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Volatile anaesthetic effects on Na⁺-Ca²⁺ exchange in rat cardiac myocytes

Inanc Seckin *, Gary C. Sieck *† and Y. S. Prakash *

	ABSTRACT

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1. We examined the influence of two clinically relevant concentrations (1 and 2 MAC (minimum alveolar concentration)) of halothane and sevoflurane on both efflux and reverse modes of Na⁺-Ca²⁺ exchange (NCX) in enzymatically dissociated adult rat cardiac myocytes. We hypothesised that a volatile anaesthetic-induced decrease in myocardial contractility is mediated by a reduction in intracellular calcium concentration ([Ca²⁺]_i) via inhibition of NCX.

2. Cells were exposed to cyclopiazonic acid and zero extracellular Na⁺ and Ca²⁺ to block sacroplasmic reticulum (SR) re-uptake and NCX efflux, respectively. As [Ca²⁺]_i increased under these conditions, extracellular Na⁺ was rapidly (< 300 ms) reintroduced in the presence or absence of a volatile anaesthetic to selectively promote Ca²⁺ efflux via NCX. Other cells exposed to cyclopiazonic acid and ryanodine to inhibit SR Ca²⁺ re-uptake and release were Na⁺ loaded in zero extracellular Ca²⁺. The reintroduction of extracellular Ca²⁺ was used to selectively activate Ca²⁺ influx via NCX.

3. Compared to controls, both 1 and 2 MAC halothane as well as sevoflurane reduced NCX-mediated efflux. The reduction in NCX-mediated influx was concentration dependent, but comparable between the two anaesthetics. Both anaesthetics at each concentration also shifted the relationship between extracellular Na⁺ (or extent of Na⁺ loading) and NCX-mediated efflux (or influx) to the right.

4. These data indicate that despite inhibition of NCX-mediated Ca²⁺ efflux, volatile anaesthetics produce myocardial depression. However, the inhibition of NCX-mediated Ca²⁺ influx may contribute to decreased cardiac contractility. The overall effect of volatile anaesthetics on the [Ca²⁺]_i profile is likely to be determined by the relative contributions of influx vs. efflux via NCX during each cardiac cycle.

	INTRODUCTION

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Volatile anaesthetics are known to decrease the contractility of cardiac muscle (Rusy & Komai, 1987; Lynch & Frazer, 1989; Hatakeyama et al. 1993; Graf et al. 1995; Skeehan et al. 1995; Park et al. 1996). These effects are thought to involve, at least in part, alterations in the regulation of intracellular Ca²⁺ concentration ([Ca²⁺]_i). Studies in several species have demonstrated anaesthetic effects on Ca²⁺ influx through L-type Ca²⁺ channels (Lynch, 1986; Nakao et al. 1989; Bosnjak, 1991a,b; Bosnjak et al. 1992; Schmidt et al. 1993) and sarcoplasmic reticulum (SR) Ca²⁺ release via ryanodine receptor (RyR) channels (Casella et al. 1987; Katsuoka et al. 1989; Herland et al. 1990, 1996; Wilde et al. 1991; Frazer & Lynch, 1992; Connelly & Coronado, 1994; Lynch & Frazer, 1994; Wheeler et al. 1994), both effects resulting in decreased [Ca²⁺]_i levels and an inhibition of contraction. Relatively less is known about volatile anaesthetic effects on other Ca²⁺ regulatory mechanisms.

In cardiac muscle, Ca²⁺ flux across the plasma membrane is a necessary requirement not only for the activation of Ca²⁺-induced Ca²⁺ release (during systole) but also for relaxation. In this regard, NCX is thought to play a key role in [Ca²⁺]_i regulation, especially during relaxation mediated by efflux of Ca²⁺ across the plasma membrane (for reviews see Reeves et al. 1994; Blaustein & Lederer, 1999). NCX is also thought to play a variable role in the elevation of [Ca²⁺]_i levels, in conjunction with L-type Ca²⁺ channels (Crespo et al. 1990; Leblanc & Hume, 1990; Levi et al. 1994; Lipp & Niggli, 1994). Some studies have suggested that NCX may also play a role in the pathogenesis of cardiac arrhythmias by increasing SR Ca²⁺ load and thus the instability of SR Ca²⁺ release, as well as by increasing [Ca²⁺]_i levels in the influx mode (for review see Blaustein & Lederer, 1999). Compared to the abundant information on other [Ca²⁺]_i regulatory mechanisms, there is relatively little information on the effects of volatile anaesthetics on NCX, and only relating to the influx mode of action (Haworth & Goknur, 1995; Blanck et al. 1997). Furthermore, differences in potency and mechanism of action between well-established anaesthetics such as halothane and isoflurane and newer anaesthetics such as sevoflurane have not been examined. We hypothesised that volatile anaesthetic-induced decrease in myocardial contractility is mediated by alterations in NCX. Accordingly, in the present study we compared the effects of clinically relevant concentrations of halothane and sevoflurane on NCX in isolated adult rat cardiac myocytes.

	METHODS

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Animals

Twenty-five adult male Sprague-Dawley rats (260-300 g) were used. The rats were anaesthetised with I.M. ketamine (60 mg kg^-1) and xylazine (2.5 mg kg^-1). Heart, lungs and descending aorta were removed and rapidly immersed in oxygenated (95 % O₂, 5 % CO₂) ice-cold Krebs solution (mM): NaCl, 118.3; KCl, 4.7; KH₂PO₄, 1.2; MgSO₄, 1.2; CaCl₂, 1.0; glucose, 11; pH 7.40).

All procedures involving animal use were reviewed and approved by the Institutional Animal Care and Use Committee of the Mayo Clinic, and were in strict accordance with the guidelines of the American Physiological Society.

Dissociation of cardiac myocytes

A modification of a Langendorff perfusion-based technique (De Young et al. 1989) was used to enzymatically dissociate Ca²⁺-tolerant cardiac myocytes from adult hearts. Dulbecco's modified Eagle's medium (Joklik modification; enriched with 10 mM KCl and 10 mM Hepes) with different Ca²⁺ concentrations was used in different parts of the dissociation process. All procedures in Joklik medium were performed at 37 ^oC. The aorta was cannulated using a 16-gauge blunt tip needle and the heart was perfused for 5 min with Joklik medium containing 1 mM Ca²⁺, allowing the heart to wash out the blood via contractions. Extracellular Ca²⁺ ([Ca²⁺]_o) in the Joklik medium was then removed over a 5 min period, and Type I collagenase (Worthington) and 1 % bovine serum albumin (Sigma) were then added to the medium. During collagenase perfusion, [Ca²⁺]_o was added back in six graded steps to a final concentration of 1 mM. Following collagenase perfusion, the ventricles were dissected out, minced and gently triturated to obtain single myocytes. The myocytes were finally washed and stored in Joklik medium with 1 % albumin and 1 mM Ca²⁺.

