General and Comparative Endocrinology 161 (2009) 208–215

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General and Comparative Endocrinology

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Corticosterone- or metapyrone-induced alterations in adrenal functionand expression of the arginine vasotocin receptor VT2 in the pituitary glandof domestic fowl, Gallus gallus

Dharmendra Sharma a, Lawrence E. Cornett b, Chandra Mohini Chaturvedi a,*

a Department of Zoology, Banaras Hindu University, Varanasi 221005, Indiab Department of Physiology and Biophysics, University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA

a r t i c l e i n f o a b s t r a c t

Article history:Received 21 May 2008Revised 20 December 2008Accepted 24 December 2008Available online 14 January 2009

Keywords:Vasotocin receptor VT2CorticosteroneMetapyronePOMCAdrenal

0016-6480/$ - see front matter � 2009 Elsevier Inc. Adoi:10.1016/j.ygcen.2008.12.019

* Corresponding author. Fax: +91 542 2368323.E-mail address: cmcbhu@yahoo.com (C.M. Chaturv

The avian neurohypophyseal hormone arginine vasotocin (AVT) is known to be involved in the regulationof adrenocorticotropin (ACTH) release by interacting with the VT2 receptor (VT2R), which is homologousto the mammalian vasopressin V1b receptor (V1bR). To study the role of glucocorticoid in the expressionand regulation of the VT2R, corticosterone (1 or 5 mg/bird/day) or metapyrone (10 or 50 mg/kg bodyweight/day) were administered daily for 8 days to white leghorn chickens. While low doses were ineffec-tive, a high dose of corticosterone upregulated, while metapyrone downregulated, pituitary VT2R mRNAexpression and ir-VT2 in the cephalic lobe of the anterior pituitary. Further, although no change wasobserved in the expression of POMC mRNA, adrenal activity (as judged by the changes in total cholesterol,3b HSD, cortical cord width and cortico-medullary ratio) exhibited suppression or stimulation followingtreatment with corticosterone or metapyrone, respectively. In view of the classical negative feedbackeffect of glucocorticoids at the level of hypothalamic CRH neurons and pituitary corticotrophs, high cor-ticosterone level-induced suppression of the CRH–ACTH axis may have been masked by VT2R-mediatedstimulation of corticotrophs, and hence the POMC mRNA level did not change. The same argument couldbe used for metapyrone. It is concluded that expression of the VT2 receptor is regulated by glucocorti-coids in chicken. These findings confirm a role for AVT, mediated by the VT2 receptor, in regulating ACTHsecretion during stress and suggest that corticotroph VT2 receptor levels may be dynamically regulateddepending on the HPA axis activity.

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1. Introduction

In mammals, regulation of ACTH secretion during stress is mul-tifactorial, and two hypothalamic peptides, corticotropin releasinghormone (CRH) and arginine vasopressin (AVP), are the main phys-iological regulators of its release (Antoni, 1993). Despite severalcontradictory lines of evidence regarding the role of endogenousAVP in the regulation of basal or stress-stimulated secretion anddiurnal variations of ACTH and cortisol/corticosterone (reviewedby Makara et al., 2004), AVP is considered to be an important con-tributor to stress adaptation (Engelmann et al., 1996). The neuro-peptides CRH and AVP interact with their corresponding Gprotein-coupled membranous receptors present on the pituitarycorticotrophs, CRH R1 (Perrin and Vale, 1999) and V1b (Sugimotoet al., 1994), leading to a cascade of events that result in an in-crease in intracellular cyclic AMP and calcium (reviewed by Masonet al., 2002). Glucocorticoids are the final end product of the hypo-thalamic–pituitary–adrenal (HPA) axis, which exert a negative

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edi).

feedback action to regulate this axis at both, the hypothalamicand anterior pituitary levels (Keller-Wood and Dallman, 1984).Alteration in the plasma level of glucocorticoid, either by adrenal-ectomy or by the administration of glucocorticoids, has been re-ported to affect the synthesis and release of both CRH and AVPfrom the hypothalamic paraventricular nuclei (PVN) (Spinediet al., 1991; Ma et al., 1997), while at the pituitary level, it regu-lates both the transcription of POMC as well as ACTH secretion (Le-vine and Roberts, 1991; Aguilera et al., 1994; Aguilera andRabadan-Diehl, 2000).

