Arginine Vasopressin Gene Expression Research Articles

Identification of a Functional AP1 Element in the Rat Vasopressin Gene Promoter

Arginine vasopressin (AVP) is expressed in paraventricular, supraoptic, and suprachiasmatic nuclei of the hypothalamus, where transcription of the AVP gene is activated by various forms of stress, such as hyperosmolality, inflammation, and photic stimulation. In vasopressinergic neurons, the expression of the Fos/Jun family proteins is known to be rapidly induced after these stimuli as well. However, it is still unknown whether these proteins actually mediate AVP gene expression. In this study we examined in vitro the role of Fos/Jun protein in transcriptional regulation of the AVP gene using the BE(2)M17 neuroblastoma cell line. We found that 5'-promoter activity of the rat AVP gene (-803/+26) markedly increased when all combinations of the Fos/Jun family proteins were overexpressed. Coexpression of the cAMP-responsive element-binding protein-binding protein and steroid receptor coactivator-1a further enhanced the Fos/Jun-mediated transcription. Using site-directed mutagenesis and EMSA techniques, we identified an activation protein 1 (AP1)-like element (-134/-128; TGAATCA) in the AVP gene 5'-promoter region, which is the sole responsible site for the Fos/Jun-mediated transcription. We also found that 12-O-tetradecarbonyl phorbol 13-acetate stimulates AVP gene transcription partly via the AP1 site through the activation of ERK signaling. Together, these results suggest that a variety of Fos/Jun family member proteins stimulate transcription of the AVP gene through the AP1 site we identified. Furthermore, this effect may be activated by both protein kinase A and protein kinase C signaling pathways.

Increased Arginine Vasopressin mRNA Expression in the Human Hypothalamus in Depression: A Preliminary Report

Elevated arginine vasopressin (AVP) plasma levels have been observed in major depression, particularly in relation to the melancholic subtype. Two hypothalamic structures produce plasma vasopressin: the supraoptic nucleus (SON) and the paraventricular nucleus (PVN). The aim of this study was to establish which structure is responsible for the increased vasopressin plasma levels in depression. Using in situ hybridization, we determined the amount of vasopressin messenger ribonucleic acid (mRNA) in the PVN and SON in postmortem brain tissue of nine depressed subjects (six with the melancholic subtype) and eight control subjects. In the SON, a 60% increase of vasopressin mRNA expression was found in depressed compared with control subjects. In the melancholic subgroup, AVP mRNA expression was significantly increased in both the SON and the PVN compared with control subjects. We found increased AVP gene expression in the SON in depressed subjects. This might partly explain the observed increased vasopressin levels in depression.

Amygdalar vasopressin mRNA increases in acute cocaine withdrawal: Evidence for opioid receptor modulation

In humans, stress is recognized as a major factor contributing to relapse to drug abuse in abstinent individuals; drugs of abuse themselves or withdrawal from such drugs act as stressors. In the animals, evidence suggests that centrally released arginine vasopressin in both amygdala and hypothalamus plays an important role in stress-related anxiogenic behaviors. The stress responsive hypothalamic–pituitary–adrenal axis is under tonic inhibition via endogenous opioids, and cocaine withdrawal stimulates hypothalamic–pituitary–adrenal activity. The present studies were undertaken to determine whether: (1) 14-day (chronic) “binge” pattern cocaine administration (45mg/kg/day) or its withdrawal for 3 h (acute), 1 day (subacute) or 10 days (chronic) alters arginine vasopressin mRNA levels in amygdala or hypothalamus; (2) the opioid receptor antagonist naloxone (1mg/kg) alters arginine vasopressin mRNA or hypothalamic–pituitary–adrenal hormonal responses in acute cocaine withdrawal; and (3) there are associated changes of mu opioid receptor or proopiomelanocortin mRNA levels. In amygdala, arginine vasopressin mRNA levels were unchanged after chronic “binge” cocaine, but were increased during acute cocaine withdrawal. Naloxone completely blocked this increase. Neither chronic cocaine nor its acute withdrawal altered amygdalar mu opioid receptor mRNA levels. The increase in amygdalar arginine vasopressin mRNA levels was still observed after subacute withdrawal, but not after chronic withdrawal. Although hypothalamic–pituitary–adrenal tolerance developed with chronic “binge” cocaine, there were modestly elevated plasma adrenocorticotropin hormone levels during acute withdrawal. While naloxone produced modest adrenocorticotropin hormone elevations in cocaine-naïve rats, naloxone failed to elicit an adrenocorticotropin hormone response in cocaine-withdrawn rats. In hypothalamus, neither chronic cocaine nor acute withdrawal altered arginine vasopressin, proopiomelanocortin or mu opioid receptor mRNA levels. These results show that: (1) opioid receptors mediate increased amygdalar arginine vasopressin gene expression during acute cocaine withdrawal, and (2) cocaine withdrawal renders the hypothalamic–pituitary–adrenal axis insensitive to naloxone. Our findings suggest a potential role for amygdalar arginine vasopressin in the aversive consequences of early cocaine withdrawal.

