A target-driven DNA-based molecular machine for rapid and homogeneous detection of arginine-vasopressin.

Rapid detection of physiological changes of neuropeptides is of great importance as they are involved in a wide range of physiological processes and behaviors. Abnormalities in their expression level are correlated with various neurological diseases. However, current methods such as radioimmunoassay, enzyme-linked immunosorbent assays and liquid chromatography tandem mass spectrometry relied on cumbersome operation steps and could not rapidly provide the information of their concentration fluctuations. Thus motivated, we developed a target-driven DNA-based molecular machine that could be triggered only in the presence of a specific target neuropeptide. Using arginine-vasopressin (AVP) as a model neuropeptide, we integrated the DNA-based molecular machine with fluorescence signal transduction and amplification technology. The assay was rapid and homogeneous, which offered a linear range of 75-700 pM and a limit-of-detection as low as 75 pM. It holds great potential for further applications in real-time monitoring of the variations of the AVP level in biological samples.

Estrogen receptor beta and G protein-coupled estrogen receptor 1 are involved in the acute estrogenic regulation of arginine-vasopressin immunoreactive levels in the supraoptic and paraventricular hypothalamic nuclei of female rats.

The ovarian hormone 17ß-estradiol is known to regulate the release, expression and immunoreactivity of arginine-vasopressin (AVP) in the supraoptic and paraventricular hypothalamic nuclei of rodents. Previous studies have shown that estrogen receptor α is involved in the effects of chronic estradiol administration on arginine-vasopressin immunoreactivity in the female rat hypothalamus. In this study we have examined the effect of an acute administration of estradiol or specific agonists for estrogen receptors α, ß and G protein-coupled estrogen receptor 1 on the immunoreactivity of arginine-vasopressin in the hypothalamus of adult ovariectomized female rats. Acute estradiol administration resulted in a significant decrease in the number of arginine-vasopressin immunoreactive neurons in the supraoptic and paraventricular nuclei after 24 h. The effects of the specific estrogen receptors agonists suggest that the action of estradiol on arginine-vasopressin immunoreactivity is mediated in the supraoptic nucleus by G protein-coupled estrogen receptor 1 and in the paraventricular nucleus by both estrogen receptor ß and G protein-coupled estrogen receptor 1. Thus, in contrast to previous studies on the effect of chronic estrogenic treatments, the present findings suggest that estrogen receptor ß and G protein-coupled estrogen receptor 1 mediate the acute effects of estradiol on arginine-vasopressin immunoreactivity in the hypothalamus of ovariectomized rats.

Pregnancy may favour the development of severe autoimmune central diabetes insipidus in women with vasopressin cell antibodies: description of two cases.

Recently, an increased incidence of central diabetes insipidus (CDI) in pregnancy, and less frequently in the post partum period, has been reported, most probably favoured by some conditions occurring in pregnancy. This study was aimed at investigating the influence of pregnancy on a pre-existing potential/subclinical hypothalamic autoimmunity. We studied the longitudinal behaviour of arginine-vasopressin cell antibodies (AVPcAbs) and post-pituitary function in two young women with a positive history of autoimmune disease and presence of AVPcAbs, but without clinical CDI, and who became pregnant 5 and 7 months after our first observation. The behaviour of post-pituitary function and AVPcAbs (by immunofluorescence) was evaluated at baseline, during pregnancy and for 2 years after delivery. AVPcAbs, present at low/middle titres at baseline in both patients, showed a titre increase during pregnancy in one patient and after delivery in the other patient, with development of clinically overt CDI. Therapy with 1-deamino-8-d-arginine vasopressin (DDAVP) caused a prompt clinical remission. After a first unsuccessful attempt of withdrawal, the therapy was definitively stopped at the 6th and the 7th month of post partum period respectively, when AVPcAbs disappeared, accompanied by post-pituitary function recovery, persisting until the end of the follow-up. The determination of AVPcAbs is advisable in patients with autoimmune diseases planning their pregnancy, because they could be considered good predictive markers of gestational or post partum autoimmune CDI. The monitoring of AVPcAb titres and post-pituitary function during pregnancy in these patients may allow for an early diagnosis and an early replacement therapy, which could induce the disappearance of these antibodies with consequent complete remission of CDI.

