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Vol. 281, Issue 3, 1136-1143, 1997
Department of Pharmacology, University of Colorado Health Sciences Center and Veterans Affairs Medical Center, Denver, Colorado
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Abstract |
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 Top
Abstract
Introduction
Procedures
Results
Discussion
References
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5-Hydroxytryptamine type 2A receptors (5-HT2A) are G
protein-coupled receptors that increase intracellular Ca2+
concentrations via activation of phospholipase C-
and
elevation of myo-inositol-1,4,5-triphosphate levels. In
the central nervous system, these receptors are involved in regulating
sleep and alertness. We now report that ethanol inhibited
(IC50 = 41 mM) 5-HT2A receptor-induced Ca2+-dependent Cl
currents in Xenopus
laevis oocytes. Pharmacologically relevant concentrations of
other n-alcohols (propanol to octanol) also inhibited
5-HT responses; however, longer-chain alcohols (decanol, undecanol and
dodecanol) had little or no effect. The protein kinase C inhibitor
GF109203X and the nonspecific protein kinase inhibitor staurosporine
abolished the inhibitory effects of ethanol and octanol on
5-HT2A receptors. GF109203X enhanced 5-HT2A
receptor function when administered alone. In addition, the volatile
anesthetics halothane and 1-chloro-1,2,2-trifluorocyclobutane decreased
5-HT2A responses in a concentration-dependent manner. The
inhibitory effects of the volatile anesthetics were also attenuated in
oocytes treated with GF109203X. The intravenous anesthetics propofol, ketamine, pentobarbital and etomidate did not affect 5-HT2A
receptor function. The modulation of 5-HT2A
receptor-dependent current was also investigated using two novel
halogenated compounds that do not produce anesthesia. The nonanesthetic
compound 2,3-chloro-octafluorobutane had no effects on 5-HT-induced
currents; however, the nonanesthetic compound
1,2-dichlorohexafluorocyclobutane had an inhibitory effect at lower
concentrations than the predicted anesthetic concentration. Thus,
5-HT2A receptors are inhibited by alcohols and volatile anesthetics, and these actions are dependent on protein kinase C.
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Introduction |
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Top
Abstract
Introduction
Procedures
Results
Discussion
References
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5-HT
(serotonin) is an important biogenic amine that
fulfills the role of both a neurotransmitter and neuromodulator
(Gyermek, 1996
). Seven different families of serotonin receptors have
been identified, including the 5-HT2A receptors.
5-HT2 receptors in the central nervous system are important
in regulating sleep and alertness (Huidobro-Toro and Harris, 1996;
Sharpley et al., 1990). Recently, it has been reported that
the 5-HT2A receptor is involved in alcohol tolerance and
dependence (LeMarquand et al., 1994a, 1994b; Pandey and
Pandey, 1996).
The effects of ethanol and anesthetics on voltage- and ligand-gated ion
channels have been the focus of several studies (reviewed in Franks and
Lieb, 1994; Harris et al., 1995). However, less is known
about the effects of ethanol and anesthetics on metabotropic receptors,
such as 5-HT2 receptors. Several investigators have recently studied the effects of ethanol and anesthetics on some G
protein-coupled receptors (Durieux, 1995, 1996; Lin et al., 1993, Sanna et al., 1994). The volatile anesthetic enflurane
inhibited the function of phoshatidylinositol-linked acetylcholine and
serotonin receptors (Lin et al., 1993); ethanol inhibited
the function of 5-HT2C and muscarinic m1 G
protein-linked receptors (Sanna et al., 1994) and halothane
inhibited the muscarinic m1 receptor expressed in Xenopus
laevis oocytes (Durieux, 1995). The inhibitory actions of ethanol
on 5-HT2C and m1 receptor was attributed to PKC-mediated receptor desensitization. It is interesting to note that
the angiotensin II receptor, which uses the same intracellular signaling system as the m1 muscarinic receptor, was not
affected by halothane (Durieux, 1995), and desensitization of this
receptor is not modulated by PKC (Oppermann et al., 1996).
