Interaction of Halogenated Anesthetics with Dobutamine in Rat Myocardium

We used adult Wistar rats that weighed 250-300 g. Care of the animals conformed to the recommendations of the Helsinski Declaration, and the study was performed in accordance with the regulations of the official edict of the French Ministry of Agriculture.

As previously reported, left ventricular papillary muscles were studied in Krebs-Henseleit bicarbonate buffer solution (118 mM NaCl, 4.7 mM KCl, 1.2 mM MgSO⁴, 1.1 mM KH²PO⁴, 25 mM NaHCO³, 2.5 mM CaCl², and 4.5 mM glucose) maintained at 29 [degree sign]C. [2,3,12]Preparations were field stimulated at 12 pulses/min by two platinum electrodes with rectangular wave pulses lasting 5 ms just above threshold. The bathing solution was bubbled with 95% oxygen and 5% carbon dioxide, resulting in a pH of 7.4. After a 60-min stabilization period at the initial muscle length at the apex of the length-active isometric tension curve (L^max), papillary muscles recovered their optimal mechanical performance. The extracellular calcium concentration ([Ca²⁺]^o) was decreased from 2.5 mM to 0.5 mM because rat myocardial contractility is at its nearly maximum value at 2.5 mM. [13,14]Because there are important differences in baseline values from one muscle to another, inotropic responses were expressed as a percentage of baseline values (i.e., after exposure of halogenated anesthetics), as previously reported. [2,3]We found that decreasing contractility by reducing the extracellular calcium concentration does not induce any potentiation of [Greek small letter alpha]- and [Greek small letter beta]-adrenoceptor stimulations. [2,3] 

Thus, in control groups (n = 10 in each group) we studied the effects of increasing concentrations (10^-8-10-3M) of dobutamine (Lilly, Indianapolis, IN) in a cumulative manner. The total volume of drugs did not exceed 2% of the bath volume. The inotropic and lusitropic responses were recorded 10 min after each dose was added to the bathing solution. We studied the same concentrations of dobutamine in separate groups of papillary muscles previously exposed to equianesthetic concentrations of four halogenated anesthetics agents (halothane, isoflurane, sevoflurane, or desflurane) at 1 (n = 10 in each group) or 2 (n = 6 in each group) minimum alveolar concentration (MAC). We also compared the effects of dobutamine with those of isoproterenol (10^-8-10-4M) in the presence of phentolamine (10^-6M) and phenylephrine (10^-8-10-4M) in presence of propranolol (10^-6M).

Because desflurane releases intramyocardial catecholamine stores in rat myocardium, [14]the effects of dobutamine in the presence of desflurane were also tested in rats pretreated with reserpine (4 mg/kg injected intraperitoneally 24 h before killing; n = 10). Finally, we studied the effects of dobutamine in the presence of 1 MAC isoflurane and [Greek small letter alpha]-adrenoceptor blockade (10^-6M phentolamine; n = 8) and compared them with those of dobutamine in the presence of [Greek small letter alpha]-adrenoceptor blockade (n = 8).

Halothane (Fluotec 3; Cyprane Ltd., Keighley, UK), isoflurane (Fortec 3, Cyprane Ltd.), sevoflurane (Sevotec 3; Ohmeda, West Yorkshire, UK), and desflurane (Devapor; Drager, Lubeck, Germany) were added to the carbon dioxide-oxygen mixture using calibrated vaporizers. The gas mixture bubbled continuously in the bathing solution. To minimize the evaporation of halogenated anesthetics, the jacketed reservoir was covered with a thin paraffin sheet. [2,3]Anesthetic concentrations in the gas phase were measured continuously using an infrared calibrated analyzer (Artema MM206; Taema, Antony, France). The following concentrations were used: halothane, 0.6 and 1.2 vol%; isoflurane, 0.8 and 1.6 vol%; sevoflurane, 1.4 and 2.8 vol%; and desflurane, 3.7 and 7.5 vol%. These concentrations are equivalent to 1 and 2 MAC in the adult rat at 29 [degree sign]C, respectively. [13-15]A 20-min equilibration period was allowed before baseline values of mechanical parameters were recorded.

