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Anesthesiology 2010; 112:1452– 63
Copyright © 2010, the American Society of Anesthesiologists, Inc. Lippincott Williams & Wilkins
Pungent General Anesthetics Activate Transient
Receptor Potential-A1 to Produce Hyperalgesia and
Neurogenic Bronchoconstriction
Helge Eilers, M.D.,* Fiore Cattaruzza, Ph.D.,† Romina Nassini, Ph.D.,§ Serena Materazzi, Ph.D.,§
Eunice Andre, Ph.D.,储 Catherine Chu, B.A.,# Graeme S. Cottrell, Ph.D.,†
Mark Schumacher, Ph.D., M.D.,* Pierangelo Geppetti, M.D.,** Nigel W. Bunnett, Ph.D.††
ABSTRACT
Background: Volatile anesthetics such as isoflurane and halothane have been in clinical use for many years and represent the
group of drugs most commonly used to maintain general anesthesia. However, despite their widespread use, the molecular
mechanisms by which these drugs exert their effects are not
completely understood. Recently, a seemingly paradoxical effect
of general anesthetics has been identified: the activation of peripheral nociceptors by irritant anesthetics. This mechanism
may explain the hyperalgesic actions of inhaled anesthetics and
their adverse effects in the airways.
Methods: To test the hypothesis that irritant inhaled anesthetics activate the excitatory ion-channel transient receptor potential (TRP)-A1 and thereby contribute to hyperalgesia and irritant airway effects, we used the measurement of intracellular
calcium concentration in isolated cells in culture. For our func* Associate Professor, # Assistant Researcher, Department of Anesthesia, † Assistant Researcher, Department of Surgery, †† Professor, Departments of Surgery and Physiology, University of California, San Francisco, San Francisco, California. § Assistant Researcher,
Department of Preclinical and Clinical Pharmacology, University of
Florence, Florence, Italy. 储 Assistant Researcher, Center of Excellence for the Study of Inflammation, University of Ferrara, Ferrara,
Italy. ** Professor, Center of Excellence for the Study of Inflammation, University of Ferrara, and Department of Preclinical and Clinical Pharmacology, University of Florence.
Received from Department of Anesthesia and Perioperative Care,
University of California, San Francisco, San Francisco, California. Submitted for publication October 13, 2009. Accepted for publication
January 19, 2010. Supported by Hellman Family Foundation Fellowship, San Francisco, California (to Dr. Eilers), and by DK57840,
DK39957, DK43207, DK46285, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda,
Maryland (to Dr. Bunnett). Presented in part at the Annual Meeting of
the American Society of Anesthesiologists, San Francisco, California,
October 13, 2007, and at the Annual Meetings of the International
Anesthesia Research Society, San Francisco, California, March 19, 2008,
and International Association for the Study of Pain, World Congress on
Pain, Glasgow, United Kingdom, August 20, 2008.
Address correspondence to Dr. Eilers: Department of Anesthesia
and Perioperative Care, University of California, San Francisco, 513
Parnassus Avenue, Room S-436, Box 0427, San Francisco, California
94143-0427. [email protected]. This article may be accessed for personal use at no charge through the Journal Web site,
www.anesthesiology.org.
1452
tional experiments, we used models of isolated guinea pig bronchi to measure bronchoconstriction and withdrawal threshold
to mechanical stimulation with von Frey filaments in mice.
Results: Irritant inhaled anesthetics activate TRPA1 expressed in human embryonic kidney cells and in nociceptive
neurons. Isoflurane induces mechanical hyperalgesia in mice
by a TRPA1-dependent mechanism. Isoflurane also induces
TRPA1-dependent constriction of isolated bronchi. Nonirritant anesthetics do not activate TRPA1 and fail to produce
hyperalgesia and bronchial constriction.
Conclusions: General anesthetics induce a reversible loss of
consciousness and render the patient unresponsive to painful
stimuli. However, they also produce excitatory effects such as
airway irritation and they contribute to postoperative pain.
Activation of TRPA1 may contribute to these adverse effects,
a hypothesis that remains to be tested in the clinical setting.
What We Already Know about This Topic
❖ Volatile anesthetics produce irritant airway effects and can, in
low concentrations, increase pain perception
❖ Transient receptor potential (TRP) channels are found in sensory
neurons and airway cells and could be activated by volatile
agents to cause these effects
What This Article Tells Us That Is New
❖ Using cells in cultures, bronchial rings in vitro, and behavior
tests in rats, general anesthetics activate TRPA1 channels
and cause hypersensitivity and bronchial constriction
A
NNUALLY, there are approximately 21 million general
anesthesia cases in the United States,1 most of which involve administration of volatile anesthetics for maintenance of
anesthesia. Although general anesthetics have different effect
䉫 This article is featured in “This Month in Anesthesiology.”
Please see this issue of ANESTHESIOLOGY, page 9A.
䉬 This article is accompanied by an Editorial View. Please
see: Gallos G, Flood P: Wasabi and a volatile anesthetic.
ANESTHESIOLOGY 2010; 112:1309 –10.
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profiles, they induce a reversible loss of consciousness and render
the patient unresponsive to painful stimuli. Traditionally, the
lack of withdrawal to skin incision has been used to compare
different drugs with respect to their anesthetic potency and to
test new experimental agents for their anesthetic potential. This
latter property is also used in clinical practice as a measure to
estimate the depth of anesthesia.2
Nevertheless, despite the lack of response to surgical stimulation, it is unclear whether volatile anesthetics induce analgesia by suppressing the activity of nociceptive neurons.
Paradoxically, low concentrations of volatile anesthetics
cause hyperalgesia,3– 6 which suggests excitation rather than
inhibition of nociceptive neurons. The mechanisms by
which anesthetics affect the activity of nociceptive neurons
are not fully understood.
