Corticotropin-releasing hormone expression is the major target for glucocorticoid feedback-control at the hypothalamic level.

Glucocorticoid production is controlled via the hypothalamo-pituitary-adrenal (HPA) axis by a negative feedback mechanism involving the glucocorticoid receptor (GR). A major site of regulation is the hypothalamus, where the GR is thought to repress the expression of genes such as corticotropin-releasing hormone (CRH) and arginine-vasopressin (AVP). To define the role of the GR in this feedback loop in more detail, the content of CRH, AVP and neurophysin in the median eminence of mice carrying a targeted disruption of the GR gene was studied using immunohistochemistry. GR-deficient mice were found to contain five times more CRH in the median eminence than wild-type littermates. In contrast, no significant change in the content of AVP was observed in the outer layer of the median eminence and neurophysin was also only moderately increased. Our studies suggest that, at the hypothalamic level, CRH synthesis is the major target for feedback control by the GR and that transcriptional control of AVP and neurophysin plays only a supportive role in this process.

Mesotocin gene expression in the diencephalon of domestic fowl: cloning and sequencing of the MT cDNA and distribution of MT gene expressing neurons in the chicken hypothalamus.

This study focuses on the structure and expression of the mesotocin (MT) gene in the chicken hypothalamus. Using an anchored and nested RT-PCR strategy, combined with circular RACE-PCR, the full length sequence of the chicken MT cDNA was obtained. The cDNA and derived amino acid sequences conformed to the structure of the oxytocin-like gene family. However, unlike most mammalian species, there does not appear to be frequent gene conversion between the MT and AVT cDNA sequences. A single specific hybridization signal of 1.2 kb was detected by Southern analysis of chicken genomic DNA, indicating only a single gene copy in the chicken genome. Northern analysis of hypothalamic RNA revealed a single band at approximately 0.6 kb. Using the same probe for in situ hybridization histochemistry, MT-mRNA was demonstrated to be predominantly localized in the parvocellular, magnocellular and periventricular subgroups of the paraventricular nucleus and, when compared to the distribution of neurons containing arginine-vasotocin (AVT)-mRNA in the same region, with far fewer neurons expressing the MT gene in the lateral subgroups. Only few and scattered neurons expressing the MT gene were found in the ventral and external subgroups of the supraoptic nucleus in which many neurons contain AVT transcripts, as demonstrated in consecutive sections. In all nuclei investigated, the intensity of AVT and MT hybridization signals per cell was approximately equal. No specific labelling for MT-mRNA was found in the bed nucleus of the stria terminalis, nor the nucleus accumbens. Using immunocytochemical detection of AVT and in situ hybridization for neurons expressing MT-mRNA, some neurons were found to contain both AVT and MT gene products in the paraventricular nucleus but not in the supraoptic nucleus.

Heterologous biosynthesis and processing of preprovasopressin in Neuro2A neuroblastoma cells.

To obtain a model for the sorting and processing of preprovasopressin (preproVP), rat VP cDNA was transfected in murine Neuro2A neuroblastoma cells, which do not express VP. The precursor of VP was expressed and processed into the authentic VP gene products VP, neurophysin (NP) and glycopeptide (GP) as determined with reversed phase HPLC and radioimmunoassay. In addition, Neuro2A-specific forms of NP and GP were observed, which may be produced in the constitutive secretory pathway in these cells.

Ontogeny of neuropeptidergic systems: luteinizing hormone releasing hormone (LHRH); somatostatin (SRIF) and neurophysin (NF) in the hypothalamus of the domestic pig by immunocytochemistry.

