British Journal of Anaesthesia 1998; 81: 569–577
Volatile anaesthetics have differential effects on recombinant m1 and
m3 muscarinic acetylcholine receptor function†
G. W. NIETGEN, C. W. HÖNEMANN, C. K. CHAN, G. L. KAMATCHI AND M. E. DURIEUX
Summary
Muscarinic acetylcholine signalling plays major
roles in regulation of consciousness, cognitive
functioning, pain perception and circulatory
homeostasis. Halothane has been shown to
inhibit m1 muscarinic signalling. However, no
comparative data are available for desflurane,
sevoflurane or isoflurane, nor have the anaesthetic effects on the m3 subtype (which is also
prominent in the brain) been studied. Therefore,
we have investigated the effects of these compounds on isolated m1 and m3 muscarinic receptor function. Defolliculated Xenopus oocytes
expressing recombinant m1 or m3 muscarinic or
(for comparison) AT1A angiotensin II receptors
were voltage clamped, and Ca2;-activated Cl9 currents (ICl(Ca)) induced by acetyl-␤-methylcholine
(Mch) or angiotensin II were measured in the
presence of clinically relevant concentrations of
halothane, sevoflurane, desflurane or isoflurane.
To determine the site of action of the volatile
anaesthetics we compared anaesthetic effects on
m1, m3 and AT1A receptor function and studied
the effects of volatile anaesthetics on signalling
induced by intracellular injection of the second
messenger IP3. Desflurane had a biphasic effect
on m1 signalling, enhancing at a concentration of
0.46 mmol litre91 but depressing at 0.92 mmol
litre91. A similar, although not significant, trend
was observed with m3 signalling. Isoflurane
had no effect on m1 signalling, but profoundly
inhibited m3 signalling. Sevoflurane depressed
the function of m1 and m3 signalling in a dosedependent manner. Halothane, similar to its
known effect on m1 signalling, dose-dependently
depressed m3 function. ICl(Ca) induced by intracellular injections of IP3 were unaffected by all four
anaesthetics. Similarly, none of the anaesthetics
tested affected AT1A signalling. Absence of interference with AT1A signalling and intracellular pathways suggest that the effects of anaesthetics on
muscarinic signalling most likely result from
interactions with the m1 or m3 receptor molecule.
Multiple interaction sites with different affinities
may explain the biphasic response to desflurane.
Anaesthetic-specific effects on closely related
receptor subtypes suggest defined sites of action
for volatile anaesthetics on the receptor protein.
(Br. J. Anaesth. 1998; 81: 569–577).
Keywords: theories of anaesthetic action, molecular; anaesthetics volatile; receptors, muscarinic
In addition to the well known actions in circulatory
and respiratory regulation,1 2 muscarinic acetylcholine
signalling plays an important role in the central
nervous system (CNS). Level of consciousness is
modulated significantly by brainstem muscarinic signalling.3 Cholinergic antagonists affect memory and
learning abilities through actions on the basal forebrain, cortex and hippocampus.4 5 Spinal muscarinic
receptors mediate antinociception, predominantly in
the substantia gelatinosa of the dorsal horn, and also
affect motor neurone areas.6 7
Anaesthetics cause CNS depression and alter
bronchial tone and heart rate, effects which may be
mediated in part by interactions with muscarinic
signalling.8 9 Various anaesthetics are known to alter
ligand binding to muscarinic receptors. Ether,10
halothane,11 chloroform, enflurane and isoflurane12
were shown to increase muscarinic antagonist binding. However, these initial reports investigated mixed
receptor populations and did not study receptor activation. More refined methods of investigating anaesthetic mechanisms became available when the
members of the muscarinic receptor subfamily were
cloned. Hence the influence of anaesthetics on the
function of individual receptor subtypes can now be
studied. Recently, we reported that halothane13 and
ketamine14 inhibited m1 muscarinic signalling.
This study was designed to extend the initial findings with halothane. First, we wished to investigate if
the effect of halothane on muscarinic signalling was
subtype-specific. Therefore, we studied anaesthetic
actions on the m3 muscarinic receptor, which is also
expressed widely in brain.15 16 Second, we wished to
investigate if the effects of halothane could be extrapolated to other volatile anaesthetics. Thus we studied
the muscarinic inhibitory effects of sevoflurane,
isoflurane and desflurane. The comparison between
isoflurane and desflurane was of specific interest, as
these anaesthetics are closely related structurally:
desflurane differs from isoflurane only in the substitution of fluorine for chlorine on the ␣-ethyl carbon.