Volatile anesthetics have distinct odors, and some are
irritating to the airway. They produce a pungent sensation
when inhaled and induce a strong cough reflex that can
precipitate laryngospasm, a potentially life-threatening
complication during induction and emergence from anesthesia. Although there is not always agreement with respect to the ranking of the four commonly used volatile
anesthetics according to their “irritating potential,” from
clinical observation, it is clear that isoflurane and desflurane are irritant and halothane and sevoflurane are nonirritant anesthetics.7 The molecular mechanisms for these
irritant effects are not understood, but the nature of the
clinical responses suggests that they would involve activation of a chemosensor in the airway.
Transient receptor potential (TRP) ion channels are
important receptors in sensory transduction. In particular, two members of the TRP family of ion channels,
TRPV1 and TRPA1, are sensors for noxious chemical
stimuli and are widely expressed in peripheral nociceptors.8 TRPA1, which is coexpressed with TRPV1, has
emerged as a sensor for a wide variety of irritant chemicals,
including the pungent ingredients of various plants, such
as wasabi, mustard, and garlic.9 –11 Environmental irritants, including acrolein and cigarette smoke, also activate
TRPA1.12 Given its role as a chemosensor for a wide array
of irritants and its expression on peripheral nociceptors,
TRPA1 presents a strong candidate for mediating the irritant effects of volatile anesthetics. In fact, in a recent
study, Matta et al.13 provided the first evidence for the
activation of TRPA1 by irritant anesthetics. Moreover,
TRPA1 activation on sensory nerves stimulates the release
of substance P and calcitonin gene-related peptide, mediators of neurogenic inflammation and inflammatory
pain.14
In this study, we test the hypothesis that pungent volatile anesthetics produce their irritant effects, such as
bronchoconstriction of the airway, and hyperalgesia
through activation of TRPA1 ion channels on peripheral
nociceptors.
Eilers et al.
Materials and Methods
Reagents
Isoflurane and desflurane were from Baxter Healthcare Corp.
(Deerfield, IL), sevoflurane from Abbott Laboratories (North
Chicago, IL) and halothane from Halocarbon Laboratories
(River Edge, NJ). The TRPA1 antagonist HC-030031 was purchased from Tocris Bioscience (Ellisville, MO). The neurokinin
receptor antagonists SR140333, SR48968, and SR142801
were gifts of X. Emonds-Alt (Sanofi-Aventis, France). All other
reagents were from Sigma-Aldrich (St. Louis, MO) unless noted
otherwise.
Animals
All experiments were carried out in accordance with the National Institutes of Health guidelines regarding the use of animals in experimental procedures. All protocols were reviewed
and approved by the Institutional Animal Care and Use Committees of the University of California, San Francisco, California, and the University of Florence, Florence, Italy. DunkinHartley guinea pig (250 g) and mice (male, 25 g) were housed in
a temperature- and humidity-controlled vivarium (12-h dark/
light cycle, with free access to food and water) for at least 5 days
before the experiments. TRPA1 heterozygote male mice had
been backcrossed with C57BL/6 female mice to obtain the
C57BL/6 genetic background. trpa1⫹/⫹ and trpa1⫺/⫺ mice
used in the current experiments were derived from breeding
heterozygous animals.15 Animals were killed using sodium pentobarbital (200 mg/kg, intraperitoneal) in combination with
bilateral thoracotomies.
Cell Lines and Neuronal Cultures
Using the Flp-In T-Rex-293 cell line, derived from human
embryonic kidney (HEK) cells, and following the manufacturer’s guidelines (Invitrogen, Carlsbad, CA), stable cell lines
were generated expressing TRPA1 (rat TRPA1 complementary DNA in a pcDNA5/FRT/TO expression vector) or the
empty vector, both under the control of a tetracycline-inducible
promoter. Cells were grown and maintained in Dulbecco’s
modified Eagle medium containing 10% fetal bovine serum,
100 ␮g/ml hygromycin B, and 5 ␮g/ml blasticidin. All cultures
were maintained in 5% carbon dioxide in air at 37°C. For the
calcium assay, HEK cells were plated on cover slips coated with
polylysine. TRPA1 expression was induced by overnight addition of tetracycline (0.1 ␮g/ml) to the medium. Dorsal root
ganglia (DRG) from the thoracic and lumbar spinal cord were
removed, desheathed in cold Hanks’ buffered salt solution, and
dispersed in Hanks’ buffered salt solution containing 1 mg/ml
collagenase type I and 0.1 mg/ml DNAse for 60 min under
agitation at 37°C followed by treatment with 0.25% trypsin for
30 min under agitation at 37°C. After blocking trypsin activity
with soybean trypsin inhibitor type III (5 mg/ml), the suspension was triturated using a sequence of pipettes with diminishing diameter. Neurons were pelleted and suspended in Dulbecco’s modified Eagle medium containing 5% fetal bovine serum,
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Irritant Volatile Anesthetics and TRPA1 Channels
5% horse serum, 100 U/ml penicillin, 0.1 mg/ml streptomycin,
2 mM glutamine, and 100 ng/ml nerve growth factor. After
plating on glass coverslips coated with poly-D-lysine and laminin, cells were cultured for 2–3 days before calcium imaging
experiments. Electrophysiology experiments were performed 1
day after dissociation and plating.
Measurement of Intracellular Calcium Concentration
([Ca2ⴙ]i)
HEK cells or DRG neurons were incubated in Hanks’ buffered salt solution, 0.1% bovine serum albumin, 20 nM
HEPES, pH 7.4, containing 2.5 mM fura-2-acetoxymethyl
ester for 30 – 45 min at 37°C. Fura-2 fluorescence at 510 nm
was measured in response to alternating excitation with 340
nm and 380 nm using a F-2500 spectrofluorometer (Hitachi
Instruments, San Jose, CA) for HEK cells and a microscopebased calcium imaging system consisting of a Zeiss Axiovert
microscope (Carl Zeiss, Thornwood, NY), a cooled digital
video camera (Stanford Photonics, Stanford, CA), and a software system for video acquisition and analysis (Imaging
Workbench 6,INDEC Biosystems, Santa Clara, CA) for
measurements in individual DRG neurons. Results are reported as fluorescence ratio F340/F380 to indicate relative
changes in [Ca2⫹]i. Allyl isothiocyanate (AITC), anesthetics,
and bradykinin were applied via injection with a calibrated
syringe or pipette from concentrated stock solutions. All experiments were performed at room temperature (21–22°C).
