MINERALOCORTICOID AND GLUCOCORTICOID RECEPTOR STEROID-BINDING AND LOCALIZATION IN COLONIC CELLS

In rat colon epithelium glucocorticoids and mineralocorticoids regulat e Na transport by binding to distinct receptors and stimulating differ ent pathways. The distribution and intracellular localization of miner alocorticoid (MR) and glucocorticoid (GR) receptors in colonic Na-abso rbing surface cells and Cl-secreting crypt cells is unknown. Surface a nd crypt cells were sequentially isolated from rat distal colon by EDT A chelation and mechanical dissociation. Cell viability was confirmed by trypan blue exclusion and low rates of 2',7'-bis(2-carboxyethyl)-5( 6)-carboxylfluorescein leak. Histologic examination, alkaline phosphat ase activity, and rates of [H-3]leucine incorporation confirmed separa tion of surface from crypt cells. Scatchard analysis of [H-3]aldostero ne and [H-3]triamcinolone acetonide binding demonstrated that the numb er of MR decreased from 7,228 +/- 1,067 in surface to 2,299 +/- 434 re ceptors/cell in crypt cells, whereas the number of GR increased from 2 0,857 +/- 4,241 in surface to 58,598 +/- 8,207 receptors/cell in crypt cells. The dissociation constants were 2.8 +/- 0.4 nM for the MR and 12 +/- 3 nM for the GR. Indirect immunofluorescence using the specific anti-MR antibody hMRsN and the anti-GR antibody BuGR-2 demonstrated t hat both unliganded receptors were cytoplasmic and translocated to the nucleus after hormone binding. These data indicate that both surface and crypt cells are potentially responsive to mineralocorticolds and g lucocorticolds and that both the MR and GR require hormone for nuclear translocation.