Halothane and sevoflurane inhibit Na/Ca exchange current in rat ventricular myocytes

G. Bru-Mercier1, P. M. Hopkins2 and S. M. Harrison1,*

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.


    Abstract
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Background. The electrogenic Na+/Ca2+ exchanger (NCX) represents the main extrusion pathway for Ca2+ in ventricular muscle and therefore plays an important role in the regulation of cytosolic Ca2+ and contraction. Halothane and sevoflurane modulate cytosolic Ca2+ regulation and at steady state are negatively inotropic, however, the involvement of anaesthetic-induced changes in NCX activity in these effects requires further study.

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


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
During excitation–contraction coupling in ventricular muscle (see ref.1 for review), the L-type Ca2+ current (ICa) acts as the main trigger for the release of Ca2+ from the sarcoplasmic reticulum (SR); however, Na+/Ca2+ exchange (NCX) acting in reverse mode may also contribute to Ca2+ influx during the initial phase of the ventricular action potential. Relaxation is initiated by the decline of cytosolic Ca2+; predominately Ca2+ is resequestered by the SR but under steady-state conditions, Ca2+ that entered the cell during excitation via ICa (and any via reverse mode NCX) must be extruded. This occurs predominately via NCX in forward mode with plasmalemmal Ca2+ ATPase (PMCA) playing a lesser role in Ca2+ efflux.2 At steady state, it is well established that volatile anaesthetics induce a negative inotropic effect in ventricular muscle predominately as a result of their inhibitory action on ICa39 and myofilament Ca2+ sensitivity.1014

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 current–voltage relationship of NCX; and (iii) to consider the potential role of this exchanger in the inotropic effects of halothane and sevoflurane.


    Methods
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Animal procedures conformed to regulations described in the Animals (Scientific Procedures) Act 1986, UK government Home Office. The experiments were carried out on Wistar rats (~250 g, Central Biomedical Services, University of Leeds), which were given access to food and water ad libitum and maintained under a 12-h light/dark cycle. Animals were killed by a schedule 1 procedure sanctioned by the UK government Home Office, the heart rapidly excised and ventricular myocytes isolated using a standard collagenase/protease dispersion technique.12 Myocytes were transferred to a circular chamber mounted on an inverted microscope (Nikon Eclipse) and superfused (at ~2.5 ml min–1) with a physiological salt solution (PSS) containing (in mM): NaCl 140; KCl 4; CaCl2 2.5; MgCl2 1.2; glucose 10; HEPES 5, pH 7.4 (NaOH), 30°C. Anaesthetics were delivered from stock solutions made up in dimethyl sulphoxide (DMSO) to a final concentration of 0.6 mM. [DMSO] never exceeded 0.2%, which had no effect on membrane current.

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 1–2 M{Omega} (Narishige, PP-830) and fire-polished to 2.5–4 MW (Narishige MF83 microforge). Recordings of membrane currents were made using an Axopatch 200B (Axon Instruments, USA) amplifier with a CV-203BU headstage. Normally 80–90% 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 s–1) 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 current–voltage 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 (Kolmogorov–Smirnov). All figures were prepared by using SigmaPlot (Jandel Scientific).


    Results
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Figure 1 illustrates representative Ni2+-sensitive membrane current traces (i.e. putative INCX) in response to the ramp clamp protocol (A) in the absence and presence of 0.6 mM halothane (B) and 0.6 mM sevoflurane (C). These illustrate that at membrane potentials positive to the reversal potential, outward current was generated that was reduced in magnitude by anaesthetic. Similarly, inward current (evoked at potentials negative to the reversal potential) was also inhibited by exposure to halothane and sevoflurane.



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Fig 1 Representative recordings of INCX elicited during the voltage clamp protocol shown in (A) under control conditions (CON) and in the presence of 0.6 mM halothane (B; HAL) and sevoflurane (C; SEVO).