The ontogenic development of some hypothalamic neuropeptides: luteinizing hormone releasing hormone (LHRH); somatostatin (SRIF) and neurophysin (NF) and their localization in the hypothalamus of fetuses in different stages of the fetal life were studied by immunoperoxidase method. It was found that differentiation of the neurons which produce the examined hormones begins in the midstage of pregnancy. LHRH is stored in the nerve terminals of the median eminence (ME) and organum vasculosum of the lamina terminalis (OVLT) since 72 day of gestation and its amount gradually increases with the development of the embryo. In this stage a few immunoreactive (ir) LHRH perikarya appear but they are most numerous in the last days of pregnancy (110 day). They are localized in the most anterior periventricular parts of the hypothalamus, area preoptica, diagonal band of Broca and very rare in the medial-basal hypothalamus. Somatostatin is produced in the separate neuronal system and appears in the last days of fetal life. Neurophysin is present in both magnocellular nuclei in 72 day-old fetuses, but at the end of gestation it is seen also in some preoptico-septal region.

Putative precursors of vasopressin, oxytocin, and neurophysins in the rat hypothalamus.

The biosynthesis of [arginine8]vasopressin (AVP) and oxytocin (OT) was studied, and the results obtained have been compared with a study of their associated neurophysins (NP), AVP-NP and OT-NP. Rat hypothalamic extracts obtained at acid pH were subjected to Sephadex G-75 gel filtration chromatography and sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE). Fractions of gel chromatography effluent and extract of SDS-PAGE gel slices were subjected to RIA for immunoreactive precursors of AVP, AVP-NP, OT, and OT-NP using specific antisera fro each molecule. Tritiated bovine serum albumin, ovalbumin, and cytochrome c were used as internal standards during gel filtration chromatography and SDS-PAGE. Molecular weight estimates obtained for precursors of AVP were more than 70K, 31K, 13K, 5K, and less than 5K (AVP) by G-75 chromatography and more than 70K, 39K, 15K, and less than 5K (AVP) by SDS-PAGE. Molecular weight estimates obtained for precursors of AVP-NP were more than 70K, 35K, 24K, and 12K (AVP-NP) by G-75 chromatography and 19K and 10K (AVP-NP) by SDS-PAGE. Molecular weight estimates of OT were more than 70K, 35K, 19K, 8K, and less than 5K (OT) by G-75 chromatography and 60K, 35K, 19K, 9K, and less than 5K (OT) by SDS-PAGE. Precursors of OT-NP were estimated to be more than 70K, 17-18K, and 10K (OT-NP) by G-75 chromatography and 28K, 18K and 10K (Ot-NP) by SDS-PAGE. These studies show that there are small differences in precursor processing among AVP, AVP-NP, and OT, OT-NP, but allow for the existence of common precursors for AVP and AVP-NP and for OT and OT-NP.

[Biosynthesis of some hypothalamic neuropeptides. Identification of precursor forms (author's transl)]. / Mécanismes de biosynthèse de quelque neuropeptides hypothalamiques. Identification de formes précurseurs.

The current knowledge on the biosynthetic mechanisms of three hypothalamic neuropeptides, neurophysins, vasopressin and somatostatin, is reviewed. Both neurophysin and vasopressin appear to be first synthesized as higher molecular weight precursors. The methodology elaborated allows to characterize essentially two main forms with apparent Mr similar to or approximately 25 000 and 80 000 respectively. It can be demonstrated that both neurophysin and vasopressin are derived from common precursors.

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

The biosynthetic origin of the 10,000 molecular weight neurophysins, carriers of the peptide hormones oxytocin and vasopressin, has been studied by cell-free synthesis, Poly(A)-RNA was isolated from bovine hypothalamus and translated in a wheat germ system containing (35)S- or (3)H-labeled amino acids. A number of unique [(35)S]cysteine- but few [(35)S]-methionine-labeled proteins were coded by hypothalamic mRNA. A single, major, isotopically labeled protein (molecular weight 23,000-25,000) was immunoprecipitated from these translation mixtures by addition of purified antibodies against bovine neurophysin II and subsequent addition of Cowan I strain of Staphylococcus aureus. Specificity of the immunoprecipitation was demonstrated by competition with unlabeled authentic neurophysins and the absence of competition with structurally unrelated ovalbumin. Furthermore, neither nonimmune serum nor purified antibodies against ribonuclease immunoprecipitated the protein. The [(35)S]cysteine-labeled protein that was specifically immunoprecipitated was oxidized with performic acid and digested with trypsin in the presence of unlabeled, authentic bovine neurophysin II. Peptide mapping revealed that most of the major [(35)S]cysteine-labeled peptides (of the translation product) were identical to major cysteine-containing peptides of authentic neurophysin. The data show that hypothalamic mRNA directs the translation of several unique cysteine-rich proteins in an in vitro cell-free system. Furthermore, one of these proteins, which has a higher molecular weight than authentic neurophysin, is recognized by purified antibodies to bovine neurophysin II and has cysteine-containing tryptic peptides in common with those of authentic neurophysin. The data suggest that this protein is the primary translation product, pre-pro-neurophysin.

