Different effects of progesterone and estradiol on chimeric and wild type aldosterone synthase in vitro

Andrea Vecchiola, Carlos F. Lagos, Cristóbal A. Fuentes, Fidel Allende, Carmen Campino, Carolina Valdivia, Alejandra Tapia-Castillo, Tadashi Ogishima, Kuniaki Mukai, Gareth Owen, Sandra Solari, Cristian A. Carvajal, Carlos E. Fardella

Research output: Contribution to journalArticle

4 Citations (Scopus)

Abstract

Background: Familial hyperaldosteronism type I (FH-I) is caused by the unequal recombination between the 11beta-hydroxylase (CYP11B1) and aldosterone synthase (CYP11B2) genes, resulting in the generation of a CYP11B1/B2 chimeric gene and abnormal adrenal aldosterone production. Affected patients usually show severe hypertension and an elevated frequency of stroke at a young age. Aldosterone levels rise during pregnancy, yet in pregnant women with FH-1, their hypertensive condition either remains unchanged or may even improve. The purpose of this study was to investigate in vitro whether female sex steroids modulate the activity of chimeric (ASCE) or wild type (ASWT) aldosterone synthase enzymes.Methods: We designed an in vitro assay using HEK-293 cell line transiently transfected with vectors containing the full ASCE or ASWT cDNAs. Progesterone or estradiol effects on AS enzyme activities were evaluated in transfected cells incubated with deoxycorticosterone (DOC) alone or DOC plus increasing doses of these steroids.Results: In our in vitro model, both enzymes showed similar apparent kinetic parameters (Km = 1.191 microM and Vmax = 27.08 microM/24 h for ASCE and Km = 1.163 microM and Vmax = 36.98 microM/24 h for ASWT; p = ns, Mann-Whitney test). Progesterone inhibited aldosterone production by ASCE- and ASWT-transfected cells, while estradiol demonstrated no effect. Progesterone acted as a competitive inhibitor for both enzymes. Molecular modelling studies and binding affinity estimations indicate that progesterone might bind to the substrate site in both ASCE and ASWT, supporting the idea that this steroid could regulate these enzymatic activities and contribute to the decay of aldosterone synthase activity in chimeric gene-positive patients.Conclusions: Our results show an inhibitory action of progesterone in the aldosterone synthesis by chimeric or wild type aldosterone synthase enzymes. This is a novel regulatory mechanism of progesterone action, which could be involved in protecting pregnant women with FH-1 against hypertension. In vitro, both enzymes showed comparable kinetic parameters, but ASWT was more strongly inhibited than ASCE. This study implicates a new role for progesterone in the regulation of aldosterone levels that could contribute, along with other factors, to the maintenance of an adequate aldosterone-progesterone balance in pregnancy.

Original languageEnglish
Article number76
JournalReproductive Biology and Endocrinology
Volume11
Issue number1
DOIs
Publication statusPublished - Aug 13 2013

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Cytochrome P-450 CYP11B2
Progesterone
Estradiol
Aldosterone
Steroid 11-beta-Hydroxylase
Enzymes
Desoxycorticosterone
Steroids
Pregnant Women
Genes
Hypertension
Pregnancy
In Vitro Techniques
HEK293 Cells
Enzyme Inhibitors
Mixed Function Oxygenases
Genetic Recombination
Complementary DNA
Stroke

All Science Journal Classification (ASJC) codes

  • Reproductive Medicine
  • Endocrinology
  • Developmental Biology

Cite this

Vecchiola, A., Lagos, C. F., Fuentes, C. A., Allende, F., Campino, C., Valdivia, C., ... Fardella, C. E. (2013). Different effects of progesterone and estradiol on chimeric and wild type aldosterone synthase in vitro. Reproductive Biology and Endocrinology, 11(1), [76]. https://doi.org/10.1186/1477-7827-11-76

Different effects of progesterone and estradiol on chimeric and wild type aldosterone synthase in vitro. / Vecchiola, Andrea; Lagos, Carlos F.; Fuentes, Cristóbal A.; Allende, Fidel; Campino, Carmen; Valdivia, Carolina; Tapia-Castillo, Alejandra; Ogishima, Tadashi; Mukai, Kuniaki; Owen, Gareth; Solari, Sandra; Carvajal, Cristian A.; Fardella, Carlos E.