 
Figure 2A illustrates the mean current–voltage relationship for INCX in the absence (open symbols) and presence (filled symbols) of 0.6 mM sevoflurane. The reversal potential was very close to –40 mV and was not shifted by the introduction of sevoflurane. At voltages equal or positive to 0 mV (P<0.05) and negative to –60 mV (P≤0.05), sevoflurane significantly reduced INCX; for example, at +50, +20, –80, and –120 mV, current was reduced by 48, 53, 53, and 65% of control, respectively. Sevoflurane-sensitive INCX reversed close to –40 mV and was effectively linear over the voltage range (Fig. 2B).



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Fig 2 (A) Relationship between mean INCX density and membrane voltage in the absence and presence of 0.6 mM sevoflurane. *P<0.05, **P<0.01 control vs sevoflurane, n=13. (B) Sevoflurane-sensitive INCX derived by subtraction of the two traces in (A).

 
Similar current–voltage relationships were observed for experiments conducted with halothane (not shown). As for sevoflurane, halothane did not induce a shift in the reversal potential and both outward and inward INCX were inhibited; for example, at +50, +20, –80, and –120 mV, current was reduced by 64, 76, 57, and 65% of control, respectively.

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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Fig 3 Voltage-dependence of block of INCX by 0.6 mM halothane and 0.6 mM sevoflurane. Data are expressed as the per cent decrease in mean INCX induced by each anaesthetic at each voltage (sevoflurane, n=13; halothane, n=10).

 

    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
These data describe the effects of halothane and sevoflurane on the current–voltage relationship of NCX and for the first time illustrate in adult ventricular myocytes that (i) both halothane and sevoflurane significantly inhibit inward and outward INCX to a similar extent at the concentration tested and (ii) show that the inhibition of INCX by both anaesthetics is not voltage-dependent. Previous data on the effects of volatile anaesthetics on INCX is sparse. One previous study15 reported a 66% reduction in outward membrane current at +60 mV by halothane 3% in rabbit neonatal ventricular cells, however in that study, inward current was not measured. This compares favourably with data from the present study where 0.6 mM halothane inhibited outward INCX by 64% at +50 mV. Our data also support previous conclusions from experiments using radioisotope flux measurement in which 0.91 mM halothane reduced Ca2+ influx (i.e. outward INCX) by 50%.17

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


    Acknowledgments
 
This work was supported by The British Heart Foundation, London UK.


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
1 Bers DM. Cardiac excitation-contraction coupling. Nature 2002; 415: 198–205[CrossRef][ISI][Medline]

2 Bassani RA, Bassani JWM, Bers DM. Mitochondrial and sarcolemmal Ca2+ transport reduce [Ca2+]i during caffeine contractures in rabbit cardiac myocytes. J Physiol 1992; 453: 591–608[Abstract]

3 Lynch C III, Vogel S, Sperelakis N. Halothane depression of myocardial slow action potentials. Anesthesiology 1981; 55: 360–8[ISI][Medline]

4 Ikemoto Y, Yatani A, Arimura H, Yoshitake J. Reduction of the slow inward current of isolated rat ventricular cells by thiamylal and halothane. Acta Anaesthesiol Scand 1985; 29: 583–6[ISI][Medline]

5 Terrar DA, Victory JGG. Effects of halothane on membrane currents associated with contraction in single myocytes isolated from guinea-pig ventricle. Br J Pharmacol 1988; 94: 500–8[ISI][Medline]

6 Bosnjak ZJ, Supan FD, Rusch NJ. The effects of halothane, enflurane, and isoflurane on calcium current in isolated canine ventricular cells. Anesthesiology 1991; 74: 340–5[ISI][Medline]

7 Eskinder H, Rusch NJ, Supan FD, Kampine JP, Bosnjak ZJ. The effects of volatile anesthetics on L- and T-type calcium channel currents in canine Purkinje cells. Anesthesiology 1991; 74: 919–26[ISI][Medline]

