The intravenous anesthetic etomidate exhibits structural similarities to specific alpha2-adrenoceptor agonists of the type such as dexmedetomidine. The current study was performed to elucidate the possible interaction of etomidate with alpha2-adrenoceptors in mice lacking individual alpha2-adrenoceptor subtypes (alpha2-KO).
Sedative and cardiovascular responses to etomidate and the alpha2-agonist, dexmedetomidine, were determined in mice deficient in alpha2-receptor subtypes. Inhibition of binding of the alpha2-receptor antagonist [3H]RX821002 to recombinant alpha2-receptors by etomidate was tested in human embryonic kidney (HEK293) cells in vitro.
In vivo, loss and recovery of the righting reflex required similar times after intraperitoneal injection of etomidate in wild-type and in alpha2A-receptor-deficient mice, indicating that the hypnotic effect of etomidate in mice does not require the alpha2A-receptor subtype. Intravenous injection of etomidate resulted in a transient increase (duration 2.4 +/- 0.2 min) in arterial blood pressure in wild-type mice (17 +/- 3 mmHg). Etomidate did not affect blood pressure in alpha2B-KO or alpha2AB-KO mice. In membranes from HEK293 cells transfected with alpha2-receptors, etomidate inhibited binding of the alpha2-antagonist, [3H]RX821002, with higher potency from alpha2B- and alpha2C-receptors than from alpha2A-receptors (Ki alpha2A 208 microm, alpha2B 26 microm, alpha2C 56 microm). In alpha2B-receptor-expressing HEK293 cells, etomidate rapidly increased phosphorylation of the extracellular signal-related kinases ERK1/2.
These results indicate that etomidate acts as an agonist at alpha2-adrenoceptors, which appears in vivo primarily as an alpha2B-receptor-mediated increase in blood pressure. This effect of etomidate may contribute to the cardiovascular stability of patients after induction of anesthesia with etomidate.
ETOMIDATE (R-(+)ethyl-1-(1-phenylethyl)-1H-imidazole-5-carboxylate) is a potent, short-acting hypnotic that was introduced as an anesthetic in 1972. 1Major advantages described with the use of etomidate are its hemodynamic stability and the minimal respiratory depression. 2,3This pharmacologic profile renders etomidate particularly suitable for induction of anesthesia in critically ill patients and patients with cardiovascular disease. The anesthetic effect is thought to be mediated primarily through an action on γ-aminobutyric acid receptors. 4–6In addition, interactions of etomidate with second messenger systems such as the nitric oxide metabolism have been shown. 7However, similar to other general anesthetics, the exact mechanism of its action remains to be shown.
The carboxylated imidazole etomidate exhibits structural similarities to specific α2-adrenoceptor agonists that belong to the class of imidazole compounds, such as clonidine and dexmedetomidine. 8,9Besides the chemical structure, etomidate and α2-adrenoceptor agonists share some clinical similarities, such as inducing sedation/hypnosis with high cardiovascular stability and only minor respiratory depression. 2,10,11In accordance with these findings, previous in vitro and in vivo studies indicate that the anesthetic action of etomidate might at least be partially mediated by α2-adrenoceptors. 12
Three subtypes of α2-adrenoceptors, termed α2A, α2B, and α2C, have been cloned from several species including mice and humans. 13–15In both species, α2-receptor subtypes are encoded by distinct genes which are localized on separate chromosomes. To further elucidate the role of α2-adrenoceptors in the effects of etomidate, we studied the interaction of etomidate with α2-adrenoceptor subtypes in vivo in mice carrying targeted deletions of α2-receptor genes 16and in vitro in human embryonic kidney (HEK293) cells expressing individual murine α2-receptor subtypes. 17
Materials and Methods
Generation and Genotyping of α2-Adrenoceptor–deficient Mice
The generation of the mouse lines lacking single α2-adrenoceptor subtypes has been described previously. 18,19Mice lacking α2A- and α2B-receptors (α2AB-KO) were generated by crossing of single gene knockout lines to first obtain double heterozygous mice, which were further intercrossed until homozygous α2AB-deficient mice were born. Genotypes were confirmed by subtype-specific polymerase chain reactions performed with genomic DNA isolated from small tail biopsies as described in detail. 20Mice were maintained in a specified pathogen-free facility. The University of Würzburg and the Government of Unterfranken (Würzburg, Germany) approved all animal procedures (protocol No. 621–2531.01–28/01).
