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REGIONAL ANESTHESIA AND PAIN MANAGEMENT

Synergistic Inhibition of Lysophosphatidic Acid Signaling by Charged and Uncharged Local Anesthetics

Lisa M. Sullivan, MD^*, Christian W. Hönemann, MD^*,, John A. Arledge, BS^*, and Marcel E. Durieux, MD^*

*Department of Anesthesiology, University of Virginia Health System Charlottesville, Virginia; and Department of Anesthesiology, Wilhelms-Universität Muenster, Muenster, Germany

Address correspondence and reprint requests to Dr. Durieux, Department of Anesthesiology, University of Virginia HSC, PO Box 10010, Charlottesville, VA 22906-0010. Address e-mail to med2p{at}virginia.edu

	Abstract

Top Abstract Introduction Methods Results Discussion Appendix 1 References

 
We investigated the mechanism of benzocaine (permanently uncharged) and QX314 (permanently charged) inhibition of lysophosphatidic acid (LPA) signaling. To determine their site of action, we studied effects of these drugs, alone and in combination, on LPA-induced Ca^{2 +}-dependent Cl currents (I_Cl(Ca)) in Xenopus oocytes. After 10 min exposure to benzocaine, QX314 (10^-6–10^-2 M), or both, we measured effects on I_Cl(Ca) induced by LPA (with and without protein kinase [PKC] activation/inhibition) and on I_Cl(Ca) induced by the intracellular injection of IP₃ and GTPS. LPA application to oocytes resulted in I_Cl(Ca) (50% effective concentration approximately 10^-8 M). Both anesthetics inhibited LPA signaling concentration-dependently (50% inhibitory concentration [IC₅₀] benzocaine 0.9 mM, QX314 0.66 mM). The combination acted synergistically (IC₅₀ benzocaine 0.097 mM/QX314 0.048 mM). Intracellular signaling pathways were not affected. This study shows that benzocaine and QX314 inhibit LPA signaling and act synergistically, which is most easily explained by the existence of two different binding sites. Lack of inhibition of IP₃ or GTPS-induced I_Cl(Ca) identifies the receptor as a target. Activation of PKC can be excluded as a potential mechanism.

Implications: Lysophosphatidic acid may play a role in wound healing, and its signaling is inhibited by local anesthetics. We identified the membrane receptor as the local anesthetic site of action and showed that charged (QX314) and uncharged (benzocaine) local anesthetics inhibit lysophosphatidic acid signaling synergistically, which can be explained by the presence of different binding sites.

	Introduction

Top Abstract Introduction Methods Results Discussion Appendix 1 References

 
Although best known for their ability to block Na channels, local anesthetics also interact with other cellular systems. The interactions of local anesthetics with ion channels other than Na channels have been studied in some detail (1); examples are effects on Ca channels (2–4) and nicotinic acetylcholine receptors (3, 5). In contrast, interactions with second messenger-linked systems, such as G-protein–coupled receptors, have not been investigated in depth.

One G-protein–coupled receptor that is inhibited in its function by local anesthetics is the lysophosphatidate (LPA) receptor (6). LPA is released by activated platelets and fibroblasts (7), suggesting a role in wound repair. Indeed, it has been reported to be generated after corneal injury (8), and we have shown it to be released after acute skin injury (unpublished data). Local anesthetics are frequently injected in high concentrations around surgical wounds and may delay wound healing (9–11); this could result from interference with LPA signaling. In a previous study, Nietgen et al. (6) demonstrated that, at concentrations such as those present when injected for postoperative analgesia, lidocaine and bupivacaine inhibit LPA signaling in Xenopus oocytes, but the mechanism of action was not investigated in detail.

In the present study, we continued this investigation. First, we determined in detail the site of local anesthetic action on the signaling cascade, including potential effects on protein kinase C (PKC) functioning. Second, we determined whether a charged group in the local anesthetic molecule is necessary for the inhibitory effect. The local anesthetics used in our previous study (6), lidocaine and bupivacaine, are both partially charged, depending on pH. Therefore, no conclusions could be drawn about the active species involved in LPA signaling blockade. To resolve this issue, in the present study, we investigated the effects on LPA signaling of two other local anesthetics: QX314 (N-(2,6)dimethylphenylcarbamoylmethyl triethylammonium bromide, a quaternary local anesthetic, 99.99% permanently charged) and benzocaine (permanently uncharged).