The electromagnetic lever system was described previously. [12]Briefly, the load applied to the muscle was determined using a servomechanism-controlled current through the coil of an electromagnet. Muscular shortening induced a displacement of the lever, which modulated the light intensity of a photoelectric transducer. All analyses were made from digital records of force and length obtained using a computer, as previously described. [2,3] 

Conventional mechanical parameters at L^maxwere calculated from three twitches. The first twitch was isotonic and was loaded with the preload corresponding to L^max; we determined maximum shortening (^max) Vc) and lengthening (^max) Vr) velocities from this twitch. The second twitch was clamped abruptly to zero-load just after the electric stimulus, with a critical damping to slow the first and rapid shortening overshoot resulting from the recoil of series passive elastic components [16]; the maximum unloaded shortening velocity (V^max) was determined from this twitch. The third twitch was fully isometric at L^max; the maximum isometric active force normalized per cross-sectional area (active isometric force [AF]), and the peak of the positive (+dF/dt) and the negative (-dF/dt) force derivatives at L^maxnormalized per cross-sectional area were determined from this twitch. V^maxand AF tested the inotropic state under low (isotony) and high (isometry) loads, respectively. Because changes in the contraction phase induce coordinated changes in the relaxation phase, (^max) Vr and -dF/dt cannot assess lusitropy, and thus variations in contraction and relaxation must be considered simultaneously to quantify drug-induced changes in lusitropy. [17]The coefficient R1 =^maxVc/(^max) Vr evaluated coupling of the lusitropy under low load. [17]During isotonic conditions, the amplitude of sarcomere shortening is greater than that observed during isometric conditions. [18]Because of the lower sensitivity of myofilaments for calcium when the cardiac muscle is markedly shortened under low load, relaxation proceeds more rapidly than contraction, apparently because of the rapid uptake of calcium by the sarcoplasmic reticulum. [19]Thus, in rat myocardium, R1 tests sarcoplasmic reticulum uptake function. [13,14]The coefficient R2 = (+dF [middle dot] dt^-1/-dF[middle dot] dt^-1) evaluated the lusitropy under high load. When the muscle contracts isometrically, sarcomeres shorten very little. [18]Because of the higher sensitivity of myofilaments for calcium, [19]the time course of relaxation is determined by calcium unbinding from troponin C rather than by calcium sequestration by the sarcoplasmic reticulum. Thus, R2 indirectly reflects myofilament calcium sensitivity. [13,14]A decrease in R1 or R2 indicates a positive lusitropic effect.

Data are expressed as the mean +/- SD. Concentration-response curves were determined by fitting the data to the Hill sigmoid pharmacologic model (Origin 5.0; Microcal Software, Northampton, MA) according to the following equation: Equation 1in which Eff⁰is the observed effect at the C concentration, Eff^maxis the maximum effect, C⁵⁰is the concentration that results in 50% of Eff^max, and n is the Hill coefficient. The Hill plots are shown only for halothane and desflurane, but the derived variables are shown for all halogenated anesthetics. Several means were compared using analysis of variance and the Newman-Keuls test. All P values were two tailed, and a P value less than 0.05 was required to reject the null hypothesis. Statistical analyses were performed on a computer using NCSS 6.0 software (Statistical Solutions Ltd., Cork, Ireland).

We studied 114 left ventricular papillary muscles. The mean L^maxwas 5.2 +/- 0.6 mm; the mean cross-sectional area was 0.67 +/- 0.06 mm (²); the mean ratio of resting force to total force was 0.15 +/- 0.04; the mean contraction-relaxation coupling under low load (R1) was 0.69 +/- 0.03, at a [Ca²⁺]^oof 2.5 mM; and no significant differences were noted among the groups. Decreases in V^max(69 +/- 6%) and AF (57 +/- 7%) were observed as [Ca²⁺]^owas decreased from 2.5 to 0.5 mM, as previously reported. [2,3]Even after exposure to halogenated anesthetics, no significant differences in baseline values were noted between groups (Table 1).

