Etomidate (ET), a carboxylated imidazole, is a widely used intravenous general anesthetic due to its desirable hemodynamic characteristics. However, the molecular mechanisms underlying action of etomidate on central nervous system remain unclear. In the mammalian central nervous system, most general anaesthetics are demonstrated to act on multiple molecular sites.1-7 Both voltage-gated and ligand-gated membrane channels are suggested targets for general anesthetic action.8-19 Pharmacological action on ion channels is fundamental to effects as well as side-effects of anaesthetic agents.1,3-6 The alteration of ion channel function by anaesthetic agents consequently determines their clinical effects. Numerous studies were involved in the effects of general anesthetics on potassium channels. The inhibition of neuronal voltage-dependent K+ currents by some intravenous anesthetic agents has been reported, for example, in the frog node of Ranvier, in demyelinated axons of Xenopus laevis, in oocytes expressing rat neuronal voltagedependent K+ channels. Etomidate had been demonstrated to inhibit potassium currents in SH-SY5Y cells.20 However, few studies have addressed the specific interactions between etomidate and the voltage-dependent potassium channels in natural neurons. In this study, we investigated the action of etomidate on delayed rectifier potassium current (Ik(dr)) and transient outward potassium current (Ik(a)) in acutely dissociated rat hippocampal pyramidal neurons using whole-cell patch clamp technique.
The preparation of rat hippocampal neurons was approved by the appropriate animal care and use committee. Dissociated neurons were prepared as decribed by Kay and Wong21 with slight modifications. In brief, hippocampi were removed from 7- to 10-day-old Wistar rats and cut into 400 μm thick slices. Slices were incubated in oxygenated artificial cerebrospinal fluid (ACSF) for 1 hour at room temperature. The slices were then incubated in the ACSF containing 0.5 g/L trypsin for 30 minutes, followed by protease E (0.5 g/L) for 30 minutes at 32°C. After enzyme treatment, the slices were rinsed several times with enzyme-free solution and then dissociated mechanically with a graded series of fire-polished Pasteur pipettes. Subsequently, cells were allowed to settle for 10-15 minutes on the bottom of the recording chamber. CA1 pyramidal neurons were easily identified morphologically with pyramidal-shaped soma and one apical dendrite.22 Neurons with bright and smooth appearance and no visible organelles were selected for recording.
Solutions and drugs
The ACSF contained (in mmol/L): NaCl 126, KCl 5, NaH2PO4 1.25, MgSO4 2, NaHCO3 26, glucose 10, CaCl2 2, and was titrated to a pH of 7.20 with HCl. The pipette solution contained (in mmol/L): KCl 145, KOH 5, N-[2-hydroxyethyl]-piperazine-N'-[2-ethanesulfonic acid] (HEPES) 10, Ethylene glycol-bic[2-aminoethylether] N,N,N',N'-tetraacetic acid (EGTA) 10, adenosine triphosphate disodium salt (Na2ATP) 2. The pH was adjusted to 7.2 with KOH. The standard external solution for whole-cell recordings contained: (in mmol/L): NaCl 150, KCl 5, MgCl2 1.1, CaCl2 2.0, glucose 10, HEPES 10, tetrodotoxin (TTX) 0.001, the solution was adjusted to pH 7.3.
Recording were made in whole-cell voltage-clamp configuration. The electrodes were pulled from borosilicate glass pipettes and filled with pipette solution. Etomidate was dissolved in the external solution and the bath was applied for 5 minutes. Currents were recorded with an EPC-10 amplifier, filtered at 3 kHz, sampled at 20-50 kHz, and stored on a computer by Pulse 8.5 software (HEKA, Germany). Electrode resistances were typically 3-5 Mη in the bath. Series resistance was compensated (>80%). Liquid junction potential less than 3 mV was not corrected. All experiments were performed at room temperature (21°C-24°C). Etomidate, HEPES, EGTA, Na2ATP, and TTX were purchased from Sigma Chemical Co. (St. Louis, USA). Other chemicals were obtained from Beijing Chemical Factory (Beijing, China).
The total outward current elicited consists of two components: the sustained potassium current (Ik(dr)) and the transient outward potassium channel (Ik(a)). Ik(dr) was separated from the transient outward potassium by applying a 50 ms pulse to -50 mV immediately prior to each depolarization step in order to inactivate Ik(a). Ik(a) was obtained by subtracting the total outward current with Ik(dr) (Figure 1). Ik(a) was measured at its peak and Ik(dr) was measured 200 ms after the onset of the test pulse. In the blank control without etomidate, Ik(dr) and Ik(a) were decreased by (4.2±2.7)% and (3.8±4.5)%, respectively, after 15 minutes current recording. So the run-down of current was negligible.
All data were analyzed using Pulse 8.5 software (HEKA Elektronik, Germany). Results were expressed as mean ± standard error (SE). Student's paired two-tail t-test was employed for data analysis. Significance was assigned at a P< 0.05.
