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The Mechanisms of the Direct Action of Etomidate on Vascular Reactivity in Rat Mesenteric Resistance Arteries

Shirozu, Kazuhiro MD; Akata, Takashi MD, PhD; Yoshino, Jun MD, PhD; Setoguchi, Hidekazu MD, PhD; Morikawa, Keiko MD, PhD; Hoka, Sumio MD, PhD

doi: 10.1213/ane.0b013e3181902826
Anesthetic Pharmacology: Research Reports
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BACKGROUND: Etomidate minimally influences hemodynamics at a standard induction dose in young healthy patients, but can cause significant systemic hypotension at higher doses for induction or electroencephalographic burst suppression (i.e., cerebral protection) in patients with advanced age or heart disease, and during cardiopulmonary bypass. However, less is known about its action on systemic resistance arteries.

METHODS: Using an isometric force recording method and fura-2-fluorometry, we investigated the action of etomidate on vascular reactivity in small mesenteric arteries from young (7–8 wk old, n = 179) and aged (96–98 wk old, n = 10) rats.

RESULTS: In the endothelium-intact strips from young rats, etomidate enhanced the contractile response to norepinephrine or KCl (40 mM) at 3 μM but inhibited it at higher concentrations (≥10 μM). The enhancement was still observed after treatment with NG-nitro l-arginine, tetraethylammonium, diclofenac, nordihydroguaiaretic acid, losartan, ketanserin, BQ-123, or BQ-788, but was not observed in aged rats. In the endothelium-denuded strips from young rats, etomidate (≥10 μM) consistently inhibited the contractile response to norepinephrine or KCl without enhancement at 3 μM. In the fura-2-loaded, endothelium-denuded strips from young rats, etomidate inhibited norepinephrine- or KCl-induced increases in both intracellular Ca2+ concentration ([Ca2+]i) and force. Etomidate still inhibited the norepinephrine-induced increase in [Ca2+]i after depletion of the intracellular Ca2+ stores by ryanodine, which was sensitive to nifedipine. Etomidate had little effect on norepinephrine- or caffeine-induced Ca2+ release from the intracellular stores or Ca2+ uptake into the intracellular stores. During stimulation with norepinephrine or KCl, etomidate had little effect on the [Ca2+]i-force relation at low concentrations (≤30 μM) but caused its downward shift at 100 μM.

CONCLUSIONS: In small mesenteric arteries, etomidate influences the contractile response to norepinephrine or membrane depolarization through endothelium-dependent enhancing and endothelium-independent inhibitory actions. The enhancement is at least in part independent of nitric oxide, endothelium-derived hyperpolarizing factor, cyclooxygenase products, lipoxygenase products, angiotensin II, serotonin, or endothelin-1, but may involve some signaling pathway that is impaired by aging. The endothelium-independent inhibition is due to decreases in both the [Ca2+]i and myofilament Ca2+ sensitivity in vascular smooth muscle cells. The decrease in [Ca2+]i would be due mainly to inhibition of voltage-gated Ca2+ influx. The observed inability of lower concentrations (1–3 μM) of etomidate to cause significant vasodilation is consistent with minimal changes in hemodynamics during induction of anesthesia with etomidate in young subjects, whereas the observed vasodilator action of higher concentrations of etomidate might underlie systemic hypotension caused by higher doses of etomidate in the clinical setting.

IMPLICATIONS: In mesenteric resistance arteries, etomidate directly depresses the contractile response to norepinephrine or membrane depolarization by inhibiting myofilament Ca2+ sensitivity and/or intracellular Ca2+ concentration in vascular smooth muscle cells. The observed inability of low concentrations (1–3 μM) of etomidate to cause significant vasodilation is consistent with minimal changes in hemodynamics during standard induction of anesthesia with etomidate in young subjects, whereas the observed direct (i.e., endothelium independent) vasodilator action of higher concentrations of etomidate might underlie systemic hypotension associated with administration of higher doses of etomidate in the clinical setting (e.g., cerebral protection).

From the Department of Anesthesiology and Critical Care Medicine, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan.

Accepted for publication August 1, 2008.

Supported by grants-in-aid for scientific research (C-13671590, C-18591708) from Japan Society of the Promotion of Science (Tokyo, Japan).

Presented in part at International Anesthesia Research Society (IARS) 79th Clinical and Scientific Congress, Honolulu, March 11–15, 2005, IARS 81st Clinical and Scientific Congress, Orlando, March 23–27, 2007, and International Society for Anesthetic Pharmacology 16th Annual Meeting, San Francisco, USA, October 12, 2007.