Immunological detection of somatostatin in a primitive hypothalamic mouse cell line, precursor of a neurophysin cell lineage.

A primitive nerve cell line (F7 clone), obtained by SV 40 transformation of mouse fetal hypothalamic cells, has been previously shown to share some properties with a progenitor cell for neurophysin synthesizing neuron. This paper describes the immunological detection, at the light microscope level, of somatostatin in this cell line. A SV 40 transformed neurosecretory clone, synthesizing neurophysin and vasopressin, was used as control.

Neurophysin biosynthesis in normal rats and in rats with hereditary diabetes insipidus.

When [35S]cysteine was injected adjacent to the supraoptic nucleus (SON) in rats, it was rapidly incorporated into proteins in the SON. The [35S]cysteine-labeled proteins extracted from the SON were separated by isoelectric focusing on polyacrylamide gels. Twenty minutes after the injection of [35S]cysteine, two major labeled peaks (pI = 5.4 and 6.1) were found in the SON of normal rats; Brattleboro rats had only one major labeled peak (pI = 5.4). One hour after the injection, four major radioactive peaks were found in the SON of normal animals (pI = 5.1, 5.4, 5.6, and 6.1). Animals with diabetes insipidus had only two major labeled proteins (pI = 5.1 AND 5.4). Twenty-four hours after normal rats were injected with [35S]cysteine, all of the labeled peaks described above, except for the one with pI = 5.1, had decreased markedly in size and a small amount of labeled protein with pI about 4.8 was present in the SON. After 24 hr the posterior pituitary of normal animals contained two [35S]cysteine-labeled proteins with pI = 4.6 AND 4.8. The pituitaries of Brattleboro rats had only the pI = 4.6 labeled protein. These pulse-chase data, with data we have presented elsewhere, indicate that the vasopressin- and oxytocin-neurophysins are synthesized as parts of separate precursors (pI = 6.1 and 5.4, respectively). These precursors are converted into at least two intermediates (pI = 5.6 and 5.1) which, in turn, yield the vasopressin-neurophysin (pI = 4.8) and the oxytocin-neurophysin (pI = 4.6).

Biosynthesis of neurophysin proteins in the dog and their isolation.

Neurophysin (Np) is generally found in close association with vasopressin and oxytocin in the hypothalamo-neurohypophyseal complex. Dog neurophysin I and II have been isolated from fresh and frozen posterior pituitaries. The proteins were characterized on the basis of disc electrophoresis, immunological properties, amino acid composition and partial sequence determination. The amino terminal sequence of dog Np I is Ala-Ala-Leu-Asp-Leu-Asp-Val-Arg-Gln-Cys-Leu-Pro-Cys-Gly-Pro-Gly-Gly-Gln-Gly-while that of dog Np-II is Ala-Met-Ser-Asp-Leu-Glu-Leu. The dog Np I appears to be metabolically less stable than Np II. Isotope experiments with [35S]cystine or 3H-labeled amino acids using a design of "in vitro pulse and in vitro chase" as well as "in vivo pulse and in vivo chase," added further confirmation of the capability of the hypothalamic neurosecretory cells to synthesize concomitantly precursors of Np and vasopressin. The radioactively labeled precursors were converted to Np-like protein and vasopressin, both of which were isolated.

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.