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Introduction: Hypothalamic hamartoma (HH) is a rare developmental disorder presenting with gelastic seizures or precocious puberty attributed to gonadotrophin-releasing hormone expression by the hamartoma. The histogenesis of HH is uncertain, and diagnosis of HH is difficult in small biopsies due to its close resemblance to normal hypothalamic nuclei. TTF-1 and arginine vasopressin (AVP) are associated with gonadotropin-releasing hormone release., Materials and Methods: In this study, we explored the expression pattern of TTF-1 and AVP in HH and its utility, if any, in diagnosis. We reviewed the clinical, radiologic, and histopathological features of 23 HH diagnosed over the past decade at our Institute., Results: The age at presentation ranged from 11 months to 34 years with gelastic seizures (82.6%), precocious puberty (17.4%), and developmental delay (8.7%) as presenting symptoms. On imaging, all the lesions (n = 9) involved the posterior and tuberal group of hypothalamic nuclei, while 5 cases involved the anterior hypothalamus. Anatomically, the lesions involved mammillary body, arcuate and periventricular nuclei. On histopathology, 52% cases revealed nodular arrangement of small neurocytic cells separated by glial stroma. TTF-1 and AVP immunoreactivity was absent in all the cases, whereas in normal hypothalamus, AVP was expressed in periventricular nuclei., Conclusion: Our results suggest that immunoexpression of TTF-1 is absent in HH, particularly in those arising from the posterior hypothalamus, and this can be used in small biopsies to distinguish from a normal hypothalamus as well as from posterior pituitary tumors.

Diabetes insipidus is a disease in which large volumes of dilute urine (polyuria) are excreted due to vasopressin (AVP) deficiency [central diabetes insipidus (CDI)] or to AVP resistance (nephrogenic diabetes insipidus). In the majority of patients, the occurrence of CDI is related to the destruction or degeneration of neurons of the hypothalamic supraoptic and paraventricular nuclei. The most common and well recognized causes include local inflammatory or autoimmune diseases, vascular disorders, Langerhans cell histiocytosis (LCH), sarcoidosis, tumors such as germinoma/craniopharyngioma or metastases, traumatic brain injuries, intracranial surgery, and midline cerebral and cranial malformations. Here we have the opportunity to describe an unusual case of female patient who developed autoimmune CDI following ureaplasma urealyticum infection and to review the literature on this uncommon feature. Moreover, we also discussed the potential mechanisms by which ureaplasma urealyticum might favor the development of autoimmune CDI.

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.

An unusual mutation in the arginine vasopressin (AVP) gene, predicting a P26L amino acid substitution of the AVP prohormone, is associated with autosomal recessive familial neurohypophyseal diabetes insipidus (FNDI). To investigate whether the cellular handling of the P26L prohormone differed from that of the Y21H prohormone associated with autosomal dominant inheritance of FNDI, the mutations were examined by heterologous expression in cell lines. Immunoprecipitation demonstrated retarded processing and secretion of the Y21H prohormone, whereas the secretion of the P26L prohormone seemed to be unaffected. Confocal laser scanning microscopy showed accumulation of the Y21H prohormone in the endoplasmic reticulum, whereas the P26L prohormone and/or processed products were localized in secretory granules in the cellular processes. RIA analysis showed reduced amounts of immunoreactive Y21H-AVP and P26L-AVP in the cell culture medium. Thus, the recessive mutation does not seem to affect the intracellular trafficking but rather the final processing of the prohormone. Our results provide an important negative control in support of the hypothesis that autosomal dominant inheritance of FNDI is caused by mutations in the AVP gene that alter amino acid residues important for folding and/or dimerization of the neurophysin II moiety of the AVP prohormone and subsequent transport from the endoplasmic reticulum.

Magnocellular neurons of the supraoptic nucleus release the neuropeptides oxytocin and vasopressin from their dendrites to regulate their synaptic inputs. This study aims to determine the cellular mechanism by which vasopressin modulates excitatory synaptic transmission. Presumably by electroporation through perforated patch, we were able to successfully introduce biocytin into cells in which we performed an electrophysiological study. This method enabled us to determine that roughly half of the recorded neurons were immunoreactive to oxytocin-associated neurophysin and showed two characteristic features: an inward rectification and a sustained outward rectification. The remaining half showed a linear voltage-current relationship and was immunoreactive to vasopressin-associated neurophysin. Using these electrophysiological characteristics and post hoc immunohistochemistry to identify vasopressin or oxytocin neurons, we found that vasopressin decreased evoked EPSCs in vasopressin neurons while increasing EPSCs in oxytocin neurons. In both types of neurons, EPSC decay constants were not affected, indicating that desensitization of non-NMDA receptors did not underlie the EPSC amplitude change. In vasopressin neurons, both vasopressin and a V1a receptor agonist, F-180, decreased AMPA-induced currents, an effect blocked by a V1a receptor antagonist SR49059. In oxytocin neurons, AMPA-induced currents were facilitated by vasopressin, whereas F-180 had no effect. An oxytocin receptor antagonist blocked the facilitatory effect of vasopressin. Thus, we conclude that vasopressin inhibits EPSCs in vasopressin neurons via postsynaptic V1a receptors, whereas it facilitates EPSCs in oxytocin neurons through oxytocin receptors.

