A common precursor molecule, pro-opiomelanocortin (POMC), serves as a substrate for differential cleavages by proteolytic enzymes in mammalian anterior and intermediate lobe pituitary cells. It has been postulated that anterior lobe POMC peptide secretion is positively regulated by peptidergic factors (corticotropin releasing factor; CRF), synthesized in the hypothalamus and released into the anterior pituitary vasculature. Intermediate lobe corticotroph secretion appears to be tonically inhibited by dopamine (DA). In preliminary experiments using the perifusion system described herein, we have observed the following: First, the most potent stimulator of POMC-derived peptides from either anterior or intermediate corticotrophs is high concentrations of potassium ion (K+), second, low K+ is as effective as maximal doses of DA in inhibiting endorphins and Alpha-MSH secretion and third, a brief exposure of perifused, dispersed intermediate corticotrophs to high K+ (in the presence of 10 MuCi/ml 35S-methionine) results in an increase in newly synthesized POMC, and radiolabeled endorphins. These experiments have established the feasibility and methodology for our proposed studies which are focused on the role of K+ and calcium (Ca++) ions in the biosynthesis of POMC, and secretion of POMC-derived peptides. Using dispersed mouse intermediate and/or anterior lobes cells (either pre-labeled, or unlabeled) in the perifusion system described we will re-evaluate the roles of K+ and Ca++ in basal release; what is """"""""basal"""""""" release, and which ion(s) are required. We will extend our """"""""dynamic labeling"""""""" studies to include Ca++ and attempt to define the effects of repeated and/or long term ion exposure or deprevation on biosynthesis and processing of POMC. These experiments will be analyzed by RIA's for ACTH, endorphins, and Alpha-MSH, as well as immunoprecipitation, fractionation and quantitation of radiolabeled POMC products by gel electrophoresis, combined with scintillation spectrometry. Finally, in order to establish whether K+ ions are moving into or out of the intracellular compartment, we will directly measure ion fluxes during stepwise and gradual changes of secretagogues in the extra cellular compartment. In order to accomplish this we will substitute radioactive Rubidium for K+. Thus we will be able to monitor the movement of K+ during stimulation or inhibition of hormone release. These studies will extend our knowledge of the ionic mechanisms regulating the secretory event, and the effect(s) of ions on the biosynthesis, processing and release of the end products of a poly protein, in this case POMC.
| Hatfield, J M; Daikh, D I; Adelman, J P et al. (1989) In situ hybridization detection of marked differences in pre-proopiomelanocortin messenger ribonucleic acid content of individual corticotropes and melanotropes. Endocrinology 124:1359-64 |
| Allen, R G; Hatfield, J M; Stack, J (1988) Post-translational processing of pro-opiomelanocortin (POMC)-derived peptides during fetal monkey pituitary development. I. Adrenocorticotropin (ACTH) and alpha-melanotropins (alpha-MSHs). Dev Biol 126:156-63 |
| Hatfield, J M; Allen, R G; Stack, J et al. (1988) Post-translational processing of pro-opiomelanocortin (POMC)-derived peptides during fetal monkey pituitary development. II. beta-Lipotropin (beta-LPH)-related peptides. Dev Biol 126:164-72 |