These studies raise a number of questions. First, only a few anesthetic
agents have been tested on metabotropic receptors. To determine whether
inhibition of these receptors might be related to anesthetic action
in vivo, it is necessary to examine a wider range of
compounds. In particular, there are structurally related compounds in
which a small structural change converts an anesthetic into a
nonanesthetic (Koblin et al., 1994). Also, the long-chain n-alcohols produce anesthesia but do not affect some of the
ligand-gated ion channels that are sensitive to ethanol (Dildy-Mayfield
et al., 1996a). Second, as noted above, the
5-HT2 family of receptors is important in brain function,
and earlier studies (Lin et al., 1993; Sanna et
al., 1994) of the effects of ethanol and anesthetics on these
receptors used brain mRNA to express putative 5-HT2C receptors in X. laevis oocytes. However, interpretation of
these results is complicated by other proteins, including other
5-HT2 receptors, that could be expressed from a mixture of
mRNAs. Third, there is evidence that ethanol inhibition of 5-HT
responses requires PKC (Sanna et al., 1994), but it is not
known if this is also true for other anesthetics. In addition, those
results were obtained by expression of brain mRNA, and it is important
to confirm them by expression of cRNA for a single 5-HT receptor
subtype. To answer these questions, we expressed 5-HT2A
receptors in X. laevis oocytes and tested the effects of
n-alcohols as well as volatile and injectable anesthetics on
the function of these receptors.
The X. laevis oocyte expression system has been used to
express a multiplicity of brain receptors from cDNAs or cRNAs with pharmacological properties that mimic those of native brain receptors (Harris. et al., 1995). The activation of 5-HT2A
receptors by 5-HT results in an increase in intracellular free
Ca2+ concentration. The rise in intracellular
Ca2+ opens Ca2+-sensitive Cl
channels found endogenously in oocytes (Pritchett et al.,
1988). This system has been well characterized and is well suited for studying the effects of ethanol and anesthetics on G protein-coupled receptors. We now report the effects of short- and long-chain alcohols,
volatile and intravenous anesthetics and some novel nonanesthetics on
the function of 5-HT2A receptors expressed in X. laevis oocytes. We also studied the effects of PKC inhibition on
the modulation of 5-HT2A receptors by these agents.
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Experimental procedures |
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Top
Abstract
Introduction
Procedures
Results
Discussion
References
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Materials. Adult X. laevis laevis female frogs were purchased from Xenopus I (Ann Arbor, MI). 5-HT, the n-alcohols, DMSO, staurosporinee and ketamine were purchased from Sigma Chemical Co. (St. Louis, MO). Ethanol was purchased from Asper Alcohol and Chemical Co. (Shelbyville, KY). Propofol was obtained from Aldrich Chemical Co. (Milwaukee, WI). Halothane was bought from Halocarbons Laboratories (River Edge, NJ). Etomidate was obtained from Janssen Pharmaceutica (Beerse, Belgium). F3, F6 and F8 were obtained from PCR Inc. (Gainesville, FL). Spiperone and ritanserin were bought from Research Biochemicals Inc. (Natick, MA). N-(4-Bromobenzyl)-5-methoxytryptamine oxalate was obtained from Tocris Cookson (St. Louis, MO). Ultracomp Escherichia coli transformation kit was from InVitrogen (San Diego, CA). The Qiagen (Chatworth, CA) kit was used for purification of plasmid cDNA. 5-HT2A cRNA were prepared using mCAP mRNA capping kit (Stratagene, La Jolla, CA). GF109203X was bought from Calbiochem (La Jolla, CA). 5-HT2A cDNA was kindly provided by Dr. David Julius (University of California, San Francisco).
