Secretion from corticotropes after avidin-fluorescein stains for biotinylated ligands (CRF or AVP)

Viable pituitary target cells were identified with biotin-labeled corticotropin-releasing factor (1-Bio-CRF) or arginine vasopressin (Bio-AVP) and avidin-fluorescein (cell sorter grade). Stain was reduced from 8-10% of pituitary cells to less than 3% if nonbiotinylated ligands were added to compete with their respective biotinylated ligand for specific binding sites. However, AVP did not decrease the stain for 1-Bio-CRF nor did CRF decrease the Bio-AVP stain. Media adrenocorticotropin (ACTH) levels increased 4-18 times when 1-Bio-CRF or Bio-AVP were added during the staining process. One hour after staining, the cells secreted ACTH at levels identical to prestain basal levels. When challenged with CRF or AVP after this hour, the cells responded again with increases not different from those seen during staining. Tests of stained cell populations also showed that the cells were fully responsive 24 h after staining. The reverse hemolytic plaque assay for ACTH, which was used to study secretion from individual stained cells, showed that approximately 80% of plaques contained a stained cell and that plaque numbers and areas were like those of unstained populations. These data show that corticotropes are not chronically desensitized during exposure to low doses of biotinylated ligands (50-500 pM).