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 in the anterior pituitary gland, where it appears to act via cell surface binding sites to suppress peptide release. We have exploited a combination of fluorescence-activated cell (FAC) analysis/sorting and electron microscopy to detect, characterize, and localize LC1-binding sites on the surface of dispersed rat anterior pituitary cells, using human recombinant LC1 (hu-r-LC1) as a probe. High affinity (Kd = 14 +/- 3 nM) hu-r-LC1-binding sites were detected on approximately 80% of anterior pituitary cells dispersed with collagenase. The binding characteristics of the ligand resembled those observed in leukocytes, in that it was saturable; concentration, Ca2+, and temperature dependent; and abolished by trypsin. Functional studies demonstrated an excellent correlation between the presence of the cell surface binding protein and the capacity of an anti-LC1 monoclonal antibody to abrogate the inhibitory actions of dexamethasone (10 nM) on the release of ACTH initiated in vitro by CRH-41 (1 nM). Morphological analysis of cells harvested by FAC sorting showed that 1) somatotrophs, corticotrophs, lactotrophs, thyrotrophs, and gonadotrophs were all included in the population expressing LC1 binding sites; and 2) the LC1-binding sites assume a punctate distribution across the cell surface. These data show that anterior 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.
Christian
H C
HC
Department of Neuroendocrinology, Imperial College School of Medicine, Charing Cross Hospital, London, United Kingdom.
Taylor
A D
AD
Flower
R J
RJ
Morris
J F
JF
Buckingham
J C
JC
D000324:Adrenocorticotropic Hormone / Q000378:metabolism; D000818:Animals; D017305:Annexin A1 / Q000378:metabolism*; D001665:Binding Sites; D001773:Blood Cells / Q000378:metabolism; D002469:Cell Separation; D005434:Flow Cytometry; D006801:Humans; D007962:Leukocytes / Q000378:metabolism; D008297:Male; D008854:Microscopy, Electron; D010903:Pituitary Gland, Anterior / Q000166:cytology / Q000378:metabolism*; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D014018:Tissue Distribution
D017305:Annexin A1; D011994:Recombinant Proteins; D000324:Adrenocorticotropic Hormone