![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
Vol. 282, Issue 3, 1581-1590, 1997
Department of Pharmacology, Stritch School of Medicine, Loyola University Chicago, Maywood Illinois
 |
Abstract |
|---|
 Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
|
|---|
The aim of the present study was to determine whether the previously observed desensitization of hypothalamic 5-hydroxytryptamine1A (5-HT1A) receptors, during daily injections of fluoxetine, is mediated by sustained blockade of 5-HT reuptake. In the present study, we examined the time course effects of another 5-HT uptake inhibitor, paroxetine. Paroxetine reduced the oxytocin, adrenal corticotropic hormone and corticosterone responses to a challenge with the 5-HT1A agonist 8-hydroxy-2-(dipropylamino)tetralin. These reductions in hormone responses were significant after 3 daily injections and reached a maximum after 7 daily paroxetine injections. These hormone responses remained maximally suppressed after 14 daily injections of paroxetine. A single day of paroxetine treatment did not alter the hormone responses to 8-hydroxy-2-(dipropylamino)tetralin. Repeated injections of paroxetine did not reduce the density of 5-HT1A receptors in any brain region but did produce a gradual reduction in the levels of Gi and Go proteins in a region-specific manner. The time course of the paroxetine-induced reduction in the level of Gi1 and Gi3 proteins in the hypothalamus was similar to the effect previously observed with fluoxetine and was also similar to the time course of paroxetine-induced reductions in oxytocin and adrenal corticotropic hormone responses to 8-hydroxy-2-(dipropylamino)tetralin. In conclusion, these results suggest that blockade of 5-HT uptake sites produces a delayed and gradual desensitization of 5-HT1A receptors in the hypothalamus. This desensitization is not due to changes in the density of hypothalamic 5-HT1A receptors. Reduction in the hypothalamic level of Gi3 proteins may play a role in the desensitization of 5-HT1A receptor systems. However, reductions in Gi1 or Go proteins cannot be excluded as potential mediators of the desensitization of 5-HT1A receptor systems.
| |
Introduction |
|---|
|
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
|
|---|
5-HT
uptake inhibitors, such as fluoxetine and paroxetine, are a new class
of drugs that were initially introduced to treat depression. 5-HT
uptake inhibitors are also effective in additional disorders, such as
obsessive compulsive disorder and premenstrual syndrome, for which
older classes of drugs such as tricyclic antidepressants and MAO
inhibitors are ineffective or less effective (Wong et al.,
1995
; Eriksson et al., 1995). This difference in therapeutic effects between 5-HT uptake inhibitors and older antidepressants may be
due to different adaptive changes that they produce in brain 5-HT
receptors (Blier and de Montigny, 1994; Duman et al., 1994;
Gardier et al., 1996). One difference observed in our
previous studies is that repeated injections of the 5-HT uptake
inhibitor fluoxetine produce a gradual desensitization of hypothalamic
5-HT1A receptors, whereas the tricyclic antidepressant and
specific norepinephrine uptake inhibitor desipramine did not produce
this effect (Li et al., 1996b; Li et al., 1994;
Li et al., 1993). A desensitization of hypothalamic
5-HT1A receptors was also observed in humans treated with
fluoxetine (Lesch et al., 1991). Desipramine may not be able to desensitize 5-HT1A receptors because it does not inhibit
5-HT uptake and thus cannot induce adaptive changes in serotonergic neurotransmission, leading to desensitization of postsynaptic 5-HT1A receptors. These observations suggest that the
desensitization of hypothalamic 5-HT1A receptors may play a
role in some of the therapeutic effects of 5-HT uptake inhibitors.
The purpose of the present study was to determine whether the effect we
previously observed of fluoxetine on the 5-HT1A receptors in the hypothalamus is mediated by blockade of 5-HT uptake sites. We
examined the effects of another 5-HT uptake inhibitor, paroxetine, on
hypothalamic 5-HT1A receptors. Paroxetine has a chemical
structure and pharmacokinetic profile different from those of
fluoxetine (Lane et al., 1995; DeVane, 1994; Nemeroff,
1993). Because both fluoxetine and paroxetine inhibit 5-HT uptake
sites, a common effect of these two drugs is likely to be mediated by
blockade of 5-HT uptake sites. In other words, if both fluoxetine and
paroxetine induce a desensitization of 5-HT1A receptors,
then it is likely that the desensitization of 5-HT1A
receptors is mediated by blockade of 5-HT uptake sites.
