The Vasopressin V1b Receptor Modulates Plasma Corticosterone Responses to Dehydration‐Induced Stress

Vasopressin V1b receptor knockout (V1b?/?) mice were used to investigate a putative role for the V1b receptor (V1bR) in fluid regulation and in the hypothalamic‐neurohypophysial system (HNS) and hypothalamic‐pituitary‐adrenal (HPA) axis responses to osmotic stress induced by water deprivation (WD). Male wild‐type and V1b?/? mice were housed in metabolic cages to allow determination of water intake and urine volume and osmolality. When provided with food and water ad lib., spontaneous urine volume and urine osmolality did not differ between genotypes. Similarly, WD for 24 h caused comparable decreases in urine volume and increases in urine osmolality irrespective of genotype. WD resulted in an increase in plasma corticosterone concentration in wild‐type animals; however, this WD‐induced increase in plasma corticosterone was significantly attenuated in V1b?/? mice. Comparable increases in neuronal activation, indicated by increased c‐fos mRNA expression, and in vasopressin mRNA expression occurred in both the supraoptic nucleus and paraventricular nucleus (PVN) of wild‐type and V1b?/? mice following WD; however, the WD‐induced decrease in corticotrophin‐releasing hormone mRNA expression seen in the PVN of wild‐type mice was not observed in the PVN of V1b?/? mice. These data suggest that, although the vasopressin V1bR is not required for normal HNS function, it is necessary for a full HPA‐axis response to the osmotic stress of WD.     

Attenuated Stress Response to Acute Restraint and Forced Swimming Stress in Arginine Vasopressin 1b Receptor Subtype (Avpr1b) Receptor Knockout Mice and Wild‐Type Mice Treated with a Novel Avpr1b Receptor Antagonist

Arginine vasopressin (AVP) synthesised in the parvocellular region of the hypothalamic paraventricular nucleus and released into the pituitary portal vessels acts on the 1b receptor subtype (Avpr1b) present in anterior pituitary corticotrophs to modulate the release of adrenocorticotrophic hormone (ACTH). Corticotrophin‐releasing hormone is considered the major drive behind ACTH release; however, its action is augmented synergistically by AVP. To determine the extent of vasopressinergic influence in the hypothalamic‐pituitary‐adrenal axis response to restraint and forced swimming stress, we compared the stress hormone levels [plasma ACTH in both stressors and corticosterone (CORT) in restraint stress only] following acute stress in mutant Avpr1b knockout (KO) mice compared to their wild‐type controls following the administration of a novel Avpr1b antagonist. Restraint and forced swimming stress‐induced increases in plasma ACTH were significantly diminished in mice lacking a functional Avpr1b and in wild‐type mice that had been pre‐treated with Avpr1b antagonist. A corresponding decrease in plasma CORT levels was also observed in acute restraint‐stressed knockout male mice, and in Avpr1b‐antagonist‐treated male wild‐type mice. By contrast, plasma CORT levels were not reduced in acutely restraint‐stressed female knockout animals, or in female wild‐type animals pre‐treated with Avpr1b antagonist. These results demonstrate that pharmacological antagonism or inactivation of Avpr1b causes a reduction in the hypothalamic‐pituitary‐adrenal (HPA) axis response, particularly ACTH, to acute restraint and forced swimming stress, and show that Avpr1b knockout mice constitute a model by which to study the contribution of Avpr1b to the HPA axis response to acute stressors.     

Regulation of Pituitary Vasopressin Receptors during Chronic Stress: Relationship to Corticotroph Responsiveness

The relationship between vasopressin (VP) receptor levels in the anterior pituitary and VP-stimulated ACTH release in vitro was studied in rats subjected to various chronic stress paradigms. The stress models used were water deprivation for 60 h and administration of 2% NaCI in the drinking water (both of which are associated with decreased pituitary ACTH responsiveness), and repeated i.p.hypertonic saline injections or repeated daily immobilization for 14 days (associated with increased ACTH responsiveness to novel stimuli). VP receptors were measured by binding of [³H]arginine-VP to anterior pituitary membrane-rich fractions, and ACTH responses to VP in collagenase dispersed anterior pituitary cells. In control rats, binding of [³H]AVP was saturable and high affinity, with a Kd of 0.45 ± 0.05 nM and a B_max of 138.8 ± 8.1 fmol/mg. In pituitary membranes from stressed rats, binding affinity was unchanged, but B_max changed according to the type of stress. While VP binding was markedly reduced after water deprivation and 2% saline (25% and 49%, respectively), it was significantly increased after repeated i.p. hypertonic saline injections and repeated immobilization (126% and 154% of controls, respectively). The changes in VP binding were associated to parallel changes in maximum VP-stimulated ACTH production in vitro, with a 34% decrease in water deprived rats and a 25% increase in hypertonic saline injected rats. The potentiating effect of VP on corticotropin releasing hormone-stimulated ACTH was also reduced in cells from water-restricted rats, and increased in cells from rats given repeated injections of hypertonic saline. The data show a direct relationship between changes in corticotroph responsiveness and changes in pituitary VP receptors during chronic stress, suggesting that pituitary VP receptor regulation is involved in the adaptation of the HPA axis during chronic stress.     

