Department of Anesthesiology, School of Medicine, University of California, Los Angeles, California 90095-7115
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ABSTRACT |
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Volatile anesthetics modulate the function of various K+ channels. We previously reported that isoflurane induces an increase in macroscopic currents and a slowing down of current deactivation of Shaker H4 IR K+ channels. To understand the single-channel basis of these effects, we performed nonstationary noise analysis of macroscopic currents and analysis of single channels in patches from Xenopus oocytes expressing Shaker H4 IR. Isoflurane (1.2% and 2.5%) induced concentration-dependent, partially reversible increases in macroscopic currents and in the time course of tail currents. Noise analysis of currents (70 mV) revealed an increase in unitary current (~17%) and maximum open probability (~20%). Single-channel conductance was larger (~20%), and opening events were more stable, in isoflurane. Tail-current slow time constants increased by 41% and 136% in 1.2% and 2.5% isoflurane, respectively. Our results show that, in a manner consistent with stabilization of the open state, isoflurane increased the macroscopic conductance of Shaker H4 IR K+ channels by increasing the single-channel conductance and the open probability.
general anesthetics; volatile anesthetics; voltage-gated channels; mechanisms of action; single channels
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INTRODUCTION |
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GENERAL ANESTHETICS act on the central nervous system (CNS) to produce a reversible state of unconsciousness, amnesia, and analgesia. The molecular mechanism(s) of action are a matter of ongoing research, and, because of the multiplicity of systems and molecules targeted by anesthetic agents, the mechanisms are bound to be varied and complex. In terms of ion channels, it is well established that the CNS depressive action of volatile anesthetics entails modulation of neuronal activity involving GABAA-, nicotinic ACh (nACh)-, and glycine-mediated synaptic responses (1, 6, 9). Voltage-gated ion channels, on the other hand, are generally not considered to be highly sensitive sites for the action of volatile general anesthetics (10); nonetheless, it is recognized that these agents, at clinically relevant concentrations, alter signal transmission dependent on functional voltage-gated channels. Small perturbations in the function of voltage-gated channels affect patterns of neuronal firing and excitability; consequently, effects of general anesthetics on specific ion channels may, in fact, be critical to the establishment of general anesthesia.
Of the voltage-gated channels, K+ channels play an important role in neuronal excitability as they set the resting potential, keep fast action potentials short, terminate periods of intense activity, slow the rate of repetitive firing, and generally lower the excitability of the cell when open. Accumulating evidence indicates that some CNS anesthetic actions can be related to effects on K+ channels and that various anesthetic agents can affect different kinds of native and recombinant K+ channels (3, 4, 25, 28). The modulation of K+ channels by volatile anesthetics is diverse in nature, suggesting different molecular mechanisms of action. Whereas some K+ channels are unaffected by certain volatile anesthetics or alcohols (5, 20), in others some relatively specific effects have been described. Volatile anesthetics have been found to activate baseline K+ channels in rat cerebellar granule neurons (29) and rat motor neurons (25), cloned mammalian two-pore-domain K+ channels (21), and isoflurane in rat hippocampal and human neocortical neurons (2) or to block dShaw2 voltage-gated K+ channels (15). Differential effects of halothane, isoflurane, chloroform, and enflurane on voltage-gated and resting K+ channels have also been described (3, 21). In addition, cloning and mutational analysis of K+ channels have helped identify specific sites or regions (15, 21) in the channel proteins that are part of the effector sites of the anesthetic(s) or that at least participate in developing their effects, lending support to specific anesthetic-protein interactions.
Here we have used the H4 IR NH2-terminal mutant of the Shaker B K+ channel as a model to investigate the effect of the volatile anesthetic isoflurane on a specific class of K+ channels. In a previous study, Correa (3) showed that H4 IR Shaker K+ channels exhibit a differential response to three general anesthetics: halothane, chloroform, and isoflurane. In contrast to the depression of the macroscopic conductance (GK) produced by halothane and chloroform, isoflurane induced an increase in GK and a slowing down of current deactivation. It was concluded that isoflurane alters the macroscopic properties of the H4 IR channel by stabilization of the open state of the channel. To further investigate the molecular basis of the action of isoflurane, we have used the patch-clamp technique (14) to study the effect of this anesthetic agent on the single-channel properties of the Shaker H4 IR K+ channels expressed in Xenopus oocytes. We report here the results obtained under identical experimental conditions by using mean-variance analysis of a population of channels and performing single-channel studies. We have found that the increase in macroscopic current induced by isoflurane results from an increase in single-channel conductance and in the open probability, both of which are consistent with an overall higher GK and in line with the proposed isoflurane-induced stabilization of the open state of the channel (3).
