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

Abstract

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.

Figure 1

Olanzapine treatment significantly increased (* p<0.05) phosphorylation of JAK2 in the frontal cortex. AG490 treatment alone had no effect on phosphorylation of JAK2, however, inhibition of the JAK-STAT pathway with AG490 pre-treatment significantly attenuated phosphorylation of JAK2. Total JAK2 protein levels were not altered. (* indicate significantly different from olanzapine treated rats p<0.05), whereas AG490 alone had no effect on JAK2 levels. There were four animals in each treatment group and the western blots were repeated three times.

Figure 2

PLC activity in the frontal cortex. (A): 5-HT-stimulated PLC activity in the frontal cortex is significantly decreased with 7 days of daily olanzapine injections compared to vehicle-treated control rats (*p < 0.01). AG490 injections alone had no effect on the PLC activity whereas the olanzapine-induced decrease in PLC activity was significantly (*p<0.05) attenuated by pre-injections of AG490 (10 mg/kg). (B) GTPγS-stimulated-PLC activity was not altered by either the AG490 or olanzapine treatments. There were five animals used in each treatment group and the assay was performed three times with two replicates.

Figure 3

Olanzapine treatment significantly (* p<0.05) increased RGS7 mRNA in the frontal cortex (A) and membrane-bound RGS7 protein levels (B) over vehicle-treated control rats. AG490 pre-treatment completely blocked the olanzapine-induced increase in mRNA levels of RGS7. Olanzapine-induced increases in protein levels were also blocked by AG490 pretreatment. (* indicate significantly different from olanzapine treated rats p<0.05), whereas AG490 alone had no effect on RGS7 levels. This assay was performed with four animals in each treatment group, three separate times and each time in triplicate.

Figure 4

Chronic treatment with olanzapine significantly attenuated DOI-stimulated hormone responses. Oxytocin (A), ACTH (B) and corticosterone (C) responses to a challenge with DOI 1 mg/ml at 30 min post-injection. The data represent the mean ± S.E.M. of eight rats per group. Basal plasma oxytocin, ACTH and corticosterone levels were not significantly different among vehicle, AG490 and olanzapine injected rats. DOI-challenge induced a significant increase of plasma oxytocin, ACTH and corticosterone levels compared with saline-challenge groups. A significant difference amongst DOI-challenged control and treated rats is indicated by * for p < 0.001. A significant effect of chronic olanzapine and AG490 treatment compared with vehicle treatment is indicated by δ δ, p < 0.01. There were eight animals in each treatment group and each sample was examined in quadruplicate.

Figure 5

Chronic olanzapine increased JAK2 phosphorylation in the PVN. The levels of phosphorylated JAK2 were significantly increased by daily treatments of 10 mg/kg olanzapine (Olan). Total JAK2 protein levels were not altered by olanzapine. *Indicates significantly different from vehicle-treated control rats at p<0.001. There were four animals in each treatment group and each western blot assay was performed three times.

Figure 6

Chronic olanzapine treatment increased RGS7 protein levels in the PVN. RGS7 levels in the membrane fraction of the PVN were increased by daily treatment with 10.0 mg/kg olanzapine. Actin was used to verify equal loading of lanes in the SDS PAGE gel. *Indicates significantly different from vehicle-treated control rats compared to 10 mg/kg olanzapine at p < 0.001. There were four animals in each treatment group and each western blot assay was performed three times.