title: The mechanism of action of cysteamine in depleting prolactin immunoreactivity.
abstract: The thiol reagent cysteamine (CSH) depletes anterior pituitary cells of immunoreactive PRL both in vivo and in vitro. We examined the hypothesis that CSH affects either the solubility or immunoreactivity of PRL through a mechanism involving thiol-disulfide exchange. Adult female rats were treated with either CSH (300 mg/kg, sc) or an equimolar dose of ethanolamine as a control. Anterior pituitary glands were extracted in 0.1 M sodium borate buffer, pH 9.0. Treatment of pituitary extracts with beta-mercaptoethanol (BME) destroys the immunoreactivity of PRL. However, extraction in the presence of reduced glutathione or CSH of pituitaries of rats treated with CSH restores immunoreactive PRL to control levels. Extracts were also subjected to polyacrylamide gel electrophoresis (PAGE). On gels of pituitary extracts of CSH-treated rats, the band that comigrates with purified PRL is diminished compared to that in ethanolamine-treated controls. This is found regardless of whether the borate extracts are treated with BME. However, extraction of the pituitaries in sodium dodecyl sulfate-containing buffer followed by chemical reduction with BME restores the PRL band. Therefore, CSH acts on PRL through a thiol-related mechanism to yield a product that is poorly soluble in aqueous buffer at pH 9 and is poorly immunoreactive. Dispersed anterior pituitary cells in tissue culture were incubated with L-[35S]methionine to radiolabel newly synthesized peptides. These cultures were incubated in the presence of either CSH or ethanolamine. PAGE followed by autoradiography confirmed the above results obtained in vivo. Also, extracts of CSH-treated cultures were subjected to gel permeation chromatography. As determined by PAGE, at least some of the radiolabeled PRL can be recovered from void volume fractions by reduction with BME, indicating that CSH induces the formation, through disulfide exchange, of a high mol wt form of PRL, possibly PRL oligomers.
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D003543:Cysteamine / Q000494:pharmacology*; D004492:Edetic Acid / Q000494:pharmacology; D019856:Ethanolamine; D004983:Ethanolamines / Q000494:pharmacology; D005260:Female; D005978:Glutathione / Q000494:pharmacology; D010903:Pituitary Gland, Anterior / Q000187:drug effects / Q000378:metabolism*; D011388:Prolactin / Q000378:metabolism*; D011863:Radioimmunoassay; D051381:Rats; D011919:Rats, Inbred Strains
chemical_list: D004983:Ethanolamines; D019856:Ethanolamine; D003543:Cysteamine; D011388:Prolactin; D004492:Edetic Acid; D005978:Glutathione