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MATERIALS AND METHODS |
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Materials. Frogs (Xenopus laevis) were purchased from either Xenopus I or Xenopus Express. Isoflurane (Forane) was purchased from Ohmeda Carbide. All other chemicals were from Sigma.
K+ channel clones and cRNA
transcription.
The cDNA for the noninactivating mutant of the Drosophila
Shaker B K+ channel (N6-46) (17)
used in this study was modified for maximal expression: the ZH4 IR
pBSTA (26) was a generous gift of Drs. D. Starace and F. Bezanilla. The cDNA was transcribed with the commercial T7 mMessage
mMachine kit (Ambion). The integrity of the transcribed RNA was
assessed in agarose gels, and the concentrations were measured spectrophotometrically.
Oocytes. X. laevis oocytes were isolated from frog ovaries by following conventional protocols (13). Oocytes were defolliculated by collagenase treatment (collagenase type AI; GIBCO BRL) in Ca2+-free Ringer solution, followed by a progressive change to the normal frog Ringer solution containing 1.8 mM Ca2+. Defolliculated oocytes were kept in Ringer solution (standard oocyte solution) consisting of 100 mM NaCl, 2 mM KCl, 1 mM MgCl2, 1.8 mM CaCl2, 5 mM HEPES-Na, and 50 µg/ml gentamicin, pH 7.2. RNA samples (2-40 ng/µl) were injected into the oocytes within 24 h of isolation. The injected oocytes were used for study from 1-5 days after injection. Animal procedures were approved by the Animal Research Council at the University of California, Los Angeles (UCLA).
Recording solutions. Macroscopic and single-channel currents were recorded with 5 KMS [5 mM potassium methane sulfonate (KMS), 115 mM sodium methane sulfonate (NaMS), 1.8 mM CaCl2, and 10 mM HEPES-Na, pH 7.2] in the pipette and with 100 KMS (100 mM KMS, 10 mM HEPES-K, and 1 mM EGTA-K, pH 7.2) in the bath. The pipette solution in tail current experiments was 20 KMS (20 mM KMS, 60 mM NaMS, 40 mM N-methyl-D-glucamine methane sulfonate, 10 mM HEPES, and 1.8 mM CaCl2, pH 7.2). Anesthetic solutions were prepared by equilibrating the bath solutions for 30 min with isoflurane-containing air (Ohio clinical vaporizer) in an Equilibrator (R. S. Weber and Associates, Westlake Village, CA) to final concentrations of 1.2 vol% (0.4 ± 0.1 mM) and 2.5 vol% (0.8 ± 0.3 mM), approximately equivalent to 1 and 2 MAC (minimum alveolar concentration), respectively. Equilibrated solutions were collected into gas-tight syringes and delivered through Teflon tubing by a gravity-fed perfusion system. Perfusion with the bath solution alone had no effect on channel currents. Anesthetic concentrations were measured by head space sampling with a gas chromatograph (Hewlett-Packard HP 6890 with head space sampler HP 7694) from an aliquot of the anesthetic-containing solution withdrawn directly from the equilibration chamber.
Electrophysiology.
Ionic currents were recorded with the patch-clamp technique using an
Axopatch 200A amplifier (Axon Instruments) controlled by a custom-built
acquisition system. Data were acquired by sampling at 100-200 kHz.
Macroscopic and single-channel currents were recorded in cell-attached
and inside-out patches. Patch electrodes were pulled (model P-97
micropipette puller, Sutter Instruments) from borosilicate capillaries
(Warner Instrument). The shanks were coated with Sylgard (Dow Corning),
and the tips were heat-polished to tip resistances of 1.5-4.0 M
for macroscopic current recording and 15-60 M
for
single-channel current recording.
