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ANESTHETIC PHARMACOLOGY

Do Dopamine Receptors Mediate Part of MAC?

Yasumasa Tanifuji, MD^*, Yi Zhang, MD, Mark Liao, BS, Edmond I. Eger, II, MD, Michael J. Laster, DVM, and James M. Sonner, MD

From the Department of Anesthesia and Perioperative Care, University of California, San Francisco, CA; *Department of Anesthesiology, The Jikei University School of Medicine, Tokyo, Japan; and Department of Anesthesiology, Fuwai Hospital and Cardiovascular Institute, Beijing, People’s Republic of China.

Address correspondence and reprint requests to Edmond I. Eger II, MD, Departments of Anesthesia and Perioperative Care, S-455, University of California, San Francisco, CA 94143-0464. Address e-mail to egere{at}anesthesia.ucsf.edu.

	Abstract

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BACKGROUND: Depletion of central nervous system catecholamines, including dopamine, can decrease MAC (the minimum alveolar concentration of an inhaled anesthetic required to suppress movement in response to a noxious stimulus in 50% of test subjects); release of central nervous system catecholamines, including dopamine, can increase MAC; and increased free dopamine concentrations in the striatum can decrease MAC. Such findings suggest that dopamine receptors might mediate part of the capacity of inhaled anesthetics to provide immobility in the face of noxious stimulation.

METHODS: We measured the effect of blockade of D2 dopamine-mediated transmission with 0.3 mg/kg or 3.0 mg/kg droperidol on the MAC of cyclopropane, desflurane, halothane, isoflurane, or sevoflurane in rats, and the effect of 3.0 mg/kg droperidol on the dose or concentration of etomidate (an anesthetic known to act principally by enhancing the response of -aminobutyric acid_A receptors to -aminobutyric acid) required to suppress movement in response to noxious stimulation.

RESULTS: Blockade of D2 dopamine-mediated transmission with droperidol does not decrease the MAC of cyclopropane, desflurane, halothane, isoflurane, or sevoflurane or its equivalent for etomidate in rats.

CONCLUSIONS: These data, plus data from studies by others about D1 dopamine receptors, indicate that dopamine receptors do not mediate the immobility produced by inhaled anesthetics.

	Introduction

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Several reports imply conflicting conclusions regarding the contribution of dopamine and dopamine receptors to the immobility produced by inhaled anesthetics. Administration of amphetamines (1) or ipromiazid (2) (drugs that increase central nervous system, CNS concentrations of catecholamines, including dopamine) increases MAC; and administration of drugs such as -methyldopa or reserpine (2) (drugs that deplete CNS catecholamines) decreases MAC. There is a 30%–50% floor (maximum) to the decrease because of the depletion of CNS catecholamines. Such studies do not distinguish among CNS catecholamines (e.g., dopamine versus norepinephrine) as determinants of MAC.

In potential contradiction to the results of the studies described above, increases in CNS dopamine as obtained by administration of levodopa (L-DOPA) decreases the MAC of halothane (3). Selective antagonism of the D2, but not the D1, dopamine receptor reverses this effect (3). Administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine selectively decreases striatal dopamine and increases halothane MAC (3), and this report concludes that halothane MAC correlates significantly with striatal dopamine content (3). Consistent with that report, infusion of dopamine into the rat brain striatum decreases halothane MAC (4). These data thus point to the striatum as the mediator of dopamine effects on MAC. Because the spinal cord is the primary mediator of MAC (5–7), the striatal effects of dopamine might not be pertinent to an understanding of the immobility produced by inhaled anesthetics.

Finally, we have shown that administration of dizocilpine (MK-801), a blocker of N-methyl-d-aspartate (NMDA) receptors, can decrease MAC by a maximum of approximately 60% (8). But MK-801 is a "dirty" blocker, potently affecting other receptors, particularly dopamine (9). Thus, the results found with MK-801 may indicate that the dopamine receptor, rather than the NMDA receptor, is an important mediator of inhaled anesthetic-induced immobility.

The present study tested the importance of the dopamine receptor to inhaled anesthetic-induced immobility by determining the effect of droperidol administration on the MAC of five conventional anesthetics: cyclopropane, desflurane, halothane, isoflurane, and sevoflurane. In addition, we tested the effect of droperidol on the concentration of etomidate required to abolish immobility.

