Differential Effects of Thiopental on Neuronal Nicotinic Acetylcholine Receptors and P^2XPurinergic Receptors in PC12 Cells 

PC12 cell line was provided by Japanese Cancer Research Resources Bank-Cell Bank. Cells were grown in RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum and 5% horse serum plus 0.3 mg/ml glutamine, 50 U/ml penicillin, and 100 micro gram/ml streptomycin. Cells were maintained in 25-cm²flasks in a 95% air, 5% CO²atmosphere at 37 [degree sign] Celsius. For the experiment, cells were plated on collagen-coated cover slips and used after additional 2–4day culture.

Membrane currents were measured by conventional whole cell voltage clamp method. [27] Cells on the cover slips were placed in a recording bath with an approximate volume of 1.5 ml and continuously perfused at the rate of 1–2 ml/min with a standard external solution containing (in mM), NaCl, 140; KCl, 5.4; CaCl², 1.8; MgCl², 1.0; N-2-hydroxy ethylpiperazine-N'-2-ethanesulphonic acid (HEPES), 10; and glucose, 11.1 (pH was adjusted to 7.4 with NaOH). Heat-polished patch pipettes had tip resistance of 2–7 M Omega when filled with an intracellular solution containing (in mM) CsCl, 150; HEPES, 10; ethylene glycol-bis-(beta-aminoethyl ether) tetraacetic acid, 5; Mg-ATP, 2 (pH, 7.3 with CsOH). Cells were voltage clamped at -60 mV with a patch clamp amplifier (CEZ 2300, Nihon Koden, Tokyo, Japan) unless otherwise stated. Whole cell currents were filtered at 0.2 KHz with Bessel filter and digitized at 0.5 kHz in most of the cases, or they were filtered at 0.2 KHz with Gaussian filter and digitized at 1 KHz in a few cases. The currents were stored and analyzed on a computer using Pclamp software (Axon Instruments, Foster City, CA). For display of the current traces, the data were 10 times reduced by averaging 10 points to yield a single point. All experiments were performed at room temperature (22–25 [degree sign] Celsius)

Nicotine or ATP-Na2 of 30 micro Meter in the external solution was applied to cells using a rapid application technique described as the “Y-tube” method. [28] The tip of the Y-tube was made by a glass micropipette (Microcaps, 2 micro liter Drummond) with about 100 micro meter-opening and was positioned about 500 micro meter from the recorded cell. This method enabled the complete exchange of the external solution surrounding the cell around 100 ms, as estimated by recording the liquid junction current produced at an open patch pipette. The agonists with or without anesthetics were applied for 4–5 s, and each application was separated by 5 min. Thiopental was coapplied with the agonists using the same method. For preincubation with the anesthetic or the antagonists, the external solutions containing the drugs were perfused at the rate of 5 ml/min for 5 min before rapid application. Cells were perfused with the plain external solution at the same rate for 5 min to wash out the drugs from the bath after the measurement.

Nicotine of 100 or 300 micro Meter was also tested to see if higher concentration of nicotine can overcome the inhibition by thiopental.

Instantaneous current-voltage curves were obtained for nicotine 30 micro Meter alone and for nicotine with thiopental 30 micro Meter after preincubation of thiopental. A ramp pulse of +30 to -70 mV (100 mV/200 ms) was applied to a cell every 200 ms, [29] and current traces near the peak current were subtracted from those in the absence of agonist. Five cells that exhibited slow desensitization were chosen for this experiment to avoid a large decline of the current during the ramp pulse.

Drugs used in the current study included (-)-nicotine, hexamethonium, suramin (Wako, Osaka, Japan), ATP-Na², ATP-Mg (Sigma, St Louis, MO), and thiopental sodium (Tanabe, Osaka, Japan). Thiopental sodium was dissolved in distilled water to make 100 mM stock solution and diluted with the external solution to the designated concentration.