In the present study the immunohistochemical localization of proline-rich peptide, so called galarmin, was examined in the brain structures of intact and galarmin-treated rats. Galarmin (a fragment of neurophysin II C-end glycopeptide) was isolated by A.A. Galoyan and coworkers in 1997, from the neurosecretory granules of bovine neurohypophysis, produced by the hypothalamic magnocellular nuclei. In intact rats galarmin-immunoreactive neurons and nerve fibers were widely distributed in the central nervous system. Single intramuscular injection of galarmin to the rats resulted in the increase of both galarmin-immunoreactivity and the number of galarmin-immunoreactive nerve cells, fibers and capillaries. In control experiments where the antisera against the fragment of immunophilin (a receptor of immunosuppressor macrolide FK-506) and the pancreatic neuropeptide Y were used as the primary antibodies, the significant increase of neuropeptide Y-immunoreactive nerve fibers and immunophilin-positive lymphocytes was revealed in galarmin-treated rats. Based on these results and the data on the motoneurons regeneration in the spinal cord hemisectioned rats given galarmin daily for 3 weeks, galarmin has been suggested to act as an immunomodulator, neurotransmitter and neuroregulator.

The vasopressin (VP) gene is largely expressed in hypothalamic neurons, where the resultant pro-VP protein is enzymatically cleaved into its peptide hormone components, which include the neuropeptide VP, VP-associated neurophysin, and VP-associated glycopeptide (VAG). Small cell lung cancer (SCLC) tumors also express the VP gene, but the tumor pro-VP protein can remain intact and localize to the cell surface membrane. Previous studies have shown that polyclonal antibodies directed against different regions of the pro-VP protein bind specifically to the surface of cultured SCLC cells and recognize proteins of approximately 20 and approximately 40 kDa in cultured SCLC whole-cell lysate. Thus, these proteins have been designated neurophysin-related cell surface antigen (NRSA). A monoclonal antibody (mAb) designated MAG-1 was raised in this laboratory using a synthetic peptide representing the COOH-terminal sequence of VAG. The MAG-1 mAb recognizes NRSA in SCLC cell and tissue lysates by Western analysis, whereas immunofluorescent cytometric and microscopic analyses indicate that MAG-1 reacts specifically with NRSA on the surface of viable SCLC cells of both the classical and the variant subtype. Immunohistochemical analysis demonstrates that MAG-1 reacts with human SCLC tumor, but not with normal pulmonary epithelial cells in lung tissue. Additionally, a MAG-1 Fab fragment was generated that was also able to recognize NRSA. This is the first study to demonstrate that a mAb directed to the VAG region of the pro-VP protein has the potential for development into an in vivo diagnostic and therapeutic tool that targets plasma membrane-incorporated NRSA.

Thymic epithelial and nurse cells (TEC/TNC) synthesize an oxytocin (OT)-like peptide in association with a neurophysin (NP)-related protein in a way similar to in the hypothalamo-neurohypophysial (NHP) system. The central T-cell tolerance of the NHP neuroendocrine functions have been proposed to be mediated through these thymic NHP-related peptides due to their close homology with the NHP neurohormones OT and vasopressin (VP). In order to investigate their putative presentation by proteins of the major histocompatibility complex (MHC), human thymic membranes were purified and passed through an immunoaffinity column using mAb B9.12 directed to the monomorphic determinant of human MHC class I proteins. This methodology provided the following observations: (1) a NP-like protein is translocated in human thymic membranes and is retained by B9.12 on the column; (2) the MW of this NP-like material (50-55 kD) is quite different from the MW of hypothalamic NP proteins (10 kD), and (3) this thymic NP-like protein could be identified on Western blots with mAb B9.12. The precise extent of this relationship between the thymic NP-like protein and the Ig/MHC superfamily is actually investigated through the characterization of the genetic mechanisms responsible for the thymic expression of NHP-related peptides. Given the physiological importance of OT and of its binding to NP for transport along the axonal processes of the NHP tract, we postulate that, somewhat analogously, the thymic NP-/MHC class I-related protein could be involved in the presentation of the OT-like peptide to immature T-cells.

Previous studies have demonstrated that histamine primarily excites unidentified neurons in the rat supraoptic nucleus. We investigated the neuromodulatory effects of histamine on immunohistochemically identified vasopressin neurons in the rat supraoptic nucleus using intracellular recording techniques from the hypothalamo-neurohypophysial explant. Exogenous application of histamine (0.1-100 microM) to vasopressinergic neurons produced a small membrane depolarization accompanied by an increase of up to 100% in the amplitude of the depolarizing afterpotential that follows current-evoked trains of action potentials. The enhancement of the depolarizing afterpotential by histamine did not depend upon the depolarization. Further, histamine enhanced the amplitude of the depolarizing afterpotential when blocking the afterhyperpolarizing potential with d-tubocurarine or apamin, and in the presence of tetrodotoxin and d-tubocurarine or apamin, indicating a postsynaptic action of histamine on the depolarizing afterpotential that is not simply a reflection of a decrease in the afterhyperpolarizing potential. These toxins also had no effect on the histamine-induced depolarization. The enhancement of the depolarizing afterpotential by histamine was mimicked by the histamine H1-receptor agonist 2-thiazolylethylamine and was reduced or blocked by the H1-receptor antagonist promethazine, but was not blocked or reduced in the presence of the histamine H2-receptor antagonist, cimetidine. In summary, these results show that the excitatory effect of histamine on immunohistochemically identified vasopressin neurons in the supraoptic nucleus is due in part to the H1-receptor-mediated enhancement of the depolarizing afterpotential independent of any change in the afterhyperpolarizing potential or membrane potential.

