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PubMed1995

Neurophysin stimulates prolactin release from primary cultured rat pituitary cells.

The neurohypophysial hormones, oxytocin and vasopressin, are present as non-covalently bound complexes with their designated neurophysin in the secretory granules of the posterior pituitary. The neurophysins are generally considered to be biologically inert carrier proteins for oxytocin and vasopressin. We have examined the actions of bovine neurophysin-I (bNP-I), bovine neurophysin-II (bNP-II), rat neurophysin (rat NP) and oxytocin on prolactin release using primary cultured rat pituitary cells. A dynamic perifusion system was chosen to test their stimulatory actions. The rat NP and bNP-II stimulated prolactin release. It is a new observation that rat NP and bNP-II stimulate prolactin release from primary cultured rat pituitary cells. The maximum sensitivities, the lowest concentration which stimulate prolactin release, of rat NP, bNP-II, bNP-I and oxytocin in primary cultured cells were 1 nmol/1, 1 nmol/l, 1000 nmol/1 and 1 nmol/1 respectively. The maximum sensitivities of rat NP and bNP-II were within the physiologically relevant concentrations.

Animals↗

PubMed1975

Interactions of bovine neurophysins with neurohypophyseal hormones. On the role of tyrosine-49.

Reaction of tetranitromethane with the lone tyrosine residue of bovine neurophysin I and II, tyrosine-49, gave nitro derivatives of these proteins which were obtained in a highly purified form by preparative electrophoresis. Equilibrium dialysis experiments indicated clearly that oxytocin binding remained essentially unaffected by the chemical modification of tyrosine-49. However, in the case of (8-lysine)vasopressin, the nitrated protein was found to bind only 1 hormone molecule in contrast to the 2 vasopressin molecules bound by the native protein. Ultraviolet absorption difference spectroscopy measurements between 250 nm and 300 nm indicated that upon binding of (2-phenylalanine, 8-lysine)vasopressin, tyrosine-49 of native neurophysin undergoes a change of microenvironment from less to more polar surroundings. Studies of the nitrotyrosyl-49 chromophore of neurophysin by ab sorption spectroscopy in the absence and presence of oxytocin or (8-lysine)vasopressin confirmed this finding. Since dimethylsulfoxide solvent perturbation studies suggested that in the Cys(Me)-Phe-Ile-NH2-neurophysin I complex, tyrosine-49 is more exposed to solvent than in neurophysin I alone, it is concluded that this residue is unmasked by conformational changes upon complex formation.

Amino Acids↗

PubMed1977

[Somatostatin-containing fibers and neurophysin-containing fibers in the median eminence of the fox, as seen with a double staining cytoimmunoenzyme technique].

In the external layer of the median eminence of the fox, the somatostatin-containing fibers and neurophysin-containing fibers of the hypothalamo-infundibular tract are located in distinct areas. In the neural lobe, somatostatin-positive areas are simultaneously neurophysin-positive. Outside the SON and PVN, some somatostatin-positive and neurophysin-negative perikarya are scattered close to the third ventricle. These facts suggest the existence of two somatostatin systems: a hypothalamo-infundibular (neurophysin-negative) one and a hypothalamo-neurohypophyseal (neurophysin-positive) one.

Animals↗

PubMed1987

Sequence redesign and the assembly mechanism of the oxytocin/bovine neurophysin I biosynthetic precursor.

