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Molecular Actions of Propofol on Human 5-HT3A

Receptors: Enhancement as Well as Inhibition by Closely


Related Phenol Derivatives
Martin Barann, PhD BACKGROUND: 5-Hydroxytryptamine type 3 (5-HT3) receptors are excitatory ligand-
gated ion channels which are involved in postoperative nausea and vomiting. They
Isabelle Linden, PhD are depressed by the anesthetic propofol, which, in contrast, enhances the activity
of inhibitory ligand-gated ion channels such as ␥-aminobutyric acid type A
receptors and glycine receptors. To investigate the molecular mechanisms respon-
Stefan Witten, PhD sible for these contrasting actions, we examined the kinetics of the action of
propofol and its lesser hydrophobic derivatives 2-isopropylphenol and phenol on
Bernd W. Urban, PhD human 5-HT3A receptors.
METHODS: Human embryonic kidney 293 cells containing stably transfected cDNA
of the human 5-HT3A receptor subunit were patch clamped (excised outside-out
patches). Drugs were applied with a fast solution exchange system (within 2 ms) and
their concentrations were determined by high performance liquid chromatography.
RESULTS: When applied in equilibrium (60 s before and during the 5-HT pulse),
propofol inhibited human 5-HT3A receptors (IC50 ⫽ 18 ⫾ 1.0 ␮M). In equilibrium,
the less hydrophobic 2-isopropylphenol was surprisingly a similarly potent inhib-
itor of human 5-HT3A receptors (IC50 ⫽ 17 ⫾ 3.2 ␮M), whereas phenol was
considerably less potent (IC50 ⫽ 1.6 ⫾ 0.2 mM). Varying the duration of drug
application before currents were elicited, and then applying 5-HT still in the
presence of the drug revealed that fast and slow processes contributed to the
(equilibrium) effects of propofol (␶IN-1 ⫽ 35 ms and ␶IN-2 ⫽ 4.8 s), 2-isopropylphenol
(␶IN-1 ⫽ 64 ms and ␶IN-2 ⫽ 6.6 s), and phenol (␶IN-1 ⬍ 10 ms, ␶IN-2 ⫽ 20.4 s). When
applied transiently together with 5-HT (open channel application), propofol
depressed currents and accelerated the 5-HT-induced desensitization significantly,
whereas, in contrast, 2-isopropylphenol and phenol increased currents and slowed
desensitization. Slowed desensitization was also observed for 5-hydroxyindole (1
mM), a 5-HT derivative, but not for benzene. The fast effects of phenol,
2-isopropylphenol, and propofol were more pronounced when the 5-HT concen-
tration was decreased from 30 to 3 ␮M, whereas the slow effects were not sensitive
to 5-HT.
CONCLUSIONS: At least two separate inhibitory actions on 5-HT3A receptors could be
identified for propofol, whereas the enhancing action seen for the two related
smaller phenol derivatives could no longer be detected. 5-HT-dependent and
5-HT-independent interactions could be distinguished for all three drugs. Propofol
was less potent than expected from its hydrophobic properties. Underlying
mechanisms appear to involve the phenolic hydroxyl group, hydrophobic interac-
tions, and steric restrictions.
(Anesth Analg 2008;106:846 –57)

T here is good evidence that several, weak interac-


tions with proteins are characteristic of anesthetic
nonspecific hydrophobic interactions.1 Although not
differing much in their hydrophobicity, intravenous
action, involving specific polar interactions and anesthetics appear to be generally more potent than
inhaled anesthetics, suggesting that additional polar
From the Department of Anesthesiology and Intensive Care interactions are responsible for the greater anesthetic
Medicine, University of Bonn, Bonn, Germany. potency.1 As a necessary step towards rational anes-
Accepted for publication November 13, 2007. thetic drug design, functional groups that might in-
Supported by the DFG (BA 1454). crease anesthetic potency should be identified and
I.L. and S.W. contributed equally to this work. distinguished from those portions of the molecule that
Address correspondence and reprint requests to Dr. Martin have a potential of causing undesirable clinical side
Barann, Department of Anesthesiology and Intensive Care Medi-
cine, University of Bonn, Sigmund-Freud Str. 25, 53127 Bonn, effects, such as emesis. Before this can be done, all
Germany. Address e-mail to martin.barann@ukb.uni-bonn.de. chemical groups of an anesthetic molecule have to be
Copyright © 2008 International Anesthesia Research Society characterized in terms of their ability to interact with
DOI: 10.1213/ane.0b013e318162ca7c
functional proteins.

846 Vol. 106, No. 3, March 2008


Figure 1. (a) Structures of phenols and
benzene with their respective experi-
mental log P values, taken from the
PhysProp Database (Syracuse Research
Corporation; http://www.syrres.com/
esc/physdemo.htm), see also Tetko et
al.16 (b) Structure of 5-hydroxyindole
(left) and 5-HT (right).