Address correspondence and reprint requests to Takashi Akata, MD, Department of Anesthesiology and Critical Care Medicine, Faculty of Medicine, Kyushu University, Fukuoka 812-8582, Japan. Address e-mail to akata@kuaccm.med.kyushu-u.ac.jp.

Etomidate, an imidazolin-derived IV anesthetic, has often been used for induction of general anesthesia. In addition, in some institutions, etomidate has been used for cerebral protection in neurosurgical procedures.1–3 However, etomidate has never met rigorous evaluation for neuroprotective properties,4 and its effectiveness as a neuroprotectant is still a matter of debate. When administered in the standard induction dose (0.3 mg/kg), etomidate minimally or only modestly influenced hemodynamics in healthy patients.5,6 However, it caused moderate decreases in systemic arterial blood pressure at a higher induction dose (0.45 mg/kg),7 in patients with advanced age8 or heart diseases,9 and during cardiopulmonary bypass.10 In addition, when administered in much higher doses (>0.7 mg/kg) to induce electroencephalographic burst suppression for cerebral protection, etomidate caused an immediate decrease in systemic arterial blood pressure that was sustained without vasopressor support.11 It has been suggested that the etomidate-induced systemic hypotension is due to decreases in both the systemic vascular resistance9,10,12 and cardiac contractility.8,9

In previous in vitro studies, etomidate (5–300 μM) inhibited the vasodilator response to endothelium-derived nitric oxide (EDNO) or endothelium-derived hyperpolarizing factor (EDHF) in isolated human renal arteries,13 canine pulmonary arteries,14 and rat aorta.15 In addition, etomidate (1–100 μM) inhibited the vasodilator response to ATP-sensitive K+ (KATP) channel openers in isolated canine pulmonary arteries16 and rat aorta.17 These results suggest that etomidate may increase vascular tone in vivo by directly inhibiting the vasodilator response to EDNO, EDHF, or KATP channels. On the other hand, in isolated human internal mammary arteries, etomidate inhibited the contractile response to norepinephrine or KCl at concentrations possibly achieved when used for cerebral protection (≥10 μM), suggesting that etomidate-induced systemic hypotension might be due, at least in part, to its direct vasodilator action.11 It was recently reported that etomidate inhibited angiotensin (AT) II-induced Ca2+ mobilization in cultured rat aortic smooth muscle cells18; however, the precise mechanisms underlying its direct vascular action are not yet fully understood. In addition, little information is available regarding the direct action of etomidate on small resistance arteries. Thus, this study was designed to investigate the direct action of etomidate on mesenteric resistance arteries and its underlying mechanisms.

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METHODS

Force and Ca2+ Measurements

After obtaining approval from the Kyushu University Animal Care and Use Committee (Fukuoka, Japan), an endothelium-intact (+E) or −denuded (−E) strip was prepared from a small mesenteric artery (third-order branch, approximately 150–200 μm in diameter) of male Wister rats (average lifespan approximately 104 weeks19) at the age of 7–8 wk (weight, 267 ± 34 [sd] g, n = 179) or 96–98 wk (weight, 721 ± 94 [sd] g, n = 10). Isometric force was then measured by attaching the strip to a strain gauge transducer as previously detailed.20,21 Briefly, the strip was horizontally mounted in a chamber attached to the stage of a microscope, and the resting tension was adjusted to obtain a maximal response to KCl. The solution was changed by infusing it into one end while aspirating simultaneously from the other end. Endothelial intactness and denudation were verified by the presence and absence, respectively, of acetylcholine (1–10 μM)-induced relaxation during stimulation with norepinephrine (10 μM), as previously detailed.22 All experiments with KCl were conducted in the presence of guanethidine (3 μM23) to prevent norepinephrine outflow from the sympathetic nerve terminals.

In the first series of experiments with +E or −E strips, only isometric force was measured to examine the effects of etomidate on contractile response to norepinephrine or KCl (40 mM) in either the absence or presence of various pharmacological inhibitors.

In the next series of experiments with the −E strips, changes in the intracellular Ca2+ concentration ([Ca2+]i) were measured simultaneously with those in force, using fura-2, a fluorescent Ca2+-indicator dye.24 Our method on the fura-2 fluorometry was previously detailed.25,26 None of the agents influenced the fluorescence signals, and changes in F340 and F380 were constantly in opposite directions.