8 Pancrazio JJ. Halothane and isoflurane preferentially depress a slow inactivating component of Ca2+ channel current in guinea-pig myocytes. J Physiol 1996; 494: 94–103

9 Rithalia A, Hopkins PM, Harrison SM. The effects of halothane, isoflurane, and sevoflurane on Ca2+ current and transient outward K+ current in subendocardial and subepicardial myocytes from the rat left ventricle. Anesth Analg 2004; 99: 1615–22[Abstract/Free Full Text]

10 Hanley PJ, Loiselle DS. Mechanisms of force inhibition by halothane and isoflurane in intact rat cardiac muscle. J Physiol 1998; 506: 231–44[Abstract/Free Full Text]

11 Harrison SM, Robinson M, Davies LA, Hopkins PM, Boyett MR. Mechanisms underlying the inotropic action of halothane on intact rat ventricular myocytes. Br J Anaesth 1999; 82: 609–21[Abstract/Free Full Text]

12 Davies LA, Gibson CN, Boyett MR, Hopkins PM, Harrison SM. Effects of isoflurane, sevoflurane, and halothane on myofilament Ca2+ sensitivity and sarcoplasmic reticulum Ca2+ release in rat ventricular myocytes. Anesthesiology 2000; 93: 1034–44[CrossRef][ISI][Medline]

13 Jiang YD, Julian FJ. Effects of halothane on [Ca2+](i) transient, SR Ca2+ content, and force in intact rat heart trabeculae. Am J Physiol 1998; 43: H106–H114

14 Graham MD, Bru-Mercier G, Hopkins PM, Harrison SM. Transient and sustained changes in myofilament sensitivity to Ca2+ contribute to the inotropic effects of sevoflurane in rat ventricle. Br J Anaesth 2004; 94: 279–86[CrossRef][ISI][Medline]

15 Baum VC, Wetzel GT. Sodium–calcium exchange in neonatal myocardium: reversible inhibition by halothane. Anesth Analg 1994; 78: 1105–9[Abstract]

16 Prakash YS, Hunter LW, Seckin I, Sieck GC. Volatile anesthetics and regulation of cardiac Na+/Ca2+ exchange in neonates versus adults. Ann N Y Acad Sci 2002; 976: 530–4[Free Full Text]

17 Haworth RA, Goknur AB. Inhibition of sodium/calcium exchange and calcium channels of heart cells by volatile anesthetics. Anesthesiology 1995; 82: 1255–65[CrossRef][ISI][Medline]

18 Seckin I, Sieck GC, Prakash YS. Volatile anaesthetic effects on Na+–Ca2+ exchange in rat cardiac myocytes. J Physiol 2001; 532: 91–104[Abstract/Free Full Text]

19 Prakash YS, Seckin I, Hunter LW, Sieck GC. Mechanisms underlying greater sensitivity of neonatal cardiac muscle to volatile anesthetics. Anesthesiology 2002; 96: 893–906[CrossRef][ISI][Medline]

20 Convery MK, Hancox JC. Comparison of Na+–Ca2+ exchange current elicited from isolated rabbit ventricular myocytes by voltage ramp and step protocols. Pflugers Arch 1999; 437: 944–54[CrossRef][ISI][Medline]

21 Evans AM, Cannell MB. The role of L-type Ca2+ current and Na+ current-stimulated Na/Ca exchange in triggering SR calcium release in guinea-pig cardiac ventricular myocytes. Cardiovasc Res 1997; 35: 294–302[CrossRef][ISI][Medline]

22 Satoh H, Ginsburg KS, Qing K, Terada H, Hayashi H, Bers DM. KB-R7943 block of Ca2+ influx via Na+/Ca2+ exchange does not alter twitches or glycoside inotropy but prevents Ca2+ overload in rat ventricular myocytes. Circulation 2000; 101: 1441–6[Abstract/Free Full Text]





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