General anesthetics have additive actions on three ligand gated ion channels

Abstract

Background: The purpose of this study was to determine whether pairs of compounds, including general anesthetics, could simultaneously modulate receptor function in a synergistic manner, thus demonstrating the existence of multiple intraprotein anesthetic binding sites.

Methods: Using standard electrophysiologic methods, we measured the effects of at least one combination of benzene, isoflurane (ISO), halothane (HAL), chloroform, flunitrazepam, zinc, and pentobarbital on at least one of the following ligand gated ion channels: N-methyl-D-aspartate receptors, glycine receptors and gamma-aminobutyric acid type A receptors.

Results: All drug-drug-receptor combinations were found to exhibit additive, not synergistic modulation. ISO with benzene additively depressed N-methyl-D-aspartate receptors function. ISO with HAL additively enhanced glycine receptors function, as did ISO with zinc. ISO with HAL additively enhanced gamma-aminobutyric acid type A receptors function as did all of the following: HAL with chloroform, pentobarbital with ISO, and flunitrazepam with ISO.

Conclusion: The simultaneous allosteric modulation of ligand gated ion channels by general anesthetics is entirely additive. Where pairs of general anesthetic drugs interact synergistically to produce general anesthesia, they must do so on systems more complex than a single receptor.

Figure 1

Inhibition of NMDA receptor function by isoflurane and benzene. a) 2-electrode voltage clamp recordings of NMDA/glycine responses (100 μM NMDA and 10 μM glycine) in Xenopus laevis oocytes before during and after the application of 1 mM isoflurane + 150 μM benzene. Bars above the current traces indicate the duration of agonists application (black) and the duration of the modulator application (gray). Calibration bars indicate the amplitude and duration of the responses. b) Normalized concentration response relationship for the inhibition of NMDA receptor function by isoflurane and benzene. Symbols represent the mean±SEM inhibition of responses to 100 μM NMDA and 10 μM glycine determined from 6 cells. The IC₅₀s and Hill coefficients for isoflurane and benzene were 2.4 ± 0.3 mM; −1.0 ± 0.1 and 0.34 ± 0.04; −0.96 ± 0.09. respectively. c) Inhibition of NMDA receptor function by a combination of isoflurane and benzene did not differ for 300 μM benzene and a 50/50 mixture (1 mM isoflurane + 150 μM benzene). Isoflurane (2mM) alone was slightly more effective as an inhibitor than the 50/50 mixture. * P < 0.05

Figure 2

Potentiation of glycine receptor function by isoflurane, halothane and zinc. a) 2-electrode voltage clamp recordings of glycine responses EC_5–10 responses in Xenopus laevis oocytes before, during and after the application of isoflurane and/or halothane. Bars above the current traces indicate the duration of agonist application. Grey represents the duration of the anesthetic (75 μM isoflurane or 62.5 μM halothane) application and black represents the duration of the anesthetic (37.5 μM isoflurane and/or 31.2 μM halothane). Calibration bars indicate the amplitude and duration of the responses. b) Potentiation of glycine receptor function by isoflurane ([ISO] = 75 μM) and halothane, ([HAL] = 62.5 μM) did not differ from [ISO]/2+[HAL]/2. c) Potentiation of glycine receptor function by a combination of isoflurane ([ISO] = 75 μM) and zinc ([Zinc] = 0.1 μM): There was no significant difference between the potentiation by [ISO] and [ISO]/2+[Zinc]/2, but [ISO]/2+[Zinc]/2 was slightly more effective as a potentiator than [Zinc] alone. * P < 0.05.

Figure 3

Potentiation of GABA_A receptor function by isoflurane, halothane, flunitrazepam and pentobarbital. a) 2-electrode voltage clamp recordings of GABA responses EC_5–10 responses in Xenopus laevis oocytes before during and after the application of isoflurane and/or halothane. Bars above the current races indicate the duration of agonist application. The grey bars above these represent the duration of 120 μM isoflurane or 114 μM halothane application; and the black bars indicate the duration of 60 μM isoflurane and/or 57 μM halothane. b) Potentiation of GABA_A receptor function by a combination of isoflurane and halothane: [ISO] = 120 μM, [HAL] = 114 μM. The potentiation by [ISO] or [HAL] did not differ significantly from that by [ISO]/2+[HAL]/2. c) Potentiation of GABA receptor function by a combination of isoflurane and flunitrazepam: [ISO] = 120 μM, [FNZ]=500nM. There was no significant difference between the potentiation by [ISO] and [FNZ] and [ISO]/2+[FNZ]/2. d) Potentiation of GABA receptor function by a combination of isoflurane and pentobarbital: [ISO] = 120 μM, [PB]=15μM. [PB] did not differ from potentiation by [ISO]=120μM and [ISO]/2+[PB]/2, but [ISO]/2+[PB]/2 was slightly more effective as potentiator than [PB] alone. *** P = 0.007

Figure 4

Responses in HEK-293 cells voltage clamped at −60mV expressing α1β2γ2s GABA_A receptor subunits to 0.3 – 1000 μM GABA are modulated by 220 μM halothane and 900 μM chloroform (1 MAC of each). a) Halothane and chloroform enhance the amplitude of currents when applied together at lower (<10 μM) but depressed at greater concentrations of GABA. Calibration bars indicate the amplitude and duration of the responses. b) Halothane enhances GABA_A receptor function by reducing the GABA EC₅₀. c) Cloroform enhances GABA_A receptor function by reducing GABA EC₅₀. d) Halothane reduces maximal GABA_A receptor function. e) Chloroform reduces maximal GABA_A receptor function. f) & g) Halothane and chloroform are additive in their ability to decrease GABA EC₅₀. C₅₀ and n were determined using the methods described and plotted in the range 0 < θ ≤ 1. Both C₅₀ and n did not significantly deviate from unity indicating that there is no significant synergism or antagonism between the two anesthetics in the reduction of GABA_AR EC₅₀.