Distribution of angiotensin type 1a receptor-containing cells in the brains of bacterial artificial chromosome transgenic mice.

In the central nervous system, angiotensin II (AngII) binds to angiotensin type 1 receptors (AT(1)Rs) to affect autonomic and endocrine functions as well as learning and memory. However, understanding the function of cells containing AT(1)Rs has been restricted by limited availability of specific antisera, difficulties discriminating AT(1)R-immunoreactive cells in many brain regions and, the identification of AT(1)R-containing neurons for physiological and molecular studies. Here, we demonstrate that an Agtr1a bacterial artificial chromosome (BAC) transgenic mouse line that expresses type A AT(1)Rs (AT1aRs) identified by enhanced green fluorescent protein (EGFP) overcomes these shortcomings. Throughout the brain, AT1aR-EGFP was detected in the nuclei and cytoplasm of cells, most of which were neurons. EGFP often extended into dendritic processes and could be identified either natively or with immunolabeling of GFP. The distribution of AT1aR-EGFP cells in brain closely corresponded to that reported for AngII binding and AT1aR protein and mRNA. In particular, AT1aR-EGFP cells were in autonomic regions (e.g., hypothalamic paraventricular nucleus, central nucleus of the amygdala, parabrachial nucleus, nuclei of the solitary tract and rostral ventrolateral medulla) and in regions involved in electrolyte and fluid balance (i.e., subfornical organ) and learning and memory (i.e., cerebral cortex and hippocampus). Additionally, dual label electron microscopic studies in select brain areas demonstrate that cells containing AT1aR-EGFP colocalize with AT(1)R-immunoreactivity. Assessment of AngII-induced free radical production in isolated EGFP cells demonstrated feasibility of studies investigating AT1aR signaling ex vivo. These findings support the utility of Agtr1a BAC transgenic reporter mice for future studies understanding the role of AT(1)R-containing cells in brain function.

Effects of neurointermediate pituitary lobectomy and desmopressin on acute experimental autoimmune encephalomyelitis in Lewis rats.

OBJECTIVE: The role of arginine vasopressin (AVP) as a direct immune regulator has not yet been clarified, and more work is needed to assess its involvement in the immunoneuroendocrine network. In the present study, the effects of neurointermediate pituitary lobectomy (NIL) and desmopressin (DP), an agonist of AVP, on acute experimental autoimmune encephalomyelitis (EAE) in female Lewis rats were evaluated. The activity of the hypothalamic-pituitary-adrenocortical (HPA) axis was also assessed. METHODS: Five groups of rats were used, as follows: (1) sham-operated (SHAM) rats, (2) SHAM + DP rats, (3) NIL rats, (4) NIL + DP rats and (5) untreated normal control rats. DP treatment started 2 weeks after surgery, and immunization to induce EAE was carried out 1 week later. RESULTS: SHAM rats developed full-blown clinical and histological signs of EAE and activation of the HPA axis. SHAM + DP animals had mild clinical signs of EAE, inflammatory infiltrations in the spinal cord and an activated HPA axis. NIL animals developed minimal EAE, scanty spinal cord inflammation and no changes in HPA axis activity. NIL + DP rats developed severe clinical signs of EAE, extensive spinal cord inflammatory infiltrations and marked activation of the HPA axis. CONCLUSIONS: NIL decreased the cell-mediated immune response, while DP in NIL animals restored the immune response. AVP is directly involved in the maintenance of immune competence.

Idiopathic central diabetes insipidus in children and young adults is commonly associated with vasopressin-cell antibodies and markers of autoimmunity.

