1 School of Biomedical Sciences and 2 Academic Unit of Anaesthesia, University of Leeds, Leeds LS2 9JT, UK
* Corresponding author. E-mail: s.m.harrison{at}leeds.ac.uk
Accepted for publication May 16, 2005.
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Abstract |
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Methods. Ventricular myocytes were isolated using a standard collagenase/protease dispersion technique and superfused with a physiological salt solution at 30°C. Whole-cell patch-clamp technique was used to control membrane voltage. INCX (identified as Ni2+ sensitive current) was recorded using a ramp clamp protocol under conditions to inhibit contaminating currents.
Results. With 0.6 mM sevoflurane, outward INCX at positive voltages (0 mV) and inward INCX at voltages negative to 60 mV was significantly reduced (P<0.05, n=13; INCX reduced by 48% at +50 and 65% of control at 120 mV). Halothane (0.6 mM) inhibited outward INCX at voltages positive to 10 mV and inward INCX at voltages negative to 80 mV (P<0.05, n=10; INCX reduced by 64% at +50 and 65% of control at 120 mV). Anaesthetic-induced inhibition of both inward and outward current was not voltage-dependent.
Conclusions. Inhibition of Ca2+ efflux via NCX (i.e. inward INCX) during an exposure to halothane or sevoflurane would be expected to limit the negative inotropic effects of these agents and help maintain SR Ca2+ content.
Keywords: anaesthetics volatile, halothane ; anaesthetics volatile, sevoflurane ; heart, myocytes
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Introduction |
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NCX plays an important role in the regulation of cytosolic Ca2+ as it represents the main Ca2+ extrusion pathway from ventricular cells. However, the effect of volatile anaesthetics on the biophysics of this exchanger has not been studied in great detail especially in adult ventricular tissue. Previous experiments to assess the effect of volatile anaesthetics on NCX function have been carried out in both neonatal15 16 and adult cells16 18 and the majority have used flux measurements to assess anaesthetic-induced changes in Ca2+ efflux (normal mode) or Ca2+ influx (reverse mode) via NCX. The results of these studies suggest that both Ca2+ efflux and influx are inhibited by volatile anaesthetics and that for halothane and sevoflurane the effects are greater in neonatal than adult ventricular cells.19 However, in flux experiments, competing Ca2+ transport pathways are inhibited pharmacologically or by ionic substitution and therefore, the results are critically dependent upon both the efficacy of pharmacological block and whether efficacy is affected by the introduction of anaesthetics. Furthermore, flux measurements of Ca2+ efflux via NCX need to be corrected for the intracellular [Ca2+] at which the measurements were taken.18 A more direct measurement of NCX activity is to record changes in membrane current associated with the operation of NCX under conditions where ionic concentrations are well controlled.20 With every cycle, three Na+ are exchanged for one Ca2+ such that in normal (Ca2+ efflux) mode, inward current is generated and in reverse (Ca2+ influx) mode, outward current is induced. The aims of these experiments were: (i) to test the hypothesis that 0.6 mM halothane and sevoflurane inhibit both inward and outward NCX current; (ii) to describe the effects of this concentration of anaesthetic on the currentvoltage relationship of NCX; and (iii) to consider the potential role of this exchanger in the inotropic effects of halothane and sevoflurane.
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Methods |
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Electrophysiological recording
The whole-cell patch-clamp technique was used to control membrane voltage. Patch pipettes (Clark patch-clamp borosilicate capillaries) were pulled to a resistance of 12 M (Narishige, PP-830) and fire-polished to 2.54 MW (Narishige MF83 microforge). Recordings of membrane currents were made using an Axopatch 200B (Axon Instruments, USA) amplifier with a CV-203BU headstage. Normally 8090% of the pipette series resistance was compensated. Cell membrane capacitance was measured by integrating the capacitance current recorded during a 10-mV hyperpolarizing pulse from 80 mV.