Experiments with Ca2⫹-free extracellular solution were used
to demonstrate that the activation involves Ca2⫹ influx. For
sensitization assays, HEK cells were pretreated with bradykinin (10 mM) for 2 min before the anesthetic was applied.15
Electrophysiology
Whole cell membrane currents of HEK cells were recorded
using a HEKA EPC-10 amplifier (HEKA Instruments, Bellmore, NY) in the voltage clamp mode. Data were recorded as
1-s sweeps with a sampling frequency of 10 kHz and filtered
at 3 kHz. The average current for the time interval from 0.2
to 0.8 s was calculated for each sweep and plotted against
time. Patch pipettes were pulled from borosilicate glass and
had resistances between 2 and 4 M⍀. The composition of the
internal solution is KCl 140 mM, MgCl2 1 mM, EGTA 5 mM,
HEPES 5 mM, and ATPNa2 5 mM and the composition of
the external solution is NaCl 140 mM, CaCl2 2 mM, KCl 4
mM, MgCl2 0.6 mM, glucose 11 mM, HEPES 5 mM, and
CsCl 3 mM. The recording chamber was at room temperature (20 –23°C) and continuously perfused with external solution (2 ml/min). AITC and anesthetics were applied
through a capillary positioned 150 ␮m from the cell. The
holding potential was ⫺60 mV.
Organ Bath Studies
Guinea pigs were killed by cervical dislocation, the lungs
were removed, and rings from the main bronchi (⬃2 mm
width) were isolated and suspended with a resting tension of
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1.5 g in a 5-ml organ bath. The contractile forces were measured using isometric transducers (Ugo Basile, Comerio, Italy). The tissues were bathed with Krebs’ solution (composition: NaCl 119.0 mM; KCl 4.7 mM; MgSO4 1.5 mM;
CaCl2 2.5 mM; NaHCO3 25.0 mM; KHPO4 1.2 mM, and
glucose 11.0 mM; pH 7.4) containing phosphoramidon (1 ␮M)
to minimize peptide degradation and indomethacin (5 ␮M) to
prevent generation of prostanoids, aerated with carbogen (95%
O2 and 5% CO2), and maintained at 37°C. Tissues were equilibrated for 60 min before the beginning of the experiments
(washed every 5 min). In all experiments, the tissues were first
contracted with carbachol (1 ␮M) to record the maximal contractile response of each preparation, as previously reported.16
After carbachol wash-out, atropine (1 ␮M) was added to the
buffer and maintained for the remainder of the experiment.
Contractile responses for isoflurane (150 ␮M) were measured in
the absence or presence of the TRPA1 antagonist HC-030031
(50 ␮M),12,17 or the TRPV1 antagonist capsazepine (10 ␮M), or
a combination of NK1,2,3 receptor antagonists (SR140333, SR
48968, and SR142801, respectively; all 1 ␮M) and their respective vehicles, or in preparations previously desensitized by capsaicin (10 ␮M for 20 min).12 All antagonists were left in contact
with the tissue for at least 15 min before isoflurane challenge (150 ␮M). In another set of experiments, the contractile response to halothane (150 ␮M) was measured.
The results were expressed as percent of the maximal carbachol-induced response (1 ␮M).
Mechanical Hyperalgesia
Mechanical nociception was measured in trpa1⫹/⫹ and
trpa1⫺/⫺ mice using the up– down paradigm of Chaplan et
al.18 Calibrated von Frey monofilaments (0.008 – 4.0 g;
North Coast Medical, Morgan Hill, CA) were applied to the
plantar aspect of the left hind paw of the animal. If a paw
withdrawal response was not observed on stimulation with
the 0.4 g monofilament, a stronger stimulation was applied,
and if the withdrawal response was detected, a weaker stimulus was used. The responses were tabulated, and the 50%
response threshold was determined. Behavioral testing occurred between 9:00 AM and 1:00 PM. Starting 2 days before
behavioral testing, trpa1⫹/⫹ and trpa1⫺/⫺ mice were habituated (at least 1 h per day) in plastic cylinders with wire mesh
bottoms. On day 3, mice received intraplantar injections into
the left hind paw of a saturated solution of either isoflurane
(10 ␮l; ⬃15 mM) or halothane (10 ␮l; ⬃17 mM) in saline or
the same volume of normal saline (control group). One
group of trpa1⫹/⫹ mice was pretreated with the TRPA1 antagonist HC-030031 (100 mg/kg; intraperitoneal).19 The
paw injections were given without anesthesia using restraint
only to not confound the results. The mechanical nociceptive thresholds were determined before the injections (baseline, time 0) followed by measurements at 15, 30, 45, and 60
min after the injection. Data are expressed in grams and
represent the mean threshold values.
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Measurement of Anesthetic Concentrations and
Preparation of Anesthetic Solutions
Buffers saturated with anesthetics were prepared by adding
excess liquid anesthetic to the experimental buffer (bath solution for Ca2⫹ measurements, electrophysiology, and bronchoconstriction assay or normal saline for the behavioral assay) in glass vials sealed with glass or Teflon stoppers.
Solutions were equilibrated by stirring overnight. Anesthetic
concentrations in liquid samples were measured using gas
chromatography after extraction into a known gas volume
using established methods.20 –22 These gas samples were then
analyzed using a gas chromatograph (Gow-Mac, Bethlehem,
PA), and concentrations in the liquid phase were determined
using the gas equations and published data for the solubility
of the volatile anesthetics.