OBJECTIVES: Autoimmune targeting of hypothalamic-neurohypophyseal structures in children and young adults with posterior pituitary and anterior pituitary dysfunction, as well as pituitary stalk involvement, are not yet completely understood. DESIGN: We aimed to (1) evaluate the presence of circulating vasopressin-cell autoantibodies (AVPc-Abs) in young patients with central diabetes insipidus (CDI), (2) detect organ-specific autoantibodies as markers of autoimmunity, and (3) define the relationship between immune markers and neuroimaging findings. PATIENTS: Twenty patients were evaluated at a median age of 16.3 years. Twelve patients had idiopathic CDI, six had Langerhans cell histiocytosis (LCH) and two had germinoma. AVPc-Abs were evaluated in 40 healthy children. Magnetic resonance imaging (MRI) of the hypothalamic-pituitary region was performed longitudinally in all subjects. MEASUREMENTS: Circulating arginine vasopressin (AVP), protein tyrosine phosphatase (IA2), glutamic acid decarboxylase (GAD), 21-hydroxylase (21-OH), endomysium antibodies (EMA), parietal cell (PCA), thyroid peroxidase (TPO), thyroglobulin (TG) and TSH-receptor (TSHr) autoantibodies were evaluated. RESULTS: Circulating AVPc-Abs were found in 15 patients (75%), nine with idiopathic CDI, four with LCH and two with germinoma; the pituitary stalk was involved in most of them. Five patients with idiopathic CDI showed a persistence of AVPc-Abs during follow-up and one became positive subsequently. Serum IA2 autoantibodies were demonstrated in 14 patients (70%) and 21-OH autoantibodies in three of them. CONCLUSION: In idiopathic CDI, circulating AVPc-Abs suggest an autoimmune involvement of the neurohypophyseal system. The identification of AVPc-Abs in subjects who could have either idiopathic CDI or LCH or germinoma, however, indicates that AVPc-Abs cannot be considered a completely reliable marker of autoimmune CDI. Thus, close clinical and MRI follow-up are needed because AVPc-Abs may mask germinoma or LCH.

Immunohistochemical detection of NRSA on small cell lung cancer with a monoclonal antibody (MAG-1) that recognizes the carboxyl terminus of provasopressin.

A single monoclonal antibody (MAG-1) directed against the C-terminal 18-amino acid region (VAGc18) of provasopressin was examined as an agent for recognizing the tumor-specific NRSA marker common to small cell lung cancer (SCLC) in formalin-fixed tissues with ABC immunohistochemistry. SCLC tumors were obtained from several tissue locations and included primary, metastatic, and recurrent disease. Positive staining was found in 91% of cases (53/58). All five of the unreactive tumors were of the lungs or chest wall, and there did not appear to be an association of this negativity with disease stage, age, or sex. Alternatively, almost all primary lesions, almost all metastatic lesions, and all recurrent lesions examined gave a positive reaction with MAG-1. For this study, vasopressin-producing cells of the human anterior hypothalamus served as a positive control, while negative controls comprised normal lung tissue, tumor that received MAG-1 in the presence of an excess of antigen (VAGc18 peptide), or tumor reacted with a commercial IgG1 isotype as primary antibody. All of the results indicate that MAG-1 can be effectively used to selectively identify the NRSA marker on almost all SCLC tumors, at all disease stages, and at all locations. Since all four tumors tested showing no reactivity with MAG-1 gave a positive reaction for synaptophysin, it is proposed that a combined use of MAG-1 with synaptophysin antibodies could allow all SCLC tumors to be detected by ABC immunohistochemistry.

Dehydration-induced cross-regulation of apelin and vasopressin immunoreactivity levels in magnocellular hypothalamic neurons.