Production by small-cell carcinoma (SCCL) of neurophysins (HNPs) and neurophysin-related cell-surface antigen (NRSA) was examined for two cell lines, for mouse xenografts, and for a resected human tumor, using polyclonal and monoclonal antibodies to vasopressin-associated human neurophysin (VP-HNP) and polyclonal antibodies to vasopressin (VP). The nature of the mRNA responsible for giving rise to these neurophysin-related products was investigated by performing Northern analysis on preparations of poly A+RNA and cDNA probes complimentary to portions of the exon A, exon B, and exon C regions of the human VP gene. SDS-electrophoresis and Western analysis revealed two prominent proteins of 42,000 and 20,000 Da in acid extracts from all SCCL sources when the monoclonal anti-HNP or one of the two polyclonal anti-HNP preparations were used. These antibodies also disclosed the presence of a minor component of 10,000 Da. A second polyclonal anti-HNP preparation reacted with one prominent protein of 30,000 Da and, for one cell line and mouse xenografts, another protein of 32,000 Da. Both of two anti-VP preparations reacted with proteins of 42,000, 30,000, 25,000, and 20,000 Da in extracts from all SCCL source material. The immunoreactive proteins of 42,000, 30,000, and 20,000 Da were all components of a membrane fraction from SCCL cells and tissues. In Northern analysis, a single RNA of about 900 bases hybridized with exon A and exon B probes, but not with the cDNA probe complimentary to exon C of the VP gene.(ABSTRACT TRUNCATED AT 250 WORDS)

Our earlier electrophysiological work provided evidence of a direct input to the supraoptic nucleus (SON) from the olfactory bulbs; however, these experiments could not determine if the input originated in the main and/or accessory portions of the olfactory bulb. Here, a connection between the accessory olfactory bulb (AOB) and the SON of the rat was examined using a combination of anatomic techniques. We employed neurophysin immunocytochemistry to delineate the morphological boundaries of the SON and the proximal arborizations of supraoptic dendrites. Accessory olfactory bulb efferents to the SON were studied by injection of wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP) into the AOB. The distribution of retrogradely labeled cells within the AOB was also determined after injection of either rhodamine-labeled latex microspheres (rhodamine beads) or Fluoro-Gold (FG) into the SON. Neurophysin immunocytochemistry revealed that SON dendrites extended beyond the generally accepted boundaries of the nucleus, coursing ventrolaterally along the surface of the periamygdaloid cortex. Anterograde tract tracing with WGA-HRP labeled AOB efferents including a dense plexus of terminals and fibers around the ipsilateral SON along the path of the ventrally projecting dendrites. Injections of retrograde tracers into the SON resulted in rhodamine bead or FG labeling of mitral cells throughout the ipsilateral AOB. Taken together, these anatomic studies suggest a direct projection from the accessory olfactory bulb to the SON of the rat and thus a vomeronasal organ to SON pathway.

Neuropeptide signals and specific neuropeptide receptors have been described in the thymus supporting the concept of a close dialogue between the neuroendocrine and the immune systems at the level of early T-cell differentiation. In this paper, we review recent data about neurohypophysial (NHP)-related peptides detected in the thymus from different species. We suggest that we are dealing in fact with other member(s) of the NHP hormone family, which seems to exert its activity locally through a novel model of cell-to-cell signaling, that of cryptocrine communication. This model involves exchange of signals between thymic epithelial cells and developing thymocytes. The NHP-related peptides have been shown to trigger thymocyte proliferation and could induce immune tolerance of this highly conserved neuroendocrine family.

Monoclonal antibodies to oxytocin (OT) and vasopressin (VP) revealed some positively staining stromal cells in the subcapsular cortex and in the medulla of the human thymus. We further demonstrated that these cells are a subset of epithelial endocrine cells and also contain immunoreactive interleukin-1 together with the neuropeptides. In addition, the thymic cells stained by monoclonal antibodies directed to the cyclic part of oxytocin or vasopressin also contained some immunoreactive neurophysins. These data support the concept of intrathymic synthesis of neurohypophyseal-like peptides fitting the hypothalamic model. However, we observed that, contrary to the situation in the brain, OT- and VP-like peptides colocalized in the same thymic cells. Furthermore, one monoclonal antibody, specific for the tail part of oxytocin, did not label thymic cells. Therefore, thymic neuropeptide(s) could be related to, but distinct from, authentic OT and VP. These observations suggest some molecular differences between hypothalamic and thymic oxytocin biosynthetic pathways which need to be further investigated.