5-HT2A cRNA preparation. The cDNA for the 5-HT2A receptor was inserted into the pGEM vector. The receptor cDNA was linearized with HindIII, phenol chloroform-extracted and ethanol-precipitated with sodium acetate. cRNA was prepared using the Stratagene transcription kit by using T7 RNA polymerase. The cRNA was extracted using phenol-chloroform and precipitated in ethanol and sodium acetate.
Whole-cell voltage-clamp of injected oocytes.
Isolation and
microinjection of X. laevis oocytes were performed as
described by Sanna et al. (1994) and Huidobro-Toro and Harris (1996). X. laevis oocytes were injected with 50 ng of
cRNA for the 5-HT2A receptor. Oocytes were placed in a
100-µl recording chamber and perfused with MBS containing (mM): NaCl,
88; KCl, 1; NaHCO3, 2.4; HEPES, 10: MgSO4,
0.82; Ca(NO3)2, 0.33; CaCl2, 0.91, pH 7.5, at a rate of 1.8 ml/min at room temperature. Recording and
clamping electrodes (1-5 M
) were pulled from 1.2-mm o.d. capillary
tubing and filled with 3 M KCl. A recording electrode was impaled into
the animal pole; once the resting membrane potential stabilized, a
clamping electrode was inserted, and the resting membrane potential was
allowed to restabilize. The Warner oocyte clamp OC 725-B (Hampden, CT)
was used for voltage-clamping each oocyte at 
70 mV. For the poorly
water-soluble alcohols (octanol-dodecanol), stocks were prepared in
DMSO and diluted and sonicated in MBS to a final DMSO concentration not
exceeding 0.05% (Dildy-Mayfield et al., 1996A). The
alcohols, anesthetics and nonanesthetics were preapplied for 2 min to
allow complete equilibration in the bath. Solutions of volatile agents
were freshly prepared immediately before use. The alcohol, anesthetic
and nonanesthetic concentrations in the figures represent bath
concentrations. We used the previously published values to
calculate the final concentration of volatile compounds in the
recording chamber (Dildy-Mayfield et al., 1996a; Mihic
et al. 1994). Although there was no loss of short-chain alcohols during oocyte perfusion, there was a 50% to 70% loss of
n-alcohols ranging from octanol to decanol and a similar
loss of volatile anesthetics (Dildy-Mayfield et al., 1996a;
Lin et al., 1992; Mihic et al., 1994).
) or staurosporine (800 nM) in
incubation media (MBS containing 10 mg of streptomycin and 10,000 units
of penicillin G plus 50 mg of gentamycin/liter, 0.5 mM theophylline and
2 mM sodium pyruvate).
Statistical analysis. Results are expressed as percentages of control responses due to variability in oocyte expression. The control responses were measured before and after each drug application to take into account possible shifts in the control currents as recording proceeded. The n values refer to the number of oocytes studied. Each experiment was carried out with oocytes from at least two different frogs. Statistical analyses were performed with t test with Dunnett's correction for multiple comparisons or analysis of variance for repeated measures. Curve fitting and estimation of EC50 values for concentration-response curves were performed using GraphPAD Inplot Software (San Diego, CA).
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Results |
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Top
Abstract
Introduction
Procedures
Results
Discussion
References
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Ethanol inhibition of currents activated by 5-HT.
5-HT
concentration-response curves were determined in X. laevis
oocytes expressing 5-HT2A receptors. Nonlinear regression analysis of these curves yielded an EC50 value for 5-HT of
0.27 ± 0.04 µM and a Hill coefficient of 0.76 ± 0.15 (fig. 1A). Maximal currents were observed at 10 µM.
