Fiona J. Thomson

Innovative Approaches for the Treatment of Depression: Targeting the HPA Axis

Altered activity of the hypothalamic pituitary adrenal (HPA) axis is one of the most commonly observed neuroendocrine abnormalities in patients suffering from major depressive disorder (MDD). Altered cortisol secretion can be found in as many as 80% of depressed patients. This observation has led to intensive clinical and preclinical research aiming to better understand the molecular mechanisms which underlie the alteration of the HPA axis responsiveness in depressive illness. Dysfunctional glucocorticoid receptor (GR) mediated negative feedback regulation of cortisol levels and changes in arginine vasopressin (AVP)/vasopressin V1b receptor and corticotrophin-releasing factor/CRF1 receptor regulation of adrenocotricotrophin (ACTH) release have all been implicated in over-activity of the HPA axis. Agents that intervene with the mechanisms involved in (dys)regulation of cortisol synthesis and release are under investigation as possible therapeutic agents. The current status of some of these approaches is described in this review.

Effect of the glucocorticoid receptor antagonist Org 34850 on basal and stress-induced corticosterone secretion

The activity of the hypothalamic‐pituitary‐adrenal (HPA) axis is characterised both by an ultradian pulsatile pattern of glucocorticoid secretion and an endogenous diurnal rhythm. Glucocorticoid feedback plays a major role in regulating HPA axis activity and this mechanism occurs via two different receptors: mineralocorticoid (MR) and glucocorticoid receptors (GR). In the present study, the effects of both acute and subchronic treatment with the GR antagonist Org 34850 on basal and stress‐induced HPA axis activity in male rats were evaluated. To investigate the effect of Org 34850 on basal diurnal corticosterone rhythm over the 24‐h cycle, an automated blood sampling system collected samples every 10 min. Acute injection of Org 34850 (10 mg/kg, s.c.) did not affect basal or stress‐induced corticosterone secretion, but was able to antagonise the inhibitory effect of the glucocorticoid agonist methylprednisolone on stress‐induced corticosterone secretion. However, 5 days of treatment with Org 34850 (10 mg/kg, s.c., two times a day), compared to rats treated with vehicle (5% mulgofen in 0.9% saline, 1 ml/kg, s.c.), increased corticosterone secretion over the 24‐h cycle and resulted in changes in the pulsatile pattern of hormone release, but had no significant effect on adrenocorticotrophic hormone secretion or on stress‐induced corticosterone secretion. Subchronic treatment with Org 34850 did not alter GR mRNA expression in the hippocampus, paraventricular nucleus of the hypothalamus or anterior‐pituitary, or MR mRNA expression in the hippocampus. Our data suggest that a prolonged blockade of GRs is required to increase basal HPA axis activity. The changes observed here with ORG 34850 are consistent with inhibition of GR‐mediated negative feedback of the HPA axis. In light of the evidence showing an involvement of dysfunctional HPA axis in the pathophysiology of depression, Org 34850 could be a potential treatment for mood disorders.

Blockade of the V1b receptor reduces ACTH, but not corticosterone secretion induced by stress without affecting basal hypothalamic–pituitary–adrenal axis activity

Vasopressin (AVP), produced in parvocellular neurons of the hypothalamic paraventricular nucleus, regulates, together with CRH, pituitary ACTH secretion. The pituitary actions of AVP are mediated through the G protein receptor V(1b) (V(1b)|R). In man, hyperactivity of the hypothalamic-pituitary-adrenal axis has been associated with depression and other stress-related conditions. There are also clinical data suggesting a role for AVP in the dysfunctional HPA axis described in some depressed patients. In this study, we have investigated the effect of a recently synthesised selective antagonist of the V(1b)R both on exogenous AVP-induced ACTH and corticosterone secretion, and on basal and stress-induced pituitary-adrenal activity. Adult male Sprague-Dawley rats treated with the V(1b)R antagonist (Org, 30 mg/kg, s.c.) or vehicle (5% mulgofen in 0.9% saline, 2 ml/kg, s.c.). We found that blockade of the V(1b)R reduced the increase in both ACTH and corticosterone secretion induced by AVP (100 ng, i.v.). The same treatment had no effect either on basal ACTH and corticosterone levels or on the ultradian or diurnal rhythms of corticosterone secretion. Acute administration of the V(1b)R antagonist reduced ACTH secretion following both restraint and lipopolysaccharide, but did not antagonise the ACTH response to noise. The same treatment did not reduce corticosterone secretion in response to any of the three stressors used in this study. Our results confirm that this compound is an antagonist of the V(1b)R in the rat, and that its ability to reduce stress-induced ACTH responses is stressor dependent with differential modulation of pituitary and adrenal responses.