5-HT1A receptors can be classified into somatodendritic
(5-HT1A autoreceptors) and postsynaptic receptors (De Vry,
1995; Hoyer et al., 1994). Somatodendritic
5-HT1A autoreceptors are located on 5-HT neurons in the
dorsal and median raphe nuclei in the midbrain (Kia et al.,
1996). Activation of somatodendritic 5-HT1A autoreceptors by 5-HT or 5-HT1A agonists decreases the firing rate of
neurons and subsequently reduces the release of 5-HT from nerve
terminals (Artigas et al., 1996; Briley and Moret, 1993;
Adell et al., 1993; Hjorth and Auerbach, 1995).
Somatodendritic 5-HT1A autoreceptors may be coupled to
Go proteins, which increase the opening of K+
channels and consequently suppress the activity of 5-HT neurons (Sprouse and Aghajanian, 1988; Innis and Aghajanian, 1987; Innis et al., 1988; Clarke et al., 1996; Romero
et al., 1994). It has been proposed that 5-HT1A
autoreceptors may be partly responsible for the delay in the
therapeutic effects of 5-HT uptake inhibitors. The rationale for this
hypothesis is as follows: The administration of 5-HT uptake inhibitors
induces an increase in 5-HT concentration in the synaptic cleft. This
increase in 5-HT concentration activates somatodendritic
5-HT1A autoreceptors in the raphe nuclei and consequently decreases 5-HT release in forebrain regions. Therefore, clinical symptoms cannot be improved until somatodendritic 5-HT1A
autoreceptors are desensitized by repeated administration of 5-HT
uptake inhibitors. Several studies support this hypothesis, mainly with
data obtained using microdialysis and/or electrophysiological
recording. However, data regarding changes in the density of
5-HT1A receptors in the midbrain raphe are inconsistent
(Welner et al., 1989; Hensler et al., 1991; Le
Poul et al., 1995; Li et al., 1994). Our previous study demonstrated that the 5-HT uptake inhibitor fluoxetine reduces the levels of Go and Gi2 proteins in the
midbrain, which may be related to the desensitization of
somatodendritic 5-HT1A autoreceptors (Li et al.,
1996b). The current study examined whether paroxetine will reduce the
levels of these G proteins in the midbrain.
Postsynaptic 5-HT1A receptors are distributed in many
forebrain regions that receive serotonergic input, such as the
hippocampus, hypothalamus, amygdala and cortex (Kia et al.,
1996; Gozlan et al., 1995; Khawaja, 1995). Activation of
postsynaptic 5-HT1A receptors by 5-HT produces
physiological responses that depend on the function of the target
cells. Stimulation of 5-HT1A receptors in the hypothalamic paraventricular nucleus increases the secretion of several hormones, including ACTH, corticosterone and oxytocin (Bagdy, 1995; Van de Kar
and Brownfield, 1993; Bagdy and Kalogeras, 1993). Therefore, the
magnitude of the hormone responses to 5-HT1A agonists can reflect the function of 5-HT1A receptor systems in the
hypothalamus (Bagdy, 1995; Bagdy and Makara, 1994). 5-HT1A
receptors can couple to Gi and Go proteins.
Their affinity for Gi3 and Gi1 proteins is
higher than for Gi2 and Go proteins. (Raymond
et al., 1993; Mulheron et al., 1994; Bertin
et al., 1992; Butkerait et al., 1995; Fargin
et al., 1991). Our previous studies indicate that daily
injections of fluoxetine produce a gradual reduction in the levels of
Gi1 and Gi3 proteins, but not Gi2
proteins, in the hypothalamus.