Following isolation, myocytes were plated at a low density (10-25 cells mm^-2) on laminin-coated glass coverslips for 1 h. Imaging studies were completed within 4-5 h of plating. Viability and Ca²⁺ tolerance were evaluated by the presence of uniformly spaced sarcomeres, a stable low basal [Ca²⁺]_i level during the initial phase of imaging, and the presence of only infrequent spontaneous contractions. Cells with surface blebs were not included, regardless of other features.

Confocal [Ca²⁺]_i imaging

Laminin-coated coverslips with myocytes were incubated in the acetoxymethyl ester form of the fluorescent Ca²⁺ indicator fluo-3 (fluo-3 AM, 5 M; Molecular Probes) at room temperature for 45 min, and placed on an open slide chamber (Warner Instruments, Hamden, CT, USA) mounted on a Nikon Diaphot inverted microscope. Cells were perfused with normal Tyrode solution (mM): NaCl, 145; KCl, 4; MgCl₂, 1; CaCl₂, 1; glucose, 10; Hepes, 10; pH, 7.4; 25 ^oC. A fluid-level controller was used to ensure minimal changes in the level of the perfusate, which could potentially affect the time taken to rapidly exchange solutions when the rate of perfusion was momentarily increased during activation of NCX (see below).

Detailed descriptions of real-time confocal imaging of [Ca²⁺]_i have been previously published (Prakash et al. 1997). Briefly, fluo-3- loaded cells were imaged using an Odyssey XL real-time confocal system (Noran Instruments, Middleton, WI, USA) attached to the inverted Nikon microscope and equipped with a 3-line Ar-Kr laser. In preliminary studies, we determined that 30 frames s^-1 was sufficient for the present study to determine the dynamic [Ca²⁺]_i responses of cardiac myocytes, without introducing frequency aliasing. An Olympus 40/1.3 oil-immersion objective lens was used and frame size was set to 640 480 pixels (0.06 m² per pixel). Optical section thickness was set to 1 m by adjusting the width of the confocal slit. One large region of interest (ROI) was used to measure the [Ca²⁺]_i response of an entire cell. Two to three myocytes were sampled from each coverslip.

A potential concern with measuring [Ca²⁺]_i from the entire cell is that differences in cell size may contribute to differences in observed influx or efflux rates. Therefore, cell dimensions were also measured from the confocal images. The length and width of the cell were measured from planar images through the maximum girth of the cell; cylindrical morphology was assumed by calculating the total cell surface area. The absolute influx and efflux rates were then normalised for the cell surface area for each individual cell.

Ca²⁺ calibrations

Since fluo-3 is a non-ratiometric Ca²⁺ indicator, differences in dye loading and photobleaching may affect the estimation of [Ca²⁺]_i based on fluorescence intensity. Several studies have used in vitro calibrations where fluorescence levels are measured at known Ca²⁺ concentrations; however, the dissociation constant (K_d) of the fluorescent dye differs in vitro compared with in vivo (for review see Takahashi et al. 1999) . Therefore, as in previous studies in other cell types (Prakash et al. 1997, 1999), we used an empirical in vivo calibration technique based on the measurement of intracellular fluorescence levels at known [Ca²⁺]_i levels. A fixed combination of laser intensity (20 % of maximum) and photomultiplier gain (1700 from a maximum of 4096) was set a priori to ensure pixel intensities between 25 and 255 grey levels (GL). Cardiac myocytes loaded with 5 M fluo-3 AM were sequentially exposed to solutions containing 10 known Ca²⁺ concentrations (0 nM to 1.25 M; Molecular Probes Calcium Calibration Buffer Kit), and the Ca²⁺ ionophore A-23187 at a concentration of 10 M . This technique allowed the equilibration of intra- and extracellular [Ca²⁺].

Previous studies have used the equation:

The protocol for examining the interactions between volatile anaesthetics and efflux mode of NCX is illustrated in Fig. 1. Cells were initially perfused at 2-3 ml min^-1. The resting [Ca²⁺]_i level was recorded for 1 min to ensure cell stability. In stable cells, perfusion was then switched to a Tyrode solution containing 0 Na⁺ and 0 Ca²⁺ with 10 M cyclopiazonic acid (CPA; Sigma), a selective inhibitor of the sarco(endo)plasmic reticulum Ca²⁺-ATPase (SERCA) (Inesi & Sagara, 1994). As suggested from previous studies, N-methyl-D-glucamine (NMG; 145 mM) was substituted for Na⁺ to inhibit NCX (Matsuoka & Hilgemann, 1994; Li & van Breemen, 1995). The solution also contained 5 mM EGTA to remove contaminating Ca²⁺. Under these conditions, the major mechanisms for the decrease in [Ca²⁺]_i levels, namely SERCA and NCX, were inhibited by CPA and 0 Na⁺-NMG. Furthermore, Ca²⁺ influx across the plasma membrane was minimised. Accordingly, [Ca²⁺]_i levels were expected to rise, due to continued 'leak' from the SR. The [Ca²⁺]_i levels were allowed to rise, plateau and then slowly decrease (see Discussion - 'Methodological issues'). At this point, perfusion was rapidly switched to a Tyrode solution containing 0 Ca²⁺ (with EGTA) and normal Na⁺ levels along with CPA, thus selectively activating the efflux mode of NCX (Fig. 1A). Complete replacement of the perfusate volume in the chamber was accomplished in < 300 ms. The rate of fall in [Ca²⁺]_i levels was recorded as an index of efflux rate via NCX. When [Ca²⁺]_i levels reached baseline values, the cells were reperfused with normal Tyrode solution with no CPA, thus allowing for replenishment of [Ca²⁺]_i stores. After 5 min, the above protocol was repeated with the introduction of 1 or 2 MAC volatile anaesthetic into solutions containing 0 Na⁺-0 Ca²⁺ and 0 Ca²⁺-normal Na⁺; their responses were recorded continuously. In control experiments the above protocol was repeated without exposure to volatile anaesthetics.

