Efonidipine, a Ca2+-Channel Blocker, Enhances the Production of Dehydroepiandrosterone Sulfate in NCI-H295R Human Adrenocortical Carcinoma Cells
Keiichi Ikeda,1 Takatoshi Saito2 and Katsuyoshi Tojo2
“Department of Molecular and Cellular Biology, Institute of DNA Medicine, Research Center for Medical Sciences, The Jikei University School of Medicine, Tokyo, Japan
2Division of Diabetes and Endocrinology, Department of Internal Medicine, The Jikei University School of Medicine, Tokyo, Japan
Steroid biosynthesis is initiated with transportation of cholesterol along with steroidogenic acute regulatory protein (StAR) into the mitchondria and is achieved with several steroidogenic enzymes. It has been reported that Ca2+ channel blockers (CCBs), such as azelnidipine, efonidipine and nifedipine, suppress the biosynthesis of aldosterone and cortisol, but the overall effects of CCBs on steroid biosynthesis remain to be clarified. The present study was designed to evaluate the effects of CCBs on the expression of steroidogenic enzymes and the production of adrenal androgen, dehydroepiandrosterone sulfate (DHEA-S) that has anti-atherosclerotic actions. NCI-H295R human adrenocortical carcinoma cells and HepG2 human hepatoma cells were cultured for 24 hours with or without a CCB (amlodipine, efonidipine, nifedipine, azelnidipine R(-)-efonidipine, verapamil or diltiazem). HepG2 hepatoma cells were used to confirm the effects of CCBs on the expression of StAR. In fact, efonidipine and nifedipine increased the expression of StAR in HepG2 cells. Efonidipine and nifedipine, but not other examined CCBs, also increased the N6, 2’-O-dibutyryladenosine 3’,5’-cyclic monophosphate (dbcAMP)-induced StAR mRNA, which reflects the action of adrenocorticotropic hormone, and efonidipine and R(-)-efonidipine enhanced the dbcAMP-induced DHEA-S production in NCI-H295R adrenocortical carcinoma cells. Therefore, efonidipine and nifedipine might increase the expression of StAR and, in turn, efonidipine enhanced the dbcAMP- induced DHEA-S production, independent of Ca2+ channel blockade. These results indicate that such effects are not associated with Ca2+ influx. Moreover, only efonidipine enhanced the angiotensin II-induced expression of StAR mRNA (P < 0.01 vs. angiotensin II alone). In conclusion, efonidipine might exert an additional action beyond anti-hypertensive actions.
Keywords: Ca2+ antagonists; dehydroepiandrosterone-sulfate; HepG2 human hepatoma cell; NCI-H295R human adrenocortical carcinoma cell; steroid biosynthesis Tohoku J. Exp. Med., 2011, 224 (4), 263-271. @ 2011 Tohoku University Medical Press
The adrenal cortex synthesizes various bioactive ste- roids, such as mineralocorticoids (e.g., aldosterone), gluco- corticoids (e.g., cortisol and corticosterone), and adrenal androgens (e.g., dehydroepiandrosterone [DHEA], testos- terone, and estrone). Some adrenal steroids, such as aldo- sterone, cortisol, and corticosterone, are involved in cardio- vascular diseases such as hypertension. Recent studies showed that some Ca2+ channel blockers (CCBs) have inhibitory actions on the expression of 11ß-hydroxylase and steroid 18-hydroxylase, leading to the suppression of secre- tion of cortisol and aldosterone in NCI-H295R human adre- nocortical carcinoma cells (Imagawa et al. 2006; Isaka et al. 2009), in addition to their primary role involving their anti- hypertensive action. However, adrenal steroid biosynthesis
is initiated with transport of cholesterol by steroidogenic acute regulatory protein (StAR), which is a rate-limiting enzyme of steroid biosynthesis (Christenson and Strauss 2000), and detailed evidence for the effect of CCBs on the expression of steroidogenic enzymes has not been eluci- dated. In addition, another group recently reported that nifedipine, which is a widely used dihydropyridine CCB, enhance the expression of StAR in MA-10 mouse Leydig cells (Pandey et al. 2010). The present study was, there- fore, designed to evaluate the effects of L-type and/or T-type CCBs, such as amlodipine, efonidipine, nifedipine, azelnidipine, and R(-)-efonidipine, on the expression of StAR using NCI-H295R human adrenocortical carcinoma cells, in which the L-, T-, and N-type Ca2+ channels are all
expressed (Isaka et al. 2009: Aritomi et al. 2011). We also used HepG2 human hepatoma cells to confirm the effects of CCBs on the expression of StAR, because the HepG2 human hepatoma cell imports cholesterol into its mitochon- dria by the StAR protein, like NCI-H295R human adreno- cortical carcinoma cells, and in turn, synthesizes bile acid (Hall et al. 2005). In addition, we have attempted to evalu- ate the involvement of the effects of CCBs on the expres- sion of other steroidogenic enzymes and on the production of DHEA sulfate (DHEA-S), which has been reported to have several actions against atherosclerosis and vascular remodeling (Alexandersen et al. 1996; Ii et al. 2009).