Our data indicate that LPA signaling inhibition by local anesthetics takes place at the membrane receptor. Both charged and uncharged local anesthetics inhibit LPA signaling, and they act synergistically when combined, which suggests the presence of two local anesthetic binding sites on the LPA receptor molecule, one of them intracellularly, and each of them able to inhibit LPA signaling when occupied by a local anesthetic molecule.

	Methods

Top Abstract Introduction Methods Results Discussion Appendix 1 References

 
The study protocol was approved by the Animal Research Committee at the University of Virginia. Xenopus oocytes were removed as described previously (12). Oocytes were incubated for 10 min in benzocaine and diluted in modified Barth's solution (MBS) to four concentrations (0.01, 0.1, 1, and 10 mM). The pH of the MBS containing 10 mM benzocaine was adjusted to pH 5 to achieve solubility. Each oocyte was then voltage-clamped and tested alternately with a control cell incubated in MBS only. The control cells for the 10 mM benzocaine group were incubated in MBS at a pH of 5. Agonist application and voltage clamp techniques were performed as described previously (12). Intracellular Ca release in response to LPA was assessed by measuring Ca-activated Cl currents (I_Cl(Ca)) (Fig. 1A). Responses were quantified by integrating the current trace.

View larger version (14K): [in this window] [in a new window]   Figure 1. Lysophosphatidic acid (LPA) signaling in Xenopus oocytes. A, Sample trace of 10-6 M LPA-induced ICl(Ca). The arrow indicates LPA application. Charge movement was 8.2 µC. B, LPA activates the LPA receptor in Xenopus oocytes in a concentration-dependent manner. ICl(Ca) induced by 10-6 M LPA (ymax) was 8.91 ± 0.99 µC. Curve-fitting using the Hill equation revealed a half-maximal effect concentration of 0.5 x 10–8M ± 0.3 x 10–8 m and a Hill coefficient of 0.6 ± 0.1. n 7 for each data point.

View larger version (25K): [in this window] [in a new window]   Figure 2. Responses to lysophosphatidic acid (LPA) are inhibited by QX314 and benzocaine. A, Examples of responses induced by LPA (10-6 M) in the absence and presence of extra- or intracellularly applied QX314 (0.5 mM). The charge movement in response to LPA is 36.3 µC under control conditions (50 nL of KCl 150 mM injected), 15.9 µC in the presence of intracellularly microinjected QX314 (diluted in 50 nL of KCl 150 mM), and 32.4 µC in the presence of 0.5 mM OX314 applied extracellularly. B, QX314 acts intracellularly (mean ± SEM). Control = 50 nL of KCl 150 mM intracellular, intra = 50 nL of KCl 150 mM containing 5 mM QX314 intracellular, extra = cells injected with 50 nL of KCl 150 mM exposed to 0.5 mM QX314 extracellular. C, QX314 inhibits LPA receptor signaling in a concentration-dependent manner. Curve-fitting using the Hill equation revealed a half-maximal effect concentration of 0.66 mM ± 0.15 and a Hill coefficient of 0.44 ± 0.04. n 7 for each data point. D, Examples of responses induced by LPA (10-6 M) in the absence and presence of benzocaine (0.1 mM). The charge movement in response to LPA is 9.7 µC in the absence and 4.0 µC in the presence of benzocaine. E, Benzocaine inhibits LPA receptor signaling in a concentration-dependent manner. Curve-fitting using the Hill equation revealed a half-maximal effect concentration of 0.91 ± 0.23 mM and a Hill coefficient of 0.66 ± 0.1. n 7 for each data point.

PKC was activated by 5 min incubation in phorbol ester (50, 500, 1000 nM) before measuring LPA-induced I_Cl(Ca). PKC was inhibited by microinjecting 50 nL of 500 µM chelerythrine diluted in 150 mM KCl or, as a control, 150 mM KCl, into oocytes 2 h before measuring LPA-induced I_Cl(Ca).

