Abstract
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 V1b (V1b|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 V1bR 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 V1bR 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 V1bR 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 V1bR 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 V1bR in the rat, and that its ability to reduce stress-induced ACTH responses is stressor dependent with differential modulation of pituitary and adrenal responses.
Introduction
Vasopressin (arginine vasopressin, AVP) and CRH are the two main neuropeptides regulating the hypothalamic–pituitary–adrenal (HPA) axis. AVP is a nonapeptide synthesised in parvocellular neurons of the paraventricular nucleus (PVN) of the hypothalamus, which, in synergy with CRH activates the release of pituitary ACTH. ACTH, in turn, potently stimulates the secretion of glucocorticoids (corticosterone in rats and cortisol in humans) from the adrenal cortex (Gillies et al. 1982, Rivier et al. 1984, Antoni 1993; Aguilera et al. 1994).
The effect of AVP on pituitary corticotrope cells is mediated through the activation of AVP V1b receptor (V1bR) subtypes (Jard et al. 1987, Aguilera 1994, Aguilera & Rabadan-Diehl 2000). Evidence for the involvement of AVP in the HPA axis response to stress includes the increased secretion of AVP into the pituitary portal circulation (de Goeij et al. 1991, 1992, Chowdrey et al. 1995) increased AVP mRNA within the parvocellular part of the PVN (Makino et al. 1995, Ma et al. 1997a,b, Ma & Lightman 1998) and increased V1bR density in the pituitary (Rabadan-Diehl et al. 1995, Aguilera & Rabadan-Diehl 2000) in rats exposed to chronic stress.
Dysfunction of the HPA axis is well described in the pathophysiology of stress-related disorders (Michael & Gibbson 1963, Holsboer 2000, 2001). Several clinical studies suggest that AVP could play an important role in the aetiology of these conditions and elevated plasma levels of AVP have been described in subjects suffering from depression (van Londen et al. 1997, 1998, 2001, de Winter et al. 2003) or post-traumatic stress disorders (de Kloet et al. 2007). Post-mortem studies have also shown increased numbers of AVP-expressing neurons in the PVN of depressed patients (Purba et al. 1996, Merali et al. 2006). In addition to evidence for abnormalities in AVP, there is also a suggestion that vulnerability to the development of depression could be due to genetic variation in the human V1bR gene (van West et al. 2004). Moreover, hyperactivity of the V1bR has been found in melancholic depression (Dinan et al. 1999, 2004).
A number of preclinical studies support the role of AVP in the affective disorders. Studies where high (HAB) or low (LAB) anxiety-related behaviour rat lines were used also suggested that AVP could contribute to the marked dysregulation of the HPA system in the HAB rat (Keck et al. 2002). In these rats, overactivity of AVP was associated with increased anxiety-like behaviour that could be reduced with chronic treatment with the antidepressant citalopram (Jochum et al. 2007). The AVP-deficient Brattleboro rat has been shown to display an attenuated depression-like behaviour in forced swimming and sucrose preference tests (Mlynarik et al. 2007).
Although V1bR knockout (KO) mice exhibit a normal corticosterone response to the acute psychological stress of restraint (Lolait et al. 2007a), they do have a reduction in the pituitary–adrenal response to more severe stressors, including forced swim and insulin-induced hypoglycaemia (Lolait et al. 2007a). Similarly, it has been shown using KO mice that the V1bR is necessary for a normal HPA response to lipopolysaccharide (LPS) and ethanol (Lolait et al. 2007b).
There is therefore considerable interest in the possible role of a AVP antagonist in the treatment of mood disorders.
Blockade of the V1bR using the V1bR antagonist SSR149415 (Serradeil-Le Gal et al. 2002) has been shown to induce antidepressant and anxiolytic-like activity in several behavioural models (Griebel et al. 2002, Serradeil-Le Gal et al. 2002, 2005, Shimazaki et al. 2006, Ramos et al. 2006, Iijima & Chaki 2007, Breuer et al. 2008). Besides being localised in the anterior pituitary, V1bR are also found in the central nervous system, in regions involved in the modulation of the stress response such as hippocampus, lateral septum and cortex (Lolait et al. 1995, Vaccari et al. 1998) suggesting that the anxiolytic and antidepressant effect of SSR149415 could also be mediated through the activation of central V1bR.
SSR149415 is able to reduce, in rats, ACTH release induced by endogenous administration of V1bR agonists (alone or in combination with CRH) and by various stressors (Serradeil-Le Gal et al. 2002, Ramos et al. 2006). However, an effect of SSR149415 on corticosterone release has not been reported.
The compound used in this study is a recently synthesised V1bR antagonist that is able to bind to the human recombinant V1bR with a>1000 fold selectivity over other members of the AVP receptor subfamily and also over a broad range (>60) of other receptors and enzymes (Presland et al. 2007, Craighead et al. 2008). Recent work from Craighead and colleagues has reported that this compound is an effective V1bR antagonist in vitro, as demonstrated by its ability to reduce AVP-induced ACTH release from isolated rat anterior pituitary cells. Furthermore, the in vivo efficacy of Org was confirmed by the ability of both acute and chronic administration of Org to reduce, in rat, the ACTH release induced by various agonists of the V1bR (Craighead et al. 2008). However, the effect of Org on both V1bR agonist- and stress-induced corticosterone secretion has not been investigated. To further investigate whether this compound is an effective AVP antagonist in vivo we have investigated the effect of this compound on the time course of ACTH and corticosterone release following i.v. AVP administration in rat. We then studied its effect on basal ACTH and corticosterone levels, as well the circadian and ultradian patterns of corticosterone release. Finally, we investigated the effect of this V1bR antagonist on the pituitary–adrenal response to various acute stressors.
