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.
| Im, Young Jun; Hurley, James H (2008) Integrated structural model and membrane targeting mechanism of the human ESCRT-II complex. Dev Cell 14:902-13 |