Statistics
Data were analyzed using one-way ANOVA with StudentNewman–Keuls post test as well as t test (Prism 5, GraphPad
Software, La Jolla, CA) as indicated in the Results section
and in the figure legends of the respective experiments. The
results of the behavioral experiments were analyzed with twoway ANOVA followed by a Bonferroni post test to compare
each group with the saline control. A value of P less than 0.05
was considered statistically significant for all statistical comparisons. The data are presented as mean ⫾ SEM unless
otherwise noted. For the calculation of the nonlinear regression of the concentration response data, we used a sigmoid
dose–response curve model with a standard slope (Hill equation with Hill coefficient of 1). The equation used is
Ex ⫽ E0 ⫹ (Emax ⫺ E0)/共1 ⫹ 10^共log EC50 ⫺ X)
where Ex ⫽ predicted effect for concentration X, E0 ⫽ minimal
effect, bottom plateau of the curve, Emax ⫽ maximal effect, top
plateau of the curve, EC50 ⫽ concentration that produces the
half maximal effect (Prism 5, GraphPad Software).
Results
Irritant Volatile Anesthetics Activate TRPA1
Heterologously Expressed in HEK293 Cells
To test the hypothesis that the irritant effects of certain volatile anesthetics are mediated through activation of TRPA1,
we first examined whether isoflurane produces an increase in
the intracellular calcium concentration ([Ca2⫹]i) in
HEK293 cells expressing rat TRPA1 under control of a tetracycline-inducible promoter. Functional expression of
TRPA1 was confirmed by the [Ca2⫹]i increase in response to
AITC, a known TRPA1 agonist (fig. 1A).10 When HEKTRPA1 cells were challenged with isoflurane at clinically
relevant concentrations (300 ␮M, approximately equivalent
to 1 minimal alveolar concentration [MAC]),2,23 we observed a similar increase in [Ca2⫹]i that was dependent on
induction of TRPA1 expression by tetracycline and also on
extracellular Ca2⫹ (fig. 1B). The increase in [Ca2⫹]i, induced
by isoflurane, was concentration-dependent over the techniEilers et al.
cally feasible concentration range (5 ␮M–5 mM) with an estimated EC50 of 534 ␮M (fig. 1C).
To answer the question whether anesthetic activation of
TRPA1 correlates with the pungency of the anesthetic, we
then compared the effects of the four most common clinically used volatile anesthetics in the same assay. Because the
tested anesthetics induce general anesthesia at different molar
concentrations, it is important to compare their effects at the
target site of interest at concentrations that are equivalent
with respect to the anesthetic effect. Therefore, we measured
the increase in [Ca2⫹]i for each anesthetic at a concentration
equivalent to 1 MAC and found that all four anesthetics
differ significantly in this aspect (fig. 1D). The ranking of the
anesthetics according to their TRPA1 activation at 1 MAC
corresponds to the clinically observed potential of the anesthetic to produce airway irritation (fig. 1D).7 Next, we obtained full concentration response curves for all four agents.
We found that the irritant volatile anesthetics isoflurane
(EC50 of 534 ␮M) and desflurane (EC50 1.15 mM) both
activated TRPA1. In contrast, the nonirritant volatile anesthetics sevoflurane and halothane had no concentration-dependent effect, even when applied at maximal concentrations (fig. 1E). Whole cell patch clamp recordings from
HEK-TRPA1 cells showed comparable inward current responses from AITC (50 ␮M)10,15 and isoflurane (300 ␮M; fig.
1F), whereas the nonirritant anesthetic halothane (300 ␮M) did
not induce any currents (data not shown). Thus, the pungent
volatile anesthetics isoflurane and desflurane but not the nonirritant sevoflurane and halothane activate heterologously expressed TRPA1 in a concentration-dependent manner. The potency of TRPA1 activation seems to correlate with the clinically
observed pungency of the tested volatile anesthetics.
Irritant Anesthetics Activate TRPA1 in Peripheral Nociceptors
Because activation of TRPA1 in heterologous overexpression
systems may be an artifact of overexpression, we determined
whether anesthetics are capable of activating TRPA1 endogenously expressed in primary sensory neurons. Using calcium imaging, we examined the effects of bath-applied volatile anesthetics on cultured neurons prepared from DRG of
wild-type mice (trpa1⫹/⫹). When neurons were challenged
with clinically relevant concentrations of the irritant volatile
anesthetics desflurane and isoflurane (300 ␮M and 150 ␮M,
respectively), we identified a subpopulation of small to medium diameter neurons sensitive to such anesthetics as demonstrated by the increase in [Ca2⫹]i. The same neurons were
also sensitive to the TRPA1 agonist AITC (100 ␮M) and the
TRPV1 agonist capsaicin (1 ␮M; fig. 2A). Other subpopulations of neurons were sensitive only to capsaicin, indicating
expression of only TRPV1 and not TRPA1, and others were
insensitive to all the three stimuli (fig. 2A, dotted and dashed
traces). Confirming our results from the HEK cell expression
system, the nonirritant anesthetic halothane did not increase
[Ca2⫹]i in neurons from trpa1⫹/⫹ mice (data not shown). To
further investigate the specificity of the detected effect and to
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Irritant Volatile Anesthetics and TRPA1 Channels
Fig. 1. Irritant volatile anesthetics activate heterologously expressed transient receptor potential (TRP)-A1 in a dose-dependent
manner. (A) Representative traces of fura-2-fluorescence ratios indicating increases in intracellular calcium concentration [Ca2⫹]i
induced by allylisothiocyanate (AITC, 20 ␮M) in human embryonic kidney (HEK) cells after tetracycline-induced (tet) TRPA1 expression
(TRPA1 ⫹ tet). Control traces show cells without tetracycline induction of TRPA1 expression (TRPA1 – tet) and TRPA1 expressing
cells in the absence of extracellular calcium. The horizontal line indicates application of AITC. (B) Typical traces of isoflurane-induced
increase in [Ca2⫹]i in TRPA1-expressing HEK cells. Same controls as in (A). The horizontal line indicates application of isoflurane (300
␮M). (C) Dose–response curve for the changes in intracellular calcium (change in fura-2-fluorescence ratio) induced by isoflurane in
TRPA1-expressing HEK cells. N ⫽ 3–5 coverslips per data point. The calculated EC50 is 534 ␮M (⬃1.6 MAC, E0 ⫽ 0.52, Emax ⫽ 2.58).
