Demonstration of neurophysin in the hypothalamo-neurohypophysial system of the normal and dehydrated rat by the use of cross-species reactive anti-neurophysins

Summary Antiserum raised against porcine neurophysin-II and ovine neurophysin-III has been shown to cross react with the three neurophysins of the rat. The antisera were used in the immunohistological demonstration of neurophysin in the posterior pituitary gland and the hypothalamic nuclei of a normal rat and a rat subjected to varying degrees of osmotic stress. Depletion of neurosecretory material from the neural lobe of the stimulated rat is also accompanied by a concomitant reduction in the neurophysin and vasopressin content of the gland. Neurophysin is present in the cytoplasm of both the supraoptic and paraventricular nuclei.This work was financed by the Zealand Medical Research Council and the Auckland Medical Research Foundation. We thank Miss V. Bailey for help with the photographs.     

The effects of ligands on enzymic carboxyl-methylation of neurophysins

The methyl-acceptor activities of bovine neurophysins I and II for the enzyme protein carboxymethylase (EC 2.1.1.24) were found to be similar and as high as for other previously identified, biologically active protein substrates. Effects on the rate of methylation of these neurophysins were investigated with the posterior pituitary hormone ligands, oxytocin and vasopressin, and the hormone-related tripeptide ligand, methionyl-tyrosyl-phenylalaninamide. An increase in the rate of neurophysin II methylation was observed with both oxytocin and tripeptide. This ligand-induced response did not occur with either native neurophysin I or disulfide-scrambled neurophysin II.     

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     

Subcellular organization of neurophysins, oxytocin, [8-lysine]-vasopressin and adenosine triphosphatase in porcine posterior pituitary lobes

Posterior pituitary lobes from young pigs were fractionated by differential and sucrose-density-gradient centrifugation. The distributions of oxytocin and [8-lysine]-vasopressin were measured by bioassay and the distributions of neurophysin-I and -II by radioimmunoassays specific for each of these two proteins. Most of the hormone and neurophysin applied to the density gradient was localized in particles with the density expected of neurosecretory granules. However, the neurosecretory granules were separated into two bands (D and E). A close statistical correlation between the distributions of [8-lysine]-vasopressin and neurophysin-I, and of oxytocin and neurophysin-II on the gradients, suggested that in vivo porcine neurophysin-I binds [8-lysine]-vasopressin within one population of granules and porcine neurophysin-II binds oxytocin within another type of granule. However, there was no significant separation of oxytocin and vasopressin in fractions D and E. The molar ratios of hormones and neurophysins indicated that there was insufficient of either neurophysin to bind the [8-lysine]-vasopressin in the granule fractions or in the whole gland. Polyacrylamide-gel electrophoresis showed that only bands corresponding in mobility to porcine neurophysins-I, -II and -III were present in large amounts in the whole gland and in the granule fractions. The component with the mobility of neurophysin-III was, however, relatively enriched in whole young glands and granule fractions compared with adult gland extracts. It is suggested that the vasopressin that cannot be assigned to neurophysin-I may occur in (a) vesicles containing vasopressin but no neurophysin, (b) vesicles containing vasopressin and a protein that cannot be quantified by the radioimmunoassays used, such as porcine neurophysin-III, or (c) normal vasopressin–neurophysin granules which have accumulated extra vasopressin. Band E of the gradient was rich in adenosine triphosphatase activity, whereas band D possessed very little of this enzyme.     

The isolation of three neurophysins from porcine posterior pituitary lobes

1. Three neurophysins, proteins that bind the polypeptide hormones oxytocin and vasopressin, have been isolated from acetone-dried porcine posterior pituitary lobes. The proteins have been named porcine neurophysins-I, -II and -III in order of their electrophoretic mobilities at pH8.1. 2. Electrophoretic comparison of the purified proteins, which are homogeneous on starch-gel electrophoresis, with the soluble proteins of fresh porcine posterior pituitary lobes extracted in 0.1m-HCl and in buffer pH8.1 suggests that the isolated proteins are native to the fresh tissue. 3. Neurophysins-I and -II are present in similar amounts in the tissue, whereas neurophysin-III is present only in small quantities. Acetone-dried tissue also contains traces of other hormone-binding neurophysin components. 4. All the neurophysins can bind both oxytocin and [8-lysine]-vasopressin. 5. The apparent molecular weights of the neurophysins increase with increasing protein concentration as measured by equilibrium sedimentation in the ultracentrifuge. 6. Neurophysins-I and -III are of similar molecular dimensions, contain one residue of methionine per molecule and lack histidine. The minimum molecular weight of neurophysin-I obtained by amino acid analysis is 9360. Neurophysin-II is of larger molecular dimensions than neurophysins-I and -III and can be separated from these by gel filtration on Sephadex G-75. It contains no histidine or methionine, and its minimum molecular weight has been estimated as 14020 by amino acid analysis. 7. Each of the three neurophysins possesses N-terminal alanine. 8. The possible biological significance of the existence of several neurophysins within one species is discussed.     

