Decreased apparent affinity of corticosterone for colonic crypt glucocorticoid receptors is dependent on the cellular milieu and is distinct from corticosterone metabolism.
[3H]Steroid binding in intact colonic crypt cells and cytosol was compared to determine if 11beta hydroxysteroid dehydrogenase (11betaHSD) activity modulates access of corticosterone (B) to both glucocorticoid receptors (GR) and mineralocorticoid receptors (MR). Cytosol from non-adrenalectomized rat colonic crypt cells showed no 11betaHSD activity, and B bound with high affinity to both MR (Kd=0.47+/-0.03 nM; Bmax=177+/-34 fmol/mg protein) and GR (Kd=4.5+/-0.3 nM; Bmax=279+/-40 fmol/mg protein). In contrast, intact colonic crypt cells incubated with 25-30 nM [3H]B for 90 min converted 62% of B to 11-dehydrocorticosterone, with little binding to MR (14+/-3 fmol/mg protein) and GR (22+/-5 fmol/mg protein). When 11betaHSD activity was inhibited with carbenoxolone, and the same concentration of [3H]B used, binding of [3H]B to MR increased 10-fold to 122+/-12 fmol/mg protein, not significantly different from MR levels in colonic crypt cytosol. In contrast, [3H]B binding to GR in intact cells increased only 1.6-fold to 36+/-9 fmol/mg protein, significantly less than to GR in cytosol (212+/-24 fmol/mg protein). Scatchard analysis showed both lower levels of GR and an apparently lower affinity for [3H]B in colonic crypt cells (Kd=31+/-3 nM; Bmax=130+/-21 fmol/mg protein) compared with cytosol (Kd=4.5+/-0.3 nM; Bmax=279+/-40 fmol/mg protein. [3H]Dexamethasone similarly showed an apparently lower affinity and capacity for GR (Kd=8.8+/-1.3 nM; Bmax=232+/-32 fmol/mg protein) in intact cells compared with cytosol (two separate determinations, Kd=2.6 and 2.9 nM; Bmax=369 and 300 fmol/mg protein). In contrast, [3H]aldosterone displayed similar affinity and capacity for MR in both intact cells (Kd=2.0 nM; Bmax=121 fmol/mg protein) and cytosol (Kd=1.5 and 1.4nM; Bmax=115 and 93 fmol/mg protein). These findings demonstrate not only that 11betaHSD modulates binding to both MR and GR in colonic crypt cells, but also that an additional mechanism(s) operating in whole cells but not in cytosol selectively reduces the affinity and capacity of colonic GR for both natural and synthetic ligands.
Sheppard
K E
KE
Baker Medical Research Institute, Prahran, Victoria, Australia. karen.sheppard@baker.edu.au
D042842:11-beta-Hydroxysteroid Dehydrogenases; D000450:Aldosterone / Q000378:metabolism; D000818:Animals; D002229:Carbenoxolone / Q000494:pharmacology; D003106:Colon / Q000166:cytology / Q000378:metabolism*; D003345:Corticosterone / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism; D003907:Dexamethasone / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006913:Hydroxysteroid Dehydrogenases / Q000037:antagonists & inhibitors / Q000378:metabolism; D066298:In Vitro Techniques; D007700:Kinetics; D008297:Male; D051381:Rats; D017207:Rats, Sprague-Dawley; D011965:Receptors, Glucocorticoid / Q000378:metabolism*; D018161:Receptors, Mineralocorticoid / Q000378:metabolism
D004791:Enzyme Inhibitors; D011965:Receptors, Glucocorticoid; D018161:Receptors, Mineralocorticoid; D000450:Aldosterone; D003907:Dexamethasone; D006913:Hydroxysteroid Dehydrogenases; D042842:11-beta-Hydroxysteroid Dehydrogenases; D002229:Carbenoxolone; D003345:Corticosterone