Propofol is clinically the most extensively used IV actions of propofol on human 5-HT3A receptors. Using
anesthetic. Compared with other IV anesthetics, such the patch-clamp technique (excised outside-out patch
as barbiturates or etomidate, the molecular structure mode) and a fast solution exchange system, we aimed
of propofol is simple and symmetric (Fig. 1), consist- (a) to test whether the human 5-HT3A receptor is also
ing of a plain benzene ring and a phenolic hydroxy- a sensitive target for propofol; (b) to identify separate
group located between two identical alkyl residues. components of action by, first, applying propofol to
Thus, any systematic study of IV anesthetic interac- different conformations of the 5-HT3 receptor and,
tions may start with this clinically important drug. For second, by comparing its actions with structurally
reasons not yet understood, propofol, on the molecu- related derivatives (Fig. 1); and, thus, (c) to shed light
lar level, suppresses ion flux through excitatory ion on the different responses to propofol by excitatory
channels but it enhances ion flow through inhibitory and inhibitory ligand-gated ion channels.
ion channels1– 4 Propofol has the advantage over in-
haled anesthetics of causing less emesis, if any at all.5
The 5-HT3 receptor is a ligand-gated excitatory ion
METHODS
channel6,7 which is located in the periphery and Cell Culture
within the central nervous system. It plays a major A human embryonic kidney (HEK)-293 cell line
role in the modulation of nausea and vomiting,8,9 containing stably transfected human 5-HT3A receptors
consistent with the fact that 5-HT3 receptor antago- was used.12 HEK-293 cells were grown as monolayer
nists are commonly used antiemetic drugs.10 The on culture plates (Nunc) in DMEM Nutrient Mix F12
amino acid sequences of the pentameric 5-HT3 recep- medium containing 10% heat inactivated fetal calf
tors show homologies with excitatory nicotinic acetyl- serum, penicillin (100 IU/mL), streptomycin (100
choline receptor channels,11 and, to some extent, even ␮g/mL), geneticin (0.75 mg/mL), and glutamine (292
with inhibitory ␥-aminobutyric acid type A (GABAA) ␮g/mL). Cells were cultured at 37°C in humidified
receptors and glycine receptors. 5-HT3A receptors atmosphere (5% CO2). For patch clamp experiments,
are the only functional homopentameric 5-HT3 re- cells were subcultured in monodishes (Nunc, 35 mm
ceptors, and because of their well defined stoichi- diameter) 7–11 days before an experiment.
ometry, they provide a useful model studying the
molecular actions of drugs such as general anesthet- Drugs and Solutions
ics and cannabinoids.12,13 5-HT (the creatinine sulfate salt) was obtained
A previous study has shown that propofol sup- from Sigma (München, Germany). Propofol (free sub-
presses 5-HT3 receptors (mouse) by more than one stance, without vehicle) was obtained from RBI (MA,
action, reducing the peak current amplitude and ac- USA), propofol derivatives, 5-hydroxyindole, and
celerating the desensitization process.14 In the present benzene from Sigma (München, Germany). 5-HT so-
study, we used an experimental approach that al- lutions were prepared daily from 25 mM aqueous
lowed us to differentiate kinetically between different stock solutions stored at ⫺20°C. Propofol and

