Comparison of Aldosterone Production Among Human Adrenocortical Cell Lines

Wang, T.; Rowland, Jane; Parmar, Jeniel; Nesterova, Maria; Seki, Tsugio; Rainey, William E.

doi:10.1055/s-0031-1298019

Cited by 42 publications

(39 citation statements)

References 43 publications

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“…Human adrenocortical carcinoma H295R cells (ATCC, Manassas, VA, USA) and its clone HAC15 (kindly provided by Dr W Rainey, Medical College of Georgia, GA, USA; described in Wang et al (2012)) were cultured in 75 cm 2 flasks (Corning Costar, Schiphol-Oost, The Netherlands) in D-MEM/F12 (GIBCO Biocult Europe, Invitrogen) containing 5% FCS, L-glutamine, and penicillin 10 5 U/l (Bristol-Myers Squibb, Woerden, The Netherlands) at 37 8C in a 5% CO 2 incubator. Once a week, the medium was refreshed and cells of both cell lines were harvested with trypsin (0 .…”

Section: Cell Culturementioning

confidence: 99%

Fluconazole inhibits human adrenocortical steroidogenesis in vitro

Pas¹,

Hofland²,

Hofland³

et al. 2012

Journal of Endocrinology

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The antifungal agent ketoconazole is often used to suppress cortisol production in patients with Cushing's syndrome (CS). However, ketoconazole has serious side effects and is hepatotoxic. Here, the in vitro effects of ketoconazole and fluconazole, which might be less toxic, on human adrenocortical steroidogenesis were compared. The effects on steroidogenesis were examined in primary cultures of nine human adrenocortical tissues and two human adrenocortical carcinoma cell lines. Moreover, the effects on mRNA expression levels of steroidogenic enzymes and cell growth were assessed. Ketoconazole significantly inhibited 11-deoxycortisol (H295R cells; maximum inhibition 99%; EC 50 0 . 73 mM) and cortisol production (HAC15 cells; 81%; EC 50 0 . 26 mM and primary cultures (mean EC 50 0 . 75 mM)). In cultures of normal adrenal cells, ketoconazole increased pregnenolone, progesterone, and deoxycorticosterone levels, while concentrations of 17-hydroxypregnenolone, 17-hydroxyprogesterone, 11-deoxycortisol, DHEA, and androstenedione decreased. Fluconazole also inhibited 11-deoxycortisol production in H295R cells (47%; only at 1 mM) and cortisol production in HAC15 cells (maximum inhibition 55%; EC 50 35 mM) and primary cultures (mean EC 50 67 . 7 mM). In the cultures of normal adrenals, fluconazole suppressed corticosterone, 17-hydroxypregnenolone, and androstenedione levels, whereas concentrations of progesterone, deoxycorticosterone, and 11-deoxycortisol increased. Fluconazole (1 mM) slightly increased STAR mRNA expression in both cell lines. Neither compound affected mRNA levels of other steroidogenic enzymes or cell number. In conclusion, by inhibiting 11b-hydroxylase and 17-hydroxylase activity, pharmacological concentrations of fluconazole dose dependently inhibit cortisol production in human adrenocortical cells in vitro. Although fluconazole seems less potent than ketoconazole, it might become an alternative for ketoconazole to control hypercortisolism in CS. Furthermore, patients receiving fluconazole because of mycosis might be at risk for developing adrenocortical insufficiency.

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Section: Cell Culturementioning

confidence: 99%

Fluconazole inhibits human adrenocortical steroidogenesis in vitro

Pas¹,

Hofland²,

Hofland³

et al. 2012

Journal of Endocrinology

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“…qRT-PCR analysis showed that the NCI-H295 cell line had higher expression levels of CYP11B2. In the NCI-295 cell line, mRNA expression of CYP11B2 was almost two-fold compared to that was observed in the HAC 15 cell line [54]. However, aldosterone production was lower than in other cell lines, including the HAC 15 cell line, because of the lower expression of HSD3B2 observed in the NCI-H295 cell line [54].…”

Section: Hac15 Cell Linementioning

confidence: 72%

“…Wang et al compared CYP11B2 mRNA levels and aldosterone production in human adrenocortical cell lines [54]. qRT-PCR analysis showed that the NCI-H295 cell line had higher expression levels of CYP11B2.…”

Section: Hac15 Cell Linementioning

confidence: 99%

Aldosterone biosynthesis in the human adrenal cortex and associated disorders

Nakamura

Yamazaki

Konosu-Fukaya

et al. 2015

The Journal of Steroid Biochemistry and Molecular Biology

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“…To assess effects of CACNA1H WT and CACNA1H M1594V on aldosterone production, we transfected HAC15 cells, a subclone of NCI-H295R that has previously been used to assess the effect of mutant channels on aldosterone production (15)(16)(17)(18). Cells were serumstarved, and aldosterone levels were measured in the supernatant by ELISA (Figure 3 A).…”

Section: Resultsmentioning

confidence: 99%

CACNA1HM1549V Mutant Calcium Channel Causes Autonomous Aldosterone Production in HAC15 Cells and Is Inhibited by Mibefradil

et al. 2016

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We recently demonstrated that a recurrent gain-of-function mutation in a T-type calcium channel, CACNA1H(M1549V), causes a novel Mendelian disorder featuring early-onset primary aldosteronism and hypertension. This variant was found independently in five families. CACNA1H(M1549V) leads to impaired channel inactivation and activation at more hyperpolarized potentials, inferred to cause increased calcium entry. We here aimed to study the effect of this variant on aldosterone production. We heterologously expressed empty vector, CACNA1H(WT) and CACNA1H(M1549V) in the aldosterone-producing adrenocortical cancer cell line H295R and its subclone HAC15. Transfection rates, expression levels, and subcellular distribution of the channel were similar between CACNA1H(WT) and CACNA1H(M1549V). We measured aldosterone production by an ELISA and CYP11B2 (aldosterone synthase) expression by real-time PCR. In unstimulated cells, transfection of CACNA1H(WT) led to a 2-fold increase in aldosterone levels compared with vector-transfected cells. Expression of CACNA1H(M1549V) caused a 7-fold increase in aldosterone levels. Treatment with angiotensin II or increased extracellular potassium levels further stimulated aldosterone production in both CACNA1H(WT)- and CACNA1H(M1549V)-transfected cells. Similar results were obtained for CYP11B2 expression. Inhibition of CACNA1H channels with the T-type calcium channel blocker Mibefradil completely abrogated the effects of CACNA1H(WT) and CACNA1H(M1549V) on CYP11B2 expression. These results directly link CACNA1H(M1549V) to increased aldosterone production. They suggest that calcium channel blockers may be beneficial in the treatment of a subset of patients with primary aldosteronism. Such blockers could target CACNA1H or both CACNA1H and the L-type calcium channel CACNA1D that is also expressed in the adrenal gland and mutated in patients with primary aldosteronism.

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Comparison of Aldosterone Production Among Human Adrenocortical Cell Lines

Cited by 42 publications

References 43 publications

Fluconazole inhibits human adrenocortical steroidogenesis in vitro

Fluconazole inhibits human adrenocortical steroidogenesis in vitro

Aldosterone biosynthesis in the human adrenal cortex and associated disorders

CACNA1HM1549V Mutant Calcium Channel Causes Autonomous Aldosterone Production in HAC15 Cells and Is Inhibited by Mibefradil

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