Previous studies have demonstrated that NCX-mediated Ca²⁺ efflux is also dependent on membrane potential, with a reversal potential, E_Na-Ca, of -10 to -50 mV (for review see Blaustein & Lederer, 1999). In order to account for the confounding effect of varying membrane potential during the efflux protocol, a second set of experiments was performed under conditions of clamped membrane potential. Cells were initially exposed to normal Tyrode solution as in the previous protocol. Cells were then exposed to a solution containing 40 mM KCl, corresponding to a membrane potential of ~-30 mV. The remainder of the protocol described above was then performed in the continued presence of KCl, except during the intervening wash with normal Tyrode solution.

In a third set of experiments, the effect of volatile anaesthetics on the relationship between Na⁺ concentration and efflux mode of NCX was examined. The above protocol was modified by introducing Tyrode solution containing 0 Ca²⁺ and one of three different concentrations of Na⁺ (35, 70 and 145 mM) during rapid activation of NCX. The same cell was used for all three concentrations. The cells were exposed to volatile anaesthetics as in the previous protocol. Cell viability was tested at the end of the protocol by repeating the protocol with 145 mM Na⁺.

Volatile anaesthetic effects on influx mode of NCX

The protocol used to examine interactions between volatile anaesthetics and influx mode of NCX is shown in Fig. 1B. Cells were initially perfused at 2-3 ml min^-1 with normal Tyrode solution and resting [Ca²⁺]_i level was recorded. The perfusate was then changed to Tyrode solution containing 0 Ca²⁺ (5 mM EGTA) and normal Na⁺ along with 10 M CPA and 10 M ryanodine (Sigma) in order to 'Na⁺ load' the cells. The combination of ryanodine and CPA ensured the inhibition of SR Ca²⁺ release as well as re-uptake, thus functionally isolating the plasma membrane. Under these conditions, [Ca²⁺]_i levels were expected to remain stable, since both major mechanisms of Ca²⁺ influx across the plasma membrane were blocked, namely L-type Ca²⁺ channels and NCX. Cells were loaded with Na⁺ for 1 min, after which perfusion was rapidly switched (< 300 ms) to Tyrode solution with 0 Na⁺ and normal Ca²⁺ along with CPA and ryanodine. Under these conditions, the influx mode of NCX was selectively activated, resulting in Ca²⁺ influx. The rate of rise of [Ca²⁺]_i was measured as an index of the influx mode of NCX. After 1 min, cells were reperfused for 5 min with normal Tyrode solution containing no CPA or ryanodine. This procedure avoided Ca²⁺ overload and contraction of the cell. The above protocol was then repeated with cells pre-exposed to volatile anaesthetics prior to rapid activation of NCX (Fig. 1B). In control experiments, the protocol was repeated without exposure to volatile anaesthetics.

Previous studies have demonstrated that NCX-mediated Ca²⁺ influx is also dependent on membrane potential, especially during depolarisation (for review see Blaustein & Lederer, 1999). In order to account for the confounding effect of varying membrane potential, a second set of experiments was performed under conditions of clamped membrane potential also using 40 mM KCl. Although this KCl concentration did not produce sufficient depolarisation to maximally facilitate Ca²⁺ influx via NCX, it permitted examination of influx without the modulating effect of membrane potential. Cells were initially exposed to normal Tyrode solution as in the previous protocol. The remainder of the protocol was then performed in the continued presence of KCl, except during the intervening wash with normal Tyrode solution.

In a third set of experiments, the effect of volatile anaesthetics on the relationship between Na⁺ gradient and influx mode of NCX was examined. The above protocol was modified by Na⁺ loading the cells with Tyrode solution containing 0 Ca²⁺, CPA, ryanodine and one of three Na⁺ concentrations (35, 70 or 145 mM).

Statistical analysis

Quality control was maintained by excluding cells from further analysis under the following conditions: (1) cells displaying > 50 nM variation in resting [Ca²⁺]_i levels at the start of a protocol; (2) cells displaying more than five spontaneous [Ca²⁺]_i transients per minute (such transients were noted in 15 % of all cells); (3) cells displaying greater than 10 % change in resting [Ca²⁺]_i during intervening washes; and (4) cells displaying > 10 % dye bleaching over a 5 min period.

At least 20 cells were analysed for each protocol. These cells were obtained from at least five animals. The specific number of samples analysed for each protocol are provided in the results as the number of animals, since data from each animal were averaged and statistical comparisons made using only animals (repeated measures). Data were compared using ANOVA with experimental conditions, anaesthetic and concentration as grouping variables. Bonferroni corrections were used for multiple comparisons. Statistical significance was tested at a 0.05 level. All data are expressed as means ± S.E.M.

There were no significant differences between vehicle control groups for either halothane or sevoflurane. Accordingly, the results of these groups were pooled in the creation of figures. However, it must be emphasised that statistical analyses were performed separately for the two anaesthetics using only their corresponding controls.

	RESULTS

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Cell surface area

The length of dissociated myocytes ranged from 126 to 139 m (131 ± 4 m) and the width ranged from 28 to 33 m (31 ± 2 m). The calculated surface area, assuming cylindrical morphology ranged from 12 073 to 13 511 m² (12 855 ± 1334 m²). The overall coefficient of variation in the surface area data was ~11 %. Closer examination of the data set revealed that in ~88 % of the cells, the coefficient of variation was less than 8 %.

Volatile anaesthetic effects on efflux mode of NCX

As indicated in Fig. 1A, inhibition of SERCA by CPA with simultaneous inhibition of NCX by a 0 Na⁺,0 Ca²⁺ Tyrode solution resulted in an initial and rapid increase in [Ca²⁺]_i levels followed by a slow decline, most likely due to continued activity by the plasma membrane Ca²⁺ ATPase, which was not inhibited by these pharmacological manipulations. The rapid reintroduction of extracellular Na⁺ resulted in a steep decline in [Ca²⁺]_i to levels similar to those in normal Tyrode solution. A straight line passing through the steepest portion of the descending curve was used for the calculation of the rate of efflux. This efflux rate of NCX ranged from 11.5 to 78.2 nM s^-1 during the first run of the efflux protocol (pooled data from control data for both anaesthetics; n = 10). When normalised for cell surface area, efflux rates ranged from 0.88 to 6.00 M s^-1 m^-2 (2.53 ± 0.11 M s^-1 m^-2). Accordingly, variations in cell size did not account for the range of Ca²⁺ efflux rates.