Effects of Novelty Stress on Vasopressin and Oxytocin Secretion by the Pituitary in the Rat

Effects of novel environmental stimuli on vasopressin and oxytocin secretion by the pituitary were studied in dehydrated male rats. As the novel environmental stimuli, rats were transferred to an experimental room, placed in a box painted black and given a pure tone auditory stimulus of 2 kHz. Exposure of rats to the novel environmental stimuli for a period of 2 min decreased plasma concentrations of vasopressin and increased plasma levels of adrenocorticotrophic hormone (ACTH) and prolactin, but did not significantly change the plasma level of oxytocin. The stimuli, however, became ineffective for producing the suppressive vasopressin response as the period of exposure was prolonged to more than 5 and up to 30 min, although the prolonged stimuli were still effective for inducing facilitatory ACTH and prolactin responses. After repeated exposures of rats to the environmental stimuli once a day for 5 or 10 days, the stimuli became disabled from producing the suppressive vasopressin response. However, the rats were still capable of responding to the novel stimuli of another kind. All these data suggest that novelty stress suppresses vasopressin secretion but does not change oxytocin secretion. In order to test the possibility that glucocorticoids expectedly secreted by the adrenals in response to the stress might have suppressed vasopressin secretion, a large amount of dexamethasone was administered to the rat before testing. Dexamethasone pretreatment depressed plasma levels of ACTH and vasopressin as reported previously and blocked the facilitatory ACTH response to the novelty stress. However, dexamethasone treatment did not affect the suppressive vasopressin response to the novelty stress. Thus, it is likely that the suppressive vasopressin response to novelty stress does not primarily depend upon endogenous glucocorticoids.     

Altered Pituitary Sensitivity to Corticotropin-Releasing Factor and Arginine Vasopressin Participates in the Stress Hyporesponsiveness of Lactation in the Rat

The regulation of the activity of the hypothalamic-pituitary-adrenal (HPA) axis is modified during lactation, wherein a blunted stress-induced adrenocorticotropic hormone (ACTH) and glucocorticoid secretion is coupled with elevated basal secretion of these hormones. The involvement of pituitary modifications in lactation-induced stress hyporesponsiveness has yet to be established. In this study we tested the hypothesis that the pituitary sensitivity to corticotropin-releasing factor (CRF) and arginine vasopressin (AVP) is altered in lactation in the rat. We examined the effect of exogenous CRF (0.1-5 microg/rat), AVP (0.01-0.5 microg/rat), and AVP (0.01-0.5 microg/rat)+CRF (0.1 microg/rat) on the ACTH response of virgin, mid-lactating (lactation day 10-12) females, as well as nursing females separated from their pups for 48 h. Additionally, to determine if changes in CRF- or AVP-receptor densities might mediate alterations in pituitary sensitivity, we compared pituitary CRF- and AVP-receptor binding by autoradiography in pregnant, mid-lactating, and virgin female rats. While both virgin and lactating female rats exhibited significant ACTH responses to CRF, the responses to the highest doses of CRF (2.0 and 5.0 microg/rat) were greater in virgin than in lactating females. Separation of the litter for 48 h partially restored pituitary responsiveness to 2.0 microg of CRF. Conversely, whereas lactating females displayed robust ACTH secretion following a high dose of AVP or following a combination of AVP+CRF, the response of virgin females was much smaller. These modifications in pituitary responsiveness were not accompanied by significant differences in pituitary CRF-and AVP-receptors levels between female groups. Our results demonstrate that a reduction in pituitary sensitivity to CRF, but not to AVP occur during lactation in the rat which mediates, at least in part, the stress hyporesponsiveness of lactation.     

