METABOLIC REGULATION OF VASOPRESSIN AND OXYTOCIN

加压素和催产素在下丘脑和 通过神经垂体中的轴突分泌，调节水的保存，以及 平滑的肌肉收缩。这两种多肽都是以 与神经物理素肽共价连接的前体，称为 前加压素-神经物理素原和前催产素神经物理素类。的研究 这项提议的核心是加压素和催产素的生物合成 加压素和催产素基因表达的两个方面的研究： (1)调控的分子机制研究 以及(2)阐明基因中的分子缺陷 表达在Brattleboro大鼠，一个品系的大鼠 后叶加压素产生的先天缺陷。在初步研究中，我们 使用了与加压素互补的合成寡核苷酸和 催产素mRNAs特异性启动cDNA的合成 大鼠下丘脑RNA。这些寡核苷酸探针还用于 杂交试验评估细胞的稳态水平 特定的加压素和催产素的mRNAs。我们建议研究 血管加压素和催产素基因表达的调控 渗透压应激对大鼠在体下丘脑的影响 下丘脑在体外对渗透压变化的反应 孵化介质。我们将使用斑点杂交技术来测量 下丘脑对刺激的稳态信使核糖核酸水平 聚合酶-径流法测定基因转录速率的尝试 用脉冲标记技术对RNA周转率进行实验和评价 下丘脑的体外实验。我们发现Brattleboro的下丘脑 大鼠含有编码前加压素-神经物理蛋白的可杂交RNA，但 比同基因正常大鼠的含量要低得多。我们 建议克隆制备的重组DNA并对其结构进行鉴定 从Long-Evans和Brattleboro大鼠下丘脑中提取mRNAs 核苷酸测序和S-1核酸酶图谱实验确定 是否存在缺陷(碱基突变、序列插入/缺失)。我们 计划从重组噬菌体制备基因组文库 Brattleboro和Long Evans大鼠的DNA及其结构的选择 确定加压素基因的特征。我们对实现这些目标的兴趣 研究表明，编码加压素和加压素的基因 催产素有两个功能，因为它们都表达为 内分泌激素和作为局部细胞间神经调节剂或 神经递质。

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.