	METHODS

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Determination of MAC of Inhaled Anesthetics
With approval of the committee on animal research of the University of CA, San Francisco, we studied male (Crl:CD(SD)BR) rats weighing 250–450 g obtained from Charles River Laboratories (Hollister, CA). Rats were housed in rooms with daily cycles of 12 h of light and 12 h of dark and had water, and standard rat chow ad lib. Desflurane and isoflurane were obtained from Baxter Healthcare (New Providence, NJ); cyclopropane from Specialty Gases of America (Toledo, OH), halothane from Halocarbon (River Edge, NJ), and sevoflurane and etomidate from Abbott Laboratories (North Chicago, IL).

MAC for desflurane, halothane, isoflurane, or sevoflurane was determined concurrently in four rats placed in individual clear plastic cylinders. A rectal temperature probe was inserted, and the temperature probe and the tail of the rat were separately drawn through holes in the rubber stopper used to seal one end of the cylinder. Ports through the rubber stoppers in each end of the cylinder allowed gas delivery at the head end of the cylinder and exit of gas at the tail. A total flow rate of 4 L/min of oxygen and the potent inhaled anesthetic were delivered (average 1 L/min per cylinder), and the exiting gases were scavenged. Cyclopropane studies differed in that the total delivered gas flow was <1 L/min and the gases were recirculated through a carbon dioxide absorbent system. The volatile anesthetics were delivered from agent-specific vaporizers, and cyclopropane from a tank through a rotameter.

We administered one of the above inhaled anesthetics at a concentration estimated to be less than MAC for 40 min, after which the tail was clamped and the clamp on the tail rotated back and forth at approximately 1 Hz for up to 1 min (less if the rat moved; at this concentration, all rats moved). After certifying that movement had occurred, the concentration was increased by 20%–25%, and after a 20–30 min period of equilibration, the tail clamp was again applied and movement or lack of movement determined. This process continued until all rats failed to move in response to application of the tail clamp. MAC was calculated as the average of the largest concentration that permitted movement and the smallest concentration that suppressed movement.

The rats then were divided into two groups. The first (droperidol) group received an intraperitoneal injection of either 0.3 mg/kg droperidol or 3.0 mg/kg droperidol, each in approximately 4–5 mL of olive oil. The second (control) group received an intraperitoneal injection of the same volume of olive oil. The investigator determining MAC was blinded. MAC was then redetermined as above. If the injection of droperidol was 0.3 mg/kg, a third MAC determination might be made after injection of 3.0 mg/kg droperidol in olive oil.

Effect of Droperidol on the MAC of Etomidate
We asked if the effect of droperidol on MAC with inhaled anesthetics would differ for an anesthetic whose effect was clearly not mediated (at least directly) by dopamine receptors. Etomidate is such an anesthetic, acting primarily by enhancing the response of -aminobutyric acid receptors (GABA_A-R) to GABA (10). At least 24 h before study, IV catheters made of polyethylene (PE 10 tubing Portex Limited, Hythe, Kent, England CT21 6JL) were placed in the right internal jugular vein under isoflurane anesthesia, and the open end of the catheter was tunneled to the ear where it exited and could be accessed. The rats were divided into two groups. In the control group, 8 mg/h etomidate was infused for 5 min and then at 2 mg/h for 30 min. Stimulation of the tail at the end of this infusion evoked movement in all rats. The infusion was increased to 3 mg/h for 20 min and again stimulation produced movement. Further 20-min step increases of approximately 20% greater than the previous infusion rate were made until immobility was produced in one or more rats. When immobility was produced in a given rat, an arterial blood sample and the brain were taken and later analyzed for etomidate as described earlier (11). In the droperidol group, after intraperitoneal administration of 3 mg/kg droperidol, step-wise increases in etomidate were given by infusion through the IV catheter in the same manner as described for the control group. As with the control group, when a rat exhibited immobility to noxious stimulation, an arterial blood sample was obtained for etomidate analysis.

Analyses of Inhaled Anesthetics
We monitored the concentrations of the volatile anesthetics with a RGM monitor (Datex Ohmeda, Louisville, CO). We used a Gow-Mac gas chromatograph (Gow-Mac Instrument, Bridgewater, NJ) equipped with a flame ionization detector to measure concentrations of inhaled anesthetics at intervals, particularly immediately before stimulation of the tail. The 4.6 m-long, 0.22 cm (ID) column was packed with SF-96. The column temperature was 100°C. The detector was maintained at temperatures approximately 50°C warmer than the column. The carrier gas flow was nitrogen at a flow of 15–20 mL/min. The detector received 35–38 mL/min hydrogen and 240–320 mL/min air. Primary standards were prepared for each compound, and the linearity of the response of the chromatograph was determined. We often used secondary (cylinder) standards referenced to primary standards for some anesthetics (e.g., isoflurane).

