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.