This is s request for an ADAMHA Research Scientist Award. The overall goal of this research is to gain a better fundamental understanding of the regulatory processes affecting peptidergic neurons and andocrine cells in general and opioid peptide producing cells in particular. With the basic skills of molecular biology acquired during the tenure of my RSDA Level II Award, I plan to continue directing my professional growth toward the application of these tools to the study of opioid peptide producing cells. Further professional growth will be achieved through collaborative projects with outstanding molecular biologists at Johns Hopkins, researchers at the Addiction Research Center, several active collaborations with noted scientists at other institutions. Participation in scientific meetings and my association with the Neuroscience graduate program. Primary anterior and intermediate pituitary cultures maintained in serum free medium will be used to evaluate the effect of secretagogues on specific steps in the post-translational processing of Pro- ACTH/endorphin and to identify key factors regulating corticotrope and melanotrope function. The global responses of Pro- ACTH/endorphin-producing cells to secretagogues of various classes will be compared by 2-dimensional SDS-PAGE of biosynthetically labeled proteins and responsive mRNA species will be identified by screening AtT-20 and melanotrope cDNA libraries with radiolabeled cDNA probes prepared from secretagogue treated tissues. Neuroendocrine specific cDNAs or cDNAs associated with D-2 type dopamine receptors will be cloned, sequenced, and analyzed for function. The cDNA and gene encoding rat peptidyl-glycine alpha- amidating monooxygenase (PAM), a key enzyme in the biosynthesis of many neuropeptides, will be cloned and sequenced. Its tissue distribution will be determined by enzyme assay, Western and Northern analysis. Membrane-associated PAM will be purified and characterized. Expression vectors will be used to investigate the subcellular localization of PAM and for structure/function studies. Regulatory factors affecting PAM mRNA, protein and enzyme activity in melanotropes, corticotropes and cardiocytes will be investigated; the 5'-regulatory region of the gene encoding PAM will be linked to a reporter gene for further studies of factors controlling tissue specific and regulated expression of this key peptide biosynthetic enzyme.