Arginine vasotocin (AVT), the avian homologue of mammalianAVP, is also involved in the regulation of water balance, behavioral,cardiovascular and stress responses (Acher et al., 1970; Romeroet al., 1998; Romero, 2006; Gray et al., 1990; Nephew et al.,2005). As with AVP in mammals, during stress the role of AVT ineliciting corticosterone release is mediated through the HPA axis.The avian HPA axis is architecturally similar to that of mammals,but its regulation is not as well understood as that of mammals.In addition to CRH (Carsia et al., 1986), AVT also stimulates pitui-tary corticotrophs to secrete ACTH (Castro et al., 1986). AVT elicitsACTH release by interacting with its pituitary receptor VT2R, a se-

Table 1Sequence of primers used for RT-PCR.

Gene Primer sequence Amplicon size

VT2 F 5’-GCG AGA TCT GCA AGA ACC-3’ 565 bpR 50-GGA AGC AGT GAC TGA ATC-3’

POMC F 5’-GAG AGC ATC CGC AAG TAC GTO-3’ 511 bpR 50-CTG ATG ACT CTG TTC AAA ACG-30

GAPDH F 50-AGT CAT CCC TGA GCT GAA TG-3’ 330 bpR 50-ACC ATC AAGTCC ACA ACA CG-30

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ven-transmembrane-domain receptor that is coupled to protein ki-nase C-calcium-dependent mechanisms (Cornett et al., 2003; Jur-kevich et al., 2005). This receptor has been found to behomologous to mammalian V1bR.

Acute stress leads to the rapid release of CRH and AVP into thepituitary portal circulation (Plotsky, 1991). However, duringchronic stress involving repeated stimulation of the HPA axis, therate of release of CRH is maintained, while AVP secretion rate ismarkedly increased (Tilders et al., 1989). It has also been foundthat during repeated stress, the regulation of HPA axis activityswitches from CRH to AVP, and changes in vasopressin receptorexpression are the determining factor in corticotroph responsive-ness (see Aguilera et al., 2008). Although certain reports in mam-mals indicate a role for glucocorticoids in the regulation of theV1b receptor expression (Koch and Lutz-Bucher, 1985; Todd andLightmann, 1987; Rabadan-Diehl et al., 1997; Rabadan-Diehl andAguilera, 1998), there is a complete lack of such knowledge inbirds.

In the present study, experiments were designed to analyze theexpression of the VT2 receptor and the POMC gene in the pituitarygland and to measure the adrenal function of chicken followingdaily administration of corticosterone or metapyrone (an inhibitorof adrenal steroidogenesis). Total adrenal cholesterol (used as aprecursor during adrenal steroidogenesis) content and the activityof 3b-hydroxysteroid dehydrogenase isomerase (3b-HSD), the en-zyme responsible for the conversion of pregnenolone to progester-one, were measured to assess adrenal function. An increase in theactivity of this enzyme indicates stimulation of steroidogenesis(Samuels et al., 1951; Dupont et al., 1990; Singh and Chaturvedi,2006). Moreover, cytometric analysis (cortical cord width as wellas cortico-medullary ratio), which has been widely used as a mea-sure of adrenocortical activity (Siller et al., 1975; Chaturvedi andThapaliyal, 1979; Chaturvedi and Kumar, 2007), was also em-ployed here. Expression of POMC was also studied since this geneis a polypeptide precursor of ACTH (the key mediator of the stressresponse) and its expression is under the control of hypothalamicfactors (CRH and AVT).

