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NEUROPHARMACOLOGY
q and G11 Proteins in Rats Undergoing Withdrawal
Center for Serotonin Disorders Research and Department of Pharmacology Loyola University of Chicago, Stritch School of Medicine, Maywood, Illinois
Received for publication
April 8, 2004
Accepted
June 1, 2004.
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Abstract |
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q and G11 proteins. Unfortunately, the exact duration of cocaine treatment necessary to induce alterations in G protein levels during cocaine withdrawal is unknown. The present study investigated the minimum cocaine treatment period required to produce changes in protein levels of membrane- and cytosol-associated Gq and G11 proteins in the hypothalamic paraventricular nucleus, amygdala, and frontal cortex. Rats were injected with cocaine (15 mg/kg i.p., b.i.d.) for 0, 1, 3, 5, and 7 days and tested after 2 days of withdrawal. The levels of Gq and G11 proteins were increased in the paraventricular nucleus and the amygdala but not in the frontal cortex. Although 1 and 3 days of cocaine treatment were sufficient to maximally elevate the protein levels of G11 and Gq proteins in the amygdala, 5 days of treatment were required to maximally increase the levels of G11 and Gq proteins in the paraventricular nucleus. The data suggest that the amygdala shows a faster neuroadaptation to the effects of cocaine than the hypothalamic paraventricular nucleus. These findings provide insight into the relative importance of individual components of 5-HT2A receptor signal transduction system in regulating the overall sensitivity of this signaling in cocaine-treated rats.
). Cocaine binds with high affinity to neurotransmitter uptake sites on monoaminergic (serotonergic, dopaminergic, and noradrenergic) neurons in the brain and peripheral tissues, blocking the reuptake of serotonin, dopamine, and norepinephrine into the presynaptic neuron (Levy et al., 1994a).
The influence of cocaine on serotonergic neurotransmission has received increasing attention in recent years. The cocaine-induced reuptake blockade leads to an increased concentration of 5-HT in the synapse and thus to stimulation of postsynaptic 5-HT receptors (Hanson et al., 1987). Furthermore, cocaine reduces the activity of 5-HT neurons in the dorsal raphe (Cunningham and Lakoski, 1988), presumably as a consequence of increased stimulation of somatodendritic 5-HT1A autoreceptors in the dorsal raphe nucleus (Pan et al., 1989).
5-HT2A and 5-HT2C receptors, which activate the dopamine mesoaccumbens pathway, play an important role in the behavioral effects of cocaine (McMahon et al., 2001). In addition, 5-HT2A receptors are expressed by neurons in the hypothalamic paraventricular nucleus (Zhang et al., 2002). Activation of 5-HT2A receptors in the hypothalamic paraventricular nucleus increases the secretion of ACTH, corticosterone, oxytocin, and prolactin (Van de Kar et al., 2001). Using neuroendocrine responses to the 5-HT2A/2C receptor agonist DOI, we found increased sensitivity of 5-HT2A receptors that stimulate the secretion of ACTH, corticosterone, and prolactin after 42-h withdrawal from repeated cocaine treatment (15 mg/kg i.p., twice a day for 7 days) (Levy et al., 1992). The mechanism of this cocaine-induced supersensitivity of 5-HT2A receptors in the paraventricular nucleus might be an altered expression of proteins associated with the 5-HT2A receptor signaling pathway, including Gq and G11 proteins, and regulators of G protein signaling proteins, such as regulators of G protein signaling (RGS)4 and RGS7 proteins. Our previous study (Carrasco et al., 2003) showed that withdrawal (2 days) from chronic cocaine treatment (14 days, 15 mg/kg twice a day or using a binge protocol) produces a transient and region-specific increase in the levels of membrane-associated Gq and G11 proteins in the hypothalamic paraventricular nucleus and the amygdala, but not in the frontal cortex (Carrasco et al., 2003). Exposure to chronic cocaine does not produce changes in the levels of membrane- or cytosol-associated 5-HT2A receptors, RGS4 or RGS7 proteins in frontal cortex, amygdala, or paraventricular nucleus (Carrasco et al., 2003). These results support the conclusion that withdrawal from chronic cocaine treatment increases 5-HT2A receptor function by altering postreceptor signal transduction mechanisms, rather than increasing 5-HT2A receptor density.