Results are reported as mean ± SEM. Because variability among batches of oocytes is common, responses were, at times, normalized to same-day controls for each batch. Differences between treatment groups were analyzed by using analysis of variance and Student's unpaired t-test, appropriately corrected for multiple comparisons (Bonferroni). P < 0.05 was considered significant. Concentration response curves were fit to the following logistic function, derived from the Hill equation (13): y = y_min + (y_max –y_min) (1 - xⁿ/[x₅₀ⁿ - xⁿ]) where y_max and y_min are the maximal and minimal responses obtained, n is the Hill coefficient, and x₅₀ is the EC₅₀ for agonist or the half-maximal inhibitory effect concentration (IC₅₀ for antagonists). These were used for both algebraic and isobolographic analysis of drug interaction (14) (Appendix 1). Statistical analysis of the isobologram was used to establish the superadditive, subadditive, or simple additive effects of benzocaine and QX314 in combination. The drugs were tested in a 2:1 ratio based on their relative IC₅₀ values (equiinhibitory concentrations). Concentration-effect data were plotted with 95% confidence intervals. Statistical analysis was then performed to determine the significance of the difference between the line of additivity and the intersection of the combination IC₅₀ values.

LPA was obtained from Avanti Polar Lipids (Alabaster, AL) and was diluted in 0.1% fatty acid-free bovine serum albumin (ICN Pharmaceuticals, Costa Mesa, CA) in Tyrode's solution to the appropriate concentration. QX314 was a gift from Astra Inc. (Wayne, PA). All other chemicals were from Sigma Chemical Company (St. Louis, MO).

	Results

Top Abstract Introduction Methods Results Discussion Appendix 1 References

 
In agreement with earlier studies (6, 15), we found that LPA induced transient inward currents in Xenopus oocytes (Fig. 1A). In contrast, the vehicle (BSA) was without effect (data not shown). We have previously shown that the oocyte response to LPA is a Ca-activated Cl current (I_Cl(Ca)) (16). It is induced via IP₃-mediated Ca release, as we have shown the IP₃ receptor antagonist heparin to be able to abolish responsiveness to LPA (17). Expanding our previous findings (6), we determined the concentration-response relationship for LPA. We found the oocyte response to LPA to be concentration-dependent, with an EC₅₀ of 5.1 ± 0.3 x 10^–9 M and a Hill coefficient of 0.6 ± 0.1 (Fig. 1B, Table 1). Maximal I_Cl(Ca) (8.9 ± 1.0 µC) was induced by 10^-6 M LPA, and this concentration was used for the remainder of the study.

When applied extracellularly, QX314 was unable to inhibit LPA-induced I_Cl(Ca) in oocytes, even when the local anesthetic was applied at high concentrations (50 mM) (Fig. 2, A and B). In contrast, when the compound was microinjected intracellularly, it inhibited LPA signaling effectively (Fig. 2, A and B). LPA signaling inhibition was concentration-dependent (Fig. 2C). Curve-fitting using the Hill equation revealed an IC₅₀ of 660 ± 154 µM with a Hill coefficient of 0.4 ± 0.05 (Table 1). Maximal inhibition (using 5 mM QX314 intracellular concentration) was 28% of control.

These findings clarify two issues. First, they indicate that the site of QX314 block of LPA signaling is located intracellularly. Second, they indicate that a charged local anesthetic molecule is able to interact with LPA signaling.

Our finding that QX314 inhibits LPA signaling does not prove that only the charged local anesthetic molecule can exert inhibitory effects on LPA signaling. To determine whether an uncharged local anesthetic also interacts with LPA signaling, we studied the ability of benzocaine, a permanently uncharged (and, therefore, highly membrane-permeant) local anesthetic, to inhibit LPA signaling in oocytes.

LPA signaling was inhibited in a concentration-dependent manner when oocytes were exposed to benzocaine (Fig. 2D). Curve-fitting using the Hill equation revealed an IC₅₀ of 909 ± 231 µM and a Hill coefficient of 0.7 ± 0.1 (Fig. 2E, Table 1). Thus, the compound was approximately half as potent as QX314. Maximal inhibition (using 10 mM benzocaine) was 20% of control. These findings show that charged and uncharged local anesthetics can inhibit LPA signaling. Two hypotheses can account for this finding: 1) the charged and uncharged compounds act at different sites, with different degrees of polarity; or 2) the compounds act at a single site, in which case the hydrophobic portion of the local anesthetic is likely to be the active moiety.

