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Anesth Analg 2002;95:893-899
© 2002 International Anesthesia Research Society

ANESTHETIC PHARMACOLOGY

General Anesthetic Actions on Norepinephrine, Dopamine, and {gamma}-Aminobutyric Acid Transporters in Stably Transfected Cells

Saira K. Shahani, MD, Ratnakumari Lingamaneni, PhD, and Hugh C. Hemmings, Jr, MD PhD

Departments of Anesthesiology and Pharmacology, Weill Medical College of Cornell University, New York, New York

Address correspondence to Hugh C. Hemmings Jr, MD, PhD, Box 50, LC-203A, Weill Medical College of Cornell University, 525 E. 68th St., New York, NY 10021. Address e-mail to hchemmi{at}med.cornell.edu Reprints will not be available from the authors.


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
The effects of general anesthetics on neurotransmitter uptake by plasma membrane transporters are controversial. We analyzed the effects of representative volatile and IV general anesthetics on recombinant transporters for norepinephrine (human NET), dopamine (rat DAT), or {gamma}-aminobutyric acid (rat GAT-1) stably expressed in a porcine kidney cell line (LLC-PK1). This approach avoids complicating factors associated with neuronal preparations, such as the involvement of multiple transporters and the indirect effects of membrane potential. At clinical concentrations, human NET was inhibited only by halothane (50% inhibitory concentration [IC50] = 0.54 mM), rat DAT was sensitive to both halothane and isoflurane (IC50 = 0.60 and 0.64 mM, respectively), and rat GAT-1 was insensitive to both volatile anesthetics. Human NET was inhibited in a dose-dependent fashion by propofol (IC50 = 41 µM), ketamine (IC50 = 150 µM), and etomidate (IC50 > 200 µM), but not by pentobarbital. Only propofol inhibited NET at a clinically relevant concentration (5 µM). Rat DAT was inhibited in a dose-dependent fashion by propofol (IC50 = 120 µM), etomidate (IC50 = 100 µM), and ketamine (IC50 = 210 µM), but not by pentobarbital. None of these anesthetics was predicted to inhibit DAT at concentrations that produce anesthesia. Propofol inhibited rat GAT-1, but only at the largest concentration tested. General anesthetics have drug- and subtype-selective actions on neurotransmitter transporters. We conclude that effects on catecholamine, but not {gamma}-aminobutyric acid, transporters may contribute to secondary synaptic actions of certain anesthetics but are unlikely to be essential to their anesthetic properties.

IMPLICATIONS: Previous studies have implicated neurotransmitter transporters as targets for general anesthetic effects on synaptic transmission. Recombinant transporters for norepinephrine and dopamine were sensitive to certain volatile and IV anesthetics, whereas {gamma}-aminobutyric acid transporters were insensitive. These anesthetic- and neurotransmitter-specific effects may underlie some of the secondary effects of general anesthetics.


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
Considerable evidence indicates that modulation of synaptic transmission is the principal mechanism of general anesthetic action (1). High-affinity Na+/Cl--dependent transporters present on neural and glial cells mediate the uptake of monoamine and amino acid neurotransmitters, thus terminating their actions at the synapse (2). However, the role of neurotransmitter transporters as targets for presynaptic anesthetic effects is unclear. This is due in large part to difficulties in analyzing anesthetic effects on voltage- and ion gradient-dependent transporters in excitable cells. We have attempted to circumvent some of these problems by assessing the effects of representative general anesthetics on the function of transporters for norepinephrine (NET), dopamine (DAT), and {gamma}-aminobutyric acid (GABA) (GAT-1) heterologously expressed in stable porcine kidney cell lines (LLC-PK1 cells).