(D) Comparison of the change in [Ca2⫹]i induced by equianesthetic concentrations of the four most commonly used volatile
anesthetics. Bars represent mean ⫾ SEM, numbers in parentheses indicate the number of observations per group. All intergroup
comparisons are statistically significant* (one-way ANOVA followed by Student–Newman–Keuls post hoc test for all intergroup
comparisons). (E) Comparison of the concentration–response relationships for the four most commonly used volatile anesthetics.
N ⫽ 3–5 coverslips per data point. Parameters calculated for the concentration–response curve of desflurane: EC50 ⫽
1150 mM, E0 ⫽ 0.38, Emax ⫽ 3.45. (F) Inward current responses induced by AITC (50 ␮M) and isoflurane (300 ␮M) in
TRPA1-expressing HEK cells. Responses were measured by whole cell patch clamp in a voltage clamp configuration with
a holding potential of ⫺60 mV.
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Fig. 2. Irritant volatile anesthetics activate transient receptor potential (TRP)-A1 expressed in peripheral nociceptors. (A) Typical
traces of fura-2-fluorescence ratios representing changes in intracellular calcium concentration [Ca2⫹]i induced by desflurane
(des, 1.15 mM–2 MAC), allyl-isothiocyanate (aitc, 100 ␮M), and capsaicin (cap, 1 ␮M) in dorsal root ganglion (DRG) neurons
isolated and cultured from TRPA1 wild-type mice. Application of desflurane, mustard oil, and capsaicin is indicated by
horizontal lines. Two cells respond to all the three substances, one cell only to capsaicin (bold black trace), and two cells do
not respond to any of the stimuli (dashed traces). (B) Representative traces of changes in [Ca2⫹]i in response to desflurane,
mustard oil, and capsaicin in DRG neurons isolated and cultured from TRPA1 knock-out mice. Labeling of the traces is similar
to that described for (A). (C) Quantitative analysis of responses to desflurane in DRG neurons from TRPA1 wild-type (trpa1⫹/⫹)
and knock-out (trpa1⫺/⫺) mice. A total of 35.5% of wild-type cells responded to desflurane but only 3% of cells isolated from
knock-out mice responded (n ⫽ 28 [trpa1⫹/⫹] and n ⫽ 42 [trpa1⫺/⫺]). (D) Representative traces of changes in [Ca2⫹]i in response
to isoflurane, mustard oil, and capsaicin in DRG neurons from TRPA1 wild-type mice. Experimental setup and labeling of traces
is similar to (A) but isoflurane (iso) was used instead of desflurane.
tile anesthetics to induce [Ca2⫹]i influx, we cultured neurons
derived from knockout mice (trpa1⫺/⫺). A total of 35.5% of
neurons from trpa1⫹/⫹ mice responded to the anesthetic
with an increase in calcium influx. In contrast, only 3% of
cells derived from trpa1⫺/⫺ mice responded to desflurane
exposure (figs. 2B and C). Thus, the activation of peripheral
sensory neurons by the pungent anesthetics desflurane (fig.
2A) and isoflurane (fig. 2D) is dependent on TRPA1 expression and is largely absent in mice lacking TRPA1.
Isoflurane-induced [Ca2ⴙ]i Increase Is Augmented by
Pretreatment with Bradykinin
Surgical procedures under general anesthesia cause tissue injury and inflammation. One mechanism by which inflammatory agents cause pain involves the sensitization of nociceptive neurons. Bradykinin, a prominent inflammatory
peptide, sensitizes ion channels including TRPA1, which
magnifies neuronal responses to TRPA1 agonists, and
TRPA1 is also required for bradykinin-induced pain. We
evaluated whether bradykinin also sensitizes anesthetic-inEilers et al.
duced activation of TRPA1. We examined sensitization in
HEK-TRPA1 cells that naturally express the bradykinin B2
receptor and compared the anesthetic-induced increase in
[Ca2⫹]i without (fig. 3A) and with preincubation with bradykinin (fig. 3B). HEK-TRPA1 cells were exposed to bradykinin (10 nM) or vehicle (control) for 2 min and were then
challenged with isoflurane (100 ␮M ⬃ 0.3 MAC). Pretreatment with bradykinin augmented the isoflurane-induced
[Ca2⫹]i increase by 70% (fig. 3C). Thus, the inflammatory
mediator bradykinin sensitizes the activation of TRPA1 by
the pungent anesthetic isoflurane.
Irritant Volatile Anesthetics Cause Neurogenic
Contractions of the Airway
The best known and most commonly accepted adverse effect
of irritant volatile anesthetics is their irritating effect on the
airway, which is clinically apparent as an unpleasant sensation, cough, or laryngospasm. The effect of anesthetics on
the bronchomotor tone is controversial: the general notion
that anesthetics are bronchodilators is in contrast with airway
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Irritant Volatile Anesthetics and TRPA1 Channels
Fig. 3. Bradykinin sensitizes transient receptor potential (TRP)-A1 activation by irritant volatile anesthetics. Representative
traces of fura-2-fluorescence ratios showing changes in intracellular calcium concentration [Ca2⫹]i in human embryonic kidney
(HEK) cells expressing TRPA1. (A) A trace in response to isoflurane (100 ␮M, application indicated by horizontal line) and (B) a
trace in response to isoflurane (100 ␮M, application indicated by gray line) after pretreatment with bradykinin (10 nM, application
indicated by black line). (C) Average responses to isoflurane (100 ␮M) in cells without (iso) and with (bk ⫹ iso) bradykinin
pretreatment. The average response to 100 ␮M isoflurane is increased by 70% after bradykinin. The asterisks symbol indicates
significant difference (t test, n ⫽ 4 coverslips per group).