The 5-HT2A receptor antagonists spiperone (100 nM) (Leysen
et al., 1978), ritanserin (100 nM) (Leysen et
al., 1985) and N-(4-bromobenzyl)-5-methoxytryptamine oxalate (100 nM) (Glennon et al., 1994) reduced the current induced by 10 nM 5-HT to 0%, 28 ± 5% and 25 ± 8% of control,
respectively. Ethanol (25-200 mM) inhibited currents elicited by 0.01 and 0.1 µM 5-HT (fig. 1B). The concentration of ethanol producing
half-maximal inhibition of 5-HT-induced currents was 41 ± 5 mM
when a 10 nM concentration of 5-HT was used. The inhibition by 200 mM
ethanol was 44 ± 2%, 40 ± 5%, 37 ± 3% and 36 ± 5% when 10 nM, 100 nM, 1 µM and 10 µM 5-HT were used (fig. 1C).
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n-Alcohol inhibition of currents activated by
5-HT.
n-Alcohols of increasing chain length were tested
next. As shown in figure 2A, the long-chain alcohols
decanol, undecanol and dodecanol did not significantly inhibit the
current produced by 10 nM 5-HT. Higher doses were not used due to
solubility problems. The shorter-chain alcohols inhibited the currents
produced by 10 nM 5-HT (fig. 2A). The ED50 values in
tadpoles for loss of righting reflex at room temperature are 190, 10.8, 0.57, 0.057, 0.037, 0.0126, 0.0081 and 0.0047 mM for ethanol, propanol,
butanol, hexanol, octanol, decanol, undecanol and dodecanol,
respectively (Alifimoff et al, 1989). We plotted the
percentage of inhibition of the 5-HT2A receptor function
produced by the alcohols as a function of the carbon chain length using
the concentration that produces loss of righting reflex in the tadpoles
(fig. 2B). Although short-chain alcohols (ethanol to hexanol) inhibited
5-HT responses at anesthetic concentrations, long-chain alcohols
(decanol, undecanol and dodecanol) had little or no effect on the 5-HT
response at anesthetic concentrations.
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Inhibition by halothane, F3, F6 and F8 of currents activated by
5-HT.
The effects of the volatile anesthetics halothane and F3 on
5-HT-induced currents are shown in figure 3A. Halothane
inhibited the 5-HT-induced currents in a concentration-dependent
manner. The 5-HT responses were decreased to 44 ± 7%, 14 ± 5% and 5.5 ± 2.7% of the control by 0.25, 1 and 2 mM halothane,
respectively. Similar to halothane, F3 also inhibited the 5-HT
responses in a concentration-dependent manner (fig. 3A). We also looked
at the effects of the nonanesthetics F6 and F8 on the 5-HT response. Although F8 had no effect on 5-HT-induced currents, F6 produced ~30%
inhibition at 1.1 µM (0.06 MAC) (fig. 3A) and similar inhibition at 1 MAC (fig. 3B). Single concentrations of intravenous anesthetics propofol (10 µM), ketamine (100 µM), pentobarbital (100 µM) and etomidate (100 µM) did not affect the 5-HT response (table
1).
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Ethanol, octanol and anesthetic inhibition of
5-HT2A receptor-mediated current and the role
of PKC.
Because PKC plays an important role in the regulation of G
protein-coupled receptors (Kato et al., 1988; Manzoni
et al., 1990; Moran and Dascal, 1988; Sakuta et
al., 1991; Sanna et al., 1994; Singer, 1990), we
investigated whether the inhibitory effects of ethanol and octanol on
5-HT2A could be modulated by this protein kinase. For this
purpose, the effects of ethanol on 5-HT2A receptors were
studied with X. laevis oocytes that were pretreated with the
PKC inhibitor GF109203X (200 nM) (Toullec et al., 1991) and the nonspecific kinase inhibitor staurosporine (800 nM). As shown in
figure 4, GF109203X treatment (200 nM) enhanced
5-HT2A receptor function. After a 20-min incubation of
GF109203X, the response of 5-HT increased to 2.5 times the initial
current, and this enhancement continued for 3 hr. Sanna et
al. (1994) previously reported that 12- to 16-hr incubation of
staurosporine (800 nM) blocks the inhibitory effects of ethanol on
5-HT2C. We investigated whether the effects of GF109203X
(200 nM) and staurosporine (800 nM) blocked the inhibitory effects of
ethanol on 5-HT2A. The inhibition by 200 mM ethanol on
5-HT2A receptor function was blocked in oocytes exposed to these inhibitors (fig. 5A). The inhibitory effects of
octanol (0.05 and 0.1 mM) were also abolished by the treatment with
GF109203X (table 2), as were the inhibitory effects of
halothane and F3. However, GF109203X did not affect F6 modulation of
5-HT2A receptors (fig. 5B).