Methods
Cells and Chemicals
NCI-H295R human adrenocortical adenoma cells were pur- chased from American Type Culture Collection and HepG2 human hepatoma cells from Dainippon Sumitomo Pharma Co., Ltd. (Osaka, Japan). Dulbecco’s Modified Eagle’s Medium (DMEM, Ca2+-free [Catalog No. 21068-028] and low glucose), DMEM/Ham’s F12 (DMEM/F12), fetal bovine serum (FBS), GlutaMAX Supplement I, and Invitrolon PVDF were purchased from Life Technologies Co. (Carlsbad, CA, USA). Bovine serum albumin (BSA), Nº, 2’-O-Dibutyryladenosine 3’,5’-cyclic monophosphate sodium salt (dbcAMP), amlodipine (2-[(2-Aminoethoxy)-methyl]-4-(2- chlorophenyl)-1,4-dihydro-6-methyl-3,5-pyridinedicarboxylic acid 3-ethyl-5-methyl ester benzene sulfonate) besylate, nifedipine (1,4-Dihydro-2,6-dimethyl-4-(2-nitrophenyl)-3,5-pyridinedicar- boxylic acid dimethyl ester), diltiazem ((2S,3S)-(+)-cis-3-Acetoxy-5- (2-dimethylaminoethyl)-2,3-dihydro-2-(4-methoxyphenyl)-1,5-ben- zothiazepin-4(5H)-one) hydrochloride, and verapamil (5-[N-(3,4- Dimethoxyphenylethyl)methylamino]-2-(3,4-dimethoxyphenyl)- 2-isopropylvaleronitrile) hydrochloride were from Sigma-Aldrich, Inc. (St. Louis, MO, USA). Efonidipine (5-(5,5-Dimethyl-1,3,2- dioxaphosphorinan-2-yl)-1,4-dihydro-2,6-dimethyl-4-(3-nitro- phenyl)-3-pyridinecarboxlic acid 2-[phenyl(phenylmethyl)amino] ethyl ester) hydrochloride was generously provided by Shionogi & Co., Ltd. (Osaka, Japan). Azelnidipine (2-Amino-1,4-dihydro-6- methyl-4-(3-nitrophenyl)-3,5-pyridinedicarboxylic acid 3-[1- (diphenylmethyl)-3-azetidinyl] 5-(1-methylethyl) ester) was gener- ously provided by Daiichi-Sankyo Co., Ltd. (Tokyo, Japan). R(-)- efonidipine, which is an R(-)-enantiomer of efonidipine and a selec- tive blocker for T-type, but not L-type, Ca2+ channels (Tanaka et al. 2004), was generously provided by Nissan Chemical Industries, Ltd. (Tokyo, Japan). Angiotensin II was purchased from Peptide Institute, Inc., (Minoh, Japan), Prime Script RT Reagent Kit and SYBER PremixEX Taq were from Takara Bio, Inc. (Otsu, Japan), ISOGEN was from Nippon gene Co. (Tokyo, Japan), CaCl2 and ethylene glycol bis (B-aminoethyl ether)-N,N,N’,N’-tetraacetic acid (EGTA) were from Nacalai Tesque, Inc. (Kyoto, Japan), and M-PER Mammalian Protein Extraction Reagent and the BCA protein assay kit were from Thermo Fisher Scientific, Inc. (Waltham, MA, USA). Goat anti-StAR anti- body, donkey anti-goat IgG-HRP, and goat anti-rabbit IgG were pur- chased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA), rabbit anti-CYP11A1 and anti-CYP21A2 polyclonal antibodies from Abgent Inc. (San Diego, CA, USA), rabbit anti-HSD3B2 was from Abcam plc (Cambridge, UK), mouse monoclonal anti-body for human B-actin, Anti-Human ACTB was from Cosmo Bio Co., Ltd.