LLC-PK1 cells stably transfected with cloned complementary DNA encoding individual transporters have been used in characterizing their pharmacological properties. Because the common parental LLC-PK1 cell does not express these neurotransmitter transporters, technical difficulties associated with multiple transporter systems having overlapping substrate and inhibitor specificities are eliminated. This approach has been used to demonstrate the overlapping substrate and inhibitor specificities of the rat dopamine transporter (rDAT) and human norepinephrine transporter (hNET) (3). In this study, we used LLC-PK1 cells that stably express hNET, rDAT, and rat brain GABA transporter (rGAT-1) to study the effects of representative general anesthetics on neurotransmitter uptake. Our results indicate both transporter- and drug-specific effects of anesthetics on NET and DAT, but not on GAT-1, consistent with the involvement of these transporters in the secondary effects of anesthetics. These actions may be relevant to potential drug interactions with antidepressants and psychostimulants.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
LLC-PK1 porcine kidney cells stably transfected with the corresponding complementary DNA (LLC-hNET, LLC-rDAT, and LLC-rGAT-1) (3,4) were generously provided by Dr. Gary Rudnick (Yale School of Medicine, New Haven, CT). L-[2,5,6-3H)]-Norepinephrine, 3,4-[7-3H)]dihydroxyphenylethylamine, and [2,3-3H)]GABA were obtained from NEN Life Science Products (Boston, MA); pargyline, L-ascorbic acid, dimethyl sulfoxide (DMSO), HEPES, nipecotic acid, GBR-12935, desipramine, pentobarbital, and ketamine were obtained from Sigma Chemical Co. (St. Louis, MO); {alpha}-modified Eagle’s medium, L-glutamine, fetal bovine serum, penicillin, streptomycin, and Geneticin were obtained from Life Technologies, Inc. (Gaithersburg MD); and bovine serum albumin (fraction 5) was obtained from J. T. Baker (Phillipsburg, NJ). Halothane (thymol free) was obtained from Halocarbon Products (River Edge, NJ); propofol was purchased from Aldrich Chemicals (Milwaukee, WI) or was a gift from AstraZeneca Pharmaceuticals (Macclesfield, UK); and etomidate was a gift from Janssen Research Products (Flanders, NJ).

Cells were maintained in modified Eagle’s medium supplemented with 2 mM L-glutamine, 10% (vol/vol) fetal bovine serum, 50 U/mL of penicillin, 50 µg/mL of streptomycin, and 450 µg/mL of Geneticin at 37°C in 95% oxygen/5% CO2 and grown in 24-well plates to confluence or for at least 48 h after plating.

Assays were performed in 24-well polycarbonate plates. Cells were washed twice with assay buffer (in mM: HEPES 25, NaCl 125, CaCl2 1.3, MgSO4 1.2, KH2PO4 1.2, KCl 5, and D-glucose 5, pH 7.4) and were preincubated for 5 min at 22°C (3,4) with 0.2 mL of assay buffer containing anesthetic (5–200 µM), vehicle (0.1% [vol/vol] DMSO), or inhibitor—5 µM desipramine (a selective norepinephrine uptake inhibitor) (3), 5 µM GBR-12935 (a selective dopamine uptake inhibitor) (5), or 1 mM nipecotic acid (a selective GABA uptake inhibitor) (6). NET and DAT assays also contained pargyline (200 µM) and L-ascorbic acid (50 µM) to inhibit oxidation. Uptake was initiated by adding 40 nM [3H]transmitter. Kinetic experiments revealed that 5-min time points were within the initial linear range of uptake for all 3 cell lines. Therefore, transport mea-surements were made at 5 min to calculate initial transport rates. After 5 min at 22°C, uptake was terminated by rapid removal of medium followed by two washes with ice-cold assay buffer. Cells were lysed for 30 min with 0.25 mL of 0.3% (vol/vol) Triton X-100, and radioactivity was estimated in the cell lysate by liquid scintillation spectrometry (LS 6000IC; Beckman Coulter, Inc., Fullerton, CA) in 3-mL of Bio-Safe 2 scintillation cocktail (RPI, Mt. Pleasant, IL). Protein concentration was determined by the bicinchoni-nic acid method (7) with bovine serum albumin as a standard. Specific uptake was defined as inhibitor-sensitive uptake. Results are expressed as a percentage of control (vehicle) specific uptake.

Halothane and isoflurane were prepared as saturated solutions in assay buffer at room temperature (10–12 mM). Required volumes were diluted into assay wells to give 0.15 to 1.5 mM (the largest concentration possible with this method) and were quickly covered. Final anesthetic concentrations in the assays were determined by gas chromatography by withdrawal of 0.2-mL aliquots of the buffer by using a gas-tight Hamilton glass microsyringe and extraction into 0.1-mL n-heptane. An aliquot (5 µL) of heptane was injected into a gas chromatograph (GC-8A; Shimadzu Corp., Kyoto, Japan) equipped with a thermal conductivity detector. Separation was achieved on a 1.8 m/6-mm-inner-diameter glass column packed with Porapack Q (Supelco, Bellefonte, PA). Column temperature was 210°C, injector temperature was 230°C, and carrier gas (He) flow was 40 mL/min. The aqueous concentration of halothane and isoflurane corresponding to 1 minimum alveolar anesthetic concentration (MAC) in the rat is 0.35 mM (8).