Vol. 106, No. 3, March 2008 © 2008 International Anesthesia Research Society 847
2-isopropylphenol were prepared daily from ethanol the stability of the excised patches and the reproduc-
stock (1 M) by dilution (e.g., 1:10,000 resulting in 100 ibility of results.
␮M) in extracellular buffer and stirring for 3 h in glass
bottles. The highest resulting ethanol concentrations at Fast Solution Exchange System
the highest drug concentrations were 2.2 mM (propo- A multitube perfusion system with an exchange
fol) and 6.6 mM (2-isopropylphenol); they had no rate (solution exchange) below 2 ms (RSC 200, Bio-
effect on 5-HT control currents. Ethanol concentra- logic, France) was used.12 The drug application sys-
tions were proportionally lower at lower drug concen- tems were equipped with five separate tubes and inert
trations. Benzene, phenol, and 5-hydroxyindole were materials, such as Teflon tubing and glass, to avoid
soluble in the extracellular buffer without any further loss of hydrophobic drugs.15
vehicle. All concentrations of the lipophilic drugs
propofol and 2-isopropylphenol (log P ⫽ 3.79 and 2.88, Drug Application Modes
respectively) were measured by high performance Three different protocols of drug application were
liquid chromatography [HPLC,15]. For this purpose, used12:
samples of drug solution were collected from the
1. Equilibrium application: Continuous exposure to
tubes directed to the patches of the application system.
the drug 60 s before and during the application
It was found, for example, that near the IC50 value an
of 5-HT;
expected concentration of 25 ␮M propofol resulted in
2. Open channel application: No drug application
(mean ⫾ standard deviations) 14 ⫾ 2.5 ␮M (n ⫽ 40)
prior to the 5-HT pulse, only simultaneous ap-
propofol and 30 ␮M 2-isopropylphenol resulted in
plication of drug and 5-HT;
21 ⫾ 4.7 ␮M (n ⫽ 11).
3. Closed channel application: Pre-exposure to the
drug 60 s before but not during the 5-HT
Electrophysiology application.
In previous studies, we described human 5-HT3A
receptors as activated by 5-HT in a concentration- Limitations of Data Aquisition
dependent manner (EC50 values, 5–9 ␮M) and that the Although more than one measurement could be
resulting currents were sensitive to the 5-HT3 receptor performed on a typical patch, the interval between
antagonist, ondansetron. For the experiments in this measurements had to be at least 1 min because of the
study, we used identical conditions, including electro- slow kinetics of recovery from desensitization. To
physiological methods, solution application systems,12,13 correct for small rundown of currents when present
and a concentration of 30 ␮M 5-HT (unless specified and to discard inconsistent data, the measurement of
otherwise) because it elicits a reproducible, nearly each data point involved a three-fold repetition of the
maximal (85%) signal. sequence: control, drug test, recovery (⫽control for
Before starting patch-clamp recordings, the culture next drug test), lasting a minimum of six min. The
medium was replaced by “extracellular” solution of limited but varying life times of such patches (nor-
the following composition: NaCl 150 mM; KCl 5.6 mally ranging from 5 to 25 min, in a few instances 1 h)
mM; CaCl2 1.8 mM; MgCl2 1 mM; HEPES 10 mM; resulted in inhomogeneous data sets with regard to
d-Glucose 20 mM; pH 7.4. d-Glucose was omitted in the number of different data points measured on each
the extracellular solution used for superfusion of the patch. However, each data point was measured on at
excised patches. Patch pipettes were filled with intra- least three different patches. As the recording of entire
cellular solution containing: KCl 140 mM; EGTA 10 concentration–response curves on single patches was
mM; MgCl2 5 mM; HEPES 10 mM; pH 7.4 and had not possible, mean IC50 values and their standard
resistances of 2– 4 M⍀. Experiments were performed deviations could not be calculated. Instead, IC50 val-
at room temperature (20°C–26°C). ues and estimates of their standard errors were calcu-
Patch pipettes were manufactured from Borosili- lated from fitting a single concentration–response
cate glass capillaries (Kwik-Fil™, World Precision curve to the entire data set (Graph Pad Prism 3.02).
Instruments, USA) using a pipette puller (List L/M- Highly hydrophobic substances have a tendency to
3P-A, List Electronic, Darmstadt, Germany). The seal absorb in the tubing,15 and, therefore, drug concentra-
resistances (excised outside-out patches) were 1– 6 G⍀. tions could not be randomized. Most experiments
For current measurements, we used a patch-clamp were started with the lowest hydrophobic drug con-
amplifier (EPC-7, List) in combination with an exter- centrations and continued with increasingly larger
nal low pass filter set at 1 kHz (Frequency Devices, concentrations. Otherwise, the superfusion system
MA). Data were digitally recorded at a sampling rate would have had to be extensively rinsed and re-
of 2 kHz with a Digidata 1200 (Axon Instruments, checked for remaining drug after each experiment,
Foster City, CA) interface. Clampex-6 software (Axon) requiring unrealistic experimental time for each ex-
was used for the recording protocols. Five-hundred periment. Instead, a second, very different superfu-
milliseconds before 5-HT exposure, the membrane sion system and set-up was used to check whether
potential was stepped from a holding value of 0 mV to there was any indication that the sequence (history) in
⫺100 mV. These conditions were chosen to optimize which experiments were performed had any impact

848 Propofol Derivatives and 5-HT3A Receptors ANESTHESIA & ANALGESIA


Figure 2. (a, c, e; left panels) Effects of propofol, 2-isopropylphenol, and phenol (equilibrium application) on 5-HT (30
␮M)-induced currents (original traces from three patches). Note that all three drugs caused suppression of the peak current;
however, propofol speeded current decay, whereas 2-isopropylphenol and phenol slowed current decay (statistical analysis
in Fig. 5). (b, d, f; right panels) Time courses of wash-in of drugs. Currents induced by 5-HT (30 ␮M) in the presence of drug
are plotted against the time duration for which the 5-HT3A receptors were pre-exposed to the respective drug. Data points
(circles) represent percent of the control currents after 60 s wash with propofol-free buffer (means ⫾ sd, n ⫽ 4 –10 different
experiments). A fast and slow inhibitory process was observed for propofol, and a fast potentiating and a slow inhibitory
effect was detected for 2-isopropylphenol. Phenol also showed a slow inhibitory effect, whereas potentiation was immediate.
Wash-in curves for propofol and 2-isopropylphenol were fit biexponentially: i ⫽ imax1 * exp(⫺t/␶IN-1) ⫹ (100-imax1 ⫺ Plateau) *
exp(⫺t/␶IN-2) ⫹ Plateau. The wash-in curve for phenol was fit monoexponentially: i ⫽ imax * exp(⫺t/␶IN) ⫹ Plateau.

on the outcome. Whenever evidence for dependence could not be estimated in advance with a power
on “history” was found, the underlying reasons were analysis.
investigated. Only in artifactual situations was a de-
pendence on history found, otherwise there was no Partition Coefficients and Predictions of Potency from
evidence or suggestion that the sequence in which Meyer-Overton Correlations
experiments were performed matters. The following Meyer-Overton correlations1 have
As this was an exploratory study, the number of been used to estimate anesthetic potencies arising
experiments needed to reach statistical significance only from nonspecific, hydrophobic actions. For