Sedation/Hypnosis Induced by Etomidate
Mice were given an intraperitoneal injection of etomidate (5–50 mg/kg body weight), thiopental (50 mg/kg body weight), or dexmedetomidine (500 μg/kg or 1 mg/kg), and the time after injection at which the righting reflex of mice was lost and the recovery time of this reflex were monitored. Because dexmedetomidine at these doses did not induce a loss of the righting reflex, its sedative effect was also assessed by placing mice on a rotating wheel (30 rpm) and determining the time for which mice stayed on the rod (Rotarod; Ugo Basile, Varese, Italy). For these series of experiments, male and female mice (3–4 months old, 5–10 mice per genotype) were used. The observer was blinded with respect to the genotype of the mice.
Blood Pressure Recording and Cardiac Catheterization
For hemodynamic measurements, two groups of male mice were used at the age of 3–5 months. In the first group, the cardiovascular effects of intravenous dexmedetomidine (5 μg/kg) were determined (eight mice per genotype). The second group was used to measure hemodynamic parameters after etomidate injection (6–10 mice per genotype). For aortic and left ventricular catheterization with a 1.4F pressure-volume catheter, 21mice were anesthetized with tribromoethanol (13 μl of 2.5% solution per g of body weight) and placed on a 37°C table. The microtip catheter was inserted into the right carotid artery and the pressure tip was advanced into the aorta or into the left ventricle. For injection of drugs, a PE-10 polyethylene tubing was inserted into the left jugular vein. Etomidate or dexmedetomidine were injected in 0.9% saline into the jugular vein catheter. Hemodynamic data were digitized via a MacLab system (AD Instruments, Castle Hill, Australia) connected to an Apple G4 PowerPC computer (Apple Computer, Inc., Cupertino, CA). 21
Stable cell lines of human embryonic kidney (HEK293) cells with high expression levels of either the cloned murine α2A-adrenoceptor (14.6 pmol receptor/mg protein) or α2C-adrenoceptor (16.8 pmol receptor/mg protein) subtype were grown to near confluency in cell culture at 37°C. 17In addition, because a stable cell line expressing the α2B-adrenoceptor subtype was not available, native HEK293 cells were transiently transfected with the cloned mouse α2B-adrenoceptor subtype by the calcium phosphate precipitation technique 22and used 48 h after transfection. Transient transfection of α2B-adrenoceptors resulted in expression levels that were similar to the stable cell lines.
Initially, HEK293 cells were washed with 5 ml of phosphate-buffered saline. The cells were then scraped from the culture dishes into 5 ml of ice-cold hypotonic buffer (5 mm Tris, 2 mm EDTA, pH 7.4) and washed again with 5 ml of ice-cold hypotonic buffer. Cells were homogenized with an Ultra-Turrax (Janke & Kunkel, Staufen, Germany) and cell nuclei and whole cells were removed by centrifugation at 1,700 g for 10 min at 4°C. Membrane vesicles were prepared by centrifugation of the supernatant at 145,000 g for 30 min at 4°C. The resulting final membrane pellet was resuspended in binding buffer (75 mm N -methyl-d-glucamine, 25 mm glycine, 40 mm HEPES, 5 mm EGTA, 10 mm MgCl, pH 7.4). Protein concentrations were determined using a colorimetric assay based on the method of Bradford 23with bovine serum albumin as a reference standard.
Competition Binding Studies
Competition binding of etomidate to α2-adrenoceptors was determined in a 200-μl reaction containing 10 nm to 1 mm etomidate, the α2-adrenoceptor antagonist [3H]RX821002 (3 nm), and binding buffer. Nonspecific binding was determined by addition of the α2-receptor antagonist atipamezole (1 μm). The 200-μl reactions containing only the solvent propylene glycol without etomidate were used as controls. Membranes (10–20 μg protein) were incubated at room temperature for 1 h in the binding mixture assay. The binding reaction was stopped by filtration using a cell harvester and three washes of buffer. Membrane-bound [3H]RX821002 was determined by scintillation counting.
Mitogen-activated Protein Kinase Phosphorylation
To determine the agonist activity of etomidate, untransfected or α2B-receptor–transfected cells were stimulated for 20 min with 10 μm etomidate. Before stimulation, HEK cells were incubated overnight in serum-free Dulbecco's Modified Eagle Medium. Stimulated HEK cells were rapidly scraped off the plates and frozen in liquid nitrogen. Cell lysates were prepared and used for Western blotting as described previously. 20Antibodies against the extracellular signal-related kinases ERK1/2 and phospho-ERK1/2 were purchased from Cell Signaling (Beverly, MA).
