Mounting electrophysiological evidence indicates that certain general anesthetics, volatile anesthetics in particular, depress excitatory synaptic transmission by presynaptic mechanisms. We studied the effects of representative general anesthetics on voltage-gated Na ϩ currents (I Na ) in nerve terminals isolated from rat neurohypophysis using patch-clamp electrophysiological analysis. Both isoflurane and propofol inhibited I Na in a dose-dependent and reversible manner. At holding potentials of Ϫ70 or Ϫ90 mV, isoflurane inhibited peak I Na with IC 50 values of 0.45 and 0.56 mM, and propofol inhibited peak I Na with IC 50 values of 4.1 and 6.0 M, respectively. Isoflurane (0.8 mM) did not significantly alter the V 1/2 of activation; propofol caused a small positive shift. Isoflurane (0.8 mM) or propofol (5 M) produced a negative shift in the voltage dependence of inactivation. Recovery of I Na from inactivation was slower from a holding potential of Ϫ70 mV than from Ϫ90 mV; isoflurane and propofol further delayed recovery from inactivation. In conclusion, the volatile anesthetic isoflurane and the intravenous anesthetic propofol inhibit voltage-gated Na ϩ currents in isolated neurohypophysial nerve terminals in a concentration-and voltage-dependent manner. Marked effects on the voltage dependence and kinetics of inactivation and minimal effects on activation support preferential anesthetic interactions with the fast inactivated state of the Na ϩ channel. These results are consistent with direct inhibition of oxytocin and vasopressin release from the neurohypophysis by isoflurane and propofol. Inhibition of voltage-gated Na ϩ channels may contribute to the presynaptic effects of general anesthetics on nerve terminal excitability and neurotransmitter release.
BACKGROUND AND PURPOSECardiac toxicity is a major concern in drug development and it is imperative that clinical candidates are thoroughly tested for adverse effects earlier in the drug discovery process. In this report, we investigate the utility of an impedance-based microelectronic detection system in conjunction with mouse embryonic stem cell-derived cardiomyocytes for assessment of compound risk in the drug discovery process. EXPERIMENTAL APPROACHBeating of cardiomyocytes was measured by a recently developed microelectronic-based system using impedance readouts. We used mouse stem cell-derived cardiomyocytes to obtain dose-response profiles for over 60 compounds, including ion channel modulators, chronotropic/ionotropic agents, hERG trafficking inhibitors and drugs known to induce Torsades de Pointes arrhythmias. KEY RESULTSThis system sensitively and quantitatively detected effects of modulators of cardiac function, including some compounds missed by electrophysiology. Pro-arrhythmic compounds produced characteristic profiles reflecting arrhythmia, which can be used for identification of other pro-arrhythmic compounds. The time series data can be used to identify compounds that induce arrhythmia by complex mechanisms such as inhibition of hERG channels trafficking. Furthermore, the time resolution allows for assessment of compounds that simultaneously affect both beating and viability of cardiomyocytes. CONCLUSIONS AND IMPLICATIONSMicroelectronic monitoring of stem cell-derived cardiomyocyte beating provides a high throughput, quantitative and predictive assay system that can be used for assessment of cardiac liability earlier in the drug discovery process. The convergence of stem cell technology with microelectronic monitoring should facilitate cardiac safety assessment. AbbreviationsBRI, beating rhythm irregularity; hERG, human ether a go go; MEA, multi elelctrode array; mESCC, mouse embryonic stem cell
Cardiac safety testing of lead drug candidates is an important part of the drug discovery and development process. All new chemical entities need to be subjected to extensive preclinical assessment for cardiac liability, especially for a potentially fatal form of ventricular arrhythmia referred to as Torsades de Pointes. We have developed an innovative label-free, real-time system, the xCELLigence RTCA Cardio System, which is designed to monitor contractility of cardiomyocytes based on impedance measurement. The assay is performed using specially designed microtiter plates that are integrated with gold microelectrodes. The system was validated using mouse embryonic stem cell-derived cardiomyocytes, human-induced pluripotent stem cell-derived cardiomyocytes, and rat neonatal primary cardiomyocytes by applying a variety of tool compounds and drugs with known mechanisms of action. Our data show that the time resolution in the assay can provide important information about compound action. Furthermore, the impedance-based beating profile in response to compound treatment can provide mechanistic toxicity information regarding the target being modulated and may be able to flag pro-arrhythmic compounds. We believe the real-time and kinetic aspect of this technology combined with beat-to-beat measurement of cardiomyocyte contraction would make this instrument an important part of preclinical cardiac safety assessment.
