Characterization of a substance in rat serum which modulates prolactin release in vitro

Abstract

A prolactin (PRL) inhibitory substance(s) present in rat serum has previously been shown to inhibit the release of PRL from rat pituitary cells in vitro (Hymer and Signorella, 1981). In this study, addition of rat serum to rat pituitary cells cultured in serum-free medium inhibited the release of PRL in dose-dependent manner. Neither the form of the released PRL molecule, nor its biological or immunological activity, was altered by rat serum treatment. Serum prepared from clotted blood previously incubated at 37°C always contained more PRL inhibitory activity than serum from blood incubated at 4°C; whole blood serum, but not plasma-derived serum, contained inhibitory activity. The inhibitor therefore comes from a formed element in the blood. Results of several experiments indicated that buffy coat cells, adherent blood cells and peritoneal cells released the inhibitor, but platelets and RBCs did not; it is suggested that a macrophage/monocyte is probably the cellular source of the PRL inhibitor. Gel permeation high-performance liquid chromatography (HPLC) of rat serum indicated that the inhibitory activity was associated with high molecular weight material, i.e. 115000–215000. The PRL inhibitory substance was (a) trypsin sensitive, (b) not extractable with butanol/diisopropyi ether, (c) precipitated at 40% saturated ammonium sulfate, (d) associated with material having an isoelectric point of 4.96 ± 0.26, and (e) not adsorbed with protein-A. These characteristics suggest the proteinaceous nature of the inhibitory substance. The inhibitor did not suppress PRL release until 9 h after addition to cultured cells, but decreased PRL synthesis was evident within 4 h of treatment. The inability of the dopanrine antagonist, haloperidol, to block the inhibitory effect of rat serum indicated that the material does not exert its effect through the dopamine receptor.