The rate of fall in [Ca²⁺]_i levels was correlated with the [Ca²⁺]_i level just prior to the reintroduction of Na⁺ ('peak' [Ca²⁺]_i) with the rate being higher for higher [Ca²⁺]_i. Accordingly, the efflux rate of NCX displayed a linear correlation to 'peak' [Ca²⁺]_i levels (Fig. 2). Repetition of the efflux protocol in control experiments resulted in a 10 ± 2 % increase in efflux rate. Accordingly, time-related bias in the protocol was ignored.

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Figure 1 A, protocol to examine the effect of volatile anaesthetics on the efflux mode of Na+-Ca2+ exchange (NCX) in single rat cardiac myocytes loaded with the Ca2+ indicator fluo-3. Following initial exposure to normal Tyrode solution containing 145 mM Na+ and 1 mM Ca2+, cells were exposed to 0 Na+,0 Ca2+ Tyrode solution to inhibit the efflux mode of NCX, and cyclopiazonic acid (CPA) to inhibit sarcoplasmic reticulum (SR) Ca2+ re-uptake. Under these conditions, [Ca2+]i levels were allowed to rise. Extracellular Na+ was then rapidly introduced (in the presence or absence of volatile anaesthetic) selectively activating efflux via NCX. The rate of fall in [Ca2+]i levels was measured as an index of NCX activity. B, protocol to examine the effect of volatile anaesthetics on the influx mode of NCX. Following initial exposure to normal Tyrode solution, a 0 Ca2+ Tyrode solution was used to 'Na+-load' under conditions of blocked SR Ca2+ release (ryanodine) and re-uptake (CPA). Ca2+ influx via NCX was then selectively activated by reintroducing extracellular Ca2+ and simultaneously removing Na+, in the presence or absence of volatile anaesthetic. The rate of rise in [Ca2+]i was measured as an index of NCX activity.

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Figure 2. Relationship between 'peak' [Ca2+]i level and efflux via NCX The rate of Ca2+ efflux via NCX was positively correlated to the 'peak' [Ca2+]i level just prior to the reintroduction of [Na+]o. The overall correlation between [Ca2+]i level and efflux rate was shown by a regression coefficient (r2) of 0.85. Data shown are from control animals (n = 10).

Exposure to both 1 and 2 MAC halothane appeared to result in a slower rate of rise in [Ca²⁺]_i levels following exposure to CPA. However, this trend was not analysed further. The reintroduction of Na⁺ resulted in a significantly slower efflux rate of NCX (example for 2 MAC halothane in Fig. 3A), both in absolute terms (Fig. 3B; P < 0.05 for both 1 and 2 MAC and for concentration dependence; n = 5 for each concentration) and when normalised for the 'peak' [Ca²⁺]_i to account for the correlation between [Ca²⁺]_i and efflux rate (Fig. 3C; P < 0.05). Compared to the control data, both 1 and 2 MAC halothane significantly blunted any correlation between 'peak' [Ca²⁺]_i levels and efflux rate (Fig. 4A).

Compared to halothane, both 1 and 2 MAC sevoflurane had a significantly smaller effect on both absolute and normalised efflux rates (Fig. 3; P < 0.05; n = 5 for each concentration), and no significant effect on the correlation between [Ca²⁺]_i level and efflux rate (Fig. 4B).

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Figure 3. Effect of volatile anaesthetics on efflux via NCX The protocol shown in Fig. 1A was performed in the same cell in the absence or presence of volatile anaesthetic. Representative tracings of the effects of 2 MAC halothane and sevoflurane are shown in panel A. The break lines represent a period of washing with normal Tyrode solution. Compared to control, both 1 and 2 minimum alveolar concentration (MAC) halothane as well as sevoflurane significantly decreased the absolute rate of Ca2+ efflux via NCX (B). Since efflux rates were found to be correlated to [Ca2+]i levels, rates were also normalised for 'peak' [Ca2+]i values. Under these conditions also, both anaesthetics decreased the rate of Ca2+ efflux (C). The effects of sevoflurane were generally less pronounced compared to halothane. *,† Significant differences (P < 0.05) from control for halothane and sevoflurane, respectively. ‡ Significant difference between 1 and 2 MAC. § Significant difference between halothane and sevoflurane. For each anaesthetic and concentration n = 5.

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Figure 4. Effect of volatile anaesthetics on the relationship between [Ca2+]i and efflux rate Both 1 MAC (r2 = 0.10) and 2 MAC (r2 = 0.15) halothane blunted the correlation between [Ca2+]i and efflux rate (A). In comparison, sevoflurane had no significant effect on the correlation (r2 = 0.80 and 0.82 for 1 and 2 MAC, respectively) (B).

In a second set of experiments, the confounding influence of membrane potential was limited by performing the above efflux protocol in the presence of 40 mM KCl. There was no qualitative difference in the [Ca²⁺]_i profile between the experiments performed in the presence or absence of KCl, for either control or anaesthetic exposure. However, under control conditions, the average efflux rate was 89 ± 6 % of that in the absence of KCl (n = 5; P < 0.05). With exposure to halothane, efflux rates were 85 ± 4 % for 1 MAC (n = 5; P < 0.05) and 80 ± 3 % (n = 5; P < 0.05) for 2 MAC, compared to efflux rates obtained in the absence of KCl. With exposure to sevoflurane, efflux rates were 86 ± 3 % for 1 MAC (n = 5; P < 0.05) and 82 ± 3 % for 2 MAC (n = 5; P < 0.05), compared to experiments in the absence of KCl. It should be noted that these comparisons were not within the same cell, unlike the protocol in the absence of KCl.

In a third set of experiments, the effect of [Na⁺]_o on efflux rate was examined. Decreasing [Na⁺]_o resulted in a slower efflux rate (Fig. 5A). In order to minimise the confounding influence of different 'peak' [Ca²⁺]_i levels on efflux rate, measurements were made by purposely selecting cells displaying comparable 'peak' [Ca²⁺]_i levels. Exposure to 1 MAC halothane resulted in considerably slower efflux rates in the presence of anaesthetic at 145, 70 and 35 mM Na⁺ compared to control (Fig. 5B; P < 0.05; n = 5 for each concentration of Na⁺). Exposure to 2 MAC halothane resulted in quantitatively greater slowing of efflux, with complete inhibition of efflux via NCX at 35 mM Na⁺.