Localization of vasopressin (V1a) receptor binding and mRNA in the rhesus monkey brain

Centrally released arginine vasopressin (AVP) has been associated with various behavioural and cognitive effects, such as scent marking, aggression, and memory, which are believed to be mediated by the V1a subtype of the vasopressin receptor. Although the distribution of V1a receptors is conserved in a few brain regions, the pattern of expression of this receptor is, in general, highly species-specific. We have used receptor autoradiography with the linear V1a receptor ligand (125I-Phenylacetyl-D-Tyr(Me)-Phe-Gln-Asn-Arg-Pro-Arg-Tyr-NH2) to characterize the pattern of receptor binding in the rhesus monkey brain. Brain sites of V1a receptor synthesis were defined using in-situ hybridization. The regions of highest V1a receptor density included the prefrontal, cingulate, pyriform, and entorhinal cortex, as well as the presubiculum and mamillary bodies. In addition, V1a receptor binding and mRNA were detected in several regions reported to have V1a receptor in most rodents, including the amygdala, bed nucleus of the stria terminalis, lateral septum, hypothalamus and the brainstem. The distribution is consistent with a role for vasopressin in higher cognitive functions, especially memory, in primates.     

The discovery of novel vasopressin V1b receptor ligands for pharmacological, functional and structural investigations

Until recently, pharmacological studies dealing with vasopressin receptor isoforms were severely hampered by the lack of selective agonists or antagonists that recognize the pituitary V(1b) vasopressin receptor. By contrast, many selective vasopressin-related compounds are available for characterization of the vasopressor (V(1a)) or antidiuretic (V(2)) vasopressin receptor subtypes. Recently, SSR149415, a selective nonpeptide molecule, was discovered with nanomolar affinity for mammalian V(1b) receptors and good selectivity for the other vasopressin and oxytocin receptor isoforms. This molecule exhibits potent antagonist properties both in vitro and in vivo. We also designed synthetic peptides derived from [deaminocysteine(1),arginine(8)]vasopressin (dAVP), modified in position 4 by various amino acid residues. Some of these, d[cyclohexylalanine(4)]AVP or d[lysine(4)]AVP, have a high affinity and an excellent selectivity for the human V(1b) receptor subtype. However, they exhibit a mixed V(1b)/V(2) pharmacological profile for the rat vasopressin receptor isoforms. Whatever the species considered, these peptides behave as agonists both in bioassays performed in vitro and in vivo. The d[cyclohexylalanine(4)]AVP was tritiated and represents the first selective radiolabelled ligand available for studying the human V(1b) receptors. The discovery of these new selective V(1b) agonists and V(1b) antagonist allows an accurate pharmacological characterization of all the vasopressin receptor isoforms. As emphasized in this review, attention to the vasopressin and oxytocin receptor species differences is of critical importance in studies with all vasopressin and oxytocin ligands.     

Central V1b Receptor Antagonism in Lactating Rats: Impairment of Maternal Care But Not of Maternal Aggression

Maternal behaviour in rodents is mediated by the central oxytocin and vasopressin systems, amongst others. The role of vasopressin, acting via the V1a receptor (V1aR), on maternal care and maternal aggression has recently been described. However, a potential involvement of the V1b receptor (V1bR) in maternal behaviour has only been demonstrated in knockout mice. The present study aimed to examine the effects of central pharmacological manipulation of the V1bR on maternal behaviour in lactating Wistar rats. On pregnancy day 18, female rats were implanted with a guide cannula targeting the lateral ventricle. After parturition, dams received an acute central infusion of a specific V1bR agonist (d[Leu4,Lys8]VP) or V1bR antagonist (SSR149415) once daily, followed by observations of maternal care [lactation day (LD) 1], maternal motivation in the pup retrieval test (LD 2), anxiety‐related behaviour on the elevated plus‐maze (LD 3) and maternal aggression in the maternal defence test followed by maternal care monitoring (LD 4). Our data demonstrate that, under nonstress conditions, the V1bR antagonist decreased the occurrence of both nursing and mother–pup interaction, whereas the V1bR agonist did not affect either parameter. Under stress conditions (i.e. after the maternal defence test), mother–pup interaction was decreased by infusion of the V1bR antagonist. During the maternal defence test, neither treatment affected aggressive or non‐aggressive behaviour. Finally, neither treatment altered maternal motivation or anxiety. In conclusion, central V1bR antagonism modulates aspects of maternal care but not of maternal aggression or maternal motivation in lactating rats. These findings further extend our knowledge on the vasopressin system as a vital mediator of maternal behaviour.     

