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Anesth Analg 2001;92:767-774
© 2001 International Anesthesia Research Society

GENERAL ARTICLES

The Effects of Ethanol on Ca2+ Sensitivity in Airway Smooth Muscle

Motohiko Hanazaki, MD, Keith A. Jones, MD, William J. Perkins, MD, and David O. Warner, MD

Departments of Anesthesiology and Physiology and Biophysics, Mayo Clinic and Mayo Foundation, Rochester, Minnesota

Address correspondence and reprint requests to David O. Warner, MD, Mayo Clinic and Foundation, 200 First St. SW, Rochester, MN 55905. Address e-mail to warner.david{at}mayo.edu


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
Halothane and other volatile anesthetics relax air-way smooth muscle (ASM) in part by decreasing the amount of force produced for a given intracellular Ca2+ concentration (the Ca2+ sensitivity) during muscarinic receptor stimulation. To determine whether this is a unique property of the volatile anesthetics, we tested the hypothesis that ethanol, another compound with anesthetic properties, also inhibits calcium sensitization induced by muscarinic stimulation of ASM. A ß-escin permeabilized canine tracheal smooth muscle preparation was used. Ethanol was applied to permeabilized muscles stimulated with calcium in either the absence or presence of acetylcholine. In intact ASM, ethanol produced incomplete relaxation (approxi-mately 40%) at concentrations up to 300 mM. Ethanol significantly increased Ca2+ sensitivity both in the presence and the absence of muscarinic receptor stimulation. Although ethanol did not affect regulatory myosin light chain (rMLC) phosphorylation during stimulation with Ca2+ alone, it decreased rMLC phosphorylation by Ca2+ during muscarinic receptor stimulation. Ethanol, like volatile anesthetics, inhibits increases in rMLC phosphorylation produced by muscarinic receptor stimulation at constant [Ca2+]i. However, despite this inhibition, the net effect of ethanol is to increase Ca2+ sensitivity (defined as the force maintained for a given [Ca2+]i) by a mechanism that is independent of changes in rMLC phosphorylation.

Implications: In permeabilized airway smooth muscle, ethanol, like volatile anesthetics, inhibits increases in regulatory protein phosphorylation caused by stimulation of the muscle when intracellular calcium concentration is constant. However, unlike volatile anesthetics, ethanol causes a net increase in force through a process not dependent on protein phosphorylation, an action favoring bronchoconstriction.


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
The contraction of airway smooth muscle (ASM) is produced in part by increases in intracellular calcium concentration ([Ca2+]i). Stimulation with contractile agonists, such as acetylcholine, also increases force developed for a given [Ca2+]i (i.e., the calcium sensitivity) (1,2). Volatile anesthetics relax ASM by decreasing both [Ca2+]i and calcium sensitivity (1,3,4). Halothane decreases calcium sensitivity in the airways by inhibiting the activity of heterotrimeric guanine-binding proteins (G-proteins) that link muscarinic receptors to intracellular effectors (5). Ethanol, like many other alcohols, has anesthetic properties (6,7) and affects G-protein-mediated signal transduction processes (8–10). Both inhibitory and stimulatory effects have been described in various cell types, with the inhibitory effects in some tissues similar to those exhibited by halothane. If, like volatile anesthetics, ethanol also inhibits the G-proteins that mediate Ca2+ sensitivity, it too may relax ASM in part by this mechanism. Ethanol could thus be a useful experimental tool to explore anesthetic effects on signal transduction mediated by G-proteins in smooth muscle.

The aim of our study was to test the hypothesis that ethanol, like volatile anesthetics, inhibits calcium sensitization induced by muscarinic stimulation of ASM. We used a permeabilized canine tracheal smooth muscle (CTSM) preparation in which Ca2+ sensitivity can be measured directly.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
Tissue Preparation
After Institutional Animal Care and Use Committee approval, mongrel dogs (15–20 kg) of either sex were anesthetized with an IV injection of pentobarbital sodium (30 mg/kg) and exsanguinated. The trachea was excised and immersed in chilled physiological salt solution (PSS) of the following composition (in millimolar): 110.5 NaCl, 25.7 NaHCO3, 5.6 dextrose, 3.4 KCl, 2.4 CaCl2, 1.2 KH2PO4, and 0.8 MgSO4. Muscle strips of 0.1–0.2-mm width, 1-cm length, and 0.2–0.3-mg wet weight were mounted in 0.1-mL cuvettes, and isometric force was measured at optimal length as described previously (1).