irritation and reports of bronchoconstriction.24 –26 To examine the effects of anesthetics on the musculature of the airway, we measured constriction of isolated bronchial rings
from guinea pigs. Exposure to isoflurane (150 ␮M ⬃ 0.5
MAC) produced a significant contractile response in the
bronchial ring that was 49 ⫾ 5% of the effect induced by
carbachol (1 ␮M). The response to isoflurane was abolished
by desensitization of sensory nerves with a high concentra-
tion of capsaicin (10 ␮M for 20 min) and by pretreatment
with the TRPA1 selective antagonist HC-030031 (50 ␮M;
fig. 4) but not by the TRPV1 selective antagonist capsazepine
(10 ␮M; fig. 4). To determine whether neuropeptides released from primary sensory neurons mediate isoflurane-induced contraction, we repeated the experiment after preincubation of tissues with a combination of SR140333,
SR48968, and SR142801 antagonists of neurokinin recep-
Fig. 4. Isoflurane induces a contractile response in guinea pig bronchus. (A) Typical traces and (B) pooled data of the motor
responses of guinea pig isolated bronchus to halothane (Hal; 150 ␮M; open triangle) and isoflurane (Iso; 150 ␮M; filled circles) or their
vehicle (Veh) either alone or after pretreatment with capsaicin (CAP, 10 ␮M for 20 min), the combination of neurokinin receptor
antagonists SR140333, SR48968, and SR142801 (NK, all 1 ␮M), the TRPA1 antagonist HC-030031 (HC, 50 ␮M), or the TRPV1
antagonist capsazepine (CPZ, 10 ␮M). Each column in (B) represents the mean ⫾ SEM, and the number of observations for each
group is listed in parentheses. * P ⬍ 0.05 vs. Veh (one-way analysis of variance, followed by Student–Newman–Keuls post hoc test).
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Fig. 5. Locally applied isoflurane but not halothane induces mechanical hyperalgesia. (A) Mechanical nociceptive threshold (von
Frey hair) after injection of isoflurane (iso, 10 ␮l, 15 mM in saline) or saline (10 ␮l) into the hind paw of trpa1⫹/⫹ and trpa1⫺/⫺ mice.
Only the wild-type animals injected with isoflurane develop a transient hyperalgesia. * P ⬍ 0.05 vs. trpa1⫹/⫹ saline, trpa1⫺/⫺ iso,
and trpa1⫺/⫺ saline. (B) Mechanical nociceptive thresholds after halothane injection (hal, 10 ␮l, 15 mM in saline). No change in
nociceptive threshold develops after halothane injection. (C) Development of hyperalgesia after isoflurane injection is blocked
by systemic pretreatment with HC-030031 (100 mg/kg intraperitoneal). Mice injected with isoflurane and pretreated with vehicle
only (iso-veh[HC]) develop hyperalgesia. * P ⬍ 0.05 vs. trpa1⫹/⫹ iso-HC, trpa1⫹/⫹ saline-HC, and trpa1⫹/⫹ saline-veh(HC). The
number of observations per data point is listed in parentheses. Data were analyzed using a two-way analysis of variance
followed by Bonferroni post hoc test.
tors types 1, 2, and 3, respectively. After pharmacologic
blockade of neurokinin receptors, the contractile response
induced by isoflurane was significantly reduced (fig. 4). In
contrast to isoflurane, halothane (up to 150 ␮M) did not
produce any measurable contraction of guinea pig bronchi
(fig. 4). Thus, clinically useful concentrations of the pungent
anesthetic isoflurane produce significant contraction in isolated bronchial rings by a neurogenic mechanism involving
TRPA1 expressed in capsaicin-sensitive neurons as well as
the release of neuropeptides from these neurons.
Isoflurane Causes Mechanical Hyperalgesia
The transmission of nociceptive signals is associated with the
release of substance P and calcitonin gene-related peptide
from the central endings of peptidergic nociceptors. Given
that volatile anesthetics are capable of exciting such a subset
of peptidergic primary sensory neurons, we sought to determine their effects on nociception. To assess mechanical hyperalgesia, we measured hind paw withdrawal threshold to
mechanical stimulation with Von Frey filaments. Intraplantar injection of the irritant volatile anesthetic isoflurane (10
␮l, 15 mM in 0.9% NaCl) significantly decreased the nociceptive threshold in trpa1⫹/⫹ mice 15–30 min after injection, indicative of mechanical hyperalgesia (fig. 5A, trpa1⫹/⫹
iso and trpa1⫹/⫹ iso-veh[HC]). In contrast, intraplantar injection of the nonirritant volatile anesthetic halothane (10
Eilers et al.
␮l, 17 mM in 0.9% NaCl) did not affect the nociceptive
threshold (fig. 5B, trpa1⫹/⫹ hal). To further confirm that the
hyperalgesic effect of isoflurane was mediated by a TRPA1dependent mechanism, we examined isoflurane-induced mechanical hyperalgesia in both mice lacking the gene that encodes for TRPA1 (trpa1⫺/⫺) and in wild-type mice
pretreated with the specific TRPA1 antagonist HC-030031
(100 mg/kg; intraperitoneal). As predicted, trpa1⫺/⫺ mice
(fig. 5A, trpa1⫺/⫺ iso) as well as trpa1⫹/⫹ mice pretreated
with systemic administration of HC-030031 (fig. 5C,
trpa1⫹/⫹ iso-HC) failed to develop mechanical hyperalgesia
after intraplantar injection of isoflurane, suggesting that the
mechanism of isoflurane-induced hyperalgesia involves activation of TRPA1 ion channels. Thus, the pungent anesthetic
isoflurane, when injected locally into the mouse hind paw,
produces mechanical hyperalgesia by a mechanism requiring
the expression of functional TRPA1.