Medial Septal Area Vasopressin Receptor Subtypes in the Regulation of Urine and Sodium Excretion in Rats

The present study aimed to determine the effects of selective antagonists of V_1a, V₂, and V_1a/V₂ (Conivaptan; Astellas Pharma Inc., Tokyo, Japan) arginine vasopressin (AVP) receptors on the flow of urine and sodium excretion induced by AVP, by means of microinjections into the medial septal area (MSA) of the rat brain. Male Holtzman rats had a guide cannula implanted into the dorsal surface of the MSA. Intravenous infusion of hypotonic saline was used to promote urinary flow, which was collected for 4 h. Pretreatment with the V_1a antagonist decreased, and the V₂ antagonist and Conivaptan (a V_1a/V₂ antagonist) increased, the urinary flow induced by AVP. Administration of AVP increased sodium excretion. Pretreatment with V₂ or V_1a antagonists decreased, and Conivaptan abolished, the sodium excretion induced by AVP. These results indicate that the V_1a and V₂ receptors of the MSA are important in the central regulation of urine and sodium excretion.     

Inhibition of vasopressin V1b receptor translation by upstream open reading frames in the 5'-untranslated region

The 5'-UTR of the vasopressin V1b receptor (V1bR) mRNA contains small open reading frames (ORF) located upstream (u) of the main ORF encoding the V1bR. The ability of the three proximal uORFs to be translated into peptides and their influence on V1bR translation was examined using fusion constructs of uORFs and V5 epitope, or ATG/ATA uORF mutations in the V1bR cDNA. In vitro translation and western blot analysis after transfection of uORF1-V5 or uORF2-V5 into cells revealed that uORF1 can be translated. As predicted by computer analysis, in vitro translation using a rabbit reticulocyte/canine microsome system, immunohistochemistry and western blot in membranes of transfected cells with uORF1-V5 revealed translocation of the uORF1 peptide into membrane fractions. In vitro translation of V1bR cDNA with mutations of the two uORFs proximal to the initiating methionine, uORFs 1 and 2 (Mut 1-2), or uORF2 (Mut 2) showed significantly increased translation of a 46 kDa band corresponding to the V1bR, compared with wild-type (WT) V1bR, an effect that was attenuated by cotranslation of uORF1-V5. Consistently, VP-induced inositol phosphate formation was higher in Chinese hamster ovay cells transfected with Mut 1-2 than with WT V1bR. Immunohistochemical and western blot analysis, using an antibody against uORF1, revealed peptide immunoreactivity in rat pituitary but not in liver. Pituitary uORF immunoreactivity increased following glucocorticoid administration. The present study shows that uORFs in the 5'-UTR of the V1bR mRNA inhibit V1bR translation, and suggests that translation of a 38-amino acid membrane peptide encoded by uORF1 exerts tonic inhibition of V1bR translation.     

Binding Sites for Vasopressin in the Human Pituitary are Associated with Corticotrophs and may Differ from Other Known Vasopressin Receptors

In view of the fact that Vasopressin can induce pituitary adrenocorticotrophin release, we performed an autoradiographical study of [³H]arginine vasopressin binding in human pituitary tissue obtained post-mortem from adults and foetuses. Sites of specific, high affinity binding (IC₅₀ 3 to 5 nM) were detected as patches in the anterior lobe and at the junction between the anterior and neural lobes. The neural lobe was not labelled. Immunocytochemical studies performed on human pituitary tissue showed that [³H]arginine vasopressin only marked zones which correspond to areas rich in cells immunoreactive to adrenocorticotrophin. We conclude that in the human pituitary, corticotrophs bear vasopressin binding sites. Since non-radioactive synthetic structural analogues of vasopressin acting as V₁ and V₂ agonists or antagonists failed to compete for binding of radioligand in the human pituitary, while a V₁-type agonist displaced [³H]arginine vasopressin binding in the rat pituitary, we postulate that binding sites in the human pituitary may differ from the previously known vasopressin receptors.     