2. Materials and methods

Day-old white leghorn chicks were procured from a local hatch-ery (Shreeji Hatchery, Shivpur, Varanasi, India) and reared underlaboratory conditions in a 12L:12D photoregime. Chicks were pro-vided with a high protein diet: chicken starter ration up to the ageof 6 weeks. Thereafter, these birds were provided with a broiler fin-isher diet. All of the birds were provided food and water ad libitum.Experiments were performed in accordance with institutionalpractice and within the framework of the Revised Animals (scien-tific procedures) Act of 2002 of Government of India on animalwelfare.

Experiments were performed to study corticosterone- or meta-pyrone-induced alterations in the expression of the VT2 receptor,POMC and adrenal function in domestic fowl. In two different setsof experiments, performed at different times, 8- to 9-week-oldchickens were divided into three groups (n = 14 in each) and wereinjected with a low (1 mg/bird/day) or high dose (5 mg/bird/day)of corticosterone subcutaneously (s.c.), or its inhibitor metapyrone(10 and 50 mg/kg body weight/day, i.m.). Corticosterone (Sigma,USA) was first dissolved in a small amount of ethanol, and the finalvolume was reached by the addition in normal saline so that each0.1 ml contained the desired concentration of corticosterone. Met-apyrone (Ciba Gayegi, India) was dissolved in normal saline to ob-tain the required concentration in 0.1 ml. In both sets, the thirdgroup received normal saline (i.m.) and served as a control. Allinjections were given daily at the same time (between 9:00 and9:30 A.M.) for a period of 8 days. Thirty to 60 min after the last

injection, eight birds from each of the six groups were weighedand sacrificed by decapitation; blood was collected in heparinizedtubes and centrifuged at 1000g for 20 min. Plasma was separatedand stored at �20 �C for the measurement of plasma osmolality.Pituitaries were immediately dissected out, snap-frozen in liquidnitrogen and kept at �70 �C until RNA isolation and RT-PCR analy-sis. Adrenal glands were also removed and stored at �20 �C for thebiochemical estimation of ascorbic acid (Schaffert and Kingsley,1955), cholesterol content (Rosenthal et al., 1960) and 3b HSDactivity (Shivanandappa and Venkatesh, 1997). The remaining sixbirds from all the groups were anaesthetized by injection with so-dium pentobarbitol (25–30 mg/kg body weight, i.v.), and werethen perfused intracardially with 0.02 M PBS containing 0.01% hep-arin, followed by Zamboni fixative (4% PFA in PBS + Picric acid).After whole body perfusion, pituitaries and adrenals were dis-sected out and post-fixed in fresh Zamboni fixative (4–6 h for pitu-itary and 24–48 h for adrenal). After fixation, tissues weredehydrated in a graded series of alcohol, cleared in xylene andembedded in paraffin wax. Eight-micrometer-thick sections ofthe pituitary and 6 lm-thick sections for the adrenal were cut witha rotary microtome (Weswox, Ambala Cant. India), attached to gel-atin-coated slides and processed for the histochemical studies(in situ hybridization and immunohistochemistry for pituitary sec-tions) and routine haematoxylin–eosin staining for the adrenalsections.

2.1. Semi-quantitative RT-PCR

Reverse transcriptase polymerase chain reaction (RT-PCR) wasused to analyze VT2R and POMC mRNA levels in the pituitary. Ineach set, four pituitaries were pooled and total RNA was isolatedusing TRI reagent (Sigma, USA). The integrity of the RNA was ver-ified by 1.5% denaturing agarose gel electrophoresis. To rule out thepossibility that PCR products would result from the amplificationof genomic DNA, RNA samples were treated with DNAse. Onemicrogram of total RNA was used to synthesize cDNA by reversetranscription using the first strand cDNA synthesis kit (Fermentas,USA) according to the manufacturer’s guidance. For PCR reactions,1/10th of the reaction volume was taken from the RT reaction andPCR was carried out using the 2� PCR Master Mix kit (Fermentas,USA). After initial denaturation by incubating at 94 �C for 5 min, 35cycles were performed including denaturation for 1 min at 94 �C,annealing of the primers for 1 min at 55 �C (for VT2), 52 �C (forPOMC) and 58 �C for GAPDH, and extension for 1 min at 72 �C, a fi-nal elongation step was then performed at 72 �C for 5 min. Primersequences for each gene and the size of amplicon are given sepa-rately in the Table 1. PCR products were analyzed on a 2% agarosegel containing ethidium bromide (0.5 lg/ml). Stained bands werecaptured digitally and densitometric analysis was conducted usingimage analysis software Alpha Imager.