We hypothesized that repeated injections of paroxetine also will produce a delayed and gradual desensitization of 5-HT1A receptors in the hypothalamus. The time course of the effect of paroxetine on the hormone responses to 8-OH-DPAT was examined to assess the function of 5-HT1A receptors in the hypothalamus. To determine which components of the 5-HT1A receptor system may be involved in the desensitization, we further examined the time course of the effect of paroxetine on the density of 5-HT1A receptors and on the levels of Gi and Go proteins in the hypothalamus, midbrain and frontal cortex.
| |
Materials and Methods |
|---|
|
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
|
|---|
Animals. Male Sprague-Dawley rats (225-275 g) were purchased from Harlan Sprague-Dawley Inc. (Indianapolis, IN). The rats were housed two per cage in a lighting- (12-hr light/dark; lights on at 7 A.M.), humidity- and temperature-controlled room. Food and water were available ad libitum. All procedures were conducted in accordance with the NIH Guide for the Care and Use of Laboratory Animals as approved by the Loyola University Institutional Animal Care and Use Committee.
Experimental procedure.
The rats were injected with
paroxetine (10 mg/kg, ip) once daily for 1, 3, 7 or 14 days. The
control rats received saline injections for 14 days. Eighteen hours
after the last injection, the rats were challenged with saline or
8-OH-DPAT (50 or 500 µg/kg, sc) and decapitated 15 min later. These
doses represent the ED50 and Emax doses of
8-OH-DPAT-induced increase on plasma oxytocin (Li et al.,
1993). Trunk blood was collected in centrifuge tubes containing 0.5 ml
of a 0.3 M EDTA (pH 7.4) solution. After centrifugation at 2500 rpm,
4°C, for 15 min, plasma aliquots were stored at 
70°C until they
were used for hormone assays. The brains from saline-challenged rats
were quickly and carefully removed and frozen on powdered dry ice until
the brains were completely frozen. The brains were then wrapped with
plastic wrap, parafilm and aluminum foil and stored at 70°C until
they were sectioned for autoradiography. The brains of the other rats
were dissected, and the hypothalamus, midbrain and frontal cortex were
stored at 70°C for immunoblots of Gi and Go
proteins.
Drugs and reagents.
The following drugs and chemicals were
used in this study: 8-OH-DPAT (Research Biochemicals Inc., Natick, MA),
ACTH antiserum (IgG1) (IgG Corp, Nashville, TN). ACTH (1-39) standards
were obtained from Calbiochem (San Diego CA). Bovine serum albumin and
aprotinin (Sigma Chemical Co., St. Louis, MO), normal rabbit serum and
goat anti-rabbit-
-globulin (Calbiochem, San Diego, CA),
125I-ACTH (INCSTAR, Stillwater, MN), corticosterone
antiserum (ICN Biochemicals, Irvine, CA), ultima gold scintillation
fluid (Packard Instrument Co., Inc., Downers Grove, IL), acetone
(Spectranalyzed A-19) and petroleum ether (Fisher, Pittsburgh, PA),
3H-corticosterone, 125I-oxytocin,
3H-8-OH-DPAT and anti Gi1/2 (AS/7) and anti
Go (GC/2) sera (DuPont-NEN, Boston, MA), anti
Gi3 serum (Upstate Biotechnology Inc., Lake Placid, NY),
rabbit peroxidase-antiperoxidase (Organon Teknika Co., Durham, NC), the
chemiluminescence substrate solution LumiGlo (Kirkegaard & Perry
Laboratories, Inc., Gaithersburg, MD) and NP-40 (Nonidet P-40 or Igepal
CA-630) (Sigma Chemical Co., St. Louis, Mo). Paroxetine was a gift from
Smith-Kline Beecham (Philadelphia, PA). All the drugs were dissolved in
saline and injected in a volume of 2 ml/kg for paroxetine and 1 ml/kg
for 8-OH-DPAT.
Radioimmunoassay for plasma hormone concentrations.
Plasma
ACTH and corticosterone were measured by radioimmunoassays as described
in detail in our previous paper (Li et al., 1993).