The structural organization of neurohypophysial hormone biosynthetic precursors and the interdependence between intramolecular folding and precursor self-association were examined using sequence-engineered mutants of the semisynthetic oxytocin/bovine neurophysin precursor (pros-OT/BNPI). In [N alpha 1-Ac,N epsilon 30,71-diacetimidyl, Ala2,des-His106] Pro-Ot/BNPI or [N alpha 1-Ac,Ala2]pros-OT/BNPI), two structural elements (Tyr2 and free alpha-amino group) were eliminated which were predicted to be critical for intramolecular conformation by stabilizing contact between hormone and neurophysin domains. This mutant was used to test the dependence of precursor self-association on intramolecular conformation. In the second mutant precursor, [N alpha 30,71-diacetimidyl,D-Pro7,D-Leu8,des-His106]p ro-OT/BNPI (or [D-Pro7,D-Leu8]pros-OT/BNPI), the stereochemistry at L-Pro7-L-Leu8 was changed to test the extent to which precursor conformation depends on ordered structure in the processing/spacer sequence which connects the interacting hormone and neurophysin I domains. Intramolecular conformation was characterized for the precursor and mutants by analytical affinity chromatography on immobilized hormone analog Met-Tyr-Phe and by circular dichroism. Data obtained by both methods showed that, while pros-OT/BNPI is folded, with hormone domain occupying the hormone-binding site of the neurophysin domain, the alpha-acetyl-Ala2 mutant is not so organized intramolecularly. When pros-OT/BNPI and the alpha-acetyl-Ala2 mutant were eluted on immobilized BNPII to measure self-association propensity, the native-like precursor was found to bind with 12-15-fold higher affinity than the assembly mutant. Thus, while pros-OT/BNPI assumes a molecular structure containing a high-affinity self-association surface induced by intramolecular hormone domain-neurophysin domain interaction, [N alpha 1-Ac,Ala2]pros-OT/BNPI does not. The results with the alpha-acetyl-Ala2 mutant show that intramolecular domain-domain interaction is the obligatory "trigger" which induces the high-affinity precursor self-association that likely drives precursor to aggregated forms in the concentrated intragranular environment that exists in peptide hormone-synthesizing cells. In contrast, affinity chromatographic and circular dichroism properties of the D-Pro7,D-Leu8 mutant show that this intramolecular trigger is dependent, but only weakly, on the conformation of the peptide sequence between domains, as judged by native-like interaction properties below 40 degrees C but lowered stability to elevated temperature.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗

PubMed1985

Neurophysin in the hypothalamo-neurohypophysial system. II. Immunocytochemical studies of the ontogeny of oxytocinergic and vasopressinergic neurons.

Two anti-neurophysin monoclonal antibodies (MABs), PS 36 and PS 41, described in the preceding paper (Ben-Barak, Y, J.T. Russell, M.H. Whitnall, K. Ozato, and H. Gainer (1985) J. Neurosci. 5:000-000), allowed us to specifically stain for oxytocin-associated neurophysin (NP-OT) or vasopressin-associated neurophysin (NP-AVP) in the hypothalamus of developing rats. Staining with these MABs specific for NP-OT or NP-AVP showed that both types of neurophysin appeared in cells in the developing hypothalamus as early as embryonic day (E16) and continued to increase in immunoreactivity throughout fetal life. The literature indicated that oxytocin appears in the system between E20 and E22, much later than vasopressin (E16 to E17), which we confirmed in immunocytochemical experiments using affinity-purified antisera to these hormones. Since the MABs recognize the specific prohormones as well as the specific mature neurophysins (Ben-Barak, Y., J. T. Russell, M.H. Whitnall, K. Ozato, and H. Gainer (1985) J. Neurosci. 5: 81-97), we conclude that there is a developmental delay between the synthesis of the oxytocin prohormone (pro-oxyphysin) and its processing to form oxytocin and NP-OT. The delay in prohormone processing in the oxytocin cells was correlated with a delay in immunocytochemically detectable neurites as compared to the vasopressin cells. This reduced level of axonal and dendritic immunoreactivity was still obvious in the oxytocin cells at 9 days after birth. In contrast, the clustering of cells to form adult-like hypothalamic nuclei appeared to follow similar time courses for the two types of cells. Adult-like distributions of cells staining for NP-OT and NP-AVP were already apparent in the supraoptic and paraventricular nuclei by E17.

Animals↗

PubMed1978

Integrated morphology of neuronal catecholamines and neurophysin in the aged macaque.

A new method for the simultaneous visualization of brain peptides and monoamine neurotransmitters was employed to analyze the integrated morphology of hypothalamic catecholamines and neurophysins in young and old monkeys. Immunocytochemical analysis using bovine neurophysin revealed a dual papulation of light and dark stained cells in the paraventricular nucleus in young and old macaques. In general, both populations of neurons stained with less density in old macaques indicating the possibility of a reduced content of neurophysin. Further analysis using specific neurophysin antisera for vasopressin or oxytocin revealed an appreciable decrease in the number of vassopressin-containing perikarya in the 20 year monkey whereas oxytocin-synthesizing neurons did not show a similar change in numbers with age. Qualitatively, terminal innervation patterns of hypothalamic catecholamines remained strikingly constant in spite of marked reductions in dark-stained neurophysin perikarya of the paraventricular nucleus.

