Cannabinoid 2 receptor- and beta Arrestin 2-dependent upregulation of serotonin 2A receptors.

Recent evidence suggests that cannabinoid receptor agonists may regulate serotonin 2A (5-HT(2A)) receptor neurotransmission in the brain, although no molecular mechanism has been identified. Here, we present experimental evidence that sustained treatment with a non-selective cannabinoid agonist (CP55,940) or selective CB2 receptor agonists (JWH133 or GP1a) upregulate 5-HT(2A) receptors in a neuronal cell line. Furthermore, this cannabinoid receptor agonist-induced upregulation of 5-HT(2A) receptors was prevented in cells stably transfected with either CB2 or ß-Arrestin 2 shRNA lentiviral particles. Additionally, inhibition of clathrin-mediated endocytosis also prevented the cannabinoid receptor-induced upregulation of 5-HT(2A) receptors. Our results indicate that cannabinoid agonists might upregulate 5-HT(2A) receptors by a mechanism that requires CB2 receptors and ß-Arrestin 2 in cells that express both CB2 and 5-HT(2A) receptors. 5-HT(2A) receptors have been associated with several physiological functions and neuropsychiatric disorders such as stress response, anxiety and depression, and schizophrenia. Therefore, these results might provide a molecular mechanism by which activation of cannabinoid receptors might be relevant to some cognitive and mood disorders in humans.

Estradiol induces partial desensitization of serotonin 1A receptor signaling in the paraventricular nucleus of the hypothalamus and alters expression and interaction of RGSZ1 and Gαz.

Hyperactivity of hypothalamic-pituitary mediated hormone responses, such as to stimulation with a serotonin 1A (5-HT(1A)) receptor agonist, are a feature of depression which are normalized with clinical improvement during drug therapy. We previously reported that SSRIs induce desensitization of 5-HT(1A) receptor signaling in the paraventricular nucleus of the hypothalamus (PVN) while estradiol benzoate (EB) produces a more rapid, partial desensitization. In the current study, time course and dose-response experiments demonstrated that two once daily doses of EB is the minimum needed to induce the desensitization response as indicated by 5-HT(1A) receptor-stimulated release of oxytocin and that 10 µg/kg/day EB produces the maximal response, a partial desensitization of approximately 40%. The effects of two once daily injections of 10 µg/kg/day EB on Gαz and RGSZ1 proteins were examined as components of the 5-HT(1A) receptor signaling system, which mediates the release of oxytocin and adrenocorticotropic hormone. RGSZ1 appears to be a major target for EB-mediated responses in the 5-HT(1A) receptor signaling system. A 55 kD membrane-associate RGSZ1 protein was greatly increased in the PVN and rest of the hypothalamus and moderately increased in the dorsal hippocampus and amygdala after EB treatment as well as after an acute dose of a 5-HT(1A) receptor agonist. These results suggest that EB is a candidate for adjuvant therapy with SSRIs to hasten the therapeutic response and that RGSZ1 is a major target of EB therapy which could be explored as a target for novel therapeutic approaches for the treatment of depression.

Activation of the JAK-STAT pathway by olanzapine is necessary for desensitization of serotonin2A receptor-stimulated phospholipase C signaling in rat frontal cortex but not serotonin2A receptor-stimulated hormone release.

Chronic treatment with olanzapine causes desensitization of serotonin 2A receptor signaling. The purpose of the current study was to further understand the mechanisms underlying this desensitization response of serotonin 2A receptor signaling in vivo. We report that desensitization of serotonin 2A receptor stimulated-phospholipase C activity in rat frontal cortex induced by olanzapine is dependent on the activation of the JAK-STAT pathway. Olanzapine treatment for 7 days significantly increased the levels of the regulator of G protein signaling (RGS7) protein, RGS7 mRNA levels, and activation of JAK2 in rat frontal cortex. Pre-treatment with a JAK2 inhibitor AG490, significantly attenuated the olanzapine-induced reductions in serotonin 2A receptor-stimulated phospholipase C activity and prevented the olanzapine-induced increases in RGS7 mRNA and protein levels. In contrast, inhibition of the JAK-STAT pathway with AG490 did not reverse the olanzapine-induced desensitization of the serotonin 2A receptor pathway in the hypothalamic paraventricular nucleus mediating increases in plasma hormone levels. AG490 dose-dependently inhibited serotonin 2A receptor-stimulated oxytocin and corticosterone release. These results suggest that the olanzapine-induced increase in RGS7 expression is mediated by the activation of JAK-STAT and is necessary for olanzapine-induced desensitization of serotonin 2A receptor-stimulated phospholipase C activity in the frontal cortex but not serotonin 2A receptor-stimulated hormone release.

