CHARACTERIZATION AND LOCALIZATION OF LIPOCORTIN 1-BINDING SITES ON RAT ANTERIOR-PITUITARY-CELLS BY FLUORESCENCE-ACTIVATED CELL ANALYSIS SORTING AND ELECTRON-MICROSCOPY/

Lipocortin 1 (LC1) is an important mediator of glucocorticoid action i n the anterior pituitary gland, where it appears to act via cell surfa ce binding sites to suppress peptide release. We have exploited a comb ination of fluorescence-activated cell (FAG) analysis/sorting and elec tron microscopy to detect, characterize, and localize LC1-binding site s on the surface of dispersed rat anterior pituitary cells, using huma n recombinant LC1 (hu-r-LC1) as a probe. High affinity (K-d = 14 +/- 3 nM) hu-r-LC1-binding sites were detected on approximately 80% of ante rior pituitary cells dispersed with collagenase. The binding character istics of the ligand resembled those observed in leukocytes, in that i t was saturable; concentration, Ca2+, and temperature dependent; and a bolished by trypsin. Functional studies demonstrated an excellent corr elation between the presence of the cell surface binding protein and t he capacity of an anti-LC1 monoclonal antibody to abrogate the inhibit ory actions of dexamethasone (10 nM) on the release of ACTH initiated in vitro by CRH-41 (1 nM). Morphological analysis of cells harvested b y FAC sorting showed that 1) somatotrophs, corticotrophs, lactotrophs, thyrotrophs, and gonadotrophs were all included in the population exp ressing LC1 binding sites; and 2) the LC1-binding sites assume a punct ate distribution across the cell surface. These data show that anterio r pituitary cells express high affinity surface LC1-binding protein(s) ; they thus provide further evidence for a specific membrane mechanism of action of LC1 in regulating the endocrine function of the anterior pituitary.