Regulation of Vasopressin Gene Expression by cAMP and Glucocorticoids in Parvocellular Neurons of the Paraventricular Nucleus in Rat Hypothalamic Organotypic Cultures

Arginine vasopressin (AVP) in the parvocellular neurons of the paraventricular nucleus (PVN) is known to play an important role in the hypothalamo-pituitary-adrenal axis. In the present study, we examined how cAMP and glucocorticoids regulate AVP gene expression in the parvocellular neurons of the PVN in rat hypothalamic organotypic cultures with in situ hybridization. AVP heteronuclear (hn) RNA, an indicator for gene transcription, was induced in the PVN with incubation of forskolin as reported previously, and AVP mRNA was increased by forskolin in the presence of the gene transcription inhibitor 5,6-dichloro-1-D-ribofuranosylbenzimidazole (DRB). These data indicate that cAMP could increase not only gene transcription but also mRNA stability. Dexamethasone treatment, in contrast, significantly decreased AVP mRNA expression levels in the PVN, but this inhibitory action was abolished in the presence of DRB or the sodium channel blocker tetrodotoxin (TTX). However, when the hypothalamic slices were treated with forskolin, dexamethasone decreased AVP mRNA expression even in the presence of DRB and/or TTX. Furthermore, AVP hnRNA expression induced by forskolin was attenuated by dexamethasone treatment in the presence of TTX. These data indicate that dexamethasone could act on AVP cells independently of action potentials to decrease mRNA stability and to suppress AVP gene transcription during stimulation by cAMP. Thus, it was demonstrated that: (1) cAMP upregulates AVP gene transcriptionally and post-transcriptionally, (2) the mode of action of glucocorticoids was dependent on whether the cells were stimulated by cAMP, and (3) the interactions between cAMP and glucocorticoids encompass both gene transcription and mRNA stability.

Vasopressin receptor expression in the placenta.

The arginine vasopressin (AVP) type 1a receptor (V1a) is well known to mediate vasoconstriction. In pregnancy, blood flow in the placenta is crucial for sustaining normal growth and development of the fetus. This is the first AVP receptor study in the placenta and fetal membranes. The aim was to compare, quantitatively, the level of V1a gene expression with that of a known marker for vascularization, aquaporin 1 (AQP1). V1a and AQP1 gene expression did not correlate; placental V1a mRNA levels were significantly upregulated at 45 and 66+/-1 compared with 27, 100+/-4, and 140 days (term approximately 150 days). V1a mRNA levels were much lower in fetal membranes in which no significant difference across gestation was observed. In situ hybridization histochemistry localized V1a gene expression in the maternal component of the placenta similar to the receptor-binding studies using 125I-labeled [d(CH2)5, sarcosine7] vasopressin. No AVP gene expression was observed in the placenta and fetal membranes, which eliminates local AVP production. This increase in V1a expression at 45 and 66+/-1 days of gestation correlates with the period of maximal placental growth in the sheep and suggests that AVP and V1a receptors may play a hitherto unrecognized role in placental growth, differentiation, and/or function, particularly in the deleterious effects of heat stress, early in pregnancy, on fetal growth.

Prolonged prenatal hypernatremia alters neuroendocrine and electrolyte homeostasis in neonatal sheep.