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Solutions and Drugs

The ionic concentrations of 4-(2-hydroxyethyl) piperazine-1-ethanesulfonic acid (HEPES)-buffered PSS were as follows: 138 mM NaCl, 5.0 mM KCl, 1.2 mM MgCl2, 1.5 mM CaCl2, 10 mM HEPES, and 10 mM glucose. The pH was adjusted with NaOH to 7.35 at 35°C. The high K+ (40 mM) solution was prepared by replacing NaCl with KCl isoosmotically. The Ca2+-free solution was prepared by removing CaCl2 and adding 2 mM EGTA.

Acetylcholine, albumin (bovine), diclofenac, guanethidine, ketanserin tartrate, NG-nitro l-arginine (LNNA), nifedipine, niflumic acid, nordihydroguaiaretic acid (NDGA), and norepinephrine were obtained from Sigma Chemical Co. (St. Louis, MO). Etomidate was purchased from Tocris Cookson (Ellisville, MO). HEPES and tetraethylammonium (TEA) were purchased from Nacalai Tesque Co. (Kyoto, Japan). BQ-123 (cyclo(d-a- aspartyl-l-propyl-d-valyl-l-leucyl-d-tryptophyl), BQ-788 (N-[N-[N-[2,6-dimethyl-1-piperidinyl]carbonyl]-4- methyl-l-leucyl]-1-(methoxycarbonyl)-d-tryptophyl)- d-norleucine monosodium), losartan, and ryanodine were obtained from Calbiochem (Darmstadt, Germany). Fura-2-AM was purchased from Dojindo Laboratories (Kumamoto, Japan). All other reagents were of the highest grade commercially available.

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Experimental Protocol

In the fura-2-nonloaded strips, we first examined the effects of etomidate on half-maximal and maximal contractile responses to norepinephrine or contractile response to KCl (40 mM). In preliminary experiments to construct concentration-response curves for norepinephrine, the half-maximal effective concentrations (EC50) in the +E and −E strips were 1.99 ± 0.9 μM (n = 6) and 0.48 ± 0.19 μM (n = 6), respectively, whereas the maximal effective concentration [ECmax] was 10 μM both in the +E and −E strips. Thus, the concentrations of norepinephrine tested in the +E strips were 2 and 10 μM, whereas those tested in the −E strips were 0.5 and 10 μM. In addition, in preliminary experiments, 1 min was sufficient for etomidate to exert its maximal effects on the contractile response to norepinephrine or KCl. Furthermore, 3 min were sufficient for the norepinephrine or KCl response to reach a plateau, and reproducible responses to either stimulant were obtained with 7-min intervals. Thus, in formal experiments, norepinephrine (EC50 or ECmax) or KCl was applied for 3 min at 7-min intervals so as to obtain reproducible responses, and then etomidate was applied for 5 min before and during subsequent applications of either stimulant. However, in experiments with the aged rats, because of their limited number, the effects of etomidate were examined only on maximal contractile response to 10 μM norepinephrine in the +E strips.

We next examined possible involvement of EDNO, EDHF, cyclooxygenase (COX) products, lipoxygenase (LOX) products, AT-II, serotonin (5-HT), or endothelin-1 (ET-1) in the endothelium-dependent action of etomidate observed in the earlier experiments. Specifically, we examined the effects of etomidate on norepinephrine response in the +E strips pretreated with 100 μM LNNA (NO synthesis inhibitor), 10 mM TEA (K+ channel blocker known to inhibit the EDHF-mediated hyperpolarization27), 10 μM diclofenac (COX inhibitor28), 1 μM NDGA (LOX inhibitor28), 1–3 μM losartan (selective AT-II Type 1 receptor antagonist), 0.03–0.1 μM ketanserin (5-HT2/5-HT1C receptor antagonist), or a cocktail application of 1 μM BQ-123 (selective ETA receptor antagonist) and 1 μM BQ-788 (selective ETB receptor antagonist). The concentrations of these inhibitors and the preincubation times were decided on the basis of the results of our previous experiments and others reported in the literature.28–30 Namely, the strips were pretreated with these inhibitors at concentrations (see earlier) and for a time considered sufficient for all of them to exert their maximal effects (60 min for LNNA, TEA, diclofenac, and NDGA; 25 min for losartan, ketanserin, BQ-123, and BQ-788), and the inhibitors were then applied throughout the remainder of the experiment. Because both LNNA and TEA significantly enhanced the response to 10 μM norepinephrine, the norepinephrine concentration was decreased to produce approximately the same level of force as the control response to 10 μM norepinephrine in the LNNA or TEA-treated strips. These low concentrations of norepinephrine were detected in each strip.

