Agonist induced-phosphorylation of Galpha11 protein reduces coupling to 5-HT2A receptors.

We previously demonstrated that 24-h treatment with (-)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane HCl (DOI) causes phosphorylation of Galpha11 protein at serine 154 and that this phosphorylation causes desensitization of serotonin (5-HT) 2A receptor signaling in A1A1v cells (Shi et al., 2007). We now report that treatment of A1A1v cells with DOI for 24 h produces a greater reduction in the Bmax of [125I](+/-)-DOI-labeled high-affinity binding sites (46%) than the reduction of [3H]ketanserin binding sites (25%). Although the KD values are not altered, there is a smaller amount of GTPgammaS [guanosine 5'-3-O-(thio)triphosphate]-sensitive [125I](+/-)-DOI binding in DOI-treated cells. These results suggest that DOI treatment causes down-regulation of 5-HT2A receptors and reductions in G protein-coupled 5-HT2A receptors. In contrast, in cells transfected with the phosphorylation state mimic G(alpha11)S154D, GTPgammaS-sensitive [125I](+/-)-DOI binding was decreased by 48%; however, there was no significant difference in the KD and Bmax values of [125I](+/-)-DOI-labeled receptors. The receptor binding experiments suggest that phosphorylation of Galpha11 on serine 154 reduces coupling of 5-HT2A receptors, whereas DOI causes down-regulation of 5-HT2A receptors in addition to the phosphorylation-induced uncoupling of Galpha11 to 5-HT2A receptors. To determine whether DOI increases phosphorylation of Galphaq/11 protein in vivo, rats were treated with 1 mg/kg/day DOI or saline for 1 to 7 days. Seven days of DOI treatment significantly decreased phospholipase C activity stimulated by an Emax concentration of 5-HT by 40% and increased phosphorylation of Galphaq/11 proteins by 51% in the frontal cortex. These data suggest that DOI causes phosphorylation of Galphaq/11 in vivo and could thereby contribute to the desensitization of 5-HT2A receptors.

Alterations in 5-HT2A receptor signaling in male and female transgenic rats over-expressing either Gq or RGS-insensitive Gq protein.

Serotonin 2A (5-HT2A) receptors are coupled to Galphaq and Galpha11 proteins to activate phospholipase C (PLC). Regulators of G-protein signaling proteins (RGS) modulate G-protein signaling by accelerating the intrinsic GTPase activity of Galphaq and Galpha11. This study investigated the effects of over-expression of wild-type Galphaq proteins (Gq-Tg) and over-expression of RGS-insensitive Galphaq proteins (G188S, RGSi-Tg) on 5-HT2A receptor mediated signaling in transgenic rats. Over-expression of wild-type Galphaq and RGS insensitive mutant Galphaq did not produce significant alterations in the levels of Galpha11, RGS2, RGS4, RGS7, RGS16 or 5-HT2A proteins. RGSi-Tg rats had higher oxytocin and corticosterone responses to (-)DOI, a 5-HT2A/2C receptor agonist, compared to Gq-Tg rats. RGSi-Tg and Gq-Tg rats had higher ACTH responses to (-)DOI compared to control rats. Similarly, 5-HT-stimulated PLC activity in the frontal cortex was higher in RGSi-Tg and Gq-Tg rats compared to control rats. In contrast, GTPgammaS-stimulated PLC activity was higher in Gq-Tg rats but not in RGSi-Tg rats compared to control rats. There was a small but statistically significant increase in the affinity of [125I]-DOI labeled 5-HT2A receptors in RGSi-Tg rats and Gq-Tg rats compared to controls. There were no significant differences in Bmax and Kd of [3H] ketanserin labeled 5-HT2A receptors among the three groups. These data suggest that the effect of RGS proteins on 5-HT2A receptor signaling is cell type specific. In transgenic rats over-expressing Galphaq, endogenous RGS proteins have a negative effect on 5-HT2A receptor-mediated oxytocin release. In contrast, endogenous RGS protein had no impact on 5-HT2A receptor-mediated ACTH release in transgenic rats.

Tandospirone activates neuroendocrine and ERK (MAP kinase) signaling pathways specifically through 5-HT1A receptor mechanisms in vivo.