Arginine vasopressin (AVP) is a neuroendocrine hormone synthesized in the hypothalamus, and is stored and secreted by the posterior pituitary gland in response to stimuli such as plasma hypertonicity and hypotension. The primary physiologic roles of AVP include plasma osmolality and blood pressure regulation. We have previously demonstrated that chronic prenatal plasma hypertonicity alters the AVP regulatory pathway in newborn lambs. The objectives of the present study were to evaluate prolonged effects of antenatal plasma hypertonicity on neonatal plasma osmoregulation. Pregnant ewes at 119 +/- 3 days of gestation were water restricted to achieve and maintain hypertonicity until normal-term delivery. After delivery, ewes were provided food and water ad libitum and lambs were allowed maternal nursing. At the age of 28 days, blood samples were obtained for the analysis of plasma osmolality, electrolytes, and AVP levels from study (n= 5) and age-matched control (n= 6) lambs. Subsequently, lambs were euthanized, and the pituitary and hypothalamus were processed for the determination of pituitary AVP content by radioimmunoassay, and AVP gene expression by Northern analysis. In response to water restriction, maternal plasma osmolality significantly increased (306 +/- 1.1 to 326 +/- 1.2 mOsm/kg, P< 0.001). At the age of 28 days, plasma sodium level was higher in study (prenatally dehydrated) than control lambs (144.6 +/- 0.4 vs 142.6 +/- 0.3,P< 0.05). Study lambs had higher plasma AVP concentrations than the control lambs (4.1 +/- 0.4 vs 1.7 +/- 0.4 pg/ml,P< 0.05). Similarly, total pituitary AVP content was higher in thein utero hypertonic lambs than in the control lambs (6.5 +/- 1.0 vs 2.8 +/-1.2 microg, P< 0.05). However, there was no difference in hypothalamic AVP mRNA levels between the two groups. The present study demonstrates that chronic maternal and fetal plasma hypertonicity has prolonged effects on pituitary and plasma AVP, as well as plasma sodium in neonatal lambs, providing further evidence suggesting prenatal imprinting of osmoregulation through at least 1 month of age.

Estrogen receptor-β regulates transcript levels for oxytocin and arginine vasopressin in the hypothalamic paraventricular nucleus of male mice

Estrogen receptor (ER)-β, unlike ER-α, is localized in the hypothalamic paraventricular nucleus (PVN) which also contains neuropeptide synthesizing neurons, such as oxytocin (OT), arginine vasopressin (AVP) and corticotropin-releasing hormone (CRH). Although it is known that some ER-β containing neurons co-express OT and AVP, but not CRH, in the PVN, it is not yet determined whether ER-β activation may indeed play a role in estrogenic regulation on syntheses of these neuropeptides. In the present study, we tested this hypothesis by comparing the effects of estrogen on the levels of OT, AVP and CRH messenger RNA (mRNA) between ER-β knockout (βERKO) and wild type (WT) control male mice. Mice were gonadectomized and implanted with either a pellet containing estradiol benzoate (2.5–5.0 μg/day) or a placebo pellet for 21 days. In situ hybridization histochemistry revealed that estrogen treatment resulted in a significant increase in OT transcripts (151.6±6.0%) and a decrease in AVP transcripts (77.8±5.2%) in the PVN of WT mice, compared to the placebo control group. This estrogenic regulation of OT and AVP mRNA levels in the PVN was completely abolished in βERKO mice. Similar genotype differences in the effects of estrogen on the numbers of OT- and AVP-containing cells were found in immunocytochemical studies performed in a separate set of mice. On the other hand, the expression of CRH mRNA in the PVN was not affected by estrogen treatment in either WT or βERKO mice. Furthermore, estrogen did not cause any changes in the levels of OT or AVP mRNA, regardless of genotype, in the supraoptic nucleus where, unlike in rats, ER-β containing neurons are rarely found in mice. Finally, estrogen significantly increased OT mRNA levels in both βERKO and WT in the medial preoptic area, which contains both ER-α and ER-β. These results suggest that ER-β activation may play a critical role in estrogenic regulation of OT and AVP gene expression in the PVN.

Upregulation of CRH gene expression and downregulation of arginine vasopressin gene expression in the hypothalamus of bilateral nephrectomized rats

The expression of the corticotropin-releasing hormone (CRH) gene and the arginine vasopressin (AVP) gene in the hypothalamus examined in bilateral nephrectomized rats by in situ hybridization histochemistry. The expression of the CRH gene was significantly increased in the parvocellular part of the paraventricular nucleus (PVN) 12 and 20 h after bilateral nephrectomy in comparison with that after sham operation. The plasma concentration of adrenocorticotropic hormone (ACTH) in nephrectomized rats was significantly higher than that in sham operated rats 20 h after surgery. In contrast, the expression of the AVP gene in both the parvocellular and magnocellular parts of the PVN and throughout the supraoptic nucleus (SON) was significantly decreased 20 h after bilateral nephrectomy in comparison with that after sham operation. These results suggest that nephrectomy-induced upregulation of the CRH gene with elevation of plasma ACTH may be due to the activation of the hypothalamo-pituitary adrenal (HPA) axis.

