PRONEUROPEPTIDE-Y PROCESSING IN LARGE DENSE-CORE VESICLES - MANIPULATION OF PROHORMONE CONVERTASE EXPRESSION IN SYMPATHETIC NEURONS USING ADENOVIRUSES

The efficient delivery of foreign genes into postmitotic cells is beco ming very important for studies of nervous system functions. Cultured sympathetic neurons synthesize neuropeptide Y (NPY) in addition to cat echolamines, providing an experimental model for studying neuronal pep tide biosynthesis. In this work, we have studied the biosynthetic proc essing of NPY in primary cultures of rat superior cervical ganglion (S CG) neurons. NPY activation is complex, requiring sequential actions o f a prohormone convertase (PC), carboxypeptidase H, and peptidylglycin e alpha-amidating mono-oxygenase. Northern analyses established that S CG neurons in the animal contain mRNAs for both PC1 and PC2, and simul taneous immunocytochemistry for NPY and PC1 or PC2 established a 1:1 c orrespondence between NPY and PC2 expression in two thirds of the neur ons that express NPY, both in the animal and in tissue culture. Biosyn thetic studies on proneuropeptide Y (pro-NPY) processing to mature NPY established a close similarity to the rates seen in endocrine cells e xpressing PC2 and established clear differences between the patterns i n SCG neurons and in endocrine cells expressing PC1. Recombinant adeno viruses were used to increase the level of PC1 in the cultured neurons from negligible to a level comparable with the level of PC1 in the an terior pituitary, and pro-NPY processing was markedly accelerated. Whe n the viruses were used to lower the endogenous PC2 levels, using an a ntisense construct, pro-NPY processing was retarded, Taken together, t hese results support a major role for PC2 as the pro-NPY converting en zyme, and they establish the cultured SCG neurons as a model to study neuronal peptide biosynthesis.