Discussion
We report, for the first time, that the activation of TRPA1 by
pungent volatile anesthetics plays a pivotal role in mediating
their irritant effects in the airways and their proposed hyperalgesic effects. Specifically, our studies suggest a mechanism
by which irritant volatile anesthetics produce their characteristic pungent sensation and cause neurogenic bronchoconstriction through activation of TRPA1 expressed in nocicepAnesthesiology, V 112 • No 6 • June 2010
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Irritant Volatile Anesthetics and TRPA1 Channels
tors. Moreover, our findings also suggest that TRPA1
activation may play a role in the development of hyperalgesia
produced by irritant volatile anesthetics.
TRPA1 detects a wide variety of chemical irritants, including
endogenous alimentary and environmental substances. Given
the proposed role of TRPA1 as a mediator of protective airway
reflexes in response to chemical stimulation,12,27–29 it seems
likely that the irritant effects of certain volatile anesthetics are
also mediated through the same pathway.
Several observations support the hypothesis that irritant volatile anesthetics activate TRPA1. First, isoflurane and desflurane
activated TRPA1 expressed in HEK cells. Isoflurane-induced
calcium influx was entirely dependent on TRPA1 expression
because no calcium influx was observed in control cells without
induced TRPA1 expression. Irritant anesthetics induced a robust, concentration-dependent effect that is comparable with
that of AITC, a specific TRPA1 agonist. Our results confirm a
recent report that irritant anesthetics directly activate TRPA1.13
The EC50 reported for isoflurane in this study (180 ␮M, ⬃0.6
MAC) is lower than the value we determined (534 ␮M, ⬃1.6
MAC), possibly related to the use of electrophysiology instead of
measurement of [Ca2⫹]i. However, both reported potencies are
within the clinically useful concentration range. Sevoflurane
and halothane, both nonirritant volatile anesthetics, did not
produce increases in the influx of calcium at any tested dose,
leading us to conclude that they do not activate TRPA1.
Second, irritant anesthetics caused calcium influx in DRG
neurons from trpa1⫹/⫹ but not trpa1⫺/⫺ mice, which confirms
that these agents activate TRPA1 in nociceptive neurons. The
activation induced by irritant volatile anesthetics is restricted to
a neuronal subpopulation sensitive to AITC and capsaicin, indicating TRPA1 and TRPV1 coexpression. This coexpression
of TRPA1 and TRPV1 in a subpopulation of peripheral nociceptors has been previously reported.30
The activation of sensory neurons by anesthetics has been
previously reported by others. Halothane, isoflurane, and
enflurane increase the discharge of C-fibers innervating the
rabbit cornea.31 The effects of these anesthetic mechanosensitive A␦-fibers are more complex: halothane increases and
isoflurane and enflurane decrease spike latency, and all three
anesthetics slightly decrease spike amplitude.31 These findings confirm and extend previous reports of sensorineuronal
activation by volatile anesthetics in a variety of models.32–34
However, the underlying molecular mechanisms for any of
these findings have not been elucidated.
TRP channels detect noxious stimuli in peripheral nociceptors and are likely targets that may mediate the irritant
effects of volatile anesthetics in C-fibers. TRPA1 and
TRPV1 are likely candidates given their sensitivity to irritant
chemicals. In preliminary studies, we did not observe irritant
anesthetics activating TRPV1 in HEK cells (unpublished
observations, July and August 2007, Helge Eilers, M.D., San
Francisco, CA, experiments). However, a recent report suggests that volatile anesthetics sensitize TRPV1 to capsaicin,
protons, and heat. Inflammatory mediators such as bradyki1460
nin and downstream targets such as protein kinase C enhanced these effects of volatile anesthetics on TRPV1.35
We report that the inflammatory mediator bradykinin
sensitizes isoflurane-induced calcium signals in TRPA1 expressing HEK cells. Because isoflurane mobilizes calcium in
these cells by a TRPA1-dependent mechanism, these results
suggest that bradykinin sensitizes TRPA1. Surgery induces
tissue injury, resulting in the formation of multiple inflammatory mediators, including bradykinin, which can sensitize
TRPA1. This sensitization would act in synergy with the
effects of irritant anesthetics, resulting in amplified neurogenic inflammation, hyperalgesia, and pain.
Although anesthetics are widely considered to be bronchodilators and are even suggested as therapeutics in the setting of
status asthmaticus refractory to other interventions,36 this concept is at odds with their well-described pungency and ability to
cause airway irritation. To address this paradox and further elucidate the effects of TRPA1 activation by irritant volatile anesthetics, we examined bronchoconstriction of the guinea pig
bronchi. Irritant anesthetics caused constriction of the bronchi
that was abolished by antagonism of neurokinin receptors and is
thus mediated by the release of tachykinins from nociceptive
neurons. This effect is entirely dependent on C- or A␦-fiber
neurons expressing TRPV1 because selective desensitization of
these neurons by high concentrations of capsaicin abolished
bronchoconstriction.
These neurogenic bronchoconstrictor effects of isoflurane are in contrast to its widely conceived bronchodilator
effect and suggest two distinct cellular mechanisms, one
that mediates the neurogenic bronchoconstriction by
TRPA1-dependent activation of sensory neurons and another that mediates bronchodilation by direct action on
calcium homeostasis in smooth muscle.37 One possible
explanation for the contradictory findings from functional studies may be that one or the other mechanism
predominates depending on the experimental model and
the anesthetic concentrations used. Alternatively, the duration and or timing of the anesthetic exposure may determine the effect. The sensorineuronal transduction
mechanism may desensitize during prolonged exposure,
leading to a dominance of the irritant effects during the
initial exposure.38 Because airway resistance is measured
at steady state, the initial bronchoconstriction may be
missed in many experimental models.