Neuronal activity is required for the circadian rhythm of vasopressin gene transcription in the suprachiasmatic nucleus in vitro.

Arginine vasopressin (AVP) is synthesized in and secreted by the suprachiasmatic nucleus (SCN) in a circadian pattern. Transcription of the AVP gene in organotypic cultures of rat SCN was studied by using an intronic in situ hybridization. AVP gene transcription in the cultured SCN maintained a daily rhythm with a peak in the daytime. Inhibition of spontaneous activity by the sodium channel blocker, tetrodotoxin (TTX), dramatically decreased AVP heteronuclear RNA levels and suppressed rhythmicity, indicating that ongoing neural activity was required for the AVP gene transcription. In the presence of TTX, the adenylate cyclase stimulator, forskolin, increased AVP transcription in the SCN. In contrast, the protein kinase C activator, phorbol 12-myristate 13-acetate, greatly increased AVP transcription in the absence of TTX, but this effect was blocked by TTX, indicating that the phorbol 12-myristate 13-acetate acted indirectly via synaptic input. Neither protein kinase A nor protein kinase C pathways appear to be involved in the rhythmicity of AVP transcription in the SCN because selective inhibitors of these protein kinases were without effect. In contrast, the MAPK pathway inhibitor, PD98059, profoundly decreased AVP transcription and abolished its daily rhythm. Hence, a functional MAPK signaling pathway appears to be critical for AVP gene expression in the SCN.

Chronic in utero plasma hyperosmolality alters hypothalamic arginine vasopressin synthesis and pituitary arginine vasopressin content in newborn lambs

Objective: Arginine vasopressin is synthesized in the hypothalamus and secreted by the posterior pituitary gland in response to plasma hypertonicity. Previous studies suggest that in utero and neonatal exposure of rat pups to prolonged alterations of plasma osmolality may permanently alter (imprint) arginine vasopressin synthesis and secretion, thus adult responses to osmotic challenges. Little is known, however, of the potential for imprinting of neuroendocrinologic systems in precocial species. In view of the frequent occurrence of altered maternal and fetal plasma tonicity (eg, maternal dehydration, hyperemesis), we sought to determine the effect of prolonged maternal hypertonicity on arginine vasopressin gene expression and pituitary gland content in neonatal sheep. Study Design: Pregnant ewes at 119 ± 3 days of gestation were water restricted to achieve and maintain plasma hypertonicity (10-20 mOsm/kg above baseline level) until normal term delivery. Newborns were provided maternal nursing ad libitum. Within 24 hours after birth, study neonatal lambs (n = 6) and age-matched control neonatal lambs (n = 5) were killed, and the pituitary gland and hypothalamus were removed and frozen immediately. Pituitary arginine vasopressin content was determined by radioimmunoassay, and hypothalamic arginine vasopressin gene expression was quantified with Northern blot. Differences in pituitary arginine vasopressin content and hypothalamic arginine vasopressin gene expression (arginine vasopressin/ β-actin ratio) between study and control newborn lambs were analyzed by unpaired t test. Results: In response to maternal water restriction, maternal plasma osmolality increased from 307 ± 0.9 mOsm/kg to 325 ± 1.3 mOsm/kg, and plasma sodium increased from 147 ± 1.3 mEq/L to 156 ± 1.2 mEq/L. The maternal plasma hyperosmolality and hypernatremia were maintained until normal term delivery. At the time of death, study (in utero dehydrated) lambs had higher plasma sodium (150 ± 0.4 mEq/L vs 146.5 ± 1.5 mEq/L; P <.05) and chloride (112.8 ± 1.0 mEq/L vs 108.5 ± 1.5 mEq/L; P <.05) levels, and lower potassium (4.5 ± 0.2 mEq/L vs 5.5 ± 0.3 mEq/L; P <.05) concentrations than control newborn lambs. Both newborn groups had similar plasma osmolality (320.0 ± 1.3 mOsm/kg vs 318.0 ± 3.4 mOsm/kg). Total pituitary arginine vasopressin content was significantly greater in the study than in the control newborn lambs (8.3 ± 2.8 μg vs 1.6 ± 1.3 μg; P <.05). Conversely, hypothalamic arginine vasopressin messenger RNA levels were lower in the study newborn lambs than in the control newborn lambs (arginine vasopressin/β-actin ratio: 0.29 ± 0.01 vs 0.68 ± 0.15; P <.05). Conclusion: Despite the presence of plasma hypernatremia, prolonged elevation of fetal plasma tonicity increases newborn pituitary arginine vasopressin content yet decreases hypothalamic arginine vasopressin gene expression. The present study suggests that prolonged prenatal exposure to plasma hypertonicity may imprint the hypothalamic-pituitary arginine vasopressin regulatory system. (Am J Obstet Gynecol 2002;187:191-6.)