In the fura-2-loaded strips, we first examined the effects of etomidate on increases in [Ca2+]i and force caused by norepinephrine (10 μM) or KCl (40 mM), using protocols identical to those used in the earlier experiments with fura-2-nonloaded strips. To investigate the effects of etomidate on norepinephrine (10 μM)-induced plasmalemmal Ca2+ influx, we next examined the effects of etomidate on norepinephrine (10 μM)-induced increases in [Ca2+]i and force after treatment with ryanodine (10 μM, 20 min), which depletes the intracellular Ca2+ stores (i.e., sarcoplasmic reticulum, SR) in this artery.31 We also attempted to characterize norepinephrine (10 μM)-induced Ca2+ influx by evaluating the effects of nifedipine (0.01–10 μM, a selective blocker of the L-type voltage-gated Ca2+ channels [VGCCs]32) and niflumic acid (10–300 μM, a selective blocker of the Ca2+-activated Cl [ClCa] channels33) on norepinephrine (10 μM)-induced increases in [Ca2+]i and force in the ryanodine-treated strips. Because etomidate was recently reported to modulate the adenosine 5′-triphosphate-sensitive K+ (KATP) channel activity in vascular smooth muscle (VSM) cells,17 we also examined the effects of etomidate on the norepinephrine- induced increases in [Ca2+]i and force after treatment with glibenclamide (1 μM, a selective blocker of the KATP channels34). In preliminary experiments, 5 min were sufficient for all of these blockers to exert their maximal effects on the response to norepinephrine or KCl. Thus, in the experiments with nifedipine, niflumic acid, or glibenclamide, the strips were incubated with each blocker for 5 min before and during subsequent application of norepinephrine.

In the fura-2-loaded strips, to investigate the effects of etomidate on either Ca2+-induced Ca2+ release or inositol 1,4,5-triphosphate-induced Ca2+ release from SR, we also examined its effects on increases in [Ca2+]i caused by caffeine (20 mM) or norepinephrine (10 μM), respectively, in the absence of extracellular Ca2+. Specifically, after loading Ca2+ into the SR, etomidate was applied for 1 min before and during application of either caffeine or norepinephrine in the Ca2+-free solution. In addition, we examined the effects of etomidate on Ca2+ uptake by the SR. For these experiments, after depletion of the SR by caffeine, etomidate was applied during Ca2+ loading (1.5 mM Ca2+, 8 min), and the amount of Ca2+ in SR was estimated from an increase in either force or [Ca2+]i caused by caffeine 2 min after removal of extracellular Ca2+.

To investigate the effects of etomidate on myofilament Ca2+ sensitivity, we examined its effects on increases in [Ca2+]i and force evoked by stepwise incremental increases in the extracellular Ca2+ concentrations ([Ca2+]e) during stimulation with KCl (40 mM) and/or norepinephrine (10 μM) in the fura-2-loaded strips, as detailed previously.22,26 The strip was exposed to etomidate (10, 30, and 100 μM) for 10 min before removal of extracellular Ca2+, and throughout the remainder of the experiment.

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Calculation and Data Analysis

The ratio (R340/380) of fura-2 fluorescence intensities excited by 340 nm (F340) to those excited by 380 nm (F380) was calculated after subtracting the background fluorescence, and used as an indicator of [Ca2+]i. The concentration-response data were fitted according to a logistic model,35 and the EC50 or half-maximal inhibitory concentration (IC50) values were derived from the least-squares fit using the aforementioned model.

Changes in the R340/380 and force were expressed as the percent value of the reference. The basal values in normal PSS were assumed to be 0% in all experiments. The effects of etomidate on norepinephrine or KCl response in normal PSS were evaluated 3 min after application of either stimulant, whereas its effects on the norepinephrine or caffeine response in the Ca2+-free solution, which was transient, were evaluated on the maximal response to either stimulant.

