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1Department of Anesthesia and Critical Care and 2Department of Neurobiology, Pharmacology, and Physiology, The University of Chicago, Chicago, Illinois
Submitted 30 May 2006; accepted in final form 31 August 2006
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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; Franks and Lieb 1994; Hemmings et al. 2005). Known targets include 
-aminobutyric acid type A (GABAA), glycine, neuronal nicotinic acetylcholine (nAChR), 
-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), N-methyl-D-aspartate (NMDA), hyperpolarization-activated cyclic nucleotide-gated (HCN) family of channels, 2P "background" K+ channels, presynaptic Na+ channels, and serotonin type-3 (5-HT3) receptors (Campagna et al. 2003; Hemmings et al. 2005; Krasowski and Harrison 1999). Other voltage-dependent channels appear to be sensitive to anesthetics at concentrations higher than used clinically (Campagna et al. 2003; Franks and Lieb 1994; Krasowski and Harrison 1999).
Isoflurane is a commonly used volatile anesthetic that acts by depressing CNS activity. Isoflurane has been shown to potentiate GABAA receptor current (Jones and Harrison 1993; Jones et al. 1992; Liao et al. 2005). We previously showed that activation of GABAA receptors produces excitation in bovine chromaffin cells. Activation of GABAA receptors depolarizes the cells, opens voltage-dependent Ca2+ channels, elevates the intracellular concentration of Ca2+ ([Ca2+]i), and promotes the release of catecholamines (Xie et al. 2003, 2004). The extrapolated anion reversal potential in chromaffin cells is about –28 mV, indicating a resting intracellular anion concentration of about 50 mM. This is different from the situation in the adult brain where GABA is the primary inhibitory neurotransmitter. Even so, depolarizing GABAA actions have also been observed in some adult neurons (Alvarez-Leefmans et al. 1988; Staley et al. 1996).
We explore herein the chromaffin cell response to isoflurane and then compare these results to previously published responses produced by etomidate (Xie et al. 2004). Etomidate is a widely used intravenous general anesthetic that is particularly useful in patients who have myocardial dysfunction because it produces minimal hemodynamic change in these patients (Angelini et al. 2001; Rothermel 2003). We showed that etomidate depolarizes chromaffin cells by activating their GABAA receptors. This depolarization activates voltage-gated Ca2+ channels, the activation of which elicits catecholamine release (Xie et al. 2004). If isoflurane mimics the effects of etomidate on the GABAA receptors, then why does isoflurane produce hemodynamic changes while etomidate does not? In this paper we show that exposing chromaffin cells to isoflurane caused a large and rapid elevation in [Ca2+]i, which required Ca2+ influx from the extracellular space. The elevation of [Ca2+]i required the activation of GABAA receptors; most of this response was blocked by the selective GABAA antagonist bicuculline. Exposing chromaffin cells to isoflurane, at clinically relevant concentrations, elicited catecholamine secretion. These excitatory responses of isoflurane were extremely similar to those produced by etomidate. Unlike etomidate, isoflurane suppressed nAChR-mediated current. In the intact adrenal gland ACh released by the "presynaptic" splanchnic nerve activates nAChRs, which elicit action potentials and catecholamine release. Thus we hypothesize that isoflurane blocks the "sympathetic drive" but that etomidate does not. This difference and others outlined in the manuscript may underlie, in part, the dissimilar cardiovascular responses produced by the two anesthetics. Some of these data were previously presented at the 2004 meeting of the American Society of Anesthesiologists and the 2005 meeting of the International Anesthesia Research Society.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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Juvenile bovine adrenal glands, from animals 4–6 mo old, were obtained from a local slaughterhouse and chromaffin cells were prepared by digestion with collagenase followed by density gradient centrifugation as previously described (Artalejo et al. 1992). The cells were plated onto collagen-coated coverslips (at a density of 0.3–0.4 x 106 cells/ml for [Ca2+]i measurements) and maintained in an incubator at 37°C in an atmosphere of 93% air and 7% CO2 with a relative humidity of 90%. Fibroblasts were effectively suppressed with cytosine-arabinoside (10 µM), leaving relatively pure chromaffin cell cultures. Half of the culture medium was exchanged every day. This medium consisted of DMEM/F12 (1:1) (Gibco) supplemented with fetal bovine serum (10%), glutamine (2 mM), penicillin/streptomycin (100 units ml–1/100 µg ml–1), cytosine arabinoside (10 µM), and 5-fluorodeoxyuridine (10 Symbol" /s 11µM).