Aging↗

PubMed1968

The isolation of neurophysin-I and-II from bovine pituitary neurosecretory granules separated on a large scale from other subcellular organelles. Demonstration of slow equilibration of neurosecretory granules during centrifugation in a sucrose density gradient.

1. An improved procedure for the isolation of neurosecretory granules from the posterior lobe of the bovine pituitary gland is described. 2. Of the total oxytocic and pressor activities present in the original tissue 80% was sedimentable. 3. The granules were separated from mitochondria by prolonged centrifugation in a sucrose density gradient. During a sedimentation period of 5hr. the granules moved progressively into denser regions of the gradient and the mitochondria remained at the top. 4. The biological activities of the granules were measured: the oxytocic activity was 11.56+/-1.63 and the pressor activity was 15.60+/-3.91 units/mg. of protein. 5. A protein was isolated from a lysate of granules prepared from 40 pituitary glands. Amino acid analysis showed that it consisted of a mixture of neurophysin-I and neurophysin-II in equal proportions. It accounted for 60% of the soluble granule protein and for 50% of the total granule protein. 6. The neurophysins present in the granules are associated with 19.1 units of oxytocic and 21.1 units of pressor activity/mg. of protein. 7. Starch-gel electrophoresis revealed the presence of both neurophysins in extracts of 15 pituitary glands studied individually. 8. We conclude that the polypeptide hormones, oxytocin and [8-arginine]-vasopressin, are normally closely associated with the two neurophysins within neurosecretory granules of the pituitary gland.

Acid Phosphatase↗

PubMed1996

Crystals of ligand-free bovine neurophysin II.

A modified neurophysin, des 1-6 bovine neurophysin II, has been crystallized in the absence of bound hormone or hormone analogue. These crystals represent the first crystals of ligand-free neurophysin, and are essential for understanding neurophysin-hormone recognition as well as hormone-induced neurophysin dimerization. The crystals diffract to beyond 1.8 A resolution, belong to space group P3(1)21 (or P3(2)21) with a = 48.86, c = 78.61 A, and contain one molecule per asymmetric unit.

Journal Article↗

PubMed1975

The hypothalamic magnocellular neurosecretory system of the guinea pig. I. Immunohistochemical localization of neurophysin in the adult.

With the use of the unlabeled antibody enzyme technique and antiserum against bovine neurophysin I, neurophysin was localized in the hypothalamic magnocellular neurosecretory system of the adult guinea pig. Immunoreactive deposits were found in the perikarya of the supraoptic and paraventricular nuclei, their fiber projections and terminals in the posterior pituitary. No parvicellular neurophysin-positive components were observed. In the median eminence neurophysin was seen in the zona interna where axons of the supraoptico-hypophysial tract pass on their way to the neural lobe. The peptide was also present in axons projecting into zona externa which terminate on the primary portal plexus.

Animals↗

PubMed1987

Immunocytochemical localization of oxytocin and neurophysin in human corpora lutea.

Corpora lutea, corpora albicantia, and ovarian stroma from normal human premenopausal ovaries were examined for the presence of oxytocin and neurophysin by using highly specific antisera and peroxidase-antiperoxidase light-microscopic immunohistochemistry. Oxytocin and neurophysin immunoreactivity was found in some but not all cells of the corpora lutea obtained on days 19 to 24 of the menstrual cycle. Stromal tissue and corpora albicantia did not give a positive reaction for either of these peptides, and negative results were also obtained with corpora lutea of mid- and term-pregnancy and preovulatory follicles. Specificity of the immunohistochemical reaction was confirmed by immunoabsorption tests. The specific localization of immunoreactive oxytocin and neurophysin in corpora lutea of the human menstrual cycle directly demonstrates the presence of oxytocin- and neurophysin-positive cells within the human corpus luteum.

Adult↗

PubMed1990

Direct immunoblotting from histological sections of brain onto nitrocellulose: evaluation with the protein neurophysin.

A method for the isolation and localization of proteins and peptides from histological sections of rat and human brain by immunoblotting is described. For validation, the well-characterized protein neurophysin was electrophoretically transferred from formaldehyde-fixed or fresh tissue sections onto a nitrocellulose membrane. Neurophysin on the nitrocellulose membrane was detected by a specific antibody reaction. The antibody against neurophysin was visualized either by using secondary antibodies, conjugated with peroxidase or by protein A gold, followed by enhancement with silver. With this simple and fast method, neurophysin (or other proteins and peptides) can be identified on nitrocellulose membranes in areas that correspond to anatomically defined regions. Since the procedure combines the advantages of precise regional localization of polypeptides with the specificity of antibody-antigen reactions, the method may prove useful for rapid screening of the distribution of peptides or proteins in (brain) tissue.