Stock solutions of pentobarbital and ketamine were prepared in water. Stock solutions of propofol and etomidate were prepared in DMSO. Appropriate dilutions were made into assay buffer immediately before use to give 5–200 µM; larger concentrations of anesthetics were limited by solubility. DMSO alone, used as a vehicle at a final concentration up to 0.1% (vol/vol), did not affect uptake (data not shown). Propofol concentrations in the assays were analyzed by high-performance liquid chromatography (9). Free aqueous anesthetic concentrations reported to produce general anesthesia in 50% of subjects (EC50) are 2.2 µM for propofol (10), 10 µM for etomidate (11), 50 µM for ketamine (12), and 25 µM for pentobarbital (13).

Concentration-effect data were fitted by nonlinear regression to the Hill equation—Y = [L]h/([L]h - IC50h), where Y is the effect, [L] is the drug concentration, IC50 is the drug concentration at 50% inhibition, and h is the slope or Hill coefficient—by using GraphPad Prism, Version 3.02 (GraphPad Software, Inc., San Diego, CA). Results are expressed as mean ± SD. Statistical significance was assessed by analysis of variance with the Newman-Keuls multiple comparison test.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
Specific [3H]norepinephrine uptake into LLC-PK1 cells expressing hNET was 2.0 ± 0.35 pmol · mg-1 · min-1. Desipramine inhibited total hNET transport activity by 99%. Specific [3H]dopamine uptake into LLC-PK1 cells expressing rat DAT (rNET) was 1.7 ± 0.40 pmol · mg-1 · min-1. GBR-12935 inhibited total rDAT transport activity by 99%. Specific [3H]GABA uptake into LLC-PK1 cells expressing rat neuronal GAT-1 (rGAT-1) was 5.0 ± 0.13 pmol · mg-1 · min-1. Nipecotic acid inhibited total rGAT-1 transport activity by 95%.

[3H]Dopamine uptake by rDAT was inhibited in a concentration-dependent manner by halothane (IC50 = 0.60 mM; 95% confidence interval [CI], 0.47–0.75 mM; h = -1.2) and isoflurane (IC50 = 0.64 mM; 95% CI, 0.42–0.97 mM; h = -2.7) equipotently (Fig. 1). [3H]Norepinephrine uptake by hNET was inhibited in a concentration-dependent manner by halothane (IC50 = 0.54 mM; 95% CI, 0.43–0.69 mM; h = -1.5), but not by isoflurane (Fig. 2). These effects were reversible after evaporation of the anesthetics (data not shown). [3H]GABA uptake by GAT-1 was relatively insensitive to both anesthetics (IC50> 1.5 mM) (Fig. 1).



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  		Figure 1. Effects of volatile general anesthetics on neurotransmitter transporters. Specific uptake of [3H]norepinephrine, [3H]dopamine, or [3H]{gamma}-aminobutyric acid (GABA) by LLC-PK1 cells stably transfected with human norepinephrine transporter (NET), rat neuronal GABA transporter (GAT-1), or rat dopamine transporter (DAT), respectively, was determined. Normalized data (±SD) were fitted to a single site competition curve by nonlinear regression. The 50% inhibitory concentration values are given in Results; the minimum alveolar anesthetic concentration of halothane and isoflurane in the rat is 0.35 mM (8).

 


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  		Figure 2. Effects of IV general anesthetics on neurotransmitter transporters. Specific uptake of [3H]norepinephrine (NE), [3H]dopamine (DA), or [3H]{gamma}-aminobutyric acid (GABA) by LLC-PK1 cells stably transfected with human norepinephrine transporter, rat dopamine transporter, or rat neuronal GABA transporter, respectively, was determined. Normalized data (±SD) were fitted to a single site competition curve by nonlinear regression. The 50% inhibitory concentration values are given in Results; the anesthesia 50% effective concentration values in serum are 2.2 µM for propofol (10), 10 µM for etomidate (11), 50 µM for ketamine (12), and 25 µM for pentobarbital (13).