Tandospirone, an azapirone, is a selective serotonin(1A) (5-HT(1A)) receptor agonist. The effects of tandospirone on plasma hormones and on mitogen-activated protein (MAP) kinase activity in the brain of male rats were studied. Tandospirone produced a time- and dose-dependent increase in plasma levels of oxytocin, adrenocorticotropin (ACTH), corticosterone, and prolactin. The minimal dose of tandospirone that led to a significant elevation of plasma oxytocin, ACTH, and prolactin levels was 1.0 mg/kg (s.c.), while the minimal dose for corticosterone release was 3.0 mg/kg (s.c.). The ED(50) of tandospirone was 1.3 mg/kg for oxytocin, 1.2 mg/kg for ACTH, 3.0 mg/kg for corticosterone, and 0.24 mg/kg for prolactin. Pretreatment with the specific 5-HT(1A) receptor antagonist WAY 100,635 (0.3 mg/kg, s.c.) completely blocked the effects of tandospirone on plasma levels of oxytocin, ACTH, and corticosterone but shifted the dose-response curve for prolactin to the right. Tandospirone injection (10 mg/kg, s.c.) stimulated the MAP kinase signaling cascade, specifically the phosphorylation of p42/44 extracellular signal-regulated kinase (ERK). Western blot analysis revealed a significant increase in phosphorylated ERK (p-ERK) levels in the hypothalamic paraventricular nucleus (PVN) as well as the dorsal raphe nucleus 5 min following tandospirone injection. These increases were blocked by pretreatment with WAY 100,635 (0.3 mg/kg). The results are the first evidence that systemic 5-HT(1A) receptor agonist administration produces a rapid increase in p-ERK levels in vivo, providing further insight into the signaling mechanisms of the 5-HT(1A) receptor.

Chronic mild stress induces behavioral and physiological changes, and may alter serotonin 1A receptor function, in male and cycling female rats.

RATIONALE: Interactions among stress, serotonin 1A (5-HT(1A)) receptors, and the hypothalamic-pituitary-adrenocortical (HPA) system have been proposed to influence the development of depression in humans. The investigation of depression-relevant behaviors and physiological responses to environmental stressors in animal models of depression may provide valuable insight regarding these mechanisms. OBJECTIVES: The purpose of these experiments was to investigate the interactions among central 5-HT(1A) receptors, endocrine function, and behavior in an animal model of depression, chronic mild stress (CMS). METHODS: The current study examined behavioral responses to a pleasurable stimulus (sucrose), estrous cycle length (in female rats), and plasma hormone levels following systemic administration of a selective 5-HT(1A) receptor agonist [(+)8-hydroxy-N,N-dipropyl-2-aminotetralin hydrobromide (8-OH-DPAT); 40 mug/kg, s.c.; administered 15 min prior to sacrifice], in male and female rats exposed to 4 weeks of CMS. RESULTS: Four weeks of CMS produced a reduction in the intake of 1% sucrose (anhedonia), as well as attenuated adrenocorticotropic hormone (ACTH) responses to 8-OH-DPAT in both male and female rats (22 and 18% lower than the control groups, respectively). Corticosterone and oxytocin responses to 8-OH-DPAT were not altered by exposure to CMS. In female rats, CMS induced a lengthening of the estrous cycle by approximately 40%. CONCLUSIONS: CMS produces minor HPA disruptions along with behavioral disruptions. Alterations in 5-HT(1A) receptor function in specific populations of neurons in the central nervous system may be associated with the CMS model. The current findings contribute to our understanding of the relations that stress and neuroendocrine function have to depressive disorders.

Estrogen reduces serotonin-1A receptor-mediated oxytocin release and Galpha(i/o/z) proteins in the hypothalamus of ovariectomized rats.