To differentiate between these two possibilities, we studied the inhibitory action of a combination of benzocaine (applied extracellularly) and QX314 (microinjected intracellularly). If the compounds acted on a single site, the combination would be anticipated to act in an additive manner. In contrast, if two sites in the signaling pathway were involved, the combination could act synergistically.

When applied in combination, benzocaine and QX314 (2:1 molar ratio, in view of their respective IC₅₀ values) inhibited LPA signaling in a concentration-dependent manner (Fig. 3A). Curve-fitting using the Hill equation revealed an IC₅₀ of 97 ± 12 µM benzocaine and 48 ± 6 µM QX314. The Hill coefficient was 0.5 ± 0.03 (Fig. 3B, Table 1). Thus, when applied in combination, the IC₅₀ values for both local anesthetics are less than those obtained when the drugs were studied in isolation, which suggests a synergistic action. We therefore performed both algebraic and isobolographic analysis of our data to determine whether synergism was indeed present (Table 2). Algebraic analysis yielded an R of 5.56 (P = 0.035); isobolographic analysis (Fig. 3C, Appendix 1) similarly revealed evidence of superadditivity or synergism.

View larger version (24K): [in this window] [in a new window]   Figure 3. Responses to lysophosphatidic acid (LPA) are inhibited by benzocaine and QX314 in combination. A, Examples of responses induced by LPA in the absence and presence of benzocaine (0.1 mM) and QX314 (0.05 mM microinjected). Control cells were injected with 50 nL of KCl 150 mM. The charge movement in response (injected with 50 nL of KCl 150 mM) to LPA is 27.6 µC in the absence and 11.9 µC in the presence of the local anesthetics in combination. B, The combination of benzocaine and QX314 inhibits LPA receptor signaling in a concentration-dependent manner. = concentration response-curve of benzocaine in combination with intracellular applied QX314, = QX314 concentration response curve in combination with benzocaine. Curve-fitting using the Hill equation revealed a half-maximal effect concentration of benzocaine 0.097 ± 0.01 mM and QX314 0.048 ± 0.006 mM and a Hill coefficient of 0.48 ± 0.03. n 7 for each data point. C, Benzocaine and QX314 synergistically inhibit LPA signaling. The solid line of additivity is drawn between the 50% inhibitory concentration (IC50) values for QX314 and benzocaine (•). A dotted line parallel to each side of the line of additivity represents the 95% confidence interval for the IC50 values. A second solid line connects the midpoint of the line of additivity to the IC50 values for the combination of benzocaine and QX314. The length of this line is 0.45, and the significance of this distance can be calculated using isobolographic analysis (Appendix 1).

To study the effect of local anesthetics on segments of the signaling pathway, we activated various segments of the intracellular signaling pathway by an intracellular microinjection of IP₃ or GTPS.

We first tested the effects of the anesthetics on currents induced by IP₃. As we (6,12,17) have demonstrated previously, IP₃ induces I_Cl(Ca) indistinguishable from that induced by LPA application to oocytes. However, these previous studies were performed with a single concentration of IP₃. We now sought to determine whether the effect was concentration-dependent. As shown in Figure 4A, the oocyte response to microinjected IP₃ is concentration-dependent; 2 mM IP₃ induces currents with an average charge movement of 11.3 ± 1.5 µC.

View larger version (29K): [in this window] [in a new window]   Figure 4. Benzocaine and QX314 have no effect on IP3 or GTPS signaling. A, ICl(Ca) generated by the injection of 50 nL of 0.2, 2, and 20 mM IP3 into oocytes. B, No significant inhibition of IP3-induced ICl(Ca) was observed when cells were incubated with 1.0 mM benzocaine or injected with 0.50 mM QX314. The injection of 50 nL of water induced no response. C, ICl(Ca) generated by the injection of 50 nL of 6 and 10 mM GTPS into oocytes. D, No significant inhibition of GTPS-induced ICl(Ca) was observed when cells were incubated with 1.0 mM benzocaine or microinjected with 0.5 mM QX314. The injection of 50 nL of water induced no response.