Animals↗

PubMed1983

Structural organization of the rat gene for the arginine vasopressin-neurophysin precursor.

The rat arginine vasopressin-neurophysin precursor gene has been isolated from a genomic library cloned in lambda phage Charon 4A. Restriction mapping and nucleotide sequence analysis demonstrated that the gene is 1.85 kilobase pairs long and contains two intervening sequences located in the protein coding region. Exon A encodes a putative signal peptide, the hormone arginine vasopressin and the variable N terminus of the carrier protein neurophysin, exon B encodes the highly conserved middle part of neurophysin and exon C its variable C terminus together with glycoprotein. Thus, the three functional domains of the percursor - arginine, vasopressin, neurophysin, glycoprotein - are encoded on three distinct exons.

Animals↗

PubMed1975

Drug-biomolecule interactions: proton magnetic resonance studies of complex formation between bovine neurophysins and oxytocin at molecular level.

Proton magnetic resonance spectroscopy was used to monitor individual amino acid residues in bovine neurophysin, in the nonapeptide hormone oxytocin, and in the complex formed between them. For neurophysin I alone, a normal titration curve for the C-2 proton resonance of the lone histidine residue was obtained with an apparent ionization constant of 6.9 addition of oxytocin to a solution of neurophysin I at pH 6.5 resulted in several changes in the spectrum. The effect on the histidine C-2 proton resonance signal indicated a slow exchange process between two states, probably representing a conformational change in the protein. The apparent pK of the histidine residue in the hormonal complex was shifted to 6.7, indicating a slightly more positive (less electron dense) environment for the histidine residue. Resonances of the single tyrosine residue of oxytocin were observed to broaden significantly, but not to shift appreciably, on the addition of neurophysin II. These observations may indicate involvement of the tyrosyl residue of oxytocin in the hormone-"carrier protein" interaction.

Animals↗

PubMed1982

Neurophysin pathways in the normal and hypophysectomized rat.

The hypothalamo-extrahypophyseal neurophysin pathways (HEH) and the three hypothalamic nuclei secreting neurophysins, the supraoptic (SON), paraventricular (PVN) and suprachiasmatic (SCN) nuclei, of normal and hypophysectomized rats were studied by application of the immunoperoxidase procedure. Eight well-defined HEH pathways were recognized. Their main sites of projection were: lateral septum and subfornical organ (1 and 2); tractus diagonalis (3); medial nucleus of the amygdala and lateral ventricle (4); nucleus periventricularis thalami, nucleus habenulae lateralis and periaqueductal gray (5); periaqueductal gray, pineal organ, collicular recess and subependymal region of the fourth ventral (6); dorsomedial nucleus and premammillary area (7); perimammillary region, corpus trapezoideum, ventral surface of medulla oblongata, nucleus tractus solitarii, nucleus commissuralis, substantia gelatinosa and formatio reticularis lateralis of the medulla oblongata and spinal cord (8). Neurophysin fibers of unknown origin were found in the frontal cerebral cortex. It was noted that in pathway 5 the amount of immunostainable material undergoes changes with age. The three neurophysin-secreting nuclei reacted differently following hypophysectomy. Among the HEH pathways the only one that seemed to be affected by hypophysectomy was that innervating the lateral septum. It is suggested that the neurons that survive hypophysectomy either do not project to the neural lobe or, alternatively, display axon collaterals projecting outside the neural lobe. Such a neuronal population could be the origin of the HEH pathways.

Aging↗

PubMed1975

Time of fixation and the localization of Gomori-positive and neurophysin-containing structures in the rat hypothalamus.