Apelin, a neuropeptide recently identified as the endogenous ligand for the G protein-coupled receptor APJ, is highly concentrated in brain structures involved in the control of body fluid homeostasis including the supraoptic (SON) and paraventricular (PVN) hypothalamic nuclei. To clarify the implication of apelin in the regulation of water balance, we sought to determine whether apelin colocalized with arginine vasopressin (AVP) in the rat SON and PVN. We also investigated the effects of water deprivation on the levels of apelin within these two nuclei by comparison with those of AVP. Using dual immunolabeling confocal microscopy, we found that a large proportion of apelin-immunoreactive neurons colocalized AVP within both the SON and PVN, but that the two peptides were segregated within distinct subcellular compartments inside these cells. Both the number and labeling intensity of magnocellular apelin-immunoreactive cells increased significantly after 24- or 48-h dehydration, whereas the number and labeling density of AVP-immunoreactive neurons significantly decreased. The dehydration-induced increase in apelin immunoreactivity was markedly diminished by central injection of a selective vasopressin-1 receptor antagonist. Conversely, the effect of dehydration was mimicked by a 16-min intracerebroventricular infusion of AVP, again in a vasopressin-1 receptor antagonist-reversible manner. These results provide additional evidence for the involvement of the neuropeptide apelin in the control of body fluid homeostasis. They further suggest that the dehydration-induced release of AVP from magnocellular hypothalamic neurons may be responsible for the observed increase in immunoreactive apelin levels within the same neurons and thus that the release of one peptide may block that of another peptide synthesized in the same cells.

Spatial and temporal regulation of mitogen-activated protein kinase phosphorylation in the mouse suprachiasmatic nucleus.

Circadian and photic regulation of mitogen-activated protein kinase (MAPK) has been shown to associate closely with the function of the circadian clock in vertebrate clock tissues such as the mouse suprachiasmatic nucleus (SCN). Here we show that, in the central region of the mouse SCN, MAPK exhibited circadian and daily rhythms in phosphorylation with a peak at (subjective) night, and this activation was sustained for at least 8 h. In contrast, in the dorsomedial region of the SCN, MAPK showed an overt rhythm in phosphorylation with a transient peak at early subjective day, which was antiphase to that in the central region. Noticeably, the phospho-MAPK-immunoreactive cells observed in the dorsomedial region were distributed from the rostral to the caudal end of the SCN, whereas those observed in the central region were localized within the middle SCN along the rostral-caudal axis. Furthermore, a 15-min light pulse given at subjective night transiently evoked MAPK phosphorylation throughout the ventrolateral region of the SCN peaking within 15 min after the light onset, whereas nighttime-phosphorylated MAPK signals in the central-middle SCN become undetectable within 60 min after the light onset. Thus, the mode of circadian and photic regulation of MAPK phosphorylation varies remarkably among the three subregions within the SCN, suggesting divergent and cell type-specific roles of MAPK in the clock system of the mouse SCN.

Space flight affects magnocellular supraoptic neurons of young prepuberal rats: transient and permanent effects.

Effects of microgravity on postural control and volume of extracellular fluids as well as stress associated with space flight may affect the function of hypothalamic neurosecretory neurons. Since environmental modifications in young animals may result in permanent alterations in neuroendocrine function, the present study was designed to determine the effect of a space flight on oxytocinergic and vasopressinergic magnocellular hypothalamic neurons of prepuberal rats. Fifteen-day-old Sprague-Dawley female rats were flown aboard the Space Shuttle Columbia (STS-90, Neurolab mission, experiment 150) for 16 days. Age-matched litters remained on the ground in cages similar to those of the flight animals. Six animals from each group were killed on the day of landing and eight animals from each group were maintained under standard vivarium conditions and killed 18 weeks after landing. Several signs of enhanced transcriptional and biosynthetic activity were observed in magnocellular supraoptic neurons of flight animals on the day of landing compared to control animals. These include increased c-Fos expression, larger nucleoli and cytoplasm, and higher volume occupied in the neuronal perikaryon by mitochondriae, endoplasmic reticulum, Golgi apparatus, lysosomes and cytoplasmic inclusions known as nematosomes. In contrast, the volume occupied by neurosecretory vesicles in the supraoptic neuronal perikarya was significantly decreased in flight rats. This decrease was associated with a significant decrease in oxytocin and vasopressin immunoreactive levels, suggestive of an increased hormonal release. Vasopressin levels, cytoplasmic volume and c-Fos expression returned to control levels by 18 weeks after landing. These reversible effects were probably associated to osmotic stimuli resulting from modifications in the volume and distribution of extracellular fluids and plasma during flight and landing. However, oxytocin levels were still reduced at 18 weeks after landing in flight animals compared to controls. This indicates that space flight during prepuberal age may induce irreversible modifications in the regulation of oxytocinergic neurons, which in turn may result in permanent endocrine and behavioral impairments.