The α2-antagonist [3H]RX821002 was used as a radioligand with a specific activity of 67 Ci/mmol (Amersham Pharmacia Biotech, Freiburg, Germany). Nonspecific binding was determined using the antagonist atipamezole (Orion Corp., Turku, Finland). Etomidate was either used as Hypnomidate® (2 mg etomidate/ml dissolved in 35% propylene glycol in water; Janssen-Cilag, Neuss, Germany) or as Etomidate®-Lipuro (2 mg etomidate/ml liposome suspension; Braun, Melsungen, Germany). Propylene glycol was used as the inactive control solution.
The results of the competition binding were analyzed using a nonlinear regression program. A binding curve was fitted according to a one-site competition model (Prism; GraphPad, San Diego, CA). Data are presented as geometric mean with 95% confidence intervals. From the competition curves, Kivalues were calculated from EC50values using the Cheng Prusoff equation. 24Hemodynamic data were analyzed using Student t test for unpaired samples. A P value of less than 0.05 was considered as statistically significant. Results are displayed as means ± SEM.
In vivo , the sedative effect of α2-receptor activation is almost exclusively mediated by the α2A-receptor subtype. 25Thus, to test whether the sedative/hypnotic effect of etomidate is mediated via the α2A-receptor, the times for the loss and for recovery of the righting reflex were determined in wild-type mice and in mice lacking α2A- or α2B-receptors (α2-KO). For these experiments, intraperitoneal etomidate doses between 5 mg/kg and 50 mg/kg were tested in wild-type mice. Whereas 5 mg/kg etomidate did not result in loss of the righting reflex, 27% of the mice lost the reflex at 10 mg/kg etomidate and all mice transiently lost the righting reflex at 20-mg/kg and higher etomidate doses. Thus, we chose a dose of 30 mg/kg intraperitoneal etomidate to investigate sedative effects of etomidate in α2-receptor-KO mice. After injection of etomidate, the righting reflex disappeared in wild-type and α2A-deficient mice at similar times after intraperitoneal injection (fig. 1a). The duration of etomidate-induced sleep was not affected by the α2A-deletion and the righting reflex recovered at similar times in wild-type and α2A-KO mice. Similarly, hypnotic properties of intraperitoneal thiopental (50 mg/kg) did not differ between wild-type and α2A-KO mice (fig. 1b). Deletion of the α2B-receptor gene did not affect the sedative effects of etomidate or thiopental in mice (fig. 1). Intraperitoneal injection of the non–subtype-selective α2-agonist dexmedetomidine up to 1 mg/kg did not result in a loss of the righting reflex. Thus, the sedative effect of dexmedetomidine was assessed on a rotating rod. Dexmedetomidine exerted a sedative effect in wild-type mice and in α2B-KO mice, as evidenced by the observation that the mice were unable to stay on a rotating rod for longer than 10–20 s (wild-type, 9 ± 2 s; α2B-KO, 14 ± 6 s; n = 8 mice per genotype, 30 min after 500 μg/kg intraperitoneal dexmedetomidine). In contrast, α2A-KO mice did not show any sedative effects in response to dexmedetomidine (α2A-KO stayed on the rod for more than 60 s, n = 10 mice).
Hemodynamic Effects of Etomidate in α2-Receptor-deficient Mice
To test potential effects of etomidate on cardiovascular α2-receptor subtypes, we first generated double-knockout mice deficient in α2A- and α2B-receptors (fig. 2 a and b). The α2AB-deficient mice were generated by crossing single receptor knockouts to obtain double heterozygotes that were further crossed to yield mice with the α2A−/−B−/− genotype (fig. 2c). At weaning age, α2AB-KO mice were detected at a frequency that was close to the expected Mendelian ratio, indicating that genetic deletion of these receptors did not interfere with embryonic development. After weaning, α2AB-deficient mice developed normally and showed no obvious phenotypes.
First, we determined basal cardiovascular parameters in anesthetized α2AB-KO mice. Mean arterial pressure did not differ between α2AB-deficient animals (85 ± 4 mmHg, n = 9) and wild-type mice (89 ± 6 mmHg, n = 11). However, α2AB-KO mice had a higher resting heart rate (493 ± 13 min−1, n = 9) than wild-type control mice (446 ± 13 min−1, n = 11). The cardiovascular response to the α2-receptor agonist was tested in anesthetized mice of different genotypes. As reported previously, rapid intravenous injection of dexmedetomidine resulted in a transient hypertension in wild-type mice that was followed by a long-lasting hypotension (fig. 3a). Deletion of the α2A-receptor disrupted the hypotensive response, whereas the initial hypertension was absent in α2B-deficient animals. Interestingly, α2AB-KO mice showed a small but significant decrease in systolic blood pressure (−13 ± 4 mmHg). This pressure effect was accompanied by a decrease in heart rate (−25 ± 8 min−1, fig. 3b).