In this article, we focused on the minimum number of days of cocaine treatment required to change the levels of 5-HT2A receptor signaling proteins in the hypothalamic paraventricular nucleus, amygdala, and frontal cortex. These regions were selected because of their prominent role in stress, anxiety, neuroendocrine function, and addiction (Carrasco and Van de Kar, 2003; Yun and Fields, 2003). The hypothalamic paraventricular nucleus plays a central role in mediating neuroendocrine responses to serotonergic activation (Bagdy, 1996). The hypothalamic paraventricular nucleus receives serotonergic projections from the raphe nuclei, which also send collaterals to other limbic structures, notably the amygdala (Liposits et al., 1987; Petrov et al., 1994). The amygdala is a limbic structure with interconnections to the cortex and the nucleus accumbens and plays a central role in the reinforcing effects of drugs of abuse (Yun and Fields, 2003).
Various drugs of abuse require repeated administration before withdrawal effects can be observed (Levy et al., 1994a). Because the immediate effect of cocaine is to reduce serotonergic firing rate (Cunningham and Lakoski, 1988), there is a good likelihood that the reduction in 5-HT release in the hypothalamus will gradually lead to supersensitive postsynaptic 5-HT2A receptors. However, the minimum duration of cocaine exposure required to induce the adaptive mechanisms leading to an increase in the levels of Gq and G11 proteins in the hypothalamic paraventricular nucleus and amygdala during withdrawal from cocaine has not been determined. In the present study, we evaluated the minimum number of days of cocaine treatment that will produce an increase in the levels of Gq and G11 proteins in rats withdrawn from cocaine.
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Materials and Methods |
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Tissue Preparation. Rat brains were placed in a cryostat at -10°C, and coronal sections were cut to obtain a 700-µm-thick section containing the paraventricular nucleus and rostral amygdala and a 1200-µm-thick section containing the caudal amygdala. The paraventricular nucleus and amygdala were microdissected from these frozen sections with the aid of a dissecting stereomicroscope. Plasma membranes of frontal cortex, amygdala, and paraventricular nucleus of the hypothalamus were prepared as described previously (Carrasco et al., 2003). All procedures were conducted at 4°C. Briefly, the frontal cortex, hypothalamic paraventricular nucleus, and amygdala were homogenized in 50 mM Tris buffer (pH 7.4) containing 150 mM NaCl, 10% sucrose, and 0.5 mM phenylmethylsulfonyl fluoride, and additional protease inhibitors purchased as a cocktail [containing 4-(2-aminoethyl)benzenesulfonyl fluoride, pepstatin A, trans-epoxysuccinyl-L-leucyl-amido(4-guanidino)butane, bestatin, leupeptin, and aprotinin] from Sigma-Aldrich (St. Louis, MO; 1.5 µl/30 mg of tissue). After centrifugation at 20,000g for 60 min, the supernatant was collected and stored at -80°C for further analyses of cytosol-associated protein levels. The pellets were collected and resuspended by sonication in a 20 mM Tris buffer (pH 8), containing 1 mM EDTA, 100 mM NaCl, 1% sodium cholate, and 1 mM dithiothreitol, plus the protease inhibitory cocktail (1.5 µl of cocktail/30 mg of tissue). The resuspended pellets were incubated while shaking for 1 h at 4°C and then centrifuged at 100,000g for 60 min. The supernatant was collected for the Western blot analyses of membrane-associated protein levels. Protein concentration was measured using a bicinchoninic acid protein assay kit (Pierce Chemical, Rockford, IL). The membrane proteins were stored at -80°C for Western blot analyses.