Materials and Methods
Subjects
All experiments were conducted on male Sprague-Dawley adult rats (Harlan-Olac, Bicester, UK) weighing 250–300 g at the time of surgery. Animals were grouped, housed 4 to a cage and allowed to acclimatise to the housing facility for a minimum of 1 week prior to the start of experiments. Rats were maintained under standard environmental conditions (21±1 °C) under a 14h light:10h darkness schedule (lights on at 0515 h) and food and water were provided ad libitum throughout the experiment. All animal procedures were approved by the University of Bristol Ethical Review Group and were conducted in accordance with Home Office guidelines and the UK Animals (Scientific Procedures) Act, 1986. All possible efforts were made to minimise the number of animals used and their suffering.
Surgery and blood sampling
Animals were anaesthetised with a combination of Hypnorm (0.32 mg/kg fentanyl citrate and 10 mg/kg fluanisone, i.m.; Janssen Pharmaceuticals, Oxford, UK) and diazepam (2.6 mg/kg i.p.; Phoenix Pharmaceuticals, Gloucester, UK). The right jugular vein was exposed and a silastic-tipped (i.d. 0.50 mm, o.d. 0.93 mm, Merck) polythene cannula (Portex, Hythe, UK) was inserted into the vessel until it lay close to the entrance of the right atrium. The cannula was pre-filled with pyrogen-free heparinised (10 IU/ml) isotonic saline. During the same surgery, a s.c. cannula, for drug administration, was inserted under the skin between the shoulder blades. The free ends of both cannulae were exteriorised through a scalp incision and then tunnelled through a protective spring that was anchored to the parietal bones using two stainless steel screws and self-curing dental acrylic. Following recovery, animals were housed in individual cages in a soundproof room. The end of the protective spring was attached to a mechanical swivel that rotated through 360 °C in a horizontal plane and 180° through a vertical plane allowing the rats to maximise freedom of movement. The cannulae were flushed daily with the heparinised saline to maintain patency. Blood samples were collected through the jugular vein cannula either by hand or using an automated blood sampling (ABS) system as previously described (Windle et al. 1998). Blood samples collected by hand (0.2 ml) were stored in ice-cold eppendorf tubes containing 10 μl EDTA (0.5 M; pH 7.4) and 10 μl Trasylol (Aprotinin, 500 000 KIU/ml, Roche). Plasma was separated by centrifugation and then stored at −80 °C until processed for ACTH and corticosterone measurements. Where the volume of the blood sample was less than 0.2 ml, only corticosterone was measured. At the end of each experiment rats were overdosed with 0.5 ml sodium pentobarbitone (Euthatal, 200 mg/ml; Merial, Harlow, UK).
Drug treatments
The V1bR antagonist (Org, provided by Schering–Plough Corporation, Newhouse, UK,) was administered through the s.c. cannula in a 0.9% saline solution with 5% mulgofen (a detergent that improves solubility; GAF Ltd, Manchester, UK) at the dose of 10 mg/kg (Exp. 1) or 30 mg/kg (Exp. 1–4) using a volume of 2 ml/kg. In our study, we chose an administration route (via an s.c. cannula) that minimises any stress induced in the rat by the injection. Vehicle controls were injected with 0.9% saline solution with 5% mulgofen. Arginine (AVP, Sigma) was administered via the jugular vein cannula at 100 ng (∼ 400 ng/kg) dissolved in 0.1 ml 0.9% saline. Both s.c. and i.v. injections were followed by injection of 0.2 ml heparin–saline to flush out the cannula and ensure that the entire volume of drug had been received by the animal.
Stress
Restraint was performed by enclosing the rats in cylindrical perspex restrainers (55 mm diameter) containing air holes to prevent overheating. The length of the restrainer was adjusted for the length of the rat in order to limit movement. Noise stress consisted of 10 min of white noise (96 dB). (LPS, Escherichia coli; 055:B5; 250 μg/ml, Sigma) was dissolved in sterile saline (0.1 ml/rat) and injected via the jugular cannula.
Experimental procedures
Experiment 1. Effect of acute treatment with the V1bR antagonist Org on AVP-induced ACTH and corticosterone secretion
Experiments were performed between 0900 and 1300 h. Rats received either Org (10 and 30 mg/kg) or vehicle 30 min prior to injection with AVP or saline (n=6–11 for each group). Blood samples were collected by hand at the time points described in Fig. 1. Blood samples were processed for ACTH and corticosterone measurements as described below.
Effect of the V1bR antagonist Org on ACTH and corticosterone secretion induced by administration of exogenous AVP. Rats were injected with Org (10 and 30 mg/kg, s.c.) or vehicle (5% mulgofen in 0.9% saline, 2 ml/kg) 30 min prior administration of AVP (100 ng, i.v.) or saline (0.1 ml; n=6–11/group). Data are expressed as mean±s.e.m. of plasma ACTH (pg/ml, A) or plasma corticosterone (ng/ml, B) with respect to the time of AVP administration. Black arrows indicate Org or vehicle injection; grey arrows indicate AVP or saline injection. #P<0.05, effect of AVP, compared with saline, in rats treated with vehicle or Org. *P<0.05, effect of Org, compared with vehicle, in rats treated with AVP or saline.
Citation: Journal of Endocrinology 200, 3; 10.1677/JOE-08-0421
Experiment 2. Effect of acute treatment with the V1bR antagonist Org on basal ACTH and corticosterone secretion
Experiments were performed between 1600 and 1900 h, the time of expected ACTH and corticosterone secretory peak levels. Rats received either Org (30 mg/kg, n=7) or vehicle (n=7). Blood samples were collected by hand as described above at the time points indicated in Fig. 2. Blood samples were processed for ACTH and corticosterone measurements as described below.
