COX-2 disruption leads to increased central vasopressin stores and impaired urine concentrating ability in mice.

It was hypothesized that cyclooxygenase-2 (COX-2) activity promotes urine concentrating ability through stimulation of vasopressin (AVP) release after water deprivation (WD). COX-2-deficient (COX-2(-/-), C57BL/6) and wild-type (WT) mice were water deprived for 24 h, and water balance, central AVP mRNA and peptide level, AVP plasma concentration, and AVP-regulated renal transport protein abundances were measured. In male COX-2(-/-), basal urine output and water intake were elevated while urine osmolality was decreased compared with WT. Water deprivation resulted in lower urine osmolality, higher plasma osmolality in COX-2(-/-) mice irrespective of gender. Hypothalamic AVP mRNA level increased and was unchanged between COX-2(-/-) and WT after WD. AVP peptide content was higher in COX-2(-/-) compared with WT. At baseline, plasma AVP concentration was elevated in conscious chronically catheterized COX-2(-/-) mice, but after WD plasma AVP was unchanged between COX-2(-/-) and WT mice (43 ± 11 vs. 70 ± 16 pg/ml). Renal V2 receptor abundance was downregulated in COX-2(-/-) mice. Medullary interstitial osmolality increased and did not differ between COX-2(-/-) and WT after WD. Aquaporin-2 (AQP2; cortex-outer medulla), AQP3 (all regions), and UT-A1 (inner medulla) protein abundances were elevated in COX-2(-/-) at baseline and further increased after WD. COX-2(-/-) mice had elevated plasma urea and creatinine and accumulation of small subcapsular glomeruli. In conclusion, hypothalamic COX-2 activity is not necessary for enhanced AVP expression and secretion in response to water deprivation. Renal medullary COX-2 activity negatively regulates AQP2 and -3. The urine concentrating defect in COX-2(-/-) is likely caused by developmental glomerular injury and not dysregulation of AVP or collecting duct aquaporins.

Effects of water restriction on gene expression in mouse renal medulla: identification of 3betaHSD4 as a collecting duct protein.

To identify novel gene targets of vasopressin regulation in the renal medulla, we performed a cDNA microarray study on the inner medullary tissue of mice following a 48-h water restriction protocol. In this study, 4,625 genes of the possible approximately 12,000 genes on the array were included in the analysis, and of these 157 transcripts were increased and 63 transcripts were decreased by 1.5-fold or more. Quantitative, real-time PCR measurements confirmed the increases seen for 12 selected transcripts, and the decreases were confirmed for 7 transcripts. In addition, we measured transcript abundance for many renal collecting duct proteins that were not represented on the array; aquaporin-2 (AQP2), AQP3, Pax-8, and alpha- and beta-Na-K-ATPase subunits were all significantly increased in abundance; the beta- and gamma-subunits of ENaC and the vasopressin type 1A receptor were significantly decreased. To correlate changes in mRNA expression with changes in protein expression, we carried out quantitative immunoblotting. For most of the genes examined, changes in mRNA abundances were not associated with concomitant protein abundance changes; however, AQP2 transcript abundance and protein abundance did correlate. Surprisingly, aldolase B transcript abundance was increased but protein abundance was decreased following 48 h of water restriction. Several transcripts identified by microarray were novel with respect to their expression in mouse renal medullary tissues. The steroid hormone enzyme 3beta-hydroxysteroid dehydrogenase 4 (3betaHSD4) was identified as a novel target of vasopressin regulation, and via dual labeling immunofluorescence we colocalized the expression of this protein to AQP2-expressing collecting ducts of the kidney. These studies have identified several transcripts whose abundances are regulated in mouse inner medulla in response to an increase in endogenous vasopressin levels and could play roles in the regulation of salt and water excretion.

Altered plasma and pituitary arginine vasotocin and hypothalamic provasotocin expression in flounder (Platichthys flesus) following hypertonic challenge and distribution of vasotocin receptors within the kidney.

