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NAD-dependent 11 beta-hydroxysteroid dehydrogenase in cultured human colonic epithelial cells
1Division of Nephrology, University of Arkansas College of Medicine, Little Rock 72205, USA.
Abstract:
The inactivation of physiological glucocorticoids by 11 beta-hydroxysteroid dehydrogenase (11 beta-HSD) confers mineralocorticoid specificity to certain aldosterone target tissues. However, 11 beta-HSD activity in a human mineralocorticoid-responsive tissue has never been characterized. The present studies describe the features of 11 beta-HSD in the cultured human colonic epithelial cell line, T84. The 11 beta-HSD activity of T84 cells resided in the microsomal fraction and showed a marked preference for NAD rather than NADP as cofactor. NAD or NADP (200 microM) increased the conversion of corticosterone to 11-dehydrocorticosterone by 24.1 +/- 2.1 and 0.5 +/- 0.7 pmol.mg protein-1.20 min-1, respectively, indicating a > 40-fold preference for NAD vs. NADP. The Michaelis constant values for corticosterone and cortisol were 11.3 +/- 1.5 and 79.8 +/- 10 nM, respectively. The T84 11 beta-HSD was inhibited by 11-dehydrocorticosterone in a noncompetitive fashion [inhibition constant (Ki) = 180 +/- 9.6 nM] and by carbenoxolone in a competitive fashion (Ki = 17.4 +/- 1.3 nM). The expression of mineralocorticoid receptors in these cells was demonstrated by reverse transcriptase-polymerase chain reaction of mRNA isolated from T84 cells and by [3H]aldosterone binding studies. The coexpression of this NAD-dependent isoform of 11 beta-HSD and mineralocorticoid receptors is consistent with the view that the NAD-dependent isoform is responsible for the specificity of mineralocorticoid responses.
Insights
This study characterizes 11 beta-hydroxysteroid dehydrogenase (11 beta-HSD) in human T84 cells, revealing a NAD-dependent isoform crucial for mineralocorticoid specificity. This finding clarifies the enzyme
Area of Science:
- Endocrinology
- Molecular Biology
- Cell Biology
Background:
- 11 beta-hydroxysteroid dehydrogenase (11 beta-HSD) is vital for glucocorticoid inactivation, ensuring mineralocorticoid specificity in target tissues.
- Characterization of 11 beta-HSD activity in human mineralocorticoid-responsive tissues remains incomplete.
Purpose of the Study:
- To characterize the features of 11 beta-HSD in cultured human colonic epithelial T84 cells.
- To investigate the cofactor preference, substrate kinetics, and inhibition patterns of T84 cell 11 beta-HSD.
- To determine the coexpression of 11 beta-HSD and mineralocorticoid receptors in T84 cells.
Main Methods:
- Microsomal fraction isolation from T84 cells.
- Enzyme kinetic studies measuring the conversion of corticosterone and cortisol.
- Inhibition assays using 11-dehydrocorticosterone and carbenoxolone.
- Reverse transcriptase-polymerase chain reaction (RT-PCR) for mineralocorticoid receptor mRNA.
- [3H]aldosterone binding studies.
Main Results:
- T84 cell 11 beta-HSD demonstrated a significant preference for NAD over NADP as a cofactor (>40-fold).
- Michaelis constant values for corticosterone and cortisol were determined (11.3 nM and 79.8 nM, respectively).
- 11 beta-HSD activity was inhibited by 11-dehydrocorticosterone and carbenoxolone.
- Expression of mineralocorticoid receptors was confirmed in T84 cells via RT-PCR and binding assays.
Conclusions:
- The NAD-dependent isoform of 11 beta-HSD is present in the human colonic epithelial cell line T84.
- Coexpression of this NAD-dependent 11 beta-HSD isoform and mineralocorticoid receptors supports its role in mediating mineralocorticoid responses.
- These findings provide insights into the molecular mechanisms underlying mineralocorticoid action in human colon.