Thyroxine modulates corticotropin-releasing factor but not arginine vasopressin gene expression in the hypothalamic paraventricular nucleus of the developing Rat.

Neonatal rats were daily injected with 100 microg/kg T4 and killed at 4, 8 or 15 days. Circulating corticosterone and corticosteroid binding globulin concentrations increased in 8- and 15-day-old rats after T4 treatment. Plasma adrenocorticotropic hormone (ACTH) concentrations, pituitary ACTH content and pro-opiomelanocortin mRNA expression were unaffected in T4-treated rats. T4 treatment induced an increase in corticotropin-releasing factor (CRF) mRNA expression in the whole population of CRF synthesizing cells of the paraventricular nucleus (PVN) that became significant at day 8 and disappeared at day 15. Double labelling in situ hybridization revealed that CRF gene expression in the CRF+/arginine vasopressin (AVP)+ subpopulation was increased at days 4 and 8 and decreased at day 15. CRF immunoreactivity in the zona externa of the median eminence increased with age but was not affected by the experimental hyperthyroidism. The degree of CRF and AVP colocalization, the concentration of AVP mRNA in the parvo and magnocellular cell bodies of the PVN and the density of immunoreactive AVP in the zona interna or zona externa of the median eminence did not change after T4 treatment. Our data demonstrate that experimental hyperthyroidism accelerates the maturation of hypothalamic CRF gene expression, including in particular in the CRF+/AVP+ subpopulation, during the stress hyporesponsive period. These observations suggest that the physiological peak of plasma thyroxine that occurs between days 8-12 may participate in the maturation of hypothalamic CRF cells.

Corticotropin-releasing hormone and arginine vasopressin: mRNA and secretion are differentially regulated according to the pattern of exposure to noradrenaline in rat hypothalamic neurones.

Hypothalamic corticotropin-releasing hormone (CRH) and arginine vasopressin (AVP) are secreted from the median eminence in a pulsatile manner and regulated by noradrenaline during stress. This study investigated the effect of pulsatile noraderanaline on CRH/AVP mRNAs and secretion. Foetal hypothalamic neurones were cultured on plastic coverslips, inserted into perifusion chambers and noraderanaline pulses given at various doses or pulse intervals for 24 h. CRH and AVP release rose in a dose dependant manner; however, maximal increases in mRNAs were seen with an intermediate noraderanaline pulse dose. The effect of noraderanaline pulse frequency was determined by giving noraderanaline pulses at intervals of 15-120 min vs continuous noraderanaline. Both pulsatile and continuous noraderanaline increased CRH and AVP release, but secretion was reduced after 22 h of treatment in the continuous noraderanaline and rapid pulse groups. CRH mRNA levels were maximally increased by medium interval pulses and AVP mRNA by rapid interval pulses. Neither CRH nor AVP mRNAs were stimulated by continuous noraderanaline. To determine noraderanaline specificity, pulses of veratridine (VER; 15-120 min intervals) vs continuous VER were examined. Only pulsatile VER increased CRH and AVP mRNAs, with maximal effects seen with the 60 min pulse interval for both. Thus, noraderanaline pulse pattern regulates CRH and AVP gene expression in both a coordinate and differential manner. Since noraderanaline plays an important role during stress, the pattern of noraderanaline signals may be critical to the observed changes in CRH and AVP expression.