Volatile anesthetics inhibit mammalian voltage-gated Na ϩ channels, an action that contributes to their presynaptic inhibition of neurotransmitter release. We measured the effects of isoflurane, a prototypical halogenated ether volatile anesthetic, on the prokaryotic voltage-gated Na ϩ channel from Bacillus halodurans (NaChBac). Using whole-cell patch-clamp recording, human embryonic kidney 293 cells transfected with NaChBac displayed large inward currents (I Na ) that activated at potentials of Ϫ60 mV or higher with a peak voltage of activation of 0 mV (from a holding potential of Ϫ80 mV) or Ϫ10 mV (from a holding potential of Ϫ100 mV). Isoflurane inhibited I Na in a concentration-dependent manner over a clinically relevant concentration range; inhibition was significantly more potent from a holding potential of Ϫ80 mV (IC 50 ϭ 0.35 mM) than from Ϫ100 mV (IC 50 ϭ 0.48 mM). Isoflurane positively shifted the voltage dependence of peak activation, and it negatively shifted the voltage dependence of end steady-state activation. The voltage dependence of inactivation was negatively shifted with no change in slope factor. Enhanced inactivation of I Na was 8-fold more sensitive to isoflurane than reduction of channel opening. In addition to tonic block of closed and/or open channels, isoflurane enhanced use-dependent block by delaying recovery from inactivation. These results indicate that a prokaryotic voltage-gated Na ϩ channel, like mammalian voltage-gated Na ϩ channels, is inhibited by clinical concentrations of isoflurane involving multiple state-dependent mechanisms. NaChBac should provide a useful model for structure-function studies of volatile anesthetic actions on voltage-gated ion channels.
Background Inhibition of voltage-gated Na+ channels (Nav) is implicated in the synaptic actions of volatile anesthetics. We studied the effects of the major halogenated inhaled anesthetics (halothane, isoflurane, sevoflurane, enflurane and desflurane) on Nav1.4, a well characterized pharmacological model for Nav effects. Methods Na+ currents (INa) from rat Nav1.4 α-subunits heterologously expressed in Chinese hamster ovary cells were analyzed by whole cell voltage-clamp electrophysiological recording. Results Halogenated inhaled anesthetics reversibly inhibited Nav1.4 in a concentration- and voltage-dependent manner at clinical concentrations. At equi-anesthetic concentrations, peak INa was inhibited with a rank order of desflurane > halothane ≈ enflurane > isoflurane ≈ sevoflurane from a physiological holding potential (−80 mV). This suggests that the contribution of Na+ channel block to anesthesia might vary in an agent-specific manner. From a hyperpolarized holding potential that minimizes inactivation (−120 mV), peak INa was inhibited with a rank order of potency for tonic inhibition of peak INa of halothane > isoflurane ≈ sevoflurane > enflurane > desflurane. Desflurane produced the largest negative shift in voltage-dependence of fast inactivation consistent with its more prominent voltage-dependent effects. A comparison between isoflurane and halothane showed that halothane produced greater facilitation of current decay, slowing of recovery from fast inactivation, and use-dependent block than isoflurane. Conclusions Five halogenated inhaled anesthetics all inhibit a voltage-gated Na+ channel by voltage- and use-dependent mechanisms. Agent-specific differences in efficacy for Na+ channel inhibition due to differential state-dependent mechanisms creates pharmacologic diversity that could underlie subtle differences in anesthetic and nonanesthetic actions.
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