Plasma AVT concentration, pituitary AVT content, hypothalamic provasotocin mRNA expression and other osmoregulatory parameters were measured in euryhaline flounder 4, 8, and 24 h after the hypertonic challenge of transfer from fresh water (FW) to seawater (SW). Osmolality and the concentration of major plasma ions, sodium and chloride, were significantly higher in fish transferred to SW by comparison with time matched controls, an effect evident within 4 h. By comparison with time matched controls, pituitary store of AVT was lower while plasma AVT concentration was higher 8 and 24 h after transfer to SW. Higher provasotocin mRNA expression in the hypothalamus was also seen at 4 and 8 h in flounder transferred from FW to SW compared with time matched controls. The lower pituitary store and higher circulating levels imply substantial AVT secretion occurs in the early phase response to this hypertonic challenge. Changes in the regulation of AVT synthesis and secretion appeared quickly following movement to SW, consistent with the rapid osmoregulatory response, including reduced urine production that fish require to accommodate the dehydrative water losses and salt loading on exposure to the new hyperosmotic environment. qPCR measures of whole kidney vasotocin receptor mRNA expression indicated similar levels in SW and FW. Immunohistochemistry for the vasotocin receptor in flounder kidney showed localisation on the afferent and efferent arterioles of the glomerulus and on the capillary bed that extends from the efferent arteriole to the smooth muscle surrounding the collecting duct. Localisation of the vasotocin receptor was comparable in SW and FW fish.

Hypothalamic vasopressin gene expression increases in both males and females postpartum in a biparental rodent.

In previous studies, the closely related neuropeptide hormones oxytocin and vasopressin have been implicated in the central mediation of parental behaviour. Several studies in rats and sheep have demonstrated a role for oxytocin in the initiation of maternal behaviour. Recently, a few studies in a biparental species, the prairie vole (Microxytocinus ochrogaster) have suggested that vasopressin is important for paternal care. The present study investigated this latter possibility by measuring changes in vasopressin and oxytocin hypothalamic gene expression 1 day and 6 days following parturition in prairie voles which show paternal care and in montane voles (M. montanus) which lack paternal care. In prairie voles, vasopressin gene expression increased in both males and females postpartum, relative to sexually naive controls. In the non-paternal montane vole, no change in vasopressin gene expression was observed in either sex. In contrast to this species difference in vasopressin gene expression, hypothalamic oxytocin gene expression increased in both prairie and montane vole females, but not in males of either species. To augment measures of gene expression, we assessed vasopressin (V1a) and oxytocin receptor binding in both species. Although forebrain vasopressin V1a receptor binding was not altered following parturition in either species, oxytocin receptor binding increased in the ventromedial nucleus of the hypothalamus in females, but not males, in both prairie and montane voles. In summary, vasopressin gene expression increases in both males and females postpartum in a biparental species and oxytocin gene expression and receptor binding increase selectively in females. These results are consistent with earlier reports of a role for vasopressin in paternal care and for oxytocin in maternal behaviour.

Vasopressin receptors in the mouse (Mus musculus) brain: sex-related expression in the medial preoptic area and hypothalamus.

We have investigated the distribution of vasopressin binding sites in the brain of male and female adult mice using a radio-iodinated ligand and film autoradiography. Vasopressin receptors were uncovered in various regions of the brain including the basal nucleus of Meynert, the substantia innominata, the hypothalamic paraventricular nucleus, the substantia nigra pars compacta and the hypoglossal nucleus. A sex-related difference in the expression of vasopressin receptors was seen in the medial preoptic area/anterior hypothalamus corresponding to the rat sexually dimorphic nucleus in the rat and in the hypothalamic mammillary nuclei. In both structures the autoradiographic labeling is more intense in females than in males. These observations confirm that vasopressin binding sites are present in the hypothalamic preoptic area of most species examined so far and that sex-related expression of neuropeptide receptors could trigger sex-related behavioral differences.

Expression cloning of an AVP-activated, calcium-mobilizing receptor from rabbit kidney medulla.

