Christian H C, Flower R J, Morris J F, Buckingham J C
Department of Neuroendocrinology, Division of Neuroscience and Psychological Medicine, Imperial College School of Medicine, Charing Cross Hospital, Fulham Palace Road, London, UK.
J Neuroendocrinol. 1999 Sep;11(9):707-14. doi: 10.1046/j.1365-2826.1999.00389.x.
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.
脂皮质素1（LC1，也称为膜联蛋白1）是一种结合钙离子和磷脂的蛋白质，是糖皮质激素在前脑垂体中发挥作用的重要介质。以往基于免疫沉淀和蛋白质印迹分析的研究表明，LC1存在于细胞内的细胞质中，与细胞膜相关联，也存在于细胞表面，通过钙离子依赖机制附着于细胞膜。然而，目前尚不清楚前脑垂体的哪些细胞类型表达该蛋白。因此，我们开发了一种基于荧光激活细胞（FAC）分析/分选和电子显微镜相结合的方法，用于检测和定量大鼠前脑垂体细胞内的LC1，并鉴定表达该蛋白的细胞类型。此外，我们还测量了细胞表面的LC1，并研究了糖皮质激素对该蛋白细胞分布的影响。用胶原酶分散前脑垂体细胞。对于测量细胞内LC1的实验，最初研究了三种细胞固定/通透方法，即：（1）赞博尼氏液（30分钟）和曲拉通-X100（0.12%，1或12小时）；（2）多聚甲醛（2%，1小时）和曲拉通-X100（0.2%，10分钟）；（3）多聚甲醛（0.2%，15分钟）和皂角苷（0.1%，5分钟）。使用多聚甲醛/曲拉通-X100的方案在有效使细胞通透的同时，能最佳地保存细胞超微结构和LC1免疫反应性（ir-LC1）；因此在后续研究中使用该方案。使用抗LC1单克隆抗体作为探针，FAC分析显示，在前脑垂体细胞异质制备物中，82±5%的分泌细胞显示出细胞内ir-LC1的特异性荧光。通过FAC分选分离的细胞的形态学分析和免疫金组织化学鉴定出显示LC1免疫荧光的细胞群体中的促肾上腺皮质激素细胞、催乳素细胞、生长激素细胞和促性腺激素细胞。通过FACS分析也在前脑垂体细胞表面检测到了LC1。在固定和分析之前，用 dexamethasone 或皮质酮（0.1和1.0 microM）孵育前脑垂体细胞，导致细胞内ir-LC1显著降低且呈浓度依赖性，同时细胞表面检测到的ir-LC1量相应增加；两种类固醇的作用在数量上无法区分。总之，我们报告了一种新方法，该方法能够：（1）检测和半定量测量前脑垂体细胞内和表面的LC1；（2）鉴定发现该蛋白的细胞类型。