Western Blot Analysis. Samples containing 2 µg (hypothalamic paraventricular nucleus), 3 µg (amygdala), and 4 µg (frontal cortex) of protein were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis containing 0.1% SDS, 12.5% acrylamide/bisacrylamide (30:0.2), 4.6 M urea, and 275 mM Tris, pH 8.7. Gels were transferred electrophoretically by semi-dry blot to nitrocellulose membranes. After incubation with a blocking buffer (phosphate-buffered saline containing 0.2% casein and 0.1% Tween 20), the nitrocellulose membranes were probed overnight at 4°C with polyclonal antisera. Immunodetection was performed with either anti-G11 (1:500; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), anti-Gq (1:500; Santa Cruz Biotechnology, Inc.), or anti-Gz (1:6000; Santa Cruz Biotechnology, Inc.). The overnight incubation with Gq, G11, and Gz antibodies was followed by incubation with peroxidase-labeled anti-rabbit antibody (1:20,000; 1 h at room temperature; Santa Cruz Biotechnology, Inc.). Finally, the membranes were incubated with the enhanced chemiluminescence substrate solution (Amersham Biosciences Inc., Piscataway, NJ) and then exposed to X-ray film (Eastman Kodak, Rochester, NY). Protein loading for each lane was verified using an anti-actin antibody (1:20,000; Santa Cruz Biotechnology, Inc.). Negative controls included either omission of primary antibody or addition of preimmune rabbit immunoglobulins.
Film Analysis. Films were analyzed densitometrically using Scion Image software (Scion Corporation, Frederick, MD). The gray scale density readings were calibrated using a transmission step-wedge standard. The integrated optical density (IOD) of each band was calculated as the sum of the optical densities of all the pixels within the area of the band outlined. An adjacent area was used to calculate the background optical density of the film. The IOD for the film background was subtracted from the IOD for each band. Each sample was measured on three independent gels. All samples were standardized to controls and normalized to its respective actin level.
Statistics. All data are expressed as the mean ± S.E.M., where n indicates the number of rats per group. A one-way analysis of variance (ANOVA) was used to analyze the Western blot data, considering p < 0.05 as statistically significant. Group means were compared by Newman-Keuls multiple range test (Steel and Torrie, 1960). GBSTAT software (Dynamic Microsystems, Inc., Silver Spring, MD) was used for all statistical analyses.
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11 Protein. G11 protein was detected as a single band at approximately 40 kDa (Fig. 1A). The level of membrane-associated G11 protein in the hypothalamic paraventricular nucleus was elevated in rats treated with cocaine for 1 to 7 days and withdrawn for 2 days. The ANOVA revealed that cocaine treatment [F(4,49) = 39.42315, p < 0.0001] had a significant main effect on the level of membrane-associated G11 protein. The increase in the levels of G11 protein ranged from 24.5% over the control levels (p < 0.01) in rats that received cocaine treatment for 1 day to 95% over the control levels (p < 0.01) in rats that received cocaine treatment for 5 days (Fig. 1A). Rats that received 7 days of cocaine treatment showed an increase of 69% over the control levels (p < 0.01) (Fig. 1A). The levels of membrane-associated G11 protein were significantly higher in rats which received 5 days of cocaine treatment than those which received 1, 3, or 7 days of cocaine treatment (p < 0.01) (Fig. 1A). Cocaine treatment did not have a significant effect on the levels of cytosol-associated G11 protein in the hypothalamic paraventricular nucleus [F(4,49) = 0.25323, p > 0.9] (Fig. 1A).
