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.     

Structure, processing and evolution of the neurohypophysial hormone-neurophysin precursors

Neurohypophysial hormones and neurophysins are derived from common precursors processed during the axonal transport from the hypothalamus to the neurohypophysis. Two neurohormones, an oxytocin-like and a vasopressin-like, on one hand, two neurophysins, termed VLDV-and MSEL-neurophysins according to residues in positions 2, 3, 6 and 7, on the other, are usually found in vertebrate species. In contrast to placental mammals that have oxytocin and arginine vasopressin, marsupials have undergone a peculiar evolution. Two pressor peptides, lysipressin and vasopressin for American species, lysipressin and phenylpressin for Australian macropods, have been identified in individual glands and it is assumed that the primordial vasopressin gene has been duplicated in these lineages. On the other hand, the reptilian mesotocin is still present in Australian species instead of the mammalian oxytocin, while the North American opossum has both hormones and South American opossums have only oxytocin. The neurophysin domain of each precursor is encoded by 3 exons and different evolutionary rates have been found for the 3 corresponding parts of the protein. The central parts, encoded by the central exons, are evolutionarily very stable and nearly identical in the 2 neurophysins of a given species. Recurrent gene conversions have apparently linked the evolutions of the 2 precursor lineages. In mammals, the 3-domain precursor of vasopressin is processed in 2 stages: a first cleavage splitting off vasopressin and a second cleavage separating MSEL-neurophysin from copeptin. Two distinct enzymatic systems seem to be involved in these cleavages. Processing is usually complete at the level of the neurohypophysis, but an intermediate precursor encompassing MSEL -neurophysin and copeptin linked by an arginine residue has been characterized in guinea pig. In vitro processing of this intermediate through trypsin--Sepharose reveals cleavages only in the interdomain region. In non-mammalian tetrapods, such as birds and amphibians, mesotocin and vasotocin are associated with neurophysins in precursors similar to those found in mammals. However, processing of the vasotocin precursor seems to be different from the processing of the vasopressin precursor, with a single cleavage leading to the hormone release.     

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.     

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.     

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.     

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.     

A monoclonal antibody to vasopressin: preparation, characterization, and application in immunocytochemistry

The hypothalamo-neurohypophysial system, containing the hormones oxytocin (OT) and vasopressin (VP) and their associated carrier proteins, the neurophysins (NPS), has been the subject of extensive investigation for more than 40 years. This system has been reinvestigated during the last decade by application of immunocytochemical methods employing the rabbit antisera to the hormones and NPS. In this study we describe the preparation and characterization of a monoclonal antibody to VP and its application in immunohistochemistry. The antibody did not cross-react with OT or arginine vasotocin (AVT). Its antigenic determinants as characterized by absorption with various VP analogs included two aromatic amino acids: Phe in position 3, and to a lesser extent Tyr in 2. Tissue fixation with formaldehyde resulted in inadequate immunostaining as compared to glutaraldehyde, most likely due to interference with the aromatic amino acid determinants by the former fixative.     

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.     

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.     

Ontogeny of the bovine neurohypophysial hormone precursors. III. Identification of neurohormones, neurophysins and copeptin in the early bovine fetus

Neurohypophysial hormone precursors are small proteins processed into several fragments during axonal transport from hypothalamus to neurohypophysis. From 3-month-old fetal bovine pituitaries the three fragments of vasopressin precursor, arginine vasopressin, MSEL-neurophysin and copeptin, and the two fragments of oxytocin precursor, oxytocin and VLDV-neurophysin, have been isolated and characterized. These polypeptides are identical to those previously identified in the late fetus (7-9 months old) and in the adult. It is concluded that the same genes are expressed during fetal and adult lives, the vasopressin gene appearing roughly four times more active than the oxytocin gene in the early fetus. Vasotocin, mesotocin and additional neurophysin have not been detected in the early fetus.     

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.     

Ontogeny of bovine neurohypophysial hormone precursors. II. Foetal copeptin, the third domain of the vasopressin precursor

Vasopressin, MSEL-neurophysin and a glycopeptide, here referred to as copeptin, are three fragments of a common protein precursor processed during axonal transport from hypothalamus to neurohypophysis. Neurohormones and neurophysins purified from 7-9-month-old bovine foetuses have previously been shown to be identical with those found in the adult. Copeptin has now been isolated from 7-9-month and 3-month-old bovine foetuses and chemically characterized. It can be concluded from the nature of the three precursors that the same vasopressin gene is expressed in the adult and the 7-9-month-old foetus.     

Cloning and sequence analyses of cDNAs encoding vasotocin and isotocin precursors of chum salmon,Oncorhynchus keta: evolutionary relationships of neurohypophysial hormone precursors

Summary The nucleotide sequences of cloned cDNAs were used to determine the primary structures of the precursors of vasotocin (sVT) and isotocin (sIT) from the hypothalamus of the chum salmon,Oncorhynchus keta. Two different cDNAs were obtained for each of sVT and sIT precursors (sVT-I and sVT-II; sIT-I and sIT-II). Both sVT and sIT precursors were found to contain a signal peptide and hormone that is connected to a neurophysin by a Gly-Lys-Arg sequence. Northern and Southern blot analyses showed that the sVT and sIT genes are expressed by the same chum salmon hypothalamus, but not by the liver and kidney. Microheterogeneity was found in the nucleotide and amino acid sequences of sVT precursors between our results and the previously reported data (Heierhorst et al. 1990). The conspicuous difference is the occurrence of a stop codon in the middle of sVT-II cDNA. The carboxyl termini of both sVT and sIT neurophysins are about 30 amino acids longer than neurophysins of toad and mammalian neurohypophysial hormone precursors. Although these extended regions do not contain a glycosylation site, they show striking similarity with the glycopeptide moiety (copeptin) of toad vasotocin and mammalian vasopressin precursors. The central portion of the neurophysins shows highest homology among corresponding regions of sVT and sIT precursors. Moreover, calculation of nucleotide substitution rates suggests that a recent gene conversion may have occurred which encompasses the exon that encodes the central segment of the sVT and sIT precursors. A possible pathway for the evolution of precursor molecules of neurohypophysial hormones is discussed.Abbreviations AVP vasopressin - C carboxyl - h human - IT isotocin - MT mesotocin - N amino - OXT oxytocin - S chum salmon - SDS sodium dodecyl sulfate - t toad - VT vasotocin     

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.     