2.2. In situ hybridization

In situ hybridization of VT2R mRNA was performed using adigoxigenin (DIG)-labeled 250 bp fragment of the VT2R cDNA

Fig. 1. Effect of corticosterone (Cort) and metapyrone (Met) administration on thebody weight and plasma osmolality in chicken. Values are the means ± SEM of thedata obtained from six birds per group in each experiment. *P < 0.05, significance ofdifference from control receiving normal saline (NS). L, low dose; H, high dose.

Fig. 2. RT-PCR analysis of VT2R, POMC and GAPDH in chicken following admin-istration of corticosterone (Cort) and metapyrone (Met). Upper panel shows therepresentative agarose gels. Lower panel shows the relative densitometric values ofamplified product of VT2 and POMC normalized to that of GAPDH. Values are themeans ± SEM of the data obtained by a pool of four pituitaries (two pools) in eachgroup from the two experiments. *P < 0.05; ***P < 0.001, significance of differencefrom respective control receiving normal saline (NS). L, low dose; H, high dose.

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(GenBank Accession No. AY008272). Hybridization was carried outin hybridization solution (50% DI formamide, 10% dextran sul-phate, 1� Denhart’s solution, 4� SSC, 10 mM DTT, 1 mg/ml ssDNA)containing 300–400 ng/ml of the labeled probe at 42 �C for 16 h.Post-hybridization washing and immunological detection wereperformed as described previously (Srivastava et al., 2007). Controlsections were incubated in hybridization buffer lacking DIG-la-beled probe.

2.3. Immunohistochemistry

After initial dewaxing in xylene and rehydration in a graded ser-ies of alcohol, slides were processed for immunohistochemistryusing rabbit-VT2R antisera (1:6000) for 24 h (Jurkevich et al.,2005), followed by incubation in HRP-conjugated secondary anti-body. Diaminobenzidine hydrocholoride (DAB) was used as a chro-mogen molecule for the immunological detection. For the negativecontrol, primary antiserum was omitted, and sections were incu-bated in normal goat serum only.

2.4. Quantitative analysis

For image analysis of in situ hybridization and immunohisto-chemical signals in sections of the pituitary gland, a relative quan-tification method was employed as described by Sakharkar et al.(2005). An image analysis system consisting of a Leitz LaborLux Smicroscope and a CCD video camera (JVC, Japan) connected to acomputer was used. The microscope images were digitized andanalyzed using Leica Qwin standard software (version 3). Five ran-domly selected square areas (21389 lm2 each) in each saggital sec-tion of the pituitary (five sections from each pituitary and total fourpituitaries) from the region of cephalic lobe were subjected toanalysis.

2.5. Morphometry

For adrenal morphometry, five randomly selected transversesections from the mid-region of the adrenal (four adrenals pergroup) were chosen. The widths of five randomly selected cortical(steroidogenic) cords as well as the total area occupied by light col-ored steroidogenic cords (cortical) and dark patches of chromaffintissue (medulla) from each section were measured to calculate thecortico-medullary (C:M) ratio.

2.6. Statistical analysis

Data are presented as the means ± SEM of the values obtainedfrom the number of birds indicated in the figure legends. Statisticalsignificance of the differences (P < 0.05) between control andexperimental groups in each set was determined by one-way AN-OVA followed by Dunnet post hoc analysis.

3. Results

With the exception of a high dose of corticosterone, corticoste-rone or metapyrone had no effect on body weight. Birds treatedwith a high dose of corticosterone exhibited a decrease in bodyweight as compared to controls (Fig. 1). On the other hand, plasmaosmolality did not change after glucocorticoid or metapyroneadministration (Fig. 1).