We measured the peak current and the steady current, which was defined as the average of the preceding 50 points at 4 s during agonist application. Because nicotine-elicited currents declined with each application of agonist, the response in the presence of thiopental was compared with the average of the elicited current before and after thiopental application. To verify this procedure, nicotine was applied successively three times with an interval of 5 min, and the second response was compared with the average of the first and third responses. The same procedure was applied for the ATP-induced current.

The decaying phases of the nicotine-induced current were fitted either to a single or a double exponential function of the following form by simplex method using Axograph software (Axon Instruments, Foster City, CA):Equation 1where I is the total peak current, I^finalis the residual current during the steady state condition, Ii is the peak current amplitude of the each component, and tauⁱis the time constant of the corresponding component. Goodness of fit was compared by chi-square test between a single and a double exponential models. The time constants of the decay were measured for the baseline responses just before the thiopental administration (Baseline) and the responses in the presence of thiopental (Thiop). These measurements were done for the experiments with thiopental preincubation and for the ones wherein nicotine alone was applied successively. The ratios of the time constants for two successive responses (Thiop/Baseline) were calculated to see the time-dependent changes and the effects of thiopental. Desensitization was also evaluated by calculating the percent decay of the current (% current decay) defined by the equation [30]:Equation 2where Ipeak denotes the peak current and I^steadydenotes the steady current.

Data are expressed as mean +/- SEM; unpaired t test and analysis of variance (ANOVA) followed by Dunnett's test were used to estimate the significance when appropriate. P less than 0.05 was considered to be significant.

Nicotine elicited inward currents at -60 mV. The nicotine-induced current decayed rapidly during agonist application, consistent with other studies, as a result of desensitization. [13,29,31] The peak current varied greatly with recorded cells, from 50 to 800 pA. When nicotine was applied successively, the peak and steady current declined with each application, so that the third response accounted for 72 +/- 9% for the peak current and 66 +/- 13% for the steady current of the first response (n = 7). The magnitude of decline seemed to depend on the interval between the applications of the agonist, i.e., the short interval enhanced the decline. Therefore, some reversible processes such as desensitization and reversal from desensitization should contribute to this phenomenon at least in part. Because the second response was close to the average of the first and the third response, 100 +/- 3% for the peak current and 102 +/- 6% for the steady current (n = 7), the effects of thiopental were evaluated by comparison with the average of the control responses before and after administration of thiopental.

The nicotine-induced current was strongly depressed by 100 micro Meter hexamethonium, a competitive antagonist for neuronal nAch receptors. The peak and steady current was 17 +/- 3% and 10 +/- 2% of the average of the control responses when 100 micro Meter hexamethonium was preperfused and coapplied (n = 4).

Thiopental alone induced no current responses at 100 or 300 micro Meter in tested cells (n = 6 for both). 300 micro Meter is about 10 times higher than reported EC50 for general anesthesia and the maximum concentration used in the present study. [32] Thiopental, 3–300 micro Meter, when coapplied with nicotine, inhibited the peak and steady current in a dose-dependent manner (Figure 1). The inhibition was much stronger for the steady current than for the peak current. Thiopental, 100 micro Meter, almost completely abolished the steady current. Regarding reversibility of inhibition, the ratio of the postcontrol responses after thiopental to the precontrol values was not different from that of three successive nicotine applications, indicating that nicotine responses recovered fully from thiopental's inhibition after a 5-min interval (Figure 2). IC50s for the peak and steady current were 56.7 +/- 10.1 and 7.4 +/- 1.4 micro Meter, respectively (Figure 3(A)).

When cells were preincubated with thiopental and then nicotine and thiopental were coapplied, inhibition of the peak current was still much smaller than that of the steady current. The relative peak currents in the presence of thiopental with or without preincubation were not different from each other at any dose of thiopental except for 3 micro Meter, wherein the relative peak current without preincubation was significantly smaller than that with preincubation. The magnitudes of blockade of the steady currents were also similar regardless of the presence or absence of preincubation. IC50s for the peak and steady current were 49.2 +/- 11.1 and 6.1 +/- 0.6 micro Meter, when thiopental preincubation was performed (Figure 3(B)). There were no statistically significant differences in IC50 or Hill coefficient for the peak and steady current measured with thiopental preincubation when compared with the values obtained without pre-incubation.