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.     

Ultrastructural studies on the localization of neurohypophysial hormones and their carrier proteins.

Neurophysin, vasopressin and oxytocin were localized in different portions of the supraopticohypophysial tract (SHT) using the unlabeled antibody enzyme technique at the ultrastructural level. In vasopressin-positive supraoptic perikarya, vasopressin and neurophysin were present in all neurosecretory granules. Within the zona interna of the median eminence, vasopressin and neurophysin were present in two populations of axons, one with granules of 1300-1500 A and one with granules of 900-1300 A. Following exposure of thin sections of median eminence to antiserum to neurophysin, reaction products were present in granules and in the extragranular cytoplasm in the axons with larger granules; in all other cases reaction product was confined to the granules. Vasopressin-positive fibers were also presented in large numbers of the zona externa of the median eminence and many terminated on the pituitary primary portal plexus. A few oxytocin fibers were present on the portal capillaries in the infundibular stalk. In the posterior pituitary all axon profiles were neurophysin positive. Neurophysin was present as both a granular and cytoplasmic pool. Vasopressin-containing axons account for 90% of the neuronal elements in the posterior pituitary and oxytocin for the remaining 10%. Findings on the subcellular distribution of these peptides are related to current theories on transport and release of neurohormones.     

Neurophysins of Mammals: evolution and biological signification]

Neurohypophysial hormone-Neurophysin complexes have been prepared from posterior pituitary glands of Artiodactyla (ox, sheep, pig), Perissodactyla (horse) and Cetacea (whale), by fractionated salt precipitation. The components have been separated by molecular sieving in 0.2 M acetic acid and neurophysins have been purified by ion-exchange chromatography on DEAE-Sephadex A-50. Two types of neurophysins, MSEL-neurophysins and VLDV-neurophysins, can be distinguished according to the amino acid residues in positions 2, 3, 6 and 7. MSEL-neurophysins of sheep, ox and pig have been characterized by the amino acid sequence. Ovine and bovine MSEL-neurophysins are nearly identical (one substitution out of 95 residues) and porcine MSEL-neurophysin is very similar (four substitutions and an apparent 3-residue C-terminal deletion). The biological function of neurophysins might be the carriage of neurohypophysial hormones but in this respect, each type of neurophysin is not clearly specific for a given hormone. On the other hand, each neurophysin might share a common precursor with a neurohypophysial hormone, the two parts remaining associated after cleavage. However, in the sheep posterior pituitary gland, the molar proportions of the two types of neurophysins, oxytocin and arginine vasopressin, are not equal, MSEL-neurophysin being more abundant than the other components. If a common precursor exists, neurophysins and neurohypophysial hormones are not merely produced by a simple cleavage mechanism.     

The distribution of vasopressin-, oxytocin-, and neurophysin-producing neurons in the guinea pig brain

Summary The location, cytology and projections of vasopressin-, oxytocin-, and neurophysin-producing neurons in the guinea pig were investigated using specific antisera against vasopressin, oxytocin or neurophysin in the unlabeled antibody enzyme immunoperoxidase method. Light microscopic examination of the neurons of the supraoptic and paraventricular nuclei shows that hormone is transported not only in axons, but also in processes having the characteristics of dendrites. Neurons were found to contain only vasopressin or oxytocin; all neurons containing neurophysin appear to contain either vasopressin or oxytocin. In the neural lobe, vasopressin and oxytocin terminals are intermingled. In the median eminence, vasopressin and oxytocin fibers are intermingled in the internal zone. In a caudal portion of the median eminence, a number of vasopressin and neurophysin (but few oxytocin) axons enter the external zone from the internal zone, and surround portal capillaries. In the supraoptic nucleus, vasopressin neurons outnumber oxytocin neurons with a ratio of at least 5:1. The paraventricular nucleus is separated into two distinct groups of neurons, a lateral group consisting of only vasopressin neurons, and a medial group consisting of only oxytocin neurons. In addition to axons passing to the neurohypophysis, a number of axons appear to interconnect the supraoptic and paraventricular nuclei.Supported by the Deutsche Forschungsgemeinschaft (SFB 51, C/21 and C/27), (We 608/3)Acknowledgements. The authors are greatly indebted to Mmes. R. Köpp-Eckmann, B. Reijerman, A. Scheiber, I. Wild and Mr. U. Schrell for technical assistance, to Mmes. P. Campbell and U. Wolf for editorial assistance, and to Dr. R.R. Dries and Ferring Pharmaceuticals, Kiel, for the generous provision of high quality peptides     

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.     