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Action potential simulations. Simulations were performed with the use of the program Nerve (http://pb010.anes.ucla.edu), written by Dr. Francisco Bezanilla (UCLA), used in the membrane action potential and propagated action potential modes. The program solves sets of differential equations developed by Hodgkin and Huxley from data obtained experimentally from the squid giant axon. The equations include the voltage dependence of nerve Na+ (fast inactivating) and K+ (delayed rectifier) conductances as described by Hodgkin and Huxley (16). Individual action potentials were elicited by brief stimuli: 0.25-ms, 10-µA current pulses. When paired stimuli were applied, channels were allowed to recover before the second stimulus of equal length and amplitude was applied. Trains of impulses were elicited by a sustained depolarizing current, large enough to initiate continuous spontaneous activity: 2 µA, with default GNa, or 5 µA, with reduced GNa. Changes in GK and GNa were introduced as percent changes in the conductances relative to the default values: 1%, 2%, 3%, 5%, 10%, 15%, 20%, and 30% increases in GK and 10% and 20% decrease in GNa. Except for the temperature, which was either the default value (6.3°C) or higher (10 and 15°C), all other variables and parameters were maintained at default values.
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RESULTS |
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Macroscopic currents.
Isoflurane, at 1.2% and 2.5%, induced an increase in macroscopic
K+ currents measured in patches from oocytes injected with
the cRNA for the Shaker H4 IR K+ channel. The
increase was specific to the expressed channels because it was not
observed in uninjected oocytes or in silent patches of injected
oocytes, and it was specific to isoflurane treatment because simple
exchange of the bath solution did not produce the effect. Figures 1 and
2 illustrate the effect of isoflurane on currents elicited by
depolarizing pulses from a holding potential of 90 mV. In external 5 KMS (Fig. 1), the noninactivating
K+ currents are outward throughout the voltage range tested
(from
80 to 80 mV). The macroscopic current-voltage (I-V)
plot in Fig. 1 is the average of six experiments performed before,
during, and after treatment with 2.5% isoflurane. Treatment with
isoflurane induced an increase in current amplitude for all test
potentials positive to
40 mV; the effects, however, were greater and
significant only at 45 mV and above, a range at which the control
currents show evidence of reduced conductance. Negative to 45 mV, the
average change (13 ± 0.3%, n = 6) was constant
between
5 and 45 mV. The current magnitudes measured at the end of
6-ms pulses to 70 mV varied between 0.6 and 1.3 nA (n = 6) in the controls. Isoflurane (1.2% and 2.5%) significantly
increased macroscopic current amplitudes, averaging 12% (n = 5, not shown) and 30% (n = 6, Table
1), respectively. The effect on the
currents is shown throughout the pulse, and the increase was
partially reversed by wash (Figs. 1 and 2).
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Nonstationary noise analysis of macroscopic currents.
Noise analysis is a powerful tool for use in investigating
single-channel properties. It yields reliable quantitative estimates of
single-channel parameters (24). For a homogeneous
population of statistically independent channels, the mean current,
I(t), and the current variance,
I(t)2, are given by
the following equations: I(t) = N · I · Po(t)
and
I(t)2 = N · i2 · Po(t)[1
Po(t)]. Provided that
I and
2 are estimated from various
Po, i and N can be
determined by fitting mean-variance data according to
2 = i · I
I2/N. Figure
3 provides an example of the
nonstationary macroscopic current mean-variance analysis performed to
investigate the influence of isoflurane on single-channel properties.
In this experiment, 2.5% isoflurane significantly increased the
single-channel current from 1.09 pA to 1.35 pA. The limiting maximum
open probabilities, obtained from the equation I = N · i · Po, were
also increased by 23% (0.69 vs. 0.85, patch 6). The results
from six such experiments are given in Table 1.
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Single-channel recording.