Exposure to 1 or 2 MAC sevoflurane also resulted in slowing of NCX-mediated efflux (Fig. 5C; P < 0.05; n = 5). However, the effect of either sevoflurane concentration was significantly smaller than that induced by halothane (P < 0.05).

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Figure 5. Effect of volatile anaesthetics on the Na+ dependence of efflux via NCX The rate of Ca2+ efflux was determined with three different [Na+]o. The rate of efflux decreased with decreasing [Na+]o (A). Both 1 and 2 MAC halothane (B), and sevoflurane to a lesser extent (C), significantly blunted the relationship between Na+ concentration and Ca2+ efflux rate. *,† Significant differences (P < 0.05) from control for halothane and sevoflurane, respectively. ‡ Significant difference between 1 and 2 MAC. § Significant difference between halothane and sevoflurane. || Significant difference in the trend of Na+ dependence. For each anaesthetic and concentration n = 5.

Volatile anaesthetic effects on influx mode of NCX

As indicated in Fig. 1B, the Na⁺ loading of cells by inhibition of both RyR channels and SERCA by ryanodine and CPA, respectively, and simultaneous inhibition of L-type Ca²⁺ channels, did not induce any significant change in resting [Ca²⁺]_i levels. The rapid reintroduction of [Ca²⁺]_o, with simultaneous removal of [Na⁺]_o, resulted in a monotonic increase in [Ca²⁺]_i levels that reached a plateau upon reintroduction of normal Tyrode solution. A straight line passing through the steepest portion of the ascending curve was used for calculation of the rate of influx. This influx rate of NCX ranged from 10.2 to 155.3 nM s^-1 during the first run of the influx protocol (pooled data from control data for both anaesthetics; n = 10). When normalised for cell surface area, influx rates ranged from 0.74 to 13.04 M s^-1 m^-2 (3.97 ± 0.10 M s^-1 m^-2). Accordingly, variations in cell size also did not account for the range of Ca²⁺ influx rates. Repetition of the influx protocol in control experiments resulted in a 5 ± 2 % decrease in influx rate. Accordingly, time-related bias in the influx protocol was also ignored.

Both 1 and 2 MAC halothane resulted in a slower rate of Ca²⁺ influx via NCX following reintroduction of [Ca²⁺]_o (Fig. 6; P < 0.05; n = 5 for each concentration). Compared to control, both 1 and 2 MAC sevoflurane had a significant effect on the influx rate via NCX (Fig. 6; P < 0.05; n = 5 for each concentration). However, there was no significant difference in effects of halothane vs. sevoflurane for either 1 or 2 MAC.

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Figure 6. Effect of volatile anaesthetics on influx via NCX Representative samples of the effects of 2 MAC halothane and sevoflurane are shown in panel A. Break lines represent a period of exposure to 0 Ca2+ Tyrode solution with CPA and ryanodine. Compared to control, both concentrations of halothane significantly decreased the absolute rate of Ca2+ influx via NCX (B) as well as influx normalised for [Ca2+]i (C). The effects of sevoflurane were generally comparable to those of halothane. *,† Significant differences (P < 0.05) from controls for halothane and sevoflurane, respectively. ‡ Significant difference between 1 and 2 MAC. § Significant difference between halothane and sevoflurane. For each anaesthetic and concentration n = 5.

A second set of experiments was performed in the presence of 40 mM KCl to limit the influence of membrane potential. As with the similar protocol in the efflux studies, there was no qualitative difference in the [Ca²⁺]_i profile between experiments performed in the presence or absence of KCl, for either control or anaesthetic exposure. However, under control conditions, the average influx rate was 105 ± 3 % of that in the absence of KCl, and was not significantly different (n = 5). With exposure to halothane, influx rates were 98 ± 4 % for 1 MAC (n = 5) and 93 ± 4 % for 2 MAC (n = 5; P < 0.05), compared to influx rates obtained in the absence of KCl. With exposure to sevoflurane, efflux rates were 102 ± 5 % for 1 MAC (n = 5) and 98 ± 4 % for 2 MAC (n = 5), compared to experiments performed in the absence of KCl. Again, these comparisons were not within the same cell, unlike the protocol in the absence of KCl.

In a third set of experiments, the relationship between the amount of Na⁺ loading and subsequent Ca²⁺ influx rate was established. The extent of Na⁺ loading was assumed to be proportional to [Na⁺]_o since the time for Na⁺ loading was fixed. No attempt was made to determine the actual Na⁺ concentration in the cell. Decreasing Na⁺ loading resulted in a slower influx rate (Fig. 7). Exposure to 1 MAC halothane significantly slowed NCX-mediated Ca²⁺ influx with Na⁺ loads of 145, 70 and 35 mM (Fig. 7; P < 0.05; n = 5 for each concentration of Na⁺). Exposure to 2 MAC halothane further slowed influx, with complete inhibition at 35 mM Na⁺.

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Figure 7. Effect of volatile anaesthetics on the Na+ dependence of influx via NCX Representative samples of the effects of decreasing [Na+]o are shown in panel A. Break lines represent a period of exposure to 0 Ca2+ Tyrode solution with CPA and ryanodine. The rate of Ca2+ influx was determined after Na+ loading cells with 1 of 3 different [Na+]o for a fixed period of time. Both 1 and 2 MAC halothane (B), and sevoflurane to a comparable extent (C), significantly blunted the relationship between Na+ concentration and Ca2+ influx rate. *,† Significant differences (P < 0.05) from control for halothane and sevoflurane, respectively. ‡ Significant difference between 1 and 2 MAC. § Significant difference between halothane and sevoflurane. || Significant difference in the trend of Na+ dependence. For each anaesthetic and concentration n = 5.

Exposure to 1 or 2 MAC sevoflurane also resulted in the slowing of NCX-mediated Ca²⁺ influx, which was comparable to that induced by halothane (Fig. 7; P < 0.05 for sevoflurane effects only; n = 5).

	DISCUSSION

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In the present study, we examined the influence of clinically relevant concentrations of volatile anaesthetics on both efflux and influx modes of NCX in enzymatically dissociated adult rat cardiac myocytes. Compared to controls, both 1 and 2 MAC halothane and sevoflurane to a lesser extent, reduced NCX-mediated efflux. NCX-mediated influx was reduced by both anaesthetics to a comparable extent. Both halothane and sevoflurane also blunted the relationship between [Na⁺]_o (or extent of Na⁺ loading) and NCX-mediated efflux as well as influx. These data demonstrate that myocardial depression induced by volatile anaesthetics is mediated, at least in part, by inhibition of NCX, especially NCX-mediated Ca²⁺ influx.