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Cocaine treatment had a significant main effect on the levels of membrane-associated G11 protein in the amygdala [F(4,49) = 26.19591, p < 0.0001p] (Fig. 1B). The peak in the levels of membrane-associated G11 protein in the amygdala was found after 1 day of cocaine treatment (107% over control levels; p < 0.01). The levels of membrane-associated G11 protein in the amygdala stayed elevated during the first 5 days of cocaine treatment (p < 0.01) (Fig. 1B). No significant differences were observed for the levels of membrane-associated G11 protein among 1 to 5 days of cocaine treatment (p > 0.05). After 7 days of cocaine treatment, the levels of membrane-associated G11 protein (76% over control levels) were significantly lower than after 1, 3, or 5 days of cocaine treatment (p < 0.05). No significant effect of cocaine was found on the levels of cytosol-associated G11 in the amygdala [F(4,49) = 0.79477, p > 0.5] (Fig. 1B).
In the frontal cortex, cocaine treatment did not produce a significant change in the levels of membrane-associated [F(4,49) = 0.55749, p > 0.69] or cytosol-associated [F(4,49) = 0.3706, p > 0.8283] G11 protein (Fig. 1C) across the different treatment durations.
Gq Protein. Gq proteins were detected in the hypothalamic paraventricular nucleus, amygdala, and frontal cortex as a single band at approximately 42 kDa (Fig. 2). The levels of membrane-associated Gq protein in the paraventricular nucleus were also affected by cocaine treatment [F(4,49) = 23.63318, p < 0.0001]. Cocaine treatment gradually increased the levels of membrane-associated Gq (Fig. 2A): 60% over control levels after 1 day and 86% over control levels after 3 days of cocaine treatment (Fig. 2A). The peak levels of membrane-associated Gq protein in the paraventricular nucleus were detected after 5 days of cocaine treatment (105% over control levels; p < 0.001). The levels of membrane-associated Gq protein were significantly higher in rats which received 5 days of cocaine treatment than those which received 1, 3, or 7 days of cocaine treatment (p < 0.05) (Fig. 2A). After 7 days of cocaine treatment, the levels of membrane-associated Gq protein were 60% over control levels (p < 0.01) (Fig. 2A). The levels of cytosol-associated Gq protein were also significantly altered by cocaine [F(4,49) = 13.76312, p < 0.0001]. However, whereas the levels of cytosol-associated Gq protein were not significantly altered (p > 0.05) during the first 5 days of cocaine treatment (Fig. 2A), a 30% reduction (p < 0.01) was detected after 7 days of cocaine treatment.
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In the amygdala, the ANOVA indicated a significant main effect of cocaine treatment [F(4,49) = 28.35248, p < 0.0001] on the levels of membrane-associated Gq protein. In rats that received cocaine for 1 day, the levels of membrane-associated Gq proteins were increased by approximately 74% over the saline control (p < 0.01) (Fig. 2B). In rats that received cocaine for 3 days, the levels of membrane-associated Gq protein were even higher (103% over control levels; p < 0.01). The levels of membrane-associated Gq protein in rats that received 3 days of cocaine treatment were higher than those which received 1, 5, or 7 days of cocaine treatment (p < 0.05) (Fig. 2B). After 5 and 7 days of cocaine treatment, the levels of membrane-associated Gq protein were only elevated by 57% and 43% over control levels, respectively. Levels of cytosol-associated Gq proteins in the amygdala were also affected by cocaine treatment [F(4,49) = 15.18487, p < 0.001]. The levels of cytosol-associated Gq proteins were increased by 25% over the control levels in rats that received cocaine for 1 day (p < 0.005) and 55% in rats treated with cocaine for 3 days (p < 0.001) (Fig. 2B). The levels of cytosol-associated Gq stayed elevated after 5 and 7 days of cocaine treatment by 50% and 45% higher over control levels (p < 0.01) (Fig. 2B). In rats treated with cocaine for 7 days, the levels of cytosol-associated Gq proteins were still 43% higher (p < 0.01) than control levels (Fig. 2B).
In the frontal cortex, cocaine treatment did not alter the levels of membrane-associated Gq protein [F(4,49) = 1.43207, p > 0.24] or the levels of cytosol-associated Gq protein [F(4,49) = 0.55094, p > 0.69] (Fig. 2C).
