μM halothane was preperfused in the bath and also coapplied with ACh. The current reduction observed with combined preperfusion and coapplication of halothane (current 2) represents the resting receptor block because the receptor was not activated during halothane preperfusion. When halothane was coapplied with ACh without preperfusion, the current exhibited time-dependent decay. If the current decay in the presence of halothane mainly reflects the time course of open channel block, the decay time constant was estimated to be approximately 300 ms in the presence of 250 μM halothane. Because the steady-state level after coapplication of halothane (current 1) reaches the same level as that achieved by a combination of preperfusion and coapplication (current 2), halothane has an identical affinity for both the activated and the resting receptors.

Comparison of Time Course of Recovery from Halothane Block with Desensitized α4β2-Type Receptors.

To assess whether halothane inhibition of α4β2-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).

Fig. 11A shows another protocol used to measure the time course of recovery from halothane block in the activated receptors. Three currents activated by 300 μM ACh are superimposed. Current 1 was generated when halothane was only preperfused for 2 min before ACh test pulse devoid of halothane. Current 2 was generated when halothane was preperfused and also coapplied with ACh during test pulse. Preperfused halothane inhibited initial peak currents. This inhibition was removed by halothane-free ACh solution (current 1) exhibiting the time-dependent rise to the control level with a time constant of 285 ± 51 ms, which was significantly faster than halothane dissociation from the resting state (p < 0.05).

To monitor recovery from receptor desensitization, a high concentration of ACh (1 mM) was applied for 10 s to induce desensitization and its recovery was tested by a short test ACh pulse (Fig. 11B). The time course of the recovery of the receptor from the ACh-induced desensitization was 5.65 ± 0.29 s, a time much longer than the recovery from halothane block. This suggests that halothane inhibition of receptor is not caused by receptor desensitization.

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.

To examine the effect of unstirred layers on the drug action, the experiment protocol of Liu and Dilger (1991) was adapted here to determine solution exchange within the unstirred layer. As shown in Fig. 12, the patch electrode was capable of detecting junction potential change with a rise of time of 60 ms and in response to half-Na solution ACh current exhibited a peak before settling at the steady-state level within 215 ms. The transient peak has been attributed to a delay of solution exchange because of the effect of the unstirred layer. Thus, the onset and offset rate of halothane action was largely rate-limited by solution exchange because the onset time constants were similar to that for solution exchange. Thus, it is entirely possible that a fast open channel block could cause the resting receptor block and activated receptor block. The simulation based on such a notion could indeed reproduce the major effects of halothane on ACh dose-response curve, desensitization, and chlorisondamine open channel block (see Discussion).

Interaction of Halothane with Chlorisondamine.

To get insight into the nature of halothane block of the α4β2-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 inX. 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 α4β2-type receptor from chlorisondamine block.

The decay phase accelerated by chlorisondamine coapplication was compared in the absence and presence of 250 μM halothane (Fig.14). The chlorisondamine-induced acceleration of decay was slowed in the presence of halothane (Fig.14A). The decay could be fit with a single exponential function, and the time constant was significantly increased in the presence of halothane (Fig. 14B). These results are consistent with a model in which the receptor can be blocked by either halothane or chlorisondamine. Halothane blocks and unblocks the open state of α4β2-type receptors more quickly than chlorisondamine does. Consequently, the halothane-blocked receptors became a sink for ACh receptors initially and became a source for chlorisondamine block as the open channels are depleted because of chlorisondamine block. Again, a simulation based on a simplified scheme will illustrate this point under Discussion.

Discussion

The present study demonstrated that halothane reversibly inhibited both α7-type and α4β2-type currents of nnAChRs in a concentration-dependent manner at clinically relevant concentrations. Furthermore, halothane block of α7-type currents was independent of ACh concentration, whereas the block of α4β2-type currents became less with decreasing ACh concentration.

The decay phases of α4β2-type and α7-type currents were differentially affected by halothane. Halothane accelerated the decay phase of α7-type currents while slowing the decay of α4β2-type currents (Figs. 4 and 7). This acceleration of the decay phase of the α7-type currents suggest that halothane accelerates desensitization or causes open channel block. However, the fact that the halothane block of α7-type currents is independent of ACh concentration is not consistent with open channel block. On the other hand, halothane block of α4β2-type currents is dependent on ACh concentration, suggesting open channel block. The ACh concentration-dependent block of α4β2-type currents will be addressed later underDiscussion.

