The purpose of this work is to elucidate the biochemical and molecular mechanisms responsible for prolactin (PRL) and growth hormone (GH) storage in pituitary, and the relationship between hormone storage and secretion. We found PRL and GH are stored in bovine secretory granules primarily as high molecular weight oligomers (mol wts as much as 106); isolated oligomers are reduced to lower molecular weight forms by exposure to thiols. Conversion can be monitored by size fractionation via gel chromatography or HPLC, but is more easily done by measurement of tissue hormone immunoactivity. The latter is feasible since oligomers are poorly detected by anti-monomer antibodies. We have found that hormone release from isolated secretory granules (largely as monomer) is profoundly increased by thiols and alkaline PH and decreased after exposure to aminothiols and divalent cations. We hypothesize that one site of control of secretion is the conversion from oligomeric storage forms to releasable hormone. This would increase the number of osmotically active particles within granules resulting in an increased intragranular osmotic pressure, thereby promoting water entry, granule swelling, granule membrane rupture, and hormone release. Experimentally, we found that hormone release from isolated granules can be inhibited by counterbalancing the increased osmotic pressure with hyperosmotic medium. We plan to study the kinetics of monomer and oligomer formation in dispersed bovine pituitary cells by labeling newly synthesized protein with 14C-leucine and by distinguishing different hormone forms by HPLC (gel and reverse phase) isoelectric focusing, gel electrophoresis, and RIA. We will determine whether changes in tissue hormone forms occur in cells when secretion is manipulated (eg., exposure to TRH, GHRH, dopamine, or somatostatin). In addition, we will develop a cell free model system for exocytosis in which granules will be incubated with plasma membranes; the requirements for granule: plasma membrane binding, hormone release, and changes in storage forms will be determined and compared to results with dispersed cells. These studies on biochemical characteristics of GH and PRL storage forms, and on factors affecting their conversion to releasable hormones should help define more precisely the final steps of secretion of GH and PRL at the molecular level.
| Lorenson, M Y; Cuccaro, M L; Jacobs, L S (1990) Calcium release from pituitary secretory granules: modulation by thiols, disulfides, and dihydropyridine calcium channel blockers. Endocrinology 126:2671-8 |
| Greenan, J; Lorenson, M Y; Conconi, M V et al. (1990) Alterations in in situ prolactin secretory granule morphology and immunoactivity by thiols and divalent cations. Endocrinology 126:512-8 |
| Lorenson, M Y; Jacobs, L S (1988) Preferential interaction of [35S]cysteamine with pituitary secretory granule storage forms of prolactin. Neuroendocrinology 47:358-64 |