The decay phases of the nicotine-induced current with and without thiopental were well fitted to single exponential functions. Because fitting to double exponential curve did not improve the adequacy of the fit, single exponential models were used. Although the absolute values of the time constants of the decay varied greatly cell to cell for the control responses (from 0.73 to 10.79 s), they did not change significantly with the repeated nicotine applications as judged by the ratio of the time constants of two successive nicotine responses. There was a tendency that the time constants decreased with increasing concentration of thiopental, although these changes did not reach a statistical significance because of the great variability. When changes in the ratio of the time constants were analyzed with respect to thiopental concentrations, thiopental clearly produced a dose-dependent decrease in the ratio of Thiop/Baseline, indicating that the anesthetic accelerated the decay rate in a dose-dependent manner (Table 1). These changes were fitted to the empirical Hill equation (ratio = IC50ⁿ/(Cⁿ+ IC50ⁿ), where C is thiopental concentration; n is Hill coefficient). Fitting was found to be very good (r²= 0.96) and gave a IC50 of 13.3 micro Meter +/- 3.2 and an n of 1.05 +/- 0.27.

The % current decay remained unchanged with the repeated applications of nicotine. Thiopental dose-dependently increased % current decay (Table 2). Increases in % current decay from the value for nicotine alone were analyzed by fitting to the following equation: Equation 3 where 54.8 is the mean value at the control condition, 45.2 is the maximal value, C is thiopental concentration, and n is Hill coefficient. The equation described the thiopental-induced increase in % current decay very well (r²= 0.98). EC50 and Hill coefficient were estimated to be 9.1 +/- 1.9 micro Meter and 0.73 +/- 0.14, respectively.

These analyses showed that thiopental dose-dependently accelerated the current decay and that thiopental concentrations producing the half maximal effects were slightly higher but comparable to IC50 for inhibition of the steady current.

When cells were preincubated with thiopental, 100 micro Meter, and then only nicotine was applied, the peak current was slightly reduced, but inhibition of the steady current was minimal (Figure 4(A and B)). Blockade by preapplied thiopental was significantly smaller for the peak and steady current than that by preincubation plus coapplication of thiopental, suggesting that channel opening by nicotine enhanced thiopental's effect (Figure 4(C)).

The nicotine-induced current exhibited strong inward rectification and reversed around 0 mV, as previously described. [13,31,33] Thiopental depressed the inward current without any obvious changes in the reversal potential (Figure 5). The magnitudes of depression of the inward current were 68 +/- 4, 77 +/- 2, and 71 +/- 4% at -30, -50, and -70 mV from the analysis of five pairs of the current-voltage curves. These values were statistically insignificant among three membrane potentials, suggesting that the thiopental's inhibition is voltage-independent from -30 to -70 mV. However, thiopental's effects seemed small at the more depolarizing potential.

When the dose of nicotine was increased to 100 or 300 micro Meter, the magnitudes of depression of the peak or steady current by thiopental, 30 micro Meter, the magnitudes of depression of the peak or steady current by thiopental, 30 micro Meter were not significantly different from those observed at the nicotine dose of 30 micro Meter. Therefore, the inhibition by thiopental was not reversed by the increased doses of nicotine (Figure 6).

ATP, 30 micro Meter, evoked a slowly desensitizing inward current mainly carried by sodium in this condition at -60 mV. The ATP-induced current was almost completely abolished by 100 micro Meter of suramin, a nonselective purine P²antagonist. [22] In the presence of suramin, the peak and steady current was 1.3 +/- 0.7 and 1.4 +/- 0.7 and 1.4 +/- 0.8% of the average of the control responses (n = 4).

