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.

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.

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.

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.

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.

Endotoxin decreases corticotropin-releasing factor receptor 1 messenger ribonucleic acid levels in the rat pituitary.

Bacterial endotoxins produce profound activation of the hypothalamo-pituitary-adrenal axis, mediated by stimulation of hypothalamic CRF neurons. Although a number of studies have described direct pituitary actions of inflammatory mediators, the effects of inflammatory stimuli on the sensitivity of corticotropes to CRF remain to be elucidated. The aim of this study was to determine the effects of inflammatory stress on the CRF receptor 1 (CRF-R1) messenger RNA (mRNA) levels in the rat pituitary. The systemic injection of endotoxin [lipopolysaccharide (LPS); 50 microg/kg, i.v.] increased plasma concentrations of ACTH and corticosterone. Ribonuclease protection analysis of total RNA isolated from individual whole pituitaries indicated that LPS produced a significant decrease in CRF-R1 mRNA that was evident by 2 h after injection (to 57% of control) and more marked by 6 h (to 38% of control). To evaluate whether the decrease in CRF-R1 mRNA was dependent upon increased exposure to CRF and/or vasopressin (AVP), LPS was injected with an anti-CRF antiserum, a CRF receptor antagonist (Astressin), or anti-AVP antiserum. A strong inhibition of the ACTH response to LPS was produced by pretreatment with anti-CRF antiserum, Astressin, or anti-AVP antiserum. However, these treatments had no effect on the decrease in CRF-R1 mRNA produced by LPS, indicating that neither CRF nor AVP are obligatory mediators of this pituitary response. The hypothesis that LPS might have direct pituitary effects on CRF-R1 mRNA levels was tested in vitro. Indeed, decreases in CRF-R1 mRNA to 43% and 53% of the control level were observed in rat anterior pituitary cell cultures that were treated with either LPS itself or the inflammatory mediator interleukin-1beta, respectively. Collectively, these results show that CRF receptor mRNA levels in the pituitary of the rat are markedly reduced by systemic LPS treatment and that this decrease is not dependent upon increased exposure of the pituitary to CRF or AVP, but may involve direct effects within the pituitary of either LPS itself or ensuing cytokine production.

Evidence from confocal fluorescence microscopy for a dense, reciprocal innervation between AVP-, somatostatin-, VIP/PHI-, GRP-, and VIP/PHI/GRP-immunoreactive neurons in the rat suprachiasmatic nucleus.

The rat suprachiasmatic nucleus (SCN) consists of several classes of neurons which can be identified by their transmitter content. Knowledge of putative interaction between these different cell types is essential in order to understand the possibilities of information processing within the SCN. The aim of the present study was therefore to obtain more information about the mutual innervation between the main cell classes in the rat SCN, viz. those containing the neuropeptides arginine vasopressin (AVP), vasoactive intestinal peptide (VIP), peptide histidine isoleucine (PHI), gastrin-releasing peptide (GRP) and somatostatin respectively. For this purpose, vibratome sections were double-immunolabelled for seven different peptide combinations and subsequently analysed by high-resolution confocal laser scanning fluorescence microscopy. Attention was focused on axosomatic appositions, the occurrence and frequency of which were quantitatively estimated. Our analysis of double-immunolabelled sections demonstrated that some of the VIP- and some of the GRP-immunoreactive nerve cells and endings showed colocalization. Assuming, on the basis of literature data, that VIP and PHI are always colocalized at the cellular level, the five main cell classes in the SCN appeared to be interconnected, at least axosomatically, in the following reciprocal way: AVP <--> VIP/PHI, AVP <--> GRP, AVP <--> somatostatin, somatostatin <--> VIP/PHI, somatostatin <--> GRP, VIP/PHI <--> GRP, VIP/PHI/GRP <--> GRP, VIP/PHI/GRP <--> VIP/ PHI. In addition to this heterologous axosomatic innervation, these cell groups also showed substantial homologous innervation. Supported by electron microscope data from the literature showing the existence of axodendritic synapses for some of these peptide combinations, our findings strongly suggest that the rat SCN comprises a complex synaptic network with strong interactive capabilities, which is probably a requisite for its biological clock function.