Vol. 106, No. 3, March 2008 © 2008 International Anesthesia Research Society 849
ligand-gated ion channels pooled for a wide range
of anesthetics (Fig. 12b in Ref. 1), the parameters
were: log (IC50) ⫽ ⫺1.568 to 0.9898 * log (Poctanol/water);
for ligand-gated ion channels pooled for inhaled an-
esthetics only (Fig. 12d in Ref. 1): log (IC50) ⫽ ⫺1.178
to 0.8975 * log (Poctanol/water); for voltage-gated ion
channels pooled for IV anesthetics only (Fig. 12c,
published in Ref. 1): log (IC50) ⫽ ⫺0.8064 to 0.9306 *
log (Poctanol/water). Experimental octanol/water parti-
tion coefficients, Poctanol/water, are taken from the
PhysProp Database (Syracuse Research Corporation;
http://www.syrres.com/esc/physdemo.htm), see also
Figure 3. Concentration–response curves for three phenols
Tetko et al.16 on 5-HT (30 ␮M)-induced peak-currents. The bottoms and
tops of the fits were set constant at 0% and 100%, respec-
Data Analysis and Statistics tively. Drugs were applied in equilibrium (means ⫾ sd, n ⫽
Analysis of the original current traces (baseline 4 –10 different patches).
adjustment, determination of peak currents and cur-
rent kinetics) was performed with Clampfit 8 software
(Axon Instruments, Foster City, CA). Graph Pad Prism both drugs inhibited 5-HT-induced currents as can be
3.03 software (Graph Pad, CA) was used to create seen in the traces of Figure 2c and e. As a third
graphics. The concentration–response curves were fit- similarity with propofol, wash-in experiments (drug
ted by the Hill equation, i ⫽ [ICn50/(cn ⫹ ICn50)], i is application for varying durations before 5-HT and
the remaining peak current as fraction of the maximal during 5-HT exposure) revealed that fast and slow
(control) current, c is the drug concentration, n is the processes (Fig. 2d and f) contribute to the effects of
Hill coefficient, and IC50 is the drug concentration 2-isopropylphenol (␶IN-1 ⫽ 64 ms and ␶IN-2 ⫽ 6.6 s) and
causing half-maximal effect. Potencies are expressed phenol (␶IN-1 ⬍10 ms, ␶IN-2 ⫽ 20.4 s).
as IC50 values plus standard error as calculated by The concentration–response curves of all three drugs
Graph Pad Prism 3.03. Differences between single data
could be fitted to Hill-equations (Fig. 3 and Table 1). The
points were tested for significance with either paired
IC50 values for propofol and 2-isopropylphenol were
or unpaired t-tests (Excel and Prism 3.03). Differences
about the same (18 ⫾ 1, 17 ⫾ 3.2 ␮M, respectively),
were considered significant when P values for the re-
whereas the slope of the concentration–response curve
spective test were ⬍0.05. Values are reported as means ⫾
for propofol (nHill ⫽ 2 ⫾ 0.2) was about two times
standard deviations (sd), unless stated otherwise.
steeper than that of 2-isopropylphenol (nHill ⫽ 1 ⫾ 0.2,
Table 1). Phenol was considerably less potent (IC50 ⫽
RESULTS
1.6 ⫾ 0.2 mM).
Propofol (0.5–160 ␮M) did not cause an activation However, propofol (ⱖ6 ␮M) increased the speed of
of human 5-HT3A receptors in the absence of 5-HT
the current desensitization (e.g., by 51% at 18 ␮M) as
when it was applied to patches which before had
did many other anesthetics before,12 whereas both
shown a pronounced response to 5-HT. Propofol
2-isopropylphenol (ⱖ20 ␮M) and phenol (ⱖ1 mM)
inhibited 5-HT-induced currents reversibly with an
caused an extensive slowing of current decay (e.g., by
IC50 ⫽ 18 ⫾ 1 ␮M (Figs. 2a and 3) when applied under
214% at 21 ␮M 2-isopropylphenol and by 188% at 1
equilibrium condition (see Methods). Next, we exam-
mM phenol). This can be seen in the traces of Figure 2
ined the kinetics of washing in the propofol effect.
Pre-exposing the patch to propofol for various durations and it is substantiated by the subsequent statistical
(32 ms to 60 s) before applying 5-HT in the presence of analysis (see below).
propofol resulted in a wash-in time course of propofol As these observations may suggest different pro-
that was characterized by a fast (␶IN-1 ⫽ 35 ms) and a cesses, an attempt was made to separate these (Fig. 4).
slow (␶IN-2 ⫽ 4.8 s) process (Fig. 2b). Fast actions were separated by coapplying propofol
In an attempt to further characterize the slow and and the phenol derivatives simultaneously with 5-HT
the fast wash-in component of the propofol effect, but not before (open channel application). In this
2-isopropylphenol and phenol were examined. Both manner, their effects on peak currents could be inves-
substances are straight derivatives of propofol, in tigated within a time frame of approximately 20 ms,
which the balance of polar and hydrophobic proper- whereas their effects on current desensitization could
ties is shifted towards lesser hydrophobicity. In the be observed for a period of 50 ms to several 100 ms.
absence of 5-HT, neither 2-isopropylphenol (0.01–1 Slow drug actions were separated by exposing drugs
mM) nor phenol (0.1–3 mM) caused direct activation to the patches for 60 s exclusively before 5-HT was
of 5-HT3A receptors in patches which in a preceding applied (closed channel application). The combined
control had shown pronounced response to 5-HT. effects of open channel and closed channel application
When applied continuously (equilibrium condition), (fast and slow actions of propofol) were studied by

850 Propofol Derivatives and 5-HT3A Receptors ANESTHESIA & ANALGESIA


Table 1. Parameters of the Hill Equations Derived from the Concentration–Response Curves (Inhibition of Peak-Currents Induced by
30 ␮M 5-HT) of Phenols
IC50 (␮M)/Hill coeff.