On intravenous injection of etomidate, wild-type mice showed a rapid and transient hypertensive response that was completely absent in mice lacking α2B-receptors (fig. 4). During the hypertensive phase, heart rate decreased by 18 ± 6 min−1in wild-type mice. No significant alterations in heart rate were observed in α2B-KO or α2AB-KO mice. After intravenous etomidate, mean arterial blood pressure remained above the preinjection value for 2.4 ± 0.2 min. The hypertensive effect of etomidate in wild-type mice was dose dependent: 1 mg/kg etomidate caused a small increase in blood pressure (systolic pressure, +6.7 ± 2.6 mmHg; diastolic pressure, +5.7 ± 2.2 mmHg; n = 6), whereas a dose of 2 mg/kg etomidate elicited significantly larger pressure responses (fig. 4; systolic pressure, +15.7 ± 2.7 mmHg; diastolic pressure, +17.0 ± 2.6 mmHg).
Two different preparations of etomidate, one in propylene glycol and the other in liposome suspension, showed identical effects on blood pressure in wild-type mice. Etomidate did not alter cardiac contractility as assessed by direct catheterization of the left ventricle. Maximal left ventricular contraction rate after intravenous injection of 2 mg/kg etomidate was identical (dp/dtmax101.6 ± 2.1% of control) to the value before etomidate.
Binding of Etomidate to Recombinant α2-Adrenoceptors
To confirm the in vivo data, we next tested whether etomidate interacts with recombinant α2-receptor subtypes expressed in HEK293 cells. For these experiments, cells expressing 14–18 fmol/mg of the three α2-receptor subtypes were used. Etomidate inhibited binding of the specifically bound α2-receptor antagonist [3H]RX821002 in a concentration- and subtype-dependent manner with greater potency for α2B- and α2C-receptors than for α2A-adrenoceptors (fig. 5).
To test whether etomidate acted as an agonist or antagonist at α2B-receptors, the effect of etomidate on α2-receptor–mediated mitogen-activated protein kinase activation was assessed in HEK293 cells. Among other intracellular signaling pathways, α2-receptors can effectively activate the mitogen-activated protein kinase cascade. 20In untransfected or α2B-receptor–expressing HEK293 cells, basal phosphorylation of the ERK1/2 kinases was hardly detectable (fig. 6). However, short-term exposure to etomidate increased ERK1/2 phosphorylation in HEK293 cells transfected with α2B-receptors but not in untransfected cells (fig. 6).
The present results indicate that etomidate interacts with α2-adrenoceptors and displaces the α2-adrenoceptor antagonist [3H]RX821002 in HEK293 cells from all subtypes in a concentration-dependent manner. In addition, etomidate leads to a transient increase in blood pressure in wild-type mice, but not in α2B-KO or α2AB-KO mice, suggesting an interaction of etomidate with α2B-adrenoceptors in the peripheral vasculature.
α2-Adrenoceptors have been implicated in a variety of physiologic functions. Pharmacologic studies and the development of genetic mouse models elucidated the physiologic effects mediated by the different α2-adrenoceptor subtypes. 16,18,26,27The α2A-adrenoceptor subtype has been reported to be the predominant subtype involved in the antinociceptive, sedative, hypotensive, hypothermic, and behavioral actions of α2-adrenoceptor agonists. 25,26,28,29Stimulation of α2B-adrenoceptors in vascular smooth muscle leads to vasoconstriction, which causes the initial hypertension after administration of α2-adrenoceptor agonists. 18In addition, the α2B-adrenoceptor subtype is involved in mediation of the antinociceptive action of nitrous oxide. 30,31The α2C-receptor has been shown to modulate dopaminergic neurotransmission, various behavioral responses, and to induce hypothermia. 32,33In addition, this subtype contributes to spinal antinociception of the imidazoline moxonidine in mice. 34
The exact molecular targets by which etomidate mediates its anesthetic effects remain under discussion. Clinically relevant plasma concentrations of etomidate to induce hypnosis vary considerably. During induction of anesthesia in humans, initial plasma concentrations of etomidate may be as high as 12–24 μm. 35These concentrations are similar to the Kivalue of etomidate for α2B-receptors expressed in HEK293 cells in our study. Etomidate concentrations from 1.2–8.2 μm have been reported as the minimal hypnotic plasma levels for anesthesia in humans. 35It has been shown that etomidate at these concentrations exhibits γ-aminobutyric acid–modulatory effects, and at higher concentrations exhibits γ-aminobutyric acid–mimetic effects at γ-aminobutyric acid receptor type A receptors. 4,5,36,37Interestingly, mice with a point mutation in the β3 subunit of the γ-aminobutyric acid receptor type A receptor did not show any suppression of noxious-evoked movements in response to etomidate. 6In this study, 5 mg/kg etomidate was injected intravenously in wild-type mice to elicit a loss of the reflex, which lasted for approximately 10 min. 6In our study, we observed a significant transient hypertensive response to intravenous injection of 2 mg/kg etomidate. Thus, higher doses of etomidate may even cause a more pronounced hypertensive phase in mice. However, in the other study, the mice were already anesthetized with tribromoethanol for arterial catheterization and we did not use higher doses of etomidate to prevent toxicity. The low affinity of etomidate for the α2A-adrenoceptor subtype determined in the current study challenges the assumption that the anesthetic effects of etomidate could be partially mediated by an action on cerebral α2-adrenoceptors. Indeed, we found no difference in the sedative/hypnotic effect of etomidate between normal mice and animals lacking functional α2A-adrenoceptors.
