Thiol Regulation of Protein, Growth Hormone, and Prolactin Release from Isolated Adenohypophysial Secretory Granules*

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Abstract
We describe a preparation of isolated, anterior pituitary secretory granules suitable for in vitro study of GH and PRL release. Hormone release, monitored by immunoassay or protein measurement, was dependent on time, pH and temperature. Release was higher at alkaline pH and the calculated activation energies of 10.1 and 16.1 Kcal/mol were consistent with physiological energy requirements. Glutathione (GSH) and other thiols, but not nonthiol reducing compounds, stimulated hormone release, whereas disulfides inhibited. The high potency of GSH (50% protein release at 0.94 mM) suggested that it might be physiologically relevant for GH and PRL release. Associated with major release caused by 5 mM GSH (85 ± 5% of the total protein; n = 10) was nearly complete disruption of granule structure as visualized by electron microscopy. Granules treated with thiols displayed an increased number of hormonal antigenic sites, since immunoassays of GH and PRL revealed more than 100% of the hormone measured without thiols. Electrophoretic characterization of released proteins demonstrated mobilities identical to those of purified GH and PRL, regardless of preincubation conditions. When total granule proteins were electrophoresed in nondenaturing gels or in sodium dodecyl sulfate without mercaptoethanol, some high molecular weight protein failed to enter the gels. This band was abolished in sodium dodecyl sulfate gels containing mercaptoethanol, in which the only stainable proteins had hormonal mobility. The sulfhydryl reagents N-ethylmaleimide and 5,5′-dithio-bis-(2-nitrobenzoic acid) inhibited release but could not prevent GSH stimulation. These data suggest that thiols may influence GH and PRL release from isolated secretory granules at multiple sites, including the disulfide-bonded hormone oligomers (granule storage forms) and/or thiol-sensitive granule membrane proteins.