THE DISTRIBUTION OF NEUROPHYSINS AND POSTERIOR PITUITARY HORMONES IN THE PORCINE NEUROHYPOPHYSEAL SYSTEM

Specific, homologous porcine neurophysin I and II radioimmunoassays were established together with specific oxytocin and vasopressin radioimmunoassays. The levels of each of these proteins and peptides were measured in acid extracts of individual paraventricular nuclei, supraoptic nuclei, neurohypophyseal stalks and posterior pituitary lobes of 12 pigs in order to quantitate the neurophysin-hormone relationships in the porcine neurohypophyseal system. Neurophysin III was found to be immunologically identical to neurophysin I. Neurophysin measurements by radioimmunoassay were quantitatively validated by scanning densitometry of polyacrylamide gels stained with 0.5% amido schwarz. In the hypothalamic nuclei vasopressin was in 3–4 M excess of oxytocin but in the neurohypophyseal stalk and posterior pituitary lobe the hormones were equimolar suggesting that the rate of formation of vasopressin differs from that of oxytocin. Neurophysin I immunoreactivity was present in a 3:1 molar ratio with neurophysin II throughout the porcine neurohypophyseal system. In posterior pituitary lobes total neurophysins were equimolar to total hormone concentrations. The specific activity (pmol/mg extracted protein) of oxytocin increased 1800 times, vasopressin 560 times and neurophysins about 360 times from the paraventricular nucleus to the posterior pituitary lobe. In the hypothalamic nuclei relationships between immunoreactive neurophysin I and vasopressin, and between neurophysin II and oxytocin were highly significant. In the posterior pituitary lobe each immunoreactive neurophysin level correlated with both hormone levels. Quantification of densitometric scans of stained polyacrylamide gels from neurophypophyseal extracts and immunoreactivity patterns of neurophysins in eluates of sliced, duplicate gels indicated that neurophysin III decreased distally within the neurohypophyseal tract while neurophysin I increased. The results demonstrated that vasopressin was associated with porcine neurophysin I. However, oxytocin may be associated with both immunoreactive neurophysin I and neurophysin II in the porcine neurohypophyseal system if a 1:1 molar ratio of neurophysin to hormone is to be maintained. Neurophysin III contributed to the stoichiometry of this relationship.     

Secretion of neurophysins during luteal regression in the goat

In non-pregnant goats, ovariectomy on day 12 of oestrous cycle resulted in parallel decrease of oxytocin and progesterone jugular concentration. Similarly, luteolysis, indicated by decreasing progesterone concentration, was accompanied by simultaneous release of oxytocin and oxytocin-associated neurophysins (mean of neurophysin I and II). It is suggested that the neurophysins are secreted concomitantly with oxytocin by the ovary during luteal regressions in the goat.     

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

Summary 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 with 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.This investigation was supported by a grant from the Belgian Nationaal Fonds voor Geneeskundig Wetenschappelijk Onderzoek     

Binding studies of polypeptide hormones to bovine neurophysins.

Experimental binding isotherms of [9-glycinamide-1-(14)C]oxytocin and [9-glycinamide-1-(14)C]arginine vasopressin to purified neurophysins I and II at pH = 4.4, 5.4, 6.5, 7.4, and 8.5 and 6 degrees, 22 degrees, and 37 degrees in aqueous buffers are reported. For purposes of comparison, binding isotherms for [4-glycine-1-(14)C]oxytocin to neurophysin II and I in aqueous buffer, and [9-glycinamide-1-(14)C]oxytocin to neurophysin II in dimethylsulfoxide under selected conditions are also reported. A brief discussion of the interpretation of binding isotherms is entered into and apparent binding constants are derived. The results indicate that the interpretations presented in the literature up to now are much too simple. There are, in contrast, multiple binding sites of oxytocin and vasopressin to the neurophysins and large temperature dependences of the number of sites and their binding constants. We find, in fact, that at 37 degrees the binding of neurohypophysial hormones to the supposed storage proteins is rather weak even at the pH of maximum binding.     