In: Reproductive Biology and Endocrinology, Vol. 11, No. 1, 76, 13.08.2013.

Research output: Contribution to journalArticle

Vecchiola, A, Lagos, CF, Fuentes, CA, Allende, F, Campino, C, Valdivia, C, Tapia-Castillo, A, Ogishima, T, Mukai, K, Owen, G, Solari, S, Carvajal, CA & Fardella, CE 2013, 'Different effects of progesterone and estradiol on chimeric and wild type aldosterone synthase in vitro', Reproductive Biology and Endocrinology, vol. 11, no. 1, 76. https://doi.org/10.1186/1477-7827-11-76
Vecchiola, Andrea ; Lagos, Carlos F. ; Fuentes, Cristóbal A. ; Allende, Fidel ; Campino, Carmen ; Valdivia, Carolina ; Tapia-Castillo, Alejandra ; Ogishima, Tadashi ; Mukai, Kuniaki ; Owen, Gareth ; Solari, Sandra ; Carvajal, Cristian A. ; Fardella, Carlos E. / Different effects of progesterone and estradiol on chimeric and wild type aldosterone synthase in vitro. In: Reproductive Biology and Endocrinology. 2013 ; Vol. 11, No. 1.
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abstract = "Background: Familial hyperaldosteronism type I (FH-I) is caused by the unequal recombination between the 11beta-hydroxylase (CYP11B1) and aldosterone synthase (CYP11B2) genes, resulting in the generation of a CYP11B1/B2 chimeric gene and abnormal adrenal aldosterone production. Affected patients usually show severe hypertension and an elevated frequency of stroke at a young age. Aldosterone levels rise during pregnancy, yet in pregnant women with FH-1, their hypertensive condition either remains unchanged or may even improve. The purpose of this study was to investigate in vitro whether female sex steroids modulate the activity of chimeric (ASCE) or wild type (ASWT) aldosterone synthase enzymes.Methods: We designed an in vitro assay using HEK-293 cell line transiently transfected with vectors containing the full ASCE or ASWT cDNAs. Progesterone or estradiol effects on AS enzyme activities were evaluated in transfected cells incubated with deoxycorticosterone (DOC) alone or DOC plus increasing doses of these steroids.Results: In our in vitro model, both enzymes showed similar apparent kinetic parameters (Km = 1.191 microM and Vmax = 27.08 microM/24 h for ASCE and Km = 1.163 microM and Vmax = 36.98 microM/24 h for ASWT; p = ns, Mann-Whitney test). Progesterone inhibited aldosterone production by ASCE- and ASWT-transfected cells, while estradiol demonstrated no effect. Progesterone acted as a competitive inhibitor for both enzymes. Molecular modelling studies and binding affinity estimations indicate that progesterone might bind to the substrate site in both ASCE and ASWT, supporting the idea that this steroid could regulate these enzymatic activities and contribute to the decay of aldosterone synthase activity in chimeric gene-positive patients.Conclusions: Our results show an inhibitory action of progesterone in the aldosterone synthesis by chimeric or wild type aldosterone synthase enzymes. This is a novel regulatory mechanism of progesterone action, which could be involved in protecting pregnant women with FH-1 against hypertension. In vitro, both enzymes showed comparable kinetic parameters, but ASWT was more strongly inhibited than ASCE. This study implicates a new role for progesterone in the regulation of aldosterone levels that could contribute, along with other factors, to the maintenance of an adequate aldosterone-progesterone balance in pregnancy.",
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AU - Vecchiola, Andrea

AU - Lagos, Carlos F.

AU - Fuentes, Cristóbal A.

AU - Allende, Fidel

AU - Campino, Carmen

AU - Valdivia, Carolina

AU - Tapia-Castillo, Alejandra

AU - Ogishima, Tadashi

AU - Mukai, Kuniaki

AU - Owen, Gareth

AU - Solari, Sandra

AU - Carvajal, Cristian A.

AU - Fardella, Carlos E.

