Central facts about nerve growth factor (NGF) have yet to be established. Despite numerous studies elucidating the protein's unique ability to promote and maintain peripheral nerve function, the site of NGF's synthesis within the body remains unknown. Cells from many different species and organs synthesize and secrete NGF-like molecules in culture, giving rise to the theory that all peripheral tissues produce the protein in vivo. The first goal of our work will be to investigate that hypothesis. Selected tissues in the mouse which are innervated by sympathetic, sensory, and motor neurons will be denervated in order to stimulate NGF production, and radioimmunoassays and bioassay will measure levels of the protein. NGF-containing cells will be identified by light and electron microscopy using antibodies to NGF coupled with fluorescent dyes and colloidal gold. Secondly, we seek to establish whether peripheral tissues in vivo and mouse cells in culture produce similar forms of NGF and whether these proteins are identical to the well-characterized NGF in mouse salivary glands. Tissue extracts and conditioned medium will be electrophoresed on polyacrylamide gels in dissociating and non-dissociating solvents. Cells will be analyzed by the """"""""western blot"""""""" technique, utilizing as probes antibodies raised against the Alpha, Beta and Gamma subunits of the 7S NGF complex from submandibular glands. Results from these experiments should provide a more complete understanding of NGF's distribution in peripheral tissues and its molecular properties. These data will fill significant gaps in our knowledge as we move towards an ultimate understanding of NGF's role in nerve development, function, and repair.