A monoclonal antibody (mAb L6) to a small-cell lung carcinoma surface antigen recognizes a common epitope of vasopressin-neurophysin and oxytocin-neurophysin in hypothalamic nuclei. We now report on the identification of a neurophysin-like precursor in human lung carcinoma (LX-1) cell membrane. mAb L6 immunoaffinity chromatography of solubilized membranes resulted in a single band of approximately 45 kDa. Western blot analysis demonstrated immunoreactivity of this band with mAb L6, anti-vasopressin, and an antibody to the vasopressin precursor, pro-pressophysin. N-terminal sequencing of this band demonstrated a 21-amino acid homology with the N terminus of human pro-pressophysin, and substitution of a Cys33 residue in the tumor antigen with Arg33. Absence of immunoreactivity with the antibodies described above in cytosolic extracts and culture medium suggests nonsecretion of processed or intact pro-pressophysin-like peptide. Northern analysis of LX-1 mRNA with a 30-mer to the C terminus of rat pro-pressophysin resulted in a band of approximately 1000 base pairs, 250 base pairs larger than hypothalamic message. In situ hybridization of LX-1 tumor-bearing nude rat brain with the same probe demonstrated specific hybridization in rat hypothalamus and xenografted tumor. These findings suggest expression of a pro-pressophysin-like protein in this tumor cell line that is preferentially targeted to the cell membrane.

1. The development of the hypothalamo-neurohypophysial system was studied using immunocytochemistry with various antisera : anti-neurophysin, anti-oxytocin, anti-vasopressin, anti-vasotocin and anti-somatostatin. 2. Immunocytochemical investigation shows that anti-vasopressin and anti-vasotocin sera react with both vasopressin and vasotocin, whereas the anti-oxytocin serum specifically reveals the oxytocin-containing structures (Fig. 1g, h, i). 3. Perikarya stained with anti-neurophysin, anti-vasopressin and anti-vasotocin sera can be seen from the 11th week of fetal life (Fig. 1a) first in the supra-optic nucleus (SON), then in the para-ventricular nucleus (PVN). Their axons reach the neural lobe as early as the 11th week (Fig. 1b, c). 4. Oxytocin-containing perikarya appear simultaneously in the PVN (Fig. 1e) and SON during the 13th week. 5. From the 16th week, neurons stained with the anti-somatostatin serum can be seen among the neurophysin-positive cell-bodies of the SON and PVN. 6. The various populations of magnocellular neurons show a significant increase in number, especially up to the 19th week, and an increase in their hormonal content up to birth.

The immunohistochemical localization of CRF- and neurophysin-containing neurons in the hypothalamus of the Mongolian gerbil was studied by means of the PAP technique. The CRF-immunoreactive fibers were detected mainly in the outer layer of the median eminence of intact adult male gerbils. The CRF-positive neurons respond to aminoglutethimide (Elipten, Ciba) administration by showing increased immunoreactivity and an increase in the number of stained cell bodies in the parvocellular division of the paraventricular nucleus. Aminoglutethimide treatment results also in an increase in the number of neurophysin-immunoreactive nervous fibers localized in the internal layer of the median eminence. However, CRF-immunoreactive fibers are observed mainly in the outer layer of the median eminence while neurophysin-immunopositive axons are seen predominantly in the internal layer of this region. Since the axons of paraventricular neurons run to the median eminence and their staining ability is changed due to aminoglutethimide, their involvement in the endocrine control of hypophysial ACTH release is postulated.

The aim of the study was to investigate CRF- and neurophysin-immunoreactive neurocytes in hypothalamo-pituitary system of the hamster. CRF-immunoreactive nerve fibers were observed mainly in the outer layer of the median eminence and pituitary stalk and also in the neurohypophysis. On the contrary, neither intermediate lobe nor anterior pituitary contained CRF-immunoassayable substance. The pattern of distribution of neurophysin-immunoreactive fibres was different from CRF-immunoreactive fibres as far as a median eminence, pituitary stalk and neurohypophysis are concerned. Between the tannocytes of the III ventricle and nervous fibres forming the internal layer of the median eminence a CRF- and neurophysin-immunoreactive perikaryons of neurocytes were found. Results of the study suggest regulatory function of CRF-immunoreactive neurons of the hamster hypothalamo-pituitary system in controlling of ACTH secretion. Moreover, the distribution of CRF-immunoreactive substances in hamster hypothalamo-pituitary system shows some peculiarities if compared with other rodents.

The distribution of atrial natriuretic factor (ANF)-like reactivity was examined in rat brain and heart by immunohistochemistry. Immunostaining in heart was confined to atrial myocytes. In the hypothalamus, ANF-absorbable immunoreactivity was observed in magnocellular perikarya of the paraventricular and supraoptic nuclei, and in their projections to the neural lobe of the pituitary gland. No staining was seen in the preoptic or arcuate hypothalamic nuclei or in brain stem nuclei as previously reported by other investigators. The patterns of reactivity for ANF reported here is similar to that observed for neurophysins (NPs). Comparison of sequence data between rat ANF-28 and bovine NPs revealed three regions of 3 amino acid homology between these hypothalamic peptides. Preabsorption of the ANF antiserum with Affigel-coupled bovine NP I also resulted in complete elimination of all "ANF-immunoreactivity" in both atrium and hypothalamus. Cross-reactivity of the ANF antiserum with bovine NP I and II was further confirmed by Western blot analysis. Our findings suggest that ANF antisera can cross-react with NPs if they are directed against the shared antigenic epitopes; complete elimination of staining by preabsorption of the antibody with the immunogen, therefore, does not guarantee authenticity of localization. These observations may have relevance to an earlier study which reported on the existence of ANF-immunoreactivity in oxytocin neurons of the hypothalamus.