Permeabilization Procedure
When appropriate, muscle strips were permeabilized with ß-escin (11) by a method validated for CTSM in our laboratory (1). ß-escin creates pores in the smooth muscle cell plasma membrane, thus allowing substances of small molecular weight such as Ca2+ to diffuse freely across the cell membrane. Muscle strips were superfused for 20 min with relaxing solution containing 100 µM ß-escin. The relaxing solution was prepared in the following composition using the algorithm of Fabiato and Fabiato (12): 7.5 mM Mg adenosine 5'-triphosphate , 4 mM EGTA, 20 mM imidazole, 1 mM dithiothreitol, 1 mM free Mg2+, 1 nM free Ca2+, 10 mM creatine phosphate, and 0.1 mg/mL creatine phosphokinase. The pH was adjusted to 7.0 at 25°C with KOH or HCl. The calcium ionophore A23187 (10 µM) was added to the relaxing solution and all subsequent experimental solutions to deplete the sarcoplasmic reticulum Ca2+ stores.

Regulatory Myosin Light Chain Phosphorylation (rMLC) Measurements
rMLC phosphorylation was measured in separate sets of permeabilized CTSM strips as described previously (13). After an equilibration period of 30 min in aerated PSS at 25°C, the strips were incubated in Ca2+ free PSS containing 2 mM EGTA for 15 min. Intracellular Ca2+ stores were then depleted by exposing the strips to 10 µM acetylcholine (ACH) for 10 min. After permeabilization and experimental interventions, muscle strips were flash-frozen by rapid immersion in acetone containing 10% trichloroacetic acid and 10 mM dithiothreitol cooled to -80°C. rMLC was extracted and phosphorylation was determined by glycerol-urea gel electrophoresis followed by Western blotting. Unphosphorylated and phosphorylated bands of rMLC were visualized by phosphorimage analysis (PhosphorImager; Molecular Dynamics, Sunnyvale, CA), and fractional phosphorylation was calculated as the density ratio of the sum of mono- and diphosphorylated rMLC to total rMLC.

Experimental Protocols
Effect of Ethanol on Ach-Induced Contraction in Intact Smooth Muscle.
Experiments in intact strips were designed to determine the concentrations of ethanol to be used in subsequent studies because there are no previous data on the effects of ethanol on isolated airways. This protocol was performed at 37°C. Muscle strips were contracted for 10 min with 0.03 µM ACh, which produces approximately 50% of maximal force (induced by 10 µM ACh). After stable contractions were obtained, cumulative concentrations of ethanol (0.01 to 1 M) were applied and concentration response curves generated.

Effects of Ethanol on Ca2+ Sensitivity in Permeabilized Smooth Muscle.
Experiments in permeabilized strips determined the effect of ethanol on Ca2+ sensitivity either in the absence or presence of muscarinic receptor stimulation. A set of four permeabilized strips was prepared from the same dog for each experiment. All strips were first maximally contracted with 10 µM Ca2+; all subsequent force measurements were normalized to these contractions. Each set of four strips was divided into two pairs. All four strips were then contracted with 0.3 µM Ca2+ for 10 min. One pair was then stimulated with 10 µM ACh and 10 µM guanosine 5'-triphosphate (GTP); the other pair continued to be exposed to Ca2+ alone. After 10 min, 240 mM ethanol was added to one strip of each pair for 15 min. The remaining strip of each pair was not exposed to ethanol, and served as a time control. In the concentrations studied, ethanol did not change the pH of solutions (data not shown).

To explore the mechanism of ethanol’s effect, other strips were treated as described above, then exposed to guanosine 5'-O-(2-thiodiphosphate) (GDPßS) (1 mM) to inhibit G-proteins, or tyrphostin A-25 (50 µM) to inhibit tyrosine kinases.