 
[3H]Norepinephrine uptake into LLC-PK1 cells expressing hNET was most sensitive to propofol (IC50 = 41 µM; 95% CI, 24–69 µM; h = -0.82) among the anesthetics tested (Fig. 1). Propofol produced significant inhibition at 5 µM (P < 0.05 by analysis of variance). Ketamine (IC50 = 150 µM; 95% CI, 110–200 µM) and etomidate (IC50 > 200 µM) also inhibited hNET; pentobarbital was inactive. [3H]Dopamine uptake into LLC-PK1 cells expressing rDAT was most sensitive to propofol (IC50 = 120 µM; 95% CI, 64–220 µM; h = -1.0) and etomidate (IC50 = 100 µM; 95% CI, 52–200 µM; h = -0.74) among the anesthetics tested (Fig. 1). Ketamine (IC50 = 210 µM; 95% CI, 77–550 µM; h = -1.0) and pentobarbital (IC50 > 200 µM) also inhibited rDAT. [3H]GABA uptake into LLC-PK1 cells expressing rGAT-1 was insensitive to all IV anesthetics tested at concentrations up to 100 µM (Fig. 2).


   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
Effects on synaptic transmission mediated by norepinephrine, dopamine, and GABA have been implicated in the actions of various general anesthetics (1). The neurotransmitter transporters for these transporters form a gene family with considerable structural homology; they are distinguished by their overall sequence homologies, substrate specificities, and sensitivities to a wide range of therapeutic drugs. For example, monoamine transporters are important targets for antidepressants and psychostimulants, and their functional deletion results in increased extracellular transmitter concentrations, prolongation of transmitter actions, and supersensitivity to psychostimulants (14,15). Both volatile and IV general anesthetics have major effects on synaptic transmission in the central nervous system (CNS) (1). Potentiation of inhibitory GABAergic responses mediated by postsynaptic GABAA receptors and inhibition of excitatory glutamate release are established mechanisms of anesthetic action (13,16). There is also evidence for anesthetic effects on catecholaminergic synaptic transmission (1). Our results show that monoamine, but not GABA, transporters are sensitive to certain anesthetics.

Most previous studies of the actions of general anesthetics on neurotransmitter uptake used semi-intact preparations, such as brain slices, synaptosomes, and isolated adrenal chromaffin cells. In slices of rat cerebral cortex, clinical concentrations of volatile anesthetics inhibited KCl-evoked [3H]norepinephrine release (17), but not uptake (18). In contrast, halothane, but not isoflurane, inhibited uptake of [3H]dopamine into whole rat brain synaptosomes (19), whereas several IV anesthetics inhibited [3H]dopamine uptake into striatal synaptosomes (20). Similarly, several IV anesthetics inhibited [3H]norepinephrine uptake into isolated bovine adrenal chromaffin cells (21,22) or rat PC-12 pheochromocytoma cells (23). GABA uptake was inhibited by IV anesthetics in rat cortical slices (24) or striatal synaptosomes (25), but not by volatile anesthetics (25–28). In contrast, GABA uptake was not inhibited by IV anesthetics in rat cortical (29) or thalamic (30) slices. Ketamine consistently inhibits norepinephrine, dopamine, and serotonin transporters in the CNS (31,32). These actions have been proposed to mediate some of the sympathomimetic and excitatory CNS phenomena seen with ketamine administration.

Use of heterologous expression systems allows pharmacological analysis of transporters independently of each other and overcomes problems associated with preparations such as synaptosomes or brain slices, which contain multiple transporters (3,4). Direct comparisons between transporters are facilitated because each transporter is expressed under the same conditions and cellular environment. Nishimura et al. (33) constructed stable cell lines of human embryonic kidney 293 cells transfected with hNET, rDAT, or rat serotonin transporters to demonstrate more directly their inhibition by ketamine. Toxic concentrations of halothane and isoflurane, but not thiamylal and thiopental, were reported to inhibit uptake by mouse GAT-1, rDAT, and rat glutamate/aspartate transporters acutely transfected into COS cells (34).