Fluorescence studies of native and modified neurophysins. Effects of peptides and pH.

The effect of neurophysin-hormone interaction on the environment of the single tyrosine of bovine neurophysin (Tyr-49) and on that of the tyrosine of oxytocin and vasopressin was studied by fluorescence; tyrosine-free peptides were used to determine effects on Tyr-49, and acetylated neurophysin was used to determine effects on the hormone tyrosine. Binding increases the fluorescence intensity of Tyr-49 by 130% while the fluorescence of the hormone tyrosine is almost completely quenched. Correlation of these results with those obtained on binding oxytocin or vasopressin to native neurophysin indicates that in the hormone complexes less than half of the fluorescence of Tyr-49 is lost by F?rster energy transfer to the quenched hormone tyrosine. These results support spin-label studies in indicating that the distance between Tyr-49 and the tyrosine of hormone bound to the strong hormone binding site is greater than 5 A. In the absence of peptides, the fluorescence of Tyr-49 increases by 40% on lowering the pH from 6.2 to 2. Titration of the acid fluorescence transition in bovine neurophysins-I and -II, and in bovine neurophysin-II treated with carboxypeptidase B to remove the Arg-Arg-Val sequence at the carboxyl terminus, indicates that this transition is due to titration of a side-chain carboxyl with an intrinsic pK of 4.6. The effects of guanidine, glycerol, and disulfide cleavage on the magnitude of the acid transition indicate that the conformational information necessary for the transition resides within the amino acid sequence adjacent to Tyr-49. Accordingly, the fluorescence acid transition is attributed to decreased quenching by Glu-46 or Glu-47 upon protonation. Glycerol is shown to perturb the glutamate-tyrosine interaction in the absence of general conformational effects. Comparison of the fluorescence low-pH transition with that of the low-pH circular dichroism transition of nitrated neurophysins suggests that the fluorescence and CD transitions reflect related, but not necessarily identical, phenomena. In an appendix, evidence is presented which suggests that the products of carboxy-peptidase digestion of bovine neurophysin-II are the same as two minor bovine neurophysin components, one of which is neurophysin-C.     

Immunocytochemical localization of peptidergic cells in the neuro-endocrine system of the Colorado potato beetle, Leptinotarsa decemlineata, with antisera against vasopressin, vasotocin and oxytocin

Antisera against vasopressin, vasotocin, oxytocin, neurophysin-1 and neurophysin-2 were used to investigate immunocytochemically the presence of neurons containing substances antigenically related to these peptides in the nervous system of the Colorado potato beetle. Ten different antisera were used, four against vasopressin, three against oxytocin and one against vasotocin, neurophysin-1, and neurophysin-2. Immunoreactivity was shown by all antisera except those against the neurophysins. The vasopressin antisera all gave different results. One antiserum revealed only a single neuron pair, whereas others revealed in addition one or two other different cell groups. The oxytocin antisera likewise revealed different neurons. The fixation procedure influenced the outcome of the immunocytochemical reaction. Immunoreactivity as revealed by vasopressin, vasotocin and oxytocin antisera is often co-localized in the same neurons; solid phase adsorptions showed that this is due to cross-reactivity of the antisera. Some of the immunoreactive neurons are identical to those recently described to contain a bovine pancreatic polypeptide/FMRFamide-like peptide. This co-localization is probably not due to a cross-reaction. These findings indicate the presence of several vasopressin-like and oxytocin-like substances which in the Colorado potato beetle all have a different degree of immunocytochemical resemblance to vasopressin and oxytocin.     

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.     

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.     

Neurohormonal Regulation of Tumor Growth

Neurohormones vasopressin and oxytocin are synthesized in the hypothalamus and are transported along the axons to the neurohypophysis as a part of equimolar complexes with hormone-specific neurophysins. The tumors of epithelial origin synthesize ectopic vasopressin and have an ability to express all types of receptors of neurohypophysis hormones. Vasopressin and oxytocin receptors provide the transduction of signals to protein kinases A, B, and C and activate intracellular cascades of the CREB, MDM2, and TORC1/2 proteins and mitogen-activated protein kinases. Central endocrine and autocrine neurohormonal contours are involved in the regulation of proliferative, migration, and angiogenic processes accompanied by tumor progression. Tumor growth and development occur in close cooperation with the supporting stroma. The interstitial tissue is involved in signal communication of tumor cells by integrins and integral CD44 glycoproteins formulating hyaluronic acid. Hyaluronic acid metabolites modulate the effect of neurohormones and peptide growth factors; intermediate hyaluronan fragments with molecular weight of approximately 20 kDa elicit the most significant angiogenic effect. Platelets expressing AVPR1 vasopressin receptors are an important source of hyaluronidase 2 hydrolyzing macromolecular hyaluronan to fragments of intermediate length. The AVPR2 receptors localized in endothelium and AVPR1-AVPR2 vasopressin receptors expressing themselves in the tumor cells are involved in the mechanisms controlling local hemostasis. Neurohormonal regulatory contours are involved in optimization of the balance of inducing and inhibiting signals generated by the tumor and stroma in the process of progressive growth.