We have reported previously that autoantibodies in sera from patients with multiple sclerosis (MS) were reactive with rat brain, including pituitary, and with swine pituitary in areas thought to contain peptides of the somatotropin family and/or vasopressin/oxytocin. We have now tested the same patient sera for their specificity to antigenic determinants which are common to animal and human peptides. Localization of the binding sites of the MS sera was demonstrated in rat pituitaries and brains using the double immunofluorescence staining method, employing anti-bovine somatotropin (STH), anti-ovine prolactin (PRL), anti-neurophysin I and II, anti-somatostatin, and anti-vasopressin as reference antibodies. In the pituitary, the positive MS sera reacted specifically with cells which were also reactive with anti-bSTH. In the brain, positivity of MS sera was mainly localized in structures reactive with anti-neurophysin I and II and anti-vasopressin. Absorption experiments, immunocytochemical model assays, and radioimmunoassays, however, did not show specific binding of the MS sera to any of the above-mentioned peptides. Therefore, while these data present additional evidence on the localization of the immunocytochemical reaction sites of the MS autoantibodies, they do not enable us to identify the specificity of these antibodies.

Extracts of bovine neurohypophysis made in acid/ethanol solution containing protease inhibitors were fractionated by two successive filtrations on Sephadex G-75 columns equilibrated in the presence and then in the absence of 4 M urea. Analysis of the pattern of neurophysin-like immunoreactivity in the eluate, with two different antibodies, indicated the presence of high M(r) forms of neurophysin (apparent sizes, [unk]70,000 and 20,000-25,000, respectively) besides the M(r) 10,000 neurophysin. [8-Arginine]vasopressin-like immunoreactivity was also detected, coeluting with the neurophysin-like species, in the material recovered in the exclusion and M(r) 20,000-25,000 elution volumes of the same molecular sieve fractionation of neurohypophyseal extracts. Upon subsequent Sephadex G-150 filtration, the immunoreactive material recovered in the exclusion volume of the Sephadex G-75 filtration showed an apparent M(r) of approximately 140,000. Both neurophysin-like and vasopressin-like immunoreactivities coeluted in the same volume. The elution profile of this M(r) 140,000 material was unmodified when reanalyzed by the same molecular sieve filtration after exposure to 8 M urea. When these M(r) 140,000 immunoreactive forms of vasopressin and neurophysin were submitted to affinity chromatography on anti-neurophysin antibodies immobilized on Sepharose, both immunoreactivities were selectively coadsorbed to the immunoadsorbent. Similarly, the neurophysin and vasopressin immunoreactivities associated with M(r) approximately 25,000 were retained together on the same anti-neurophysin immunoadsorbent. The M(r) 140,000 and M(r) 25,000 species having both neurophysin and [8-arginine]vasopressin antigenic determinants generated the two neurosecretory components when exposed to proteolytic activities. This in vitro processing was inhibited in acid medium, at low temperature, and in the presence of a mixture of protease inhibitors. It is concluded that these two large forms of proteins containing both neurophysin and vasopressin may represent common biosynthetic precursors of these two neurohypophyseal components.

The distribution of fibres reacting with neurophysin antisera has been studied in the frontal cortex and anterior basal forebrain of young and adult rats. Fibres reacting with antisera raised against neurophysin A (vasopressin-neurophysin) were observed in the medial bank of frontal cortex, the anterior hippocampal rudiment, the rostral extension of substantia innominata lying between nucleus accumbens and olfactory tubercle, bed nucleus of stria terminalis, ventral pallidum, substantia innominata, lateral preoptic and magnocellular preoptic area. Axons were also identified lying in the anterior tip of the lateral ventricle and amongst the cells of the proliferative subependymal layer lining the ventricle. The significance of these findings is discussed. Particular attention is paid to the known connections of these, and associated regions, with limbic dopaminergic cell groups.

Seven mouse monoclonal antibodies (IgGs) were produced against rat neurophysins (NPs). Three were specifically directed against vasopressin-associated NP (NP-AVP), and four were specific for oxytocin-associated NP (NP-OT). These specificities were observed in liquid phase assays, immunoblot, and immunoprecipitation experiments. Homozygous Brattleboro rat tissues and extracts, which do not contain vasopressin or NP-AVP, did not react with the anti-NP-AVP antibodies but reacted with high affinity to the anti-NP-OT antibodies. In immunoprecipitation assays the antibodies brought down the appropriate NPs as well as their precursor molecules synthesized in vivo with no detectable cross-reactivity. In solid phase assays where the antigens were presented in a different manner, there was a significant cross-reactivity of the anti-NP-AVP antibodies with NP-OT. The extent of this cross-reactivity in solid phase correlated with the cross-reactivities of the antibodies observed in immunocytochemical studies. These solid phase (and immunocytochemical) data demonstrated that liquid phase specificities and absorption controls of antibodies are inadequate to assess their immunocytochemical (solid phase) specificities. Posterior pituitary extracts from the mouse and frog, as well as purified NPs from the rat, cow, and human were studied for their cross-reactivities to two of the antibodies, PS 36 and PS 45. In liquid phase assays the anti-rat NP-OT antibody, PS 36, reacted only with rat and mouse NPs and did not cross-react with NPs from any of the other species. In contrast, the anti-rat NP-AVP antibody, PS 45, was cross-reactive across species lines including an NP-like antigen extracted from frog posterior pituitaries. Immunoblot staining with these antibodies showed heterogeneity of NP-AVP and NP-OT in the rat posterior pituitary. Analysis of the epitopes for PS 36 and PS 45 indicated the antigenic determinants were located near amino acid positions 80 to 81 in NP-OT and 75 to 86 in NP-AVP, respectively.