[Ca2+]i measurements
Experiments were conducted 1–5 days after chromaffin cells were prepared. Cells were incubated in Hank’s balanced salt solution (HBSS) with 2 µM fura-2 AM and 1 mg/ml albumin for 45 min and then washed in a fura-2–free solution for 1 h. Fura-2 loading and wash were carried out at 37°C. The coverslip was transferred to an experimental chamber for recording. Backgrounds at 340 and 380 nm were obtained using an area of the coverslip devoid of cells. Data were continuously collected throughout the experiment. On each coverslip, 10–40 chromaffin cells were selected and individually imaged. Image pairs (one at 340 and one at 380 nm) were obtained every 2 s by averaging 16 frames at each wavelength. Backgrounds were subtracted from the individual wavelengths and the 340-nm image was divided by the 380-nm image to provide a ratiometric image. Ratios were converted to free [Ca2+]i by comparing data to fura-2 calibration curves made in vitro by adding fura-2 (50 µM free acid) to solutions that contained known concentrations of calcium (0–2000 nM). The recording chamber was continually perfused with fresh solution from gravity-fed reservoirs.
Measurement of isoflurane concentration
Isoflurane solution was prepared and measured as previously described (Jones and Harrison 1993; Jones et al. 1992). Briefly, isoflurane solutions were prepared by injection of pure liquid form of isoflurane with gastight syringes into evacuated saline bags containing defined volumes of extracellular solution. Isoflurane was then applied to the chromaffin cell preparation by the extracellular solution. Because isoflurane can slowly leak from the bags they were used for only a short period before they were discarded and replaced with fresh bags. The bath isoflurane concentration was found to be remarkably constant when measured in representative experiments. Isoflurane, etomidate, and muscimol all activate the GABAA receptors found in chromaffin cells and elevate [Ca2+]i. The variance observed in the [Ca2+]i response to isoflurane was quite similar to that of etomidate or muscimol (data not shown); the comparability in responses of the different drugs suggests that the bath concentration of isoflurane was reliable.
To determine the concentrations of isoflurane, control samples were obtained by sampling isoflurane from the bags at various time points during a period of 90 min after allowing sufficient time for equilibration. Experimental samples were withdrawn directly from the bath in a condition similar to calcium-imaging recordings at various time points. Control samples (2 µl) and experimental samples (2 µl) were measured immediately after the collection with gas chromatography (GC). GC analysis was carried out by use of a Shimadzu model 17A (Shimadzu, Kyoto, Japan) with a flame-ionization detector and a capillary column of Elite 5 (30 m x 0.32 mm ID x 1.0 µm, Elite 5; Perkin-Elmer Instruments, Norwalk, CT) using nitrogen as a carrier gas. Samples were injected (100°C) onto the column (35°C) and detected by flame ionization (200°C). Peak areas were used to quantify concentrations. The control experiments showed that losses of isoflurane in fluid bags were <5% over a period of 90 min (most of our experiments were completed within 60 min). In addition, isoflurane concentrations in the experiment samples collected from the recording bath were very close (<5% difference) to those in the control samples obtained from the fluid bags. Overall, the losses of isoflurane from the perfusion system were relatively small (<10%). All anesthetic concentrations reported herein are provided in millimoles. The MAC [minimum alveolar concentration required for immobility in response to a noxious stimulus in 50% of trials (Eger et al. 1965)] equivalents of the isoflurane used in this study were previously reported to be in the range of about 0.3 mM (Franks and Lieb 1996) to about 0.51 mM (Jones and Harrison 1993) at 25°C.