However, in contrast with isoflurane, desflurane has
been noted to induce a relative sympathetic activation,
resulting in significantly increased heart rate and
mean arterial pressure compared with isoflurane.17–19
GREGOR W. NIETGEN, MD, CHRISTIAN W. HÖNEMANN, MD, CARRIE K.
CHAN, MS, GANESAN L. KAMATCHI, PHD, MARCEL E. DURIEUX, MD,
Department of Anaesthesiology, University of Virginia Health
Sciences Center, PO Box 10010, Charlottesville, VA 22903–0010,
USA. Accepted for publication: May 19, 1998.
Correspondence to M. E. D.
†Presented in part at the American Society of Anesthesiologists
Annual Meeting, New Orleans, USA, October 19–23, 1996.
570
We hypothesized that this effect might result in part
from muscarinic inhibition, and that, despite their
structural similarities, these anaesthetics might show
divergent effects on muscarinic receptor function.
Therefore, we have determined the action of desflurane, isoflurane and sevoflurane on m1 and m3
muscarinic and, as a comparison, on angiotensin II
receptor function. In addition, the effects of
halothane on m3 signalling were investigated.
Specifically, we investigated if the anaesthetics, at
clinically comparable concentrations, influenced m1
and m3 muscarinic signalling, and where in the
signalling pathway their effects were localized.
Materials and methods
XENOPUS OOCYTES
The study was approved by the Animal Research
Committee of the University of Virginia. Adult
female Xenopus laevis frogs were obtained from
Xenopus I (Ann Arbor, MI, USA) and housed in an
established frog colony. To obtain oocytes, frogs were
anaesthetized with 1% tricaine (3-aminobenzoic acid
ethyl ester) until unresponsive, and operated on ice.
Oocytes were harvested via a 5-mm incision in the
lower lateral abdominal wall and placed immediately
in modified Barth’s solution containing (mmol
litre91): NaCl 88, KCl 1, NaHCO3 2.4, CaCl2 0.41,
MgSO4 0.82, Ca(NO3)2 0.3, gentamicin 0.1, HEPES
15, pH adjusted to 7.6. Frogs were returned to the
main tank after recovering for 2 days in isolation.
Oocytes (Dumont’s stages V and VI) were defolliculated by gentle shaking for 2 h in a solution of 1 mg
ml91 collagenase type Ia in calcium-free OR2 solution, containing (mmol litre91): NaCl 82.5, KCl 2,
MgCl2 1, HEPES 5, pH adjusted to 7.5, and were
afterwards incubated in modified Barth’s solution at
18⬚C.
RECEPTOR EXPRESSION
The rat m1 and m3 muscarinic acetylcholine receptors were a gift from T. I. Bonner (National Institutes
of Mental Health, Bethesda, MD, USA). Its complementary DNA (cDNA) consists of a 2.8-kilobasepair
(kpb) fragment in a commercial vector (pGEM1;
Promega, Madison, WI, USA). The construct for m1
expression was linearized by digestion with the
nuclease HindIII, and complementary RNA (cRNA)
was transcribed in vitro using the bacteriophage RNA
polymerase T7. A capping analogue (7mGpppG) was
included in the reaction to generate capped transcripts, as these are translated more efficiently in the
oocyte. The resulting cRNA was quantified by spectroscopy and diluted in water to a concentration of
0.1 mg ml91. Oocytes were each injected with 5 ng of
cRNA using an automated microinjector (Nanoject;
Drummond Scientific, Broomall, PA, USA).
Adequacy of injection was confirmed by noting the
slight increase in cell size during injection. m3 muscarinic cDNA was subcloned in the pCMV vector.
cDNA 10 ng in H2O 9.5 nl was injected directly into
the germinal vesicle (nucleus) of the oocyte. This
technique bypasses in vitro cRNA preparation and
therefore yields faster and more convenient, although
less reliable, expression.
British Journal of Anaesthesia
A 1.2-kbp cDNA in the CDM8 vector (Invitrogen,
San Diego, CA, USA), encoding the rat AT1A
angiotensin II receptor, was obtained from Dr. K. R.
Lynch (University of Virginia, Charlottesville, VA,
USA). The construct was linearized with the nuclease XhoI and transcribed in vitro by T7 RNA polymerase in the presence of a capping analogue.
Oocytes were injected as described above. All injected
cells were cultured for 72 h at 18⬚C in modified
Barth’s solution before study.