Increased Colocalization of Corticotropin‐Releasing Factor and Arginine Vasopressin in Paraventricular Neurones of the Hypothalamus in Lactating Rats: Evidence from Immunotargeted Lesions and Immunohistochemistry

AbstractIn lactating female rats, tonically elevated glucocorticoid secretion is accompanied by blunted stress responsiveness, reduced expression of hypothalamic corticotropin‐releasing factor (CRF) mRNA and modest increases in arginine vasopressin (AVP) expression in the paraventricular nucleus (PVN). To determine the relative contribution of CRF and AVP to parvocellular function, we performed selective CRF (CRF‐Tx) or AVP (AVP‐Tx) lesions in the PVN neurones of ovariectomized virgin or lactating females (day 2 of lactation) by using ricin A associated with monoclonal antibodies directed towards CRF or AVP. We also performed double immunohistochemical labelling of CRF and AVP in the PVN of control rats injected with immunoglobulin (Ig)Gs associated with the ricin A (IgG‐Tx). Brains were collected 12 days after the lesion and processed for in situ hybridization of CRF and AVP mRNA or for double fluorescence CRF and AVP immunohistochemistry. We found that lactating females exhibit a high degree of CRF and AVP colocalization in parvocellular PVN neurones, hypothalamic processes and median eminence terminals compared to virgins. While CRF mRNA is significantly reduced in lactating rats, AVP mRNA and protein levels are greatly enhanced in parvocellular PVN neurones during lactation. Hypothalamic CRF or AVP ricin‐A lesions significantly reduced both CRF and AVP expression (15–35% decrease) as well as peptide immunoreactivity in PVN neurones in both groups of females. The specificity of the lesions varied between virgins and lactators since in virgin females, AVP‐Tx did not affect CRF mRNA expression whereas in lactating females, this same lesion significantly reduced CRF mRNA expression, suggesting that parvocellular PVN neurones are more sensitive to the effects of the lesions during lactation. In both virgins and lactators, lesion with CRF‐Tx tended to increase AVP mRNA expression; however, in virgins, parvocellular PVN neurones were possibly compensating for the loss of CRF synthesis by increasing AVP expression and immunoreactivity. We conclude that lactation is associated with a high degree of CRF and AVP colocalization in parvoPVN neurones and that the increased AVP production in these neurones increases their sensitivity to immunotargeted lesions. The opposite regulation of CRF and AVP gene expression during lactation might provide a useful model to study differential sensitivity to glucocorticoid feedback or hypothalamic activation of transcription factors.

Increased colocalization of corticotropin-releasing factor and arginine vasopressin in paraventricular neurones of the hypothalamus in lactating rats: evidence from immunotargeted lesions and immunohistochemistry.

In lactating female rats, tonically elevated glucocorticoid secretion is accompanied by blunted stress responsiveness, reduced expression of hypothalamic corticotropin-releasing factor (CRF) mRNA and modest increases in arginine vasopressin (AVP) expression in the paraventricular nucleus (PVN). To determine the relative contribution of CRF and AVP to parvocellular function, we performed selective CRF (CRF-Tx) or AVP (AVP-Tx) lesions in the PVN neurones of ovariectomized virgin or lactating females (day 2 of lactation) by using ricin A associated with monoclonal antibodies directed towards CRF or AVP. We also performed double immunohistochemical labelling of CRF and AVP in the PVN of control rats injected with immunoglobulin (Ig)Gs associated with the ricin A (IgG-Tx). Brains were collected 12 days after the lesion and processed for in situ hybridization of CRF and AVP mRNA or for double fluorescence CRF and AVP immunohistochemistry. We found that lactating females exhibit a high degree of CRF and AVP colocalization in parvocellular PVN neurones, hypothalamic processes and median eminence terminals compared to virgins. While CRF mRNA is significantly reduced in lactating rats, AVP mRNA and protein levels are greatly enhanced in parvocellular PVN neurones during lactation. Hypothalamic CRF or AVP ricin-A lesions significantly reduced both CRF and AVP expression (15-35% decrease) as well as peptide immunoreactivity in PVN neurones in both groups of females. The specificity of the lesions varied between virgins and lactators since in virgin females, AVP-Tx did not affect CRF mRNA expression whereas in lactating females, this same lesion significantly reduced CRF mRNA expression, suggesting that parvocellular PVN neurones are more sensitive to the effects of the lesions during lactation. In both virgins and lactators, lesion with CRF-Tx tended to increase AVP mRNA expression; however, in virgins, parvocellular PVN neurones were possibly compensating for the loss of CRF synthesis by increasing AVP expression and immunoreactivity. We conclude that lactation is associated with a high degree of CRF and AVP colocalization in parvoPVN neurones and that the increased AVP production in these neurones increases their sensitivity to immunotargeted lesions. The opposite regulation of CRF and AVP gene expression during lactation might provide a useful model to study differential sensitivity to glucocorticoid feedback or hypothalamic activation of transcription factors.