Gz Protein. Gz is a 40-kDa protein that has not been reported to associate with the 5-HT2A receptor signaling cascade. The determination of Gz protein was used to verify the specificity of the effects of cocaine treatment on G proteins. Because Gz proteins are not involved in 5-HT2A receptor signaling during withdrawal from chronic cocaine treatment, changes in the levels of membrane and cytosol-associated Gz proteins were also evaluated as negative controls. The ANOVA for the levels of membrane and cytosol-associated Gz protein in the paraventricular nucleus showed no significant main effect of cocaine treatment [F(4,49) = 0.60125, p > 0.66 and F(4,49) = 0.28564, p > 0.88, for membrane and cytosol, respectively; Fig. 3A]. In the amygdala, the statistical analysis for the levels of membrane and cytosol-associated Gz proteins showed no significant main effects of cocaine treatment [F(4,49) = 0.55889, p > 0.69 and F(4,49) = 0.2732, p > 0.89, for membrane and cytosol, respectively; Fig. 3B]. Additionally, cocaine treatment did not affect the levels of membrane or cytosol-associated Gz protein in the frontal cortex [F(4,49) = 1.21265, p > 0.31 and F(4,49) = 1.24992, p > 0.30, for membrane and cytosol, respectively; Fig. 3C].
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). Cocaine administration (15 mg/kg i.p, twice a day for 7 days) enhances the head shake response elicited by a specific 5-HT2A/2C receptor agonist (DOI) (Baumann et al., 1993). We have also previously reported that DOI-mediated increases in plasma levels of prolactin, corticosterone, and ACTH become supersensitive after an identical cocaine paradigm (Levy et al., 1992). Interestingly, in both the behavioral and the neuroendocrine studies, there was no increase in the maximal responses to DOI but instead a leftward shift in the dose-response curve (Levy et al., 1994a; Baumann and Rothman, 1998), suggesting alterations in the coupling state or postreceptor signal transduction mechanisms. Because no differences in the density of 5-HT2A receptors have been found in the frontal cortex, amygdala, nucleus accumbens, caudate putamen, and thalamus of cocaine-treated rats, when measured by quantitative receptor autoradiography and binding assays (Perret et al., 1998), the increased sensitivity must be due to changes in the efficiency of 5-HT2A receptor transduction mechanisms. 5-HT2A receptors are coupled via Gq/11 and G
proteins to phospholipase C and A2 signaling cascades (Roth et al., 1998).
The present results indicate that the increase in the levels of Gq and G11 proteins in the amygdala peaks during withdrawal from 1 to 3 days of cocaine treatment. In the hypothalamic paraventricular nucleus, the increase in the levels of Gq and G11 proteins is more gradual and peaks after withdrawal from 5 days of cocaine treatment. Thus, cocaine induced a more rapid increase in the membrane-associated levels of Gq and G11 proteins in the amygdala than in the hypothalamic paraventricular nucleus, suggesting that the amygdala shows a faster neuroadaptation to the effects of cocaine. Although the neuroadaptations occurring in both of these brain regions may not be related, there are three lines of evidence supporting a role of the amygdala in the function of the hypothalamic paraventricular nucleus. First, injection of ketanserin, a 5-HT2 receptor antagonist into the amygdala inhibited the effect of photic stress on the release of ACTH and corticosterone (Feldman et al., 1998). Second, as mentioned earlier, activation of 5-HT2A receptors expressed by neurons in the hypothalamic paraventricular nucleus increases the secretion of ACTH, corticosterone, oxytocin, and prolactin (Van de Kar et al., 2001). We recently reported (Zhang et al., 2002) that when MDL 100,907, a 5-HT2A receptor antagonist, is injected directly into the hypothalamic paraventricular nucleus, it completely blocks the prolactin and oxytocin responses to DOI, whereas the same dose of MDL 100,907 produces an incomplete inhibition of the ACTH response to DOI, suggesting a role of extrahypothalamic structures in the regulation of ACTH release. Last, 5-HT2A receptors are found in oxytocin and corticotrophin-releasing factor-immunoreactive cells in the hypothalamic paraventricular nucleus, whereas in the amygdala, 5-HT2A receptors are found in corticotrophin-releasing factor-immunoreactive cells (Zhang et al., 2001; Gray et al., 2003). 5-HT2A receptors are not found on enkephalin neurons, which are part of an inhibitory local circuit in the amygdala, further emphasizing their excitatory role in this brain area (Gray et al., 2003). Interestingly, a number of studies suggest that amygdaloid corticotrophin-releasing factor plays an important role in the expression of cocaine-induced behavior (Yun and Fields, 2003).