(Tokyo, Japan), and Amersham ECL Anti-Mouse IgG Horseradish Peroxidase-Linked Species-Specific Whole Antibody and the ECL Plus Western Blotting Detection System were from GE Healthcare UK Ltd. (Buckinghamshire, UK). NCI-H295R human adrenocortical carcinoma cells were purchased from the American Type Culture Collection (ATCC, Manasas, VA, USA) and HepG2 human hepatoma cells from Dainippon Sumitomo Pharma Co., Ltd. (Osaka, Japan). The DHEA-S-specific EIA kit was purchased from Immunospec Co. (Chatsworth, CA, USA) and the cortisol-specific enzyme EIA kit from Oxford Biomedical Research Inc. (Oxford, MI, USA).
Cell culture of NCI-H295R human adrencocortical carcinoma cells and HepG2 human hepatoma cells
NCI-H295R human adrenocortical carcinoma cells were cul- tured as previously described (Isaka et al. 2009). Briefly, cells were initially cultured in 10 cm culture dishes with DMEM/F12 containing 5% FBS (v/v), 6.25 mg/l transferrin, 6.25 mg/l insulin, 1.25 g/l BSA and antibiotics (100 U/ml penicillin G and 10ug/ml streptomycin) (maintenance medium) and passed into 6-well plates at a density of 2.0×106 cells/well, incubated with maintenance medium for 48 hours prior to each experiment. HepG2 human hepatoma cells were also cultured in 6-well plates after seeding at a density of 4.0 × 106 cells/ well for 48 hours with DMEM containing 10% FBS (v/v).
Isolation of mRNA from NCI-H295R human adrenocortical cells and HepG2 human hepatoma cells and preparation of cDNA
Isolation was carried out after culture of NCI-H295R human adrenocortical carcinoma cells in stimulation medium (DMEM/F12 containing 0.1% BSA and a stimulation agent, such as dbcAMP (10-3 mol/l), which stimulates adrenocorticotropin (ACTH)-dependent ste- roidogenic gene expression (Ozbay et al. 2004), or angiotensin II (10-7 mol/l) with or without CCBs (amlodipine besylate, efonidipine hydrochloride, nifedipine, azelnidipine, R(-)-efonidipine, diltiazem hydrochloride, or verapamil hydrochloride, 10-6 mol/l) for 6 h and 24 h. The vehicle medium contained a maximum concentration of 0.1% dimethyl-sulfoxide (DMSO), in which all examined CCBs are dis- solved. The concentration of each CCB was unified as 10-6 mol/l, because azelnidipine, efonidipine, or nifedipine at this concentration exerted maximal effects on steroid biosynthesis in the previous study (Isaka et al. 2009). NCI-H295R human adrenocortical carcinoma cells were also cultivated in Ca2+-free DMEM, which was supple- mented with 0.1% BSA (w/v) and 1% GlutaMAX Supplement I (v/v), containing 10-3 mol/l CaCl2, 0.5 x 10-3 mol/l EGTA, or 10-3 mol/l EGTA with or without 10-3 mol/l dbcAMP for 24 hours. HepG2 human hepatoma cells were also cultured in FBS-free DMEM with or without a CCB (amlodipine, nifedipine, efonidipine, azelnidipine, or R(-)-efonidipne, 10 6 mol/l) for 24 hours. The cells were then washed with phosphate-buffered saline and lysed with ISOGEN. Total RNA was extracted using the isothiocyanate-acid-phenol-chloroform method. cDNA was then synthesized by Prime Script RT reagent Kit with the oligo-dT primer.
Quantitative real-time PCR for StAR mRNA
Synthesized cDNA was used for real-time PCR using SYBR PremixEX Taq and Thermal Cycler Dice (Takara Bio, Inc.). The spe- cific primers for StAR and glyceraldehyde-3-phosophate dehydroge- nase (GAPDH) were as follows: StAR (GenBank accession No. U17280), forward: 5’-TGGCATGGACACAGACTT-3’, reverse: 5’-GTGAAGCACCATGCAAGTGG-3’; GAPDH (Isaka et al. 2009),
forward: 5’-GAAGGTGAAGGTCGGAGTC-3’, reverse: 5’-GAAGATGGTGATGGGATTTC-3’. Quantitative data were nor- malized to the amount of GAPDH mRNA. Quality of PCR products was confirmed by dissociation curves and data were analyzed by the 2-44CT method (Livak and Schmittgen 2001) using the 2nd derivative curve of amplification plots (Thermal Cycler Dice Real Time System TP800 software version 2.10B, TaKaRa Bio, Inc.). Data are repre- sented as mean ± S.D.