In the control group, dobutamine induced a significant positive inotropic effect in isotonic and isometric conditions. Peak enhancement of V (^max) and AF occurred at 10^-4M dobutamine (Table 2). Furthermore, dobutamine induced a significant positive lusitropic effect under low load but not under high load (Table 2). When a higher (10^-3M) concentration of dobutamine was tested, we observed a decrease in the positive inotropic effect (AF: 141 +/- 48% of baseline) compared with 10^-4M and the disappearance of the positive lusitropic effect under low load (R1: 100 +/- 21% of baseline, not significant), which suggested a toxic effect, as previously reported. [10]Consequently, the concentration-response curves were analyzed between 10^-8and 10^-4M. The magnitude of the positive inotropic effect of dobutamine was comparable to that of isoproterenol and significantly greater than that of phenylephrine (Table 2). Nevertheless, the concentration-response curve of dobutamine was shifted to the right; that is, it was less potent compared with that of isoproterenol (AF, C⁵⁰: 3.4 +/- 2.3 vs. 0.5 +/- 0.4 [micro sign]M, P < 0.05). Dobutamine did not induce any significant lusitropic effect under high load (Table 2).

(Figure 1) shows the absolute values of AF recorded in response to increasing concentrations of dobutamine, during control conditions, and in the presence of 1 MAC halothane and desflurane. At 1 MAC, halothane induced a greater decrease in AF (56 +/- 18% of baseline) compared with those of isoflurane (90 +/- 10%), sevoflurane (92 +/- 7%), and desflurane (96 +/- 12%). However, because the baseline value of AF differs markedly from one papillary muscle to another, it is difficult to show a pharmacologic effect when using absolute values (Figure 1). When measured as a percentage of baseline halogenated anesthetic response, the positive inotropic effect of dobutamine was not significantly enhanced in the presence of halothane, isoflurane, and sevoflurane (Table 3, Figure 2). We could not accurately study the effects of 2 MAC halothane, because of excessive contractile depression (AF: 37 +/- 18% of baseline). In contrast, desflurane significantly potentiated the positive inotropic effect of dobutamine (Table 3, Figure 2).

Halothane, isoflurane, sevoflurane, and desflurane did not significantly modify the positive lusitropic effect under low load of dobutamine (Figure 3A). Under high load (R2), the halogenated anesthetics, except halothane, did not significantly modify the lusitropic effects of dobutamine (Figure 3B).

Because desflurane enhanced the positive inotropic effect of dobutamine, we investigated the mechanism of this effect. As previously reported, [14]we studied an additional group of rats pretreated with reserpine (n = 10). In this group, desflurane abolished the potentiation of the positive inotropic effect of dobutamine (Figure 4).

Because the absence of potentiation of the inotropic effect of dobutamine by most halogenated anesthetic agents contrasts with previous results obtained using a pure [Greek small letter beta]- or pure [Greek small letter alpha]-adrenoceptor stimulation, we studied the interaction of isoflurane and dobutamine in the presence of [Greek small letter alpha]-adrenoceptor blockade. In the presence of [Greek small letter alpha]-adrenoceptor blockade, the positive inotropic effect of dobutamine was slightly greater, but the difference was not statistically significant (AF, Eff^max: 208 +/- 25% vs. 185 +/- 36% of baseline; P = 0.07). Even in the presence of [Greek small letter alpha]-adrenoceptor blockade, isoflurane did not potentiate the positive inotropic effect of dobutamine (Figure 5).

Dobutamine exhibited a maximum positive inotropic effect that occurred at 10^-4M, as previously reported. [20,21]The magnitude of this positive inotropic effect of dobutamine was comparable to that of isoproterenol and significantly greater than that of phenylephrine (Table 2), and the concentration-response curve of dobutamine was shifted to the right compared with that of isoproterenol, as previously reported. [20]The lusitropic effects of dobutamine differed slightly from those of isoproterenol (Table 2). [2,3]Indeed, whereas [Greek small letter beta]-adrenergic stimulation induces positive lusitropic effects under low and high load, dobutamine did not exhibit significant lusitropic effects under high load. This results suggests that dobutamine did not modify the calcium myofilament sensitivity. It should be recognized that, despite its wide use in clinical practice, little information is available about the precise effects of dobutamine on intrinsic myocardial contractility [10,20,21]and its lusitropic effects. [22,23]Indeed, several in vivo studies have shown that dobutamine improved left ventricle relaxation, [22,23]but the magnitude of the lusitropic effect of dobutamine is difficult to analyze because of changes in afterload. Furthermore, the fact that relaxation depends on contraction was not accounted for in these in vivo studies. Our results are consistent with the knowledge that dobutamine is an agonist at the [Greek small letter alpha]^1-and [Greek small letter beta]^1-myocardialadrenoceptors. [21]The differences observed in the effects of dobutamine and isoproterenol (Table 2) are also consistent with several studies indicating that the cardiac effects of dobutamine differ from those of isoproterenol. Indeed, dobutamine increases heart rate and oxygen consumption less than isoproterenol does. [24,25] 