Amperometric measurement of catecholamine release
Carbon-fiber electrodes were fabricated and prepared for recording as previously described by Grabner et al. (2005). The electrode was pressed gently against the cell during the recording for the highest possible collection efficiency (Bruns et al. 2000; Schroeder et al. 1992). The electrode was held at +700 mV versus a ground electrode using an NPI VA-10 amplifier to oxidize catecholamine transmitter. The amperometric signal was low-pass filtered at 2 kHz (eight-pole Bessel; Warner Instruments, Hamden, CT). A 16-bit A/D converter (National Instruments, Austin, TX) was interfaced with custom data-acquisition software. The amperometric signal was acquired at 5 kHz and stored on a personal computer. Amperometric spike features and kinetic parameters were analyzed using a series of macros written in Igor Pro (Wavemetrics) kindly supplied by Dr. Eugene Mosharov (Columbia University, New York, NY). The detection threshold for an event was set at four to five times the baseline root-mean-squared noise and the spikes were automatically detected.
Solutions
Experiments were carried out in HBSS (Gibco), which contained (in mM): 138 NaCl, 5 KCl, 1.3 CaCl2, 0.3 KH2PO4, 0.8 MgSO4, 0.3 Na2HPO4, 5.6 D-glucose, and 20 HEPES (pH 7.35). Nominally Ca2+-free HBSS was prepared by treating Ca2+-free, Mg2+-free, HCO3-free HBSS with Chelex-100, then adding MgCl2 to a final concentration of 1 mM. Muscimol hydrobromide (Calbiochem) was prepared as a 25 mM stock in H2O. Muscimol was diluted to a final concentration with HBSS. (–)-Bicuculline methiodide (Sigma) was prepared as a 100 mM stock in H2O and then diluted to a final concentration of 100 µM with HBSS. Lanthanum chloride (Sigma) was prepared as a 100 mM stock in H2O and then diluted to a final concentration of 1 mM with HBSS. The solution containing sodium thiocyanate (Sigma) contained (in mM): 140 NaSCN, 1.3 CaCl2, 5 KCl, 1 MgCl2, 5.6 D-glucose, and 20 HEPES (pH 7.35). For the amperometry experiments the bath contained HBSS. All experiments were carried out at room temperature (–23°C).
Statistics
A Student’s t-test was used to assess differences between populations of cells.
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RESULTS |
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Chromaffin cells were tested after 1–5 days in primary culture. A coverslip that contained cells was transferred to an experimental chamber for each experiment. The microscope was adjusted until a field of view was obtained in which dozens of chromaffin cells were visible. [Ca2+]i measurements from 10–40 cells per coverslip were collected every 2 s.
Figure 1 shows representative data from an experiment that monitored [Ca2+]i with fura-2. Data from 31 cells were averaged and then plotted. Responses to isoflurane (0.5 mM) were obtained in the presence or absence of extracellular Ca2+. Isoflurane was perfused into the bath (as indicated by the bar), producing an elevation in [Ca2+]i of about 130 nM. After recovery, the cells were perfused with a Ca2+-free solution and then isoflurane was reapplied without any significant change in [Ca2+]i observed. Subsequently, Ca2+ was reintroduced into the bath and then isoflurane was applied once again, this time producing a response that was indistinguishable from that of the original response. Although isoflurane could be applied at 
0.5 mM several times without obvious change in the response, higher concentrations of the anesthetic appeared to produce some desensitization.