Rats were decapitated and the complete head stored at 4 degrees C. At various time intervals, up to a maximum of 14 days, the brains and pituitary glands were removed and fixed in formalin-picric acid. Neurosecretory material (NSM), as revealed with crotonaldehyde fuchsin, and neurophysin as demonstrated by means of immunoperoxidase histochemistry were localized in the hypothalamic magnocellular nuclei, median eminence and posterior pituitary gland. NSM and neurophysin were present in the paraventricular (PVN) and supra-optic (SON) nuceli up to periods of 14 days although the cellular morphology was disrupted after about 4 days. After 4 days there was an abundance of positive staining "droplets" in both the SON, PVN and along the nerve fibers of the supraoptico-neurohypophysial tract. Pituitary gland NSM and neurophysin were still present 14 days post mortem. The slab gradient polyacrylamide gel electrophoresis pattern of soluble proteins extracted from the posterior pituitary gland from the experimental and normal animals showed almost complete identity confirming the minimal action of proteolytic enzymes at 4 degrees C up to a period of 6 days. It is concluded that as long as the tissue is cooled, rapid fixation of hypothalamic tissue is not essential for the localization of NSM and neurophysin.

Animals↗

PubMed1976

Immuno-cytochemical demonstration of the inability of the homozygous Brattleboro rat to synthesize vasopressin and vasopressin-associated neurophysin.

Immuno-enzyme cytochemical investigations have shown that, (1) the hypothalamic supraoptic and paraventricular nuclei of the Brattleboro rat, as in the normal rat, contain separate neurons which produce oxytocin + neurophysin; (2) the hereditary inability of the Brattleboro rat to synthesize vasopressin and its associated neurophysin is due to a biochemical defect of separate "neurophysin-vasopressin" neurons in the supraoptic and the paraventricular nuclei. These observations strongly support the hypotheses that (1) vasopressin and its associated neurophysin are formed via a common precursor, and (2) the initial point of intracellular appearance of the hereditary defect in the Brattleboro rat lies in the synthesis of this precursor, which occurs on ribosomes. Moreover, observations have demonstrated that, in the Brattleboro rat, in addition to the hereditary inability of the hypothalamic magnocellular neurosecretory system to synthesize vasopressin, there also exists a similar hereditary defect in the hypothetical parvicellular suprachiasmatic-median eminence neurosecretory system.

Animals↗

PubMed1975

Presence of neurophysin and vasopressin in the hypothalamic magnocellular nuclei of rats homozygous and heterozygous for diabetes insipidus (Brattleboro strain) as revealed by immunoperoxidase history.

The 3-layer immunoperoxidase-bridge technique was used to study the distribution of neurophysin and vasopressin in the neurosecretory neurons of rats homozygous and heterozygous for diabetes insipidus (Brattleboro strain). In the homozygous rats there was a marked hypertrophy of the hypothalamic magnocellular structures when stained either for neurosecretory material or neurophysin-like antigens. Neurophysin was present in both the paraventricular (PVN) and supraoptic nuclei (SON) of homozygous and heterozygous animals. Less than half of the cells in the PVN and SON were stained for neurophysin. This observation was less apparent when histochemical stains were used to visualize the distribution of neurosecretory material. Although it is generally considered that the homozygous Brattleboro rat does not synthesize vasopressin, a positive reaction was observed in the PVN and SON when anti-[8-lysine]-vasopressin serum was employed in the immunohistochemical procedure.

Animals↗

PubMed1980

Ultrastructure and immunocytochemistry of neurons in the supraoptic and paraventricular nuclei of the lizard Liolaemus cyanogaster. Evidence for the intracisternal location of the precursor of neurophysin.

The supraoptic (SON) and paraventricular (PVN) nuclei of the lizard Liolaemus cyanogaster c. were studied by use of histochemical, immunocytochemical and electron microscopic methods. The immunofluorescence staining for neurophysin was applied to methacrylate-embedded material before and after treatment of the sections with urea and trypsin. Pseudoisocyanine was applied to sections previously used for immunocytochemistry. The ultrastructural study showed that the SON and PVN neurons posses neurosecretory granules (nsg), distributed throughout the perikaryon, and large (2 to 12 micrometers) electron-dense droplets located within dilatations of the cisternae of the rough endoplasmic reticulum. Whereas the perikaryon (nsg) and the secretory droplets are stainable with pseudoisocyanine, only the former displays immunoreactivity for neurophysin. However, after treating the sections with urea and trypsin, the same secretory droplets become immunoreactive. It is suggested that the secretory droplets are sites of storage for the precursor of neurophysin, and that the tryptic digestion either triggers its conversion into neurophysin or exposes its immunoreactive sites. Based on the ultrastructure and the histochemical behavior of the secretory droplets, it is also postulated that they contain, in addition to peptides, a glycoprotein component.

Animals↗