Relative densitometric analysis of the RT-PCR reaction as well asrelative quantitative analysis of ISH and IHC showed that neitherVT2R mRNA transcript levels and ir-VT2 nor the expression ofPOMC mRNA were altered in chickens receiving low dose of corti-costerone or metapyrone (Fig. 2). However, a high dose of cortico-sterone significantly increased while metapyrone decreased the

expression of VT2R as compared to the control. Again, the expres-sion of POMC mRNA was unaltered (Fig. 2). Results of relativequantitative analysis of the in situ hybridization and immunohisto-chemical signals did not show any change in the VT2R expression,following low doses of glucocorticoid (corticosterone) or metapy-

Fig. 3. In situ hybridization (upper row) and immunohistochemical localization (lower row) of VT2R in the anterior pituitary gland of chickens treated with low doses ofcorticosterone (Cort) and metapyrone (Met). Histograms represent relative quantitative analysis of the hybridization and immunoreactive signals. Values are themeans ± SEM of the data obtained by relative quantitative analysis of signals in the pituitaries of four birds per group.

Fig. 4. In situ hybridization and Immunohistochemical localization of vasotocin receptor VT2 in the anterior pituitary gland of chickens treated with high doses ofcorticosterone (Cort) and metapyrone (Met). Histograms represent relative quantitative analysis of the hybridization and immunoreactive signals. Values are themeans ± SEM of the data obtained by relative quantitative analysis of signals in the pituitaries of four birds per group.*P < 0.05; ***P < 0.001 significance of difference fromcontrol receiving normal saline (NS).

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rone (Fig. 3); however, a high dose of corticosterone increased,while metapyrone decreased, levels of VT2R transcript and ir-VT2R as compared to their respective controls (Fig. 4).

Administration of a low dose of corticosterone or metapyroneresulted in no significant change in adrenal weight or ascorbic acidor cholesterol contents, but 3b HSD activity in the adrenal glanddecreased and increased, respectively, as compared to control birds(Fig. 5). However, following administration of high doses of corti-costerone or metapyrone, these parameters showed a significantchange. When compared with the control, a high dose of cortico-sterone resulted in significant reductions in adrenal weight and3b HSD activity, while adrenal cholesterol content increased. Incontrast, a high dose of metapyrone resulted in significant in-creases in adrenal weight and 3b HSD activity, whereas adrenalcholesterol content decreased compared to controls (Fig. 5). How-ever, adrenal ascorbic acid did not show any change irrespective ofthe treatment and the doses (Fig. 5). Histologically, transversesections of the adrenal gland showed typical avian patterns, i.e.,

Fig. 5. Effect of corticosterone (Cort) and metapyrone (Met) administration on theadrenal activity of chicken. Values are the means ± SEM of the data obtained fromfour birds per group in each experiment. *P < 0.05; ***P < 0.001 significance ofdifference from control receiving normal saline (NS). L, low dose; H, high dose.

intermingling of the steroidogenic (cortical) cords and catecholam-inergic (medullary) patches. Cytometric analysis of the adrenalgland revealed no change following administration of low dosesof either glucocorticoid or metapyrone, but the high dose of corti-costerone significantly reduced the cortical cord width and cortico-medullary ratio while metapyrone increased these parameters(Fig. 6).

4. Discussion

Glucocorticoids are known to be the end product as well as thekey regulator of the HPA axis by exerting a negative feedback con-trol. It has also been shown in rat and horse that CRH-stimulatedACTH release is more susceptible to glucocorticoid inhibition thanvasopressin-stimulated release (Dallman, 1993; Evans et al., 1993;Oki et al., 1991). In the present study, repeated injections of corti-costerone or the steroid synthesis inhibitor metapyrone wereadministered in two different doses to study the response of theHPA axis. Repeated injections of a low dose of both corticosteroneand metapyrone were found to be ineffective in changing eitherthe adrenal parameters (adrenal weight, cholesterol, 3b HSD, corti-cal cord width as well as cortico-medullary ratio), or the pituitaryVT2R and POMC mRNA expression. These results suggest that thisdose of corticosterone (1 mg/day for 8 days) was not sufficient toinduce a negative feedback effect, nor could metapyrone (10 mg/kg body weight) induce an effect similar to chemical adrenalec-tomy. However, the second group of birds treated with a high doseof corticosterone exhibited decreased activity of the adrenal gland,while the high dose of metapyrone produced an overall increase inadrenal activity.