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Responses were observed at concentrations of isoflurane as low as 0.1 mM. Figure 2A shows the significant elevation in [Ca2+]i observed at an isoflurane concentration of 0.3 mM. For this experiment, data from 22 cells were averaged and then plotted. Figure 2B provides a more detailed analysis of the response of chromaffin cells to isoflurane. This figure plots the average change in [Ca2+]i observed in response to different isoflurane concentrations. The change in [Ca2+]i observed at each concentration of isoflurane was normalized to that observed at 1 mM (see figure legend for more detail). The data were fit with a standard dose–response equation. The EC50 provided by the fitting function (see legend) was about 0.78 mM. This can be compared with a MAC equivalent in the range of about 0.3 to 0.51 mM at 25°C (Franks and Lieb 1996; Jones and Harrison 1993). These data suggest that there is a statistical and likely biologically important effect at an isoflurane concentration that induces immobility.
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GABAA receptors are involved in the response to isoflurane
Isoflurane is thought to modulate GABAA receptor currents (Jones and Harrison 1993; Jones et al. 1992; Liao et al. 2005). In an earlier study we showed that activation of GABAA receptors, with the specific agonist muscimol, produced an increase in [Ca2+]i (Xie et al. 2003) and that this response could be mimicked by etomidate (Xie et al. 2004). To verify the involvement of GABAA receptors in the isoflurane response, the specific GABAA receptor antagonist bicuculline (100 µM) was applied to chromaffin cells. The response to muscimol (5 µM; n = 17) was completely suppressed by bicuculline (Fig. 3A). Interestingly, although bicuculline inhibited most of the response to isoflurane, roughly 35% of the response remained even in the presence of the antagonist (Fig. 3A). After the bicuculline was washed from the bath both muscimol and isoflurane produced large elevations in [Ca2+]i when applied (Fig. 3A). In this experiment isoflurane produced an average [Ca2+]i of 158.2 ± 2.9 nM (n = 25) when applied in the absence of bicuculline, although this increase was inhibited, to 61.1 ± 5.5 nM by bicuculline (Fig. 3B). These data demonstrate that isoflurane and muscimol increase [Ca2+]i in chromaffin cells by a common mechanism: activation of GABAA receptors. Furthermore, these results suggest that isoflurane can directly activate GABAA receptors.
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Previously we showed that the depolarization produced by activation of GABAA receptors in chromaffin cells results in the activation of Ca2+ channels (Xie et al. 2003). To verify that isoflurane operated through the activation of voltage-dependent Ca2+ channels, Cd2+ was used to block the channels. Figure 4A shows an experiment where Cd2+ (0.1 mM) blocked the Ca2+ influx elicited by isoflurane (n = 40). The bar at the bottom shows the time interval that Cd2+ was introduced into the bath. In the presence of Cd2+ isoflurane could not elicit a significant change in [Ca2+]i. Washing Cd2+ from the bath and then reapplying isoflurane subsequently produced a substantial change in [Ca2+]i. In this experiment isoflurane produced an average elevation in [Ca2+]i of 211.85 ± 15.8 nM (n = 65) when applied in the absence of Cd2+, although this increase was inhibited, to 22 ± 3 nM, by Cd2+ (Fig. 4B). These data demonstrate that voltage-dependent Ca2+ channels most likely play a role in the response to isoflurane.
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For GABA to depolarize a cell sufficiently to activate voltage-dependent Ca2+ channels the anion equilibrium potential must be considerably depolarized. To test this hypothesis we altered the Cl– reversal potential by replacing most of the extracellular Cl– with SCN–, an anion that is more permeant in GABAA receptors and other chloride channels than Cl– itself (Bormann et al. 1987; Currie et al. 1995). Replacing Cl– with SCN– anion typically results in a negative shift in the anion equilibrium potential. If the shift in the anion reversal potential were large enough it would preclude activation of voltage-dependent Ca2+ channels in response to isoflurane. When cells were exposed to isoflurane in the presence of SCN– (140 mM), the elevation of [Ca2+]i observed was less than half that produced by isoflurane alone (Fig. 5A). The mean response of chromaffin cells to isoflurane in this experiment was an elevation of [Ca2+]i of 288 ± 8.2 in HBSS but only 137.4 ± 5.7 nM in the presence of SCN– (Fig. 5B). These data support the idea that the anion equilibrium potential of the cells is such that application of isoflurane produces a depolarization that is strong enough to activate voltage-dependent Ca2+ channels.