Neural Regulation of Corticotropin Releasing Hormone (CRH) and CRH Receptor mRNA in the Hypothalamic Paraventricular Nucleus in the Rat

The role of afferent innervation to the hypothalamic paraventricular nucleus (PVN) on CRH mRNA and CRH receptor mRNA levels was studied in control and stressed rats. Groups of rats were subjected to unilateral transection of the stria terminalis (ST), the medial forebrain bundle at the rostral hypothalamic level (RMFB), or the lower brainstem through the medulla oblongata between the obex and the locus coeruleus (CBs). Twelve days after surgery, each group of rats was further divided into controls (basal conditions) and stressed (1 h immobilization), before collecting brains for mRNA analysis by in situ hybridization histochemistry. While ST and RMFB cuts had no effect on basal CRH mRNA levels in the PVN, CBs cut decreased CRH mRNA in the PVN ipsilaterally to the knife cut but it was without effect on the contralateral side (– 40% and –37%vs contralateral and sham-operated, respectively, P&0.01). Acute stress (rats were killed 3 h after immobilization) increased CRH mRNA levels by about 30% bilaterally, an effect which was unchanged by any of the three hemisections. Under basal conditions, CRH receptor mRNA levels in the PVN were indistinguishable from the surrounding areas in sham-operated controls, ST and RMFB operated rats. However, brainstem hemisection resulted in clear expression of CRH receptor mRNA in areas consistent with the dorsal, medial-ventral and lateral parvicellular subdivisions of the PVN, ipsilateral to the transection. CRH neurons in these subdivisions project to the lower brainstem and the spinal cord. Expression of CRH receptor mRNA in the medial-dorsal and anterior parvicellular divisions (CRH neurons with median eminence projections) was not affected by CBs cut. In these subdivisions, immobilization stress markedly increased CRH receptor mRNA levels but it did not influence CBs cut-induced CRH receptor expression. ST and RMFB hemisections were without effect on PVN CRH receptor mRNA levels under basal or stress conditions. Oxytocin (OT) and vasopressin (VP) mRNA levels in the magnocellular subdivision of the PVN were unchanged after immobilization, or following ST, RMFB or CBs cuts, whereas OT mRNA in the medial-ventral and caudal parvicellular subdivisions was decreased by 52% after CBs cut. The data demonstrate that: 1) basal CRH mRNA levels in the PVN are under tonic stimulatory influence of the lower brainstem (and/or spinal cord) afferents; 2) CRH receptor mRNA expression in PVN subdivisions (pituitary vs lower brainstem/spinal cord projecting neurons) is under different control mechanisms, and 3) immobilization-induced changes in CRH mRNA and CRH receptor mRNA levels are mediated either by neural inputs from brain areas other than those investigated here, or by humoral factors.     

Distribution of Corticotrophin‐Releasing Factor Type 1 Receptor‐Like Immunoreactivity in the Rat Pituitary

Corticotrophin‐releasing factor (CRF) regulates the hypothalamic‐pituitary‐adrenal axis response to stress through its type 1 receptor (CRF₁) in the corticotrophs of the anterior pituitary. Although CRF₁ mRNA expression has been confirmed in the rat pituitary, the distribution pattern of CRF₁ protein in the pituitary has not been reported. Therefore, we generated an antiserum against the amino acid fragment corresponding to the 177–188 sequence of the first extracellular loop of the rat CRF₁. Using the antiserum, CRF₁‐like immunoreactivity (CRF₁‐LI) was detected in the anterior lobe cells of the rat pituitary where some of them expressed intense signals. CRF₁‐LI also appeared in the intermediate lobe cells and on the fibre‐like elements of the posterior lobe of the pituitary. Dual immunofluorescence labelling showed that corticotrophs exhibited the highest percentage of CRF₁ (male: 27.1 ± 3.0%, female: 18.0 ± 3.0%), followed by lactotrophs (male: 6.7 ± 3.0%, female: 12.1 ± 1.3%), gonadotrophs (male: 2.6 ± 1.0%, female: 7.5 ± 0.5%), thyrotrophs (male: 2.9 ± 0.1%, female: 5.3 ± 1.2%) and somatotrophs (male: 1.1 ± 0.3%, female: 1.2 ± 0.5%). The percentage of CRF₁‐LI‐positive cells that were corticotrophs was significantly higher in male rats than in female rats, whereas CRF₁‐LI‐positive lactotrophs and gonadotrophs were significantly higher in female rats than in male rats. Almost all of the melanotrophs were positive for CRF₁ in the intermediate lobe (98.9 ± 0.2%). CRF₁‐LI and the percentage of CRF₁‐LI in corticotrophs were decreased in the anterior pituitary, and the distribution patterns were altered from a diffuse to punctate one by adrenalectomy; the changes were restored by treatment with dexamethasone (100 μg/kg bw). These results suggest that CRF₁ is involved in the modulation of the functions of the pituitary; moreover, protein expression and the distribution patterns of CRF₁ are regulated by glucocorticoids in the rat anterior pituitary.