The overall objectives of the proposed research deal with how neurons and endocrine cells produce and secrete biologically active substances -- primarily peptides, but also conventional neuro- transmitters. One set of questions concerns what forces act upon the cells to produce the final mixture of agents secreted; these forces may be direct interactions with other cells, molecules arriving in the blood, nerve impulses in a certain pattern, or the summation of multiple influences acting on the cell. Another set of questions concerns what post-transcriptional or post- translational processes occur within a particular neuron or endocrine cell to give the final pattern of bioactive peptides produced; variables include synthesis of peptide precursor, enzymes that process precursor into final peptides, necessary cofactors, storage of peptide, alterations at the cell membrane during secretion. The production of neuropeptide Y (NPY) and pro- ACTH/endorphin after transfection of normal and site-directed mutant cDNA into various cell lines will be examined with biosynthetic labeling, antibodies, peptide analyses, and microsequencing. The apparent competition between transfected and endogenous peptides for transit through the secretory pathway will be probed. Specific endoproteases involved in the biosynthesis of peptides from their precursors will be sought. How cells sort proteins into the secretory pathway vs other fates will be investigated after transfection of cell lines with cDNAs for hybrid proteins. Electrophysiological events during secretion will be recorded by patch clamping. Expression vectors to study wild type and mutant forms of peptidyl-glycine alpha-amidating monooxygenase (PAM) will be constructed and expressed in various cell types; the consequences of expressing excess or nonfunctional PAM will also be studied. The develop mental loss of plasticity in peptide processing known to occur in the anterior pituitary will be examined, as will influences of maternal drugs on peptide processing in offspring. Regulatory effects of drugs and targets for innervation on production of NPY and conventional neurotransmitters will be studied in sympathetic neurons.