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The effects of ethanol on desensitization
5-HT2A receptor responses.
Activation of
PKC promotes desensitization of G protein-coupled receptors (Sakuta
et al., 1991; Singer et al., 1990; Tan and Marty,
1991). Sanna et al. (1994) showed that ethanol enhances the
rate of current desensitization induced by repeated applications of
5-HT on 5-HT2C receptors. We investigated the effects of
ethanol on desensitization of 5-HT2A receptors and further
examined whether inhibition of PKC activity would reduce
desensitization. Desensitization of 5-HT2A receptors was
studied using repeated 200 nM 5-HT applications at 3-min intervals
(fig. 6). The current amplitude decreased gradually and
reached 14.5 ± 0.5% of the maximum response after 21 min (eight applications of 5-HT). Continuous application of ethanol (100 mM)
accelerated the desensitization of the 5-HT response. In agreement with
previous reports (Sanna et al., 1994), treatment with a PKC inhibitor, GF109203X, decreased desensitization produced by repeated application of 5-HT.
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Discussion |
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Top
Abstract
Introduction
Procedures
Results
Discussion
References
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Effects of ethanol and kinase inhibitors on
5-HT2A receptors.
Our results demonstrate
that acute exposure to ethanol inhibits 5-HT2A receptor
function. Stimulation of the 5-HT2A receptor leads to the
activation of PLC, a process that is mediated by a G protein, resulting
in the formation of IP3 and DAG. IP3 promotes, through its interaction with IP3 receptors, the release of
Ca2+ from the endoplasmic reticulum, and this in turn
triggers the opening of Cl channels in oocytes (Pritchett
et al., 1988). The direct injection of either
IP3 or GTP
S also induces currents via the
Ca2+-activated Cl channel; however, ethanol
has no effect on these currents, suggesting that ethanol does not
interfere with IP3-induced Ca2+ release, with
the binding of guanine nucleotides to G proteins or with the coupling
between G proteins and PLC (Sanna et al., 1994). The PKC
inhibitor GF109203X and the nonspecific kinase inhibitor,
staurosporine, abolished the ethanol-induced inhibition of
5-HT2A receptor function. Moreover, ethanol increased the
desensitization produced by repeated stimulations with 5-HT. The
inhibition of ethanol was not reversed by increased 5-HT concentration.
These data are similar to those obtained by Sanna et al.
(1994) for the 5-HT2C receptor and suggest that the action
of ethanol on both subtypes of 5-HT2 receptors involves PKC
modulation.
; Sanna et al., 1994). Thus, blockade of this
negative feedback loop would be expected to enhance actions of 5-HT, as observed in our study.
Our results suggest several mechanisms by which ethanol inhibits
5-HT2A receptor function: (1) ethanol may inhibit
5-HT2A function by activating PKC or (2) ethanol may
directly inhibit the 5-HT2A receptor and PKC may alter the
receptor sensitivity to ethanol indirectly. Recently, another
possibility was raised by Mitchell et al. (1996), who
reported that a series of alcohols, which include ethanol, modulates
the activation of a G protein-coupled receptor (rhodopsin) by a
lipid-mediated mechanism. In support of the first hypothesis, several
studies suggest that ethanol activates PKC activity in vivo
by promoting its translocation from the cytosol to the membrane
fraction (DePetrillo and Swift, 1992; Skwish and Shain, 1990; Tuominen
et al., 1992). Previously, Sanna et al. (1994)
suggested that ethanol inhibits the 5-HT2C receptor by
enhancing PKC-dependent desensitization, perhaps due to phosphorylation
of the 5-HT2C receptor. Moreover, 5-HT2A
receptors have 11 serine/threonine residues, which may be possible
targets for phosphorylation by this protein kinase (Bach et
al., 1993; Boess and Martin, 1994). Mutation of these amino acids
is required to test hypothesis that receptor phosphorylation is
responsible for actions of ethanol.