Peptidergic innervation of the mesencephalic trigeminal nucleus in the cat.

BACKGROUND: The trigeminal processing of proprioceptive information is unique and very little is known about the neurochemical organization of trigeminal primary afferent neurons which mediate the sensory aspects of proprioception. In studies using immunocytochemicalretrograde tracing techniques, some classical neurotramsitters mediating the afferent modulation of the mesencephalic trigeminal nucleus (MTN) have been investigated. This paper summarizes our current understanding of the peptidergic innervation of the cat MTN. METHODS: The distribution of immunoreactive substances was studied using specific antisera against 11 major neuropeptides. Light and electron microscopic peroxidase-antiperoxidase immunocytochemical staining techniques in colchicine-treated animals were used to clarify the distribution of peptide-identified fibers related to the MTN. RESULTS: Immunoreactivity to any of the tested neuropeptides could not be detected in the MTN cell bodies. Numerous fibers containing various peptides such as substance P, bombesin, enkephalins, cholecystokinin, vasoactive intestinal polypeptide, vasopressin, and neuropeptide Y were present in the nucleus, however. These thin positive fibers covered the neuronal surface of the MTN cell bodies and some of the immunoreactive varicosities appeared to be in close proximity to profiles of MTN neurons. Electron microscopic observations revealed that perisomatic fibers were in direct apposition to perikarya of unstained large cells and some of them made synaptic contacts with their cell bodies and dendrites. CONCLUSIONS: The present results demonstrate that the MTN neurons receive dense basket-like innervation from peptidergic neurons on somata and processes and have supported earlier evidence that the MTN of the cat is under influence of peptidergic input. Results of this study provide further evidence that the neuropeptides examined may play an important role in the integration and transmission of trigeminal proprioceptive information. Most likely they may co-exist with a classical but hitherto unknown neurotransmitter(s), that is unique for this region and whose release can be modulated by peptides.

Multiple gonadotropin-releasing hormone (GnRH)-immunoreactive systems in the brain of the dwarf gourami, Colisa lalia: immunohistochemistry and radioimmunoassay.

The present study characterizes gonadotropin-releasing hormone (GnRH) neuronal groups that are located in several different brain regions by investigating GnRH molecular species and projection patterns in an anabantid fish, Colisa lalia. First, we examined the molecular species of GnRHs in extracts of the brain and the pituitary by reverse-phase high-performance liquid chromatography followed by radioimmunoassays. We found salmon GnRH (sGnRH), chicken GnRH-II (cGnRH-II), and an unfamiliar GnRH-like substance. Next, to examine the distribution of each GnRH molecule in different GnRH neuronal groups, we performed immunohistochemistry using four kinds of antisera and an antibody. Furthermore, we performed brain lesioning experiments of terminal nerve (TN) cells, the most conspicuous GnRH-immunoreactive cells in Colisa lalia. Comparisons of immunoreactive structures between TN-lesioned fish and untreated fish elucidated the projection area of each neuronal group. Three major neuronal groups were observed. TN-GnRH cells, which are located in the transitional area between the olfactory bulb and the telencephalon, showed strong sGnRH and weaker cGnRH-II immunoreactivity. TN-GnRH cells projected to wide areas of the central nervous system from the olfactory bulb to the spinal cord. The second group, located in the preoptic area, showed only sGnRH immunoreactivity and projected only to the pituitary. The third one, located in the midbrain tegmentum, exhibited strong cGnRH-II and weaker sGnRH immunoreactivity. This cell group projected mainly to brain regions posterior to the hypothalamus and the spinal cord. These different projection patterns suggest functional differentiation of each GnRH neuronal group.

In vivo evidence that arginine vasopressin is involved in the adrenocorticotropin response induced by interleukin-6 but not by tumor necrosis factor-alpha in the rat.