Effect of vasopressin 1b receptor blockade on the hypothalamic-pituitary-adrenal response of chronically stressed rats to a heterotypic stressor.

Exposure to chronic restraint (CR) modifies the hypothalamic-pituitary-adrenal (HPA) axis response to subsequent acute stressors with adaptation of the response to a homotypic and sensitization of the response to a heterotypic stressor. Since vasopressin (AVP) activity has been reported to change during chronic stress, we investigated whether this was an important factor in HPA facilitation. We therefore tested whether vasopressin 1b receptor (AVPR1B) blockade altered the ACTH and corticosterone response to heterotypic stressors following CR stress. Adult male rats were exposed to CR, single restraint, or were left undisturbed in the home cage. Twenty-four hours after the last restraint, rats were injected with either a AVPR1B antagonist (Org, 30 mg/kg, s.c.) or vehicle (5% mulgofen in saline, 0.2/kg, s.c.) and then exposed to either restraint, lipopolysaccharide (LPS) or white noise. CR resulted in the adaptation of the ACTH and corticosterone response to restraint and this effect was not prevented by pretreatment with Org. Although we found no effect of CR on LPS-induced ACTH and corticosterone secretion, both repeated and single episodes of restraint induced the sensitization of the ACTH, but not corticosterone response to acute noise. Pretreatment with Org reduced the exaggerated ACTH response to noise after both single and repeated exposure to restraint.

The priming effect of luteinizing hormone-releasing hormone (LHRH) but not LHRH-induced gonadotropin release, can be prevented by certain protein kinase C inhibitors.

The priming effect of LHRH in vitro (which results in increased responsiveness of gonadotropes to both LHRH receptor-mediated and receptor-independent stimuli) is brought about by an unknown mechanism. The present results indicate that induction of the LHRH priming effect is inhibited in a concentration-dependent manner by the protein kinase C (PKC) inhibitors staurosporine, K252a, H7 and by the novel highly-selective PKC inhibitor, Ro 31-8220. In contrast, a range of other compounds that are relatively selective inhibitors of other kinases such as tyrosine kinases and Ca2+/calmodulin-dependent kinases were unable to prevent priming. The PKC inhibitors prevented priming without affecting initial LHRH-induced gonadotropin secretion. Thus, the priming-elicited increment in secretion was selectively removed, restoring hormone release to the level measured during an initial response to LHRH. Similar results were obtained on different days of the estrous cycle where the magnitude of the priming effect varies. Experiments on the time course of PKC inhibitor action revealed that the critical period was in the induction of the priming effect, not its expression. The PKC inhibitors had neither acute nor delayed effects on gonadotropin secretion induced by ionomycin. Staurosporine, K252a and Ro 31-8220 inhibited LHRH priming with identical potencies to their inhibition of phorbol ester-induced gonadotropin secretion. The reduced potency of H7 seen on LHRH priming compared to phorbol ester-induced gonadotropin release parallels results seen with this inhibitor on phorbol ester-induced secretion of growth hormone (Johnson and Mitchell (1989) Biochem. Soc. Trans. 17, 751-752) and on the pharmacological characteristics of PKCs partially purified from anterior pituitary tissue. In all aspects of this study, effects on luteinizing hormone (LH) and follicle-stimulating hormone (FSH) secretion appeared to be entirely similar.