Effect of acute administration of the V1bR antagonist Org on basal ACTH and corticosterone secretion. Sample collection started at 1600 h, the time of the expected hormone peak, immediately before administration of Org (30 mg/kg, s.c., n=7) or vehicle (5% mulgofen in 0.9% saline, 2 ml/kg, n=7). Samples were collected for 3 h every 15 or 30 min. Data are expressed as mean±s.e.m. of plasma ACTH (pg/ml, A) or plasma corticosterone (ng/ml, B) with respect to the time of day.
Citation: Journal of Endocrinology 200, 3; 10.1677/JOE-08-0421
Experiment 3. Effect of acute treatment with theV1bR antagonist Org on corticosterone diurnal rhythm and pulsatile pattern
Experiments were performed between 1600 and 0700 h. Four days after surgery, rats were connected to the ABS and blood samples were collected every 10 min. After collection of basal samples for 1 h, rats were injected with either Org (30 mg/kg, n=7) or vehicle (n=7). Blood samples were processed for corticosterone measurements as described below.
Experiment 4. Effect of acute treatment with the V1bR antagonist Org on stress-induced ACTH and corticosterone secretion
All experiments were performed between 0900 and 1300 h. Rats were injected with either Org (30 mg/kg) or vehicle 30 min prior to restraint (n=9–10 for each group), or noise (n=8 for each group) or acute administration of LPS (n=6–8 for each group). For each stressor a different group of animals was used. Blood samples were collected by hand prior to Org or vehicle administration and then before, during and after restraint or noise and before and after LPS administration. Blood samples were processed for ACTH and corticosterone measurements as described below.
Hormone measurements
ACTH levels were measured by IRMA using commercially available kits (DiaSorin Ltd, Wokingham, UK). For ACTH measurement, 80 μl plasma were diluted in 120 μl saline. Intra- and inter-assay coefficients of variation of the ACTH assay were 2.84 and 6.41% respectively.
Corticosterone levels were measured using RIA as previously described (Spiga et al. 2007). For blood samples collected by hand, 5 μl of each plasma sample was diluted in 500 μl of a citrate buffer and processed in triplicate. For blood samples collected using the ABS, samples were partially diluted by the ABS so that each sample consisted of 37.7 μl blood diluted 1:5 in heparinised saline. For the assay, 50 μl of each blood sample was further diluted into 50 μl of a citrate buffer (pH 3.0) and samples were processed in duplicate. Samples were incubated overnight at 4 °C with 50 μl of 125I corticosterone tracer (Oxford Bio Innovation DSL Ltd, Oxford, UK) and 50 μl of rabbit anti-rat corticosterone primary antibody (kindly donated by G. Makara, Hungary). On day 2, a charcoal/dextran solution was added to the samples, which were then centrifuged (15 min, 4000 r.p.m., 3.120 g, 4 °C) and aspirated before being loaded onto a γ-counter. Intra- and inter- assay coefficients of variation of the corticosterone assay were 16.65 and 13.30% respectively.
Statistical analysis
All statistical analyses were performed using SPSS 11.5 for Windows (SPSS Inc., Chicago, IL, USA). Data are expressed as mean±s.e.m. One-way or two-way ANOVA with repeated measures was used to estimate the effect of Org (time×Org effect, exp.2 and exp.4) or Org and AVP (time×AVP effect, time×Org effect and time×AVP×Org effect, exp 1) on the time course of ACTH and corticosterone release. When appropriate, further comparisons were made with Fisher's least significant difference post hoc test. In exp. 4, where a significant effect of time occurred (indicating a significant effect of the stressor) a paired sample t-test was used to identify the time at which the stressor effect occurred within each group. Moreover, in exp. 4, the area under curve (AUC) for the ACTH and corticosterone responses to stress were calculated for each group and analysed using Student's t-test. In exp. 3, the hormone profile for each animal was analysed for variation in pulse characteristics (AUC, mean daily corticosterone concentration and the number, amplitude and frequency of corticosterone pulses) using the PULSAR algorithm (Merriam & Wachter 1982). For the PULSAR algorithm, the following G values were employed: G1=5, G2=3, G3=2, G4=1.5 and G5=0.8, together with a peak splitting parameter of 5 (s.d. units). These values were obtained from visual inspection of the data, as recommended (Windle et al. 1998). Each PULSAR parameter were analysed using Student's t-test. Statistical significance was set at P≤0.05, except on the paired samples t-test where Bonferroni correction was used (exp. 4: P≤0.006, P≤0.007 and P≤0.0055 respectively for restraint, noise and LPS experiments).
Results
Experiment 1. Effect of acute treatment with the V1bR antagonist Org on AVP-induced ACTH and corticosterone secretion
The effect of the acute administration of the V1bR antagonist Org on the ACTH response to i.v. AVP is shown in Fig. 1A.
Two-way ANOVA revealed a significant effect of AVP (time×AVP effect: F (6,258)=101.72; P<0.00001), a significant effect of Org (time×Org effect: F (6,258)=17.88; P<0.00001) and a significant interaction between the two factors (time×AVP×Org effect: F (6, 258)=16.20; P<0.00001) over the time course of ACTH release.
Acute administration of AVP induced a robust increase on ACTH secretion in rats treated with vehicle or Org. Pretreatment with Org had a significant inhibitory effect on the ACTH response to AVP and this effect was significant 5 min (P<0.0001) and 15 min (P<0.005) after AVP administration for both doses of Org. No effect of Org on ACTH release in rats treated with saline was observed.
The effect of the acute administration of the V1bR antagonist Org on the corticosterone response to i.v. AVP is shown in Fig. 1B.
