The studies described in this proposal are designed to investigate the course and intracellular locations of conversion of a neuropeptide precursor into its products. The experimental preparation is the bag cell cluster of the CNS of the mollusk Aplysia californica. These neurons are known to synthesize a precursor protein that contains the sequences for 5 secreted peptides. We have found in pulse-chase kinetic studies that two of these peptides are not coordinately committed to axonal transport or secretion. In additional studies we have found two morphologically distinct neurosecretory granules in these cells and noncovariant distribution of the two secretory peptides on isosmolar density gradients. Taken together these data suggest the hypothesis that the bag cell neurons produce two granule types that differ structurally and by virtue of their peptide contents. The evaluation of this hypothesis is the major focus of this proposal. Studies are described to follow precursor and precursor-derived species through the intracellular pathway from endoplasmic reticulum to secretory granule. A key determination will be the locations of major precursor conversion processes. The methods employed will include centrifugal density-gradient separation of organelles, in vitro RNA translation, use of organic cross linkers, electrophoresis, and high-pressure liquid chromatography. If separate granule enclosure is found for the peptides of interest, the way will be open for investigation of the possibility that the cells can regulate not only the quantity of their secretory product but its specific composition as well. This would represent an hitherto undescribed form of neural plasticity.
