Prolactin secretory bypath exposed in cultured lactotrophs

In the present report, the prolactin secretory pathways were re-examined in cultured lactotrophs submitted to various experimental conditions of stimulation, inhibition and/or alteration of the intracellular flow of the synthesis and release of prolactin. Primary cultures of rat pituitary cells stimulated with thyrotropin-releasing hormone, or inhibited with either cycloheximide or dopamine in the presence or absence of 0.1 mu g/ml brefeldin A, were used. The radioimmunoassay quantification of released and intracellular prolactin was correlated with ultrastructural and immunocytochemical studies. Brefeldin A diminished significantly the secretion and the intracellular content of prolactin 4 h after application, while morphological effects were seen starting from 30 min. The drug did not modify the response to thyrotropin-releasing hormone (120% increment). The simultaneous incubation of brefeldin A with cycloheximide or dopamine diminished the released prolactin concomitant with a lower (cycloheximide) or greater (dopamine) hormonal intracellular prolactin content with respect to brefeldin A. The combined treatment cycloheximide-dopamine inhibited prolactin secretion. The ultrastructural and immunocytochemical features of lactotrophs supported these radioimmunoassay data. These results revealed that prolactin release in vitro in the presence or not of brefeldin A is dependent on either: the neo-synthesized hormone that can be inhibited by cycloheximide, and the hormone stored in granules, the exocytosis of which was blocked by dopamine, indicates the contribution of both constitutive and regulated pathways in the secretory process. The brefeldin A blockade of the intracellular transport also disclosed morphological evidence of an alternative pathway of prolactin secretion through vesicles originated in the rough endoplasmic reticulum bypassing the Golgi complex.