We examined whether arginine vasopressin (AVP) is involved in the adrenocorticotropin (ACTH) response induced by interleukin (IL)-6 or tumor necrosis factor (TNF)-alpha in the rat. To accomplish this, we employed immunoneutralization of brain AVP by injecting anti-AVP antiserum intracerebroventricularly (i.c.v., 3rd ventricle). For comparison, we also tested the effect of immunoneutralization of corticotropin-releasing hormone (CRH) in the brain. Anti-CRH antibody, anti-AVP antibody, or normal rabbit serum (control) was given i.c.v. 15 min before an i.c.v. administration of human recombinant IL-6 (100 ng) or TNF-alpha (100 ng). Both IL-6 and TNF-alpha significantly elevated plasma ACTH levels. The IL-6-induced ACTH response was significantly suppressed by both anti-CRH and anti-AVP antibodies. On the other hand, the TNF-alpha-induced ACTH response was not significantly affected by anti-AVP antibody, although anti-CRH antibody could suppress the response. These results suggest that the IL-6-induced ACTH response may be mediated by both CRH and AVP, whereas the ACTH response to TNF-alpha is only via CRH.

Retinohypothalamic projections and immunocytochemical analysis of the suprachiasmatic region of the desert iguana Dipsosaurus dorsalis.

Two separate and distinct retinal projections to the hypothalamus in the iguanid lizard Dipsosaurus dorsalis were described using horseradish peroxidase and cobalt-filling techniques. Both of the projections were unilateral and completely crossed; one terminated in the supraoptic nucleus and the other in the suprachiasmatic nucleus. Immunocytochemical analysis showed that the supraoptic nucleus contained cell bodies and fibers that cross-react with antibodies raised against arginine vasopressin, while the suprachiasmatic nucleus contained arginine vasopressin-like immunoreactive fibers emanating from cells in the nearby paraventricular nucleus. The suprachiasmatic nucleus contained a dense plexus of fibers that cross-reacted with neuropeptide-Y antibody. Antiserum against vasoactive intestinal polypeptide showed no reactivity in any part of the forebrain, while antiserum against serotonin showed sparse and uniform reactivity throughout the forebrain, including the suprachiasmatic nucleus. These results, together with other data, indicate that the suprachiasmatic nucleus of D. dorsalis is homologous to the suprachiasmatic nuclei of rodents, structures known to contain circadian pacemakers. We suggest that the suprachiasmatic nucleus may play a similar role in the circadian system of D. dorsalis.

Atrial natriuretic factor and arginine vasopressin production in tumor cell lines from patients with lung cancer and their relationship to serum sodium.

Patients with lung cancer (n = 263) were studied to determine the relationship among ectopic production of atrial natriuretic factors (ANF) and arginine vasopressin (AVP), serum sodium, and patient outcome. Of 133, 21 (16%) patients with small cell lung cancer (SCLC) had hyponatremia (serum sodium, < 130 mmol/liter), compared to none of 130 (0%) patients with non-small cell lung cancer (P < 0.0001). Patients with extensive-stage SCLC and hyponatremia had shorter survival than patients with extensive stage SCLC and normal serum sodium values (P = 0.012). Of the 11 hyponatremic patients with SCLC and tumor cell lines available for study, 9 produced ANF mRNA, 7 of 11 produced AVP mRNA, and 5 of 11 produced both ANF mRNA and AVP mRNA. All 11 cell lines produced either ANF mRNA and ANF peptide or AVP mRNA and AVP peptide, or both. The quantity of AVP peptide in the tumor cell lines was more closely associated with hyponatremia in the patients (P = 0.0026, r2 = 0.28) than was the production of ANF peptide (P = 0.066, r2 = 0.12), although neither association was strong. All tumor cell lines studied from SCLC patients with hyponatremia produce ANF and/or AVP mRNA and peptides.

Lack of interaction of vasopressin with its antisense peptides: a functional and immunological study.

The peptide encoded in the 5' to 3' direction by rat vasopressin complementary RNA, rat PVA (H-Ser-Ser-Trp-Ala-Val-Leu-Glu-Val-Ala- OH) and the corresponding bovine PVA (H-Ala-Pro-Trp-Ala-Val-Leu-Glu-Val-Ala-OH) were investigated with respect to their interaction with [8-arginine] vasopressin (AVP) and V2 vasopressin receptor binding and function. Rat or bovine PVA did neither affect the binding of the hormone to the V2 receptor of bovine kidney membranes and LLC-PK1 pig kidney cells nor influence the AVP-induced cAMP-production in LLC-PK1 cells. Rat PVA was further investigated by the use of vasopressin-specific polyclonal and monoclonal antibodies with different affinity and epitope specificity. Consistent with receptor binding studies no inhibition of [3H]AVP-binding in fluid- or solid-phase antibody binding tests after preincubation with PVA was found. Direct interaction of rat PVA and [3H]AVP measured on solid surface was not observed in contrast to specific binding of the hormone with NP II and antibodies. In our study no evidence for an interaction of AVP and its antisense peptides was found.