Effects of Ethanol on Ca2+ Concentration-Response Curves in Permeabilized Smooth Muscle.
To determine whether the effects of ethanol are related to the increases in osmolarity because of its relatively large concentration, the effect of mannitol on Ca2+ sensitivity also was examined. In preliminary experiments, 300 mM mannitol increased the osmolarity of the 0.3 µM Ca2+ solution by an amount equal to that produced by 300 mM ethanol (data not shown). A set of three ß-escin-permeabilized strips was prepared from the same dog for each experiment. All strips were first maximally contracted with 10 µM Ca2+. Free Ca2+ concentration-response curves (0.01–10 µM) were generated for each strip in the absence or presence of ethanol (300 mM) or mannitol (300 mM).

Effects of Ethanol on rMLC Phosphorylation.
This protocol determined how ethanol affects rMLC phosphorylation. A set of eight permeabilized strips was prepared from the same dog for each experiment. One strip was not stimulated and was flash-frozen in relaxing solution (baseline). The remaining seven strips were exposed to 0.3 µM free Ca2+. After 20 min, one strip was exposed to 300 mM ethanol and frozen 15 min later. Another strip did not receive ethanol and was frozen at the same time (after 35 min of Ca2+ stimulation). Five other strips were exposed to 10 µM ACh and 10 µM GTP beginning 10 min after Ca2+ stimulation. Three of these strips received ethanol (30, 100, or 300 mM) beginning 10 min after ACh stimulation, then were frozen 15 min later. One of these strips did not receive ethanol and was frozen at the same time (after 25 min of ACh stimulation). Finally, to allow for comparisons with our previous work, the final strip was exposed to halothane (0.71 ± 0.12 mM in solution measured by gas chromatography) (14) beginning 10 min after ACh stimulation, then was frozen 15 min later. Thus, all strips were frozen at the same time after the beginning of Ca2+ stimulation (35 min).

Materials
The polyclonal affinity-purified rabbit anti-20-kDa rMLC antibody was a generous gift of Dr. Susan J. Gunst. Ethanol was purchased from AAPER Alcohol, Shelbyville, KY. Adenosine 5'-triphosphate disodium salt was purchased from Research Organics, Cleveland, OH. All other drugs and chemicals were purchased from Sigma Chemical, St. Louis, MO. A23187 was dissolved in dimethyl sulfoxide (0.05% final concentration).

Data are expressed as mean ± SD; n represents the number of dogs. In protocols using permeabilized strips, forces were expressed as a percentage of the maximal force induced by 10 µM Ca2+ determined in each individual strip before the experimental protocol. In the protocol using intact strips, relaxation was expressed as a percent of the initial force, adjusted for the effect of time by using the change in force of the time-matched control strip as described previously (15). Concentration-response curves were parameterized by using nonlinear regression analysis (Sigma Stat; Jandel Scientific, San Rafael, CA), and parameter coefficients compared by using paired t-tests. rMLC phosphorylation measurements were compared by using repeated measures analysis of variance, and the Student-Newman-Keuls test was used for post hoc comparisons. A value of P < 0.05 was considered to be statistically significant.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
Effects of Ethanol in Intact Tracheal Smooth Muscle
Ethanol produced a concentration-dependent relaxation of intact CTSM strips contracted with 0.03 µM ACh ( Fig. 1). However, the relaxation produced by even large concentrations of ethanol was incomplete (maximal relaxation of approximately 40%). At larger concentrations (>300 mM), ethanol produced progressively less relaxation (data not shown). Based on these results, the concentrations of ethanol chosen for further study in the permeabilized preparation ranged up to 300 mM, a value that approximates 50% effective concentration (EC50) values for anesthetic action in other species (6,7).



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  		Figure 1. Effect of ethanol on isometric force in intact canine tracheal smooth muscle precontracted with 0.03 µM acetylcholine. Relaxation is expressed as a percent decrease from initial active force. Values are mean ± SD, n = 6.

 
Effects of Ethanol on Ca2+ Sensitivity in Permeabilized Tracheal Smooth Muscle
In permeabilized strips, 0.3 µM Ca2+ produced stable contractions ( Fig. 2, upper panel). The subsequent addition of 10 µM ACh and 10 µM GTP further increased force, indicating an increase in Ca2+ sensitivity (Fig. 2, lower panel). Ethanol (240 mM) significantly increased force in the permeabilized preparation when applied either in the presence or absence of receptor stimulation ( Figs. 2 and 3), demonstrating that ethanol increased Ca2+ sensitivity.