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In chromaffin cells, ACh release from presynaptic splanchnic neurons activates nAChRs, which elicit action potentials in chromaffin cells. Both voltage-dependent Ca2+ channels as well as nicotinic receptors mediate Ca2+ influx into chromaffin cells. More recent experiments suggest that transient receptor potential (TRP) channels may also allow Ca2+ into cells (Obukhov and Nowycky 2002).
It was previously reported that isoflurane inhibits nAChRs (Charlesworth and Richards 1995; Pocock and Richards 1988) and that this action may involve the reduction of agonist affinity at the nAChRs (Rada et al. 2003). We sought to determine whether isoflurane inhibited nAChRs under the conditions of our experiments. Nicotine (20 µM) was used to selectively activate nAChRs. Figure 6A shows that nicotine by itself elevated [Ca2+]i in this experiment by nearly 750 nM, but that the response was only about 350 nM when nicotine was applied in the presence of isoflurane (0.5 mM). Subsequently, nicotine was reapplied, this time in the absence of isoflurane, and produced an elevation in [Ca2+]i that was indistinguishable from that of the first response. Nicotine was applied again in the presence of isoflurane and produced a response almost identical to that of the first one (Fig. 6A).
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; Minami et al. 1994; Rada et al. 2003), suggest that isoflurane can inhibit Ca2+-influx into chromaffin cells elicited by the activation of nAChRs.
To test whether isoflurane could activate nAChRs while simultaneously inhibiting them, we tested for the involvement of these receptors in the elevation of [Ca2+]i produced by isoflurane. In the presence of a cocktail of nicotinic antagonists [methyllycaconitine (1 µM), mecamylamine (20 µM), and dihydro-
-erythroidine (20 µM), all from Research Biochemicals International, Natick, MA] the response to nicotine (100 µM) was effectively blocked, but isoflurane still produced an elevation in [Ca2+]i not different from that observed in control conditions (data not shown). Our data suggest that the elevation of [Ca2+]i observed on application of isoflurane does not involve the activation of neuronal nicotinic receptors.
Isoflurane stimulates catecholamine release in chromaffin cells
We previously showed that activation of GABAA receptors in chromaffin cells could depolarize the cells sufficiently to trigger neurotransmitter release (Xie et al. 2003, 2004). We sought to determine whether isoflurane produced a sufficiently large depolarization to mimic this effect. Figure 7A shows that isoflurane does promote secretion. A carbon-fiber electrode was gently pressed against a cell to record catecholamine release. The amperometric current recorded from a cell is shown in Fig. 7A. The trace shows that application of isoflurane for 3 min elicited a small number of amperometric events that were not observed in the absence of the anesthetic. Averaging data from 18 such cells (Fig. 7B) shows that isoflurane stimulated chromaffin cells to release a modest amount of catecholamine. Control cells (unstimulated) released on average 4.9 x 106 molecules of transmitter in a 3-min measurement window, whereas isoflurane elicited the release of 1.19 x 108 molecules of transmitter (a 24-fold increase) in the same time frame. On average there was 0.49 events per 3-min epoch in control conditions and 11.85 events in isoflurane-stimulated conditions. Therefore in addition to elevating [Ca2+]i, isoflurane also promotes the release of catecholamines.
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DISCUSSION |
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; Jones et al. 1992; Liao et al. 2005); at clinically relevant doses, isoflurane was thought to both prolong and reduce inhibitory postsynaptic currents (Banks and Pearce 1999; Jones and Harrison 1993). Isoflurane was previously shown to inhibit responses to saturating GABA concentrations in both native and recombinant receptors (Benkwitz et al. 2004; Hall et al. 1994). Our results, in contrast, suggest that isoflurane can directly activate the GABAA receptors in chromaffin cells, which then sufficiently depolarize the cells to activate voltage-gated Ca2+ channels. The resulting Ca2+ influx elicits a modest amount of catecholamine release. At the same time nAChRs are suppressed by isoflurane.