Propofol 2-Isopropylphenol Phenol


Equilibrium (human) (fast and slow effects) 18 ⫾ 1/2.0 ⫾ 0.2 17 ⫾ 3.2/1.0 ⫾ 0.2 1620 ⫾ 223/1.1 ⫾ 0.2
Equilibrium (mouse) 14 ⫾ 1/1.5 ⫾ 0.2a
Open channel (fast effect) 28 ⫾ 2.6/1.2 ⫾ 0.1 Potentiation Potentiation
Closed channel (slow effect) 22 ⫾ 2.5/1.2 ⫾ 0.2 14 ⫾ 1.1/1.1 ⫾ 0.1 1035 ⫾ 108/1.1 ⫾ 0.1
Octanol/water partition coefficient 6170 760 29
Three different drug-application modes were used.
a
The data for equilibrium (mouse) are from Barann et al.14 For octanol/water partition coefficients see Methods.

Figure 4. Effects of three phenols on 5-HT (30 ␮M)-induced currents, using three different drug application modes (original
current traces). Traces for propofol were obtained from two patches (separate patch for the closed channel application); those
for 2-isopropylphenol and phenol from single patches, respectively.

applying drugs 60 s before and also during the acti- mM, respectively (Fig. 5). The slow action (closed-
vation of the 5-HT3A receptor by 5-HT (equilibrium channel application) of 2-isopropylphenol and phenol
application). left the time constants of desensitization unchanged
The fast actions (open-channel) of 2-isopropylphenol (Fig. 5), whereas both substances suppressed the peak
and phenol (Fig. 4), in contrast to propofol (which amplitudes in a concentration-dependent manner
caused significant peak amplitude reduction and ac- (IC50 values ⫽ 14 ⫾ 1.1 ␮M and 1.04 ⫾ 0.1 mM,
celeration of the desensitization at concentrations ⱖ6 respectively).
␮M, Fig. 5), consisted of an increase of the peak However, whenever the fast process was visible
amplitude and a pronounced slowing of the desensi- (both in the open-channel and in the equilibrium
tization process at drug concentrations ⱖ20 ␮M and 1 application), the desensitization time constants changed

Vol. 106, No. 3, March 2008 © 2008 International Anesthesia Research Society 851
Figure 5. Effects of phenols on the current decay-kinetics of 5-HT (30 ␮M)-induced currents using three different drug
application modes (note the different scaling of the y-axes). Results are expressed as percentage of the time constants ␶off of
the control currents in the absence of drug (100%, indicated by dashed lines); the mean for all ␶off values of the respective
control currents was 117 ⫾ 73 ms, n ⫽ 43, which is a typical value for human 5-HT3A receptors, see discussion. Data are shown
as means ⫾ sd, n ⫽ 3–10 different patches. *Significant difference from the control value in the absence of drug (P ⬍ 0.05,
paired t-test).

with concentration in the same manner (Fig. 5): they was changed (Fig. 6, bottom row), in contrast to the
accelerated with increasing propofol concentration fast (open channel application) actions of the three
(ⱖ6 ␮M) and they were successively slowed by in- drugs. Here, in the case of propofol, 3 ␮M 5-HT-
creasing concentrations of either 2-isopropylphenol evoked currents were much more potently suppressed
(ⱖ20 ␮M) or phenol (ⱖ1 mM). During closed-channel (by 58%) than 30 ␮M 5-HT-evoked currents (by 14%),
application (slow action), desensitization time con- whereas 2-isopropylphenol or phenol produced much
stants were not affected by any of the three drugs (Fig. larger current increases at 3 ␮M 5-HT (by 206% and
5). No significant effects of propofol, 2-isopropylphenol, 197%, respectively) than at 30 ␮M 5-HT (by 11% and
and phenol on the current activation kinetics were 13%, respectively (Fig. 6, middle row).
observed in any application mode. In the equilibrium application, the inhibition by 14
An additional series of experiments tested for each ␮M propofol was larger than in either of the other
application mode separately whether the drug effects application modes (open channel and closed channel),
depended on the agonist (5-HT) concentration (Fig. 6). irrespective of the 5-HT concentration (Fig. 6, left
We compared the effects of propofol and its deriva- panels). Although increases in current amplitudes by
tives when currents were elicited either by a nearly 2-isopropylphenol and phenol were still present for
saturating concentration of 5-HT (30 ␮M) or a concen- current peaks evoked with the lesser 5-HT concentra-
tration well below the EC50 for 5-HT (5–9 ␮M) but tion in the equilibrium application (Fig. 6, upper
high enough (3 ␮M) to evoke currents amenable to trace), they were reduced compared with the open
reproducible curvefitting. The slow (closed channel channel application. However, at the higher 5-HT (30
application) inhibitions of current amplitudes by ␮M) concentration in the equilibrium application, in
any of the three substances, propofol (14 ␮M), 2- the presence of either 2-isopropylphenol (21 ␮M) or
isopropylphenol (21 ␮M), or phenol (1 mM), were not phenol (1 mM), inhibition of current amplitudes (by
significantly different when the 5-HT concentration 37% and 30%, respectively) was observed (Fig. 6,