The acute inhibition of 5-HT2A and 5-HT2C
receptor function by ethanol may be related to the effects of chronic
alcohol treatments on these receptors. For example, chronic consumption
of ethanol changes the density and function of 5-HT2C
receptors in choroid plexus of rats (Pandey et al., 1993;
Pandey and Pandey, 1996). Thus, it is possible that the initial
inhibition of 5-HT receptor function leads to a compensatory
upregulation of receptor density with chronic exposure. This could
contribute to ethanol tolerance and dependence, as discussed by others
(Pandey et al., 1993; Pandey and Pandey, 1996).
Effects of long-chain alcohols on 5-HT2A
receptors.
The effects of alcohol on receptor-gated ion channels
display a clear dependence on chain length. Recently, several
investigators reported that the effects of long-chain alcohols
distinguish between several classes of receptor-gated ion channels. For
example, long-chain alcohols (e.g., decanol and dodecanol)
enhance both GABAA receptor function (Dildy-Mayfield
et al., 1996a; Kurata et al., 1993; Nakahiro et al., 1991) and glycine receptor activity (Mascia et
al., 1996) while having no effect on NMDA, AMPA or kainate
(Dildy-Mayfield et al., 1996a; Peoples and Weight, 1995) or
ATP (Li et al., 1994) receptor function. This lack of effect
of long-chain alcohols has been termed "cutoff." Unlike these
receptor-gated ion channels, the effects of alcohol chain length on G
protein-coupled receptors have not been studied. Our results
demonstrate that the 5-HT2A receptor exhibits a cutoff like
that seen with NMDA, AMPA and kainate receptors (Dildy-Mayfield
et al., 1996a; Peoples and Weight, 1995).
), but
receptors formed by rho subunits are not. These
rho, or GABAc, receptors are inhibited by alcohols with a
cutoff similar to that found for the 5-HT2A receptor (Mihic
and Harris, 1996), and these receptors are also inhibited by PKC
activators (Kusama et al., 1995). Thus, there may be a
common mechanism of action for alcohols on some metabotropic and
ionotropic receptors.
As discussed by Franks and Lieb (1985), the concept of the cutoff for
these receptor-gated ion channels implies that a small hydrophobic
pocket allows short-chain alcohols to bind to the protein and modulate
receptor function, whereas long-chain alcohols would be unable to fit
and therefore unable to alter receptor function. In our results, the
inhibitory effect of octanol on the 5-HT2A receptor was
blocked in the oocytes that were treated by GF109203X. These results
suggest that octanol may inhibit the 5-HT2A receptor by
modulating PKC activity. These results raise the possibility that PKC
is a target for n-alcohol action and the PKC may play an important role
in the cutoff for the 5-HT2A receptor.
Inhibition by anesthetics of 5-HT2A receptor function. Both halothane and F3 inhibit currents induced via 5-HT2A receptors at a 1 MAC concentration. Pretreatment with the protein kinase inhibitor GF109203X abolished the inhibitory effects of these anesthetics. These results suggest that these anesthetics inhibit the 5-HT-induced current by a PKC-sensitive pathway similar to the one found with ethanol and octanol.
It is interesting that the nonanesthetic F6 has inhibitory effects and the protein kinase inhibitor did not abolish the inhibitory effects of F6. These results suggest that the inhibitory mechanism dose not involve PKC and is different from that of ethanol, F3 and halothane. It has been reported that F6 and F8 have little effect on receptor-gated ion channels. For example, Dildy-Mayfield et al. (1996B)
showed that F6 and F8 have no effects on AMPA or kainate responses, and
Mihic et al. (1994) showed that F6 and F8 do not affect the
GABAA receptor. Recently, Kandel et al. (1996) reported that a low concentration of F6 abolishes learning and memory.
