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From the *Department of Anesthesia and Perioperative Care, University of California, San Francisco, California, and the Department of Anesthesiology, Fuwai Hospital and Cardiovascular Institute, Beijing, China.
Address correspondence and reprint requests to Edmond Eger, II, MD, Department of Anesthesia, S-455, University of California, San Francisco, CA 94143-0464. Address e-mail to egere{at}anesthesia.ucsf.edu.
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Abstract |
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METHODS: In the present study, we indirectly tested this possibility by measuring the capacity of acetylcholine receptor blockade to decrease the anesthetic requirement for etomidate, an anesthetic thought to act solely by enhancing the effect of 
-aminobutyric acid on -aminobutyric acidA receptors.
RESULTS: Administration of 10 mg/kg atropine plus 5 mg/kg mecamylamine did not change the infusion rate of etomidate, or the blood or brain concentrations of etomidate required to produce immobility in rats.
CONCLUSION: Acetylcholine receptors do not mediate the capacity of anesthetics to produce immobility in the face of noxious stimulation.
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Introduction |
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Suppose administration of a blocker of a given receptor does not affect MAC. Obviously, blockade of the receptor by the inhaled anesthetic cannot be the sole cause of anesthesia, or the blocker would have produced anesthesia. That is, at a minimum, another receptor must be blocked (or enhanced if it is a receptor mediating inhibitory impulses). Suppose that blockade of both receptors is needed to produce anesthesia. Thus, injection of a blocker (e.g., atropine to block muscarinc acetylcholine receptors; mecamylamine to block nicotinic acetylcholine receptors) of acetylcholine receptors that are already blocked by the inhaled anesthetic might not decrease the need for the anesthetic to block the second receptor, leaving the concentration required for anesthesia unchanged.
One test of this possibility would apply an anesthetic whose effect is mediated by a receptor other than a neuronal acetylcholine receptor; etomidate, for example, which acts solely by enhancing the action of -aminobutyric acid on -aminobutyric acidA receptors (5). If neuronal acetylcholine receptors can contribute to the immobility produced by anesthetics, then blockade of such receptors should decrease the requirement for etomidate. The present study examined this possibility.
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METHODS |
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Studies of MAC in Rats
With approval of the Committee on Animal Research of the University of CA, San Francisco, we studied 16 male Sprague Dawley rats (Crl:CD(SD)BR) weighing 300–450 g obtained from Charles River Laboratories (Hollister, CA). Each animal was caged alone, and all had continuous access to standard rat chow and tap water before study. IV catheters (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. Studies were performed after allowing at least 24 h for recovery to occur.
The infusion rates of etomidate, and the associated concentrations of etomidate in blood and brain needed to produce immobility were determined concurrently in two groups of eight rats placed in individual clear plastic cylinders, each cylinder receiving approximately 1 L/min oxygen. An infusion of etomidate was initiated at 4 mg/h via the previously placed IV catheters. After induction of anesthesia, a rectal temperature probe was inserted. Half of the rats were given 10 mg/kg atropine and 5 mg/kg mecamylamine intraperitoneally. The other half were given an injection of normal saline intraperitoneally. The investigator making the determination of anesthetic effect was blinded to the contents of the injections.
After administration of etomidate for 50 min, the tail was clamped and moved by rolling the clamp at 1–2 Hz for up to 1 min (less if the rat moved). After certifying that movement had occurred, the infusion was increased by 1 mg/h, and after a 40 min period of equilibration the tail clamp was again applied and movement or lack of movement determined. This process continued until one or more rats failed to move in response to application of the tail clamp. The ED50 was calculated as the average of the largest infusion that permitted movement and the smallest infusion that suppressed movement. When a given rat failed to move in response to stimulation, the abdomen was entered, the aorta canulated with a 20-gauge catheter, and approximately 10 mL of arterial blood drawn into a heparinized syringe (the exact volume was noted). Immediately after this exsanguination, the brain was removed and weighed. Etomidate was immediately extracted from both the blood and brain.