The present study examined the effect of estradiol on hypothalamic serotonin-1A (5-HT(1A)) receptor signaling in female rats. We first examined the time-course effects of a single injection of the 5-HT(1A) receptor agonist (+/-)8-OH-DPAT (5, 15 or 30 min prior to decapitation), and dose response of (+)8-OH-DPAT (50, 100, 200 or 500 microg/kg, s.c.) on plasma hormones in ovariectomized rats that received a daily injection of beta-estradiol 3-benzoate (10 microg/day, s.c.) or vehicle (sesame oil) for 2 days. In vehicle- and estrogen-treated rats, the peak response of hormones occurred at 15 min after injection and the time-course of oxytocin and adrenocorticotropic hormone (ACTH) responses to an injection of 8-OH-DPAT were comparable. However, only the oxytocin response was reduced by estrogen treatment. A second experiment compared the ACTH and oxytocin responses with doses of 50 or 200 microg/kg, s.c. of (+)8-OH-DPAT vs. (+/-)8-OH-DPAT in ovariectomized rats that were treated with oil or beta-estradiol 3-benzoate (10 microg/day, s.c.) for 2 days. (+)8-OH-DPAT and (+/-)8-OH-DPAT produced a similar magnitude of increase in plasma levels of ACTH and oxytocin. Treatment with beta-estradiol 3-benzoate produced a significant and comparable reduction in the oxytocin response to the highest dose (200 microg/kg, s.c.) of both (+)8-OH-DPAT and (+/-)8-OH-DPAT but did not alter the ACTH response to either (+)8-OH-DPAT or (+/-)8-OH-DPAT. In the dose-response experiment, a dose of 50 microg/kg of (+)8-OH-DPAT produced a maximal increase in plasma levels of ACTH, while the maximal oxytocin response was achieved with a dose of 200 microg/kg, s.c. Treatment with beta-estradiol 3-benzoate reduced the maximal oxytocin response to (+)8-OH-DPAT (by 29%) but did not alter the ACTH response to any doses of (+)8-OH-DPAT. To examine potential mechanisms mediating the effects of estrogen on 5-HT(1A) receptor signaling, we measured the levels of Galpha(i), Galpha(o) and Galpha(z) proteins, which couple 5-HT(1A) receptors to their effector enzymes, in two subregions of the hypothalamus. The levels of Galpha(z) protein were reduced in the mediobasal hypothalamus (containing the ventromedial and arcuate nuclei), which mainly expresses estrogen receptor-alpha, but not in the paraventricular hypothalamus, which mainly expresses estrogen receptor-beta. Estradiol reduced the levels of Galpha(i2) and Galpha(i3 )proteins in both hypothalamic regions but did not affect Galpha(i1) levels in either area. Combined, the data suggest that racemic and stereoselective 8-OH-DPAT have similar neuroendocrine effects and that both estrogen receptor-alpha and estrogen receptor-beta mediate the reduction in levels of Galpha(i2,3) proteins.

Short-term cocaine treatment causes neuroadaptive changes in Galphaq and Galpha11 proteins in rats undergoing withdrawal.

One of the characteristics of drug dependence is that a drug has to be administered repeatedly before withdrawal effects can be observed. We have previously shown that withdrawal after 14 days of cocaine treatment produces a supersensitivity of hypothalamic 5-hydroxytryptamine (serotonin) 2A (5-HT(2A)) receptors, which is accompanied by increases in the levels of Galpha(q) and Galpha(11) 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 Galpha(q) and Galpha(11) 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 Galpha(q) and Galpha(11) 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 Galpha(11) and Galpha(q) proteins in the amygdala, 5 days of treatment were required to maximally increase the levels of Galpha(11) and Galpha(q) 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-HT(2A) receptor signal transduction system in regulating the overall sensitivity of this signaling in cocaine-treated rats.

Desensitization of 5-HT1A receptors by 5-HT2A receptors in neuroendocrine neurons in vivo.

An imbalance between serotonin-2A (5-HT2A) and 5-HT1A receptors may underlie several mood disorders. The present studies determined whether 5-HT2A receptors interact with 5-HT1A receptors in the rat hypothalamic paraventricular nucleus (PVN). The sensitivity of the hypothalamic 5-HT1A receptors was measured as oxytocin and adrenocorticotropic hormone (ACTH) responses to the 5-HT1A receptor agonist (+)-8-hydroxy-2-(di-n-propylamino) tetralin hydrobromide [(+)8-OH-DPAT] (40 microg/kg s.c.). The 5-HT(2A/2C) receptor agonist (-)DOI [(-)-1-(2,5-dimethoxy-4-iodophenyl)2-aminopropane HCl] (1 mg/kg s.c.) injected 2 h prior to (+)8-OH-DPAT significantly reduced the oxytocin and ACTH responses to (+)8-OH-DPAT, producing a heterologous desensitization of the 5-HT1A receptors. Microinjection of the 5-HT2A receptor antagonist MDL100,907 [(+)-alpha-(2,3-dimethoxyphenyl)-1-[2-(4-fluorophenylethyl)]-4-piperidinemethanol; 0, 10, or 20 nmol, 15 min prior to (-)DOI] into the PVN dose-dependently prevented the desensitization of 5-HT1A receptors induced by the 5-HT2A receptor agonist (-)DOI. Double-label immunocytochemistry revealed a high degree of colocalization of 5-HT1A and 5-HT2A receptors in the oxytocin and corticotropin-releasing factor neurons of the PVN. Thus, activation of 5-HT2A receptors in the PVN may directly induce a heterologous desensitization of 5-HT1A receptors within individual neuroendocrine cells. These findings may provide insight into the long-term adaptation of 5-HT1A receptor signaling after changes in function of 5-HT2A receptors; for example, during pharmacotherapy of mood disorders.