Two-way ANOVA revealed a significant effect of AVP (time×AVP effect: F (6342)=38.02; P<0.00001), a significant effect of Org (time×Org effect: F (6342)=4.95; P=0.0012) and a significant effect between the two factors (time×AVP×Org effect: F (6342)=3.56; P=0.01) over the time course of corticosterone release.
Acute administration of AVP induced a robust increase on corticosterone secretion in rats treated with vehicle or Org. Pretreatment with Org had a significant inhibitory effect on the corticosterone response to AVP and this effect was significant 30 min (10 and 30 mg/kg, P<0.05 and P<0.0001 respectively) and 45 min (30 mg/kg, P<0.0005) after AVP administration. No effect of Org on corticosterone release in rats treated with saline was observed.
Based on those results, the 30 mg/kg dose of Org was chosen for all further experiments.
Experiment 2. Effect of the V1bR antagonist Org on basal ACTH and corticosterone secretion
The effect of Org on basal ACTH and corticosterone release was investigated during the diurnal peak (Fig. 2). No effect of treatment with Org on basal ACTH (Fig. 2A) and corticosterone (Fig. 2B) secretion was found.
Experiment 3. Effect of acute treatment with the V1bR antagonist Org on basal corticosterone diurnal rhythm and pulsatile pattern
The mean corticosterone concentration of rats injected with Org or vehicle is represented in Fig. 3. The hormone profile for each animal was analysed for variation in pulse characteristics (AUC, mean daily corticosterone concentration and the number, amplitude and frequency of corticosterone pulses) using the PULSAR algorithm. No significant difference on any of the parameters analysed was found between the two experimental groups (Table 1).
Effect of acute administration of the V1bR antagonist Org on corticosterone diurnal rhythm. Data represented are mean±s.e.m. blood corticosterone concentration for rats injected with Org (30 mg/kg, s.c., n=7) or vehicle (5% mulgofen in 0.9% saline, 2 ml/kg, s.c., n=7). Rats were connected to an automated blood sampling system and samples were collected every 10 min for 15 h. Injection time is indicated by black arrows; the grey bar represents the dark phase (1915–0515 h).
Citation: Journal of Endocrinology 200, 3; 10.1677/JOE-08-0421
Mean±s.e.m. of PULSAR parameters measurements
| AUC (ng) | Mean blood corticosterone (ng/ml) | Number of pulses | Inter-pulse interval | Amplitude of pulses (ng/ml) | Frequency of pulses (pulse/h) | |
|---|---|---|---|---|---|---|
| Vehicle | 370.2±54 | 25.4±9 | 8.8±1 | 1.5±0.2 | 41.6±6 | 0.64±0.08 |
| Org | 384.3±32 | 26.1±3 | 10.8±0.5 | 1.2±0.04 | 45.3±6 | 0.79±0.04 |
Rats were treated with vehicle (5% mulgofen in 0.9% saline, 2 ml/kg, s.c., n=7) or Org (30 mg/kg, s.c., n=7). Parameters were measured between 1700 h, (time of vehicle or Org administration) and 0700 h (time of collection of the last samples; see Fig. 3).
Experiment 4. Effect of the V1bR antagonist Org on stress-induced ACTH and corticosterone secretion
In all the stress experiments the time of Org administration with respect to the time of exposure to stress was based on the results from experiment 1.
Restraint
The effect of Org on the ACTH and corticosterone response to acute restraint is shown in Fig. 4.
Effect of the V1bR antagonist Org on ACTH and corticosterone secretion induced by exposure to restraint stress. Rats were injected with Org (30 mg/kg, s.c., n=9) or vehicle (5% mulgofen in 0.9% saline, 0.2 ml/kg, n=9–10) 30 min prior exposure to restraint (60 min). Data are expressed as mean±s.e.m. of plasma ACTH (pg/ml, A) or plasma corticosterone (ng/ml, B) with respect to the time of exposure to stress. Black arrow indicates Org or vehicle injection, shaded bar indicate restraint stress. # P<0.05, effect of stress, compared with basal value (0 min), in rats treated with vehicle or Org. *P<0.05, effect of Org, compared with vehicle, in rats exposed to restraint.
Citation: Journal of Endocrinology 200, 3; 10.1677/JOE-08-0421
Exposure to restraint increased ACTH release (time effect: F (8,128)=43.844; P<0.0001) both in rats treated with vehicle and Org. There was a significant effect of Org on ACTH response to stress (time× Org effect F (8,128)=3.331; P=0.035).
Restraint stress increased corticosterone secretion both in rats treated with vehicle and Org (time effect F (8,136)=51.879; P<0.0001). In contrast to the ACTH data, no effect of Org on restraint-induced corticosterone increase was found (time×Org effect: F (8136)=0.588; P=0.641).
Noise
The effect of Org on ACTH and corticosterone secretion induced by acute noise stress is shown in Fig. 5.
Effect of the V1bR antagonist Org on ACTH and corticosterone secretion induced by exposure to noise stress. Rats were injected with Org (30 mg/kg, s.c., n=8) or vehicle (5% mulgofen in 0.9% saline, 0.2 ml/kg, n=8) 30 min prior exposure to white noise (10 min, 96 dB). Data are expressed as mean±s.e.m. of plasma ACTH (pg/ml, A) or plasma corticosterone (ng/ml, B) with respect to the time of exposure to noise. Black arrows indicate Org or vehicle injection, shaded bars indicate noise. #P<0.05, effect of stress, compared with basal value (0 min), in rats treated with vehicle or Org.
Citation: Journal of Endocrinology 200, 3; 10.1677/JOE-08-0421
Noise–stress induced a smaller (compared with restraint) increase in ACTH secretion in rats treated with both Org and vehicle (time effect: F (798)=63.394; P<0.0001). No significant effect of Org on noise-induced ACTH increase was found (time×Org effect: F (798)=0.681; P=0.481).