Extra-nuclear estrogen receptor GPR30 regulates serotonin function in rat hypothalamus.

Selective serotonin reuptake inhibitors (SSRIs), such as Prozac, are used to treat mood disorders. SSRIs attenuate (i.e. desensitize) serotonin 1A (5-HT(1A)) receptor signaling, as demonstrated in rats through decreased release of oxytocin and adrenocorticotropin hormone (ACTH) following 5-HT(1A) receptor stimulation. Maximal therapeutic effects of SSRIs for treatment of mood disorders, as well as effects on hypothalamic 5-HT(1A) receptor signaling in animals, take 1 to 2 weeks to develop. Estradiol also attenuates 5-HT(1A) receptor signaling, but, in rats, these effects occur within 2 days; thus, estrogens or selective estrogen receptor modulators may serve as useful short-term tools to accelerate desensitization of 5-HT(1A) receptors in response to SSRIs if candidate estrogen receptor targets in the hypothalamus are identified. We found high levels of GPR30, which has been identified recently as a pertussis-toxin (PTX) sensitive G-protein-coupled estrogen receptor, in the hypothalamic paraventricular nucleus (PVN) of rats. Double-label immunohistochemistry revealed that GPR30 co-localizes with 5-HT(1A) receptors, corticotrophin releasing factor (CRF) and oxytocin in neurons in the PVN. Pretreatment with PTX to the PVN before peripheral injections of 17-beta-estradiol 3-benzoate completely prevented the reduction of the oxytocin response to the 5-HT(1A) receptor agonist, (+)-8-hydroxy-2-dipropylaminotetralin (DPAT). Treatment with the selective GRP30 agonist, G-1, attenuated 5-HT(1A) receptor signaling in the PVN as measured by an attenuated oxytocin (by 29%) and ACTH (by 31%) response to DPAT. This study indicates that a putative extra-nuclear estrogen receptor, GPR30, may play a role in estradiol-mediated attenuation of 5-HT(1A) receptor signaling, and potentially in accelerating the effects of SSRIs in treatment of mood disorders.

Cocaine-mediated supersensitivity of 5-HT2A receptors in hypothalamic paraventricular nucleus is a withdrawal-induced phenomenon.

We previously reported that treatment and withdrawal from cocaine increases: (1) 5-HT2A receptor-mediated neuroendocrine responses, and (2) Galphaq and Galpha11 G-protein levels in the hypothalamic paraventricular nucleus (PVN) at 48 h post-treatment. This study investigates changes in the initial 24 h of withdrawal to discern whether 5-HT2A receptor supersensitivity is due to cocaine treatment or is induced during the withdrawal period. We report here increases in 5-HT2A receptor-mediated neuroendocrine responses only 12 or 24 h post-treatment, but not during the initial 4 h withdrawal period. Levels of membrane- or cytosol-associated Galphaq or Galpha11 proteins in PVN are not altered during the first 24 h of withdrawal. However, the density of 125I-(-)-1-(2,5 dimethoxy-4-iodophenyl)-2-amino-propane HCl (DOI)-labeled high-affinity 5-HT2A receptors in PVN increased 35% in rats withdrawn from cocaine for 24 h. These findings demonstrate that cocaine-induced increases in 5-HT2A receptor function in PVN represents a withdrawal-induced phenomena that: (1) is likely attributed to increased G-protein coupled/high-affinity conformational state of the 5-HT2A receptor, and (2) occurs in the absence of changes in the levels of associated G proteins during the first 24 h.

