11β-Hydroxysteroid dehydrogenase type 2 complementary deoxyribonucleic acid stably transfected into Chinese hamster ovary cells: Specific inhibition by 11α-hydroxyprogesterone
The 11β-hydroxysteroid dehydrogenase type 2 (11βHSD-2) enzyme is thought to confer aldosterone specificity upon mineralocorticoid target tissues by protecting the mineralocorticoid receptor from binding by the more abundant glucocorticoids, corticosterone and cortisol. We have developed a Chinese ha...
Guardado en:
| Autor principal: | |
|---|---|
| Publicado: |
1996
|
| Materias: | |
| Acceso en línea: | https://bibliotecadigital.exactas.uba.ar/collection/paper/document/paper_00137227_v137_n6_p2308_Morita http://hdl.handle.net/20.500.12110/paper_00137227_v137_n6_p2308_Morita |
| Aporte de: |
| id |
paper:paper_00137227_v137_n6_p2308_Morita |
|---|---|
| record_format |
dspace |
| spelling |
paper:paper_00137227_v137_n6_p2308_Morita2023-06-08T14:36:07Z 11β-Hydroxysteroid dehydrogenase type 2 complementary deoxyribonucleic acid stably transfected into Chinese hamster ovary cells: Specific inhibition by 11α-hydroxyprogesterone Cozza, Eduardo Néstor 11alpha hydroxyprogesterone 11beta hydroxysteroid dehydrogenase animal cell article Chinese hamster competitive inhibition enzyme activity enzyme inhibition enzyme kinetics genetic complementation genetic transfection glycosylation nonhuman potassium excretion priority journal sodium retention The 11β-hydroxysteroid dehydrogenase type 2 (11βHSD-2) enzyme is thought to confer aldosterone specificity upon mineralocorticoid target tissues by protecting the mineralocorticoid receptor from binding by the more abundant glucocorticoids, corticosterone and cortisol. We have developed a Chinese hamster ovary cell line stably transfected with a plasmid containing the rat 11βHSD-2 complementary DNA. This cell line has expressed the enzyme consistently for many generations. The 11βHSD-2 was located primarily in the microsomes, but significant amounts also existed in the nuclei and mitochondria. The enzymatic reaction was unidirectional, oxidative, and inhibited by the product, 11-dehydrocorticosterone, with an IC50 of approximately 200 nM. The K(m) for corticosterone was 9.6 ± 3.1 nM, and that for NAD+ was approximately 8 μM. The enzyme did not convert dexamethasone to 11-dehydrodexamethasone. Tunicamycin, an N-glycosylation inhibitor, had no effect on enzyme activity, 11α-Hydroxyprogesterone (11αOH-P) was an order of magnitude more potent a competitive inhibitor of the 11βHSD-2 than was glycyrrhetinic acid (GA) (approximate IC50 0.9 vs. 15 nM). 11βOH-P, progesterone, and GA were almost equipotent (IC50 = 10 and 6 nM, respectively), and 5α-pregnandione and 5β-pregnandione were less potent (IC50 = 100 and 500 nM, respectively) inhibitors of the enzyme. When the inhibitory activities were examined with intact transfected cells, 11αOH-P was more potent than GA (IC50 = 5 and 150 nM, respectively). 11αOH-P was not metabolized by 11βHSD-2. We were unable to demonstrate the presence of 11αOH-P in human urine. In conclusion, a cell line stably transfected with the rat 11βHSD-2 was created, and the enzyme kinetics, including inhibition, were characterized. 11αOH-P was found to be a potent relatively specific inhibitor of the 11βHSD-2 enzyme. Its potential importance is that it is the most specific inhibitor of the 11βHSD-2 so far encountered and would aid in the study of the physiological importance of the isoenzyme. Fil:Cozza, E.N. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. 1996 https://bibliotecadigital.exactas.uba.ar/collection/paper/document/paper_00137227_v137_n6_p2308_Morita http://hdl.handle.net/20.500.12110/paper_00137227_v137_n6_p2308_Morita |
| institution |
Universidad de Buenos Aires |
| institution_str |
I-28 |
| repository_str |
R-134 |
| collection |
Biblioteca Digital - Facultad de Ciencias Exactas y Naturales (UBA) |
| topic |
11alpha hydroxyprogesterone 11beta hydroxysteroid dehydrogenase animal cell article Chinese hamster competitive inhibition enzyme activity enzyme inhibition enzyme kinetics genetic complementation genetic transfection glycosylation nonhuman potassium excretion priority journal sodium retention |
| spellingShingle |