The records shown in Fig. 4,
top, are selected records that illustrate typical unitary
Shaker H4 IR K+ currents in a patch with two
channels during a test pulse to 70 mV. Visual inspection of the traces
reveals that opening events in 2.5% isoflurane (Fig. 4, top
right) were characterized by more stable openings, i.e., less
flicker, than in control conditions (top left). At this
potential, the amplitudes of control unitary currents averaged 1.4 pA
(n = 3 patches, average of 100-150 events each)
with 5 mM K+ in the pipette (Table
4). The unitary event sizes of these
three patches averaged 1.7 pA after perfusion with 2.5% isoflurane
(23% higher than control; Table 4). A similar result was obtained in
patches exposed to 20 mM K+ (Table 4); the average
single-channel current increased from 1.2 pA to 1.4 pA
(n = 4 patches; 100-150 events per patch), a 20%
increase.
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DISCUSSION |
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We have used the noninactivating mutant of the Shaker B K+ channel, the H4 IR, as a model to study anesthetic-induced changes in single-channel properties of a voltage-gated K+ channel. In a previous study, Correa (3) reported that isoflurane induced a significant increase in GK and a slowing down of current deactivation and proposed that the effect could be attributed to stabilization of the open state. To provide the single-channel basis for this phenomenon, we have used the patch-clamp technique to study macroscopic and single-channel currents under identical conditions.
The macroscopic currents recorded from patches containing several hundred to a few thousand channels had the same characteristics and the same response to isoflurane as the currents recorded with the cut-open oocyte voltage-clamp technique from a significantly larger area of the oocyte surface (3) and under different experimental conditions. In isoflurane, macroscopic currents were larger at all potentials at which channels open. In addition, the magnitude and the time course of the currents upon repolarization were enhanced. The effects were only partially reversible upon wash. Lack of full reversibility may result from partitioning of the anesthetic into the yolk of the oocyte, thus contributing a constant supply even after prolonged wash; this would be more critical when recording in the cell-attached mode. Limited reversibility may also arise from lack of direct access to the membrane patch and/or the pipette solution; anesthetic would thus remain in small amounts in the vicinity of the patch even after intensive exchange of bath solution.
We found that the difference in maximum current amplitude during test depolarizations after isoflurane treatment was only significant at potentials positive to 45 mV in external 5 KMS. At these potentials, control macroscopic currents show signs of rectification, which has been attributed to entry into a nonconducting state accessed from the open state (17, 22). The fact that the effect of isoflurane is more prominent at very positive potentials suggests that one action of the anesthetic could be the relief of such rectification by reducing the rate at which channels enter this nonconducting state. This effect would stabilize the open state of the channel by eliminating or making less frequent that exit route. An immediate prediction from this hypothesis is that the mean open time of the channel would be longer in the presence of the anesthetic. The single-channel records shown in Fig. 4 hinted at an increased dwell time in the open state at 70 mV in isoflurane. Preliminary analysis of single-channel records indicates that, in fact, the mean open time at 70 mV is ~30% longer in 2.5% isoflurane (1.17 ± 0.02 ms, 4,809 events) relative to the control (0.87 ± 0.01 ms, 5,580 events). Also, the mean close time is close to 9% shorter in isoflurane (0.34 ± 0.01 ms, 5,684 events in the control vs. 0.31 ± 0.01 ms, 5,051 events after isoflurane).
The effect of isoflurane appeared more prominent during repolarization, associated with channel closing, than during the depolarizing test pulses. Tail-current kinetics were slower in isoflurane, producing appreciable currents upon return to holding. The average effect in terms of the amplitude of the tail currents, however, was the same as that during the test pulse (27% vs. 31%, 30-100 mV), indicating that the change was proportional to the voltage and involved an increase in the macroscopic conductance. Because slower deactivation contributes significant current density during repolarization, this may be the most relevant physiological effect of the anesthetic, which could contribute to changes in firing patterns (see below).
An increase in macroscopic conductance could result from an increase in
any of the following single-channel parameters: the single-channel
conductance, o; the number of channels in a patch, N; or the probability of opening, Po.