NCX in [Ca²⁺]_i regulation

A number of regulatory mechanisms come into play with every heart beat in order to transiently elevate and decrease [Ca²⁺]_i levels in a stable and cyclical fashion. Elevation of [Ca²⁺]_i involves both Ca²⁺ influx across the plasma membrane, especially through L-type Ca²⁺ channels, as well as Ca²⁺ release from the SR through ryanodine receptor channels. A decrease in [Ca²⁺]_i levels involves Ca²⁺ re-uptake into the SR via SERCA as well as Ca²⁺ efflux across the plasma membrane. As with most mammalian cells, the plasma membrane of cardiac myocytes also contains the NCX protein that operates in parallel with both L-type Ca²⁺ channels, thus assisting in Ca²⁺ influx, and with ATP-sensitive Ca²⁺ efflux mechanisms (plasma membrane Ca²⁺-ATPase; PMCA). Depending on net electrochemical driving force, the NCX can thus move Ca²⁺ both in and out of cells. Indeed, studies have shown that NCX can change direction during cyclical cell activity (as in cardiac muscle), when membrane potential, [Na⁺]_i and/or [Ca²⁺]_i levels change (Kimura et al. 1986; Miura & Kimura, 1989; Hilgemann, 1990; Matsuoka & Hilgemann, 1992; Blaustein & Lederer, 1999). Furthermore, [Na⁺]_o and [Ca²⁺]_o also appear to play a role (Miura & Kimura, 1989; Matsuoka & Hilgemann, 1992; Blaustein & Lederer, 1999). During quiescent periods, i.e. no action potential, the driving force for NCX is largely determined by membrane potential and [Na⁺]_i, and may serve to regulate mean [Ca²⁺]_i levels.

In cardiac muscle, the Ca²⁺ influx mode of NCX is considered to be dependent on [Ca²⁺]_o for activation (Miura & Kimura, 1989; Matsuoka & Hilgemann, 1992; Blaustein & Lederer, 1999), especially in the low millimolar range, but actual Ca²⁺ influx is dictated by [Na⁺]_i as well as [Ca²⁺]_i levels when above resting levels (Rasgado-Flores et al. 1989; Blaustein & Lederer, 1999). In contrast, the efflux mode of NCX does not depend on [Ca²⁺]_o for activation, but is dependent on both [Na⁺]_o and [Ca²⁺]_i levels (Matsuoka & Hilgemann, 1992; Blaustein & Lederer, 1999), with [Na⁺]_i having a biphasic effect at high levels. Overall, no ATP hydrolysis is required for NCX activity, although the ATP-dependent Na⁺-K⁺ pump is eventually involved. Compared to PMCA, NCX has been found to have a 10-fold lower affinity for Ca²⁺, but a considerably higher turnover rate (DiPolo, 1979). This high turnover rate is thought to be important in the rapid extrusion of Ca²⁺ during cardiac diastole, when PMCA may become saturated. Indeed, the role of NCX as a primary Ca²⁺ efflux mechanism in the heart is now widely accepted (Crespo et al. 1990; Grantham & Cannell, 1996; Blaustein & Lederer, 1999), although there also appear to be species differences in the relative contributions of NCX to [Ca²⁺]_i regulation (Bassani et al. 1994a,b; Sham et al. 1995). Furthermore, during the plateau phase of the cardiac action potential, a decreased driving force for NCX may help reduce the net rate of Ca²⁺ extrusion, thus maintaining [Ca²⁺]_i levels for a brief period. Thus, volatile anaesthetic effects on NCX have tremendous potential for interfering with normal cardiac function.

Methodological issues

In the present study, [Ca²⁺]_i in cardiac myocytes was measured using real-time confocal imaging of a non-ratiometric [Ca²⁺]_i indicator (fluo-3). While fluo-3 cannot be used to measure [Ca²⁺]_i precisely, estimates can be reliably and reproducibly obtained using an empirical Ca²⁺ calibration curve, as in the present study. Similar calibration techniques have also been previously reported (Takahashi et al. 1999). The reliability of the calibration technique is underlined by relatively small variations in basal [Ca²⁺]_i across myocytes obtained on different days. A second potential limitation of fluo-3 is the relative lack of sensitivity of this dye at extremely high [Ca²⁺]_i values. Although it is possible that the absolute peak of the [Ca²⁺]_i responses may be underestimated when in the micromolar range, the results and conclusions from different protocols are unlikely to have been affected since statistical comparisons were made within the same cell prior to and during volatile anaesthetic exposure.

Previous studies have used various procedures to examine the relative contribution of NCX to either Ca²⁺ influx or efflux (for a review see Blaustein & Lederer, 1999). These procedures have included examining the rate of [Ca²⁺]_i decline following electrical stimulation under conditions of altered [Na⁺]_o and [Ca²⁺]_i, thus somewhat mimicking the normal cyclical pattern of cardiac myocyte activation. In the present study, we did not use electrical stimulation because a number of previous studies have demonstrated a significant effect of volatile anaesthetics, especially halothane, on both L-type Ca²⁺ channels (Lynch, 1986; Nakao et al. 1989; Bosnjak, 1991a,b; Bosnjak et al. 1992; Schmidt et al. 1993) and SR Ca²⁺ release/re-uptake (Casella et al. 1987; Katsuoka et al. 1989; Herland et al. 1990, 1996; Wilde et al. 1991; Frazer & Lynch, 1992; Connelly & Coronado, 1994; Lynch & Frazer, 1994; Wheeler et al. 1994), both of which are likely to influence the profile of the [Ca²⁺]_i transient. Addition of appropriate blockers such as nifedipine and/or ryanodine to isolate the effects of NCX would obviously result in the abolishment of the [Ca²⁺]_i transients in response to electrical stimulation. Accordingly, in the present study, we depended on pharmacological interventions to selectively inhibit or activate NCX under defined conditions of [Ca²⁺]_i, [Ca²⁺]_o, [Na⁺]_i, and [Na⁺]_o. A potential confounding factor with such protocols is their effect on membrane potential and thus one of the driving forces for NCX activity (Blaustein & Lederer, 1999). The driving force is dependent on the membrane potential as well as the reversal potential for NCX. The exclusion of [Na⁺]_o or [Ca²⁺]_o in our protocols ensured that the driving forces (estimated from the Nernst equation) were large, and in the appropriate direction. Nonetheless, since anaesthetics have been shown to also influence membrane potential, we additionally evaluated the anaesthetic effect on NCX activity under conditions of clamped membrane potential. Our results indicate that the observed effects of halothane and sevoflurane were attributable only in a small way to effects on membrane potential.