Western blot analysis of the effects of CCBs on the expression of steroidogenic enzymes in NCI-H295R human adrenocortical carcinoma cells
NCI-H295R human adrenocortical carcinoma cells stimulated by dbcAMP with or without CCBs for 24 hours in 10cm culture dishes were lysed by M-PER Mammalian Protein Extraction Reagent and proteins were electrophoresed by sodium dodecyl sulfate poly- acrylamide gel. The proteins were subsequently transferred onto a polyvinylidene difluoride membrane (Invitrolon PVDF) and probed by goat anti-StAR antibody (1:200), rabbit anti-CYP11A1 polyclonal antibody (1:80), rabbit anti-HSD3B2 polyclonal antibody (1:1,000), or rabbit anti-CYP21A2 polyclonal antibody (1:80) and, in turn, rep- robed with mouse monoclonal antibody for human B-actin, Anti- Human ACTB (1:500). Probed proteins were then treated with don- key anti-goat IgG-HRP (1:5,000) and Amersham ECL Anti-Mouse IgG Horseradish Peroxidase-Linked Species-Specific Whole Antibody, respectively, and visualized using the ECL Plus Western Blotting Detection System. Chemiluminescence was detected by LAS-4000miniEPUV luminescent image analyzer and analyzed using Multi Gauge Ver3.0 software (Fuji Photo Film, Tokyo, Japan). Each value was normalized to the amount of B-actin.
Measurement of DHEA-S and cortisol in culture medium of NCI-H295R human adrenocortical carcinoma cells
NCI-H295R human adrenocortical carcinoma cells plated in 6-well plates were cultured in DMEM/F12 containing 0.1% BSA, antibiotics and a stimulator, such as dbcAMP (10-3 mol/l) or angio- tensin II (10-7 mol/l) with or without CCBs for 24 h. Culture medium was collected, and DHEA-S and cortisol in the same culture medium were measured using the DHEA-S- and cortisol-specific enzyme immunoassay (EIA) kits according to the manufacturer’s protocol. The data were normalized with protein content, which was measured by the BCA protein assay kit following lysis of the cells using albu- min standard.
Statistical Analysis
Statistical analysis was performed using analysis of variance followed by a post hoc test for between-group comparison (StatView 5.0, SAS Institute, Inc., Cary, NC). P values less than 0.05 were con- sidered to indicate statistical significance, and all data were expressed as the mean ± standard deviation.
Results
Effects of CCBs on the expression of StAR mRNA and protein in H295R human adrenocortical carcinoma cells and HepG2 human hepatoma cells
Each CCB (106 mol/l) alone, except for nifedipine, did not exert significant effects on the expression StAR mRNA in NCI-H295R human adrenocortical cells at 24
hours (data not sown). Nifedipine alone significantly increased the expression of StAR mRNA small (about 1.5- fold increase of DMSO). dbcAMP, which increases intra- cellular cAMP like ACTH (Ozbay et al. 2004), significantly increased the expression of StAR mRNA (about 2.7 folds of DMSO, P < 0.01 vs. vehicle, Fig. 1A-E) and its protein (about 16.4 folds of DMSO, Fig. 1F). Efonidipine or nife- dipine dose-dependently and significantly enhanced the expression of the dbcAMP-induced StAR mRNA in NCI- H295R human adrenocortical carcinoma cells (P < 0.01 vs. dbcAMP alone), whereas amlodipine, azelnidipine, or R(-)- efonidipine significantly decreased dbcAMP-induced StAR mRNA (P < 0.01 vs. dbcAMP alone, Fig. 1A-E). The results of Western blot analysis on the expression of StAR protein are consistent with the results of real-time PCR of StAR mRNA. Angiotensin II, which is another stimulator of steroid biosynthesis in adrenocortical cells, also increased the expression of StAR mRNA (P < 0.01 vs. DMSO, about 1.4 fold of DMSO, Fig. 2). Efonidipine also enhanced the angiotensin II-induced expression of StAR mRNA (P < 0.01 vs. angiotensin II alone), whereas azelni- dipine dose-dependently decreased angiotensin II-induced expression of StAR mRNA (P < 0.05 vs. angiotensin II alone) (Fig. 2). In contrast, amlodipine and nifedipine did not exert significant action on angiotensin II-induced expression of StAR mRNA (Fig. 2). Efonidipine, nifedip- ine, or R(-)-efonidipine also significantly increased the expression of StAR mRNA in HepG2 human hepatoma cells, whereas amlodiopine and azelnidipine significantly decreased its expression (P < 0.01 vs. DMSO, Fig. 3A). The results of Western blot analysis were also consistent with those of the real-time PCR (Fig. 3B, C). The expres- sion of StAR mRNA was decreased by the L-type Ca2+ channel blockade with non-dihydropyridine CCBs, such as diltiazem or verapamil, and by chelation of extracellular Ca2+ with EDTA, indicating that the efonipine, nifedipine, or R(-)-efonidipine-induced increase in StAR mRNA may be independent to extracellular Ca2+ (Fig. 4).
