Abstract
The role of osmotic pressure in the exocytosis of prolactin from rat pituitary tumor (GH) cells in culture was investigated. Reducing the osmotic strength of the medium from 300 mosm to 150 mosm by removal of NaCl did not alter basal secretion of prolactin but inhibited secretion stimulated by thyrotropin-releasing hormone (TRH) and forskolin. Both basal and stimulated secretion of prolactin were inhibited by increasing the osmotic strength of the medium with NaCl (IC50 at approximately 500 mosm). The stimulated release of hormone from GH-cells was independent of sodium and unaffected by replacement of sodium ion with tetramethylammonium or choline, or by addition of 500 nM tetrodotoxin. Secretagogue-stimulated release was, however, dependent upon chloride. Exchange of medium chloride with benzoate or isethionate significantly inhibited the stimulated release of prolactin (IC50 at approximately 60 mM exchange) regardless of the secretagogue utilized (phorbol ester, forskolin, depolarization plus BAY K8644, or TRH). Exchange of medium chloride with either isethionate or benzoate reduced cell volume by 10% compared to 60% for sucrose and mannitol, suggesting that inhibition of secretion by isethionate exchange was not a result of increased intracellular osmotic pressure. Complete exchange of medium chloride with isethionate did not alter equilibrium [3H]methyl-TRH binding, resting internal [Ca2+], or the [Ca2+]i response to depolarization and TRH as measured with intracellularly trapped Fura 2. Chloride removal did not change resting internal pH and recovery from an acid load as measured by the intracellular pH-sensitive dye 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein. The stimulated secretion of prolactin was also inhibited by exchange of chloride with isethionate in normal pituitary cells in primary culture and the ability of normal cells to respond to the dopamine agonist bromocryptine was not affected by the exchange. These results suggest that exocytosis of prolactin from GH-cells and normal pituitary cells in culture is an osmotically driven process that is chloride-dependent. Stimulated release is more chloride-dependent than constitutive release. The inhibitory effect of isethionate substitution occurs after signal transduction and is distinct from the site of dopamine inhibition of prolactin release.
Highlights
From the DeDartmentof Pharmacolog” y and the Cancer Center, University of Rochester Medical Center, Rochester, N e w York 14642
Cell Culture in Exchange Media-For experiments involving growth of cells in media with chloride replaced with isethionate, The Effect of Osmotic Strength on Prolactin Secretion-The medium was made according t.o the formulation given in the Gibco osmotic strength of the medium in which GH pituitary tumor catalogue except that chloride salts were replaced with isethionate. cells were incubated was varied in order to test the effect of
Several lines of evidence suggest that the exocytosis of hormone from regulated secretory cells is dependent upon the mobilization of osmotic species within the secretory granule leading to osmotic swelling, membrane fusion, and lysis [13, 15, 29]
Summary
From the DeDartmentof Pharmacolog” y and the Cancer Center, University of Rochester Medical Center, Rochester, N e w York 14642. Exchange of medium chloride with benzoateor isethionate significantly inproteininto discrete intracellular compartments known as secretory granules or vesicles. These vesicles fuse with the plasma membrane in response toan external signal and release theircontents tothe extracellular space [3]. This mechanism allows cells to respond to a stimulus andrelease, via the regulated pathway, large amounts of their secretory hibited the stimulated releaseof prolactin
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