Peptides represents a major mode of chemical transmission in the nervous and endocrine systems. We have used the LHRH prohormone as a model system to study the regulation of biosynthesis, processing, and secretion of neuropeptides. In order to study mechanisms underlying the regulation of and the relationships among these 3 cellular processes, we have extended our studies from rat brain to an immortalized LHRH neuronal cell line. With this cell line, we have shown that the pro-LHRH has multiple molecular forms and that it is processed to various C-terminally extended LHRH intermediates with LHRH and GAP-(1-56) as the final products. Our peptide and Northern blot studies indicate that the pro-LHRH undergoes processing by the PC2 endopeptidase, carboxypeptidase E, glutaminyl cyclase, and alpha-amidating enzyme. Electron microscopy results reveal that the pro-LHRH is packaged Into secretory vesicles and all of the processing steps may be executed within this vesicle. In secretion experiments, perifusion of the cells with media alone leads to rhythmic and pulsatile secretion of LHRH and GAP. These findings indicate that the LHRH neuron contains a pulse generator which may regulate LH secretion from the pituitary. When the cells are depolarized by [K+] or stimulated with phorbol esters, prostaglandin E2, phospholipase C or, A2, secretion of LHRH and GAP is enhanced. Presently, we are attempting to describe the mechanism of phorbol ester stimulated secretion. Our Western blot and immunocytochemical studies indicate that these LHRH cells express protein kinase Calpha, beta1, betaII, gamma, delta, epsilon, and zeta. Upon continual exposure to phorbol esters, these PKC isoenzymes show differential down-regulation. Future studies will examine in detail the molecular and cellular mechanisms which regulate biosynthesis of the pro-LHRH peptide, its processing, and the various pathways of its secretion.
