METABOLIC REGULATION OF VASOPRESSIN AND OXYTOCIN

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Vasopressin and oxytocin nonapeptides synthesized in the hypothalamus and secreted via axons in the neurohypophysis, regulate water conservation, and smooth muscle contractions. Both peptides are synthesized in the form of precursors in covalent linkages with neurophysin peptides, termed preprovasopressin-neurophysin and preprooxytocinneurophysin. Studies of vasopressin and oxytocin biosynthesis in this proposal center on investigations of two aspects of vasopressin and oxytocin gene expression: (1) investigations of the molecular mechanisms involved in the regulation of gene expression and (2) elucidation of the molecular defect in gene expression manifested in the Brattleboro rat, a strain of rat with an inborn defect in the production of vasopressin. In preliminary studies we have used synthetic oligonucleotides complementary to vasopressin and oxytocin mRNAs to specifically prime the synthesis of cDNAs from hypothalamic RNA of rats. These oligonucleotide probes are also used in hybridization assays to evaluate steady-state cellular levels of the specific vasopressin and oxytocin mRNAs. We propose to study the regulation of the expression of the vasopressin and oxytocin genes in the hypothalamus of rats in vivo as a consequence of osmotic stress and in hypothalami in vitro in response to changes in the osmolality of the incubation media. We will use blot hybridization techniques to measure steady state mRNA levels in the hypothalami in response to the stimuli, attempt to measure gene transcription rates by polymerase-runoff experiments and evaluate rates of RNA turnover by pulse-labeling studies of hypothalami in vitro. We found that the hypothalamus of the Brattleboro rat contains hybridizable RNA encoding prepropressophysin-neurophysin but in much lower amounts than is found in the syngeneic normal rat. We propose to clone and structurally characterize recombinant cDNAs prepared to mRNAs from the hypothalami of Long-Evans and Brattleboro rats by nucleotide sequencing and S-1 nuclease mapping experiments to determine whether there is a defect (base mutation, sequence insertion/deletion). We plan to prepare genomic libraries of recombinant bacteriophages from the DNA of the Brattleboro and Long Evans rats and to select and structurally characterize the vasopressin gene. Our interest in carrying out these studies resides in the fact that the genes encoding vasopressin and oxytocin have a bifunctionality in that they are expressed both as endocrine hormones and as local cell-to-cell neuromodulators or neurotransmitters.