Estrogen treatment increases the levels of regulator of G protein signaling-Z1 in the hypothalamic paraventricular nucleus: possible role in desensitization of 5-hydroxytryptamine1A receptors.

Desensitization of post-synaptic serotonin1A (5-HT1A) receptors may underlie the clinical improvement of neuropsychiatric disorders. In the hypothalamic paraventricular nucleus, Galphaz proteins mediate the 5-HT1A receptor-stimulated increases in hormone release. Regulator of G protein signaling-Z1 (RGSZ1) is a GTPase-activating protein selective for Galphaz proteins. RGSZ1 regulates the duration of interaction between Galphaz proteins and effector systems. The present investigation determined the levels of RGSZ1 in the hypothalamic paraventricular nucleus of rats subjected to four different treatment protocols that produce desensitization of 5-HT1A receptors. These protocols include: daily administration of beta estradiol 3-benzoate (estradiol) for 2 days; daily administration of fluoxetine for 3 and 14 days; daily administration of cocaine for 7 or 14 days; and acute administration of (+/-)-1-(2,5 dimethoxy-4-iodophenyl)-2-amino-propane HCl (DOI; a 5-HT2A/2C receptor agonist). Estradiol treatment was the only protocol that increased the levels of RGSZ1 protein in the hypothalamic paraventricular nucleus in a dose-dependent manner (46%-132% over control). Interestingly, previous experiments indicate that only estradiol produces a decreased Emax of 5-HT1A receptor-stimulation of hormone release, whereas fluoxetine, cocaine and DOI produce a shift to the right (increased ED50). Thus, the desensitization of 5-HT1A receptors by estradiol might be attributable to increased levels of RGSZ1 protein. These findings may provide insight into the adaptation of 5-HT1A receptor signaling during pharmacotherapies of mood disorders in women and the well-established gender differences in the vulnerability to depression.

Neuroendocrine evidence that (S)-2-(chloro-5-fluoro-indol- l-yl)-1-methylethylamine fumarate (Ro 60-0175) is not a selective 5-hydroxytryptamine(2C) receptor agonist.

The 5-hydroxytryptamine(2A) and (2C) (5-HT(2A) and 5-HT(2C)) receptors are so closely related that selective agonists have not been developed until recently with the advent of (S)-2-(chloro-5-fluoro-indol-l-yl)-1-methylethylamine fumarate (Ro 60-0175), a putatively selective 5-HT(2C) receptor agonist. In the present study, Ro 60-0175 was used to analyze the importance of 5-HT(2C) receptors in hormone secretion. Injection of Ro 60-0175 (5 mg/kg s.c.) produced a maximum increase in plasma levels of adrenocorticotrophic hormone, oxytocin, and prolactin at 15 min postinjection and a maximum increase in plasma corticosterone levels at 60 min postinjection. Ro 60-0175-mediated increases in plasma hormone levels were dose-dependent (corticosterone ED(50) = 2.43 mg/kg; oxytocin ED(50) = 4.19 mg/kg; and prolactin ED(50) = 4.03 mg/kg). To assess the role of 5-HT(2C) and 5-HT(2A) receptors in mediating the hormone responses to Ro 60-0175, rats were pretreated with the 5-HT(2C) antagonist 6-chloro-5-methyl-1-[2-(2-methylpyridyl-3-oxy)-pyrid-5-yl carbonyl] indoline (SB 242084) or 5-HT(2A) antagonists (+/-)-2,3-dimethoxyphenyl-1-[2-4-(piperidine)-methanol] (MDL 100,907) before injection of Ro 60-0175 (5 mg/kg s.c.). Neither SB 242084 (0.1, 0.5, 1, and 5 mg/kg i.p.) nor MDL 100,907 (1, 5, and 10 microg/kg s.c.) significantly inhibited the Ro 60-0175-induced increases in plasma hormone levels. The data suggest that Ro 60-0175 increases hormone secretion by mechanisms independent of the activation of 5-HT(2C) and/or 5-HT(2A) receptors and suggest that Ro 60-0175 is not a highly selective 5-HT(2C) receptor agonist.