Because the distal signaling pathway was not affected by local anesthetics, we determined their effects on the more proximal pathway. GTPS is a nonhydrolyzable GTP analog that irreversibly activates G proteins. We have previously shown that this compound induces I_Cl(Ca) in oocytes (12). We now demonstrate that these responses are concentration-dependent (Fig. 4C). GTPS (10 mM) induced currents with an average charge movement of 7.1 ± 0.5 µC.

Similar to the situation with IP₃ signaling, neither benzocaine (1 mM) nor QX314 (0.5 mM injected intracellularly) affected the currents induced by 10 mM GTPS (Fig. 4D). This suggests that the LPA signaling pathway downstream from the receptor is not affected by the local anesthetics. Hence, their sites of action are most likely located on the LPA receptor molecule itself.

However, benzocaine and QX314 could exert their inhibitory effects on LPA receptors through a different mechanism. Instead of direct modulation of receptor configuration by interaction with the protein, they could act indirectly by activating PKC, which could then catalyze phosphorylation of LPA receptors and decrease receptor functioning. To determine whether PKC can indeed modulate LPA signaling, we studied the effect of PKC inhibition and activation.

Xenopus oocytes were pretreated for 2 h with the PKC inhibitor chelerythrine. Chelerythrine produced a 30% enhancement of the currents activated by 1 µM LPA (Fig. 5A) and 70% enhancement of the currents activated by 100 nM LPA (Fig. 5A). Conversely, the PKC activator PMA (50, 500, and 1000 nM for 5 min) inhibited currents evoked by 10^-6 M LPA concentration-dependently to 56%, 40%, and 10% of the initial currents, respectively (Fig. 5B). Thus, LPA receptor functioning can be modulated by PKC.

View larger version (32K): [in this window] [in a new window]   Figure 5. Effects of protein kinase C (PKC) on lysophosphatidic acid (LPA) signaling. A, Responses induced by 10-6 and 10-7 M LPA. PKC was inhibited through the microinjection of 50 nL of chelerythrine 20 µM 2 h before experiments. B, Responses of oocytes incubated in various concentrations of phorbol ester (0, 50, 500, and 1000 nM) for 5 min. C, ICl(Ca) evoked by 10-6 M LPA with and without benzocaine or intracellular QX314. PKC inhibition did not reverse the inhibitory effect of both local anesthetics on LPA signaling (*P < 0.05). Values are mean ± SEM. n > 7 for each experiment.

	Discussion

Top Abstract Introduction Methods Results Discussion Appendix 1 References

 
Our results demonstrate that benzocaine and QX314 inhibit LPA receptor function. Both a completely charged (QX314) and a completely uncharged local anesthetic (benzocaine) can inhibit LPA signaling, with a relatively similar IC₅₀ (ratio 2:1). For either local anesthetic, the site of action is localized to the LPA receptor; neither the intracellular signaling pathway nor PKC-mediated modulation of LPA signaling are involved. For the charged species, the site of action is intracellular. This rules out the possibility that local anesthetic inhibition of LPA signaling could be due to direct interactions between the local anesthetic and the LPA molecule or to competition between LPA and local anesthetic for binding to albumin. The charged and uncharged local anesthetic interact synergistically, which indicates separate binding sites on the LPA receptor molecule.

These findings are in complete agreement with data obtained in previous studies (6,18), in which other local anesthetics were found to inhibit LPA signaling in a reversible manner but were without effect on angiotensin II signaling, which shares the same signaling pathway. Our present study expands these findings by investigating different local anesthetics, localizing the site of action in more detail, and elucidating the role of charge in the local anesthetic molecule. Binding studies and mutational analysis of the LPA receptor could clarify these issues further but are not possible until a reproducible binding assay is established and a clone has been definitely identified.

One item that we did find to be different between the studies are the IC₅₀ values of the compounds. Whereas our previous study reported IC₅₀ values of 30 ± 8 mM and 5 ± 1 mM (6) for lidocaine and bupivacaine, respectively, we found IC₅₀ values of 0.66 ± 0.16 mM and 0.91 ± 0.23 mM for QX314 and benzocaine, respectively, in our present investigation. This difference may be due to pharmacological characteristics of the compounds or to pH (our previous study was performed at pH 6.4). Alternatively, small changes in experimental technique (continuous flow chamber, collagenase rather than manual defolliculation) may have yielded greater apparent potency.