It is interesting to note that the 5-HT2 receptors are closely related to muscarinic receptors (Peralta et al.,
1988), which are critical for numerous central processes, including
memory and learning (Wess, 1993). Moreover, we found inhibitory effects of F6 on the m1 receptor expressed in
oocytes.2 Thus, novel halogenated
nonanesthetics, such as F6, may have inhibitory effects on G
protein-coupled receptors, influencing central processes such as memory
and learning.
Previous studies of anesthetics on PKC activity have yielded
contradictory results. In vitro studies demonstrate that
halothane has inhibitory effects on brain PKC (Hemmings et
al., 1995; Hemmings and Adamo, 1994; Slater et al.,
1993), but halothane was reported to stimulate PKC activity in both
brain cytosol (Tsuchiya et al., 1988) and PC12 cells (Tas
and Koschel, 1991). Park et al. (1996) reported different
effects of halothane and isoflurane on PKC activity in vivo:
halothane inhibited PKC-induced contraction in rat coronary arteries,
and isoflurane enhanced it. In our studies, the PKC inhibitor GF109203X
blocked the inhibitory action of ethanol, halothane and F3 on
5-HT2A receptors. Although the effects of anesthetics on
PKC activity are controversial, our results suggest that the inhibitory
effects of halothane and F3 require PKC.
Intravenous anesthetics did not affect 5-HT2A receptor
function. However, several investigators have reported the effects of
intravenous anesthetics on PKC activity. Pentobarbital has inhibitory
effects on PKC activity in synaptosomes
(Ki = 480 µM) (Deshmukh et
al., 1989), and the activity of purified rat brain PKC was
inhibited to ~50% by 1 mM of pentobarbital (Mikawa et al., 1990). Hemmings and Adamo (1994) reported that propofol
activate purified brain PKC activity (EC50 = 240 µM).
There have been no reports demonstrating the effects of ketamine and
etomidate on PKC activity. However, the concentrations of anesthetics
required to affect the PKC activity in these studies were much higher
(~2-10-fold) than the concentration used in our study. The
EC50 values of pentobarbital, ketamine, propofol (Franks
and Lieb., 1994; Harris et al., 1995; Lin et al.,
1992) and etomidate (Fragen, 1994) for producing anesthesia in mammals
are 25, 182, 8 and 0.4 µM, respectively. Thus, clinically relevant
concentrations of these anesthetics have little or no effect on PKC
activity and no effect on the 5-HT2A receptor.
These results demonstrate that volatile anesthetics and
n-alcohols up to octanol inhibit the function of
5-HT2A receptors expressed in X. laevis oocytes.
This action requires PKC activity and is most easily explained by
activation of PKC by these agents leading to enhanced desensitization
of the 5-HT receptor response, but available data do not rule out other
possibilities. A related issue is the site of action of these
anesthetics. As discussed above, it is likely to be PKC or the 5-HT
receptor rather than downstream signaling machinery because ethanol
does not alter the release of intracellular calcium produced by stimuli
subsequent to receptor activation (e.g., GTPS or
IP3) (Sanna et al., 1994) and halothane does not
alter the action of angiotensin II receptors even though they share the
same signaling steps as other metabotropic receptors (Durieux, 1996).
It is possible that anesthetics alter the phosphorylation of the
5-HT2A receptor and thereby inhibit its function, and
future studies should directly address this hypothesis with the use of
site-directed mutagenesis or measurement of receptor phosphorylation.