Using individual transporter assays, we found that DAT is inhibited by both halothane and isoflurane (IC50 = 0.60 and 0.64 mM, respectively), whereas NET is sensitive to halothane (IC50 = 0.54 mM) but insensitive to isoflurane, a distinction that would be difficult to discern in crude brain preparations. These effects of volatile anesthetics on catecholamine transporters occur at concentrations achieved clinically; the MAC of halothane and isoflurane corresponds to an aqueous anesthetic concentration of 0.35 mM (8). Previous studies have demonstrated inhibition of catecholamine uptake by various anesthetics in a variety of intact and semi-intact cellular preparations. For example, halothane and isoflurane inhibited [3H]dopamine uptake (IC50 = 0.72 and 2.2 mM, respectively) (19) and high-affinity cocaine binding sites (Ki =0.61 and 0.75 mM, respectively) (35) in whole rat forebrain synaptosomes. However, both DAT and NET effectively use [3H]dopamine as a substrate and bind cocaine analogs (3), such that both transporters contribute to uptake or binding in measurements made with whole forebrain preparations. The greater affinity for [3H]dopamine of NET compared with DAT (3) may underlie the apparent insensitivity of [3H]dopamine uptake to isoflurane reported in a forebrain synaptosome preparation (19). Conversely, the greater affinity for cocaine analogs of DAT compared with NET in radioligand binding assays (3) could yield anesthetic Ki values in forebrain membranes (35) that more closely reflect the volatile anesthetic sensitivity of heterologously expressed rDAT than of hNET.

Sugimura et al. (34) reported comparable low-potency inhibition by halothane and isoflurane of [3H]dopamine uptake by rDAT, [3H]GABA uptake by mouse GAT-1, and [3H]glutamate uptake by glutamate/aspartate transporters in acutely transfected COS cells. These weak effects were interpreted as nonspecific toxicological actions. However, it is difficult to accurately assess actual anesthetic concentrations in this study, given the unusual methods used for anesthetic preparation and quantification. We found that the amino acid transporter rGAT-1 was less sensitive to volatile anesthetics compared with the monoamine transporters rDAT and hNET when final anesthetic concentrations were determined directly rather than only in supersaturated stock solutions before dilution. Such pharmacological differences in anesthetic sensitivity between monoamine and amino acid transporters are consistent with their classification into distinct transporter subfamilies on the basis of their overall sequence homologies. Evidence for volatile anesthetic actions on catecholamine transporters has also been obtained in cultured rat PC-12 pheochromocytoma cells (23). [3H]Norepinephrine uptake was inhibited by halothane, isoflurane, enflurane, and alkanols at concentrations comparable to their anesthetic potencies. However, identification of the anesthetic-sensitive targets is complicated by the potential presence of both DAT and NET in this cell line (36) and by a large unexplained anesthetic-insensitive component of the uptake (23).

A number of studies have identified effects of ketamine on monoamine transporters (20,31–33), in addition to its well characterized action as a noncompetitive N-methyl-D-aspartate (NMDA) receptor antagonist (37). We observed inhibition by ketamine of recombinant rDAT and hNET expressed in LLC-PK1 cells with somewhat lower potencies compared with those reported previously for the same transporters expressed in human embryonic kidney cells (IC50 = 66 and 82 µM, respectively) (33). Our results agree with uptake assays performed in rat brain synaptosomes that showed inhibition by ketamine of both [3H]dopamine (IC50 = 290 µM) and [3H]norepinephrine uptake (IC50 = 330 µM), with only weak effects on [3H]GABA uptake (IC50 = 1.3 mM) (20). Our value for rDAT is considerably higher than that reported for inhibition of [3H]dopamine uptake in rat striatal synaptosomes (IC50 = 4.6 µM) (20). This raises the possibility that membrane polarization or some other property unique to the neuronal environment increases the anesthetic sensitivity of DAT. However, we found that [3H]dopamine uptake by rDAT was somewhat more sensitive to volatile anesthetics compared with uptake into rat forebrain synaptosomes (19), making this mechanism less likely. Our data for inhibition of hNET by ketamine (IC50 = 150 µM) agree with those reported by Hara et al. (32) for inhibition of [3H]norepinephrine uptake by isolated bovine adrenal chromaffin cells (IC50 = 97 µM). Inhibition of catecholamine transporters by ketamine is considerably less potent than blockade of NMDA receptors (Ki {cong} 1 µM) (37), consistent with a principal role for NMDA receptor inhibition in the production of anesthesia by ketamine (and other phencyclidine derivatives). Inhibition of monoamine transporters by ketamine may contribute to the psychostimulant and sympathomimetic side effects characteristic of these anesthetics at large concentrations.