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 α4β2-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 α7 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 α4β2-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 α4β2-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_Areceptors 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 α4β2-Type nnAChRs.

In the present study with the α4β2-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.

R is nnAChR, A is ACh, RA₂* is an open state, RD1 is a fast desensitized state, RD2 is a slow desensitized state, H is halothane, B1 is a halothane blocked state, C is chlorisondamine, and B2 is a chlorisondamine blocked state. Halothane is assumed to interact rapidly with the open state of the receptor.

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.

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 α4β2-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. laevisoocytes (Flood et al., 1997). Halothane and isoflurane both potently inhibit the α4β2-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 α7-type receptor is much less sensitive to the anesthetics than the α4β2-type receptor. Halothane at 552 μM (2.2 MACs) inhibits the α7-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).

Violet et al. (1997) also studied the effect of halothane on nnAChRs expressed in X. laevis oocytes. They found that the halothane IC₅₀ value for the α4β2 receptor expressed in oocytes was 27 μM compared with 105 μM in the present study. In contrast to our finding, they found that halothane block was independent of ACh concentration. Because the source of material (rat) is the same in both studies, the quantitative differences must be caused by the different expression system. Consistent with this view are the findings that the ACh EC₅₀ value for the rat α4β2 receptor is estimated to be 104 μM (Violet et al., 1997), whereas the value for the α4β2-type current of native rat cortical neurons is 3 μM (Aistrup et al., 1999) or 5.5 μM (present study).

The time constant for the recovery of the α4β2-type nnAChR currents from halothane block (<300 ms) was much faster than the recovery from ACh-induced desensitization (5.65 s). This strongly suggests that halothane block is not caused by desensitization. The above simulation also suggests that the halothane-bound receptor might not undergo desensitization at all. This is in contrast with studies with the muscle type nAChRs. Several studies have suggested that general anesthetics stabilize the slowly desensitized conformational state in the muscle type nAChRs through conversion from a low-affinity state to a high-affinity state by agonist (Young and Sigman, 1983; Dilger et al., 1993; Firestone et al., 1994; Raines et al., 1995). In addition,Violet et al. (1997) found that the muscle nAChRs are 32 times less sensitive to halothane than the α4β2 nnAChRs.

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.

Halothane reversibly inhibited both α7-type and α4β2-type currents of nnAChRs with IC₅₀ values of 552 and 105 μM, respectively. The IC₅₀ value for α4β2-type currents is a subanesthetic concentration and almost equivalent to 0.4 MAC, whereas the IC₅₀ value for α7-type currents is more than surgical concentration and almost equivalent to 2 MACs. Thus, the α4β2-type receptors in rat cortical neurons are highly sensitive to halothane and may play an important role for clinical anesthesia, whereas the α7-type receptors may be less significant for anesthesia.

At subanesthetic doses, inhalational anesthetics have been shown to induce diverse behavioral effects, including suppression of learning and memory (Newton et al., 1990, Galinkin et al., 1997; Dwyer et al., 1992), drowsiness (Newton et al., 1990; Galinkin et al., 1997), and cognition impairment (Galinkin et al., 1997). ACh is one of the important neurotransmitters released from the brain stem, hypothalamus, basal forebrain, cerebral cortex that participate in cognition, memory, learning, alertness, and antinociception (McCormick and Bal, 1997;Changeux et al., 1998). Suppression of interneuronal activity in cerebral cortex is suspected to play an important role for primary hypnotic action of anesthetics (Woodforth et al., 1999). This suggests that drowsiness and unconsciousness caused by inhalational anesthetics may be caused, at least in part, by suppression of cerebral cortex. Thus the high sensitivity of nnAChRs, α-BuTX-insensitive α4β2-type receptors in particular, to halothane in interneurons of cerebral cortex may explain various behavioral effects of anesthetics such as hypnosis, amnesia, cognition impairment, and drowsiness.

In summary, halothane block of the α4β2-type nnAChRs at subanesthetic and anesthetic concentrations is deemed to play an important role in anesthesia via a direct action on the receptor and an indirect action to suppress release of various neurotransmitters.