Modulation of hippocampal synaptic transmission by the kynurenine pathway member xanthurenic acid and other VGLUT inhibitors

Xanthurenic acid (XA), an endogenous kynurenine, is a known vesicular glutamate transport (VGLUT) inhibitor and has also been proposed as an mGlu2/3 receptor agonist. Changes in these systems have been implicated in the pathophysiology of schizophrenia and other psychiatric disorders; however, little is known of how XA affects synaptic transmission. We therefore investigated the effects of XA on synaptic transmission at two hippocampal glutamatergic pathways and evaluated the ability of XA to bind to mGlu2/3 receptors. Field excitatory postsynaptic potentials (fEPSPs) were recorded from either the dentate gyrus (DG) or CA1 region of mouse hippocampal slices in vitro. Addition of XA to the bathing medium (1–10 mM) resulted in a dose-related reduction of fEPSP amplitudes (up to 52% reduction) in both hippocampal regions. In the DG, the VGLUT inhibitors Congo Red and Rose Bengal, and the mGlu2/3 agonist LY354740, also reduced fEPSPs (up to 80% reduction). The mGlu2/3 antagonist LY341495 reversed the LY354740 effect, but not the XA effect. LY354740, but not XA, also reduced DG paired-pulse depression. XA had no effect on specific binding of 1 nM [3H]LY341495 to membranes with human mGlu2 receptors. We conclude that XA can modulate synaptic transmission via a mechanism that may involve VGLUT inhibition rather than activation of mGlu2/3 receptors. This could be important in the pathophysiology of nervous system disorders including schizophrenia and might represent a target for developing novel pharmacological therapies.

Characterization of a novel and selective V1B receptor antagonist

It has been argued that hyperactivity of the hypothalamic-pituitary-adrenal (HPA) axis is a major biological abnormality in patients suffering from psychiatric conditions such as major depression. Both arginine vasopressin (AVP) and corticotrophin releasing factor (CRF) are responsible for stimulating the release of adrenocorticotropic hormone (ACTH) from the anterior pituitary. CRF is thought to be the predominant secretagogue under normal conditions but AVP may play a more important role in situations of aberrant/chronic stress. Studies in patients suffering from melancholic depression indicate a hyper-responsiveness to agonism at the vasopressin receptor type 1B (V(1B)); patients display a heightened ACTH release after challenge with the mixed V(1B)/V(2) (vasopressin receptor type 2) agonist desmopressin in comparison to control subjects. A V(1B) antagonist has been developed which has significant selectivity for the human V(1B) receptor over the other members of the vasopressin receptor sub-family. The compound acts as an effective antagonist at both the human recombinant receptor (stably expressed in Chinese hamster ovary (CHO) cells) and the native rat V(1B) receptor (using isolated anterior pituitary cells), blocking the induction of luciferase and the release of ACTH, respectively. In vivo the compound can block the release of ACTH after challenge with a variety of V(1B) agonists. It can also attenuate the ACTH response to acute stressors in rats. Interestingly, this compound does not modulate the activity of the HPA axis under normal basal conditions.

The differential effects of protein kinase C activators and inhibitors on rat anterior pituitary hormone release

