Intrapituitary distribution and effects of annexin 5 on prolactin release

Annexin 5 is expressed by rat anterior pituitary cells and a depolarizing stimulus results in increased extracellular display and, depending on local calcium concentrations, potential release into the extracellular environment. In order to further investigate the role of annexin 5 in anterior pituitary function, we have examined the intracellular distribution by immunocytochemistry and the effects of annexin 5 on the release of a major secretory product, prolactin. Prolactin was chosen because we could easily monitor effects on basal release and effects on the immediate and sustained phases of thyroid stimulating hormone releasing hormone (TRH)-stimulated release. Immunocytochemical localization of annexin 5 showed staining of the majority of anterior pituitary cells. Labeling was predominantly on the nuclear envelope and plasma membrane. For the chosen secretory product, prolactin, annexin 5 was found in most, but not all prolactin positive cells. When recombinant annexin 5 (50 ng/mL) was added to a 3 h static culture incubation of rat anterior pituitary cells, prolactin release was inhibited by about 30% (p<0.05). A lower dose had a reduced effect and higher doses had no further inhibitory effect, indicating that the effect was specific to annexin 5 and not a nonspecific toxic effect of some contaminant in the preparation. This interpretation was further strengthened in a time-course experiment demonstrating that when TRH and annexin 5 were added together, there was no effect of annexin 5 on the amount of prolactin released. After a 3 h preincubation in annexin 5, however, prolactin release, in response to TRH, was suppressed by about 30% in both the acute and sustained phases. These data suggest that annexin 5 may be a local regulator of release in the anterior pituitary, but a slow onset effect on both phases of TRH-stimulated release suggests that this is not an effect at the plasma membrane such as local extracellular calcium depletion by plasma membrane-bound annexin 5.