As in our previous study, we chose the Xenopus oocyte model. In addition to providing consistency with the other investigation, the oocyte provides great advantages for studies of this kind. In particular, its size allows easy intracellular access, allowing study of intracellular pathways and site of action by microinjection of different compounds. However, potential disadvantages should also be kept in mind. Most relevant to the present study is the fact that the LPA receptor studied is amphibian, and its behavior may therefore diverge from its mammalian ortholog. However, LPA-induced Ca signaling in oocytes and in mammalian cells has been shown to be similar (7,17,19). Nonetheless, confirmatory studies of local anesthetic effects in other models, e.g., LPA-induced platelet aggregation or fibroblast proliferation, would be of interest.

As benzocaine is freely membrane permeable, we could not determine whether its site of action is intra- or extracellular. It might interact with the lipophilic agonist binding pocket on the LPA receptor. Unfortunately, lack of an established binding assay makes it impossible to test this hypothesis directly. In any case, the synergistic interaction between the two compounds makes it likely that two separate binding sites are involved: one for the charged and one for the uncharged species. Lack of effect on GTPS and IP₃ signaling and lack of effect of PKC inhibition locates these two sites to the LPA receptor molecule itself. Our study does not rule out an effect of local anesthetics on PKC; there are many isoforms of this enzyme (20), and our results only apply to the (as yet not characterized) isoforms modulating LPA signaling. One alternative hypothesis to be considered is the presence of several subtypes of LPA receptor, one of which would be inhibited by charged, the other by uncharged, local anesthetics. Although there some evidence suggests the presence of multiple receptor subtypes in oocytes (21), definitive determination awaits molecular cloning of the receptor(s). Although some potential candidate clones have been reported (22), they have not yet been generally accepted by the scientific community. Nonetheless, it seems very unlikely that this could explain our findings, as these receptors would form parallel, rather than sequential, signaling pathways, and additive, rather than synergistic, interactions would therefore be expected.

In conclusion, the site of local anesthetic action on LPA signaling seems to be the membrane receptor. Intracellular signaling pathways and the PKC isoform modulating LPA signaling are not inhibited by local anesthetics. LPA receptors most likely contain multiple binding sites for charged and uncharged local anesthetics. Therefore, clinically used compounds, such as lidocaine (67% charged at physiological pH) and bupivacaine (88% charged at physiological pH), may influence this receptor by several means. As a result, they can be expected to modulate the effects of LPA after its release in the wound bed, as well as other LPA-mediated physiologic responses.

	Appendix 1

Top Abstract Introduction Methods Results Discussion Appendix 1 References

 
Isobolographic Analysis
An isobologram is a cartesian plot of pairs of doses that, in combination, yield a specified level of effect. It is a convenient way of graphically displaying results of a drug combination because paired values of experimental points that fall below or above the line connecting the axial points (usually the EC₅₀ values) denote a supra- or subadditive combination, respectively. During our isobolographic and statistical analysis of the isobologram, we followed the suggestions of Tallarida et al. (14).

If C is <1, our tested drug combination is superadditive; if it =1, then our combination has a simple additive effect; if it is >1, we have a subadditive effect of the drug combination. In our study, C = 0.1796; therefore, the combination of benzocaine and QX314 had superadditive effects.

Statistical confidence limits must be expressed on the isobologram if this diagram is to establish superadditive effects. For two different drugs whose regression lines have slopes that do not differ significantly, a common slope ß is determined as a weighted mean of the individual slopes. Thus, benzocaine and QX314 have their respective regression lines calculated using the equations below. We used this method to calculate the variance and SEM of the fictive simple additive EC₅₀ of the drug combination (point on the line of additivity) and analyzed the statistical significance of our findings using Student's t-tests. We used the following equations:

We used the calculated values and solved the following equation. The result of this equation is the calculated variance of the fictive simple additive point on the line of additivity, which can be used to calculate the SEM.

We then compared this mean ± SEM with the measured point (mean ± SEM) using Student's t-tests.

	Acknowledgments

 
This work was supported in part by National Institutes of Health Grant GMS 52387 to MED. CWH is supported by the Innovative Medizinische Forschung fund, Münster, Germany.

	References

Top Abstract Introduction Methods Results Discussion Appendix 1 References

 

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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