ELECTROPHYSIOLOGY
m1, m3 or AT1A receptor activation in oocytes leads
to G protein-induced phospholipase C activity and
release of inositoltrisphosphate (IP3) from phosphoinositolbisphosphate (fig. 1A). IP3 then binds to its
specific receptor on intracellular Ca2; stores to
release Ca2;, which in turn opens an endogenous
Ca2;-activated Cl9 channel. The resulting current can
be measured by voltage clamp, and this Ca2;activated Cl9 current (ICl(Ca)), integrated and expressed
as micro coulombs (␮C), is a measure of intracellular
Ca2; release.20–23 The upward deflection during
agonist application at times observed in the traces
is a slight motion artefact. All experiments were
performed at room temperature (approximately
22⬚C).
Microelectrodes were pulled in one stage from
capillary glass (BBL with fibre; World Precision
Instruments, Sarasota, FL, USA) on a micropipette
puller (model 700C; David Kopf Instruments,
Tujunga, CA, USA). Tips were broken to a diameter
of approximately 10 ␮m, providing a resistance of
1–3 MΩ, and filled with KCl 3 mol litre91. A single
oocyte was placed in a perfusable bath containing
3 ml of Tyrode’s solution containing (mmol litre91):
NaCl 150, KCl 5, MgCl2 1, CaCl2 2, glucose 10,
HEPES 10, pH adjusted to 7.4 with NaOH. Two
microelectrodes were inserted into the oocyte, and a
holding potential of 970 mV was applied to the
membrane. The voltage clamp amplifier (OC725A;
Warner Corporation, New Haven, CT, USA) was
connected to a data acquisition and analysis system
running on an IBM-compatible personal computer.
The acquisition system consisted of a DAS-8 A/D
conversion board (Keithly-Metrabyte, Taunton,
MA) and analysis was performed with OoClamp
software.24 Occasional cells that did not show a
stable holding current of less than 1 ␮A during a
1-min equilibration period were excluded from
analysis.
Membrane current was sampled at 125 Hz and
recorded for 5 s before and 55 s after application of
the agonist (acetyl-␤-methylcholine (Mch) or
angiotensin II (AT) delivered at appropriate EC50
values: Mch 1097 mol litre91 for m1 receptors13, Mch
1096 mol litre91 for m3 receptors; AT 1097 mol litre91
for AT1A receptors). Agonists were delivered in 30-␮l
aliquots over 1–2 s using a micropipettor positioned
approximately 1 mm from the oocyte. Responses
were quantified by integrating the current trace by
quadrature and are reported as ␮C as this reflects
intracellular Ca2;-release better than peak current.25
Any motion artefact on agonist delivery was
excluded from analysis. Each oocyte received a single
agonist application only.
Effects of volatile anaesthetics on muscarinic signalling
IP3 AND HEPARIN MICROINJECTION
To evaluate the influence of volatile anaesthetics on
the second messenger system, we injected IP3 or
heparin into the oocyte. Heparin inhibits the IP3
receptor on intracellular Ca2; stores, thereby inhibiting Ca2; release induced by IP3-signalling receptors.
Heparin injections were performed 30 min before
voltage clamping; heparin 120 ng (2 ng nl91) was
microinjected using the same nanoinjector mentioned
above. IP3, when injected into the cell, induces an
ICl(Ca) similar to that observed after receptor activation
in the oocyte. IP3-induced ICl(Ca) was evoked by injecting 50 nl of IP3 2 mmol litre91 in water into the oocyte.
Each oocyte was injected only once with IP3; therefore, any kinetic effects observed were not caused by
time-dependent effects or run-down after multiple
injections. Control injections with water as vehicle
were performed to assure a specific action of IP3.
STATISTICAL ANALYSIS
Results are reported as mean (SEM). As variability
between batches of oocytes is common, responses
were at times normalized to same-day controls for
each batch. Differences between treatment groups
were analysed using ANOVA and Student’s unpaired
t test, appropriately corrected for multiple comparisons (Bonferroni). P:0.05 was considered significant. Concentration–response curves were fit to the
following logistic function, derived from the Hill
equation:

xn 
y = y min + ( y max − y min ) 1 − n

x50 + x n 

571
figure 1D, curve fitting was performed on averaged
raw data. In the other graphs, because of variability of
oocyte batches from different frogs, data were normalized to the same day control value and then the
fitting was performed on the averaged normalized
values. All fitting values are given in table 1 and fits
were used only if r2 was greater than 0.95.
MATERIALS
Molecular biology reagents were obtained from
Promega (Madison, WI, USA) and other chemicals
were obtained from Sigma (St Louis, MO, USA).