. Plasma was first extracted before it was used in the
radioimmunoassay. Briefly, rat plasma (1 ml) was mixed with 2 ml
ice-cold acetone (Spectranalyzed, Fisher, Pittsburgh, PA) and
centrifuged at 2000 rpm, 4°C, for 30 min. The supernatant was then
added to 5 ml cold petroleum ether and mixed immediately. After
centrifugation at 2000 rpm, 4°C, for 15 min, the top layer was
aspirated and discarded. The remaining solution was dried by blowing
air into the tubes at 4°C. The dried extract was then dissolved in 1 ml of assay buffer (0.05 M phosphate buffer, pH 7.4, containing 0.125%
bovine serum albumin, 0.01% sodium azide and 0.001 M EDTA) and was
used for the oxytocin radioimmunoassay. This assay is a double-antibody
assay. The plasma extract (20 or 200 µl) and oxytocin standard (0-1
ng) were incubated in triplicate with 0.1 ml of rabbit anti-oxytocin
serum (1:50,000 dilution) in a total volume of 0.4 ml for 24 hr at
4°C. 125I-oxytocin (Du Pont-NEN, Boston, MA, 3000 cpm,
0.1 ml) was then added to the tubes and incubated for 72 hr at 4°C.
To the tubes was then added 0.1 ml goat anti-rabbit -globulin
(1:12.5 dilution), followed by 0.1 ml of normal rabbit serum (1:120
dilution). After incubation for 24 hr, the tubes were centrifuged at
15,000 × g, at 4°C, for 20 min. The supernatant was
decanted, and the radioactivity in the pellet was counted for 5 min by
a Micromedic 4/200 plus counter and analyzed from the standard
curve using the RIA-AID computer program (Robert Maciel Associates,
Arlington, MA). The concentration of plasma oxytocin was calculated
with a correction factor based on the recovery of the extraction. The
sensitivity limit of this assay is 1 pg/tube, and the intra- and
interassay variabilities are 8.1% and 8.6%, respectively.
Autoradiographic analysis of 3H-8-OH-DPAT
binding.
The brains from the rats that received a saline challenge
were cut into 15-µm coronal sections using a cryostat at 21°C. The sections were thaw-mounted on chromalum/gelatin-coated slides and
stored at 20°C. Sections were collected from the following levels:
frontal cortex (Bregma + 3.70 mm), medial hypothalamus (Bregma 1.80 mm), caudal hypothalamus (Bregma 3.14 mm)
and midbrain (Bregma 7.8 mm) according to the atlas of Paxinos
and Watson (1986). Sections from these levels were used for
autoradiographic analysis (fig. 1).
|
. Briefly, after a preincubation in the assay buffer
(containing 0.17 M Tris, 4 mM CaCl, 10 µM pargyline and 0.01%
ascorbic acid, pH 7.6), slide-mounted sections were incubated with
3H-8-OH-DPAT (2 nM, 135 Ci/mmol) at room temperature for 1 hr. This concentration of 3H-8-OH-DPAT is equal to its
KD in hypothalamic homogenates (Li et al.,
1993). Nonspecific binding was defined in the presence of
10
6 M 5-HT. After being washed twice with Tris buffer at
4°C for 5 min, the slides were dipped in cold H2O and
then blow-dried immediately. Then they were exposed to
tritium-sensitive Hyperfilm-3H for either 2 months or 2 weeks (for sections containing a high density of the binding sites). A
set of 3H microscales (Amersham, Arlington Heights, IL) was
exposed to each film together with the slides to calibrate the optical
density to fmol/mg tissue equivalent. The films were developed by a
Kodak developing procedure for X-ray films.
Autoradiograms were analyzed densitometrically using the NIH image
analysis program for Macintosh computers. The gray scale density
readings were calibrated to fmol/mg tissue equivalent using the
[3H]microscales. Brain regions were identified according
to the atlas of Paxinos and Watson (1986), as shown in figure 1. The layers of the cortex were identified according to the atlas of Zilles
(1985). The density of 3H-8-OH-DPAT binding in each brain
region was measured and expressed as fmol/mg tissue equivalent. An area
outside of the section was measured as the background of the film,
which was subtracted from each measurement in the section. Specific
3H-8-OH-DPAT binding sites in each brain region were
determined by subtracting the nonspecific binding sites from the total
binding sites in each region. The data for a brain region of each rat represents the mean of four adjacent brain sections.
Immunoblots.