Thiopental, when preperfused for 5 min and then coapplied with ATP, did not affect the ATP-induced current at 30 micro Meter but slightly reduced the current at 100 micro Meter. Reduction in the steady current by 100 micro Meter of thiopental was slight but statistically significant (Figure 7(A and B)). Thiopental induced no remarkable changes in the current-voltage relationship of the ATP-induced peak current at membrane potential from -70 to 30 mV (Figure 8).

The results of the present study demonstrate that thiopental at clinically relevant concentrations inhibited the neuronal nAchR-mediated current but not the P^2Xpurinergic receptor-mediated response in PC12 cells.

Thiopental at high concentrations did not induce any current consistent with the absence of functional GABA^Areceptors in PC12 cells as previously described, [16] whereas high concentration of barbiturates elicited an inward current in adrenal chromaffin cell under the similar conditions. [8,10] Therefore, a potential influence from direct effects of thiopental on GABA^Areceptor can be excluded in this study.

Thiopental reversibly depressed the nicotine-induced current and augmented desensitization in a dose-dependent manner. The reversal potential of the nicotine-induced current was not changed, and inhibition was voltage-independent at membrane potential of -30 to -70 mV. The inhibitory effects were not reversed by the higher concentration of nicotine, suggesting that the effects of thiopental are not competitive at the nicotine-binding sites.

The inhibitory effects were greater for the steady current than for the peak current, and this difference cannot be explained by the inadequate resolution of the peak current. It is possible that the apparent peak current is lowered by the slow exchange of solutions when the current decays rapidly. However, the same or greater influence should present in the presence of thiopental because the current decay is more rapid in this condition, i.e., inhibition in the peak current tends to be overestimated but not to be underestimated in the presence of thiopental. Therefore, difference in sensitivity between the peak and steady current would be even greater when a more rapid solution exchange was used. In addition, this difference cannot be explained by the slow onset of thiopental's effects as a result of the slow application because preincubation of thiopental did not potentiate the magnitude of inhibition of the peak current significantly.

Lack of significant effects of preincubation is attributable to lack of inhibition of closed channels and indicates preferential inhibition of open channels. [34] When nicotine was applied after preincubation of thiopental, inhibition of peak current was only 25 +/- 4%, much smaller than 62 +/- 9% for preincubation plus coapplication. This difference also indicates that channel opening is needed for thiopental to exert its effects fully. However, a certain degree of blockade of closed channels appeared to exist because preperfusion alone caused a small reduction in the peak current. Recovery from this small reduction in the wave form in Figure 4(A) reflects the relief of thiopental's inhibition on closed channels, although the exchange of solutions was too slow to analyze dissociation kinetics. It may be that an apparent reduction in the peak current by preincubated thiopental is caused by block of open channels occurring in the period of coexistence of preperfused thiopental and nicotine but not by block of closed channels before agonist application. A relatively slow exchange time of the drug application system allowed a sufficient time for two of these agents to coexist. Additionally, poor time resolution of the recording condition prevents the definitive conclusions. Therefore, it is not clear how much thiopental acts on closed channels, although most inhibition is exerted on open channels.

Preferential inhibition on open channels is consistent with the results of single channel recording of neuronal nAchRs using bovine chromaffin cells, which demonstrated that pentobarbitone and methohexitone principally shorten the mean open time and induce “flickering”. [8,9] The decrease in the exponential time constants of the current decay by thiopental also agree with the decrease in mean channel open time. [35] Barbiturates' inhibition of open channels has been also reported for the non-NMDA receptor-mediated current in rat cortical neuron. [36]

The decay phases of neuronal nAchRs have been analyzed by fitting to one or two exponential functions in several papers. [30,31,37,38] Several factors, such as the subunit composition of the receptors, the speed of solution exchange, and agonist concentration, seem to influence which model better describes the current decay. In our study, the rate of the current decay varied greatly among cells, and the variability may reflect heterogeneity of the nAchRs in PC12 cells. The presence of the cells whose time constants of decay were rather high seemed to favor the single exponential model. This model has been used in the previous reports using the perfusion system with the similar exchange time. [31,37] It is also possible that the fast phase of decay was obscured because of the relatively slow exchange of solutions. Despite the great variability in the baseline, thiopental was shown to accelerate the current decay by calculating the ratio of Thiop-to-Baseline, and this notion was further supported by the increases in % current decay by thiopental.