Most of the studies outlined herein used an isoflurane concentration of either 0.5 or 1 mM. How relevant is this concentration, given that the MAC equivalent is in the range of roughly 0.3 to 0.51 mM at 25°C (Franks and Lieb 1996; Jones and Harrison 1993)? The measured EC50 for the [Ca2+]i elevation by isoflurane was about 0.8 mM and we observed clear changes in [Ca2+]i at concentrations <0.5 mM. Even though they are not in common use, concentrations of isoflurane as high as 3 MAC are not clinically irrelevant. For example, high-dose isoflurane is occasionally used for induced hypotension to minimize arterial bleeding in patients who undergo surgeries, such as orthognathic surgery (Lessard et al. 1989). A good description of the hypotension arising from its actions in reducing system vascular resistance can be found in Miller’s Anesthesia (Miller 2005). Because larger concentrations of volatile agents (1.5 to 2 MAC) are more efficacious in inducing hemodynamic changes, we believe that these are the most relevant concentrations to study. In addition, there is no reason to assume that isoflurane MAC measurements for immobility (or analgesia) would directly correspond to the MAC for hemodynamic stability. Perhaps a more relevant measure for our study is MACBAR, the minimum alveolar concentration of inhalational anesthetics that blocks the stress (adrenergic) response to surgical incision (Roizen et al. 1981). Stress is typically defined as changes in blood pressure, heart rate, and so forth. In one study by Roizen et al. (1981), MACBAR for halothane was equivalent to 1.45 MAC for immobility. A different study showed that MACBAR for isoflurane and desflurane was equivalent to 1.3 MAC (Daniel et al. 1998) and for sevoflurane the value was 3.5 to 4 MAC (Ura et al. 1999). Some of the differences in the MACBAR values may be explained by the fact that the designs of these studies were not identical. However, there is no doubt that the values for MACBAR are higher than MAC for every agent.
GABA plays an important role in the physiology of the adrenal medulla; chromaffin cells possess the enzymes that synthesize and degrade GABA (Fernandez-Ramil et al. 1983; Kataoka et al. 1984); GABA is taken up by chromaffin cells and released in response to depolarizing stimuli (Kataoka et al. 1984; Oset Gasque et al. 1985, 1990); GABA immunoreactive nerve fibers are found throughout the adrenal medulla (Kataoka et al. 1986; Oomori et al. 1993); and GABA immunoreactive nerve fibers run into the medulla with varicosities often in close contact with chromaffin cells (Kataoka et al. 1986; Oomori et al. 1993). In addition, chromaffin cells express GABAA receptors (Amenta et al. 1988; Bormann and Clapham 1985; Peters et al. 1989). These earlier results raise the possibility that isoflurane potentiated the response to endogenously released GABA. We believe this possibility to be very unlikely because the cells were continuously perfused; any released GABA would be rapidly washed away. In addition, the bath volume was relatively large compared with the number of cells present, precluding any buildup of substances secreted by the cells. Rather, it appears that clinically relevant concentrations of isoflurane directly activated the GABAA receptors found in chromaffin cells.
Ionotropic GABAA receptors are usually associated with inhibition, but during development, GABAA-mediated depolarization has been observed in many regions of the CNS (Ben-Ari 2002; Chen et al. 1996; Eilers et al. 2001; Ge et al. 2006; Luhmann and Prince 1991; Mueller et al. 1984; Owens et al. 1996; Yuste and Katz 1991). The action of GABAA receptors in neurons is determined by the transmembrane Cl– gradient and the resultant ECl (Staley et al. 1996). The depolarized ECl observed is attributed to, at least in some cases, a member of the NKCC cotransporter family (Alvarez-Leefmans et al. 1988; Rohrbough and Spitzer 1996; Sun and Murali 1999), which driven by Na+ and K+ gradients, acts to accumulate intracellular Cl– (Ben-Ari 2002). Expression of another cotransporter that normally lowers intracellular Cl–, KCC2 (Payne et al. 1996), appears to mediate the developmental shift toward inhibition (Ben-Ari 2002; Owens and Kriegstein 2002; Rivera et al. 1999). We previously showed that chromaffin cells from animals 4–6 mo old did not express the KCC2 cotransporter, as expected from animals of this age (Xie et al. 2003). Rather these cells continue to express the NKCC cotransporter typically found in embryonic cells. This result provides a molecular explanation for the depolarization produced by activation of GABAA receptors by isoflurane. Data from our current study are consistent with a depolarized anion equilibrium potential.