852 Propofol Derivatives and 5-HT3A Receptors ANESTHESIA & ANALGESIA


Figure 6. Effects of three phenols on 3 ␮M and 30 ␮M 5-HT-induced currents, using three different drug application modes.
Any of the three substances propofol, 2-isopropylphenol or phenol caused suppression of current in the closed channel
application, which did not depend on the concentration of 5-HT. Note the different current scales in the open channel or in
the equilibrium applications, since propofol caused inhibition while isopropylphenol or phenol produced current potentia-
tion. Significance (P ⬍ 0.05, unpaired t-test) refers to the difference between relative drug effects on current amplitudes
elicited by either 3 ␮M or 30 ␮M 5-HT (each reduction normalized to the respective 3 and 30 ␮M 5-HT control in the absence
of drug).

upper row), in contrast to the current increases in the phenolic OH group (1 mM) was applied to the 5-HT3
open channel application. receptor in the open-channel mode, it also lead to a
When the slow and the fast effects on current slowing of the current desensitization time constant
amplitudes were multiplied for each drug, then the (Fig. 7, left panel). On the other hand, benzene (3 mM)
product obtained both for the lesser 3 ␮M 5-HT no longer showed a slowing of desensitization but
concentration (Fig. 6, upper row, dotted line on the rather an acceleration (Fig. 7, right panel).
left) and the higher 30 ␮M 5-HT concentration (Fig. 6,
upper row, dotted line on the right) came very close to
the effect measured in the equilibrium application DISCUSSION
(Fig. 6, upper row, respective bars through which Comparison with Previous Study
dotted line runs). The present study confirms that human 5-HT3A
A phenolic group is also part of the agonist of the receptors are also inhibited by 30 ␮M 5-HT are also
5-HT3 receptor. When ethylamine is removed from inhibited by propofol with a potency similar to that
5-HT, 5-hydroxindole is obtained, which no longer recorded for murine 5-HT3 receptors.14
directly activates currents through 5-HT3A receptors The slope of the concentration–response curve for
(data not shown). When this aromatic alcohol with its propofol (at 30 ␮M 5-HT) is steeper for the human

Vol. 106, No. 3, March 2008 © 2008 International Anesthesia Research Society 853
Figure 7. Effect of 5-hydroxyindole (1 mM) and benzene (3 mM) on 5-HT-induced currents in two outside-out patches.
5-Hydroxyindole or benzene was exclusively applied together with the 1.2 s 5-HT pulse. Note that the traces for control and
wash superimpose. 5-Hydroxyindole decreased the current decay kinetics almost two-fold (i.e., slow down), which remained
monoexponential (time constant ␶off). In contrast, benzene accelerated the current decay. Typical experiments from four
experiments (each) are shown.

receptor (Table 1). Other differences between human hydrophobic by the addition of purely hydrophobic
and murine receptors are the biphasic wash-in of the isopropyl-groups. By adding an isopropyl-group to
propofol effect reported here and the fact that the fast phenol, the resulting 2-isopropylphenol becomes
wash-in time constant (35 ms) is almost a factor of 3 more potent than predicted by lipophilicity. In con-
faster than the current desensitization time constant, trast, when the second isopropyl-group is added,
␶off, of the 5-HT3A receptor [this study: ␶off ⫽ 117 ⫾ 73 turning 2-isopropylphenol into propofol, the IC50 re-
ms, n ⫽ 43 patches; entire data pool from our studies: mains unchanged, although hydrophobicity alone
␶off ⫽ 112 ⫾ 100 ms, n ⫽ 1760 patches17], whereas the should have made propofol roughly nine times more
slow wash-in time constant is considerably slower potent. This observation, together with the differences
than ␶off. This latter observation goes against the in the slopes of the respective concentration–response
hypothesis proposed in an earlier paper14 that propo- curves, suggests additional interactions to be involved
fol triggers an unmodified desensitization mechanism that are antagonistic to hydrophobic actions. How-
in the absence of 5-HT with the same kinetics as does ever, as has been found before for many other anes-
5-HT. thetics,1 on average the three substances follow the
correlation with hydrophobicity very well, as propofol
Slow Action scatters by approximately the same factor to the right
Propofol and its derivatives clearly interact with as 2-isopropylphenol scatters to the left.
human 5-HT3A receptors, even when they are applied
to the receptor exclusively in the absence of any 5-HT. Fast Action
Although not with the same, but with much slower, Exclusive exposure of 5-HT3A receptors to propofol
kinetics than for the unmodified desensitization pro- or its derivatives during the 5-HT pulses (open chan-
cess hypothesized above, the compounds investigated nel application) sufficed to produce effects on the
in this study may still shift activable 5-HT3A receptors amplitudes of the elicited currents as well as on their
into an anesthetic-related desensitized state. The frac- desensitization time constants (Figs. 4 – 6). Propofol
tion of channels inhibited by the slow action (closed suppressed the amplitudes of the currents and caused
channel application) may be in this state. The actions them to desensitize more rapidly, whereas phenol and
consist of a reduction in the peak current of the test 2-isopropylphenol, in contrast, increased their ampli-
pulse for all three substances (Fig. 4), without any tudes and slowed desensitization in a concentration-
effect on the current decay time constant (Fig. 5), even dependent manner. Increases in peak amplitudes have
though the duration of superfusion by propofol or its been observed for volatile anesthetics18 –21 and alco-
derivatives had lasted long enough so that the reduc- hols,22–24 whereas retardation of desensitization has
tion in peak current had reached a steady state. been reported for morphine.25
The IC50 of phenol for slow action (closed channel The effects on the amplitudes were more clearly
application, Table 1) is very close to the one predicted seen for lower 5-HT concentrations, whereas the ef-
by the Meyer-Overton relation for nonspecific inhibi- fects on the time constants of desensitization were
tion by phenol (predicted, see Methods: 970 ␮M; obvious at both 5-HT concentrations. These observa-
measured: 1035 ␮M). For 2-isopropylphenol, the pre- tions may, at least partly, be explained by the kinetics
diction under-estimates the actual potency by a factor of wash-in (Fig. 2), as the fast action may not have
of 3 (predicted: 38 ␮M; measured: 14 ␮M), whereas for fully developed by the time the current peak is
propofol it over-estimates the potency by a factor of 4 reached (about 20 ms at 30 ␮M 5-HT), whereas desen-
(predicted: 5 ␮M; measured: 22 ␮M). This may be an sitization can be observed for a duration of several 100
indication that more than added hydrophobicity is ms. Whether or not additional competition between
involved in the actions of compounds made more these drugs and 5-HT is involved, could only be