The levels of Gi1, Gi2,
Gi3 and Go proteins in the hypothalamus,
midbrain and frontal cortex were measured using immunoblots as
described in detail in our previous paper (Li et al.,
1996b). Briefly, the solubilized proteins (10-35 µg of protein) that
were extracted from the membranes of the hypothalami, midbrains and frontal cortices (Sternweis and Robinshaw, 1984; Okuhara et
al., 1996) were resolved by SDS-polyacrylamide gel electrophoresis [containing 0.1% SDS, 12% acrylamide/bisacrylamide (30:0.2), 4 M
urea and 375 mM Tris, pH 8.4 (Mullaney and Miligan, 1990)]. The
proteins were then electrophoretically transferred to nitrocellulose membranes. The membranes were incubated with polyclonal antisera for
Gi1/2 (AS/7, 1:2500 dilution), Gi3
(Anti-Gi
3, 1:2000 dilution) and Go (GC/2,
1:2000 dilution), followed by a secondary antibody (goat anti-rabbit
serum, 1:10,000 dilution) and then rabbit peroxidase-antiperoxidase
(1:10,000 dilution). After several washes, the membranes were incubated
with the chemiluminescence substrate solution (LumiGlo) and then
exposed to Kodak X-ray film. Films were analyzed densitometrically
using the NIH image analysis program for Macintosh computers as
detailed in our previous paper (Li et al., 1996b).
Statistics.
The data from the hormone analyses were
extrapolated from standard curves using the RIA-AID computer program
(Robert Maciel Associates, Arlington, MA). The data are presented as
group means ± S.E.M. The data from the hormone assays were
analyzed by a two-way ANOVA, and the data obtained from
autoradiographic analysis of 3H-8-OH-DPAT binding and from
immunoblots for G proteins were analyzed by a one-way ANOVA. Group
means were compared by Newman-Keuls multiple-range test (Steel and
Torrie, 1960). A computer program (NWA STATPAK, Portland, OR) was used
for all the statistical analyses.
| |
Results |
|---|
|
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
|
|---|
Hormone responses to 8-OH-DPAT.
Both doses of 8-OH-DPAT (50 and 500 µg/kg) significantly increased plasma oxytocin, ACTH and
corticosterone concentrations. Pretreatment with paroxetine blunted the
increase in the levels of these hormones induced by a low dose (50 µg/kg) of 8-OH-DPAT as detailed below (figs.
2, 3, 4).
However, paroxetine had different effects on the oxytocin, ACTH and
corticosterone responses to the high dose (500 µg/kg) of 8-OH-DPAT
(table 1).
|
|
|
|
Autoradiographic analysis of 3H-8-OH-DPAT binding. Autoradiographic analysis of 3H-8-OH-DPAT binding revealed no effect of paroxetine on the density of 5-HT1A receptors in any nuclei in the hypothalamus, amygdala or hippocampus, the dorsal or median raphe nuclei or several layers of the cortex (table 2).
|
Levels of Gi and Go proteins in the
hypothalamus, midbrain and frontal cortex.
The effects of repeated
injections of paroxetine on the levels of Gi1,
Gi2, Gi3 and Go proteins are shown
in figures 5, 6, 7, 8.
The four G proteins were differentially influenced in the hypothalamus,
midbrain and frontal cortex. Figure 5 shows an example of the
immunoblots for the time course of effects of paroxetine on
the levels of G proteins in the frontal cortex, hypothalamus and
midbrain.
|
|
|
|
| |
Discussion |
|---|
|
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
|
|---|
The effects of paroxetine described here are very similar to those obtained with fluoxetine, which suggests that sustained blockade of 5-HT uptake produces a delayed and gradual desensitization of 5-HT1A receptors in the hypothalamus. This desensitization is not due to a down-regulation of hypothalamic 5-HT1A receptors and may be due to mechanisms downstream from the receptor level. Because the time course of paroxetine-induced reduction in the level of Gi3 proteins in the hypothalamus is similar to the time course of reduction in the hormone responses to 8-OH-DPAT, it is possible that the reduction in the level of Gi3 proteins may play a role in the desensitization of hypothalamic 5-HT1A receptors. However, the possibility of a role for Gi1 or Go proteins cannot be excluded.
In the present study, we used the elevation in plasma levels of
oxytocin, ACTH and corticosterone after an injection of 8-OH-DPAT to
assess the function of hypothalamic 5-HT1A receptors.