Our results also address the role of 5-HT2A receptors in
anesthesia. Although volatile anesthetics inhibited these receptors at
concentrations equivalent to MAC in vivo, the amount of
inhibition varied markedly among the agents tested in this study. For
example, halothane had a small effect, ethanol and F3 had large effects but decanol and dodecanol had no effect. The injectable anesthetics were also without effect. In addition, the nonanesthetic F6 produced effects that were as pronounced as those of halothane. From these data,
we conclude that the 5-HT2A receptor is not likely to be important for the immobility component of anesthesia. However, this
receptor may be important in other actions of some of these drugs. For
example, ethanol inhibits this receptor at subanesthetic concentrations, and this action may be important for other aspects of
intoxication. Likewise, F6 disrupts learning and memory, and this may
be related to its action on metabotropic receptors. It will be of
interest to determine whether other metabotropic receptors show similar
sensitivity to F6 and a similar cutoff for the n-alcohols.
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Acknowledgments |
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We thank Drs. M. P. Mascia, S. J. Mihic, M. Wick, C. F. Valenzuela and T. Vanderah for kind discussion and technical suggestions.
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Footnotes |
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Accepted for publication February 14, 1997.
Received for publication November 20, 1996.
1 Supported by the Department of Veterans Affairs, NIH Grants GM 47818 and AA 06399, the Yokoyama Clinical Pharmacology Foundation and the Uehara Memorial Foundation.
2 K. Minami, M. Minami and R. A. Harris, unpublished observations.
Send reprint requests to: Dr. R. Adron Harris, Department of Pharmacology, University of Colorado Health Sciences Center and Veterans Affairs Medical Center, 4200 E. 9th Ave., Box C236, Denver, Colorado, 80262. E-mail: adron.harris{at}uchsc.edu
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Abbreviations |
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AMPA, 
-amino
3-hydroxy-5-methyl-4-isoxozolepropionic acid;
DAG, diacylglycerol;
DMSO, dimethylsulfoxide;
F3, 1-chloro-1,2,2-trifluorocyclobutane;
F6, 1,2-dichlorohexafluorocyclobutane;
F8, 2,3-chloro-octafluorobutane;
GABA, -aminobutyric acid;
GF, GF109203X;
GTPS, guanosine-5
-O-(3-thio)triphosphate;
HEPES, 4-(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid;
5-HT, 5-hydroxytryptamine;
IP3, myo-inositol-1,4,5-triphosphate;
MAC, minimum alveolar
concentration;
MBS, modified Barth's solution;
NMDA, N-methyl-D-aspartate;
PLC, phospholipase
C.
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Abstract
Introduction
Procedures
Results
Discussion
References
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-aminobutyric acid actions on -aminobutyric acidA receptors expressed by Xenopus oocytes: Lack of involvement of intracellular calcium.
J. Pharmacol. Exp. Ther.
263: 569-578, 1992-aminobutyric acid type A receptor-mediated chloride currents by novel halogenated compounds correlates with their abilities to induce general anesthesia.
Mol. Pharmacol.
46: 851-857, 1994[Abstract].-aminobutyric acid receptor-channel complex by alcohols.
J. Pharmacol. Exp. Ther.
259: 235-240, 1991This article has been cited by other articles:
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), propanol (C3, 
), butanol (C4, 
), hexanol (C6,
), octanol (C8, 
), decanol (C10, 
), undecanol (C11, 
) or
dodecanol (C12, 
) in oocytes expressing 5-HT2A
receptors. Inhibition of the effects of 5-HT (10 nM) was greater when
shorter-chain alcohols were tested (ethanol to octanol). Little or no
inhibition was found when decanol, undecanol or dodecanol was tested.
Each point represents a minimum of five separate experiments. Data are
mean ± S.E.M. of 5 to 10 oocytes. B, Comparison of changes in
5-HT receptor function by alcohol concentrations that produce loss of
righting reflex in tadpoles. The percentage change of 5-HT receptor
function produced by the alcohols ethanol-dodecanol (using
EC50 value for producing loss of righting reflex in
tadpoles from Alifimoff et al., 1989
