Effects of some new antiepileptic drugs and progabide on glucocorticoid receptor-mediated gene transcription in LMCAT cells.

Antiepileptic drugs affect endocrine and immune system activity, however, it is not clear whether these effects are indirect, via interference with neurotransmitters, membrane receptors and ion channels or maybe independent of neuronal mechanisms. In order to shed more light on this problem, in the present study, we evaluated effects of some new-generation antiepileptic drugs and progabide as a GABA-mimetic on the corticosterone-induced chloramphenicol acetyltransferase (CAT) activity in mouse fibroblast cells stably transfected with mouse mammary tumor virus (MMTV)-CAT plasmid. Treatment of cells with felbamate for five days inhibited in a concentration-dependent manner (3-100 microM) the corticosterone-induced reporter gene transcription. Progabide and loreclezole also inhibited the corticosterone-induced CAT activity, but with lower potency, and significant effects were observed at 10 to 100 microM concentration. Tiagabine and stiripentol showed less potent inhibitory effect on functional activity of glucocorticoid receptors (GR). In contrast, topiramate and lamotrigine (3-100 microM) failed to affect the corticosterone-induced gene transcription. These data indicate that some new antiepileptic drugs and progabide may suppress glucocorticoid effects via the inhibition of GR-mediated gene transcription. In turn, attenuation of GR function could influence antiepileptic drug effect on seizures, neuronal degeneration and immune system activity.

Effects of neurosteroids on glucocorticoid receptor-mediated gene transcription in LMCAT cells--a possible interaction with psychotropic drugs.

Aberrant activity of hypothalamic-pituitary-adrenal (HPA) axis is often observed in psychiatric disorders and both antidepressant and antipsychotic drugs are known to ameliorate some deleterious effects of glucocorticoids on brain function. Some neurosteroids possess antidepressant and neuroleptic-like properties and attenuate the stress-activated HPA axis activity. However, intracellular mechanism of neurosteroid interaction with glucocorticoids has not been elucidated. We evaluated effects of some neurosteroids on functional activity of glucocorticoid receptor (GR) in vitro. A combined treatment with antipsychotic drugs and involvement of some protein kinases in allopregnanolone effect on GR function were also studied. The effects of allopregnanolone, its two isomers (5beta-pregnan-3alpha-ol-20-one and 5alpha-pregnan-3beta-ol-20-one) and dehydroepiandrosterone sulfate (DHEAS) on the corticosterone-induced chloramphenicol acetyl transferase (CAT) activity were evaluated in mouse fibroblast cells stably transfected with mouse mammary tumor virus (MMTV)-CAT plasmid. We found that allopregnanolone (1-100 microM) and, to a lesser extent, both its isomers inhibited the GR-mediated gene transcription in a concentration-dependent manner. In contrast, DHEAS at the concentration up to 100 microM was inactive. Further experiments revealed that allopregnanolone and antipsychotic drugs (chlorpromazine and clozapine) showed a moderate, additive inhibitory effect on the GR function. With respect to intracellular mechanism of allopregnanolone action, we showed that this neurosteroid inhibited protein kinase C (PKC) activity, decreased the level of PKCalpha isoenzyme in the membrane fraction and decreased the amount of active phosphorylated form of extracellular signal-regulated kinase-mitogen-activated protein kinase (ERK-MAPK) in LMCAT cells. Since PKC and ERK-MAPK inhibitors attenuate the corticosterone-mediated gene transcription, the above findings suggest that allopregnanolone effect on GR function involves interaction with these kinase pathways. On the other hand, allopregnanolone had no effect on protein kinase A (PKA) activity. These data indicate that pregnanolone derivatives, like antidepressants and antipsychotic drugs, may attenuate some glucocorticoid effects via inhibition of GR-mediated gene transcription. Furthermore, the inhibitory effect of allopregnanolone on the corticosterone-induced gene transcription in LMCAT cells depended on the inhibition of PKC and ERK-MAPK pathways.

Antipsychotic drugs inhibit the human corticotropin-releasing-hormone gene promoter activity in neuro-2A cells-an involvement of protein kinases.

