FUNCTIONAL ROLE OF HETEROGENETIY IN PITUITARY CELLS

在过去的两年里，我们开发了一种测量荷尔蒙的方法 单个腺垂体(AP)细胞在混合培养中的分泌物称为 反向溶血空斑试验。化验的终点是抗体 分泌激素的AP细胞周围的RBC定向溶血 与抗体相对应。AP细胞在有 蛋白A偶联绵羊红细胞；在AP激素抗体和 补体，分泌的激素与抗体结合，抗体又与 蛋白A，修复补体，并诱导红细胞溶解。空旷的区域 AP细胞周围的溶解称为斑块，可检测到 用显微镜测量的。 我们用RHPA测定催乳素、黄体生成素、促肾上腺皮质激素和生长激素。 单个AP细胞的分泌。在所有情况下，我们都找到了证据 对于优先反应的细胞亚群 下丘脑分泌物。在本申请中，我建议 进一步研究生长激素的功能异质性 反向溶血空斑试验。具体目标如下： (1)使用下丘脑GHRF和 生长抑素来确定是否存在假设的亚群 优先对这些分泌物作出反应。 (2)斑块大小以生长激素的PG表示。 (3)测定生长激素在个体生长激素中的储存量 光镜ICC和显微分光光度法。 (4)H-3亮氨酸脉冲标记法测定生长激素转化率 放射自显影检测H-3GH的释放量(以颗粒为单位 牌匾)。 (5)检测单个斑块上的生长激素释放因子和生长抑素受体 形成生长激素；这将允许建立数量 受体数目与细胞生长激素分泌量的关系。 (6)采用ICC法和显微分光光度法检测细胞内cAMP； 建立细胞内cAMP含量与GH分泌的关系。 (7)用微量荧光法测定个体生长激素对钙的摄取 奎宁2的分析：建立与生长激素分泌量的关系。 (8)将对以下亚群进行电子显微镜和EM-ICC检查 生长激素。

During the past 2 years we have developed a method for measuring hormone secretion by individual adenohypophysial (AP) cells in mixed culture called the reverse hemolytic plaque assay. The end-point of the assay is antibody directed hemolysis of RBC surrounding an AP cell secreting the hormone corresponding to the antibody. AP cells are cultured in the presence of protein A-coupled ovine RBC; in the presence of AP hormone-antibody and complement, secreted hormone binds to antibody which in turn binds to protein A, fixes complement, and induces lysis of the RBC. The clear areas of lysis surrounding AP cells are called plaques and are detected and measured microscopically. We have used the RHPA for measuring prolactin, LH, ACTH, and growth hormone secretion by individual AP cells. In all instances we have found evidence for subpopulations of cells that are preferentially responsive to hypothalamic secretagogues. In the present application, I propose to investigate further this functional heterogeneity of somatotropes using the reverse hemolytic plaque assay. The specific aims are as follows: (1) perform a somatotrope population study using hypothalamic GHRF and somatostatin to determine if putative sub-populations exist that are preferentially responsive to these secretagogues. (2) quantitation of plaque size as pg of GH. (3) determine the amount of GH stored in individual somatotropes using light-microscopic ICC and microspectrophotometry. (4) determine GH turn-over rate using pulse-labeling with H-3 leucine and autoradiography to detect amount of H-3 GH released (measured as grains in the plaque). (5) measure receptors for GHRF and somatostatin on individual plaque forming somatotropes; this will permit establishment of the quantitative relationship between receptor number and amount of GH secreted/cell. (6) measure intracellular cAMP using ICC and microspectrophometry; establish relationship between amount of intracellular cAMP and GH secreted. (7) measure Ca uptake by individual somatotropes using microfluorometric analysis of Quin 2: establish relationship with amount of GH secreted. (8) electron microscopy and EM-ICC will be performed on subpopulations of somatotropes.