Volatile anaesthetics (desflurane and isoflurane from
Ohmeda Pharmaceutical Products Division, Liberty
Corner, NJ, USA, sevoflurane from Abbott
Laboratories, North Chicago, IL, USA and halothane
from Halocarbon Laboratories, River Edge, NJ, USA)
were delivered using agent-specific vaporizers (carrier
gas:air at 500 ml min91) and equilibrated in Tyrode’s
solution by bubbling for at least 10 min. Oocytes were
exposed to anaesthetics for approximately 5 min
before testing. Bath concentrations of anaesthetics
were verified by gas chromatography (Aerograph 940,
Varian Analytical Instruments, Walnut Creek, CA,
USA) using saline–gas partition coefficients (Psol/gas) at
22⬚C for isoflurane (1.08), sevoflurane (0.52),
halothane (1.20) and desflurane (0.45) (estimations
for desflurane and sevoflurane were based on the
observation that Psol/gas is approximately twice as
great at 22⬚C as at 37⬚C).26–28
Results
where ymax and ymin:maximum and minimum response
obtained, respectively, n:the Hill coefficient, and
x50:concentration at which the half-maximal response
occurs (EC50 for agonist, IC50 for anaesthetics). In
FUNCTIONAL EXPRESSION OF MUSCARINIC AND
ANGIOTENSIN RECEPTORS IN XENOPUS OOCYTES
Control oocytes were unresponsive to Mch 1097 mol
litre91 or AT 1096 mol litre91 (data not shown). Oocytes
Table 1 Curve fitting variables (Hill equation) for Mch concentration–response relationships in Xenopus
oocytes expressing recombinant m1 or m3 muscarinic acetylcholine receptors and inhibition
concentration relationship for isoflurane, sevoflurane and halothane on m1 or m3 muscarinic receptor
signalling values. Values are given as mean (SEM).
EC50
(mol litre91)
Hill
coefficient
Emax (µC)
r2
1.3 (0.15) × 1097
0.83
(0.07)
8.77
(0.14)
0.99
1.02 (0.58) × 1096
0.75
(0.27)
9.95
(0.88)
0.96
EC50
(mol litre91)
Hill
coefficient
Emax (%)
r2
Concentration–inhibition
relationship
m3 muscarinic receptor and
isoflurane
0.45
(0.06)
1.92
(0.43)
99.12
(6.31)
0.98
m1 muscarinic receptor and
sevoflurane
0.32
(0.01)
3.18
(0.32)
97.11
(2.25)
0.99
m3 muscarinic receptor and
sevoflurane
0.75
(0.05)
7.25
(3.8)
103.7
(7.56)
0.96
m3 muscarinic receptor and
halothane
0.88
(0.02)
5.4
(0.66)
98.61
(2.79)
0.99
Concentration-response
relationship
muscarinic m1 receptor
muscarinic m3 receptor
572
injected with mRNA encoding the m1 or AT1A receptor or cDNA encoding m3 muscarinic acetylcholine
receptor, respectively, responded with a transient
inward current in a concentration-dependent manner (fig. 1B, C, D–fitting values in table 1). Agonist
concentrations were chosen to reflect approximate
EC50 values, as determined in fig. 1D. Currents developed within several seconds after agonist application,
British Journal of Anaesthesia
and returned to baseline after approximately 30 s.
Average response sizes were 3.4 (0.8) ␮C for the m1
receptor, 5.0 (1.2) ␮C for the m3 receptor and 1.6
(0.6) ␮C for the AT1A receptor. These findings are
similar to those reported in our previous studies,
where we showed that these currents were ICl(Ca).13 14
To ensure that the responses were mediated by
specific receptors, we attempted to inhibit them with
antagonists. Atropine 1 ␮mol litre91 completely inhibited responses to Mch in oocytes expressing m1 and
m3 receptors (fig. 1B), and the m3-selective antagonist 4-DAMP 100 nmol litre91 inhibited responses of
m3 receptors (data not shown). Similarly, the nonpeptide angiotensin receptor antagonist losartan
10 ␮mol litre91 inhibited AT signalling in cells
expressing AT1A receptors (fig. 1C).