The results of the present study also indicate that repeatedinjections of a high dose of corticosterone upregulated, while met-apyrone downregulated, the expression of VT2R mRNA and ir-VT2R. Increased or decreased translation of the mRNA indicates aparallel relationship between transcription and translation, andthus indicates an up- or down-regulation of the pituitary VT2Rgene expression following glucocorticoid or metapyrone adminis-tration, respectively. A transient decrease in the mRNA level ofthe V1b receptor has been reported following adrenalectomy inrats, but it returns to the normal level (no difference from the con-trol values) after six days (Rabadan-Diehl et al., 1997). It has beenshown that alterations in plasma glucocorticoid levels affect thesynthesis and secretion of hypothalamic regulators (CRH andAVP) of ACTH (Aguilera, 1994; Plotsky, 1991) and that membranereceptors for peptide hormones can be downregulated by theirown ligands (Catt et al., 1979). Therefore, a possible mechanismfor this increase or decrease in the expression of VT2R mRNA fol-lowing glucocorticoid or metapyrone administration, respectively,may involve alteration in the secretion of hypothalamic AVT intothe portal circulation in response to changes in the level of plasmaglucocorticoids (Koch and Lutz-Bucher, 1985). However, a directaction of glucocorticoids at the pituitary level cannot be ruledout, as has been proposed previously (Rabadan-Diehl et al.,1997). In this study, treatment with glucocorticoid in adrenalecto-mized rats with hypothalamic deafferantation prevented pituitaryVP receptor loss (Rabadan-Diehl et al., 1997).

Earlier studies in mammals have shown that during stressfulconditions or following adrenalectomy, vasopressin receptorV1bR mRNA content does not show a correlation with the actualreceptor level/binding (Aguilera and Rabadan-Diehl, 2000). More-over, steady state levels of the receptor mRNA are not the primaryfactor determining pituitary V1b receptor content (Aguilera andRabadan-Diehl, 2000). As discussed above, following surgical adre-nalectomy, there is a transient decrease in the expression of V1bRmRNA, but administration of dexamethasone prevents this de-crease (Rabadan-Diehl et al., 1997). Adrenalectomy does cause a

Fig. 6. Transverse section of the adrenal gland of chicken treated with low (upper row) and high dose (lower row) of corticosterone (Cort) and metapyrone (Met). Noteintermingling of lightly stained steroidogenic (cortical) cords and dark catecholaminergic (medullary) patches. Histograms represent changes in the cortical cord width andcortico-medullary (C:M) ratio. Values are the means ± SEM of the data obtained by randomly selected five sections from the mid-region of the adrenal of bird (four birds pergroup). *P < 0.05; **P < 0.01; ***P < 0.001, significance of difference from control receiving normal saline (NS).

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significant decrease in the vasopressin binding (Koch and Lutz-Bu-cher, 1985; Rabadan-Diehl and Aguilera, 1998), suggesting adown-regulation of the vasopressin receptor level. These resultsindicate a role for either post-transcriptional mechanisms or theinternalization of membrane receptors, or both. The number ofV1b receptors in the pituitary depends on the rate of receptor syn-thesis as well as receptor desensitization, internalization or degra-dation following stimulation by vasopressin (Aguilera et al., 2003).But in the present study, it was found that up- or down-regulationof VT2 receptor mRNA coincided with the increased or decreasedexpression of the immunoreactive VT2R, indicating that glucocor-ticoid or metapyrone treatment actually up- or downregulatedthe VT2 receptor synthesis, respectively. As discussed above, ithas been observed in mammals that glucocorticoid administrationdownregulates V1b receptor (in view of decreased binding). Thismay involve the phenomena of receptor internalization rather thandecreased efficiency of transcriptional and post-transcriptionalmodifications. However, present findings do not indicate a parallelrelation between receptor synthesis and the actual receptor level,and therefore further study is required. It is quite possible thatthere could be post-translational modifications that also regulatethe actual state of vasotocin receptors in the pituitary.