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Statistics

All results are expressed as the mean ± sd. The n denotes the number of preparations. Comparisons within each group were made by one-factor ANOVA for repeated measures, and post hoc comparisons were made using the contrast for multiple comparisons. Comparisons among groups were performed by two-factor ANOVA for repeated measures. When overall differences were detected, individual comparisons among groups at each concentration were performed by the Dunnett test. All other necessary comparisons between two groups were made by the Student’s t-test or Welch t-test after the homogeneity of variances was determined by the F test. P < 0.05 was considered significant.

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RESULTS

Contraction Experiments with Fura-2-Nonloaded Strips

Effects of Etomidate on Norepinephrine Response in Young Rat Arteries

In the +E strips, etomidate modestly enhanced the contractile response to norepinephrine (2 and 10 μM) at a low concentration (3 μM), but inhibited it at higher concentrations (≥10 μM), with IC50s for the 2 and 10 μM norepinephrine responses being 26 ± 19 μM (n = 7) and 61 ± 43 μM (n = 8), respectively (Fig. 1). However, in the −E strips, etomidate (≥10 μM) consistently inhibited the contractile response to norepinephrine (0.5 and 10 μM) without enhancement at 3 μM, with IC50s for the 0.5 and 10 μM norepinephrine responses being 66 ± 30 μM (n = 9) and 109 ± 23 μM (n = 8), respectively (Fig. 1).

Figure 1

Figure 1

The IC50 for inhibition of 10 μM norepinephrine response in the +E strips was lower (P = 0.01) than that in the −E strips, but identical (P = 0.81) to the IC50 for inhibition of 0.5 μM norepinephrine response in the −E strips.

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Effects of Etomidate on Norepinephrine Response in Aged Rat Arteries

In the +E strips from aged rats, etomidate (≥10 μM) consistently inhibited the contractile response to 10 μM norepinephrine without enhancement at 3 μM with an IC50 of 62 ± 26 μM (n = 9, Fig. 1), which was identical (P = 0.95) to that in the +E strips from young rats (61 ± 43 μM, n = 8).

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Effects of Etomidate on KCl Response in Young Rat Arteries

In the +E strips, etomidate slightly enhanced the contractile response to KCl at 3 μM, but inhibited it at higher concentrations (≥30 μM) with an IC50 of 87 ± 8 μM (n = 7, Fig. 2). However, in the −E strips, etomidate (≥30 μM) consistently inhibited the KCl response without enhancement at 3 μM with an IC50 of 100 ± 18 μM (n = 7, Fig. 2), which was not significantly different (P = 0.10) from the IC50 for inhibition in the +E strips.

Figure 2

Figure 2

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Experiments with Inhibitors of Endothelial Vasoactive Substances in Young Rat Arteries

In the +E strips, LNNA (100 μM) greatly enhanced the contractile response to 10 μM norepinephrine (193.8 ± 16.8%, n = 8, P < 0.001), and eliminated the etomidate (3 μM)-induced enhancement (Fig. 3A). However, etomidate (3 μM) still enhanced the submaximal response to a low concentration (1.0 ± 0.3 μM, n = 8) of norepinephrine (Fig. 3A), the amplitude of which (102.2 ± 15.8% of control) was identical (P = 0.72) to that of the 10 μM norepinephrine response before LNNA treatment. In addition, higher concentrations (≥10 or 30 μM) of etomidate still inhibited both the submaximal and maximal responses to norepinephrine with IC50s of 68 ± 22 μM (n = 8) and 133 ± 18 μM (n = 8), respectively (Fig. 3A). The IC50 for inhibition of the submaximal norepinephrine response after LNNA treatment (68 ± 22 μM, n = 8) was identical (P = 0.71) to the IC50 for control inhibition of 10 μM norepinephrine response before LNNA treatment (61 ± 43 μM, n = 8).

Figure 3

Figure 3

In the +E strips, TEA (10 mM) also enhanced the 10 μM norepinephrine response (139.4 ± 16.9%, n = 8, P = 0.001), and attenuated the etomidate (3 μM)-induced enhancement (Fig. 3B). However, etomidate (3 μM) still enhanced the submaximal response to a low concentration (1.8 ± 0.7 μM, n = 8) of norepinephrine (Fig. 3B), the amplitude of which (98.8 ± 15% of control) was identical (P = 0.82) to that of the 10 μM norepinephrine response before TEA treatment. In addition, higher concentrations (≥10 or 30 μM) of etomidate still inhibited both the submaximal and maximal responses to norepinephrine with IC50s of 33 ± 16 μM (n = 8) and 93 ± 34 μM (n = 8), respectively (Fig. 3D). The IC50 for inhibition of the submaximal norepinephrine response after TEA treatment (33 ± 16 μM, n = 8) was not significantly different (P = 0.11) from the IC50 for control inhibition of 10 μM norepinephrine response before TEA treatment (61 ± 43 μM, n = 8).