Arginine vasopressin in the cytoplasm and nuclear fraction of lymphocytes from healthy donors and patients with depression or schizophrenia.

We investigated whether cytoplasmic or nuclear extracts of human peripheral blood lymphocytes contain AVP in samples from healthy controls and patients diagnosed as depressed or schizophrenic. Both the cytoplasmic and nuclear extracts contained AVP as determined by radioimmunoassay. AVP and other peptides were detected in the purified samples by matrix-assisted laser desorption/ionization time of flight mass spectrometry. It is the first time that AVP has been characterized in human lymphocytes of patients with depression or schizophrenia. This finding demonstrates the presence of another important component within the potential regulatory loop between immune and neuro-endocrine tissues.

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.

Perturbations of arginine vasopressin secretion during inflammatory stress. Pathophysiologic implications.

Pro-inflammatory cytokines, such as interleukin-1 beta (IL-1 beta), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF alpha), released from inflammatory foci, can activate the hypothalamus to produce corticotrophin-releasing hormone (CRH) and arginine vasopressin (AVP). These hypothalamic peptides in synergy increase ACTH production by the pituitary gland and hence corticosteroid (CS) secretion by the adrenal cortices. CS dampens inflammation. The pituitary also produces prolactin (PRL), which is pro-inflammatory, and macrophage inhibitory factor (MIF), which by counteracting the anti-inflammatory and immunosuppressive effects of CS, is pro-inflammatory. Lewis rats develop a variety of induced-autoimmune inflammatory conditions, such as streptococcal cell wall arthritis, whereas the histocompatible F344 Fisher rats are resistant to this condition. Lewis rats have a defective hypothalamic-pituitary adrenal (HPA) response to a variety of hypothalamic stimuli, but have augmented systemic secretion of AVP. Patients with rheumatoid arthritis (RA) have deficient CS with exaggerated PRL responses to inflammatory stimuli. Within inflammatory foci, CRH is pro-inflammatory. AVP, which augments autologous mixed lymphocyte reactions, can replace the IL-2 requirement for gamma IFN production by T cells via V1a receptors, and potentiates primary antibody responses, is also pro-inflammatory. Lewis rats have significantly high plasma levels, hypothalamic content, and in vitro release of AVP in comparison to the inflammatory disease-resistant Fischer rats. Immunoneutralization of AVP attenuates inflammatory responses. In Sprague-Dawley rats, AVP potentiates PRL secretion. Preliminary studies in patients with RA have shown that the circulating levels of AVP are significantly increased, which might be a compensatory response to low CS levels or a result of elevated levels of IL-6 in these patients but could nevertheless contribute to rheumatoid inflammation. A similar observation has been made in patients with ankylosing spondylitis.

Inhibition of heme oxygenase in the central nervous system potentiates endotoxin-induced vasopressin release in the rat.

Previous in vitro studies have shown that increases in endogenous carbon monoxide (CO) generation via activation of the enzyme heme oxygenase (HO) within the rat hypothalamus are associated with the reduced release of the neuropeptides, vasopressin (AVP) and oxytocin, while evidence concerning corticotrophin-releasing hormone (CRH) is controversial. The present study investigated whether there is also a functional relationship between the HO-CO pathway and AVP and corticosterone (Cort) in vivo. Male Wistar rats were challenged with bacterial lipopolysaccharide (LPS) at doses producing significant activation of the hypothalamo-pituitary-adrenal (HPA) axis. LPS was given alone or after pretreatment with the HO inhibitor Sn-protoporphyrin-9 (SnPP9). The latter was injected either intraperitoneally (i.p.) or by intracerebroventricular (i.c.v.) route. SnPP9 given i.p. failed to modify either basal or LPS-stimulated levels of AVP and Cort. On the contrary, i.c.v. SnPP9 strongly potentiated LPS-induced AVP release and significantly enhanced basal serum Cort levels, although it failed to potentiate stimulation by LPS. The LPS + i.c.v. SnPP9 also significantly reduced the hypothalamic stores of AVP compared to controls, correlating with increased circulating levels of AVP. Taken collectively, these data are in concordance with previous in vitro observations showing that the HO-CO pathway acts centrally to attenuate endotoxin-stimulated AVP release, while having less effects on the pituitary-adrenal axis.