Propofol inhibited rDAT and hNET, the latter with somewhat greater potency. Inhibition by propofol of hNET at a clinical concentration supports a previous report of inhibition of [3H]norepinephrine uptake by propofol (IC50 {cong} 30 µM) (21). Significant inhibition of hNET by propofol at 5 µM despite the IC50 of 41 µM is consistent with a biphasic concentration-effect curve reported previously in isolated bovine adrenal chromaffin cells (IC50 = 0.9 and 49 µM) (22), but this was not resolved in our assays. Inhibition of NET by propofol could result in mild antinociception and/or potentiation of opioid analgesia (38). Functional deletion of mouse NET is associated with potentiation of opioid analgesia (39). Increases in synaptic dopamine in the limbic system associated with inhibition of DAT correlate with the rewarding properties of psychostimulant drugs of abuse (15). Our IC50 value for propofol inhibition of recombinant rDAT (IC50 = 120 µM) corresponds to that reported by Keita et al. (20) for inhibition of [3H]dopamine uptake by rat striatal synaptosomes (IC50 = 150 µM). However, the relative insensitivity of rDAT to IV anesthetics suggests that a direct effect on DAT does not contribute to the rewarding properties and abuse potential of propofol (40) or ketamine (41) at subanesthetic doses.

Etomidate inhibited rDAT and nNET, but these effects occurred at concentrations considerably larger than those achieved clinically (<10 µM) (11) and likely represent toxic concentrations. Our IC50 value for inhibition by etomidate of recombinant rDAT (IC50 = 100 µM) is comparable to that reported for inhibition of [3H]dopamine uptake in rat striatal synaptosomes (IC50 = 55 µM) (20).

Pentobarbital did not affect rDAT or hNET, in agreement with the results of Nishimura et al. (33). Propofol, etomidate, ketamine, and pentobarbital (up to 200 µM) did not significantly affect rGAT-1. Thus, both the volatile and IV anesthetics tested were selective for the monoamine subfamily versus the amino acid subfamily of Na+/Cl--coupled transporters. The insensitivity of rGAT-1 to anesthetics is consistent with the observation that GABA uptake is insensitive in brain slices (27,29,30) and isolated nerve terminals (25,26,28).

Neurotransmitter transporters are involved in rapidly reducing synaptic concentrations of certain neurotransmitters to terminate synaptic transmission (2) and have been considered by many investigators as potential sites of anesthetic action. Interpretation of many of transporter studies is complicated by the existence of multiple transporters with overlapping substrate specificities (3), and thus the potential role of direct anesthetic actions on transporter function is uncertain. We investigated the role of neurotransmitter transporters as targets for general anesthetics by using heterologously expressed transporters. Drug- and transporter-specific anesthetic effects were observed. Halothane and isoflurane inhibited dopamine and norepinephrine transporters at concentrations achieved during clinical anesthesia; propofol and ketamine were also inhibitory at clinical concentrations, whereas etomidate and pentobarbital were not. GAT-1 was insensitive to all drugs tested and thus does not appear to contribute to the facilitation of inhibitory synaptic transmission produced by many general anesthetics (13). Thus, inhibition of DAT or NET is not a general property of anesthetics, but it may be involved in certain drug-specific secondary actions, including psychostimulant, sympathomimetic, cardiostimulant, and arrhythmogenic effects.


   Acknowledgments
 
Supported by Grant GM-58055 from the National Institutes of Health and by the Department of Anesthesiology, Weill Medical College of Cornell University.


   Footnotes
 
Presented in part at the annual meeting of the International Anesthesia Research Society, Honolulu, HI, March, 2000.


   References
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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Accepted for publication June 14, 2002.




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Lippincott, Williams & Wilkins Anesthesia & Analgesia® is published for the International Anesthesia Research Society® by Lippincott Williams & Wilkins with the assistance of Stanford University Libraries' HighWire Press®. Copyright 2006 by the International Anesthesia Research Society. Online ISSN: 1526-7598   Print ISSN: 0003-2999 HighWire Press