In the +E strips, diclofenac (10 μM) did not significantly affect (P > 0.05) the contractile response to 10 μM norepinephrine (90.3 ± 14.4%, n = 8) or the effects of etomidate on 10 μM norepinephrine response (Fig. 3C). Similarly, in the +E strips, NDGA (1 μM) did not significantly influence (P > 0.05) the contractile response to 10 μM norepinephrine (100.4 ± 10.5%, n = 8) or the effects of etomidate on the 10 μM norepinephrine response (Fig. 3D).

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Experiments with Various Receptor Antagonists in Young Rat Arteries

Treatment with losartan (n = 5), ketanserin (n = 5), or BQ-123 plus BQ-788 (n = 6) did not affect (P > 0.05) contractile response to 10 μM norepinephrine in the +E strips (1 μM losartan, 96.5 ± 5.8%; 3 μM losartan, 105.6 ± 13.6%; 0.03 μM ketanserin, 100.7 ± 8.1%; 0.1 μM ketanserin, 95.1 ± 17.4%; 1 μM BQ-123 + 1 μM BQ-788, 98.5 ± 8.5%). In addition, each treatment did not affect (P > 0.05) the etomidate (3 μM)-induced enhancement of the norepinephrine (10 μM) response in the +E strips. Specifically, in the experiments with losartan, the enhancements in control, 1 μM losartan-treated, and 3 μM losartan-treated strips were 115.9 ± 3.4%, 111.0 ± 6.4%, and 118.6 ± 10.6%, respectively. In those with ketanserin, the enhancements in control, 0.03 μM ketanserin-treated, and 0.1 μM ketanserin-treated strips were 119.8 ± 7.2%, 118.5 ± 9.9%, and 121.8 ± 5.6%, respectively. In those with BQ-123 plus BQ-788, the enhancements before and after treatment with BQ-123 plus BQ-788 were 118.0 ± 3.9% and 124.1 ± 14.8%, respectively.

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Experiments with Fura-2-Loaded, Endothelium-Denuded Strips from Young Rats

Effects of Etomidate on KCl-Induced Increases in R340/380 and Force

Etomidate (≥10 μM) inhibited the KCl-induced increase in both R340/380 and force with IC50s of 56 ± 22 μM (n = 7) and 139 ± 46 μM (n = 7), respectively (Fig. 4). In some experiments, 300 μM etomidate eliminated only the increase in force but not the increase in R340/380; the remaining increase in R340/380 was considered sufficient for force generation (Fig. 4). This suggests that etomidate inhibits myofilament Ca2+ sensitivity.

Figure 4

Figure 4

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Effects of Etomidate on Norepinephrine-Induced Increases in R340/380 and Force

Etomidate (≥3 μM) also inhibited the norepinephrine- induced increase in both R340/380 and force with IC50s of 99 ± 39 μM (n = 8) and 66 ± 18 μM (n = 8), respectively (Fig. 5).

Figure 5

Figure 5

Treatment with ryanodine almost eliminated the initial phasic component of the norepinephrine-induced increase in R340/380 (6.6 ± 7.7%, P < 0.0001, n = 8, Fig. 5A), whereas only modestly inhibiting its later sustained component (76.7 ± 11.8%, P = 0.0008, n = 8, Fig. 5A). Identical results were obtained regarding the effects of ryanodine on the norepinephrine-induced increase in force (not shown). After ryanodine treatment, etomidate (≥3 μM) still inhibited the norepinephrine (10 μM)-induced increase in both R340/380 and force with IC50s of 76 ± 41 μM (n = 8) and 48 ± 42 μM (n = 8), respectively (Fig. 5). No significant difference was found in the IC50 for inhibition of the norepinephrine-induced increase in either R340/380 or force between before and after ryanodine treatment (P > 0.05).

