Distribution of nicotinic binding sites with respect to CRF and neurophysin immunoreactive perikarya within the rat hypothalamus.

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.

Use of a cross-species immunohistochemical procedure to demonstrate the presence of neurophysin antibodies in the serum of a patient treated with Pitressin.

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.

Neurophysin in the hypothalamo-neurohypophysial system. I. Production and characterization of monoclonal antibodies.

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.

Immunocytochemical localization of the posterior lobe hormones and of their carrier proteins.

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.

Immunological and chemical identification of a neurophysin-containing protein coded by messenger RNA from bovine hypothalamus.

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.

Immunocytochemical demonstration of separate vasopressin-neurophysin and oxytocin-neurophysin neurons in the human hypothalamus.

With the use of immunocytochemistry, it was shown that both the supraoptic and paraventricular hypothalamic nuclei in humans contain at least two different neurophysins. These two human neurophysins are immunologically related to bovine neurophysin I and neurophysin II, respectively. One human neurophysin is associated with vasopressin, the other wiht oxytocin. Human vasopressin-neurophysin and oxytocin-neurophysin are located separately in two different types of neurons, which correspond respectively to the vasopressinergic and oxytocinergic neurons of both the supraoptic and paraventricular nuclei. The neurophysin of the human vasopressinergic suprachiasmatic neurons appears to be closely related to or identical with neurophysin of the vasopressinergic neurons of the human magnocellular hypothalamic nuclei.

Preparation of antisera to three individual rat neurophysins and their use for radioimmunoassays.

A number of antisera have been raised against individual rat neurophysins. One of these proved suitable for the specific radioimmunoassay of rat vasopressin-neurophysin and data validating such a use are presented. Use of this antiserum showed that vasopressin-neurophysin is present in rat neurohypophyses in equimolar amounts to vasopressin, while in the hypothalamus the assay detected twice as much neurophysin-like material as hormone. A number of partially specific antisera for rat oxytocin-neurophysin have been obtained and the best of these was raised against the minor neurophysin.

Bovine neurophysins I, II and C: new methods for their purification and for the production of specific antibodies.

1. Bovine neurophysins were prepared by a modified method, in which a Biogel P-60 column was used. This yielded two neurophysin fractions, the first containing neurophysin I and small quantities of the other neurophysins,the second containing neurophysin II and C, and only traces of neurophysin I. 2. Antibodies against neurophysin I, II and C were prepared by an original method, 5 mug in 100 mul water of each of the two fractions were applied on a gel slab and separated by iso-electric focusing in a pH gradient 4--6. The separated bands were visualized with 8-aniline-1-naphthalene sulfonic acid, magnesium salt and strips respectively containing neurophysin I, II or C were cut out. The neurophysin-containing strips were homogenized in complete Freund's adjuvant and injected into rabbits. 3. The specificity of the antisera were tested by immunocytochemistry and by radioimmunoassay. By this latter method, it was determined that cross-reactivity was less than 1%. The cross-reaction, observed with the immunohistochemical method could be eliminated by differential absorption. 4. It was found that neurophysin C antisera were undistinguishable from the neurophysin II antisera, while showing little cross-reactivity with the neurophysin I antisera. This suggests that in vivo neurophysin C is not a real neurophysin, or at least, that it is very similar to neurophysin II. 5. Highly purified bovic focusing method. By modifying a LKB Uniphor electrophoresis apparatus, the elute the proteins without switching off the voltage. The resolution of the technique is close to that offered by analytical gel iso-electric focusing.

Establishment of a clone of mouse hypothalamic neurosecretory cells synthesizing neurophysin and vasopressin.

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.