Several studies have demonstrated that 8-OH-DPAT increases the plasma concentrations of ACTH, corticosterone and oxytocin in a dose-dependent manner. The ACTH and corticosterone responses to 8-OH-DPAT can be
inhibited by the 5-HT1A antagonists pindolol, spiperone,
NAN-190, UH-301, WAY-100135 and WAY-100635 (Cowen et al.,
1990; Lejeune et al., 1993; Pan and Gilbert, 1992; Fletcher
et al., 1995; Przegalinski et al., 1989; Kelder
and Ross, 1992; Vicentic et al., 1996). The oxytocin
response to 8-OH-DPAT can be inhibited by WAY-100635 and NAN-190
(Vicentic et al., 1996; Bagdy and Kalogeras, 1993). A lesion
in the hypothalamic paraventricular nucleus blunted the ACTH,
corticosterone and oxytocin responses to another 5-HT1A agonist, ipsapirone (Bagdy and Makara, 1994; Bagdy, 1995). These data
suggest that the 8-OH-DPAT-induced increase in plasma hormone concentrations is mediated by activation of 5-HT1A
receptors in the hypothalamus, probably in the paraventricular nucleus.
Therefore, the magnitude of hormone responses to 8-OH-DPAT can be used
as a tool to assess the function of hypothalamic 5-HT1A
receptor systems (Van de Kar, 1991; Van de Kar, 1989; Van de Kar and
Brownfield, 1993). The results of the present study show that repeated
injections of paroxetine decrease the hormone responses to 8-OH-DPAT,
which suggests that paroxetine produces a desensitization of
5-HT1A receptor systems in the hypothalamus. The
desensitization of hypothalamic 5-HT1A receptors appears
after 3 daily injections and reaches a maximum after 7 and 14 days.
These results are consistent with those observed after repeated
injections of fluoxetine (Li et al., 1996b), which suggests
that a similar adaptive mechanism leads to the desensitization of
5-HT1A receptors during blockade of 5-HT uptake sites.
A previous study on the time course of the effects of fluoxetine indicated that three daily injections produce a partial desensitization of hypothalamic 5-HT1A receptors. In the present study, we added a group that received a single injection of paroxetine to determine when the desensitization of the hypothalamic 5-HT1A receptors would start. The results of the present study indicate that desensitization of hypothalamic 5-HT1A receptors does not occur 1 day after a single injection of paroxetine.
In a result similar to our previous observations with fluoxetine, daily
injections of paroxetine decreased the ACTH and corticosterone responses to a low dose, but not a high dose, of 8-OH-DPAT, whereas the
decrease in the oxytocin response was observed at both low and high
doses of 8-OH-DPAT (table 1). This difference between ACTH and oxytocin
responses might result from differences in receptor reserve for these
hormones. There is a higher 5-HT1A receptor reserve for
ACTH and corticosterone responses than for the oxytocin response to
5-HT1A agonists (Meller and Bohmaker, 1994; Pinto et
al., 1994; unpublished data from W. Pinto, L. D. Van de Kar and G. Battaglia). These differences in receptor reserve may be related
to an amplification of the signals in each stage of the hypothalamic-pituitary-adrenal axis for the ACTH and corticosterone responses to activation of 5-HT1A receptors, whereas
oxytocin is released directly from cells in the hypothalamus
via their nerve terminals in the neural lobe of pituitary
gland. However, it is also possible that the lack of change in the ACTH
and corticosterone responses to a high dose of 8-OH-DPAT is due to
activation of other receptors, which increase ACTH and corticosterone
secretion and may be sensitized by repeated injections of paroxetine.
For example, 8-OH-DPAT has about a 10-fold lower affinity for
5-HT7 receptors than for 5-HT1A receptors
(Kawahara et al., 1994; Tsou et al., 1994).
However, a role for 5-HT7 receptors in ACTH and oxytocin
secretion has not been studied yet.