Presence of neurophysins I and II in the human pineal gland: Comparison with the content of neurohypophyseal hormones

We have previously measured the individual content of immunoreactive vasopressin (AVP), oxytocin (OT) and vasotocin (AVT) in 155 human pineal glands, and report here identification and measurement of the neurophysin (Np) content of the same glands, using specific homologous human neurophysin I (HNp I) and neurophysin II (HNp II) radioimmunoassays. Median values for HNp I were for men 47 ng/gland (range, 5 to 1360) and for women 24 ng/gland (range, 5 to 1000); median values for HNp II were respectively 7 ng/gland (range, 2 to 191) and 15 ng/gland (range, 2 to 356) with no significant difference between men and women for HNp I and HNp II but a significant difference (p<0.001) between HNp I and HNp II for both sexes. Gel filtration showed that pineal neurophysins were eluted at the same volume as both standard Np and Np from human posthypophyses used as controls. HNp I correlated both with AVP (r_s=0.54 for men and 0.55 for women) and OT (r_s=0.86 for men and 0.57 for women) but not with AVT, while HNp II correlated with AVP (r_s=0.52 for men and 0.53 for women) and OT (r_s=0.92 for men and 0.50 for women) but not with AVT. This study thus confirms the presence of two neurophysins in the human pineal gland and further indicates that they are related to AVP and OT concentrations in the same gland. The results also imply, however, that the presence of immunoreactive AVT (more probably a closely related peptide) is independent of the neurophysins.     

High-performance liquid chromatographic mapping and structural characterization of neurophysin isoforms

Both ion-exchange and reverse-phase HPLC protocols for micromapping of neurophysins have been examined and the structural relationships among the major isoforms identified in the maps have been characterized. Reverse-phase HPLC was found to be especially useful for obtaining fingerprints of the isoforms within each of the two major families of neurophysins, I (oxytocin-related) and II (vasopressin-related), for both bovine and human neurophysins from posterior pituitary sources. From fractionation of the bovine proteins on octylsilyl columns, at least four neurophysins I were identified, one of which corresponds to the intact sequence of 93 residues and three of which vary from the parent by various degrees of carboxyl-terminal truncation. For bovine neurophysin II, two isoforms were identified in the reverse-phase HPLC maps, both of 95 residues, which vary from one another by the residue, either Ile or Val, at position 89. Isoforms were also detected for human neurophysins, including a carboxyl-terminal truncated form of human neurophysin II. All of the major neurophysin isoforms and several of the minor forms were shown to be functionally active as expressed by their binding to peptide ligand affinity matrices. Reverse-phase HPLC mapping on the octylsilyl matrix allowed neurophysin fingerprinting of crude tissue extracts by providing a narrow "window" within which the neurophysins elute but many other polypeptides expected to be present are excluded. The reverse phase HPLC method provides a useful way to obtain isolated neurophysin isoforms for physicochemical characterizations now usually carried out with mixtures of isoforms obtained by ion-exchange chromatography. The method also has characteristics amenable both for high-sensitivity fingerprinting of neurophysin isoforms, from different species and anatomical sources, and as a prelude to microstructural and -functional characterization of the isoforms so isolated.     

Preparative and analytical affinity chromatography of neurophysins on methionyl-tyrosyl-phenylalanyl-aminohexyl-agarose

Methionyl-tyrosyl-phenylalanyl-ω-aminohexyl-agarose was synthesized and shown to be suitable for both the affinity chromatographic purification of neurophysins and the measurement of the ligand binding parameters of these proteins by quantitative affinity chromatography. Bovine neurophysin I binds to the tripeptidyl matrix in 0.4 m ammonium acetate, pH 5.7, conditions under which no binding occurs with the parent ω-aminohexyl-agarose. Subsequent elution can be effected with 0.2 m acetic acid. The affinity matrices obtained have capacities for neurophysin of up to 4 mg/ml gel bed volume and therein provide for the convenient purification of the neurophysins by a two-step buffer-acid elution. [Carbamoyl-¹⁴C]neurophysin I also binds to the ligand-agarose matrix. Using this labeled protein, competitive elution analysis was performed by one-step elution of zones of protein with the binding buffer in the presence of varying amounts of soluble competitive ligand, lysine vasopressin. The characteristic decrease of elution volume of labeled protein with increasing soluble, competing ligand concentration indicates that the affinity matrix interacts biospecifically with neurophysin. This analysis allows the binding affinities for both soluble vasopressin and immobilized tripeptide ligand to be quantitated.     