In previous studies, caffeine has been used to rapidly elevate [Ca²⁺]_i levels under conditions of blocked NCX, with subsequent activation of NCX to examine the dynamics of the [Ca²⁺]_i response (e.g. Sham et al. 1995; Bers et al. 1996). However, in addition to the direct effect on ryanodine receptor channels, caffeine is also known to inhibit intracellular phosphodiesterases. This is a confounding issue, especially since the effects of volatile anaesthetics on phosphodiesterases and/or cAMP are not well established. Furthermore, in the present study, the use of CPA to elevate [Ca²⁺]_i levels allowed for the repetition of efflux protocols with or without anaesthetic. Obviously, in the influx protocol, blocking concentrations of ryanodine were necessary in order to prevent any elevation in [Ca²⁺]_i via the SR.

Overall, the pharmacological manipulations used in the study allowed inclusion or exclusion of SR and membrane Ca²⁺ regulatory mechanisms, except the PMCA. This was particularly relevant in the efflux protocol, where the continued activity of PMCA may account for the slow decline in [Ca²⁺]_i even in the absence of [Na⁺]_o. However, given the large difference in the activities of PMCA vs. NCX (shown by the large change in slope of the [Ca²⁺]_i profile with reintroduction of [Na⁺]_o), this mechanism was ignored in the calculations. Further studies are required to characterise the interactions between volatile anaesthetics and the PMCA more clearly.

In the present study, we measured Ca²⁺ influx and efflux from entire cells. Since NCX proteins are distributed across the cell surface, cell size and thus cell surface area are factors that may potentially affect the calculations of absolute rates. We found that in the cells analysed, there was little variation in cell size, and thus a more or less consistent cell surface area. However, the range in absolute Ca²⁺ influx and efflux rates persisted even when the data were normalised for surface area. Therefore, it is likely that a substantial portion of the correlations reported are indeed attributable more to [Ca²⁺]_i rather than surface area. However, the contribution of surface area cannot be totally excluded since the area within membrane invaginations of the cardiac myocyte were not considered in our analysis. Nonetheless, our conclusions regarding volatile anaesthetic effects are unlikely to be affected since these comparisons were made within the same cell.

Effect of volatile anaesthetics on efflux mode of NCX

The effects of volatile anaesthetics on the efflux mode of NCX, arguably the more important aspect of NCX in cardiac function, has not been previously examined. A potential difficulty has been the lack of selective inhibitors or activators of NCX, beyond Na⁺ and Ca²⁺ themselves.

We found that at a fixed [Na⁺]_o, the rate of Ca²⁺ efflux via NCX was positively correlated with the 'peak' [Ca²⁺]_i level. This result is consistent with a number of previous studies (Matsuoka & Hilgemann, 1992; Blaustein & Lederer, 1999) that have demonstrated the dependence of NCX-mediated Ca²⁺ efflux on [Ca²⁺]_i with half-maximal activation in the range of 0.6-3 M. Mutational studies have suggested that there are two internal sites that bind Ca²⁺, with one Ca²⁺ molecule actually being extruded, while the other may serve a catalytic function (Matsuoka et al. 1995). In this respect, the effect of volatile anaesthetics is significant in that halothane blunts the correlation between [Ca²⁺]_i and the rate of Ca²⁺ efflux, while sevoflurane has negligible effects. These differences may be related to the relative lipid solubilities of the two anaesthetics and thus their target of action. However, further biochemical studies are needed to thoroughly establish the mechanisms.

Another significant finding was the effect of both halothane and sevoflurane on the [Na⁺]_o dependence of NCX-mediated Ca²⁺ efflux. Previous studies have shown that the Na⁺ dependence is sigmoidal (Matsuoka & Hilgemann, 1992; Blaustein & Lederer, 1999), with a half-maximal activation ranging from 50 to 140 mM, depending on the ATP state of the cell. In the present study, we found that at 140 mM [Na⁺]_o, the effect of both 1 and 2 MAC halothane was significantly greater than that of sevoflurane. However, the effects of the two anaesthetics were comparable at lower [Na⁺]_o. It has been speculated that the binding of three Na⁺ ions to two binding sites is necessary for the proper function of NCX in efflux mode (Blaustein & Lederer, 1999). Accordingly, it is possible that the differential effects of halothane vs. sevoflurane at different [Na⁺]_o may be related to competitive interference with Na⁺ binding. Another potential factor may be the level of ATP in the cell, which is known to influence the Na⁺ affinity of NCX (for review see Blaustein & Lederer, 1999). However, we did not examine this issue in this study.

Effect of volatile anaesthetics on influx mode of NCX

There is currently very little data on the effects of volatile anaesthetics on the influx mode of NCX in cardiac muscle. In the single study on rat cardiac myocytes, Haworth & Goknur (1995) showed that ⁴⁵Ca²⁺ uptake by suspensions of Na⁺-loaded cells was significantly inhibited in a dose-dependent fashion by clinically relevant concentrations of halothane, isoflurane and enflurane. Therefore, these investigators suggested that the influx mode of NCX was affected by volatile anaesthetics. However, in that study, the relative roles of [Ca²⁺]_i, [Na⁺]_i, [Ca²⁺]_o, and [Na⁺]_o were not examined. Blanck et al. (1997) reported that in fura-2-loaded rat cardiac myocytes, halothane had no effect on NCX. However, that study was performed under conditions of zero [Na⁺]_o, and it was speculated that sensitivity to volatile anaesthetics was reduced under these conditions. On the other hand, we also examined NCX-mediated Ca²⁺ influx under conditions of zero [Na⁺]_o and found that volatile anaesthetics have a proportionately greater inhibitory effect on influx mode of NCX, compared to efflux. Furthermore, the effects of halothane and sevoflurane were comparable. The underlying reasons for this discrepancy in the results of these two studies are not clear.