The results presented in this paper, which were obtained with two
independent techniques, support the idea that isoflurane induces an
increase in the Po and
o of the
channels already present in the membrane. Mean-variance analysis
indicated that isoflurane induces an increase in the single-channel
current amplitude. The value of i was consistently higher in
isoflurane compared with control. Because the mean-variance analysis
was done at 70 mV, a potential at which, as mentioned above, the
increase in K+ currents could involve a reduction of the
number of channels in the nonconducting state, the changes in
K+ conductance would not necessarily be observed at more
negative potentials. Evidence that the effect occurs at other
potentials as well was provided by the observed increase in single
currents of open-channel events at various potentials. Single-channel
I-V plots demonstrated that i was larger for all
potentials tested and that
o was increased by the
anesthetic. If, in fact, isoflurane increases the conductance of the
single channel, this finding would be rather unique for a voltage-gated
channel. It is likely, however, that the increase in
o
is apparent, a consequence of better resolved events, in view of the
fact that channels flickered less in the presence of isoflurane (Fig.
4) relative to control conditions.
The second line of evidence that the effect of isoflurane takes place
throughout the voltage range tested is provided by the tail-current
experiments. The curve shown in Fig. 2D gives evidence that
the conductance of the channels was affected for all potentials at
which channels open. Moreover, maximum currents during the test pulses
and peak tail currents upon repolarization were modified by the
anesthetic to the same extent (~30% increase) in a
voltage-independent manner for potentials more positive than 30 mV.
Conversely, in external 5 KMS, the increase in macroscopic currents was
smaller (13%) and was voltage independent only between 5 and 45 mV.
It is tempting to hypothesize that the external [K+] can
influence anesthetic interaction with the channel.
K+-dependent K+ current inhibition by the
intravenous general anesthetics has been reported in a study done on
sympathetic nervous system-derived human neuroblastoma cells (SH-SY5Y)
(11). Increases in the extracellular [K+]
promoted delayed rectifier K+ current block by etomidate
and propofol (but not ketamine), suggesting an active role of
K+ on the extent of anesthetic effect. The results of
mean-variance analysis reported here in Tables 1, 3, and 4, indicate
that this is unlikely, because the change in i and
Po induced by isoflurane was the same
irrespective of the [K+]. Also, although
o
was slightly larger in higher external K+, the
anesthetic-induced increase in
o was independent of
[K+]. Further experimentation is required to resolve this
apparent discrepancy.
The value of i obtained through noise analysis (Tables 1 and
3) was smaller than the unitary amplitude at 70 mV measured from
single-channel events (Table 4) even in the controls. Although not
significant, this difference could result from the estimation of
i, which is influenced by the open channel noise, normally giving an underestimate of the actual single-channel current
(8). Because the size of an opening can be better resolved
from the individual events, the magnitude of single-channel currents
and the o are more reliably determined from analysis of
single channels. The values of
o reported here
are consistent with those previously reported for this mutant
(17).
The time course of tail currents was dramatically slower in isoflurane than in control conditions irrespective of the potential visited during the pulse. From the single-channel perspective, this means that isoflurane-treated channels take longer to go from the Po attained during the depolarizing pulse to that at the holding potential. Subsequent analysis of single-channel kinetics (in progress) should provide further insight to the steps, states, and rates that are modulated by the anesthetic.
Although the Shaker B H4 IR K+ channel is a
model channel used to explore molecular mechanisms of action of
anesthetics, the findings reported in this paper have implications for
anesthesia because of the prominent role K+ channels play
in setting the resting membrane potential and the rate of
repolarization as well as determining, along with other voltage-gated
channels, the overall excitability of neuronal tissue. Small changes in
the activity of K+ channels can induce significant changes
in the behavior of excitable cells. The concentrations of anesthetics
used in this study, 1.2 and 2.5 vol%, are close to 1 and 2 MAC for
isoflurane. In both concentrations we found an increase in open channel
probability and a retardation of current decay back to baseline upon
repolarization. Although clinically relevant concentrations of
isoflurane are normally close to 1 MAC, the effects described here at
concentrations 1 MAC are likely to occur during general anesthesia.