Daily profiles of arginine vasopressin mRNA in the suprachiasmatic, supraoptic and paraventricular nuclei of the rat hypothalamus under various photoperiods

Daily rhythm of arginine vasopressin (AVP) mRNA levels in the suprachiasmatic nucleus (SCN) of rats maintained under a short, LD 8:16 photoperiod differed from that of rats maintained under a long, LD 16:8 photoperiod: under the short photoperiod the morning AVP rise occurred significantly later than under the long one. Daily profiles of AVP mRNA in the supraoptic and paraventricular nuclei were not rhythmic and AVP mRNA levels under LD 8:16 did not differ from those under LD 16:8. The data indicate that photoperiod affects selectively the clock driven AVP gene expression in the SCN.

Centrally Administered Murine-Leptin Stimulates the Hypothalamus-Pituitary- Adrenal Axis through Arginine-Vasopressin

Starvation induces a decrease in circulating leptin levels and activation of the hypothalamus-pituitary-adrenal (HPA) axis. Leptin inhibits the HPA axis in unfed rodents or genetically leptin-deficient ob/ob mice, whereas it stimulates corticotropin-releasing hormone (CRH) gene expression in the paraventricular nucleus (PVN). However, the interactions between leptin, CRH and the HPA axis are poorly understood and are likely to be complex. We recently demonstrated that central leptin administration caused increases in plasma arginine-vasopressin (AVP) and AVP gene expression of the PVN in nonstressful rats. AVP stimulates the release of adrenocorticotropic hormone (ACTH), but it also potentiates the action of CRH on ACTH release. In this study, we investigated the effects of leptin on plasma ACTH and corticosterone levels, CRH mRNA of the PVN and proopiomelanocortin (POMC) mRNA of the pituitary in nonstrained rats. Intracerebroventricularly administered leptin caused increases in plasma ACTH and corticosterone levels in dose-dependent manners. In Northern blot analyses, the leptin injection induced significant increases in the expression of CRH mRNA in the PVN and POMC mRNA in the pituitary. The increased plasma ACTH and corticosterone levels by leptin were attenuated with intracerebroventricular pretreatment of a V_1a receptor antagonist (OPC-21268) or a V_1a/V_1b receptor antagonist (dP[Tyr(Me)²]AVP), but not with that of a V₂ receptor antagonist (OPC-31260). The leptin-induced CRH mRNA expression in the PVN and POMC mRNA expression in the pituitary were also reduced by the pretreatment with OPC-21268 and dP[Tyr(Me)²]AVP. These results suggest that intracerebroventricular leptin administration activates the HPA axis by AVP receptor activation through V_1a receptors in the PVN which in turn activates CRH neurons to drive ACTH and corticosterone secretion in concert with AVP in nonstrained rats.

Glucocorticoid Negative Feedback Selectively Targets Vasopressin Transcription in Parvocellular Neurosecretory Neurons

To identify molecular targets of corticosteroid negative feedback effects on neurosecretory neurons comprising the central limb of the hypothalamo-pituitary-adrenal (HPA) axis, we monitored ether stress effects on corticotropin-releasing factor (CRF) and arginine vasopressin (AVP) heteronuclear RNA (hnRNA) expression in rats that were intact or adrenalectomized (ADX) and replaced with corticosterone (B) at constant levels ranging from nil to peak stress concentrations. Under basal conditions, relative levels of both primary transcripts varied inversely as a function of plasma B titers. In response to stress, the kinetics of CRF hnRNA responses of intact and ADX rats replaced with low B were similar, peaking at 5 min after stress. By contrast, intact rats showed a delayed AVP hnRNA response (peak at 2 hr), the timing of which was markedly advanced in ADX/low B-replaced animals (peak at 5-30 min). Transcription factors implicated in these responses responded similarly. Manipulation of B status did not affect the early (5-15 min) phosphorylation of transcription factor cAMP-response element-binding protein (CREB) but accelerated maximal Fos induction from 2 hr after stress (intact) to 1 hr (ADX). Assays of binding by proteins in hypothalamic extracts of similarly manipulated rats toward consensus CRE and AP-1 response elements supported a role for the stress-induced plasma B increment in antagonizing AP-1, but not CRE, binding. These findings suggest that glucocorticoid negative feedback at the transcriptional levels is exerted selectively on AVP gene expression through a mechanism that likely involves glucocorticoid receptor interactions with immediate-early gene products.

Analysis of the vasopressin system and water regulation in genetically polydipsic mice.