Once whole-cell clamp conditions were achieved, all external solutions were applied to the cell under study using a multi-barreled, temperature controlled superfusion device. A Cs+-based internal dialysis solution was used for all recordings. Its composition was as follows (in mM): CsCl 110; NaCl 20; HEPES 10; MgCl2 0.4; glucose 5; tetraethylammonium chloride 20; EGTA 5; MgATP 4; CaCl2 1, titrated to pH 7.2 with CsOH. For measurement of INCX, cells were superfused with a K+-free PSS (equimolar substitution with Cs+) supplemented with 10 µM nifedipine and 10 µM strophanthidin (to inhibit ICa and the Na+/K+ ATPase, respectively). Holding potential was set at 40 mV. Membrane potential was then clamped to +50 mV for 100 ms and then ramped to 120 mV over a period of 2.5 s (i.e. at 68 mV s1) before returning to 40 mV. Ramp clamps were repeated in the same cell in the presence of 5 mM Ni2+ to inhibit NCX and the putative INCX measured as the difference current (i.e. the Ni2+-sensitive current). Ni2+ was then removed and if membrane current did not return to control, then the cell was excluded from analysis. The ramp clamp protocol was then repeated in each cell in the presence of either 0.6 mM halothane or 0.6 mM sevoflurane, a dose that is clinically relevant [approximately twice the minimum alveolar concentration (MAC) for the rat] and approximately equi-anaesthetic. These experiments generated Ni2+-sensitive membrane currents in the absence and presence of anaesthetic in each cell. Current magnitude was scaled for cell capacitance and assessed at 10 mV intervals (between 120 and +50 mV) to allow construction of mean currentvoltage relationships in the absence and presence of anaesthetic. In cells where complete post-control data sets were recorded, the effects of halothane and sevoflurane on INCX were reversible.
Statistical analysis
Data are presented as mean (SEM) of n determinations (number of cells) and statistical comparisons were performed with paired Student's t-tests using SigmaStat (Jandel Scientific, Erkrath, Germany) or Wilcoxon's signed rank test if data failed a standard normality test (KolmogorovSmirnov). All figures were prepared by using SigmaPlot (Jandel Scientific).
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Results |
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Figure 3 shows a comparison of the efficacy of block of halothane and sevoflurane on inward INCX (between 60 and 120 mV) and outward INCX (between 0 and +50 mV). This illustrates that block by sevoflurane and halothane of both inward and outward INCX was not voltage-dependent and that the extent of block of both inward and outward INCX was broadly equivalent for both anaesthetics (the apparent greater inhibition of outward INCX by halothane was not significantly different to data obtained with sevoflurane).
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Discussion |
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More recent experiments18 investigated the dose-dependent effects of 1 and 2 MAC halothane and sevoflurane on the rates of both Ca2+ influx and efflux in adult rat ventricular myocytes; halothane (2 MAC) inhibited Ca2+ influx (equivalent to outward INCX) and efflux (equivalent to inward INCX) by approximately 71 and 50%, values very similar to those reported here (see Fig. 3). Data derived from Seckin and co-workers18 indicate that 2 MAC sevoflurane inhibited Ca2+ influx by approximately 67% and Ca2+ efflux by approximately 48%. In the present study, we found 0.6 mM sevoflurane inhibited outward INCX by 48% (at +50 mV) and inward INCX by 53% (at 80 mV). Although only one, high concentration of anaesthetic (2 MAC) was studied in the present report, there is very good agreement between data derived from Ca2+ flux measurements with 2 MAC anaesthetic and direct measurement of INCX for halothane and sevoflurane. Furthermore, because of the apparent lack of voltage-dependence of block of INCX by halothane and sevoflurane this minimizes the impact of changes in membrane potential on Ca2+ flux rate, which was considered to be a potential confounding variable.18
The impact of block of NCX by halothane and sevoflurane on the regulation of intracellular Ca2+ and therefore contractility will depend on the balance between normal and reverse mode of the exchanger as both modes are inhibited to a similar extent by both anaesthetics. It is generally conceded that under normal physiological conditions, Ca2+ entry via NCX (reverse mode) plays only a minor role in the Ca2+ influx, which triggers SR Ca2+ release21 22 and therefore, it follows that the normal mode of operation of NCX is to extrude Ca2+ from the cell. Given that inward current is inhibited at the resting membrane potential this would be expected to reduce Ca2+ efflux via NCX during diastole. This would offset the reduction in Ca2+ entry via ICa and help to maintain SR Ca2+ content and contractility. Furthermore, as Ca2+ efflux from the cell via NCX generates an inward depolarizing current, inhibition of this current would also contribute to the reduction in action potential duration observed in ventricular cells during exposure to halothane or sevoflurane.9
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Acknowledgments |
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References |
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