Etomidate is a widely used intravenous general anesthetic, which is particularly useful in patients who have myocardial dysfunction because it produces minimal hemodynamic change in these patients (Angelini et al. 2001; Rothermel 2003). In an earlier study we showed that etomidate directly activated GABAA receptors at clinically relevant concentrations, which depolarized chromaffin cells, elevated [Ca2+]i, and elicited catecholamine release (Xie et al. 2004). Because isoflurane can produce hemodynamic changes, whereas etomidate does not, it is instructive to compare the effects of these anesthetics in chromaffin cells. This is especially important because we proposed that catecholamine release from chromaffin cells plays an important role in the hemodynamic stability of etomidate (Xie et al. 2004).
Although both isoflurane and etomidate produce an elevation of [Ca2+]i, specific details are different. In both cases blockade of voltage-gated Ca2+ channels inhibits almost all of the response. The specific GABAA antagonist bicuculline, however, which suppressed virtually the entire response to etomidate, inhibited only roughly 65% of the response to isoflurane. It is possible that isoflurane produces a cellular depolarization by another mechanism as well, by a target that we have not yet identified, or that it activates a population of GABAA receptors that are insensitive to bicuculline. This might include direct blockade of channels that help maintain the resting potential. This latter hypothesis is given weight by the observation that replacing extracellular Cl– with SCN– almost completely suppressed the response to etomidate (Xie et al. 2004), but inhibited only a little more than half of the response to isoflurane.
The most interesting difference between isoflurane and etomidate is that isoflurane inhibits nAChRs (Matsuura et al. 2002; Minami et al. 1994; Rada et al. 2003), whereas etomidate does not [Flood and Krasowski 2000; Xie et al. 2004; but see the study by Charlesworth and Richards (1995), where they describe a modulation of nAChRs by etomidate]. The sympathetic splanchnic nerve innervates chromaffin cells. Release of ACh from presynaptic nerve terminals opens nAChRs, resulting in a strong depolarization of the chromaffin cells, activation of Ca2+ channels, and subsequent catecholamine release. Our results suggest that isoflurane, even at 0.5 mM, effectively inhibits this "sympathetic drive," whereas etomidate does not, and may help explain the cellular mechanisms underlying the autonomic reaction caused by isoflurane. This difference between etomidate and isoflurane may give rise to the alterations in hemodynamic properties observed with the two anesthetics. It is possible that in the case of etomidate, which does not block nAChRs, both low-level sympathetic drive and direct depolarization of chromaffin cells combine to maintain circulating catecholamine levels. In the case of isoflurane, the anesthetic will depolarize the chromaffin cells to elicit catecholamine release, although the sympathetic drive component is at the same time blocked. Our data suggest that the block of the sympathetic drive is the larger effect, especially at higher isoflurane concentrations, which explains why blood pressure drops in the presence of isoflurane. Our data, however, do not preclude other direct effects of isoflurane on the vascular system. Interestingly, the catecholamine release elicited by the direct activation of chromaffin cells by isoflurane, although modest, may help ameliorate part of response that would otherwise be larger.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Address for reprint requests and other correspondence: Z. Xie, The University of Chicago, Dept. of Anesthesia and Critical Care, 5841 S. Maryland, MC 4028, Chicago, IL 60637 (E-mail: jxie{at}dacc.uchicago.edu)
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