Polydipsic mice, STR/N, which show extreme polydipsia and polyuria, were discovered in 1958. In the STR/N, urine outputs are much higher than in control mice. The possibility of an abnormal regulation of the arginine vasopressin (AVP) system, or an abnormality in the renal susceptibility to AVP, should be considered. In this study we investigated the AVP system and water regulation in STR/N. We sequenced the AVP and the AVP V(2)-receptor genes of the STR/N by direct sequencing. No mutation was found in either of them. AVP gene expression examined by in situ hybridization and plasma sodium in 8-wk-old STR/N was significantly lower than in control mice, whereas it was significantly higher at 20 wk. Renal sensitivity to injected AVP was attenuated in 20-wk-old STR/N. The suppression of AVP synthesis due to excessive water retention in 8-wk-old STR/N suggests that polydipsia may be the primary cause in this strain. The 20-wk-old STR/N became dehydrated with the acceleration of AVP synthesis, which might have resulted from secondary desensitization to AVP.

Increase of vasopressin mRNA in the hypothalamus of inbred polydipsic mice

The genetically inbred polydipsic mice, STR/N strain, are characterized by extreme polydipsia and polyuria without arginine vasopressin (AVP) deficiency. The expression of AVP gene in the hypothalamus of polydipsic and non-polydipsic mice was examined by Northern blot analysis and in situ hybridization histochemistry. Northern blot analysis revealed that the total amount of AVP mRNA in the hypothalamus of the STR/N mice was approximately three-fold of that in the control, ICR mice. In situ hybridization histochemistry showed that the signals of AVP mRNA in the paraventricular (PVN) and supraoptic nuclei (SON) of the STR/N were stronger than those in the ICR. Although AVP gene transcripts were detected in the anteroventral parts of the PVN (avPVN) in the STR/N, there was a few AVP transcripts in the same area (avPVN) in the ICR. There were no differences in plasma osmolality and hematocrit between STR/N and ICR mice. These results suggest that upregulation of AVP mRNA in the hypothalamus of STR/N may be involved in the central mechanism responsible for the polydipsia in genetically polydipsic mice.

The effects of estrogen and progesterone on corticotropin-releasing hormone and arginine vasopressin messenger ribonucleic acid levels in the paraventricular nucleus and supraoptic nucleus of the rhesus monkey.

Ovarian steroids increase hypothalamic-pituitary-adrenal (HPA) axis activity and sensitize the hypothalamic-pituitary-ovarian (HPO) axis to stress-induced inhibition. The present study investigated the effect of ovarian steroids on CRH and arginine vasopressin (AVP) messenger RNA (mRNA) levels in the rhesus monkey hypothalamus, as both neuropeptides have been shown to stimulate the HPA axis and inhibit the HPO axis in this species. This was accomplished by measuring CRH and AVP mRNA in the paraventricular nucleus (PVN) and supraoptic nucleus (SON) by in situ hybridization histochemistry. Menstrual cycles were simulated in ovariectomized (OVX) rhesus monkeys by sequential addition and removal of SILASTIC brand (Dow Corning Corp.) tubing containing either 17beta-estradiol (E2) or progesterone (P4). On the morning of day 11 of the simulated follicular phase (E2 alone) or day 21 of the luteal phase (E2 + P4), animals were anesthetized, and the brains were perfused with paraformaldehyde via the carotid artery. Coronal sections (30 microm) were cut, and mRNA for CRH and AVP in the paraventricular nucleus (PVN) and supraoptic nucleus (SON) were semiquantified by in situ hybridization. CRH mRNA in the PVN of E2-replaced OVX animals (n = 7) was 2-fold greater than that in untreated OVX controls (n = 4), whereas CRH mRNA after E2 + P4 (n = 4) was no different from that in controls (optical density + SEM, 0.38 +/- 0.06, 0.13 +/- 0.08, and 0.14 +/- 0.09 for OVX + E2, OVX + E2 + P4, and OVX, respectively; P = 0.02). CRH in the SON was undetectable. In contrast to CRH, AVP mRNA in the PVN and the SON was similar in the three treatment groups. We conclude that E2 and E2 + P4 replacement to OVX monkeys exert different effects on CRH and AVP gene expression, as estrogen stimulation of CRH mRNA in the PVN was abrogated by progesterone, whereas no effect of ovarian steroids on AVP mRNA in either the PVN or SON was observed. We postulate that ovarian steroid regulation of CRH synthesis and release may in part explain the central nervous system mechanisms by which ovarian steroids affect the HPA and HPO axes during basal and stress conditions.