DESFLURANE AFFECT M1 AND M3 MUSCARINIC
SIGNALLING IN A CONCENTRATION-DEPENDENT BIPHASIC
MANNER
We then tested the ability of desflurane at various
concentrations to influence ICl(Ca) induced by appropriate Mch concentrations in oocytes expressing m1
(fig. 2A) or m3 receptors. Signalling of m1 receptors
was enhanced significantly when desflurane
0.46 mmol litre91 was present in the bath: responses
were 71 (28) % greater than control (P:0.001,
t test). Desflurane, at a concentration of 0.70 mmol
litre91, induced no significant change in response
(decrease of 12 (17) %; P:0.47, t test) compared
with control. At higher concentrations, desflurane
inhibited muscarinic signalling: desflurane 0.92 and
1.66 mmol litre91 reduced responses by 43 (11) %
(P:0.003, t test) and 75 (5) % (P:0.001, t test),
respectively (fig. 2B). Similar results were obtained
for m3 signalling (fig. 2C). Thus desflurane had
a biphasic, concentration-dependent effect on
muscarinic receptor function.
ISOFLURANE DID NOT AFFECT M1, BUT INHIBITED M3
MUSCARINIC SIGNALLING
Figure 1 Muscarinic and angiotensin signalling in Xenopus laevis
oocytes. A: Signalling pathway linking activation of the muscarinic
receptor to chloride (Cl9) channel opening. The double line
represents the cell membrane. The agonist acetyl-␤-methylcholine
(Mch) binds to a G protein-coupled receptor, which in turn
activates phospholipase C (PLC). PLC releases
inositoltrisphosphate (IP3) from cell membrane-bound
phosphatidylinositolbisphosphate (PIP2). Calcium (Ca2;) is
released from IP3-sensitive intracellular stores and activates a Ca2;dependent Cl9 channel in the cell membrane. Angiotensin
receptors signal over the same pathway. B: Left: Example of inward
Ca2;-activated Cl9 current (I(CICa)) induced by acetyl-␤methylcholine (Mch) 1097 mol litre91 in an oocyte expressing m1
muscarinic rectors. The current is integrated and expressed as
charge movement (1.9 ␮C). Right: The response to Mch can be
blocked completely by the antagonist atropine (1 ␮mol litre91).
Similar observations were obtained with m3 receptors and their
specific antagonist 4-DAMP (100 nmol litre91; data not shown). C:
Left: Example of inward Ca2;-activated Cl9 current (I(CICa)) induced
by angiotensin II (ATII) 1097 mol litre91 in an oocyte expressing
AT1A receptors. Charge movement is 1.7 ␮C. Right: The response
can be blocked completely by the angiotensin II receptor
antagonist losartan (10 ␮mol litre91). D: Concentration–response
relationship for m1 and m3 muscarinic receptors. Fitting values
using the Hill equation are given in table 1.
In contrast with desflurane, isoflurane had no effect
on Mch-induced m1 muscarinic signalling (fig. 3A).
At very high concentrations (0.75–0.96 mmol litre91)
an inhibitory trend was noted (fig. 3B). However,
these effects were not statistically significant. The
effects of isoflurane on m1 signalling were therefore
different from those of desflurane, especially when
the actions of the two compounds were compared at
MAC-equivalent concentrations. In contrast with its
lack of effect on m1 signalling, isoflurane inhibited
m3 signalling (IC50 0.45 mmol litre91) (fig. 3C).
SEVOFLURANE AND HALOTHANE INHIBITED M1 AND M3
SIGNALLING
Sevoflurane and halothane inhibited m1 and m3
muscarinic signalling. We have reported previously
that halothane inhibits m1 signalling with an IC50 of
0.3 mmol litre91. 13 In this study, we found that it
inhibited m3 signalling with an IC50 of 0.88 mmol
litre91 (fig. 4C). Sevoflurane inhibited m1 and m3 signalling in a similar manner (IC50 0.32 mmol litre91 for
m1 muscarinic inhibition (fig. 4A) and 0.75 mmol
litre91 for m3 muscarinic signalling (fig. 4B)). Thus,
Effects of volatile anaesthetics on muscarinic signalling
573
Figure 2 Influence of desflurane on m1 and m3 muscarinic signalling. A: ICl(Ca) induced by Mch 1097 mol litre91 in
oocytes expressing m1 muscarinic receptors at various concentrations of desflurane (desflurane 0 mmol litre91 (1.5 ␮C),
0.46 mmol litre91 (2.2 ␮C), 0.70 mmol litre91 (1.4 ␮C) and 0.92 mmol litre91 (0.7 ␮C)). B: Desflurane has a biphasic effect
on Mch-induced ICl(Ca) in oocytes expressing m1 receptors: it enhances signalling by 77% at concentrations of 0.46 mmol
litre91, had no significant effect at 0.70 mmol litre91 and reduces signalling by 43% and 75% at desflurane concentrations
of 0.92 mmol litre91 and 1.66 mmol litre91, respectively. C: Desflurane has a biphasic effect on Mch-induced ICl(Ca) m3
muscarinic signalling: it has no significant effect at concentrations of 0.46 mmol litre91 but enhances signalling by 78%
(P:0.03) at concentrations of 0.70 mmol litre91. No effect is seen at 0.92 mmol litre91, whereas signalling is inhibited at
1.66 mmol litre91 (P:0.001). **P:0.01, ***P:0.001 (ANOVA, t test).