Highly purified neurophysin proteins: Method of isolation based on their acidic properties

The isolation of highly purified bovine neurophysins I and II from freshly frozen posterior pituitaries is reported. The method can also be used for the isolation of neurophysins from other species, and acetone-desiccated preparations may serve as starting material as well. Crude posterior pituitary extract was obtained as described by Hollenberg and Hope (1967, Biochem. J., 104, 122–127). Basic and neutral proteins were then separated from the acidic neurophysins by cation-exchange chromatography on Cellex-CM (carboxymethyl). Neurophysin I was separated from neurophysin II by anion-exchange chromatography on DEAE-(diethylaminoethyl)-Sephadex with a continuous sodium chloride gradient (0 to 0.4 m). Highly purified bovine neurophysin I was also secured with a stepwise sodium chloride gradient (0.22 m starting gradient followed by a steep gradient from 0.22 to 0.4 m). The current method yields neurophysin proteins in a higher overall yield than previous procedures, as determined by single radial immunodiffusion and concentration-dependent absorption after disc electrophoresis. The method also gives neurophysins of greater purity than standard procedures currently in use. The proteins are characterized by a single, sharp precipitation band on immunodiffusion and immunoelectrophoretic analysis against antiporcine neurophysin antibody, by single bands on analytical gel disc electrophoresis at a running pH of either 8.8, 5.9, or 4.0. Isoelectric focusing on polyacrylamide gel gave an apparent pI value of 4.31 ± 0.07 for neurophysin I and a value of 4.79 ± 0.11 for neurophysin II. Radioimmunoassay revealed barely detectable levels of adrenocorticotropin-like material in neurophysin I (12 pg/100 μg of neurophysin) and no detectable levels in neurophysin II. Both proteins were devoid of avian vasodepressor activity in the conscious chicken, melanotropic activity in vitro in frog skin, and did not effect electrolyte excretion in hydropenic rats.     

Enzymatic Protein Carboxyl Methylation in Rat Posterior Pituitary: Neurophysins in Rapid-Turnover Pool Determine Methyl Accepting Capacity

In vitro stimulation of intact rat posterior pituitary by either veratridine or K+ depolarization results in the concomitant release of neurophysins and in a decrease (70-80%) in their carboxyl methylation as measured either with L-[methyl-3H]methionine in the intact lobes after stimulation or in their homogenates with [methyl-3H]S-adenosyl-L-methionine and purified protein carboxyl methyltransferase. A similar reduction in neurophysin methylation (60%) was observed when the arrival of newly synthesized neurophysins at the posterior pituitary was blocked by colchicine. Experimental data indicate that the reduction in neurophysin content of the lobes after 12 h of colchicine treatment (less than 7%) or after in vitro stimulation (about 10%) cannot account for the marked reduction in neurophysin methylation. The results suggest that the granule pool characterized by rapid turnover of neurophysins probably represents the major source of methyl acceptor proteins in the lobe. In spite of the marked reduction in neurophysin methyl accepting capacity observed after stimulation, there was no parallel increase in methyl accepting capacity of the released neurophysins. We propose that a neurophysin subfraction that might be associated with the membrane of releasable granules participates in the methylation reaction in situ.     

Use of reverse-phase high-performance liquid chromatography in structural studies of neurophysins,photolabeled derivatives,and biosynthetic precursors

The neurophysins are a class of hypothalamo-neurohypophyseal proteins that function as carriers of the neuropeptide hormones oxytocin and vasopressin. Currently, we are using reverse-phase high-performance liquid chromatography for structural characterization of the neurophysins, their chemically modified derivatives, and biosynthetic precursors. A cyanopropylsilyl (Zorbax CN) matrix has been found to be efficient and convenient for separation of major tryptic peptides of performic acid, oxidized or reduced, and alkylated neurophysins. Using this peptide mapping system we have studied the site of modification of a photoaffinitylabeled derivative of bovine neurophysin II by separation and identification of covalently modified peptides. In addition, this system has been used for mapping subfemtomole amounts of radioactively labeled biosynthetic precursors of the neurophysins. This procedure has allowed identification of neurophysin sequences within both pre-pro-neurophysins produced by in vitro translation and rat pro-neurophysins produced by in vivo pulse labeling.     