Single stress induces long-lasting elevations in vasopressin mRNA levels in CRF hypophysiotrophic neurones, but repeated stress is required to modify AVP immunoreactivity.

Repeated stress is known to induce an increased vasopressin (AVP) expression in paraventricular corticotrophin-releasing factor (CRF) neurones which is supposed to enhance the ACTH-releasing capacity of these cells. To test the hypothesis that a single stress is sufficient to produce these changes, we used quantitative in-situ hybridization analysis to measure steady state CRF and AVP mRNA. Moreover the colocalized AVP and CRF immunoreactive sites were assessed in the dense core vesicle compartment of CRF axon terminals in the external zone of the median eminence with quantitative immunoelectron microscopy. Acute immobilization produced a significant increase in the average AVP and CRF mRNA levels (145% and 65%, respectively, above control values) in the medial parvocellular subdivisions of the paraventricular nucleus (PVN), and these changes persisted for over 4 days after stress. In contrast to these changes in AVP mRNA levels, there were no concomitant changes in AVP immunostaining in CRF terminals and axons during the 4-day period. However, when immobilization stress was repeated daily, the number of CRF terminals containing AVP increased progressively. Moreover, the ratio of AVP and CRF immunoreactivity in the dense core vesicle compartment was increased. Taken together, these results provide evidence that single stress experience can cause long-lasting changes in AVP and CRF mRNA steady state expression that is not apparently accompanied by changes in peptide levels. They also suggest that repeated stress is required for developing progressive shifts in the neurohormone storage pattern of these neurones.

CRF type I receptor-deficient mice exhibit a pronounced pituitary-adrenal response to local inflammation.

Recent studies indicate that the regulation of adrenocorticotropin (ACTH) secretion by corticotropin-releasing factor (CRF) is mediated predominantly by the type I CRF receptor (CRF-R1). Indeed, CRF-R1-deficient (CRF-R1 -/-) mice show marked impairment of the pituitary-adrenal axis. However, the plasma ACTH concentrations of unstressed CRF-R1 -/- mice are similar to those in wild-type mice. We show here that arginine vasopressin (AVP) is a major ACTH secretagogue in CRF-R1 -/- mice in resting conditions, since administration of anti-AVP serum, but not anti-CRF serum, markedly reduced (by 60%) resting plasma ACTH concentrations in these mutants. We also investigated the pituitary-adrenal response to turpentine-induced local inflammation in CRF-R1 -/- mice. Administration of turpentine into the hind-limb of CRF-R1 -/- mice produced a slightly (15-25%) smaller swelling of the limb, but a 10 fold greater rise in plasma IL-6 levels, compared to CRF-R1 +/+ controls. Turpentine-induced local inflammation produced pronounced elevations in the plasma concentrations of both ACTH and corticosterone in both CRF-R1 -/- and wild-type mice, but ACTH secretion could be inhibited by anti-CRF and anti-AVP sera only in wild-type mice. These data indicate that resting ACTH secretion in CRF-R1 -/- mice is in part attributable to AVP-dependent mechanisms. Furthermore, while in normal mice the pituitary-adrenal response to local inflammation is mediated largely via CRF-dependent mechanisms, mice deficient in CRF-R1 are still able to mount a pituitary-adrenal response via mechanisms that do not depend critically on either CRF or AVP action.