Antipsychotic drugs can regulate transcription of some genes, including those involved in regulation of hypothalamic-pituitary-adrenal (HPA) axis, whose activity is frequently disturbed in schizophrenic patients. However, molecular mechanism of antipsychotic drug action on the corticotropin-releasing hormone (CRH) gene activity has not been investigated so far. This study was undertaken to examine the influence of conventional and atypical antipsychotic drugs on the CRH gene promoter activity in differentiated Neuro-2A cell cultures stably transfected with a human CRH promoter fragment linked to the chloramphenicol acetyltransferase (CAT) reporter gene. It has been found that chlorpromazine (0.1-5.0 microM), haloperidol (0.5-5.0 microM), clozapine (1.0-5.0 microM), thioridazine (1.0-5.0 microM), promazine (5.0 and 10 microM), risperidone (5.0 and 10.0 microM), and raclopride (only at the highest used concentrations, ie 30 and 100 microM) present in culture medium for 5 days inhibited the CRH-CAT activity. Sulpiride and remoxipride had no effect. Since CRH gene activity is most potently enhanced by cAMP/protein kinase A pathway, the effect of antipsychotics on the forskolin-induced CRH-CAT activity was determined. Chlorpromazine (1.0-5.0 microM), haloperidol (1.0-5.0 microM), clozapine (1.0-5.0 microM), thioridazine (3.0 and 5.0 microM), and raclopride (30 and 100 microM), but not promazine, sulpiride, risperidone, and remoxipride, inhibited the forskolin-stimulated CRH gene promoter activity. A possible involvement of protein kinases in chlorpromazine and clozapine inhibitory action on CRH activity was also investigated. It was found that wortmannin (0.01 and 0.02 microM), an inhibitor of phosphatidylinositol 3-kinase (PI3-K), significantly attenuated the inhibitory effect of chlorpromazine and clozapine on CRH gene promoter activity. In line with these results, a Western blot study showed that these drugs increased phospho-Ser-473 Akt level, had no effect on total Akt, and decreased glycogen synthase kinase-3beta level. Additionally, we found that clozapine decreased protein kinase C (PKC) level and that its action on CRH activity was attenuated by PKC activator (TPA, 0.1 microM). The obtained results indicate that inhibition of CRH gene promoter activity by some antipsychotic drugs may be a molecular mechanism responsible for their inhibitory action on HPA axis activity. Clozapine and chlorpromazine action on CRH activity operates mainly through activation of the PI3-K/Akt pathway. Moreover, PKC-mediated pathway seems to be involved in clozapine action on CRH gene activity.

Effects of some new antidepressant drugs on the glucocorticoid receptor-mediated gene transcription in fibroblast cells.

Antidepressant drugs are thought to counteract effects of hypercortisolemia, frequently associated with depression, by lowering cortisol level and by modifying the function of glucocorticoid receptors (GR). Indeed, classical antidepressants inhibit corticosteroid-induced gene transcription in cell cultures. The aim of the present study was to investigate effects of new generation antidepressant drugs on GR function in mouse fibroblast cells (L929), stably transfected with mouse mammary tumor virus-chloramphenicol acetyltransferase (MMTV-CAT) plasmid (LMCAT cells). It has been found that reboxetine (at 10 and 30 microM), venlafaxine, citalopram and mirtazapine (at 30 microM), but not milnacipran, in statistically significant manner inhibited corticosterone-induced gene transcription. However, the effects of new generation antidepressant drugs were weaker than those evoked by imipramine, which was active already at 3 microM concentration. Further studies on the mechanism of antidepressant action on GR function revealed that protein kinase C, but not mitogen-activated protein kinases (MAPK), glycogen synthase kinase (GSK-3) and protein kinase B (PKB, Akt) play a role in this phenomenon.

Effect of lipopolysaccharide and antidepressant drugs on glucocorticoid receptor-mediated gene transcription.

It has been hypothesized that pro-inflammatory response and hyperactivity of hypothalamic-pituitary-adrenocortical axis (HPA) are involved in the pathogenesis of depression. Hyperactivity of HPA axis results probably from deregulation of glucocorticoid receptor function and impairment of the control mechanism of glucocorticoid secretion. Previously, we found that antidepressants inhibited glucocorticoid receptor (GR) function under the in vitro condition. In order to study a role of some mediators of pro-inflammatory response in this process, presently, we investigated the effect of lipopolysaccharide (LPS) on imipramine- or fluoxetine-induced inhibition of GR-mediated gene transcription in fibroblast cells, stably transfected with mouse mammary tumor virus promoter (LMCAT cells). Two days of incubation of the cells with imipramine (3-10 microM), fluoxetine (10 microM) or LPS (1 microg/ml) inhibited the corticosterone-induced gene transcription. Concomitant incubation of the cells with LPS and fluoxetine or imipramine had stronger inhibitory effect than that evoked by each compound alone. Moreover, we found that fluoxetine (10 microM) but not imipramine (3-10 microM) significantly inhibited the LPS-stimulated interleukin-6 (IL-6) production in these cells. These data suggest that pro-inflammatory agents facilitate antidepressant-induced inhibition of glucocorticoid receptor function.