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N2 - Background: Familial hyperaldosteronism type I (FH-I) is caused by the unequal recombination between the 11beta-hydroxylase (CYP11B1) and aldosterone synthase (CYP11B2) genes, resulting in the generation of a CYP11B1/B2 chimeric gene and abnormal adrenal aldosterone production. Affected patients usually show severe hypertension and an elevated frequency of stroke at a young age. Aldosterone levels rise during pregnancy, yet in pregnant women with FH-1, their hypertensive condition either remains unchanged or may even improve. The purpose of this study was to investigate in vitro whether female sex steroids modulate the activity of chimeric (ASCE) or wild type (ASWT) aldosterone synthase enzymes.Methods: We designed an in vitro assay using HEK-293 cell line transiently transfected with vectors containing the full ASCE or ASWT cDNAs. Progesterone or estradiol effects on AS enzyme activities were evaluated in transfected cells incubated with deoxycorticosterone (DOC) alone or DOC plus increasing doses of these steroids.Results: In our in vitro model, both enzymes showed similar apparent kinetic parameters (Km = 1.191 microM and Vmax = 27.08 microM/24 h for ASCE and Km = 1.163 microM and Vmax = 36.98 microM/24 h for ASWT; p = ns, Mann-Whitney test). Progesterone inhibited aldosterone production by ASCE- and ASWT-transfected cells, while estradiol demonstrated no effect. Progesterone acted as a competitive inhibitor for both enzymes. Molecular modelling studies and binding affinity estimations indicate that progesterone might bind to the substrate site in both ASCE and ASWT, supporting the idea that this steroid could regulate these enzymatic activities and contribute to the decay of aldosterone synthase activity in chimeric gene-positive patients.Conclusions: Our results show an inhibitory action of progesterone in the aldosterone synthesis by chimeric or wild type aldosterone synthase enzymes. This is a novel regulatory mechanism of progesterone action, which could be involved in protecting pregnant women with FH-1 against hypertension. In vitro, both enzymes showed comparable kinetic parameters, but ASWT was more strongly inhibited than ASCE. This study implicates a new role for progesterone in the regulation of aldosterone levels that could contribute, along with other factors, to the maintenance of an adequate aldosterone-progesterone balance in pregnancy.

AB - Background: Familial hyperaldosteronism type I (FH-I) is caused by the unequal recombination between the 11beta-hydroxylase (CYP11B1) and aldosterone synthase (CYP11B2) genes, resulting in the generation of a CYP11B1/B2 chimeric gene and abnormal adrenal aldosterone production. Affected patients usually show severe hypertension and an elevated frequency of stroke at a young age. Aldosterone levels rise during pregnancy, yet in pregnant women with FH-1, their hypertensive condition either remains unchanged or may even improve. The purpose of this study was to investigate in vitro whether female sex steroids modulate the activity of chimeric (ASCE) or wild type (ASWT) aldosterone synthase enzymes.Methods: We designed an in vitro assay using HEK-293 cell line transiently transfected with vectors containing the full ASCE or ASWT cDNAs. Progesterone or estradiol effects on AS enzyme activities were evaluated in transfected cells incubated with deoxycorticosterone (DOC) alone or DOC plus increasing doses of these steroids.Results: In our in vitro model, both enzymes showed similar apparent kinetic parameters (Km = 1.191 microM and Vmax = 27.08 microM/24 h for ASCE and Km = 1.163 microM and Vmax = 36.98 microM/24 h for ASWT; p = ns, Mann-Whitney test). Progesterone inhibited aldosterone production by ASCE- and ASWT-transfected cells, while estradiol demonstrated no effect. Progesterone acted as a competitive inhibitor for both enzymes. Molecular modelling studies and binding affinity estimations indicate that progesterone might bind to the substrate site in both ASCE and ASWT, supporting the idea that this steroid could regulate these enzymatic activities and contribute to the decay of aldosterone synthase activity in chimeric gene-positive patients.Conclusions: Our results show an inhibitory action of progesterone in the aldosterone synthesis by chimeric or wild type aldosterone synthase enzymes. This is a novel regulatory mechanism of progesterone action, which could be involved in protecting pregnant women with FH-1 against hypertension. In vitro, both enzymes showed comparable kinetic parameters, but ASWT was more strongly inhibited than ASCE. This study implicates a new role for progesterone in the regulation of aldosterone levels that could contribute, along with other factors, to the maintenance of an adequate aldosterone-progesterone balance in pregnancy.

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