Polyclonal rabbit antisera raised against oxytocin, bovine neurophysin I and vasopressin were used, together with an immunogold complex, to localise the peptides in ultrathin sections of ovine corpus luteum. The only organelle which consistently showed gold labelling was the secretory granule of the large luteal cell. In non-consecutive sections of the same large luteal cell all the granules showed a similar level of labelling after oxytocin or neurophysin I antisera: however no immunolabelling was detected for vasopressin. Oxytocin and neurophysin seem to be rapidly lost after secretion since exocytosed granule cores showed no labelling above background levels.

Serum from a patient who had been treated with Pitressin for extended periods was evaluated for the presence of autoantibodies. When this immune serum was used with either an immunofluorescence or an heterologous immunoperoxidase technique, positive staining was observed in the neurophysin-containing cells of the rat supraoptic, paraventricular and suprachiasmatic nucleus. Whereas the immune serum may also be expected to contain antibodies against oxytocin, vasopressin and the anterior pituitary hormones, this could not be substantiated with the immunohistochemical procedures.

These studies determined the differential autoradiographic distribution of [125I]alpha-bungarotoxin versus [3H]nicotine relative to the histochemically defined perikarya for neurophysin and corticotropin releasing factor (CRF). Specific [3H]nicotine binding sites occurred in relatively greater density within the neuropil surrounding PVN and SON compared to within the nuclei. In contrast, the highest density of [125I]alpha-BTX sites codistributed with neurophysin immunoreactive perikarya within these nuclei.

Preliminary studies are reported on the use of 131I-labelled antibodies against vasopressin associated human neurophysin to image tumors in patients with small cell carcinoma of the lung (SCCL). The rabbit polyclonal antibodies used in these studies were affinity purified on columns of neurophysin-Sepharose. Patients were pre-screened for the presence of neurophysin producing tumors by plasma RIA. Six patients who screened positive received approximately 1 mCi/70 kg body weight of radioiodinated antibody preparation, and scintigraphy was subsequently performed at 24 h, 48 h, and 72 h using a subtraction technique based on simultaneous imaging with radiopharmaceutical agents labelled with technetium-99m. Tumor was clearly demonstrated at 72 h in all five patients who had measurable lesions at the time of study, while no positive image was noted for one patient in complete clinical remission. Since approximately 70% of all SCCL patients have neurophysin-producing tumors, our findings suggest that neurophysin antibodies may be effectively used to scan tumors in a majority of patients with SCCL.

Analytical immuno high performance liquid affinity chromatography (analytical immuno HPLAC) was evaluated as a molecular diagnostic tool. Antibodies raised in rabbits against bovine neurophysin II were immobilized through Protein A crosslinking onto coated silica. Interaction of immobilized antibody with mobile antigen was characterized by zonal and frontal elutions of 14C-labeled bovine neurophysin II under isocratic, nondenaturing conditions. The chromatographic behavior shows that analytical immuno HPLAC with immobilized antibodies can be used to detect the number and functional nature of matrix-interacting antigens in mixtures, thus providing a quantitative chromatographic technology for "antigen mapping."

Ultrastructural post-embedding immuno-gold techniques were applied to the supraoptic nucleus and the neurohypophysis of mice and rats. The primary antibodies were three different monoclonal antineurophysins, used in protein A-gold and immunoglobulin-gold procedures. Conventional plastic embedding as well as hydrophilic media (L.R. White) were used; non-osmicated and osmicated tissues were immunolabeled; sodium metaperiodate oxidation was used, but was not essential for immunolabeling. Vasopressinergic and oxytocinergic NSGs were identified by the specific immunoreactivity of their respective neurophysins on adjacent thin sections, and by sequential double labeling on the same thin section using two different antibodies associated with gold probes of different diameters. The immunoidentification indicates that vasopressin NSGs can additionally be differentiated as larger, with more electron-dense matrix, and susceptible to damage by sodium metaperiodate. The only organelles consistently labeled were neurosecretory granules (NSGs), either intact or within lysosomal configurations. Some lysosomal dense bodies were immunoreactive even when discrete NSGs were no longer morphologically recognisable within them. Labeled NSGs were located within neuronal cell bodies, along axonal shafts and within axonal swellings and endings; occasionally immunoreactive NSGs were observed within synaptic boutons. Labeling intensity was semi-quantitatively gauged by counting gold particles in relation to numbers of NSGs per axonal varicosity. The precise localisation achieved with particulate immunogold labeling surpasses that previously obtained with diffuse electron-dense immunoreaction products.

Employing unlabeled antibody enzyme technique, the development of the hypothalamo-neurohypophysial system of the common Indian toad, Bufo melanostictus, has been studied by using antiserum raised in rabbit against bovine neurophysin. Immunoreactivity to the antiserum is first observed at stage 22 (Gosner, 1960) in the presumptive preoptic nucleus and at stage 23 in the presumptive median eminence and pars nervosa. As development proceeds, progressive increase in the immunoreactivity in all the components of the hypothalamo-neurohypophysial system is observed. The detection of neurohypophysial hormones (stages 22-23) much before the appearance of limb buds (stages 25-26) may be due to the greater sensitivity of the immunohistochemical technique.