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.     

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.     

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.     

HPLC Analysis of Neurophysin Proteins from Neurosecretory Granules

Abstract: Neurosecretory granules were obtained from neurolobes of porcine pituitary glands. From the granules, highly purified neurophysins were prepared by HPLC. According to polyacrylamide gel electrophoresis, isoelectric focusing, N- and C-terminal amino acid residue determination, and amino acid composition, the neurophysins I₁ I₂, and II were identical to the neurophysins obtained from whole posterior lobes. Since degradation could not have occurred, we conclude that neurophysin I₁ and I₂ originated in the neurosecretory granules.     

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.     

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.     

Maturation of the hypothalamo-neurohypophysial system

Summary Sections of the hypothalamus, median eminence and pituitary from fetal and neonatal rats were examined with the immunoperoxidase staining technique and light microscopy. Purified antisera raised against vasopressin and oxytocin, and antisera cross-reactive with rat neurophysin were used to localize these antigens in the hypothalamo-neurohypophysial system (HNS). Neurophysin was detected throughout the HNS of the 18-day fetal rat. Vasopressin was present in the hypothalamus and pituitary of the 19-day fetus, and in the median eminence of the 4-day neonate. Oxytocin was not detected in the pituitary until 1–2 days after birth, in the hypothalamus after 4 days, and in the median eminence after 8 days. During the first days after birth the supraoptic nucleus was more mature than the paraventricular nucleus. The HNS did not approach maturity until at least 7 days after birth. The relative maturity of the supraoptic nucleus compared with the paraventricular nucleus, and the detection of vasopressin before oxytocin are evidence for the one-neuron-one-hormone theory. The data do not exclude the possibility that the fetal hypothalamo-neurohypophysial system, and perhaps the fetal hormone, vasotocin, affect the initiation and course of parturition.This work was financed by the Medical Research Council of New Zealand     

Transitory coexistence of oxytocin and vasopressin in the hypothalamo neurohypophysial system of parturient rats.

We used in situ hybridization and immunocytochemistry to investigate a possible coexistence of vasopressin and oxytocin in hypothalamic neurons of parturient rats. We found that a fraction of magnocellular neurons in the paraventricular and supraoptic nuclei contained immunostaining for both peptides as well as oxytocin and vasopressin mRNA hybridization. Colocalization of immunoreactive vasopressin and oxytocin could be observed in some of the Herring bodies in the median eminence and the posterior lobe. No coexistence of vasopressin and oxytocin was found in pregnant or in lactating animals, indicating that the observed coexistence is transitory, perhaps mediated through changing hormonal conditions peri partum.     

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.     

Localization of hormone secreting pathways in the brain by immunohistochemistry and light microscopy: a review.

Immunocytochemical techniques are now being used to localize hypothalamic neurosecretory hormones and related peptides in the mammalian brain. The data are probably incomplete, due primarily to false negative results. A number of previous assumptions concerning these pathways have been confirmed while other unexpected results were obtained. As expected, vasopressin and oxytocin and their associated proteins, neurophysins, were found in the magnocellular cell bodies of the hypothalamus and in their axonal projections to the neural lobe of the pituitary. Gonadotropin-releasing hormone (Gn-RH), somatostatin, and thyrotropin-releasing hormone (TRH) were located in what appears to be parvicellular nerve terminals on portal capillaries. Gn-RH has been found in perikarya in the arcuate nucleus, which is considered a source of fibers to the portal capillary bed. An extensive network of cell bodies and fibers in the preoptic area was also found to contain Gn-RH, and others in the periventricular nucleus in the anterior hypothalamus reacted with antiserum to somatostatin. Unexpected was considerable evidence that vasopressin is secreted directly into hypophyseal portal blood. This hormone and its neurophysin were also found in parvicellular neurons in the suprachiasmatic nucleus of rodents. All the hormones were found in fibers in the organum vasculosum of the lamina terminalis and in the posterior pituitary gland.