A region-specific increase in Galphaq and Galpha11 proteins in brains of rats during cocaine withdrawal.

Serotonin 2A (5-HT2A) receptor-mediated increases in plasma hormone levels become supersensitive after 42 h of withdrawal from cocaine treatment. The present study investigated which components of the 5-HT2A receptor signaling system are associated with this supersensitivity. Rats were injected daily for 14 days with either saline or cocaine (15 mg/kg i.p.) twice a day or were injected using a "binge" protocol (three injections per day, 1 h apart). Rats were sacrificed 2 or 7 days after the last cocaine injection, and the levels of membrane and cytosol-associated 5-HT2A receptors, Galphaq, Galpha11, regulators of G protein signaling (RGS)4, and RGS7 proteins were assayed in the hypothalamic paraventricular nucleus, amygdala, and frontal cortex using Western blot analysis. Two days of withdrawal from cocaine, administered twice a day or using a binge protocol, produced an increase in membrane-associated Galphaq and Galpha11 proteins in the paraventricular nucleus and the amygdala (but not in the frontal cortex). This effect was reversible after 7 days of withdrawal. The protein levels of the 5-HT2A receptor, Galphaz protein, and RGS4 or RGS7 proteins were not altered by cocaine withdrawal in any of the above-mentioned brain regions. These findings suggest that the supersensitivity of the 5-HT2A receptors, during withdrawal from chronic cocaine, is associated with an increase in membrane-associated Galphaq and Galpha11 proteins and not with changes in the expression of 5-HT2A receptors.

Evidence that 5-HT2A receptors in the hypothalamic paraventricular nucleus mediate neuroendocrine responses to (-)DOI.

The present study determined whether the serotonin2A (5-HT2A) receptors in the hypothalamic paraventricular nucleus mediate the neuroendocrine responses to a peripheral injection of the 5-HT2A/2C receptor agonist (-)DOI [(-)1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane]. The 5-HT2A receptor antagonist MDL100,907 ((+/-)-alpha(2,3-dimethoxyphenyl)-1-[2-(4-fluorophenylethyl)]-4-piperidinemethanol), the 5-HT2C receptor antagonist SB-242084 (6-chloro-5-methyl-1-[[2-[(2-methyl-3-pyridyl)oxy]-5-pyridyl]carbamoyl]-indoline), or vehicle were microinjected bilaterally through a chronically implanted double-barreled cannula into the hypothalamic paraventricular nucleus 15 min before a peripheral injection of (-)DOI in conscious rats. (-)DOI significantly elevated plasma levels of oxytocin, prolactin, ACTH, corticosterone, and renin. Neither the 5-HT2A receptor antagonist nor the 5-HT2C receptor antagonist, injected alone, altered the basal levels of these hormones. MDL100,907 (0.748, 7.48, and 18.7 nmol) dose dependently inhibited the (-)DOI-induced increase in all of the hormones except corticosterone. In contrast, SB-242084 (10 nmol) did not inhibit (-)DOI-increased hormone levels. To confirm the presence of 5-HT2A receptors in the hypothalamic paraventricular nucleus, 5-HT2A receptors were mapped using immunohistochemistry. Densely labeled magnocellular neurons were observed throughout the anterior and posterior magnocellular subdivisions of the hypothalamic paraventricular nucleus. Moderately to densely labeled cells were also observed in parvicellular regions. Thus, it is likely that 5-HT2A receptors are present on neuroendocrine cells in the hypothalamic paraventricular nucleus. These data provide the first direct evidence that neuroendocrine responses to a peripheral injection of (-)DOI are predominantly mediated by activation of 5-HT2A receptors in the hypothalamic paraventricular nucleus.