The objectives of this proposal are to elucidate cellular and molecular mechanisms which regulate neuropeptide production in adrenal medullary tumors, and to gain insights into regulatory derangements which might occur during the development of these neoplasms. Current studies at the cellular level indicate that PC12 pheochromocytoma cells are a representative model of many spontaneously occurring rat adrenal medullary tumors, that these cells produce the peptide neurotensin (NT), and that production of this phenotypic marker is regulated by synergistic actions of nerve growth factor (NGF), glucocorticoids and activators of adenylate cyclase. To investigate the molecular mechanisms of the complex regulation, in the proposed studies, cDNA clones encoding NT will be constructed and used in hybridization assays to quantitate changes in NT mRNA levels in response to these agents. These cDNA clones will also be used to deduce the amino acid sequence of the NT precursor, and to investigate that organization of the NT gene. In addition, both the cellular and molecular studies will be expanded by testing responses of NT peptide and mRNA to other influences which might affect neuropeptide production by normal or abnormal chromaffin cells during development or in adult life. These include membrane excitation, alteration in synthesis, storage or uptake of catecholamines, and expression of specific oncogenes which mimic the morphological effects of NGF. The roles of cyclic AMP in regulating NT peptide and NT mRNA will be further investigated using PC12 cell mutants deficient in cyclic AMP dependent protein kinase type I and/or type II. The regulation of NT peptide in PC12 cells will also be compared to that in fetal, neonatal and adult rat chromaffin cells, to determine whether NT regulation in PC12 cells is characteristic of a particular stage of normal development. Information gained from studies of neuropeptide gene expression using neuroendocrine tumors of the adrenal medulla as a model might contribute significantly to understanding of pathological conditions both of the adrenal medulla itself and of other functionally similar neuroendocrine tissues. Because the characteristics of neuroendocrine tumor cells often parallel the behavior of these cells' normal counterparts, insights might also be gained into a number of developmental and functional abnormalitites.