Regulation of other steroidogenic enzymes by dihydropyri- dine CCBs in NCI-H295R human adrenocortical carcinoma cells
The CCBs did not exert significant effects on the expression of both db-cAMP- and angiotensin II-induced changes of 17a-hydroxlase/17,20-lyase (CYP17), sulfo- transferase (SULT2A1), and steroid sulfatase (STS) mRNAs analyzed by real time RT-PCR (data not shown). Therefore, we analyzed the protein expression of CYP11A1, HSD3B2, and CYP21A2 with Western blot analysis, because the expression levels of their mRNAs were marginally increased by the CCBs (about 1 to 2-fold- increases in both dbcAMP- and angiotensin II-induced lev- els of each mRNA expression). As shown in Fig. 5, the expression levels of these enzymes were not noticeably increased by dbcAMP with or without the CCBs, whereas angiotensin II increased the expression of these enzymes.
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NCI-H295R human adrenocortical carcinoma cells were cultured for 24 hours with or without a CCB (azelnidipine, amlodipine, nifedipine, efonidipine, or R(-)-efonidipine). Efonidipine and nifedipine dose-dependently enhanced the dbcAMP-induced expression of StAR at mRNA and protein levels, while amlodipine and R(-)-efonidipine suppressed the dbcAMP-induced expression of StAR both at mRNA and protein levels at 10-6 mo/l. A)-E) Dose-dependent effects of CCBs on the expression of StAR mRNA quantified with real-time PCR ((A) azelnidipine, (B) amlodipine, (C) nife- dipine, (D) efonidipine, and (E) R(-)-efonidipine). The qualification and gel image of the expression of StAR protein in Western Blot analysis at 10 6mol/l of dihydropyridine CCBs were shown in F) and G), respectively. The data were reproduced in at least two independent experiments. * P < 0.01 vs. DMSO, ** P < 0.05 vs. DMSO, “P < 0.01 vs. dbcAMP alone. D: DMSO (vehicle, 0.1%, [.]), dbcAMP: dbcAMP 10-3mol/1, dbcAMP 10-3 mol/l alone (u), Aze: azel- nidipine 10 6 mol/l (4), Aml: amlodipine 10-6 mol/l (V), Nif: nifedipine 106 mol/l (A), efonidipine 10-6 mol/l (V), R(-)- Efo: R(-)-efonidipine (o).
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Fig. 2. The dose-dependent effects of CCBs on the angioten- sin II-induced expression of StAR mRNA in NCI-H295R adrenocortical carcinoma cells. Each of the CCBs did not exert remarkable actions on the angiotensin II-induced expression of StAR mRNA at 24 hours after incubation. A)-D) Dose-dependent effects of CCBs on the expression of StAR mRNA quantified with real-time PCR ((A) azelnidipine, (B) amlodipine, (C) nifedipine, and (D) efonidipine). The data were repro- duced in at least two independent experiments. * P < 0.01 vs. DMSO, ** P < 0.05 vs. DMSO, *P < 0.01 vs. angio- tensin II alone. D: D: DMSO (vehicle, 0.1%, [.]), Ang II: Ang II 10-7mol/1, Ang II alone (), Aze: azelnidipine 106 mol/l (4), Aml: amlodipine 10-6 mol/l (V), Nif: nife- dipine 10-6 mol/l (A), efonidipine 10 6 mol/l ()
Importantly, co-treatment with each CCB further enhanced the angiotensin II-induced increase of such enzymes.