Blockade of etomidate on Ik(dr) and Ik(a)
The current (Ik(dr)) was studied with a 150-ms pulse from a holding potential of -50 mV to +40 mV, following a 50 ms interval after a 150 ms conditioning prepulse to -110 mV. The total voltage-dependent potassium currents were evoked by a 150 ms pulse from a holding potential of -50 mV to +40 mV, following a 150 ms conditioning prepulse to -110 mV. The Ik(a) was obtained by subtracting Ik(dr) from the total current. The concentration-response curve for the action of etomidate on Ik(dr) reveals an IC50 value of 5.4 μmol/L, the Hill coefficients of 2.45, with maximal effect (about 49.2%) reached at 100 μmol/L (Figure 2). At the concentration of 100 μmol/L, Ik(a) decreased only by (3.7±4.3)% (n=8, P >0.05). This result suggested that Etomidate inhibited Ik(dr) in a concentration-dependent manner, but have no effect on Ik(a).
Effects of etomidate on activation kinetics of Ik(dr)
Figure 3 shows the current density-voltage (I-V) relationship for Ik(dr) and steady-state activation curve before and after etomidate application, respectively. Current density (pA/pF) for each cell was obtained by dividing the current by cell capacitance. Etomidate decreased the current density of Ik(dr). We studied the effect of etomidate on the activation kinetics of Ik(dr). As shown in Figure 3B, the steady-state activation curve was negatively shifted by etomidate application. The current was half activated at (17.3±1.5) mV and (10.7±2.9) mV (n=8, P <0.05) in the absence and presence of etomidate, respectively. Thus, the application of 10 μmol/L etomidate produced a 6.6 mV negative shift of steady-state activation. The results suggested that etomidate inhibited Ik(dr), significantly modifying its activation properties. In contrast, the effect of etomidate on activation curve of Ik(a) have no stastistical significance (data not shown).
Effects of etomidate on inactivation kinetics of Ik(dr)
The effects of etomidate on the inactivation properties of Ik(dr) was shown in Figure 4. The half-maximal inactivation voltage was (—18.3±2.2) mV and (-45.3±9.4) mV (n=8, P <0.05) in the absence or presence of 10 μmol/L etomidate, respectively. Thus, the application of 10 μmol/L etomidate produced a 27-mV hyperpolarizing shift of steady state inactivation of Ik(dr). The results suggested that etomidate inhibited Ik(dr). significantly modifying its inactivation properties. Etomidate mainly affected the inactivation kinetics compared with activation kinetics. In contrast, the effect of etomidate on inactivation curve of Ik(a) have no stastistical significance (data not shown).
In the current study, we investigated the effects of etomidate on potassium currents in pyramidal neurons acutely isolated from rat hippocampus and found that Ik(dr) was inhibited by etomidate in a concentrationdependent manner, albeit etomidate has no effect on IK(a) at a concentration up to 100 μmol/L. IC50 value of etomidate towards Ik(dr) is 5.4 μmol/L, which is in the range of clinical relevant concentrations. It is supposed that etomidate inhibit significantly Ik(dr) at the clinical anesthetic concentrations.
In our study, etomidate at the concentration of 10 μmol/L caused a hyperpolarizing shift of the activation curve of Ik(dr) and significantly shifted the inactivation curve of Ik(dr) to negative potential. It suggested that etomidate at clinically relevant concentrations significantly enhanced activation and inactivation of Ik(dr). In rat hippocampal pyramidal neurons, we also found the enhancement of inactivation of Ik(dr) by 10 μmol/L etomidate is more potent than that of activation. So it is supposed that etomidate inhibits Ik(dr) mainly through acceleration of inactivation of Ik(dr). We also noted that, the inhibition rate of etomidate on Ik(dr) only reached about 49.2% even at the maximal concentration of drug under study. Because voltage-activated K+ channels in rat pyramidal neurons are composed of various K+ channel subtypes such as Kv1, Kv2, Kv3 and Kv4,23 etomidate is probably selective for some delayed rectifier K+ channel subtypes in hippocampal cells. Further study needs to be done to understand the channel selectivity of etomidate.
General anesthetics differentially affect various families of potassium channels, and some potassium channels are suggested to be potential targets for anesthetics and alcohols.20,24 Neuronal voltage-dependent K+ channels are important for various cellular functions in the central nervous system. IK(dr) plays an important role in repolarization of action potential and blockade of IK(dr) might depolarize the neuron. Thus the inhibition of IK(dr) in the current study would presumably lead to increased neuronal excitability. If this effect is indeed relevant to the generation of anesthesia, the most possible explanation would be by activation of inhibitory pathways within the brain. In addition, increased neuronal excitability attributable to inhibition of potassium conductance may contribute to the excitatory phase of general anesthesia induction.
Friederich reported that etomidate reversibly inhibited voltage-dependent K+ currents from human neuroblastoma cells in a concentration-dependent manner.20 Effects of etomidate already occurred at clinical concentrations and IC50 values of K+ current inhibition correlated with clinical concentrations.20 Combined with our results, it suggested that these voltage-dependent K+ channels appeared to be a biophysical target of intravenous anesthetics that may help to establish molecular determinants of anesthetic potency.
In summary, our study demonstrated that etomidate inhibited Ik(dr) but not IK(a) from acutely dissociated rat hippocampal pyramidal neurons in a concentrationdependent manner. The results of our study suggested that understanding actions of etomidate on IK(dr) might help to elucidate molecular mechanisms underlying drug action. We hypothesized that this inhibition may be associated with general anesthesia and/or the excitatory effects during anesthesia with etomidate. It has been shown that anesthetic actions may be species-specific and subtype-specific.25 The underlying detailed mechanisms of actions of etomidate need further investigation.
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