Processing and secretion of the egg laying hormone precursor in the bag cells of Aplysia californica.

摘要

Peptide hormones are commonly used by both the nervous and endocrine systems as extracellular chemical messengers and have numerous physiological roles. In the nervous system neuropeptide transmitters are often synthesized as parts of larger inactive precursor proteins which undergo significant post-translational modifications, such as proteolytic cleavages, in order to liberate their bioactive peptides. Further, prohormones are packaged into dense cored secretory vesicles which are transported to subcellular release sites where, upon the appropriate stimulus, exocytosis occurs. Lastly, through interaction with specific receptors and activation of second messenger systems, peptides can act synergistically on ensembles of target cells to mediate complex physiological responses such as behaviors.;The localization of processing reactions was investigated using various methods. Pulse-chase experiments with the Golgi transport inhibitor monensin, with bag cells fractionated over percoll density gradients and using EM autoradiography all suggest that the initial cleavage of the prohormone occurs in a Golgi derived compartment and not in mature secretory granules. Thus, segregation of peptides into separate subcellular pathways allows for the possibility of differential turnover, selective release and differing physiological actions of multiple peptides derived from the same precursor.;This thesis will focus on the biochemistry and cell biology of neuropeptides in the marine snail Aplysia californica. Firstly, a neuropeptide precursor specifically expressed in the identified and FMRFamide immunoreactive neuron L5 is characterized through molecular genetic means. The remainder of the thesis is devoted to a neuropeptide precursor expressed in the neurosecretory bag cells, the egg-laying hormone (ELH) precursor. Through the use of immunological methods (western blotting with antibodies to different portions of the prohormone) and chemical analysis, the major intermediates in the processing pathway are identified. Immunocytochemistry at both the light and electron microscope levels revealed that different portions of the prohormone were packaged into unique classes of dense cored vesicles based upon their antigenicity, size and localization. Differential labeling of the prohormone in pulse-chase experiments revealed that the bag cell peptide (BCP) side of the prohormone was not transported to the neuronal processes at nearly the rate of the ELH side.