Repeated injections of paroxetine did not alter the density of
5-HT1A receptors in any brain regions. This observation is in agreement with our observations with fluoxetine (Li et
al., 1993; Li et al., 1996a) and also is in agreement
with data reported by other investigators (Le Poul et al.,
1995; Hensler et al., 1991). Other investigators have shown
that neither fluoxetine nor paroxetine altered the density of
5-HT1A receptors in the dorsal raphe nucleus or in the
dentate gyrus of the hippocampus (Le Poul et al., 1995) and
that repeated injections of sertraline or citalopram did not alter the
density of 5-HT1A receptors in several brain regions
(Hensler et al., 1991). Together, these observations suggest
that sustained inhibition of 5-HT reuptake does not lead to
down-regulation of 5-HT1A receptors. The lack of
paroxetine-induced change in 3H-8-OH-DPAT binding in
tissues in which G protein levels have been decreased raises several
questions. 3H-8-OH-DPAT is a 5-HT1A agonist and
should bind with high affinity to the coupled state of
5-HT1A receptors. Under the conditions of our assay, using
concentrations equal to the KD (2 nM), binding is
determined by both receptor number and affinity. If there is little
surplus of G proteins, a reduction in G proteins should lead to reduced
coupling to the receptors, and thus there should have been a reduction
in 3H-8-OH-DPAT binding. This clearly was not the case,
which could suggest a surplus of G proteins. Yet there was a marked
reduction in the functional responsiveness of hypothalamic
5-HT1A receptors, which suggests that desensitization did
occur. Therefore, the mechanism responsible for paroxetine-induced
desensitization could occur downstream from the receptor G protein
level.
Although the effects of paroxetine and fluoxetine on G protein levels
are similar, they are not identical. Overall, paroxetine produces a
greater and earlier reduction in the levels of Gi and Go proteins, especially the levels of Gi1 and
Gi2 proteins. For example, paroxetine reduced the levels of
Gi1 proteins in the hypothalamus after 3 daily injections,
whereas fluoxetine did not significantly reduce the hypothalamic levels
of Gi1 protein until 7 daily injections (Li et
al., 1996b). Also, fluoxetine did not significantly alter the
frontal cortical levels of Gi1 and Gi2
proteins, but paroxetine produced a sustained decrease in their levels
beginning after one daily injection. The reason for the difference
between the effects of fluoxetine and paroxetine on the levels of
Gi and Go proteins is still unclear, because little is known about the mechanism by which these drugs decrease G
protein levels.
Despite the differential effect of fluoxetine and paroxetine on the
levels of G proteins, there are some similarities that may be important
for our understanding of the desensitization of 5-HT1A
receptor systems. The time course of paroxetine-induced reductions in
the hypothalamic levels of Gi3 proteins is similar to the
time course of fluoxetine-induced reductions in the level of
Gi3 proteins and is also similar to reduced hormone
responses to 8-OH-DPAT after repeated injections of paroxetine or
fluoxetine. Because 5-HT1A receptors have a high affinity
for Gi3 proteins (Raymond et al., 1993), this
similarity in time course suggests that the reduction in the level of
Gi3 may be involved in the desensitization of hypothalamic
5-HT1A receptors. However, it is still unclear whether
Gi3 proteins mediate 5-HT1A receptor-induced secretion of ACTH, corticosterone and oxytocin. Therefore, it seems too
early to conclude that the reduction in the level of Gi3
proteins mediates the desensitization of 5-HT1A receptor
systems. Furthermore, other G proteins may be involved in this
desensitization.
Repeated injections of paroxetine significantly decreased the levels of
Gi2 protein in the midbrain. The reduction in the levels of
Gi2 proteins appeared after 1 day and remained during 14 daily injections of paroxetine. Paroxetine reduced the levels of
Go proteins after 3 daily injections, and this reduction
was maintained after 7 daily injections. These changes are similar to
the results observed with fluoxetine injections (Li et al., 1996b). The levels of Go proteins returned to normal after
14 daily injections of paroxetine. This is in contrast with
fluoxetine-induced reduction of Go proteins, which remained
reduced during the 22 daily injections. Desensitization of
somatodendritic 5-HT1A autoreceptors in the dorsal raphe
nucleus has been detected after 3 to 14 daily injections of fluoxetine
or paroxetine (Le Poul et al., 1995). However, no data are
available on the effect of 1 daily injection of 5-HT uptake inhibitors
on somatodendritic 5-HT1A autoreceptors. Therefore, it is
difficult to determine which G proteins are involved in the
desensitization of somatodendritic 5-HT1A autoreceptors in
the raphe nuclei.