We investigated the possibility that various protein kinase C (PKC) activators and inhibitors may differentially affect luteinizing hormone (LH) and growth hormone (GH) release from rat anterior pituitary tissue, incubated in vitro. Activators of PKC induced LH release with the following order of potency: mezerein > phorbol 12,13-dibutyrate (PDBu). Mezerein and PDBu were equipotent on GH release. A range of PKC inhibitors (including compounds highly selective for PKC) potently and completely inhibited PKC activator-induced LH and GH release. Chelerythrine and 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H7) were less potent inhibitors of PDBu-induced GH release than of LH release. A component of PDBu- and mezerein-induced LH release was inhibited by H7 with high potency, but a second H7-insensitive component was detected. Mezerein- and PDBu-induced GH release consisted of an H7-resistant component only. When the regulatory domain of PKCs from different sources was investigated by displacement of [3H]PDBu binding, the affinity for mezerein was 3-5-fold greater than that for PDBu at PKCs from cerebral cortex, lung and alpha and beta isoforms extensively purified from brain. Anterior pituitary PKCs were unusual in showing closely matched affinity for mezerein and PDBu, reminiscent of their equivalent potency on GH release. In order to investigate the potency of the catalytic domain inhibitor H7 on PKCs from different sources, enzyme activity assays were carried out on partially purified cytosolic PKCs from midbrain and anterior pituitary and on extensively purified PKC alpha and PKC beta. The Ca(2+)-independent component of PDBu-induced (phosphatidylserine-dependent) activity from anterior pituitary alone showed unusually low potency of inhibition by H7 but was potently inhibited by staurosporine and Ro 31-8220. In contrast, the Ca(2+)-dependent PKC activity in anterior pituitary was inhibited by H7, staurosporine and Ro-31-8220 with high potency as in all other preparations. These results are consistent with the presence and active role in secretion of pharmacologically distinct forms of PKC (or PKC-like kinases) in rat anterior pituitary cells.

Differential effect of glucocorticoid receptor antagonists on glucocorticoid receptor nuclear translocation and DNA binding

The effects of RU486 and S-P, a more selective glucocorticoid receptor antagonist from Schering-Plough, were investigated on glucocorticoid receptor nuclear translocation and DNA binding. In the in vitro study, AtT20 cells were treated with vehicle or with RU486, S-P or corticosterone (3–300 nM) or co-treated with vehicle or glucocorticoid receptor antagonists (3–300 nM) and 30 nM corticosterone. Both glucocorticoid receptor antagonists induced glucocorticoid receptor nuclear translocation but only RU486 induced DNA binding. RU486 potentiated the effect of corticosterone on glucocorticoid receptor nuclear translocation and DNA binding, S-P inhibited corticosterone-induced glucocorticoid receptor nuclear translocation, but not glucocorticoid receptor-DNA binding. In the in vivo study, adrenalectomized rats were treated with vehicle, RU486 (20 mg/kg) and S-P (50 mg/kg) alone or in combination with corticosterone (3 mg/kg). RU486 induced glucocorticoid receptor nuclear translocation in the pituitary, hippocampus and prefrontal cortex and glucocorticoid receptor-DNA binding in the hippocampus, whereas no effect of S-P on glucocorticoid receptor nuclear translocation or DNA binding was observed in any of the areas analysed. These findings reveal differential effects of RU486 and S-P on areas involved in regulation of hypothalamic–pituitary–adrenal axis activity in vivo and they are important in light of the potential use of this class of compounds in the treatment of disorders associated with hyperactivity of the hypothalamic–pituitary–adrenal axis.

Effect of the glucocorticoid receptor antagonist Org 34850 on fast and delayed feedback of corticosterone release

We investigated the effect of the glucocorticoid receptor (GR) antagonist Org 34850 on fast and delayed inhibition of corticosterone secretion in response to the synthetic glucocorticoid methylprednisolone (MPL). Male rats were implanted with a catheter in the right jugular vein, for blood sampling and MPL administration, and with an s.c. cannula for Org 34850 administration. All experiments were conducted at the diurnal hormonal peak in the late afternoon. Rats were connected to an automated sampling system and blood samples were collected every 5 or 10 min. Org 34850 (10 mg/kg, s.c.) or vehicle (5% mulgofen in saline) was injected at 1630 h; 30 min later, rats received an injection of MPL (500 microg/rat, i.v.) or saline (0.1 ml/rat). We found that an acute administration of MPL rapidly decreased the basal corticosterone secretion and this effect was not prevented by acute pretreatment with Org 34850. However, blockade of GR with Org 34850 prevented delayed inhibition of MPL on corticosterone secretion measured between 4 and 12 h after MPL administration. Our data suggest an involvement of GR in modulating delayed, but not fast, inhibition induced by MPL on basal corticosterone secretion.