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  		Figure 2. Representative experiments showing the effect of ethanol on permeabilized canine tracheal smooth muscle. Ethanol (240 mM) increased the force maintained at a given [Ca2+] (i.e., the Ca2+ sensitivity) both in the absence (upper panel) and presence (lower panel) of acetylcholine (ACH) (10 µM) and guanosine 5'-triphosphate (GTP) (10 µM).

 


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  		Figure 3. Force developed in permeabilized canine tracheal smooth muscle during stimulation with 0.3 µM Ca2+ with (ethanol) and without (control) exposure to 240 mM ethanol, both in the absence and presence of acetylcholine (ACH) (10 µM) and guanosine 5'-triphosphate (GTP) (10 µM). All values were measured 35 min after initial stimulation with calcium, are expressed as a percent of the response to 10 µM Ca2+, and are mean ± SD, n = 6. *Significant difference by paired t-test, P < 0.05.

 
To determine whether the increases in Ca2+ sensitivity produced by ethanol could be a result of the increased osmolarity produced by these relatively large concentrations, concentration-response curves to increasing [Ca2+] were obtained in the absence and presence of ethanol (300 mM) and mannitol (300 mM). Ca2+ alone caused a concentration-dependent increase in force. Ethanol (300 mM) caused a leftward and upward shift of the free Ca2+ concentration-response curves ( Fig. 4), producing additional force at a constant submaximal [Ca2+] as compared with the force induced by free Ca2+ alone. The maximal force at large [Ca2+] was significantly increased by ethanol (from 78% ± 11% to 99% ± 9% of the response to initial stimulation with 10 µM Ca2+; P < 0.002). The EC50 for [Ca2+] was significantly decreased by 300 mM ethanol (from 0.62 ± 0.07 µM to 0.42 ± 0.09 µM; P < 0.001). Mannitol did not affect either the maximal force or EC50 (Fig. 4), showing that the effects of ethanol were not caused by increases in osmolarity.



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  		Figure 4. Relationship between force and [Ca2+] in permeabilized canine tracheal smooth muscle in the absence and presence of ethanol (300 mM) or mannitol (300 mM). Values are mean ± SD, n = 9 for ethanol and calcium alone, n = 4 for mannitol. *Significant difference by nonlinear regression, P < 0.05.

 
Neither GDPßS nor tyrphostin A-25 affected Ca2+ sensitization produced by ethanol, suggesting that this effect was mediated neither by G-proteins nor tyrosine kinases sensitive to tyrphostins ( Fig. 5).



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  		Figure 5. Representative experiments showing the effect of guanosine 5'-O-(2-thiodiphosphate) (GDPßS) (1 mM) (A), or tyrphostin A-25 (50 µM) (B), on calcium sensitization induced by ethanol (240 mM). Neither compound affected sensitization, suggesting that the effect of ethanol was mediated neither by G-proteins nor tyrosine kinases. Tracings are representative of three experiments for each compound.

 
Effects of Ethanol on rMLC Phosphorylation
Increasing free [Ca2+] from 1 nM to 0.3 µM significantly increased rMLC phosphorylation above baseline values ( Fig. 6). Ethanol (300 mM) did not change rMLC phosphorylation when added during stimulation with 0.3 µM free Ca2+ alone. Stimulation with 10 µM ACh and 10 µM GTP in 0.3 µM free Ca2+ produced a further increase in rMLC phosphorylation compared with Ca2+ alone. Ethanol produced a concentration-dependent decrease in rMLC phosphorylation when added to strips stimulated with ACh, GTP, and 0.3 µM Ca2+. The decrease produced by 300 mM ethanol was similar to that produced by halothane.



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  		Figure 6. Effect of ethanol on regulatory myosin light chain (rMLC) phosphorylation in permeabilized canine tracheal smooth muscle. Data were obtained before calcium stimulation (baseline), after stimulation with 0.3 µM Ca2+ with (EtOH [300]) or without (Ca2+ alone) 300 mM ethanol, and after stimulation with 0.3 µM Ca2+, 10 µM acetylcholine (ACH), and 10 µM guanosine 5'-triphosphate (GTP) (Ca2+ + ACh) with (EtOH) or without ethanol from 30 to 300 mM. Also shown is the effect of halothane (HAL). Values are mean ± SD, n = 5. *Significant difference, Student-Newman-Keuls test, P < 0.05; NS, not significant. For other comparisons not shown on the figure for clarity, the effect of 300 µM ethanol was significantly different from the effects of both 30 and 100 µM ethanol, and the effect of 300 µM ethanol was not significantly different from the effect of halothane.