In conclusion, the results of the present study suggest that repeated injections of paroxetine produce a delayed and gradual desensitization of hypothalamic 5-HT1A receptor systems. This desensitization is similar to that induced by fluoxetine, which suggests that sustained blockade of 5-HT uptake sites mediates this effect. Because the density of 5-HT1A receptors in hypothalamic nuclei was not altered, it is unlikely that the desensitization of hypothalamic 5-HT1A receptors is due to their down-regulation. Similarities in the time course of the decrease in both Gi3 proteins and hormone responses to 8-OH-DPAT suggests that Gi3 proteins may be involved in the desensitization of 5-HT1A receptors.
| |
Acknowledgments |
|---|
The authors thank Dr. Theresa Cabrera, Wilfred Pinto and Francisca Garcia for their excellent technical assistance with the experiments.
| |
Footnotes |
|---|
Accepted for publication May 9, 1997.
Received for publication February 7, 1997.
1 Supported in part by United States Public Health Service Grants MH45812, NS34153 (L.D.VdK.), DA 07741 (G.B.) and NS30460 (N.A.M.).
2 Present address: Laboratory of Clinical Science, NIMH, 10 Center Dr. MSC 1264, Bethesda, MD 20892-1264.
Send reprint requests to: Louis D. Van de Kar, Ph.D., Department of Pharmacology, Stritch School of Medicine, Loyola University Chicago, 2160 S. First Avenue, Maywood IL 60153.
| |
Abbreviations |
|---|
ACTH, adrenal corticotropic hormone; ANOVA, analysis of variance; 5-HT, 5-hydroxytryptamine (serotonin); 8-OH-DPAT, 8-hydroxy-2-(dipropylamino)tetralin; SDS, sodium dodecyl sulfate.
| |
References |
|---|
|
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
|
|---|
-subunits.
J. Biol. Chem.
267: 8200-8206, 1992)-tertatolol act as antagonists at both 5-HT1A autoreceptors and postsynaptic 5-HT1A receptors in vivo.
Eur. J. Pharmacol.
240: 307-310, 1993[Medline].-subunits in rat forebrain.
Synapse
23: 246-257, 1996[Medline].-subunits and lack of coupling to Gs.
Biochemistry
32: 11064-11073, 1993[Medline].This article has been cited by other articles:
|
|
 |
|
  G. A. Carrasco, L. D. Van de Kar, C. Jia, H. Xu, Z. Chen, R. Chadda, F. Garcia, N. A. Muma, and G. Battaglia Single Exposure to a Serotonin 1A Receptor Agonist, (+)8-Hydroxy-2-(di-n-propylamino)-tetralin, Produces a Prolonged Heterologous Desensitization of Serotonin 2A Receptors in Neuroendocrine Neurons in Vivo J. Pharmacol. Exp. Ther., March 1, 2007; 320(3): 1078 - 1086. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. M. la Cour, S. El Mestikawy, N. Hanoun, M. Hamon, and L. Lanfumey Regional Differences in the Coupling of 5-Hydroxytryptamine-1A Receptors to G Proteins in the Rat Brain Mol. Pharmacol., September 1, 2006; 70(3): 1013 - 1021. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Corradetti, B. Mlinar, C. Falsini, A. M. Pugliese, A. Cilia, C. Destefani, and R. Testa Differential Effects of the 5-Hydroxytryptamine (5-HT)1A Receptor Inverse Agonists Rec 27/0224 and Rec 27/0074 on Electrophysiological Responses to 5-HT1A Receptor Activation in Rat Dorsal Raphe Nucleus and Hippocampus in Vitro J. Pharmacol. Exp. Ther., October 1, 2005; 315(1): 109 - 117. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Zhang, T. S. Gray, D. N. D'Souza, G. A. Carrasco, K. J. Damjanoska, B. Dudas, F. Garcia, G. M. Zainelli, N. R. Sullivan Hanley, G. Battaglia, et al. Desensitization of 5-HT1A Receptors by 5-HT2A Receptors in Neuroendocrine Neurons in Vivo J. Pharmacol. Exp. Ther., July 1, 2004; 310(1): 59 - 66. |