Figure 3 Influence of isoflurane on m1 and m3 muscarinic signalling. A: ICl(ca) induced by Mch 1097 mol litre91 in oocytes expressing m1
receptors at various isoflurane concentrations: isoflurane 0.24 mmol litre91 (1.5 ␮C), 0.69 mmol litre91 (1.7 ␮C) and 0.75 mmol litre91
(1.4 ␮C). B: Isoflurane had no effect on Mch-induced ICl(ca) signalling in oocytes expressing m1 receptors (average control response 1.6 ␮C;
number of oocytes tested in parentheses). C: Concentration–inhibition relationship of isoflurane on m3 muscarinic signalling. Fitting values
are given in table 1.
the m1 receptor appeared to display higher sensitivity
to the inhibitory effects of halothane and sevoflurane.
ANGIOTENSIN II AND MUSCARINIC RECEPTORS SHARED
THE SAME INTRACELLULAR SIGNALLING PATHWAY
As we wished to study the effects of the anaesthetics
on angiotensin signalling to localize their site of
action, it was necessary to demonstrate that
angiotensin and muscarinic receptors signal through
the same pathway. We have shown previously that
AT1A receptors expressed in Xenopus oocytes signal
through the IP3 pathway, as signalling can be blocked
specifically by intracellular microinjection of the IP3
receptor antagonist heparin.29 To confirm the experiments reported previously, and to determine if
muscarinic signalling uses the same pathway, we
evaluated the effect on signalling of microinjection of
574
Figure 4 Influence of sevoflurane and halothane on m1 and m3
muscarinic signalling. Average control response 3.4 ␮C for m1 and
5.0 ␮C for m3 muscarinic receptors; number of oocytes tested in
parentheses. A: Concentration–inhibition relationship of
sevoflurane on m1 muscarinic signalling. Calculated IC50 is 0.32
␮mol litre91, Hill coefficient is 3.5. B: Concentration–inhibition
relationship of sevoflurane on m3 muscarinic signalling.
Calculated IC50 is 0.75 ␮mol litre91, Hill coefficient is 6.8. C:
Concentration–inhibition relationship of halothane on m3
muscarinic signalling. Fitting values are given in table 1.
heparin (2 ng nl91, 60 nl, 30 min before recording)
into oocytes expressing m1 or AT1A receptors. m1
muscarinic signalling was inhibited to the same
degree as angiotensin signalling (fig. 5A). Heparin
microinjection into the oocyte inhibited responses to
Mch by 93% compared with controls (fig. 5B). In
contrast, microinjected water had no effect on either
muscarinic or angiotensin signalling (data not
shown). As the oocyte expresses only a single isoform
of phospholipase C, coupling receptor activation to
intracellular Ca2; release,21 our findings indicate that
the muscarinic and AT1A signalling pathways are
identical downstream of the G protein (compare
fig. 1A).
SITE OF ACTION: VOLATILE ANAESTHETICS HAD NO EFFECT
ON ANGIOTENSIN SIGNALLING
As muscarinic and angiotensin signalling use the
same intracellular second messenger pathways, we
investigated the effect of desflurane, isoflurane and
sevoflurane on AT1A receptor function. In contrast
with their variable effects on m1 and m3 signalling,
British Journal of Anaesthesia
Figure 5 Heparin blocks muscarinic and angiotensin signalling.
Top: Response of a control oocyte to Mch 1097 mol litre91 (1.4
␮C), and lack of response after microinjection (30 min prior) of
heparin 120 ng in a 60-nl volume. Bottom: Angiotensin II (ATII)
signalling is similarly inhibited by prior microinjection of heparin.
B: Mch-induced ICl(Ca) is blocked by heparin microinjection 30 min
prior to agonist application. ; and 9 refer to oocytes injected and
not injected with heparin, respectively (average control response
1.6 ␮C; number of oocytes tested in parentheses) (P:0.001 (t
test). C: Desflurane, sevoflurane [S] and isoflurane [I], in common
with halothane,13 had no effect on angiotensin II signalling.