Another proposed adverse effect of pungent volatile anesthetics is their pronociceptive action.3,4,6,39,40 Although the
hyperalgesic effect of volatile anesthetics has been established
in animal experiments at low anesthetic concentrations,
these findings are not confirmed in human subjects. Low
concentrations of volatile anesthetics cause a decrease in hind
paw withdrawal latency to painful heat in rats and mice,
indicating hyperalgesia.3,4,6,39,40 Several mechanisms have
been proposed, including nicotinic and ␣-adrenergic activation. However, a consensus has not been reached, and it is
difficult to compare directly the results of these studies because the models used differ substantially.3,39,41,42 In human
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subjects, nitrous oxide and methoxyflurane have analgesic
properties at subanesthetic concentrations, whereas halothane, isoflurane, enflurane, and sevoflurane have no effect
on thermal pain.5 Interestingly, the observed effect of the
latter four drugs trended toward increased pain sensitivity
but was not statistically significant.5 Extensive studies designed to assess the analgesic effect of subanesthetic concentrations of isoflurane in human volunteers using a wide variety of experimental pain models failed to demonstrate a clear
analgesic effect.43,44 However, hyperalgesia was also not
observed.
The contradictory effects of irritant anesthetics on pain in
experimental animals (algesia) and human subjects (analgesia) may be attributable to the route of administration. Systemic administration by inhalation causes hypnotic effects in
the central nervous system as a confounding factor. Loss of
the righting reflex in combination with the tail-flick latency
has been used to separate hypnotic from analgesic effects.4
To avoid the contribution of central effects, we chose local
application of anesthetics by intraplantar injection to test for
anesthetic-induced hyperalgesia. Intraplantar injection is
routinely applied to measure hyperalgesia induced by other
irritants such as capsaicin and AITC.19,45– 47 Our results
demonstrate that intraplantar injection of the irritant volatile
anesthetic isoflurane, but not the nonirritant halothane,
caused mechanical hyperalgesia in trpa1⫹/⫹ but not
trpa1⫺/⫺ mice. Moreover, to further support the specificity
of the detected effect, we showed that pretreatment of
trpa1⫹/⫹ mice with the TRPA1 antagonist HC-030031
abolished isoflurane-induced mechanical hyperalgesia. The
isoflurane concentration was at saturation (⬃15 mM) and,
therefore, considerably higher than clinically useful concentrations. However, the absolute amount of anesthetic delivered was small (10 ␮l ⫽ 150 nmol, in the unlikely event that
the injection process can be accomplished without loss) and
even if equilibration of the anesthetic would be restricted to
the paw, the maximal achievable concentration would be
equivalent to 3 MAC, assuming an average paw volume of
150 ␮l.48 Furthermore, the observation that these effects
were absent when TRPA1 was deleted or antagonized argues
against a nonspecific effect produced by a high concentration. Furthermore, a saturated solution of halothane failed to
produce hyperalgesia. Together, we interpret this as evidence
for isoflurane-induced mechanical hyperalgesia by activating
TRPA1.
Locally administered volatile anesthetics have been reported to have anesthetic actions,49,50 which contradict
our findings of hyperalgesia after injection into the hind
paw. However, these studies used extremely high concentrations such as pure sevoflurane applied to the spinal
cord50 or lipid preparations of anesthetics at concentrations
exceeding 0.5 M.49 Although in our experiments the lack of
effect in knockout animals and the lack of effect induced by an
equimolar concentration of the nonirritant anesthetic halothane
provide convincing evidence for a specific, TRPA1-mediated
Eilers et al.
effect, other studies may lack adequate controls to assure
specificity.49,50
Our observations provide strong evidence for a mechanism
by which pungent volatile anesthetics may not only produce
their well-described airway irritation but also contribute to the
severity of postoperative pain. Unequivocal clinical evidence for
anesthetic-induced postoperative pain is currently lacking, although more severe postoperative pain has been observed in
patients anesthetized with isoflurane (an irritant volatile anesthetic) than in patients who received propofol, as determined by
morphine requirement and verbal pain scores.51 Although a
hyperalgesic effect of isoflurane is only one possible explanation
for their findings, this study emphasizes the possibility that the
hyperalgesic effects become only apparent under conditions of
inflammation after surgery.
The hypothesis that irritant volatile anesthetics, such as
isoflurane and desflurane, activate TRPA1 ion channels on
peripheral sensory neurons is supported by multiple lines of
evidence presented here as well as by recent reports of other
investigators.13,35 For the first time, we demonstrate a
TRPA1-specific hyperalgesic effect of irritant volatile anesthetics and provide a plausible mechanism not only for the
airway irritation produced by anesthetics but also for anesthetic-induced hyperalgesia.
The authors thank Michael J. Laster, D.V.M., Senior Research Associate, Edmond I Eger II, M.D., Professor, and James Sonner, M.D.,
Professor (Department of Anesthesia and Perioperative Care, University of California, San Francisco, San Francisco, California), for
their assistance with the gas chromatographic measurements and
critical discussion. They also thank David Julius, Ph.D., Professor
(Department of Physiology, University of California, San Francisco), for
allowing the use of transient receptor potential-A1– deficient mice.
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ANESTHESIOLOGY REFLECTIONS
The Morton House I by Vandam
In August of 1819, William Thomas Green Morton (1819 –1868) was born in Charlton, Massachusetts. His original birthplace burned down to its foundations, so the house depicted above is
“a successor to the original edifice.” So remarked artist-anesthesiologist Leroy D. Vandam
(1914 –2004), a retired Editor of ANESTHESIOLOGY, who painted this watercolor (see above). Championing
Morton as the first to publicly demonstrate ether for surgical anesthesia, Dr. Vandam delighted in painting
this Morton House at various seasons of the year. Only a few copies of the 100-print run autographed by
the late Dr. Vandam remain for sale as a benefit for the Wood Library-Museum. (Copyright © the American
Society of Anesthesiologists, Inc. This image appears in color in the Anesthesiology Reflections online
collection available at www.anesthesiology.org.)
George S. Bause, M.D., M.P.H., Honorary Curator, ASA’s Wood Library-Museum of Anesthesiology,
Park Ridge, Illinois, and Clinical Associate Professor, Case Western Reserve University, Cleveland, Ohio.
[email protected].
Eilers et al.
Anesthesiology, V 112 • No 6 • June 2010
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