title: Localisation and semi-quantitative measurement of lipocortin 1 in rat anterior pituitary cells by fluorescence-activated cell analysis/sorting and electron microscopy.
abstract: Lipocortin 1 (LC1, also called annexin 1), a Ca2(+)- and phospholipid-binding protein, is an important mediator of glucocorticoid action in the anterior pituitary gland. Previous studies based on immunoprecipitation and Western blot analysis suggest that LC1 is found intracellularly both in the cytoplasm and in association with membranes and also on the cell surface where it attaches to the membrane by a Ca2(+)-dependent mechanism. However, as yet it is unclear which anterior pituitary cell types express the protein. Accordingly, we have developed a method based on a combination of fluorescence activated cell (FAC) analysis/sorting and electron microscopy to detect and quantify intracellular LC1 in rat anterior pituitary cells and to identify the cell types in which it is expressed. In addition, we have measured cell surface LC1 and examined the influence of glucocorticoids on the cellular disposition of the protein. Anterior pituitary cells were dispersed with collagenase. For experiments measuring intracellular LC1, three cell fixation/permeabilisation methods were examined initially, i.e. (1) Zamboni's fluid (30 min) and Triton-X-100 (0.12%, 1 or 12 h); (2) paraformaldehyde (2%, 1 h) and Triton-X-100 (0.2%, 10 min); and (3) paraformaldehyde (0.2%, 15 min) and saponin (0.1%, 5 min). The protocol using paraformaldehyde/Triton-X-100 provided optimal preservation of cell ultrastructure and of LC1 immunoreactivity (ir-LC1) while also effectively permeabilising the cells; it was therefore used in subsequent studies. Using an anti-LC1 monoclonal antibody as a probe, 82+/-5% of the secretory cells in the heterogeneous anterior pituitary cell preparation were shown by FAC analysis to display specific fluorescence for intracellular ir-LC1. Morphological analysis and immunogold-histochemistry of cells separated by FAC sorting identified corticotrophs, lactotrophs, somatotrophs and gonadotrophs in the population displaying LC1 immunofluorescence. LC1 was also detected on the surface of anterior pituitary cells by FACS analysis. Incubation of anterior pituitary cells with dexamethasone or corticosterone (0.1 and 1.0 microM) prior to fixation and analysis produced a significant, concentration-dependent decrease in intracellular ir-LC1 and a concomitant increase in the amount of ir-LC1 detected on the surface of the cells; the effects of the two steroids were indistinguishable quantitatively. In conclusion, we report a novel method which permits (1) the detection and semi-quantitative measurement of intracellular and surface LC1 in anterior pituitary cells; and (2) the identification of the cell types in which the protein is found.
authors.affiliation: Department of Neuroendocrinology, Division of Neuroscience and Psychological Medicine, Imperial College School of Medicine, Charing Cross Hospital, Fulham Palace Road, London, UK.
authors.forename: H C
authors.initials: HC
authors.lastname: Christian
authors.forename: R J
authors.initials: RJ
authors.lastname: Flower
authors.forename: J F
authors.initials: JF
authors.lastname: Morris
authors.forename: J C
authors.initials: JC
authors.lastname: Buckingham
mesh_terms: D000818:Animals; D017305:Annexin A1 / Q000378:metabolism*; D002463:Cell Membrane Permeability; D002469:Cell Separation; D003345:Corticosterone / Q000494:pharmacology; D003907:Dexamethasone / Q000494:pharmacology; D005434:Flow Cytometry; D005938:Glucocorticoids / Q000494:pharmacology; D007425:Intracellular Membranes / Q000378:metabolism; D008297:Male; D008854:Microscopy, Electron; D010903:Pituitary Gland, Anterior / Q000166:cytology / Q000378:metabolism* / Q000648:ultrastructure; D051381:Rats; D017207:Rats, Sprague-Dawley; D014018:Tissue Distribution
chemical_list: D017305:Annexin A1; D005938:Glucocorticoids; D003907:Dexamethasone; D003345:Corticosterone