Extraction and Analysis of Etomidate
Hundred microliters sodium fluoride (10 mg/mL) was added to all samples. Each sample of blood or brain then was mixed with a volume of n-pentane twice that of the blood or brain. Blood was mixed with the n-pentane by vortexing for 60 s. The cerebral samples were homogenized and vortexed for 60 s. The blood and cerebral samples were centrifuged at 3500 rpm for 10 min and the supernatant pentane phase was then removed. Extraction of the etomidate with n-pentane was repeated a second time. The pooled pentane was evaporated under nitrogen. The dried samples were stored frozen at –80°C until analyzed.
A high-performance liquid chromatograph (Agilent 1100 series, Agilent Technologies Inc, Mountain View, CA), equipped with an autosampling system was used. Analyses were performed on a 3.5 µm C-18 Polaris column (15 cm x 4.6 mm internal diameter) operating at ambient room temperature (20–25°C). Acetonitrile, methanol, and 0.05 M dibasic sodium phosphate (25:20:55) with a pH value of 8.1 were used as mobile phase. The flow rate was 0.25 mL/min and elution was monitored at 242 nm. The frozen samples were reconstituted in 100 or 200 µL of eluent, and a 40-µL aliquot was injected. Quantitation was performed by comparing the values for these samples against values obtained from a calibration curve composed from samples covering a range of 0–200 µg/mL etomidate prepared in both blank blood and blank homogenized brain. Areas under all peaks were measured. For the calibration curves, these increased rectilinearly over the 0–200 µg/mL range with r2 > 0.99.
Samples of blood and brain were spiked with known quantities of etomidate and treated as were the experimental samples. Recovery of etomidate equaled 100%, and an internal standard was not used for either these control or the experimental samples.
Statistical Analyses
Mean values and standard deviations were determined for the ED50, and the blood and brain concentrations. These were compared using Student’s t-test. We accepted a value of P < 0.05 as significant.
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RESULTS |
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DISCUSSION |
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Our thesis depends on at least two assumptions. First, we assume that sufficient mecamylamine remained to produce blockade at the time of measurement of immobility. Given that the half-life of mecamylamine in rats exceeds an hour (7,8), this would seem to be a reasonable assumption. Second, we assume that etomidate itself does not maximally block neuronal nicotinic acetylcholine receptors. An analog of etomidate, azietomidate, a compound with anesthetic properties that parallel those of etomidate (9), can photolabel nicotinic acetylcholine receptors (10). Etomidate and azietomidate can, indeed, block acetylcholine receptors, but the concentration needed to produce 90% blockade is approximately two orders of magnitude greater than required to produce immobility (10,11), considering the 80% plasma binding of etmoidate in the rat (12). Thus, we believe the present evidence supports our contention that acetylcholine receptors play no role in the immobility produced by inhaled anesthetics. Although blockade may have no relevance to MAC, the present results do not exclude an importance of acetylcholine receptors to other important aspects of anesthesia, particularly learning and memory. Large doses of atropine, alone, can cause amnesia (13) and unconsciousness (14).
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Footnotes |
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Supported in part by NIH grant 1P01GM47818.
Dr. Eger is a paid consultant to Baxter Healthcare Corp, who donated the isoflurane used in these studies.
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  L. J. Voss, J. W. Sleigh, J. P. M. Barnard, and H. E. Kirsch The Howling Cortex: Seizures and General Anesthetic Drugs Anesth. Analg., November 1, 2008; 107(5): 1689 - 1703. [Abstract] [Full Text] [PDF]
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E. I. Eger II, D. E. Raines, S. L. Shafer, H. C. Hemmings Jr, and J. M. Sonner Is a New Paradigm Needed to Explain How Inhaled Anesthetics Produce Immobility? Anesth. Analg., September 1, 2008; 107(3): 832 - 848. [Abstract] [Full Text] [PDF]
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