Alterations in in situ prolactin secretory granule morphology and immunoactivity by thiols and divalent cations.

The mechanisms involved in PRL storage in secretory granules are generally poorly understood. Recent studies with isolated granules, however, have suggested that granule storage forms may be relatively osmotically inactive due to oligomerization involving hormonal intermolecular disulfide bonds. Thus, expenditure of metabolic energy by the cell in order to maintain granule integrity would be reduced. When secretion is stimulated, oligomer depolymerization by thiol exchange mechanisms has been proposed to occur before or even concomitant with exocytosis. The present studies were designed to investigate the influence of metabolic inhibitors, thiols, and divalent cations on PRL storage in situ, rather than in isolated granules. The results suggest that 1) PRL granules require little energy to maintain their structure, since a combination of azide (10 mM), fluoride (10 mM), and cyanide (1 mM) had no effect on PRL granule morphology in normal anterior pituitary cells in primary culture; 2) disulfide linkages are involved in the osmotic activity of the PRL granule contents, since thiols induced granule swelling in lightly fixed cells; and 3) thiols and divalent cations are capable of altering the arrangement of stored hormone molecules, since PRL immunoactivity could be modified by these agents in glycol methacrylate-embedded exposed granule cores.

Chloroquine affects prolactin secretion and Golgi morphology in the mammotroph.

Chloroquine is widely used as a lysosomal enzyme inhibitor, and while effects on DNA repair and membrane recycling have also been reported, there has been no investigation of effects on the processing of secretory products. To determine whether chloroquine had any effect on secretion and secretory organelles, we monitored the effects of low concentrations (10(-6) M) of chloroquine on a cell population which normally secretes copious amounts of a simple polypeptide (PRL) in vitro and in which there exists a subpopulation of cells which releases PRL very rapidly after synthesis. Cultured anterior pituitary cells were exposed to 10(-6) M chloroquine for 6, 12, 24, or 48 h. At these times, the culture medium was used for determination of PRL content and beta-glucuronidase activity, and the cells were used for determination of DNA content and level of beta-glucuronidase activity or for electron microscopy. Treatment with 10(-6) M chloroquine resulted in 20-40% inhibition of PRL release (maximum inhibition at any dose), no change in the total amount of beta-glucuronidase activity, and a number of ultrastructural changes in the Golgi region consistent with an accumulation of chloroquine within the cisternae and immature granules. The effects of chloroquine on Golgi morphology were unaltered by cotreatment with 50 micrograms/ml cycloheximide, and chloroquine had no affect on PRL synthesis. These results are consistent with an adverse effect of chloroquine on packaging of PRL into immature granules in the Golgi apparatus, without any effect on the release of mature secretory granules.