11alpha hydroxyprogesterone 11beta hydroxysteroid dehydrogenase animal cell article Chinese hamster competitive inhibition enzyme activity enzyme inhibition enzyme kinetics genetic complementation genetic transfection glycosylation nonhuman potassium excretion priority journal sodium retention Cozza, Eduardo Néstor 11β-Hydroxysteroid dehydrogenase type 2 complementary deoxyribonucleic acid stably transfected into Chinese hamster ovary cells: Specific inhibition by 11α-hydroxyprogesterone |
| topic_facet |
11alpha hydroxyprogesterone 11beta hydroxysteroid dehydrogenase animal cell article Chinese hamster competitive inhibition enzyme activity enzyme inhibition enzyme kinetics genetic complementation genetic transfection glycosylation nonhuman potassium excretion priority journal sodium retention |
| description |
The 11β-hydroxysteroid dehydrogenase type 2 (11βHSD-2) enzyme is thought to confer aldosterone specificity upon mineralocorticoid target tissues by protecting the mineralocorticoid receptor from binding by the more abundant glucocorticoids, corticosterone and cortisol. We have developed a Chinese hamster ovary cell line stably transfected with a plasmid containing the rat 11βHSD-2 complementary DNA. This cell line has expressed the enzyme consistently for many generations. The 11βHSD-2 was located primarily in the microsomes, but significant amounts also existed in the nuclei and mitochondria. The enzymatic reaction was unidirectional, oxidative, and inhibited by the product, 11-dehydrocorticosterone, with an IC50 of approximately 200 nM. The K(m) for corticosterone was 9.6 ± 3.1 nM, and that for NAD+ was approximately 8 μM. The enzyme did not convert dexamethasone to 11-dehydrodexamethasone. Tunicamycin, an N-glycosylation inhibitor, had no effect on enzyme activity, 11α-Hydroxyprogesterone (11αOH-P) was an order of magnitude more potent a competitive inhibitor of the 11βHSD-2 than was glycyrrhetinic acid (GA) (approximate IC50 0.9 vs. 15 nM). 11βOH-P, progesterone, and GA were almost equipotent (IC50 = 10 and 6 nM, respectively), and 5α-pregnandione and 5β-pregnandione were less potent (IC50 = 100 and 500 nM, respectively) inhibitors of the enzyme. When the inhibitory activities were examined with intact transfected cells, 11αOH-P was more potent than GA (IC50 = 5 and 150 nM, respectively). 11αOH-P was not metabolized by 11βHSD-2. We were unable to demonstrate the presence of 11αOH-P in human urine. In conclusion, a cell line stably transfected with the rat 11βHSD-2 was created, and the enzyme kinetics, including inhibition, were characterized. 11αOH-P was found to be a potent relatively specific inhibitor of the 11βHSD-2 enzyme. Its potential importance is that it is the most specific inhibitor of the 11βHSD-2 so far encountered and would aid in the study of the physiological importance of the isoenzyme. |
| author |
Cozza, Eduardo Néstor |
| author_facet |
Cozza, Eduardo Néstor |
| author_sort |
Cozza, Eduardo Néstor |
| title |
11β-Hydroxysteroid dehydrogenase type 2 complementary deoxyribonucleic acid stably transfected into Chinese hamster ovary cells: Specific inhibition by 11α-hydroxyprogesterone |
| title_short |
11β-Hydroxysteroid dehydrogenase type 2 complementary deoxyribonucleic acid stably transfected into Chinese hamster ovary cells: Specific inhibition by 11α-hydroxyprogesterone |
| title_full |
11β-Hydroxysteroid dehydrogenase type 2 complementary deoxyribonucleic acid stably transfected into Chinese hamster ovary cells: Specific inhibition by 11α-hydroxyprogesterone |
| title_fullStr |
11β-Hydroxysteroid dehydrogenase type 2 complementary deoxyribonucleic acid stably transfected into Chinese hamster ovary cells: Specific inhibition by 11α-hydroxyprogesterone |
| title_full_unstemmed |
11β-Hydroxysteroid dehydrogenase type 2 complementary deoxyribonucleic acid stably transfected into Chinese hamster ovary cells: Specific inhibition by 11α-hydroxyprogesterone |
| title_sort |
11β-hydroxysteroid dehydrogenase type 2 complementary deoxyribonucleic acid stably transfected into chinese hamster ovary cells: specific inhibition by 11α-hydroxyprogesterone |
| publishDate |
1996 |
| url |
https://bibliotecadigital.exactas.uba.ar/collection/paper/document/paper_00137227_v137_n6_p2308_Morita http://hdl.handle.net/20.500.12110/paper_00137227_v137_n6_p2308_Morita |
| work_keys_str_mv |
AT cozzaeduardonestor 11bhydroxysteroiddehydrogenasetype2complementarydeoxyribonucleicacidstablytransfectedintochinesehamsterovarycellsspecificinhibitionby11ahydroxyprogesterone |
| _version_ |
1768546703996092416 |