Isolation and localization of neurophysin-like proteins in rat uterus

Neurophysins are part of the prohormones for vasopressin and oxytocin, and are localized with these hormones in the magnocellular cells of the neurohypophysis. New techniques have identified neurophysins in other areas within and outside the central nervous system, and we report here the isolation of neurophysins from the uterus of the rat. Using immunohistology the neurophysin immunoreactivity was localized to the epithelial lining cells of the uterus, and using radioimmunoassay was also present in uterine fluid suggesting secretion into the uterine cavity. The amount of uterine neurophysin increased in response to administered estrogen and was especially elevated in the pregnant uterus. The neurophysin-like material isolated from the uterus was similar to neurophysins from the neurohypophysis by radioimmunoassay, molecular sieve chromatography, isoelectric focusing and SDS gel electrophoresis. Both neurohypophyseal hormones, vasopressin and oxytocin, were also extracted from uterine endothelium and identified by radioimmunoassay and high pressure liquid chromatography.     

Mapping and isolation of large peptide fragments from bovine neurophysins and biosynthetic neurophysin-containing species by high-performance liquid chromatography

A peptide mapping procedure suitable for rapid analysis and peptide recovery was devised for the neurophysins. Tryptic fragments of performic acid-oxidized bovine neurophysins I and II were fractionated by reverse-phase high-performance liquid chromatography using γ-cyanopropyl-bonded columns. Elutions were achieved using a gradient from triethylammonium phosphate buffer to mixtures of this buffer with increasing proportions of acetonitrile. All tryptic fragments, except for dipeptides, were separated. Assignments of eluted peaks to particular authentic neurophysin peptides were achieved by collection of peaks and determination of their amino acid compositions. The use of this peptide mapping procedure to detect subpicomole amounts of neurophysin peptides in cell-free biosynthetic products was demonstrated.     

Intra-axonal transport and turnover of neurophysins in the rat. A proposal for a possible origin of the minor neurophysin component

1. Radioactivity associated with the three neurophysins in the neural lobe of the rat was determined at intervals up to 5 weeks after an intracisternal injection of [(35)S]cysteine. 2. The radioactivity associated with the two major neurophysins (one supposedly associated with vasopressin and the other with oxytocin) increased linearly for 12h after the injection and the ratio of the rates of increase in the two proteins was very similar to the ratio of vasopressin to oxytocin in the gland. 3. From 12h onwards the radioactivity associated with each major neurophysin declined exponentially but the half-life of the supposed oxytocin-neurophysin (13.3 days) was shorter than that for the supposed vasopressin-neurophysin (19.8 days). 4. The kinetics of labelling of the minor neurophysin was quite different from that of the two major ones. It became slowly labelled during 3-5 days after injection and the radioactivity hardly decreased during the following 4 weeks. 5. The data could support the hypothesis that the minor neurophysin is a metabolic product of oxytocin-neurophysin. The exponential rate of disappearance of radioactivity from oxytocin-neurophysin and the minor component taken together has a rate constant similar to that for vasopressin-neurophysin (e.g. half-life=18.9 days).     

