AJP - Cell Physiology, Vol 266, Issue 3 C729-C740, Copyright © 1994 by American Physiological Society

ARTICLES

Mineralocorticoid and glucocorticoid receptor steroid binding and localization in colonic cells

G. Schulman, N. M. Robertson, I. B. Elfenbein, D. Eneanya, G. Litwack and C. P. Bastl
Department of Medicine, Temple University Health Sciences Center, Philadelphia 19140.

In rat colon epithelium glucocorticoids and mineralocorticoids regulate Na transport by binding to distinct receptors and stimulating different pathways. The distribution and intracellular localization of mineralocorticoid (MR) and glucocorticoid (GR) receptors in colonic Na-absorbing surface cells and Cl-secreting crypt cells is unknown. Surface and crypt cells were sequentially isolated from rat distal colon by EDTA 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 phosphatase activity, and rates of [3H]leucine incorporation confirmed separation of surface from crypt cells. Scatchard analysis of [3H]aldosterone and [3H]triamcinolone acetonide binding demonstrated that the number of MR decreased from 7,228 +/- 1,067 in surface to 2,299 +/- 434 receptors/cell in crypt cells, whereas the number of GR increased from 20,857 +/- 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 that 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 mineralocorticoids and glucocorticoids and that both the MR and GR require hormone for nuclear translocation.

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