Density separation of rat adrenocortical cells: Morphology, steroidogenesis, and P-450scc expression in primary culture

Abstract

We have developed a method that separates rat adrenocortical cells by density into populations which retain zone specific properties in primary culture. Two different parenchymal populations were obtained and designated 2FASC (1.034 g/ml, 18.0 microns cell diameter) and 7GLOM (1.069 g/ml, 11.7 microns cell diameter). In freshly isolated cell suspensions the physical characteristics and differential steroidogenic responses to adrenocorticotropin and angiotensin II suggested that 2FASC cells originated predominantly from the zona fasciculata and 7GLOM cells from the zona glomerulosa. In primary culture (Dulbecco's Modified Eagle's Medium-F12 medium with 15% horse serum and 2.5% fetal bovine serum) the two populations exhibited different morphologies. 2FASC cells retained lipid and formed cohesive epithelial monolayers that remained stationary for 3 wk. 7GLOM cells were initially epithelial but rapidly lost lipid, spread, and assumed fibroblastic shapes. Both cell types were positive for the cholesterol side-chain cleavage cytochrome P-450 by immunofluorescence. Therefore, the morphologic changes seen in 7GLOM cultures were due to modulation, not fibroblastic overgrowth. This phenotypic plasticity may reflect the mesodermal origin of the adrenal cortex, and the subcapsular location of 7GLOM cells in vivo. In contrast, cells such as 2FASC which are located deeper in the cortex seem to have a more restricted, fully committed parenchymal phenotype.

Isolation of Human Adrenocortical Aldosterone-Producing Cells by a Novel Immunomagnetic Beads Method

“…Previous methods for isolation of adrenocortical cells were based on either manual dissection of the different adrenal zones (10,16) or density gradient centrifugation (17,18). By itself, dissection cannot provide an accurate separation of the cells and carries some contamination with ZF and ZR cells.…”

Adrenocortical zonation and ACTH

“…Both of these are much more abundant in the inner zones (of the mouse and rat adrenal, respectively) Raza and Vinson, 2000) than in the glomerulosa, again consistent with the view that there is relatively little P450 activity in the glomerulosa and that it is probably only a poor de novo source of steroid. Roskelley and Auersperg, 1990;Halder et al, 1998;Mitani et al, 1999 CYP11B1 Ϫ ϩϩϩ Mitani et al, 1999;Ho and Vinson, 1993a;Halder et al, 1998CYP11B2 ϩ Ϫ Mitani et al, 1999Peters et al, 1998;Halder et al, 1998 CYP21 No Yamaguchi et al, 1990;Oda et al, 1991;Husain et al, 1987;Mulrow, 1992b;Shier et al, 1989;Vinson and Ho, 1998b Signalling MAPK: Vinson et al, 2000;Vinson, 2000 c-fos Lehoux et al, 1998;Raza et al, 1998;Vinson et al, 1998c-jun Lehoux et al, 1998Raza et al, 1998;Vinson et al, 1998 Halder et al, 1998;Whitworth and Vinson, 2000;Raza et al, 1998 Distinctions between the steroidogenic activities of fasciculata and reticularis in the rat are less clear. As in other species, including the human, the range of steroid products is essentially the same, although the response to ACTH stimulation is less, and it may be for this reason that overall steroid output appears significantly lower.…”

Rapid ras-oncogene-mediated transformation maintains steroidogenic differentiation in adrenocortical parenchymal cells

“…The resulting crude cell suspension was filtered through multilayered nitex cloth (100-km mesh size), washed by centrifugation and resuspendend in Dulbecco's modified Eagle's medium (DMEM)/F12 medium (1:l mixture, Sigma). Homogeneous populations of cells derived from the zona glomerulosa were then isolated by density gradient centrifugation as described previously [37]. Briefly, adrenocortical cell suspensions were layered upon discontinuous Percoll (Pharmacia, Uppsala, Sweden) gradients composed of 8 steps (15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% and 65% Percoll in DMEM/F12), and centrifuged at 1000 g for 20 min.…”

“…In primary culture GLOM cells are highly steroidogenic ( Fig. 7) [37]. A strict requirement for this function is the expression of the mitochondria1 P-450 side-chain cleavage enzyme (P-~~OSCC), which converts cholesterol to pregnenolone, the first step in all steroidogenic pathways [19, 431. In response to treatment with the trophic hormone second messenger CAMP, primary GLOM cells expressed P-45Oscc that was localized as punctate staining in the mitochondria of all cells.…”