Statistical Analyses
We calculated the MAC values for each rat for each of the three MAC determinations [initial (control), 0.3 mg/kg droperidol, and 3.0 mg/kg droperidol]. We calculated the percent change in MAC from the initial MAC produced by 0.3 mg/kg droperidol, and by 3.0 mg/kg droperidol, each rat serving as his own control. A Student’s t-test without correction for repeated analyses was used to compare the MAC values measured in the control group with those measured in the droperidol group or the change in MAC values. Several ANOVA models also were used to ascertain whether significant differences arose among inhaled anesthetics. For all analyses, we accepted P < 0.05 as indicating statistical significance.

	RESULTS

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The injection of droperidol did not consistently decrease MAC, either for the individual inhaled anesthetics (Table 1) or for these anesthetics collectively (Fig. 1). No dose-related effect of droperidol was obvious (Table 1, Fig. 1). The ratio of the second MAC to the initial MAC for the control group was 1.02 ± 0.09 (mean ± sd), whereas the ratio was 0.96 ± 0.10 for the 0.3 mg/kg droperidol group (n = 20; P > 0.05). Similarly, the ratio of the third MAC to the initial MAC for the control group was 1.01 ± 0.11, whereas the ratio was 0.97 ± 0.11 for the 3.0 mg/kg droperidol group (n = 18; P > 0.05). Finally, combining all ratios for the control versus droperidol groups gave respective values of 1.01 ± 0.11 vs 0.97 ± 0.10 (n = 38; P > 0.05).

View larger version (17K): [in this window] [in a new window]   Figure 1. An initial MAC was obtained for all rats. The rats then were divided into two groups, an experimental (droperidol) group that received 0.3 mg/kg droperidol and a control group that received only the vehicle for the droperidol. MAC was determined a second time in both groups. The droperidol group then received a second dose of droperidol (3.0 mg/kg) while again the control group received only vehicle. MAC was determined a third time in both groups. For both groups, the ratio of the second to the initial MAC (n = 20/group) and the ratio of the third to the initial MAC (n = 18/group) were determined and compared (Student’s t-test). Values are given as the mean, sd. Neither comparison revealed a significant difference (P > 0.05), nor was there a trend to an increasing difference with an increase in droperidol dose. Baxter Healthcare Corp. donated the desflurane and isoflurane used in these studies.

In addition, we applied two ANOVA models. In the first, we assumed that the MAC ratios could be a function of the droperidol dose (0, 0.3, or 3 mg/kg) and the anesthetic. We asked if, in the control groups getting no droperidol, does the ratio of second MAC to control versus the third MAC to control differ? The answer was no (P = 0.744, so those data were pooled.).

Next we applied a two-way ANOVA using two models. In the first, we asked, do the MAC values for 0, 0.3, or 3 mg/kg of droperidol differ? The answer was no (P = 0.223). We also asked if the MAC ratios differed by anesthetic (no, P = 0.055). Finally, we asked if the effect of droperidol depended on the choice of anesthetic (no, P = 0.067).

In the second ANOVA model, we asked if the MAC for the animals that got droperidol differed from the MAC of those that did not (no, P = 0.070). Finally, we asked if the MAC ratios by anesthetic differed (no, P = 0.125), or if there was an interaction [did the effect of droperidol depend on the anesthetic the animals were given (no, P = 0.271).]

The infusion rate of etomidate needed to produce immobility was not less for rats given 3.0 mg/kg of droperidol (9.75 ± 0.96 mg/h; n = 4) than for control rats (9.25 ± 1.26 mg/h; n = 4). No difference was found for either the cerebral (49 ± 31 µg/g of brain versus 58 ± 17 µg/g of brain) or plasma (11.1 ± 6.3 µg/mL of plasma versus 11.1 ± 0.8 µg/mL of plasma) levels of etomidate.