The primary findings of our study focused on the interaction between dobutamine and halogenated agents, which are not consistent with our previous studies of the interaction of halogenated anesthetics with [Greek small letter alpha]- or [Greek small letter beta]-adrenoceptor stimulations in rat myocardium. [2,3]These previous investigations showed that halothane, isoflurane, and sevoflurane potentiated the positive inotropic effects of [Greek small letter alpha]- and [Greek small letter beta]-adrenoceptor stimulations. Furthermore, they emphasized that, although isoflurane and sevoflurane did not modify the positive lusitropic effect of [Greek small letter beta]-adrenoceptor stimulation, [3]halothane alters the positive lusitropic effect of [Greek small letter beta]-adrenoceptor stimulation under low load. [2]These previous studies were performed using isoproterenol or phenylephrine in combination with phentolamine or propranolol, respectively. Therefore, the interaction between the halogenated anesthetics and the adrenoceptor stimulations was assessed only using a pure [Greek small letter alpha]- or [Greek small letter beta]-adrenergic stimulation. In the current experiment, we evaluated the global effect of dobutamine, a clinically useful complex sympathomimetic amine. The inotropic effect of dobutamine, which was initially attributed to stimulation of myocardial [Greek small letter beta]¹adrenoceptors, [26]is now considered to result from [Greek small letter alpha]^1-and [Greek small letter beta]-adrenoceptor stimulation. [10,20,21]Because it has been shown recently that [Greek small letter alpha]-adrenoceptor stimulation may modulate [Greek small letter beta]-adrenoceptor response, [11]we wanted to determine whether such a modulation might explain the lack of enhancement by halogenated anesthetics of dobutamine's inotropic effect. Even after blockade of [Greek small letter alpha] adrenoceptors with phentolamine, isoflurane did not potentiate the inotropic effect of dobutamine (Figure 5). Therefore, we suggest that the absence of interaction between halogenated anesthetic agents and dobutamine is related to the characteristics of dobutamine, because its myocardial effects differ from those of isoproterenol and phenylephrine (Table 2). [21]Additional studies will be needed to understand, on a molecular basis, the precise reasons for this absence of interaction.

Only desflurane potentiated the positive inotropic effect of dobutamine. To identify the mechanism of this potentiation, we studied rats treated with reserpine, which completely depletes intramyocardial catecholamine stores. After reserpine treatment, desflurane no longer potentiated the positive inotropic effect of dobutamine. Dobutamine may have enhanced the previously described release of catecholamines from intramyocardial sympathetic nerve endings elicited by desflurane. However, by itself, dobutamine has been shown to be devoid of such a presynaptic effect. [10] 

Finally, the halogenated anesthetics, except for halothane, did not modify the lusitropic effects of dobutamine. In the presence of halothane, dobutamine induced a positive lusitropic effect under high load. This result suggests an interaction of halothane and dobutamine at the myofilament level, which unmasks some of the effects of dobutamine on myofilament sensitivity. However, the effects of halothane per se on myofilament sensitivity remain controversial.

The following points must be considered when the clinical relevance of our results is assessed. First, this study was conducted at 29 [degree sign]C and at a low-stimulation frequency. Papillary muscles must be studied at this temperature because the mechanical parameters are not sufficiently stable at 37 [degree sign]C and at a low frequency because high-stimulation frequency induces core hypoxia. [27]Second, the study was performed using the rat myocardium, which differs from human myocardium. The density of [Greek small letter alpha] adrenoceptors and, consequently, the positive inotropic effect induced by their stimulation are greater in rats than in humans. [28]Nevertheless, the relative importance of [Greek small letter alpha] adrenoceptors in cardiac contractility can be increased in disease conditions. [29] 

In isolated rat myocardium, halogenated anesthetics did not modify the inotropic effects of dobutamine. Only desflurane enhanced the positive inotropic effect of dobutamine, but this effect was related to the desflurane-induced release in intramyocardial catecholamine stores.