Neuronal associations in the rat suprachiasmatic nucleus demonstrated by immunoelectron microscopy.

The synaptic associations of neurons in the suprachiasmatic nucleus (SCN) of rats were examined by single immunolabeling for somatostatin (SRIH) and arginine vasopressin (AVP), and double immunolabeling for SRIH plus AVP and vasoactive intestinal polypeptide (VIP) plus AVP. Single immunolabeling showed that SRIH neurons, which displayed some somatic and dendritic spines, formed synaptic contacts with immunonegative and positive axon terminals. AVP neurons also formed synaptic contacts with both immunonegative and positive axon terminals. The immunonegative terminals contained small, spherical clear vesicles or flattened clear vesicles. A few immunopositive AVP fibers made synapses with immunonegative somatic or dendritic spines. Double immunolabeling showed synaptic associations between SRIH axons and AVP cell bodies or dendritic processes, and between AVP axons and the somata or dendrites of SRIH neurons. These findings suggest a reciprocal relation between the two types of neurons. Synaptic contacts between AVP neurons and VIP axon terminals were also demonstrated. Previously, we found synapses between SRIH axons and VIP neurons. Thus SRIH neurons appeared to regulate AVP and VIP neurons. On the basis of these findings, two possible oscillation systems of the SCN are proposed.

Neuropeptides are chemoattractants for human tumor cells and monocytes: a possible mechanism for metastasis.

Bombesin (BN), a tetradecapeptide neuropeptide growth factor, is shown to be a potent (ED50 of 5 X 10(-12) M) chemoattractant for human monocytes and small cell lung carcinoma cells (SCCL). These effects are BN receptor-mediated since potencies of several BN analogs to induce chemotaxis and to inhibit [125I-tyr4] BN binding activity correlate well (P less than 0.001). As has been demonstrated for other BN receptor-mediated effects, carboxy-terminal amino acids are required for optimum biological activity. BN is not an exclusive chemoattractant for SCCL cells but was also active in promoting migration of other, but not all, lung tumor cells. Other neuropeptides, such as beta-endorphin, substance P, and arg-vasopressin, are also shown to be chemoattractants for SCCL cells, with EC50's also in the 10(-12) M range. The ability of these ligands to effect monocyte and some tumor cell migration suggest a role for neuropeptides in inflammation and metastasis. In the latter case, tumor cells, in response to neuropeptide chemical gradients, may become localized at specific body sites. Neuropeptide release, in response to cognitive or other stimuli, may thereby modify cell migratory patterns. Additionally, such hormones may influence early developmental events such as tissue organization and histogenesis.

Comparison of three immunoassays in the screening and characterization of monoclonal antibodies against arginine-vasopressin.

A method for screening monoclonal antibodies (McAbs) to neuropeptides was evaluated using 8-arginine-vasopressin (AVP) as a model. Mice were immunized with AVP-thyroglobulin conjugate and their spleen cells were fused with X 63-Ag8.653 mouse myeloma cells. The resulting hybridoma supernatants were screened for specific antibody production using 3 different assays: solid phase enzyme radioimmunoassay in Terasaki plates (Ter-ELISA), liquid phase radioimmunoassay (LPRIA) and an immunohistochemical technique. From 2 independent fusions, 7 McAbs specific for AVP were obtained. They belonged to the IgG1 subclass and reacted more strongly to the ring part of the nonapeptide. The screening strategy proposed relies upon a crude selection of conjugate-reacting hybridomas, followed by neuropeptide-specific hybridoma identification using both LPRIA (with radioiodinated synthetic peptide) and an immunohistochemical technique (to detect natural neuropeptide). During subcloning steps Ter-ELISA is then chosen, to select for specific clones and to eliminate those reacting with the carrier thyroglobulin.