 

   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
The major finding of this study in ß-escin-permeabilized CTSM is that ethanol, like volatile anesthetics, inhibits increases in rMLC phosphorylation produced by muscarinic receptor stimulation at constant [Ca2+]i. However, despite this inhibition, the net effect of ethanol is to increase Ca2+ sensitivity (defined as the force maintained for a given [Ca2+]i) by a mechanism that is independent of changes in rMLC phosphorylation.

In smooth muscle, contraction is primarily regulated by the phosphorylation of rMLC (16). rMLC phosphorylation is determined by the balance between the activities of myosin light chain kinase, regulated by [Ca2+]i, and smooth muscle protein phosphatase, which is functionally linked to membrane receptors via a cascade of G-proteins (17–19). G-protein-coupled Ca2+ sensitization involves two different classes of G-proteins, heterotrimeric and small (monomeric), and is produced by an increase in rMLC phosphorylation resulting from inhibition of smooth muscle protein phosphatase (18,19). Previous studies suggest that halothane and other volatile anesthetics can disrupt receptor-mediated signal transduction by interfering with G-proteins in many (but not all) types of tissues (20–25). We have shown that halothane inhibits increases in Ca2+ sensitivity induced by muscarinic receptor stimulation of CTSM primarily by interfering with activation of a heterotrimeric G-protein, possibly by inhibiting its dissociation (5). In permeabilized ASM, halothane thus decreases the force produced at a given free [Ca2+] (i.e., decreases the calcium sensitivity) during muscarinic stimulation. Because G-proteins are not activated in the absence of receptor stimulation in this preparation, halothane has no effect on calcium sensitivity in the absence of receptor stimulation (1,26). Effects on receptor-mediated increases in Ca2+ sensitivity have also been observed with other volatile anesthetics (27) but not with IV anesthetics (28), showing that this effect is not a general property shared by all anesthetic drugs.

Ethanol, like many other alcohols, has anesthetic properties (6,7) and affects G-protein-mediated signal transduction processes after both acute and chronic administration (8–10,29,30). Findings consistent with both activation and suppression of G-protein activity in different tissues have been reported. Several studies have found that the acute application of ethanol inhibits G-protein-coupled responses to muscarinic stimulation (31–33). Aronstam (34) suggested that, like halothane, ethanol might stabilize the heterotrimeric G-proteins and inhibit its activation. We thus speculated that ethanol, at concentrations similar to those reported to be approximately EC50 for anesthesia in other species (approximately 200 mM) (6,7), might also inhibit G-protein-mediated increases in Ca2+ sensitivity in the airway. Consistent with this hypothesis, we found that ethanol inhibited increases in rMLC phosphorylation produced by muscarinic receptor stimulation during conditions of constant [Ca2+]. This effect alone would tend to reduce force (i.e., reduce the Ca2+ sensitivity) during muscarinic receptor stimulation. However, ethanol actually increased force under conditions of constant free [Ca2+] in the permeabilized preparation, both in the absence and presence of muscarinic stimulation. In the absence of receptor stimulation, this increase in force was not associated with an increase in rMLC phosphorylation. Thus, although ethanol inhibits receptor-coupled mechanisms regulating rMLC phosphorylation, ethanol also increases Ca2+ sensitivity by mechanisms independent of changes in rMLC phosphorylation. The net effect of these two opposing factors is an increase in Ca2+ sensitivity.