Hypothalamic cells taken from 14-day-old mouse embryos were cultured for 6 days and transformed with simian virus 40. After cloning, a homogeneous cell population was obtained. Its morphological, Ultrastructural, biochemical, and immunochemical properties were studied. These cells possess ultrastructural features of primitive neurosecretory cells. They synthesize (35)S-labeled protein components that have the molecular weight and isoelectric focusing behavior of, and display the same immunoreactivity as, neurophysin. In addition, a (35)S-labeled peptidic fraction with a molecular weight close to 1000 is synthesized and is radioimmunologically indistinguishable from vasopressin. Immunochemical staining shows that both neurophysin and vasopressin are localized in the cytoplasm. These observations strongly suggest that a clone of mouse hypothalamic neurosecretory cells has been obtained with the synthesizing capacities of secretory neurons of the magnocellular hypothalamic nuclei.

Serial sections of vertebrate hypothalami were stained with the immunocytochemical peroxidase-antiperoxidase method. In addition to the single staining method, our double staining method was used, which enabled us to visualize two tissue antigens in single tissue sections. In both staining methods, differentially adsorbed antineurohypophysial hormone sera, anti-somatostatin serum and anti-bovine neurophysin sera were used. The results confirm the one hormone, one neuron hypothesis.

Arginine-vasopressin (AVP) and its associated neurophysin (AVP-NP) are synthesized via a precursor, propressophysin, which also contains a 39 amino acid glycopeptide at its C-terminus (C-terminus of propressophysin, or CPP). In the present study, immunocytochemical techniques were used to determine the cellular co-localization of CPP with AVP, oxytocin (OXY), AVP-NP and OXY-NP in the rat hypothalamus using colchicine pre-treatment and serial 5 micron section analysis. Extensive cross-competition studies of antisera raised against each peptide with the various antigens yielded no significant crossreactivity of the CPP, AVP, OXY and NP antisera. The NP antiserum, although directed against both AVP-NP and OXY-NP, demonstrated a preference for OXY-NP at a dilution of 1:20,000. CPP and AVP were always co-localized within the same magnocellular neurons of the supraoptic, paraventricular and circularis nuclei, and further showed very similar patterning in the suprachiasmatic nucleus as well. In contrast, no cellular overlap could be detected between CPP and OXY, in any of the above nuclei (the suprachiasmatic nucleus is devoid of OXY). Likewise, no examples of co-localization of CPP and OXY-NP were found in the magnocellular nuclei. These results are in strong agreement with a biosynthetic relationship between CPP, AVP and AVP-NP, and their separateness from the OXY and OXY-NP precursor.

Primary dissociated cultures were established from diencephalic tissue of 14-day-old fetal rats. Neurons exhibiting immunocytochemical staining for neurophysin appeared in these cultures after 6 days of cultivation. Addition of dimethyl sulfoxide (DMSO) to the culture medium resulted in a slight decrease in total neuronal cell mass as assessed by immunocytochemistry and radio-immunometric quantitation of neuron-specific enolase. In contrast, in DMSO-treated cultures the number of neurophysin-immunoreactive neurons was more than doubled as compared to control cultures. [3H]Thymidine labeling and autoradiography in conjunction with immunocytochemistry for neurophysin showed that this was not due to a mitogenic effect of DMSO on precursor cells. Time-course analysis of the action of DMSO revealed a 6-day time lag between the initiation of treatment and the appearance of increased numbers of neurophysin-immunoreactive cells. These findings suggest that DMSO, which has previously been reported to have a differentiation-inducing effect on malignant transformed cells, may also modulate cellular processes that control differentiation in specific types of neurons in primary culture.

The distribution of nerve fibers containing immunoreactive substance P (SP), estrogen-stimulated neurophysin (ESN), nicotine-stimulated neurophysin (NSN), oxytocin (OT), and vasopressin (VP) was examined in the epithalamic area of adult male and female macaques. Perfused or immersion-fixed epithalamic tissues, sectioned, and mounted on glass slides were processed through the avidin-biotin immunofluorescence method. Fibers containing immunoreactive SP were observed in the pineal organ along the periphery, in the perivascular space, and dispersed between the pinealocytes. Fibers were often observed in the pineal stalk region, and the habenular nuclei had high concentration of immunoreactive SP. Immunoreactive ESN fibers were observed in the stria medullaris, in the lateral habenula, in the pineal stalk, and in the pineal organ. Within the pineal, fibers containing ESN were present in the perivascular space, often concentrated in the walls of blood vessels, but also dispersed between pineal cells. Fibers containing OT, NSN, and VP were also present in the macaque pineal, but in lower quantities compared with fibers containing ESN. These studies show that the pineal of subhuman primates contain nerve fibers (ESN, NSN, VP, OT) of possibly hypothalamic origin. It also has a rich supply of SP fibers, which might be of habenula origin, peripheral parasympathetic ganglia origin, or both. The functional significance of these peptidergic nerve fibers remains to be determined. However, there are indications that they might be involved in regulation of blood flow and release of secretory products from the pinealocytes.