In the ryanodine-treated strips, the norepinephrine-induced increases in R340/380 and force were inhibited by nifedipine in a concentration-dependent manner with IC50s of 0.3 ± 0.2 μM and 0.3 ± 0.2 μM, respectively (n = 5; P < 0.05), and almost eliminated by 10 μM nifedipine (R340/380, 0 ± 0%, n = 5; force, 0.5 ± 1.0%, n = 5). In addition, the norepinephrine-induced increases in R340/380 and force were inhibited by niflumic acid (≥30 μM) in a concentration-dependent manner with IC50s of 83 ± 33 μM and 43 ± 16 μM, respectively (n = 4; P < 0.05), and almost eliminated by 300 μM niflumic acid (R340/380, 5.4 ± 6.3%, n = 4, P = 0.001; force, 0 ± 0%, n = 4, P = 0.001).

Treatment with glibenclamide (1 μM) did not affect the norepinephrine (10 μM)-induced increase in both R340/380 and force (Fig. 6). In addition, glibenclamide did not affect the etomidate (10–100 μM)-induced inhibition of the norepinephrine (10 μM) response (Fig. 6).

Figure 6

Figure 6

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Effects of Etomidate on Intracellular Ca2+ Stores

Etomidate did not affect the caffeine (20 mM)-induced increase in R340/380 or force in Ca2+-free solution (Fig. 7). Etomidate also did not affect the norepinephrine (10 μM)-induced increase in R340/380 or force in Ca2+-free solution at lower concentrations (≤30 μM), but slightly inhibited it at 100 μM (Fig. 7).

Figure 7

Figure 7

Etomidate, applied during Ca2+ loading, did not affect the caffeine (20 mM)-induced increase in R340/380 or force in Ca2+-free solution; the caffeine-induced increases in R340/380 after treatment with 10, 30, and 100 μM etomidate were 100.9 ± 13.2%, 96.7 ± 12.4%, and 106.0 ± 13.7% of control, respectively (n = 6; P > 0.05). Identical results were obtained in the analyses regarding the effects of etomidate on caffeine-induced increase in force (not shown).

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Effects of Etomidate on R340/380-Force Relation During Stimulation with KCl or Norepinephrine

The stepwise increment of [Ca2+]e during stimulation with KCl or norepinephrine produced concentration-dependent increases in both R340/380 and force (Figs. 8 and 9). Etomidate (10–100 μM) inhibited those increases in both R340/380 and force during stimulation with KCl or norepinephrine (Figs. 8 and 9). During stimulation with KCl or norepinephrine, etomidate did not influence the R340/380-force relation at lower concentrations (10–30 μM), but induced its downward shift at 100 μM (Figs. 8 and 9).

Figure 8

Figure 8

Figure 9

Figure 9

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DISCUSSION

Mechanisms Behind the Enhanced Contractile Response to Norepinephrine by Etomidate

The observed difference in the effects of etomidate on contractile responses to norepinephrine between the +E and −E strips indicates that etomidate has at least two distinct actions on norepinephrine response, i.e., endothelium-dependent enhancing and endothelium- independent inhibitory actions. Because the endothelial denudation is presumed to greatly enhance contractile response to norepinephrine,30,36 comparison of its inhibitory action on contractile response to norepinephrine between +E and −E strips would not be straightforward. It is thus unclear whether or not etomidate has endothelium-dependent inhibitory action on the contractile response to norepinephrine besides those two actions.

The ability of etomidate to enhance the KCl (40 mM) response or norepinephrine response after treatment with LNNA, TEA, diclofenac, NDGA, losartan, ketanserin, or BQ-123 plus BQ-788 suggests that the enhancement is at least in part independent of the EDNO, EDHF, COX, LOX, ATII, 5HT, or endothelin-1. In the LNNA or TEA-treated strips, the ability of etomidate to enhance the 10 μM norepinephrine response was greatly impaired, whereas its ability to enhance the response to lower concentrations of norepinephrine was well preserved. The impairment was probably due to prior maximal or near-maximal enhancement of the 10 μM norepinephrine response by either treatment (i.e., ceiling effects). Thus, it seems unlikely that inhibition of the EDNO or EDHF pathways is involved in the enhancement.

The inability of etomidate to enhance norepinephrine response in aged rats suggests that some signaling pathway(s) that is impaired in aged rats may account for the enhancement.

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Mechanisms Behind the Attenuated Contractile Response to Norepinephrine by Etomidate

The results obtained in experiments examining the effects of etomidate on a norepinephrine- or KCl-induced increase in [Ca2+]i or its effects on the [Ca2+]i-force relation indicate that etomidate attenuates the norepinephrine or KCl response mainly by decreasing [Ca2+]i at low concentrations (≤30 μM), while decreasing both [Ca2+]i and myofilament Ca2+ sensitivity at high concentrations (≥100 μM). The results obtained in experiments performed after removal of extracellular Ca2+ or treatment with ryanodine (i.e., depletion of SR31) further indicate that the [Ca2+]i-decreasing effect is mainly due to inhibition of plasmalemmal Ca2+ influx but not due to effects on SR Ca2+ release or uptake.