Improved extraction procedure and RIA for determination of arginine8-vasopressin in plasma: role of premeasurement sample treatment and reference values in children.

We optimized an RIA for measurement of arginine8-vasopressin (AVP) in plasma by use of 100-mg Isolute C18 columns for extraction, addition of a preincubation step, and use of maximal dilution of a commercially available antiserum. The detection limit was 0.06 ng/L when 0.5 mL of acidified plasma was extracted. The within- and between-run CVs (n = 16) at physiological concentrations were 5.8-10. 2% and 6.5-11.7%, respectively. Prolonged storage of blood at 25 degreesC, but not at 4 degreesC, led to a significant increase in measured plasma AVP concentrations. When plasma samples were prepared at several centrifugation speeds, plasma AVP was significantly correlated with the platelet count (r = 0.899; P <0. 001). This emphasizes the need for careful sample preparation to avoid contamination of plasma with platelet-bound AVP. Basal plasma AVP in 203 children and adolescents (105 males and 98 females; ages, 1 day to 18 years) averaged 1.1 +/- 0.6 ng/L. There was no significant difference between males and females and no correlation with age. In 16 healthy adult controls, plasma AVP averaged 1.0 +/- 0.5 ng/L.

Dynamic changes in the immunoreactivity of neuropeptide systems of the suprachiasmatic nuclei in golden hamsters during the sleep-wake cycle.

The sleep-wake cycle is virtually the most prominent circadian rhythm in mammals. In the timing system of sleep and wakefulness, the intrinsic neuropeptide systems of the suprachiasmatic nuclei (SCN) may play an important role. To elucidate this possible influence in the golden hamster, the immunoreactivity patterns of the suprachiasmatic gastrin-releasing peptide (GRP), vasoactive intestinal polypeptide (VIP), and arginine-vasopressin (AVP) systems were investigated in relation to the day-night and sleep-wake cycle by use of immunocytochemistry combined with semiquantitative planimetric analysis. For the GRP system, the highest level of immunoreactivity (expressed as area density) was observed in sleeping hamsters. Intermediate levels were found in awake, motorically active evening animals, whereas the lowest levels of immunoreactivity were detected in awake, motorically inactive hamsters studied in the morning. The immunoreactivity of the VIP system showed a completely opposite pattern, indicating highest area density in awake morning, intermediate area density in awake evening and lowest area density in sleeping golden hamsters. The immunoreactivity pattern of the AVP system, displaying highest levels in sleeping individuals, was virtually identical to that of the GRP system. Together with the related signs of neuronal activity, the present results favor an important role of these neuropeptide systems for the integration of central nervous information related to the sleep-wake cycle with photic information of the retinal input.

Hypothalamic-pituitary-mediated immunomodulation: arginine vasopressin is a neuroendocrine immune mediator.

Organisms respond to a variety of environmental agents, such as those that cause inflammation, by mounting a coordinated complex series of adaptive responses involving the immune, nervous and endocrine systems. These adaptations are aimed at restoring the homeostatic balance and the return to the status quo ante. This interaction is facilitated by cytokines, hormones and neurotransmitters, as well as receptors that are endogenous to the neural, immune and endocrine systems. These shared ligands and receptors provide the molecular basis of this cross-talk. Studies of animal models of autoimmune diseases have shown that defects in the neuroendocrine immune communications contribute to the development of chronic inflammatory autoimmune disease. By analogy, similar observations have now been made in patients with inflammatory rheumatic disorders. For instance, patients with rheumatoid arthritis have abnormally low cortisol responses to inflammation, whilst the production of prolactin is excessive and dysregulated. Prolactin is a pro-inflammatory neuropeptide. This paper reviews the evidence to support the viewpoint that the neuropeptide arginine vasopressin, which is also produced by the hypothalamus, should be considered to be another neuroendocrine modulator of immune and inflammatory responses. It is also being hypothesized that the production of arginine vasopressin might be dysregulated and excessive in rheumatoid arthritis, and that this could be another additional neuroendocrine factor contributing to the pathophysiology of the disease.