Volatile general anesthetics inhibit spontaneous and evoked neuronal activity in various parts of the CNS. It is well established that the molecular mechanisms of the depressive action of volatile anesthetics comprise the modulation of neuronal activity involving GABAA-, nACh-, and glycine-mediated synaptic responses and the modulation of several kinds of voltage-gated ionic channels (10). A number of publications have already indicated that volatile anesthetics modulate different kinds of native or recombinant potassium channels (12, 18, 19, 21, 27, 28, 30, 31). Our results indicate that isoflurane affects certain K+ channels during depolarization and repolarization. To evaluate the possible contribution of such changes at the cellular level, we performed simple action potential simulations with the program Nerve, which is based on the Hodgkin-Huxley model for action potential generation and propagation derived from the activity of squid nerve fast-inactivating Na+ channels and delayed rectifier K+ channels. Because the Shaker IR K+ channel has characteristics of a delayed rectifier K+ channel, we used the simulations to explore the influence of increases in GK, like those obtained in the present work, on the generation and propagation of action potentials. Increases in GK (from 1% to 30%) produced delays and amplitude reduction in successive action potentials triggered by paired stimuli or by maintained depolarizing currents. In fact, early termination of a train of impulses occurs with GK increases of 5% under sustained depolarization, producing effective filtering of action potential trains. Furthermore, the influence of increases in GK was more pronounced when a reduction of GNa, similar to that reported for isoflurane-treated mammalian Na+ channels (7), was included in the simulation. One could hypothesize that, combined, these effects could produce a significant alteration of nerve conduction and firing frequency. However interesting, this hypothesis remains to be tested in vitro.
Identification of general anesthetic binding sites on K+ channels is a matter of active research. Experiments involving mutational analysis on anesthetic-sensitive and -insensitive K+ channels have shed light on segments of the channel proteins that are required for anesthetic-channel interactions. Recent work on molecular determinants of 1-alkanol action on the dShaw2 (15) determined that the structural integrity of a 13-amino acid region in the cytoplasmic loop between membrane segments S4 and S5 is necessary for 1-alkanol action. Likewise, activation by volatile anesthetics of the human TREK-1 channel (21) depends on the COOH-terminal region of the channel. In view of the multiplicity of action of general anesthetics on various ion channels and the differential sensitivity to general anesthetics even within classes of channels, the molecular mechanisms of action are bound to be complex. Despite this complexity, the evidence clearly points toward direct anesthetic-protein interactions at specific sites, and although bilayer-mediated steps and modulation via other membrane-bound proteins may still play a crucial role, a solely bilayer-based anesthetic action is unlikely.
In conclusion, our results show that the volatile anesthetic isoflurane induces a reproducible increase of the macroscopic conductance of Shaker H4 IR K+ channels. Isoflurane modulates these K+ channels by effects on the single-channel properties. The increase in macroscopic conductance induced by isoflurane is associated with an increase in the single-channel conductance and the opening probability, which supports stabilization of the open state of the channel. The relevance of our findings at the cellular level needs yet to be addressed, but together with other data on volatile anesthetic action on voltage-gated channels, these results suggest that volatile general anesthetics may alter neuronal excitability during anesthesia by mechanisms that involve the fine modulation of the activity of voltage-gated ion channels.
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ACKNOWLEDGEMENTS |
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We thank Drs. D. Starace and F. Bezanilla for the cDNA of the ZH4 IR mutant of the Shaker B K+ channel, E. Grigorova for oocyte isolation, N. Shafaee for technical assistance, and Drs. D. Sigg and F. Bezanilla for guidance and helpful discussion.
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FOOTNOTES |
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This project was supported by National Institute of General Medical Sciences Grant GM-53781 (to A. M. Correa) and the Department of Anesthesiology, University of California, Los Angeles (UCLA).
Preliminary reports of these data have been published in abstract form (19) and presented in poster form at the Susumu Hagiwara Memorial Symposium, UCLA, 1999.
Address for reprint requests and other correspondence: A. M. Correa, Dept. of Anesthesiology, UCLA, BH-509A CHS, PO Box 951775, Los Angeles, CA 90095-7115 (E-mail: nani{at}ucla.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 19 April 2000; accepted in final form 22 November 2000.
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