Extracellular matrices play a critical role in cell growth and differentiation in part by influencing cell shape. PC 12 cells are a clonal cell line which exhibits a dramatic change in cell shape and in the release of dopamine when cultured on extracellular matrix. Since these cells also produce the peptide hormones, enkephalin and vasoactive intestinal peptide (VIP); and manifest acetylcholine and glucocorticoid receptors, they provide an excellent model for definitive studies on the role of cell shape in releasing and responding to chemical messengers, i.e., hormones. Numerous functional properties relating to hormone production and secretion will be compared in PC 12 cells which are flattened on extracellular matrix versus rounded on plastic. These functional properties include: dopamine secretion, storage, synthesis, uptake, and turnover; enkephalin secretion, synthesis and storage; vasoactive intestinal peptide secretion, synthesis and storage; acetylcholine sensitivity and receptor binding characteristics; glucocorticoid sensitivity and receptor binding characteristics; and adenylate cyclase activity and sensitivity. A radioenzymatic assay is used to measure catecholamine secretion and storage. Radioimmunoassays for enkephalin and VIP will be used to measure secretion and storage. Radiolabeled tyrosine and Dowex resin chromatography will be used to determine dopamine synthesis and turnover. Radiolabeled norepinephrine will be used in experiments examining re-uptake function. Radiolabeled leucine or methionine in combination with immunoprecipitation of enkephalin and VIP will be used to examine synthesis of the peptide hormones. Radiolabeled receptor ligands will be used to characterize the acetylcholine and glucocorticoid receptor properties in the cells which are flattened on matrix versus rounded on plastic. During development a single cell becomes a multicellular organism composed of many different cells with a complexity of functions. This process involves growth, migration and differentiation of cells. Mistakes in this process result in all forms of birth defects and death. During differentiation various parts of the genome are activated or repressed at critical times, but the cues for this process are not well understood. Cells transmit and receive information to and from their environment which must play a role in gene expression. This information appears to involve chemical signals as well as physical position. This project proposal abstracts from these concepts as it focuses on the role of cell shape in hormone secretion and processing.
| Bethea, C L; Borg, T K (1988) PC12 cell aggregation and dopamine production on EHS-derived extracellular matrix. Mol Cell Endocrinol 58:113-28 |