Our results show a higher affinity of etomidate for α2B- and α2C-adrenoceptor subtypes expressed in HEK293 cells. The role of the α2Cadrenoceptor subtype in anesthesia and cardiovascular control is currently still unknown. Previous investigations in mice have shown that disruption of the α2C-adrenoceptor subtype does not lead to hemodynamic effects. 18However, α2C-receptors operate together with α2A-receptors as presynaptic inhibitory regulators of sympathetic norepinephrine release. 21To unequivocally distinguish the cardiovascular roles of α2C- and α2B-receptors in vivo , we generated mice deficient in α2A- and α2B-receptors. Interestingly, these mice showed a small but significant decrease of the heart rate in response to the α2-agonist dexmedetomidine, which was accompanied by a decrease in aortic blood pressure. Administration of dexmedetomidine failed to produce the initial hypertension, which can be attributed to the α2B-adrenoceptor subtype 18and the central hypotensive effect related to the α2A-adrenoceptor subtype. 26These findings are consistent with the observation that activation of presynaptic sympathetic α2C-receptors can decrease norepinephrine release. 21
Etomidate elicited a transient increase in aortic blood pressure in anesthetized wild-type mice that was not observed in α2B-KO or in α2AB-KO mice. The hypertensive response was accompanied by a mild bradycardia. In humans, the most distinctive property of etomidate as compared with other anesthetics is its minimal effect on cardiovascular parameters. 2,3However, several studies in patients have shown that etomidate may cause transient increases in blood pressure and/or systemic vascular resistance. 38,39Thus, during induction of anesthesia etomidate may interact with vascular α2B-adrenoceptors to elicit a transient hypertensive effect or to counteract the hypotensive effects of other anesthetics and drugs used in anesthesia. In addition to a direct effect on vascular α2B-receptors, etomidate may elicit the transient hypertension by an indirect mechanism. It is tempting to speculate that increased levels of catecholamines (caused by etomidate) may contribute to the transient hypertension. However, wild-type or α2-deficient mice did not show any typical signs of increased norepinephrine secretion; i.e. , tachycardia (fig. 4). Further studies on isolated microvessels are required to determine whether the etomidate-mediated hypertension is indeed mediated by a direct vasoconstrictory mechanism.
Interaction of etomidate with the α2A-receptor subtype is unlikely to play an important role in mediating its anesthetic action because of the low affinity of etomidate for this receptor subtype. This result is also consistent with the clinical profile of etomidate in humans. Activation of α2A-receptors elicits analgesia and inhibits sympathetic outflow. Etomidate does not have significant analgesic effects in humans and it does not block the sympathetic activation that is usually observed during intubation. 40
In conclusion, etomidate can stimulate α2B-adrenoceptors in mice, which results in a transient hypertensive response after rapid intravenous injection. Although this study was performed with transgenic mouse models and recombinant murine α2-adrenoceptors, it is tempting to speculate about the implications for the mechanism of action of etomidate in humans. The cardiovascular stability of etomidate during induction of anesthesia even in patients with cardiac disease and during bolus application might be related to an interaction of etomidate with peripheral α2B-adrenoceptors. The α2B-mediated vasoconstriction may oppose hypotensive effects of other coadministered agents during induction of anesthesia, and thus be responsible for the known beneficial hemodynamic profile of etomidate.