A:
the anaesthetics used had no effect on AT1A signalling, even at high concentrations (fig. 5C). Similar
results were reported for halothane.13 These results
indicate that the actions of these anaesthetics on m1
and m3 signalling are not mediated by effects on the
intracellular pathways, as these are shared by muscarinic and angiotensin receptors.
SITE OF ACTION: VOLATILE ANAESTHETICS HAD NO EFFECT
9
ON IP3-INDUCED CL CURRENTS
To elucidate further the site of action of volatile
anaesthetics on muscarinic signalling, we determined
the effect of the anaesthetics on currents induced by
microinjected IP3 (50 nl of 2 mmol litre91, yielding
approximately 100 ␮mol litre91 intracellularly). IP3induced Cl9 currents resembled the responses
induced by receptor activation (fig. 6A). In contrast,
microinjected water was ineffective (fig. 6B).
Desflurane, even at high concentrations, had no
effect on IP3-induced currents, indicating that this
Effects of volatile anaesthetics on muscarinic signalling
Figure 6 Inositoltrisphospate (IP3) signalling is not inhibited by
high concentrations of desflurane. A: Left: 50 nl of IP3 injected
(final intracellular concentration 100 ␮mol litre91) induced an ICl(Ca)
resembling responses generated by Mch or AT (compare fig. 1B,
1C). Right: Desflurane 0.92 mmol litre91 had no effect on the
oocyte response to IP3. B: Desflurane 0.46 and 0.92 mmol litre91
had no effect on ICl(Ca) generated by intracellular IP3 injection
(average control response 1.3 ␮C; number of oocytes tested in
parentheses). Injection of vehicle (H2O) into the oocyte did not
induce currents.
575
Discussion
Figure 7 Reversibility of anaesthetic actions on muscarinic
receptor signalling. A: ICl(Ca) induced by Mch 1096 mol litre91 to
determine reversibility of inhibition of volatile anaesthetics on m1
muscarinic acetylcholine receptor signalling. The first bar
represents control measurements (4.83 (0.86) ␮C) and the second
bar shows the inhibitory effect of the volatile anaesthetic tested
(2.5% halothane, 3.3% isoflurane, 5% sevoflurane and 18%
desflurane). ICl(Ca) was reduced to 14% (0.7 (0.15) ␮C), 96.6%
(4.64 (1.45) ␮C), 28.15% (1.36 (0.78) ␮C) and 24% (1.16 (0.27)
␮C) by halothane, isoflurane, sevoflurane and desflurane,
respectively. The third bar shows recovery after 10 min of
perfusion with Tyrode’s solution without anaesthetic (*P:0.05,
t test). B: ICl(Ca) induced by Mch 1096 mol litre91 to determine
reversibility of inhibition of volatile anaesthetics on m3 muscarinic
acetylcholine receptor signalling. Three consecutive
measurements were made in different oocytes. The first bar
represents control measurements (15.2 (0.67) ␮C) and the second
bar shows the inhibitory effect of the volatile anaesthetic tested
(2.5% halothane, 3.3% isoflurane, 5% sevoflurane, 18%
desflurane). ICl(Ca) was reduced to 22% (3.43 (2.25) ␮C), 6.5%
(0.93 (0.33) ␮C), 25% (3.8 (1.55) ␮C) and 37% (5.58 (1.88) ␮C)
by halothane, isoflurane, sevoflurane and desflurane, respectively.
The third bar shows recovery after 10 min of perfusion with
Tyrode’s solution without anaesthetic (*P:0.05, t test).
Muscarinic signalling by m1 and m3 receptors was
inhibited in a differential manner by the volatile
anaesthetics studied. Halothane and sevoflurane, in
clinically relevant concentrations, inhibited muscarinic signalling, with the m1 receptor displaying a
higher sensitivity than the m3 receptor. Isoflurane
had no effect on m1 muscarinic signalling, even at
concentrations which would be anticipated to disrupt
membrane structure,28 but significantly inhibited m3
signalling. Desflurane showed a biphasic effect on
both receptors.
Differences in effect between desflurane and
isoflurane are remarkable, as the two compounds differ only in a single halogen atom: a chlorine atom in
isoflurane is replaced by a fluorine atom in desflurane. Thus minor changes in chemical structure can
induce significant pharmacological effects.
The biphasic action of desflurane on muscarinic
signalling is unusual, although not unprecedented.