	DISCUSSION

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Overall, the administration of 0.3 mg/kg or 3.0 mg/kg of droperidol did not affect the concentration of inhaled anesthetics needed to produce immobility (did not affect MAC). This would seem to indicate that dopamine receptors are not important mediators of the immobility produced by inhaled anesthetics. However, another interpretation is possible. If MAC of all the test inhaled anesthetics potently decreases the effect of dopamine, then no further effect might be produced by administration of a blocking drug such as droperidol. That is, the dopamine receptors might already be dormant, either because dopamine release is hindered, the receptors are blocked, or both. But isoflurane increases, rather than decreases, release of dopamine in the striatum (12), so hindered release would not seem to be an issue. And administration of L-DOPA decreases halothane MAC in mice, while selective antagonism of the D2 dopamine receptor with YM-09151–2 attenuates this effect of L-DOPA (3), indicating that dopamine receptors continue to be affected by dopamine during anesthesia with halothane.

Etomidate acts by enhancing the action of GABA on GABA_A receptors (10,13,14). Thus, our finding that droperidol does not affect etomidate requirement indirectly argues that suppression of dopamine release or blockade of dopamine receptors does not (at least need not) underlie the immobility produced by inhaled anesthetics.

Further to this point, droperidol potently binds to D2 dopamine receptors (15). Binding to D1 dopamine receptors appears not to have been studied, but may be minimal. Segal et al. (3) demonstrated that L-DOPA administration increased striatal dopamine nearly four-fold, and that this administration decreased halothane MAC by 49%. Selective blockade of the D1 dopamine receptor with SCH-23390 did not alter the L-DOPA-induced decrease in halothane MAC, but selective antagonism of the D2 dopamine receptor with YM-09151–2 attenuated the effect of L-DOPA on MAC. These results suggest a minimal role for D1 receptors as mediators of the immobility produced by inhaled anesthetics, and the present results demonstrate that D2 receptors also are of minimal importance. The combined results indicate that dopamine receptors are not important to MAC.

As noted in the Introduction, administration of drugs that increase CNS concentrations of catecholamines including dopamine [e.g., amphetamines (1) or ipromiazid (2)], increases MAC; and administration of drugs that deplete CNS catecholamines [e.g., -methyldopa or reserpine (2)] decreases MAC. The present finding that dopamine receptors do not mediate immobility suggests that other CNS catecholamine receptors (especially norepinephrine) may be important mediators.

We previously demonstrated (8) that administration of dizocilpine (MK-801), a blocker of NMDA receptors, can decrease MAC for conventional anesthetics (including cyclopropane, halothane, isoflurane, and sevoflurane) by a maximum of approximately 60 percent. The decrease in MAC did not correlate with the capacity of the anesthetics to block NMDA receptors, and we interpreted the absence of a correlation as indicating that NMDA receptors do not mediate the immobility produced by conventional inhaled anesthetics. However, because MK-801 more potently blocks dopamine receptors than NMDA receptors (9), we suggested that an effect on dopamine might have compromised our interpretation of the importance of NMDA receptors. Our present finding that blockade of dopamine receptors does not decrease MAC suggests that blockade of dopamine receptors did not compromise our interpretation.

Our interpretation of the results of the present study would likewise be compromised if the doses of droperidol we chose were insufficient to appreciably block dopamine receptors. Results from other studies suggest that even the lower dose used in the present study would be sufficient to block dopamine receptors for the period of study. For example, 0.6, 1.0, and 3.0 mg/kg of droperidol block dopaminergic mediated behavior (16), and 1.5 mg/kg produces catalepsy lasting more than 2 h (17).

Finally, two of the 10 changes in MAC in Table 1 were significant, perhaps suggesting a small effect of droperidol. However, the smallness of these changes (12% and 18%), and the finding that they were not dose-related [e.g., the 12% decrease seen with sevoflurane at the 0.3 mg/kg dose of droperidol was not seen with the 3.0 mg/kg dose of droperidol (a 7% decrease that was not significant); also see Figure 1] leads us to discount the 2 significant changes. Note that, overall, there was only a 4% difference (decrease) between control and droperidol results.

We conclude that dopamine receptors do not mediate the immobility produced by inhaled anesthetics.

	ACKNOWLEDGMENTS

 
Dr. Eger is a paid consultant to Baxter Healthcare Corp. Baxter Healthcare Corp. donated the desflurane and isoflurane used in these studies.

	Footnotes

 
Accepted for publication July 14, 2006.

Supported by National Institutes of Health Grant 1PO1GM47818.

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