Noise–stress increased corticosterone secretion in both Org and vehicle treated rats (time effect: F (798)=92.968; P<0.0001). No effect of Org on corticosterone secretion in response to noise was observed (time×Org effect: F (798)=2.251; P=0.073).
Lipopolysaccharide
The effect of Org on the ACTH and corticosterone response to LPS is shown in Fig. 6.
Effect of the V1bR antagonist Org on ACTH and corticosterone secretion induced by injection of lipopolysaccharide. Rats were injected with Org (30 mg/kg, s.c., n=7–8) or vehicle (5% mulgofen in 0.9% saline, 0.2 ml/kg, n=6–8) 30 min prior to administration of lipopolysaccharide (LPS, 250 μg, i.v.). Data are expressed as mean±s.e.m. of plasma ACTH (pg/ml, A) or plasma corticosterone (ng/ml, B) with respect to the time of LPS injection. Black arrows indicate Org or vehicle injection, grey arrows indicate LPS injection. #P<0.05, effect of stress, compared with basal value (0 min), in rats treated with vehicle or Org. *P<0.05, effect of Org, compared with vehicle, in rats injected with LPS.
Citation: Journal of Endocrinology 200, 3; 10.1677/JOE-08-0421
Intravenous injection of LPS increased ACTH in both Org and vehicle treated rats (time effect: F (9117)=38.886; P<0.0001). Acute treatment with Org reduced ACTH response to LPS (time×Org: F (9117)=7.080; P<0.0001).
Intravenous injection of LPS increased corticosterone secretion in both Org and vehicle treated rats (time effect: F (9108)=100.539; P<0.0001). In contrast to the ACTH results, Org had no effect on the LPS-induced corticosterone increase (time×Org effect: F (9108)=0.722; P=0.584).
A summary of the effect of stress on ACTH and corticosterone secretion (expressed as AUC) and its modulation by Org is summarised in Table 2.
Effect of the V1bR antagonist Org on pituitary–adrenal response to stress
| Restraint | Noise | LPS | |
|---|---|---|---|
| ACTH | |||
| Vehicle | 1276±132 | 649±53 | 7155±602 |
| Org | 865±114* | 606±36 | 4089±206* |
| Corticosterone | |||
| Vehicle | 1255±127 | 707±78 | 2634±168 |
| Org | 1187±144 | 637±55 | 2442±230 |
Data are mean±s.e.m. of area under curve (AUC) of ACTH (pg) or corticosterone (ng) of rats injected with Org (30 mg/kg, s.c.) or vehicle (5% mulgofen in saline, 0.2 ml/kg, s.c.) 30 min prior restraint (1 h), with noise (96 dB for 10 min) or lipopolysaccharide (LPS, 250 μg, i.v).
*P<0.05, effect of Org, compared with vehicle in rats exposed to stress.
Discussion
Although there are extensive data on the regulation of AVP mRNA in parvocellular PVN neurones and on the synergistic actions of AVP and CRH in stimulating ACTH release, there is remarkably little information about the role of AVP or pituitary V1b receptors in the regulation of normal circadian and ultradian activity of the HPA axis or on the integrated response to stress. We have used a novel and selective V1b antagonist to assess the importance of AVP in HPA homeostasis.
The compound described in this study is an effective V1b antagonist both in vitro and in vivo (Presland et al. 2007, Craighead et al. 2008), and this was confirmed by our data that clearly show that Org is able to reduce the effect of AVP on ACTH and corticosterone release. It was beyond the scope of the current study to perform dose-response relationships for Org or AVP, but we chose a dose of AVP that in pilot studies caused a pituitary–adrenal response of similar magnitude to that seen after noise stress. We found that the higher dose of Org used in this study (30 mg/kg) was, compared with the smaller dose of 10 mg/kg, more potent in reducing AVP-induced corticosterone secretion, whereas the ability of Org to reduce AVP-induced ACTH secretion was similar in both doses. These data are consistent with recently reported data showing an effect of this compound on reducing CRH/desmopressin (dDAVP)-induced ACTH release. Furthermore, the same authors also showed that this V1bR antagonist was able to reduce both ACTH and corticosterone response to CRH in combination with the V1bR agonist d[Cha]AVP (Craighead et al. 2008).
Effect of the V1bR antagonist Org on basal pituitary–adrenal activity
Org was injected in the late afternoon, at the time of the diurnal peak of HPA activity. We found no change in either ACTH or corticosterone secretion. This was not unexpected. Although AVP is a strong modulator of the response of the corticotrophs to CRH, AVP secretion is low under basal conditions (Gillies et al. 1982, Rivier et al. 1984, Antoni 1993). These data are consistent with the hypothesis for CRF to be primarily involved in the modulation of basal HPA axis activity, whereas AVP may be involved in conditions of stress.
We further investigated the effects of Org on basal HPA axis activity by studying the circadian (Jasper & Engeland 1991) and ultradian (Windle et al. 1998) rhythms of corticosterone secretion. Cortisol in man, and corticosterone in rodents, is released in a pulsatile manner throughout the 24-hour period and it is the variation in the amplitude and frequency of these pulses that determine the diurnal rhythm (Windle et al. 1998). Org had no effect on either the circadian rhythm or the pattern of pulsatile corticosterone release.
ACTH is also released in a pulsatile manner in the rat (Carnes et al. 1989), and shows a diurnal rhythm albeit with an amplitude which is considerably lower than that of corticosterone (Dallman et al. 1978, Kaneko et al. 1980, 1981). Unfortunately due to the large sample volumes needed for the assay of ACTH, we were limited in the frequency and duration of samples we were able to perform and thus we could not investigate the effect of Org on ACTH pulsatility or diurnal rhythm.
