FUNCTIONAL ROLE OF HETEROGENETIY IN PITUITARY CELLS

在过去的2年中，我们开发了一种测量激素的方法， 单个腺垂体（AP）细胞在混合培养物中分泌， 反向溶血空斑试验。 测定的终点是抗体 分泌该激素的AP细胞周围的RBC的定向溶血 对应于抗体。 AP细胞在以下存在下培养： 蛋白A偶联绵羊红细胞;存在AP抗体和 补体分泌的激素与抗体结合，抗体又与补体结合。 蛋白A，固定补体，并诱导RBC溶解。 空隙区域 AP细胞周围的溶解称为斑块， 用显微镜测量。 我们用RHPA测定催乳素、LH、ACTH和生长激素 单个AP细胞的分泌。 在所有案件中我们都发现了证据 对于优先响应的细胞亚群， 下丘脑促分泌素 在本申请中，我提议 进一步研究这种功能异质性的生长激素使用 反向溶血空斑测定。 具体目标如下： (1)使用下丘脑GHRF进行生长激素人群研究， 生长抑素，以确定是否存在假定的亚群， 对这些促分泌素有优先反应。 (2)将噬斑大小定量为GH的pg。 (3)测定GH的量储存在个别生长激素使用 光学显微ICC和显微分光光度法。 (4)用H-3亮氨酸脉冲标记法测定GH转化率， 放射自显影检测释放的H-3 GH的量（以 饰板）。 (5)测量单个斑块上GHRF和生长抑素的受体 形成生长激素;这将允许建立定量的 受体数量与GH分泌量/细胞的关系。 (6)用ICC和显微分光光度法测定细胞内cAMP; 建立了细胞内cAMP含量与GH分泌量关系。 (7)用显微荧光计测量个体生长激素的钙摄取 Quin 2分析：建立与GH分泌量的关系。 (8)电子显微镜和EM-ICC将对以下亚群进行 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.