In the only previous study of the effect of ethanol on Ca2+ sensitivity in any type of smooth muscle, Kuroiwa et al. (35) found that ethanol increased Ca2+ sensitivity in an {alpha}-toxin-permeabilized preparation of porcine coronary artery. This Ca2+ sensitization required GTP and was inhibited by GDPßS (which inhibits G-protein function), suggesting that it was produced by activation of G-proteins. They did not examine effects of ethanol on agonist-induced increases in Ca2+ sensitivity. In our study, the increase in Ca2+ sensitivity produced by ethanol was significant in the absence of GTP. In additional studies, GDPßS did not affect ethanol-induced sensitization. Thus, unlike the results in coronary artery, we find no evidence that ethanol activates G-proteins in CTSM. The differing permeabilization procedures in the two studies are not responsible for the discrepancy, because we have also observed Ca2+ sensitization by ethanol that is not inhibited by GDPßS in {alpha}-toxin permeabilized CTSM (data not shown). Elucidation of the mechanism responsible for this sensitization requires further study, but it does not appear to be related to changes in osmolarity produced by the relatively large concentrations of ethanol that were examined (Fig. 5). It also does not appear to involve tyrosine kinases sensitive to tyrphostins, which have been implicated in the regulation of proteins such as caldesmon that might regulate force independently of changes in rMLC phosphorylation in smooth muscle (36–38). This action of ethanol on ASM is not unique; we have found that other compounds such as phorbol esters can also increase Ca2+ sensitivity in ASM without increasing rMLC phosphorylation through a mechanism that does not involve G-proteins or protein kinase C (13).

Although it was not the focus of our study, we found for the first time that ethanol relaxes isolated ASM. This result is consistent with findings in several other types of smooth muscle (39–43), although there is a report of ethanol-induced contraction in gut smooth muscle (38). We assume that ethanol-induced relaxation of intact CTSM stimulated with ACh is produced by decreases in [Ca2+]i. Kuroiwa et al. (35) found that large concentrations of ethanol (>300 mM) produced an increase in force and [Ca2+]i when applied to porcine coronary arteries with intact endothelium, and that these effects were modulated by endothelial removal. However, other studies suggest that ethanol in smaller concentrations decreases [Ca2+]i in vascular smooth muscle (39,44), perhaps by inhibiting the influx of extracellular Ca2+ (39). Further studies are needed to determine the effect of ethanol on [Ca2+]i in the tissue used in our study, which presumably explains its ability to relax intact CTSM despite concurrent increases in Ca2+ sensitivity.

The clinical importance of these findings is limited. Although these concentrations of ethanol are similar to EC50 values for anesthesia measured in animals [approximately 200 mM (6,7)], the concentrations of ethanol used in our study approach toxic levels in humans (an intoxicating blood concentration of ethanol [0.1%] corresponds to approximately 25 mM). However, there is some evidence that inhaled ethanol, which may produce locally larger concentrations in the airways, may reduce bronchial responsiveness (45). Ethanol aerosol has in fact been used as a bronchodilator and antifoaming agent in the treatment of pulmonary edema, with variable results (46). Our findings suggest that any clinical bronchodilating properties of ethanol would be limited by increases in calcium sensitivity.

In conclusion, ethanol, like volatile anesthetics, inhibits increases in rMLC phosphorylation in ASM produced by muscarinic receptor stimulation during conditions of constant [Ca2+]i. This provides another example of a compound with anesthetic properties that interferes with G-protein coupled signaling processes, and demonstrates for the first time that these effects are not limited to volatile anesthetics. However, despite the inhibition of this signaling pathway, the net effect of ethanol is to increase Ca2+ sensitivity by an unknown mechanism that is independent of G-proteins and changes in rMLC phosphorylation.


   Acknowledgments
 
This study was supported in part by Grants HL-45532 and HL-54757 from the National Institutes of Health, Bethesda, MD, and grants from the Mayo Foundation, Rochester, MN. MH was sponsored by Dr. Masahisa Hirakawa, Department of Anesthesiology and Resuscitology, Okayama University Medical School, Okayama, Japan.

The authors thank Susan J. Gunst, PhD, Departments of Physiology and Biophysics, Indiana University School of Medicine, Indianapolis, IN, for generously providing us with polyclonal affinity-purified rabbit anti-20 kDa regulatory myosin light chain antibody, and Kathy Street and Darrell Loeffler for expert technical assistance.


   Footnotes
 
Presented in part at the American Society of Anesthesiologists annual meeting, October 9–13, 1999, Dallas, Texas.

MH is currently affiliated with the Department of Anesthesiology and Resuscitology, Okayama University Medical School, Okayama, Japan.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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Accepted for publication November 8, 2000.




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M. J. Rebecchi and S. N. Pentyala
Anaesthetic actions on other targets:protein kinase C and guanine nucleotide-binding proteins
Br. J. Anaesth., July 1, 2002; 89(1): 62 - 78.
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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