Limited tryptic fragmentation of disulfide-intact bovine neurophysins I and II (NP-I and -II, respectively) has been found to cause selective disruption of both hormone binding and neurophysin self-association. Loss of binding interactions, measured as a loss of ability to stimulate retardation of 125I-labeled neurophysin on Met-Tyr-Phe-amino-butylaminoagarose, is complete within 3 h at 37 degrees C. Reverse-phase high-performance liquid chromatography (HPLC) analysis of tryptic digests of neurophysin I allows detection of two major protein products and the peptide fragment 1-8. Release of the latter N-terminal piece occurs at about the same rate as loss of binding interactions. Reverse-phase HPLC elution behavior before and after performic acid oxidation and amino acid composition of the protein products led to their identification as NP-I-(9-93) (the 9-93 sequence) and [des-19,20]NP-I-(9-93) (the 9-93 sequence with the dipeptide 19-20 missing) for the more rapidly and more slowly formed species, respectively. NP-I-(9-93), unlike intact neurophysin I, is not retarded strongly by either Met-Tyr-Phe-amino-butylaminoagarose or neurophysin II-Sepharose. In contrast, both NP-I-(9-93) and [des-19,20]NP-I-(9-93) are equally as effective as intact NP-I in binding neurophysin I antibodies. The role of amino-terminal residues in promoting hormone binding, self-association, and antigenic recognition interactions is considered.

With the use of tissue prepared by freeze-substitution and the unlabelled antibody enzyme technique, neurophysin and vasopressin were localized at the ultrastructural level in the pofterior pituitary and median eminence of the guinea pig. In the posterior pituitary neurophysin was found in the large neurosecretory granules (1300-1500 A) of axons, Herring bodies, and nerve terminals. In some of these axons immunoreactive neurophysin was found outside of granules in the axoplasm. By light microscopy neurophysin was found in both the zona interna and zona externa of the median eminence; this was confirmed by electron microscopy. In the zona interna as in the posterior pituitary, neurophysin was localized both inside and outside the large neurosecretory granules. In the zona externa, immunoreactive deposit was primarily located in granules with a diameter of 900-1100 A in nerve terminals abutting on the primary portal plexus. The distribution of vasopressin paralleled that of neurophysin except that the hormone was rarely extragranular. These results demonstrate for the first time that both neurophysin and vasopressin are present in granules of axons that are in contact with the hypophysial portal vasculature.

The biosynthetic origin of the 10,000 molecular weight neurophysins, carriers of the peptide hormones oxytocin and vasopressin, has been studied by cell-free synthesis, Poly(A)-RNA was isolated from bovine hypothalamus and translated in a wheat germ system containing (35)S- or (3)H-labeled amino acids. A number of unique [(35)S]cysteine- but few [(35)S]-methionine-labeled proteins were coded by hypothalamic mRNA. A single, major, isotopically labeled protein (molecular weight 23,000-25,000) was immunoprecipitated from these translation mixtures by addition of purified antibodies against bovine neurophysin II and subsequent addition of Cowan I strain of Staphylococcus aureus. Specificity of the immunoprecipitation was demonstrated by competition with unlabeled authentic neurophysins and the absence of competition with structurally unrelated ovalbumin. Furthermore, neither nonimmune serum nor purified antibodies against ribonuclease immunoprecipitated the protein. The [(35)S]cysteine-labeled protein that was specifically immunoprecipitated was oxidized with performic acid and digested with trypsin in the presence of unlabeled, authentic bovine neurophysin II. Peptide mapping revealed that most of the major [(35)S]cysteine-labeled peptides (of the translation product) were identical to major cysteine-containing peptides of authentic neurophysin. The data show that hypothalamic mRNA directs the translation of several unique cysteine-rich proteins in an in vitro cell-free system. Furthermore, one of these proteins, which has a higher molecular weight than authentic neurophysin, is recognized by purified antibodies to bovine neurophysin II and has cysteine-containing tryptic peptides in common with those of authentic neurophysin. The data suggest that this protein is the primary translation product, pre-pro-neurophysin.

Monoclonal antibodies (MAbs) raised against the neurophysin (NP) specifically synthesized with vasopressin (VP, VP-NP) were injected into the paraventricular nucleus (PVN) of the rat hypothalamus. Their fate was studied by immunocytochemistry from 1 min to 3 h after the end of injection. It could be demonstrated that the VP-NP MAbs penetrated in vivo into some magnocellular neurons of the injected PVN and were transported ipsi- and contralaterally in individual neurons and in accessory magnocellular groups. When the time after injection was longer than 15 min, the VP-NP MAbs were also carried in the fibers of the median eminence. The prior treatment of rats with colchicine did not prevent the uptake of VP-NP MAb in the neurons but inhibited the transport towards the eminential fibers, the individual neurons and accessory groups. The detection of the PVN endogenous peptides (VP and oxytocin) on the same brain sections indicates that the neuronal uptake was specific. It only occurred in the neurons which synthesized VP and never appeared in the brain of rats suffering from a genetic defect of the central VP synthesis (Brattleboro rat). These data support the hypothesis of the location on the cell surface of the VP-NP precursor in magnocellular neurons which synthesize VP. This membrane signal identifies the neuron and allows the immunological recognition of the neurosecretory neurons in vivo.