Effect of the V1b antagonist on stress-induced pituitary–adrenal activation
We investigated the effect of Org on restraint – a physical and psychological stressor, exposure to white noise – a psychological stressor, and administration of LPS – an immunological stressor. Org reduced the ACTH responses to restraint and LPS but had no significant effect on the response to noise. On the other hand, Org had no effect on the corticosterone response to any of these stressors.
With respect to the differential effects on ACTH secretion, it would certainly appear that the AVP antagonist was preferentially affecting the two stressors (restraint and LPS) which have the most pronounced effect on ACTH release. This could reflect the fact that AVP release in the portal circulation is increased only when there is a major activation of the HPA axis.
Our data are consistent with other studies where the inhibitory effect of an other V1bR antagonist, SSR149415, was investigated on ACTH release in rats exposed restraint, although in the same study the effect of this compound on stress-induced corticosterone release was not described (Serradeil-Le Gal et al. 2002).
With respect to the differential effects of Org on stress-induced ACTH and corticosterone secretion, it is possible that, even following Org treatment, ACTH levels are still high enough to induce a normal corticosterone response to stress. However, a pituitary-independent modulation of adrenal corticosterone secretion cannot be excluded. There is certainly strong evidence supporting a role for sympathetic innervation in modulating adrenal glucocorticoid secretion (Engeland & Arnhold 2005). Splanchnic nerve transection results in decreased plasma corticosterone during the diurnal peak and this effect is associated with decreased adrenal sensitivity to ACTH.
Our data are consistent with evidence showing that AVP deficient rats display lower ACTH but normal corticosterone secretion in response to stress (Zelena et al. 2006, Mlynarik et al. 2007). However, our data contrast with the decrease in both ACTH and corticosterone response to LPS in V1bR KO mice (Lolait et al. 2007a,b). Moreover, the same authors showed that mice lacking theV1bR had an attenuated ACTH, but not corticosterone, response to acute restraint (Stewart et al. 2008).
Our data demonstrate that Org is an active V1bR antagonist in vivo which has no effect on basal pituitary–adrenal activity but can reduce stress-induced ACTH secretion. In man there is a close concurrence between the pituitary secretion of ACTH and the adrenal secretion of cortisol (Henley et al. 2008). Since severe depression is frequently associated with hyperactivity of the HPA axis, a V1b antagonist may have therapeutic potential in this condition and other stress-related disorders. Since any behavioural response might also be related to an effect on central V1b receptors, further studies with the use of central (intraventicular or site specific) administration of V1bR antagonists would also be of interest.
Declaration of interest
Some of the authors have conflict of interests, specifically: F Spiga, L Harrison and D Knight were funded by Schering–Plough Corporation; C MacSweeney, F Thomson and M Craighead are employed by Schering–Plough Corporation.
Funding
This research was supported by Schering–Plough Corporation, Newhouse, UK.
Acknowledgements
We would like to acknowledge Helen Atkinson for her contribution to the selection of AVP dose and Yvonne Kershaw and Emma Castrique for their technical assistance.
References
Aguilera G 1994 Regulation of pituitary ACTH secretion during chronic stress. Frontiers in Neuroendocrinology 15 321–350.
Aguilera G & Rabadan-Diehl C 2000 Regulation of vasopressin V1b receptors in the anterior pituitary gland of the rat. Experimental Physiology 85 19S–26S.
Aguilera G, Pham Q & Rabadan-Diehl C 1994 Regulation of pituitary vasopressin receptors during chronic stress: relationship to corticotroph responsiveness. Journal of Neuroendocrinology 6 299–304.
Antoni FA 1993 Vasopressinergic control of pituitary adrenocorticotropin secretion comes of age. Frontiers in Neuroendocrinology 14 76–122.
Breuer MF, van Gaalen MM, Wernet W, Claessens SE, Oosting RS, Behl B, Korte SM, Schoemaker H, Gross G, Olivier B et al. 2008 SSR149415, a non-peptide vasopressin V(1b) receptor antagonist, has long-lasting antidepressant effects in the olfactory bulbectomy-induced hyperactivity depression model. Naunyn Schmiedeberg's Archives of Pharmacology DOI: 10.1007/S00210-008-0336-1 (in press).
Carnes M, Lent S, Feyzi J & Hazel D 1989 Plasma adrenocorticotropic hormone in the rat demonstrates three different rhythms within 24 h. Neuroendocrinology 50 17–25.
Chowdrey HS, Larsen PJ, Harbuz MS, Jessop DS, Aguilera G, Eckland DJ & Lightman SL 1995 Evidence for arginine vasopressin as the primary activator of the HPA axis during adjuvant-induced arthritis. British Journal of Pharmacology 116 2417–2424.
Craighead M, Milne R, Campbell-Wan L, Watson L, Presland J, Thomson FJ, Marston HM & Macsweeney CP 2008 Characterization of a novel and selective V(1B) receptor antagonist. Progress in Brain Research 170 527–535.
Dallman MF, Engeland WC, Rose JC, Wilkinson CW, Shinsako J & Siedenburg F 1978 Nycthemeral rhythm in adrenal responsiveness to ACTH. American Journal of Physiology 235 R210–R218.
Dinan TG, Lavelle E, Scott LV, Newell-Price J, Medbak S & Grossman AB 1999 Desmopressin normalizes the blunted adrenocorticotropin response to corticotropin-releasing hormone in melancholic depression: evidence of enhanced vasopressinergic responsivity. Journal of Clinical Endocrinology and Metabolism 84 2238–2240.
Dinan TG, O'Brien S, Lavelle E & Scott LV 2004 Further neuroendocrine evidence of enhanced vasopressin V3 receptor responses in melancholic depression. Psychological Medicine 34 169–172.
Engeland WC & Arnhold MM 2005 Neural circuitry in the regulation of adrenal corticosterone rhythmicity. Endocrine 28 325–332.