We further speculate that serotonin release in the amygdala activates 5-HT2A receptors (located on corticotrophin-releasing factor-expressing neurons) and induces corticotrophin-releasing factor secretion from terminals in the brainstem that mediate anxiety and stress-like responses. Thus, 5-HT2A receptors could activate corticotrophin-releasing factor neurons in the amygdala, which excite dorsal raphe neurons to induce ACTH release via their projection to hypothalamic neurons (Commons et al., 2003). Also, direct projections from the central amygdaloid nucleus to the hypothalamic paraventricular nucleus could mediate this effect (Gray et al., 1989). In this way, the activation of 5-HT2A receptors in the amygdala could be one mechanism by which neuroendocrine (hypothalamic corticotrophin-releasing factor) and autonomic/behavioral (amygdaloid corticotrophin-releasing factor) responses are integrated during responses to stressful or aversive stimuli (Gray et al., 2003). Thus, it is possible that in rats withdrawn from cocaine, an exacerbation of 5-HT2A receptor signaling in the amygdala, produced by an increase in the membrane-associated levels of Gq and G11, could lead to a supersensitivity of the 5-HT2A receptor signaling in the paraventricular nucleus.
The increase in Gq and G11 protein levels during cocaine withdrawal may be due to three different phenomena: 1) an increased expression of the G proteins specifically at the cell membrane, 2) a general increase in G protein levels in both membrane and cytosolic cellular compartments, and 3) a translocation of the G proteins from the cytoplasmic to the membrane portion. Because no reductions in the levels of cytosol-associated Gq and G11 proteins were found, it is not likely that the supersensitivity of 5-HT2A receptor signaling in cocaine-treated rats involves a redistribution or translocation of Gq and G11 proteins from the cytosol to the plasma membrane in the amygdaloid-hypothalamic neuronal circuits. The significant increase in the cytosol-associated levels of Gq proteins in the amygdala could involve increase in the mRNA levels encoding Gq and G11 proteins or decreased rate of degradation of these proteins in this brain region. Although Gq and G11 proteins are targets for N-myristoylation but not ADP-ribosylation (Chen and Manning, 2001), there is no evidence that treatment with cocaine leads to changes in the post-translational modifications of Gq or G11 proteins.
Additionally, it is possible that other proteins involved in the function of the 5-HT2A receptor and Gq and G11 proteins could be modified by cocaine treatment. These include two families of G protein regulators important to the specificity and kinetics of the G protein signaling: the RGS proteins and the activator of G protein signaling (AGS) family. In a previous report (Carrasco et al., 2003), we did not find changes in the levels of membrane and cytosol associated-levels of RGS4 and RGS7 proteins, two RGS proteins associated with the Gq and G11 protein signaling (Hollinger and Hepler, 2002), in the hypothalamic paraventricular nucleus, amygdala, or frontal cortex. However, other RGS proteins are up-regulated by cocaine (Bishop et al., 2002; Rahman et al., 2003). RGS4 shows a biphasic response in the locus coeruleus, with decreased RGS4 mRNA levels after acute administration and increased levels of RGS4 mRNA after chronic administration (Bishop et al., 2002). Also, the protein content of RGS9 is increased by chronic cocaine in the nucleus accumbens (Rahman et al., 2003). In human cocaine overdose abusers, up-regulation of RGS3 and down-regulation of RGS12 mRNAs in the ventral tegmental area have been reported (Tang et al., 2003). On the other hand, AGS3 one member of the AGS proteins is up-regulated in the prefrontal cortex during withdrawal from cocaine (Bowers et al., 2004). AGS3 binds to Gi proteins and inhibits GDP dissociation (Bowers et al., 2004). To the best of our knowledge, there is no current evidence of AGS proteins associated to the Gq and G11 protein signaling.