854 Propofol Derivatives and 5-HT3A Receptors ANESTHESIA & ANALGESIA


decided in an additional study, which would be from Meyer-Overton correlations (see Methods). Can-
difficult and extensive, as the 5-HT activation curve didates for specific interactions are hydrogen bonds
itself is bell-shaped,26 which is typical for ligand-gated formed by the phenolic hydroxygroup, van der Waals
ion channels.27,28 interactions with the ␲-electrons of the aromatic ring
of the phenol and its derivatives, a lipophilic pocket of
Additivity of Fast and Slow Actions limited dimensions, or a combination of some or all of
The effects of propofol and its derivatives on the these possibilities.
desensitization time constants persisted as long as The phenolic OOH group seems to be involved in
they were administered together with 5-HT (Figs. 4 the effect of increasing current amplitudes and slow-
and 5), whether or not they had been given before the ing desensitization as suggested by the failure of
5-HT application as well (equilibrium or open channel benzene to cause either effect (Fig. 7). The only differ-
application). Thus, the concentration-dependent accel- ence between phenol and benzene is the OOH group,
eration (propofol) or retardation (propofol deriva- which benzene is lacking. There is other circumstantial
tives) of the desensitization time constant requires the evidence that phenolic groups of a molecule may slow
coapplication with 5-HT, i.e., an open channel confor- the desensitization of 5-HT3 receptors. Morphine,
mation during drug application. which also contains a phenolic OOH group, causes a
There is additional evidence that fast and slow pronounced slowing of 5-HT3A receptor current de-
actions appear to be independent and additive: When cay.25 5-Hydroxyindole, a 5-HT derivative lacking the
the effects of the open-channel application and the ethylamine group, has lost the intrinsic activity of
closed-channel applications on current amplitudes are eliciting currents through 5-H3A receptor, but it does
multiplied, the resulting product (see dotted lines in slow current decay of 5-HT3 receptors [Ref. 40 and
Fig. 6, upper panels) is close to the measured result Figure 7].
under equilibrium condition (bars in the same figure). When first synthesizing and exploring phenol de-
This observation supports the hypothesis of at least rivatives suitable for general anesthesia, James and
two separate and superimposing processes postulated Glen41 already noted not only the importance of their
in our previous paper.14 lipophilic character and H-bond donor/acceptor
properties but they also realized that steric consider-
Comparison with Other Ligand-Gated Ion Channels ations mattered. They found that potency and kinetics
Comparing the concentrations at which propofol appeared to be a function of both the lipophilic
interacts with other ion channels, one finds them all to character and the degree of steric hindrance exerted by
lie within a similar range, as observed in this study ortho substituents. The phenolic group, in conjunction
(IC50 ⫽ 18 ␮M; more potent at lower 5-HT concentra- with aliphatic substituents in the ortho position, has
tions, Fig. 6). IC50 values of 20 ␮M,29 between 4.6 and been implicated in the direct activation of chloride
23 ␮M,30 and between 6 and 25 ␮M31 have been currents through GABAA receptors in the absence of
reported for human brain, skeletal muscle, and rat GABA.42 Additional evidence that steric consider-
brain of sodium channels, respectively. The voltage- ations are important comes from the observation by
dependent potassium channel from SH-SY5Y human Krasowski et al.37 that 2,6-di-sec-butylphenol did en-
blastoma cell lines was suppressed with an IC50 ⫽ 44 hance GABAA receptor, whereas 2,6-di-tert-butylphenol
␮M.32 For the neuronal type ␣4␤2 nicotinic acetylcho- did not. In addition, hydrophobic groups of a drug
line receptor channel, IC50 values of 19 ␮M33 or 4.5 may show specificity, for example, when they exceed
␮M34 have been found, whereas the muscle subtype the molecular volume available in a hydrophobic
␣␤␥␦ was half-suppressed by 46 ␮M.34 Depending on pocket. Thus, fewer hydrophobic sites may be avail-
subtype, GABAA receptors are enhanced by 2.6 ␮M able to propofol than to either 2-isopropylphenol or to
(␣1␤3␥235), 4.6 ␮M (␣6␤3␥235), 8.7 ␮M (␣2␤136), or 39.7 phenol.
␮M (␣1␤2␥2S37) propofol. Enhancement is also ob- This may be why inhaled anesthetics with too large
served for the glycine receptor channel ␣1␤ with an a molecular volume no longer cause net enhancement
EC50 ⫽ 12.5 ␮M.38 All these IC50 and EC50 values of 5-HT3 receptors.43 Thus, steric limitations compro-
scatter around the value of 5 ␮M predicted by the mising the simultaneous interaction of two or more
Meyer-Overton correlation for nonspecific interac- functional groups and/or too large a hydrophobic
tions of propofol (see Methods). volume may be why propofol, in this and other
studies, does not enhance 5-HT3A receptors,22,44,45
Active Functional Groups while 2-isopropylphenol and phenol (this study) do.
Just as has been postulated for the interaction
between serotonin and its receptor site,39 the interac- Implications for Anesthetic Mechanisms
tion between propofol or its phenol derivatives and James and Glen41 finally settled for propofol to be
the 5-HT3 receptor may be a combination of hydro- tested in human trials, because of all the phenol
phobic and specific polar interactions. Evidence for derivatives they had tested, propofol had proved to be
hydrophobic contributions is the relatively close pre- the most potent hypnotic. In our study, we find that
diction of the IC50 values for closed channel inhibition propofol is about equally as potent as 2-isopropylphenol in