Arginine vasopressin (AVP) is a nonapeptide that regulates body fluid and blood pressure homeostasis. We have used expression cloning in the Xenopus laevis oocyte system to identify cDNA clones from a rabbit renal medullary expression library encoding an AVP receptor linked to Ca2+ mobilization. cRNA generated from positive clones conferred upon oocytes the capacity to mobilize intracellular Ca2+ in response to AVP. A cDNA clone encoding a protein of 780 amino acids was isolated, sequenced, and subcloned into an SV40-based expression vector. Expression of the cloned protein [designated the vasopressin-activated, calcium-mobilizing (VACM-1) protein] in COS-1 cells, resulted in increased 125I-labeled AVP binding [dissociation constant (Kd) of approximately 2 nM] and increased AVP-induced mobilization of Ca2+. Importantly, 125I-AVP could be immunoprecipitated both from detergent-solubilized membranes from COS-1 cells expressing VACM-1 protein and from an in vitro translation system, in which VACM-1 protein was synthesized, using antibodies prepared against a synthetic peptide derived from the NH2-terminal sequence of VACM-1. Interestingly, immunohistochemical staining of rabbit kidney sections with this antibody showed specific staining of collecting tubule epithelia. The deduced amino acid sequence is not homologous with any nucleic acid or amino acid sequences reported to date, including those of the V1 and V2 AVP receptors. The VACM-1 protein may represent a novel AVP receptor.

Cellular localization of vasopressin V1a receptor messenger ribonucleic acid in adult male rat brain, pineal, and brain vasculature.

Vasopressin V1a receptor (V1aR) transcripts were localized in brain, pineal, and superficial brain vascular tissues of adult male rats using hybridization histochemistry and an [35S]riboprobe complementary to the messenger ribonucleic acid (mRNA) encoding the fifth to the midseventh transmembrane regions of the receptor. V1aR mRNA was extensively distributed throughout brain and was expressed in 1) superficial cells of the granule cell layers of the main olfactory bulb, hippocampal dentate gyrus, and cerebellum; 2) numerous anatomically distinct brain nuclei; 3) isolated cells dispersed throughout the central nervous system; 4) cells of the choroid plexus, occasional blood vessels in the olfactory bulb and interpeduncular nucleus, and extraparenchymal intracranial vasculature; and 5) some white matter structures. Numerous cells expressing V1aR transcripts were found in forebrain structures, including primary olfactory (piriform) cortex, the anterior and posterior olfactory nuclei; dorsal, intermediate, and ventral lateral septal nuclei; the septo-fimbrial nucleus and accumbens nucleus; and numerous hypothalamic regions with the most intense hypothalamic labeling in the arcuate, stigmoid, suprachiasmatic, and periventricular nuclei and the lateral hypothalamic area. Cells expressing V1aR transcripts were ubiquitous throughout the midbrain, pontine, and medullary regions. A lower intensity signal was found in cells of the parvocellular paraventricular and anteroventral nucleus of the thalamus, circumventricular organs including the pineal, and the subfornical organ. V1aR transcripts were not generally detected in parenchymal vasculature, but could be found over large blood vessels in the interpeduncular nucleus and medial olfactory bulb; transcripts were commonly detected in perivascular brain cells. V1aR mRNA was abundantly expressed by choroid plexus, endothelial cells of midline blood vessels between the main olfactory bulbs, and superficial vascular tissue on all brain surfaces. These data confirm the presence of the vascular/hepatic-type V1aR gene in brain tissue and document an extensive expression. The distribution of V1aR mRNA suggests that there are at least two types of vasopressin-responsive cells in brain: one type exemplified by lateral septal ara neurons innervated by classical axodendritic/somatic synaptic vasopressinergic terminals and a second, perivascular/vascular type that would facilitate humoral vasopressinergic signaling in the brain.

Androgen manipulation and vasopressin binding in the rat brain and peripheral organs.

It is now widely recognized that there is a sexual dimorphism in the development of arginine vasopressin (AVP) immunoreactivity in certain parts of the brain, and that changes in brain AVP immunoreactivity change with manipulation of androgen status. The aim of this experiment was to determine specifically any AVP receptor changes in response to manipulation of androgen levels using a selective V1 antagonist radioligand. Following castration, plasma testosterone levels fell and AVP immunoreactivity was reduced in the lateral septum and bed nucleus of the stria terminalis. With testosterone supplementation in castrated animals, the immunoreactivity in these regions was restored to a higher degree than in sham-operated animals. Central and peripheral V1 AVP receptor binding (as determined using the selective AVP V1 antagonist radioligand [125I](d(CH2)5,sarcosine7)AVP was not changed in any of the brain regions studied or in liver or kidney membranes from the three groups. This study demonstrates that there is no change in brain AVP receptor binding despite changes in regional AVP immunoreactivity in the brain, and excludes any confounding interaction with changes in oxytocin receptors.