Despite the established/classical feedback effect of glucocorti-coids at the level of anterior pituitary (corticotrophs), in the pres-ent study no significant change was observed in the expression ofPOMC mRNA either after glucocorticoid administration or afterchemical adrenalectomy. In birds, unlike mammals, there are noreports available regarding the synthesis and secretion of AVT frommagno- and parvocellular neurons during different stressful orphysiological conditions. But we do know that increased plasmaosmolality following osmotic stress selectively stimulates magno-cellular neurons in both PVN and SON, increasing AVT release intothe peripheral circulation through the hypothalamo-neurohypo-physeal system (Acher et al., 1970; Skadhauge, 1981; Arad and

Skadhauge, 1984; Arad et al., 1985; Stallone and Braun, 1986; Cha-turvedi et al., 1994, 1997; Seth et al., 2004). However, it is not yetknown whether osmotic stress also stimulates parvocellular neu-rons, as in case of other stressors. Alteration in pituitary VT2RmRNA following osmotic stress (Sharma et al., accepted for publi-cation) as well as during glucocorticoid administration or chemicaladrenalectomy (present study) does indicate that hypothalamicAVT is also released into median eminence to regulate corticotrophsecretion; but whether the source of this AVT is magnocellular orparvocellular is not yet clear. Although it is quite reasonable to pre-sume that at least during water deprivation, magnocellular AVT isalso released into the median eminence, and this in turn modulatesor alters the expression of its own receptor. Since in the presentstudy plasma osmolality was not altered, this indicated that mag-nocellular AVT does not play a role in the HPA axis of chickenreceiving corticosterone or metapyrone. But parvocellular AVTseems to alter corticotroph activity via VT2 receptors since VT2RmRNA levels are changed following corticosterone or metapyronetreatment. Moreover, glucocorticoid is known to exert negativefeedback on parvocellular vasopressinergic neurons and not onthe magnocellular vasopressinergic neurons (Kovács et al., 2000).

Since glucocorticoids stimulate pituitary VT2R expression, itmight be expected to increase pituitary POMC and/or ACTH levels.On the other hand, a high glucocorticoid level is reported to sup-press CRH and ACTH levels through a negative feedback effect.The lack of change observed in the POMC mRNA levels of thesechickens may possibly be due to the fact that the high corticoste-rone level-induced suppression of the hypothalamo–pituitary(CRH–ACTH) axis was masked by the vasotocin receptor-inducedincrease. The same argument could be used for the metapyrone ef-fect. However, the role of other intermediary factors in the regula-tion of transcription and translation (of both VT2R and POMCmRNA) still cannot be ruled out. In all such cases the end effectof the HPA axis (i.e., adrenal activity) seems to correlate more with

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the CRH-ACTH axis than with the AVT–ACTH pathway. On the basisof present observations, specifically VT2R and adrenal activity (endpoint of HPA axis), it is concluded that high glucocorticoid level, inspite of increasing the pituitary VT2R transcript, in general has anoverall suppressive effect on the HPA axis. Further, it is suggestedthat in chicken, during glucocorticoid-induced stress conditions,AVT also plays a major role as the stress hormone modulatingthe pituitary–adrenal axis via its receptor VT2.

Acknowledgments

This work was supported in part by a research Grant (F. No. 32-472/2006 (SR)) to C.M.C., from the University Grant Commission,New Delhi, India. We also thank the Council of Scientific and Indus-trial Research, for providing financial support in the form of Juniorand Senior Research fellowships to D.S. The authors thank SandieJacobi for the preparation of VT2R cDNA (through NSF Grant IBN0111006 to L.E.C.) and Dr. N.K. Subhedar, Department of pharma-ceutical sciences, University of Nagpur, Nagpur, India for providingthe facility for image analysis.

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