Biosynthesis and axonal transport of rat neurohypophysial proteins and peptides

35S-cysteine injected adjacent to the supraoptic nucleus (SON) of the rat is rapidly incorporated into proteins. These 35S-cysteine-labeled proteins in the SON (1-24 h after injection) were separated by polyacrylamide gel electrophoresis, and the distribution of radioactive proteins on the gels was analyzed. 1 h after injection, about 73% of the radioactivity appeared in two peaks (both about 20,000 mol wt). With time, these peaks (putative precursors of neurophysin) decreased, as a 12,000 mol wt peak (containing two distinct neurophysins) increased in radioactivity. Both the 20,000- and 12,000-mol wt proteins are transported into the axonal (median eminence) and nerve terminal (posterior pituitary) regions of the rat hypothalamo-neurohypophysial system. Conversion of the larger precursor protein to the smaller neurophysin appears to occur, in large part, intra-axonally during axonal transport. Six distinct 35S-cysteine-labeled peptides (less than 2500 mol wt), in addition to arginine vasopressin and oxytocin, are also synthesized in the SON and transported to the posterior pituitary where they are released together with labeled neurophysin by potassium depolarization in the presence of extracellular calcium. These data provide support for the hypothesis that the neurohypophysial peptides (vasopressin and oxytocin) and neurophysins are derived from the post- translational clevage of protein precursors synthesized in the SON, and that the conversion process can occur in the neurosecretory granule during axonal transport.     

Precursors of mesotocin and vasotocin in birds: Identification of VLDV- and MSEL-neurophysins in chicken,goose, and ostrich

Precursors of neurohypophysial hormones are small proteins processed into nonapeptide hormones and neurophysins during axonal transport to the neurohypophysis. In mammals, oxytocin is associated with VLDV-neurophysin and vasopressin with MSEL-neurophysin. In birds, mesotocin and vasotocin are found instead of mammalian oxytocin and vasopressin. From goose, chicken and ostrich posterior pituitary glands, two types of neurophysins related to mammalian VLDV-and MSEL-neurophysins, respectively, have been identified by their N-terminal sequences. It is assumed that, as in mammals, hormonal peptide and the first 9 residues of the corresponding neurophysin are encoded by a common exon and that mesotocin and vasotocin, evolutionary predecessors of oxytocin and vasopressin, are associated in the precursors with VLDV-neurophysin and MSEL-neurophysin, respectively.     

Complete amino acid sequence of goose VLDV-neurophysin. Traces of a putative gene conversion between promesotocin and provasotocin genes

Goose VLDV-neurophysin (mesotocin-associated neurophysin) has been purified from posterior pituitary glands through molecular sieving on Sephadex G-75 and high-pressure reverse-phase liquid chromatography on Nucleosil C-18 columns. Despite apparent molecular mass of unreduced VLDV-neurophysin measured by polyacrylamide gel electrophoresis with sodium dodecylsulfate appeared near 17 kDa, this value fell to 11 kDa after reduction with mercaptoethanol, suggesting the existence of a homodimer. Complete amino acid sequence (93 residues) of goose VLDV-neurophysin has been determined. N- and C-terminal sequences of the protein have been established by Edman degradation (microsequencing) and use of carboxypeptidase Y, respectively. Peptides derived from oxidized or carboxamidomethylated neurophysin by trypsin or staphylococcal proteinase hydrolyses have been isolated by high-pressure liquid chromatography and microsequenced, allowing determination of the complete sequence. Comparison within the vertebrate VLDV-neurophysin lineage, namely goose VLDV-neurophysin to mammalian VLDV-neurophysins and to deduced toad VLDV-neurophysin, reveals a residue insertion between positions 66 and 67 in the nonmammalian VLDV-neurophysins. When goose MSEL-neurophysin (vasotocin-associated neurophysin) and goose VLDV-neurophysin are compared to their bovine counterparts, identical substitutions are found in positions 17 (Asn in both goose neurophysins instead of Gly in both ox neurophysins), 18 (Arg instead of Lys), 35 (Tyr instead of Phe), and 41 (Thr instead of Ala). Identity of the sequences 10-74 in both ox neurophysins has been explained by partial gene conversion between oxytocin and vasopressin genes, and identical substitutions in both goose neurophysins might reveal a similar gene conversion between mesotocin and vasopressin genes in birds.     