The activity of the hypothalamic-pituitary-adrenal axis before, at the time of, and subsequent to cocaine exposure seems to be an important determinant of whether individuals will engage in cocaine-seeking behavior (Mantsch et al., 2003). Whereas acute administration of cocaine stimulates the hypothalamic-pituitary-adrenal axis in rats (Rivier and Vale, 1987; Levy et al., 1991), chronic cocaine administration is associated with neuroadaptive changes that lead to attenuated responses to cocaine in rats and humans (Levy et al., 1993; Zhou et al., 2003). Rats chronically treated with cocaine showed a deficit in serotonergic nerve terminal function as seen from a reduced ACTH response to serotonin-releasing drugs p-cloroamphetamine and fenfluramine (Levy et al., 1994b; Van de Kar et al., 1995). On the other hand, withdrawal from cocaine is associated with activation of the hypothalamic-pituitary-adrenal axis in rats (Peltier et al., 2001; Zhou et al., 2003). This enhanced response of the hypothalamic-pituitary-adrenal axis during withdrawal from chronic cocaine has been reported 24 to 48 h after the last cocaine injection (Peltier et al., 2001; Zhou et al., 2003). Similar results have been reported in human cocaine addicts (Vescovi et al., 1992; Baumann et al., 1995). These observations suggest that after development of adaptation or tolerance to chronic cocaine that produces deficits in the serotonergic activity, and during the withdrawal period, there is an increase in the activity of the hypothalamic-pituitary-adrenal axis. This increased activity would be associated to the supersensitivity of the 5-HT2A receptors and can be measured as increased 5-HT2A receptor-mediated release of ACTH, corticosterone, and prolactin. This supersensitivity of postsynaptic 5-HT2A receptors in the hypothalamic-pituitary-adrenal axis may be mediated through increased levels of membrane-associated of Gq or G11 proteins in the hypothalamic paraventricular and/or amygdala. However, at this point in time we cannot conclusively show whether the changes observed in Gq or G11 proteins are due to cocaine treatment or the withdrawal period.
In summary, our results reveal unique neuroadaptive mechanisms in the hypothalamic paraventricular nucleus and the amygdala but not frontal cortex of rats withdrawn from cocaine. The mechanisms by which cocaine mediates its region-specific effects are incompletely understood, although some behavioral and neuroendocrine effects associated with 5-HT2A receptor regulation seem to involve increased levels of membrane-associated Gq and G11 proteins. These findings provide insight into the relative importance of individual components of the 5-HT2A receptor signal transduction system in regulating the overall sensitivity of this signaling transduction pathway.
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: 5-HT, 5-hydroxytryptamine (serotonin); ACTH, adrenocorticotropic hormone; DOI, (±)-1-(2,5 dimethoxy-4-iodophenyl)-2-amino-propane HCl; RGS, regulators of G protein signaling; IOD, integrated optical density; ANOVA, analysis of variance; AGS, activator of G protein signaling; MDL 100,907 (+)-
-(2,3-dimethoxyphenyl)-1-[2-(4-fluorophenylethyl)]-4-piperidinemethanol.
Address correspondence to: Dr. Louis D. Van de Kar, Department of Pharmacology, Loyola University of Chicago, Stritch School of Medicine, 2160 South First Ave., Maywood, IL 60153. E-mail: lvandek{at}lumc.edu
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