The experiments with nifedipine or niflumic acid suggest that stimulation with norepinephrine results in activation of ClCa channels, leading to membrane depolarization and hence activation of VGCCs. Because etomidate inhibited a KCl (i.e., membrane depolarization)-induced increase in [Ca2+]i with an IC50 (56 μM) almost identical to that for its inhibition of a norepinephrine-induced increase in [Ca2+]i in the ryanodine-treated strips (76 μM), it is likely that etomidate inhibits norepinephrine-induced Ca2+ influx by inhibiting VGCC activity. However, there remains a possibility that etomidate also inhibits ClCa channel activity. Further research would be necessary to clarify these issues.

Etomidate was recently reported to inhibit the KATP channel activity in VSM cells at clinical concentrations (≥1 μM).17 Thus, if KATP channels were active under basal conditions or activated in response to norepinephrine, etomidate would exert an enhancing action on the contractile response to norepinephrine by inhibiting KATP channel activity and thereby causing membrane depolarization. However, the observed inability of glibenclamide to influence the norepinephrine response indicates that KATP channels are not active during stimulation with norepinephrine, excluding the above possibility. Furthermore, glibenclamide did not influence the inhibitory action of etomidate on norepinephrine response, excluding a possibility that etomidate inhibits VGCC activity by activating KATP channels.

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Possible In Vivo Relevance

Plasma concentrations of etomidate necessary for induction of anesthesia and electroencephalographic burst suppression are 2–11 μM37 and 5–28 μM38, respectively. In this study, such clinical concentrations (3–10 μM) of etomidate affected contractile response to the sympathetic neurotransmitter norepinephrine or KCl (i.e., membrane depolarization). However, the observed degree of either the enhancement or the inhibition of norepinephrine or KCl response was relatively small. Thus, its contribution to the changes in hemodynamics associated with administration of etomidate would be modest. The inability of lower concentrations (1–3 μM) of etomidate to cause significant vasodilation would be consistent with maintained hemodynamic stability during anesthetic induction with etomidate, whereas the observed vasodilator action of higher concentrations of etomidate would underlie systemic hypotension caused by higher doses of etomidate.

In aged patients, induction of anesthesia with etomidate caused moderate decreases (20%–30%) in arterial blood pressure,8 which was larger than those observed in younger patients.5 In this study, etomidate did not inhibit the norepinephrine response at 1–3 μM (i.e., plasma concentrations possibly achieved during induction with etomidate) in both young and aged rats. However, 3 μM etomidate enhanced the norepinephrine response only in the young rats but not in aged rats. Thus, if our data can be extrapolated to humans, the larger decreases in arterial blood pressure during administration of etomidate in aged patients would not be due to its direct inhibitory action on the norepinephrine response but could be attributable in part to its inability to enhance the norepinephrine response.

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CONCLUSIONS

In mesenteric resistance arteries, etomidate influences the contractile response to norepinephrine or membrane depolarization through endothelium-dependent enhancing and endothelium-independent inhibitory actions. Endothelium-dependent enhancement is at least in part independent of the EDNO, EDHF, COX, LOX, ATII, 5HT, or ET-1 pathway but may involve some signaling pathway that is impaired in aged subjects. Endothelium-independent inhibition is due to decreases in both [Ca2+]i and myofilament Ca2+ sensitivity in VSM cells. The [Ca2+]i-decreasing action is due mainly to inhibition of Ca2+ influx through voltage-gated Ca2+ channels. The observed vascular action of etomidate might underlie circulatory changes associated with administration of etomidate either for anesthetic induction or cerebral protection in the clinical setting.

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ACKNOWLEDGMENTS

The authors are grateful to Atsushi Nakagawa (undergraduate student, Kyushu University, Fukuoka, Japan), Masae Yamakawa, BS (research assistant, Kyushu University), Shosuke Takahashi, MD, PhD (Kyushu Medical Center, Fukuoka, Japan), Katsuya Hirano, MD, PhD (Kyushu University), and Walter A. Boyle III, MD (Washington University, St. Louis, MO) for their helpful comments and kind assistance regarding this work.

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