With respect to the site of action, preferential inhibition of open channels does not necessarily mean that thiopental acts on the ion-permeating pore. Voltage-independence of inhibition at negative membrane potentials suggests a different mechanism(s) of block from charged channel blockers such as local anesthetics. [39] However, it does not eliminate the pore as a possible site of action because uncharged thiopental molecules, which account for a half of all molecules at pH 7.4, could cause the inhibition.

IC50s were 56.7 and 7.4 micro Meter for the peak and steady current in the absence of preincubation. IC50 for the steady current in this study is close to the reported IC50 (7.8 micro Meter) for thiopental's inhibition of the nicotine-induced catecholamine release in dog adrenal medulla, [6] indicating that block of nicotine receptor is responsible for inhibition of catecholamine release. EC50 for general anesthesia is reportedly 25 micro Meter. [32] Inhibition by thiopental is notable for the peak and steady current at this concentration.

Although ethanol has been reported to suppress the ATP-induced current in frog dorsal root ganglion (DRG), [40,41] thiopental exhibited little effect on the ATP-induced current in PC12 cells. Several subtypes of P^2Xreceptors have been identified and found to distribute differentially in central and peripheral nervous systems. [42,43] It is likely that the dominant subunits in frog DRG and PC12 cells are different because they exhibit different sensitivities to agonists. [21,22,42] Therefore, difference in the subunits and difference in the agents can explain this discrepancy. P sub 2X receptors in rat sympathetic neuron from superior cervical ganglia mostly resemble those in PC12 cells. [29] Therefore, it is suggested that thiopental does not much affect P^2Xreceptors in rat sympathetic neurons.

Although the P^2Xreceptors function as a ligand-gated ion channel, there is no primary sequence homology between the P^2Xreceptor and other ligand-gated ion channels such as nicotinic, glutamate, GABA^A, and glycine receptor, and the structure of the P sub 2X receptor is considered to be very different from that of other ligand-gated ion channels. [42] The finding that P^2Xreceptors are much less sensitive to thiopental than neuronal nAchRs indicates that P^2Xreceptors lack the structure wherein thiopental can act on with high affinity.

We found that neuronal nAchRs in PC12 cells are very sensitive to thiopental, whereas this is not the case for P^2Xpurinergic receptors. The former finding is consistent with the previous studies, demonstrating high sensitivity of molluscan neuronal nAchRs receptors to various anesthetics. [44] In molluscan, neuronal nAchRs exhibit different characteristics from those of mammalian receptors, i.e., some of molluscan receptors show extraordinarily high sensitivity to nicotinic agonists and are permeable to Cl sup -. [44] Neuronal nAchRs receptors in PC12 cells resemble the receptors of sympathetic ganglia neuron in terms of ion selectivity, current-voltage relationship, pharmacology, putative subunit composition. [12–14,24,25] Therefore, it is conceivable that thiopental suppresses synaptic transmission in sympathetic ganglia by inhibition of neuronal nAchRs. However, further studies are needed to define anesthetic effects on central nAchRs and the significance of such effects for general anesthesia because central nAchRs are heterogeneous and different from ganglionic counterparts in terms of subunit composition and pharmacology. [2]

The authors thank Dr. Ken Nakazawa (Division of Pharmacology, National Institute of Health Sciences) and Dr. Susumu Kawamoto (Department of Bacteriology, Yokohama City University School of Medicine) for their advice.