Evidence for the secretion of immunoreactive neurophysin I in addition to oxytocin from the ovary in cattle

In Exp. I, blood samples were collected simultaneously from the posterior vena cava and jugular vein or aorta from 7 heifers every 5-20 min for 2-5 h. Concomitant pulsatile secretion of oxytocin and immunoreactive neurophysin I was detected in the vena cava, but not in the jugular vein or aorta. Concentrations of oxytocin and immunoreactive neurophysin increased earlier and were higher in the vena cava than in the jugular vein or aorta after the injection of a luteolytic dose of prostaglandin F-2 alpha analogue during the mid-luteal phase of the oestrous cycle, demonstrating its ovarian but not pituitary origin. In Exp. II, blood samples were collected from the jugular vein every 12 h during 1 week after oestrus. Follicular growth had been stimulated during the preceding oestrous cycle with PMSG (10 heifers and cows) or with FSH (5 animals); 6 heifers served as controls. There was a high correlation between the number of follicles or CL and the increase in oxytocin and immunoreactive neurophysin I. Although PMSG had a greater luteotrophic effect than did FSH on progesterone secretion, a similar stimulation of oxytocin and immunoreactive neurophysin I was not observed. It is concluded that immunoreactive neurophysin I and oxytocin are secreted from the ovary in concentrations dependent upon the number of corpora lutea (and of follicles) present. During the mid-luteal period the secretion occurs in a concomitant pulsatile fashion.     

Preparation of Porcine Neurophysin Proteins by High Performance Liquid Chromatography

Abstract: The crude neurophysin containing extract from posterior lobes of porcine pituitaries was roughly purified by gel chromatography. 15 mg of the lyophilized neurophysin complex were completely separated by HPLC yielding in neurophysin I₁ (3.6 mg), I₂ (4.0 mg), II (4.6 mg) and III (1.9 mg). All of the neurophysins were homogenous by PAGE and SDS-electrophoresis, isoelectrofocussing, amino-acid composition and N- and C-terminal amino acid analysis. In conclusion, HPLC is a reliable and quick method for the preparation of pure neurophysins.     

EGFP-Tagged Vasopressin Precursor Protein Sorting Into Large Dense Core Vesicles and Secretion From PC12 Cells

1. Hypothalamic magnocellular neurons synthesize, store, and secrete large quantities of the neuropeptides, vasopressin (VP) and oxytocin (OT), which are synthesized as protein precursors also containing proteins called neurophysins. These protein precursors are sorted through the regulated secretory pathway (RSP), packaged into large dense core vesicles LDCVs, and their peptide products are secreted from nerve terminals in the posterior pituitary.2. It has been hypothesized that this efficient packaging is dependent on the interaction of the peptide with neurophysin in a complex that forms the granule core. To test this, PC12 cells were transfected with vasopressin precursor DNA constructs that either contained or deleted the neurophysin moiety and tagged with enhanced green fluorescent protein (EGFP) as reporters. The intracellular routing and secretion of the EGFP-tagged VP precursor proteins were studied by in differentiated PC12 cells by fluorescence microscopy, electron microscopic immunocytochemistry, and fluorescent imaging techniques.3. The data showed that only when the neurophysin was present in the VP precursor construct did the fluorescent fusion protein become routed to the RSP and get efficiently packaged into LDCVs and secreted. These data are consistent with the view that routing of the precursor to LDCVs requires the amino acids that encode the intravesicular chaperone, neurophysin.     

Isolation of a third bovine neurophysin

1. A third native hormone-binding protein, neurophysin-C, has been isolated from acetone-desiccated bovine pituitary posterior lobes. 2. This protein was detected in lysates of neurosecretory granules isolated from bovine pituitary posterior lobes. 3. The molecular weight appears to be close to 10000. 4. Neurophysin-C is similar in amino acid composition to neurophysin-I and -II; it contains a single residue of tyrosine and of methionine. The N-terminal amino acid in all three neurophysins is alanine. 5. Neurophysin-C accounts for approximately 15% of the total hormone-binding protein present in the pituitary posterior lobes. 6. The new neurophysin forms complexes with oxytocin as well as with [8-arginine]-vasopressin. The complex with vasopressin has been crystallized. 7. Bioassay of the pressor and oxytocic activities of the protein-hormone complexes shows that neurophysin-C binds one molecule of either vasopressin or oxytocin.