Gillies GE, Linton EA & Lowry PJ 1982 Corticotropin releasing activity of the new CRF is potentiated several times by vasopressin. Nature 299 355–357.
de Goeij DC, Kvetnansky R, Whitnall MH, Jezova D, Berkenbosch F & Tilders FJ 1991 Repeated stress-induced activation of corticotropin-releasing factor neurons enhances vasopressin stores and colocalization with corticotropin-releasing factor in the median eminence of rats. Neuroendocrinology 53 150–159.
de Goeij DC, Jezova D & Tilders FJ 1992 Repeated stress enhances vasopressin synthesis in corticotropin releasing factor neurons in the paraventricular nucleus. Brain Research 577 165–168.
Griebel G, Simiand J, Serradeil-Le Gal C, Wagnon J, Pascal M, Scatton B, Maffrand JP & Soubrie P 2002 Anxiolytic- and antidepressant-like effects of the non-peptide vasopressin V1b receptor antagonist, SSR149415, suggest an innovative approach for the treatment of stress-related disorders. PNAS 99 6370–6375.
Henley DE, Russell GM, Wood SA, Leendertz JA, Woltersdorf WW, Catterall JR & Lightman SL 2008 HPA axis activation in obstructive sleep apnea – preliminary findings. Abstract of the Endocrine Society's Annual Meeting, June 15–18, 2008, San Francisco, CA, USA..
Holsboer F 2000 The corticosteroid receptor hypothesis of depression. Neuropsychopharmacology 23 477–501.
Holsboer F 2001 Stress, hypercortisolism and corticosteroid receptors in depression: implications for therapy. Journal of Affective Disorders 62 77–91.
Iijima M & Chaki S 2007 An arginine vasopressin V1b antagonist, SSR149415 elicits antidepressant-like effects in an olfactory bulbectomy model. Progress in Neuro-Psychopharmacology & Biological Psychiatry 31 622–627.
Jard S, Barberis C, Audigier S & Tribollet E 1987 Neurohypophyseal hormone receptor systems in brain and periphery. Progress in Brain Research 72 173–187.
Jasper MS & Engeland WC 1991 Synchronous ultradian rhythms in adrenocortical secretion detected by microdialysis in awake rats. American Journal of Physiology 261 R1257–R1268.
Jochum T, Boettger MK, Wigger A, Beiderbeck D, Neumann ID, Landgraf R, Sauer H & Bar KJ 2007 Decreased sensitivity to thermal pain in rats bred for high anxiety-related behaviour is attenuated by citalopram or diazepam treatment. Behavioral Brain Research 183 18–24.
Kaneko M, Hiroshige T, Shinsako J & Dallman MF 1980 Diurnal changes in amplification of hormone rhythms in the adrenocortical system. American Journal of Physiology 239 R309–R316.
Kaneko M, Kaneko K, Shinsako J & Dallman MF 1981 Adrenal sensitivity to adrenocorticotropin varies diurnally. Endocrinology 109 70–75.
Keck ME, Wigger A, Welt T, Muller MB, Gesing A, Reul JM, Holsboer F, Landgraf R & Neumann ID 2002 Vasopressin mediates the response of the combined dexamethasone/CRH test in hyper-anxious rats: implications for pathogenesis of affective disorders. Neuropsychopharmacology 26 94–105.
de Kloet CS, Vermetten E, Geuze E, Wiegant VM & Westenberg HG 2007 Elevated plasma arginine vasopressin levels in veterans with posttraumatic stress disorder. Journal of Psychiatric Research.
Lolait SJ, O'Carroll AM, Mahan LC, Felder CC, Button DC, Young WS III, Mezey E & Brownstein MJ 1995 Extrapituitary expression of the rat V1b vasopressin receptor gene. PNAS 92 6783–6787.
Lolait SJ, Stewart LQ, Jessop DS, Young WS III & O'Carroll AM 2007a The hypothalamic–pituitary–adrenal axis response to stress in mice lacking functional vasopressin V1b receptors. Endocrinology 148 849–856.
Lolait SJ, Stewart LQ, Roper JA, Harrison G, Jessop DS, Young WS III & O'Carroll AM 2007b Attenuated stress response to acute lipopolysaccharide challenge and ethanol administration in vasopressin V1b receptor knockout mice. Journal of Neuroendocrinology 19 543–551.
van Londen L, Goekoop JG, van Kempen GM, Frankhuijzen-Sierevogel AC, Wiegant VM, van der Velde EA & De Wied D 1997 Plasma levels of arginine vasopressin elevated in patients with major depression. Neuropsychopharmacology 17 284–292.
van Londen L, Kerkhof GA, van den BF, Goekoop JG, Zwinderman KH, Frankhuijzen-Sierevogel AC, Wiegant VM & De WD 1998 Plasma arginine vasopressin and motor activity in major depression. Biological Psychiatry 43 196–204.
van Londen L, Goekoop JG, Kerkhof GA, Zwinderman KH, Wiegant VM & De WD 2001 Weak 24-h periodicity of body temperature and increased plasma vasopressin in melancholic depression. European Neuropsychopharmacology 11 7–14.
Ma XM & Lightman SL 1998 The arginine vasopressin and corticotrophin-releasing hormone gene transcription responses to varied frequencies of repeated stress in rats. Journal of Physiology 510 605–614.
Ma XM, Levy A & Lightman SL 1997a Emergence of an isolated arginine vasopressin (AVP) response to stress after repeated restraint: a study of both AVP and corticotropin-releasing hormone messenger ribonucleic acid (RNA) and heteronuclear RNA. Endocrinology 138 4351–4357.