Previous studies have shown that the influx mode of NCX is totally dependent on [Ca²⁺]_i level (DiPolo, 1979; Rasgado-Flores et al. 1989). No influx occurs under conditions when [Ca²⁺]_i levels are less than 10 nM, a level not typically achieved in cardiac muscle. Half-maximal activation occurs in the range 0.6-2 M [Ca²⁺]_i. However, in our protocols, [Ca²⁺]_i levels prior to selective activation of NCX were in the range 200-250 nM, and slope measurements were made in the initial time period of the [Ca²⁺]_i response. Given the already small catalytic effect of [Ca²⁺]_i on influx in this Ca²⁺ range, the sensitivity of our protocol for evaluating the effect of volatile anaesthetics on the correlation between [Ca²⁺]_i and influx was low. Therefore, in the present study, we did not directly examine the effects of volatile anaesthetics on the relationship between [Ca²⁺]_i and influx via NCX. It is also possible that for a fixed [Na⁺]_i, the measured Ca²⁺ influx rate was an underestimation of the actual value under more realistic conditions during cardiac muscle contraction, where [Ca²⁺]_i levels are higher than 600 nM. This is also suggested by a somewhat curvilinear profile of [Ca²⁺]_i once NCX was activated. Nonetheless, the overall interpretation and conclusion of volatile anaesthetic effects are unlikely to be influenced, since the starting [Ca²⁺]_i level as well as the time period of measurement with or without anaesthetic were purposely made comparable.

We also found that both halothane and sevoflurane produce considerable alterations in the relationship between [Na⁺]_i and NCX activity. It should be emphasised that the actual [Na⁺]_i was not determined. Instead, it was assumed that using a fixed and known Na⁺ gradient and a fixed period during the Na⁺ loading of cells allowed for a similar [Na⁺]_i across experiments and conditions. Previous studies (Miura & Kimura, 1989; Matsuoka & Hilgemann, 1992) have shown that the activation of influx via NCX by [Na⁺]_i is a sigmoidal function with a half-maximal activation of 15-25 mM, a level likely to be have been achieved with Na⁺ loading. As with the efflux mode, there are two binding sites for three Na⁺ ions. The apparent affinity for Na⁺ is increased by [Ca²⁺]_i (DiPolo, 1979). It is possible that, as with efflux, volatile anaesthetics interfere with Na⁺ binding, thus inhibiting influx. Again, these biochemical targets remain to be determined.

Other mechanisms of volatile anaesthetic action

The mechanisms by which volatile anaesthetics produce their clinical effects, including decreased myocardial contractility, are still being investigated. It was generally believed that since anaesthetics are lipid soluble, their action was mostly a result of non-specific perturbation of various proteins in the plasma membrane and the SR. However, recent data suggest that anaesthetics may directly target the proteins themselves (Franks & Lieb, 1994). Accordingly, it is possible that the effects observed in the present study represent more than an indirect interference of anaesthetics with NCX function. The specific targets within the NCX protein remain to be determined.

In addition to the NCX, several other regulatory mechanisms in cardiac myocytes are also affected by volatile anaesthetics, especially halothane. A number of previous studies have demonstrated the inhibition of L-type Ca²⁺ channels (Lynch, 1986; Nakao et al. 1989; Bosnjak, 1991a,b; Bosnjak et al. 1992; Schmidt et al. 1993). This may have significant impact on the sustained phase of the cardiac action potential, especially when NCX-mediated Ca²⁺ influx may also be slowed. Furthermore, volatile anaesthetics also block both inward rectifier (Stadnicka et al. 2000) and ATP-sensitive (Han et al. 1996) K⁺ channels in cardiac muscle, both effects leading to membrane depolarisation and thus complex effects on Ca²⁺ influx through L-type channels as well as NCX activity. Previous studies (Casella et al. 1987; Katsuoka et al. 1989; Herland et al. 1990, 1996; Wilde et al. 1991; Frazer & Lynch, 1992; Connelly & Coronado, 1994; Lynch & Frazer, 1994; Wheeler et al. 1994) have also shown that SR Ca²⁺ release and re-uptake are affected by anaesthetics such that Ca²⁺ stores are depleted, leading to decreased myocardial contraction. In recent studies, Prakash et al. (2000) also demonstrated that both halothane and sevoflurane may influence myocardial contractility by altering the Ca²⁺ sensitivity of the contractile apparatus and/or by interference with cross-bridge cycling. Thus, the physiological significance of volatile anaesthetic effects on NCX may be influenced by the relative contribution of other effects of anaesthetics to cardiac function. Furthermore, the effect of anaesthetics on these other mechanisms such as L-type Ca²⁺ channels and the SR may lead to decreased myocardial contractility despite the potential positive inotropy induced by inhibition of NCX-mediated Ca²⁺ influx.

Clinical relevance

Inhibition of Ca²⁺ efflux via NCX by volatile anaesthetics should produce a positive inotropy in cardiac muscle due to the accumulation of intracellular Ca²⁺. Therefore, it appears that volatile anaesthetics produce myocardial depression despite the inhibition of NCX-mediated Ca²⁺ efflux. On the other hand, inhibition of the influx mode of NCX should dampen the [Ca²⁺]_i response to electrical stimulation, especially at the peak of the action potential. The overall effect of volatile anaesthetics on the [Ca²⁺]_i profile will be determined, at least in part, by the relative contributions of influx vs. efflux via NCX during each cycle. Furthermore, it is possible that NCX has a greater role to play in the regulation of the mean [Ca²⁺]_i over extended periods of time, preventing excess accumulation of [Ca²⁺]_i. Volatile anaesthetics also have several other effects on intrinsic Ca²⁺ regulation as well as neural mechanisms controlling cardiac contraction (for reviews see Rusy & Komai, 1987; Bosnjak, 1991a). The level of compensation by these other mechanisms becomes important in the overall effect of inhibited NCX. Volatile anaesthetic effects on NCX may take on additional significance under conditions where other regulatory mechanisms are unable to compensate, such as in cardiac failure, or in the neonate, where these mechanisms are not well established.

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Acknowledgements

This work is supported by grants GM 57816 (Y.S.P.) and GM 56686 (G.C.S.) from the National Institutes of Health, Bethesda, MD, USA and from the Mayo Foundation, Rochester, MN, USA. The authors acknowledge the technical assistance of Mr Larry Hunter in the studies.

Corresponding author

Y. S. Prakash: 4-184 W. Jos SMH, Mayo Clinic, Rochester, MN 55905, USA.

Email: prakash.ys{at}mayo.edu

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