Vol. 106, No. 3, March 2008 © 2008 International Anesthesia Research Society 855
suppressing 5-HT3A receptor currents in the equilib- whereas the fast component of drug action described
rium application, although propofol is much more here (Fig. 2) may be considerably slower. When there
hydrophobic. This implies that propofol in compari- is a part of the fast component that is present only in
son with 2-isopropylphenol has lost some potential for the open channel conformation, then neither the open
interactions that cause current suppression, even channel nor the equilibrium application would allow
though propofol already lacks the counteracting abil- steady-state measurements of the current peak. In this
ity for enhancement, i.e., it causes neither increases in case, it would be extremely difficult to experimentally
current amplitudes nor in the desensitization time separate kinetic from competitive effects. On the other
constant. hand, the experiments involving drug action in the
There is other evidence for a lesser specificity of absence of the agonist clearly demonstrate that there is
propofol compared with other IV drugs. The IC50 (18 at least a large component of drug action that is not
␮M) for propofol differs little from those listed in a competitive with the agonist. In vivo it would not be
previous section for voltage-gated ion channels. It possible to overcome this component by synapses that
differs by only a factor of two from that predicted by are flooded with transmitter agonists. As to the fast
hydrophobicity for voltage-gated ion channels (see component, clearly, a synapse releasing a great deal of
Methods), and not by about an order of magnitude as agonist would be suppressed by propofol less than a
would be the case when average values for IV anes- synapse that released less 5-HT.
thetic action on ligand-gated and on voltage-gated ion In conclusion, at least two separate inhibitory ac-
channels are compared.1 tions on 5-HT3A receptors could be identified for
In fact, in some sense, propofol is not unlike inhaled propofol, whereas the enhancing action seen for the
anesthetics, which are considered less specific in their two related smaller phenol derivatives could no
molecular actions than IV anesthetics, because here we longer be detected. 5-HT-dependent and 5-HT-
find propofol to be more potent than inhaled anesthet- independent interactions could be distinguished for
ics by a factor of only 1.5 (see Methods), whereas on all three drugs. Propofol was less potent than expected
average, IV anesthetics are an order of magnitude from its hydrophobic properties. Underlying mecha-
more potent on ligand-gated ion channels than in- nisms appear to involve the phenolic hydroxyl group,
haled anesthetics.1 When the various ion channels hydrophobic interactions, and steric restrictions.
were compared in a previous section, it was evident
that they all respond to propofol within a factor of 10 ACKNOWLEDGMENTS
in the low micromolar concentration range, rather We thank Dr. J.P. Dilger for carefully reading this manu-
than in the low nanomolar or picomolar range, as script and discussions, Ms von dem Bussche for maintaining
would be expected for a specific pharmacological the cell cultures, and Ms M. Meiboom for performing
drug, supporting the empirical hypothesis that the less experiments with 5-hydroxyindole.
specific drug may be the more useful general anes-
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Vol. 106, No. 3, March 2008 © 2008 International Anesthesia Research Society 857