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

Abstract

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. 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. 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. 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.

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.

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).

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.

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).

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.

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.

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.