Ma XM, Levy A & Lightman SL 1997b Rapid changes of heteronuclear RNA for arginine vasopressin but not for corticotropin releasing hormone in response to acute corticosterone administration. Journal of Neuroendocrinology 9 723–728.
Makino S, Smith MA & Gold PW 1995 Increased expression of corticotropin-releasing hormone and vasopressin messenger ribonucleic acid (mRNA) in the hypothalamic paraventricular nucleus during repeated stress: association with reduction in glucocorticoid receptor mRNA levels. Endocrinology 136 3299–3309.
Merali Z, Kent P, Du L, Hrdina P, Palkovits M, Faludi G, Poulter MO, Bedard T & Anisman H 2006 Corticotropin-releasing hormone, arginine vasopressin, gastrin-releasing peptide, and neuromedin B alterations in stress-relevant brain regions of suicides and control subjects. Biological Psychiatry 59 594–602.
Merriam GR & Wachter KW 1982 Algorithms for the study of episodic hormone secretion. American Journal of Physiology 243 E310–E318.
Michael RP & Gibbson JL 1963 Interrelationships bethween the endocrine system and neuropsychiatry. International Review of Neurobiology 5 243–302.
Mlynarik M, Zelena D, Bagdy G, Makara GB & Jezova D 2007 Signs of attenuated depression-like behavior in vasopressin deficient Brattleboro rats. Hormones and Behavior 51 395–405.
Presland J, Thomson F, Milne R, MacSweeney C, Campbell-Wan L, Watson M & Craighead M 2007 In vitro and in vivo characterisation of a novel antagonist for the type 1B vasopressin receptor (V1B (V3)). Abstract of the 37th Annual Meeting of the Society for Neuroscience, 4–7 November 2007, San Diego, CA, USA..
Purba JS, Hoogendijk WJ, Hofman MA & Swaab DF 1996 Increased number of vasopressin- and oxytocin-expressing neurons in the paraventricular nucleus of the hypothalamus in depression. Archives of General Psychiatry 53 137–143.
Rabadan-Diehl C, Lolait SJ & Aguilera G 1995 Regulation of pituitary vasopressin V1b receptor mRNA during stress in the rat. Journal of Neuroendocrinology 7 903–910.
Ramos AT, Troncone LR & Tufik S 2006 Suppression of adrenocorticotrophic hormone secretion by simultaneous antagonism of vasopressin 1b and CRH-1 receptors on three different stress models. Neuroendocrinology 84 309–316.
Rivier C, Rivier J, Mormede P & Vale W 1984 Studies of the nature of the interaction between vasopressin and corticotropin-releasing factor on adrenocorticotropin release in the rat. Endocrinology 115 882–886.
Serradeil-Le Gal C, Wagnon J, Simiand J, Griebel G, Lacour C, Guillon G, Barberis C, Brossard G, Soubrie P & Nisato D et al. 2002 Characterization of (2S,4R)-1-[5-chloro-1-[(2,4-dimethoxyphenyl)sulfonyl]-3-(2-methoxy-phenyl) -2-oxo-2,3-dihydro-1H-indol-3-yl]-4-hydroxy-N,N-dimethyl-2-pyrrolidine carboxamide (SSR149415), a selective and orally active vasopressin V1b receptor antagonist. Journal of Pharmacology and Experimental Therapeutics 300 1122–1130.
Serradeil-Le Gal C, Wagnon J III, Tonnerre B, Roux R, Garcia G, Griebel G & Aulombard A 2005 An overview of SSR149415, a selective nonpeptide vasopressin V(1b) receptor antagonist for the treatment of stress-related disorders. CNS Drug Reviews 11 53–68.
Shimazaki T, Iijima M & Chaki S 2006 The pituitary mediates the anxiolytic-like effects of the vasopressin V1B receptor antagonist, SSR149415, in a social interaction test in rats. European Journal of Pharmacology 543 63–67.
Spiga F, Harrison LR, Wood SA, Atkinson HC, Macsweeney CP, Thomson F, Craighead M, Grassie M & Lightman SL 2007 Effect of the glucocorticoid receptor antagonist Org 34850 on basal and stress-induced corticosterone secretion. Journal of Neuroendocrinology 19 891–900.
Stewart LQ, Roper JA, Young WS III, O'Carroll AM & Lolait SJ 2008 Pituitary–adrenal response to acute and repeated mild restraint, forced swim and change in environment stress in arginine vasopressin receptor 1b knockout mice. Journal of Neuroendocrinology 20 597–605.
Vaccari C, Lolait SJ & Ostrowski NL 1998 Comparative distribution of vasopressin V1b and oxytocin receptor messenger ribonucleic acids in brain. Endocrinology 139 5015–5033.
van West D, Del-Favero J, Aulchenko Y, Oswald P, Souery D, Forsgren T, Sluijs S, Bel-Kacem S, Adolfsson R & Mendlewicz J et al. 2004 A major SNP haplotype of the arginine vasopressin 1B receptor protects against recurrent major depression. Molecular Psychiatry 9 287–292.
Windle RJ, Wood SA, Lightman SL & Ingram CD 1998 The pulsatile characteristics of hypothalamo–pituitary–adrenal activity in female Lewis and Fischer 344 rats and its relationship to differential stress responses. Endocrinology 139 4044–4052.
de Winter RF, van Hemert AM, Derijk RH, Zwinderman KH, Frankhuijzen-Sierevogel AC, Wiegant VM & Goekoop JG 2003 Anxious-retarded depression: relation with plasma vasopressin and cortisol. Neuropsychopharmacology 28 140–147.
Zelena D, Mergl Z & Makara GB 2006 The role of vasopressin in diabetes mellitus-induced hypothalamo–pituitary–adrenal axis activation: studies in Brattleboro rats. Brain Research Bulletin 69 48–56.