Ryanodine31 and adenosine32 are known to act similarly in various models. Our study was not designed
to determine the molecular mechanism of this biphasic action. However, we could rule out interactions
with the intracellular signalling pathway, as none of
the anaesthetics had effects on both AT and IP3 signalling. Hence the site of action is most likely at the
muscarinic receptor or (possibly) the associated G
protein. Biphasic responses are explained most easily
by the existence of two different binding sites at the
receptor with different affinities and different agonistic–antagonistic effects when occupied. Mutagenesis
studies may be able to delineate such various binding
sites on the muscarinic receptor.
Our findings should be interpreted in view of the
agent affects the signalling pathway before interaction between IP3 and its receptor (fig. 6B). Similarly,
we have shown that halothane, isoflurane and
sevoflurane have no effect on intracellular IP3
signalling or signalling induced by direct G-protein
activation with GTP␥S.30
REVERSIBILITY OF ANAESTHETIC ACTION
To complete this investigation, we determined the
reversibility of anaesthetic inhibition of muscarinic
signalling. Data are presented in figure 7. In all cases,
anaesthetic inhibition was reversible on wash-out.
576
artificial environment in which the receptors were
expressed. However, muscarinic and angiotensin
receptors have been expressed frequently in the
Xenopus oocyte model system, both by ourselves13 14
and others,33 34 and it is well established that they
function similarly in oocytes as they do in native
cells. Although the receptors are expressed at a lower
temperature than they are normally exposed to, this
has not been shown to result in significant changes in
kinetics or function.
Any attempt to correlate our findings to observed
clinical effects of anaesthetics is arduous, as conclusions drawn from several receptor subtypes cannot be
transferred to a complex organism and its response to
anaesthesia. Although most clinical and experimental
data comparing isoflurane and desflurane show a congruence in their CNS35 36 and circulatory effects,37–39
some unexplained differences exist. Desflurane is
known to exhibit less negative inotropic activity and to
induce smaller decreases in arterial pressure.39 When
heart rate, mean arterial pressure and sympathetic
nerve activity were compared after 10.9% desflurane
anaesthesia 2 min after induction in humans, all variables were enhanced significantly compared with
isoflurane17; after more prolonged anaesthesia there
were no differences between the anaesthetics. This initial period of excitation, when anaesthetic concentrations are relatively low, could result potentially from
interactions with muscarinic signalling pathways. It
would be of great interest to study the interactions
between desflurane and adrenergic receptor functioning, as excitatory effects of desflurane directly on the
␤-adrenergic receptor or on presynaptic ganglia might
also explain its excitatory effects.
CNS muscarinic stimulation raises the level of
consciousness and improves learning.3 It is interesting to speculate if stimulation of m1 signalling (the
most prominent cerebral muscarinic subtype) by low
desflurane concentrations might contribute to the
rapid mental recovery observed with the anaesthetic.40 If so, this might be of particular importance
in elderly patients with limited CNS muscarinic
signalling reserve (e.g. patients with Alzheimer’s
disease). The prominence of the m3 subtype in
bronchial smooth muscle41 and glands1 could explain
in part the irritating effect of desflurane during
induction and emergence from anaesthesia, when
anaesthetic concentrations are relatively low.
In brief, we have demonstrated that desflurane had
a concentration-dependent biphasic effect on m1
and m3 muscarinic receptor functioning, whereas
isoflurane had no effect on m1 signalling but pronounced effects on m3 signalling. We have also
shown that halothane and sevoflurane inhibited m1
and m3 signalling in a dose-dependent manner. This
action is most likely localized at the receptor level, as
the anaesthetics did not influence intracellular signalling pathways. In addition, the anaesthetics had
no effect on angiotensin signalling. In combination
with our findings that halothane inhibits m1 signalling in a concentration-dependent manner,13 these
results show that different volatile anaesthetics affect
m1 and m3 muscarinic signalling in highly divergent
ways, and that small changes in chemical structure of
the volatile anaesthetic or the receptor can significantly affect anaesthetic–protein interactions.
British Journal of Anaesthesia
Acknowledgements
Supported in part by National Institutes of Health grant GMS
52387/02. Dr Nietgen is supported by a grant from the German
Research Society (DFG, Ni 482/1–1) and by the Department of
Anaesthesiology, Medizinische Hochschule Hannover, Germany.
Dr Hönemann is supported by a grant from the Innovative
Medizinische Forschung (IMF, Hö-2–6-II/97–27), Germany.
We thank Prof Dr med. Hugo Van Aken (Westfälische WilhelmsUniversität Münster, Germany) for his support.
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