The effects of dihydropyridine CCBs on dbcAMP- or angiotensin II-induced DHEA-S and cortisol production
As shown in Fig. 6A, CCBs except efonidipine and R(-)-efonidipine did not exert significant actions on the production of DHEA-S in NCI-H295R human adrenocorti- cal carcinoma cells, whereas efonidipine and R(-)- efonidipine significantly increased the production of DHEA-S. On the contrary, angiotensin II alone decrease in the production DHEA-S, and treatment with amlodipine, efonidipine, or R(-)-efonidipine further decreased the pro- duction of DHEA-S, but azelnidipine and nifedipine did not exert siginificant actions on angioptensin II-induced decrease in DHEA-S (Fig. 6B). The CCBs, except for R(-)-efonidipine, decreased the production of cortisol, con- sistent with our previous results (Isaka et al. 2009).
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Discussion
The adrenal steroids were synthesized with many ste- roidogenic enzymes. Steroid biosynthesis was initiated by StAR, which is a rate-limiting enzyme of steroid biosysn- thesis and consisted with many steroidogenic enzymes, such as CYP11A1, HSD3B2, CYP21A2, CYP17, SULT2A1, and STS. Although the dihydropyridine CCB is one of major compounds to treat hypertension, recent stud- ies revealed that the dihydropyridine CCBs have various action on steroid biosynthesis and steroid actions beyond anti-hypertensive actions (Imagawa et al. 2006; Dietz et al. 2008; Isaka et al. 2009). We previously described the sup- pressive action of azelnidipine on steroid biosynthesis, such as cortisol and aldosterone by modulating the expression of CYP11B1 and CYP11B2 (Isaka et al. 2009). But the change of the expression pattern of steroidogenic enzymes by CCBs was not evaluated in our previous study. Therefore, the present study was designed to evaluate the
A
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SS 3 2 1 0 1 Dil Ver Dil Ver B dbcAMP 4 \* mRNA expression 3 *# \# 2 1 0 J Dil Ver Dil Ver C Ang II \* 7 mRNA expression 6 \* $. 5 ¥;#,§ § 4 3 2 1 0 Ca 2+ 0.5 1.0 Ca 2+ 0.5 1.0 EGTA {mmol/L) EGTA (mmol/L) dbcAMP </figure> effects of CCBs on expression patterns of other steroido- genic enzymes and synthesis of adrenal steroids. One of the novel findings in the present study is that some CCBs enhance dbcAMP-induced expression of StAR at mRNA and protein levels in NCI-H295R adrenocortical carcinoma cells and HepG2 human hepatoma cells in addi- tion to MA-10 mouse Leydig cells (Pandey et al. 2010), which may be dihydropyridine-specific and independent to Ca2+ blockade, because blockade of influx of extracellular Ca2+ by BAYK8644 (Clark et al. 1995) or the non-dihydro- pyridine Ca2+ channel antagonist and chelating the extracel- lular Ca2+ cause the decrease in the expression of StAR. The effects of CCBs on the expression of StAR in HepG2 human hepatoma cells is consistent with those in NCI- H295R human adrenocortical carcinoma cells, indicating that transportation of cholesterol into mitochondria may be facilitated by CCBs such as efonidipine or nifedipine. But such CCBs suppressed the production of aldosterone and cortisol (Isaka et al. 2009), which are final products in adre- nal cells in steroidogenic system (Miller 2008). Then, a question is arisen; "How the steroidogenic system treat the transported cholesterol?" The results of the present study also revealed that the CCBs, especially, efonidipine and R(-)-efonidipine, may enhanced dbcAMP-induced produc- tion of DHEA-S, which has potentially beneficial actions against atherosclerosis and vascular remodeling (Alexandersen et al. 1996; Ii et al. 2009), whereas the CCBs enhanced angiotensin II-induced decrease of the production of DHEA-S. Age-associated declines in adrenal androgen production may contribute to decreased immune function, osteoporosis, and atherosclerosis (Dharia and Parker 2004) and the prevalence of hypertension raised in aged popula- tion. Therefore, efonidipine, which exerts cardio- and renal protective actions via the blockade of the T-type Ca2+ chan- nel resulting in suppression of release of aldosterone from adrenal cells and the activation of nuclear factor-KB (Imagawa et al. 2006; Isaka et al. 2009; Hayashi et al. 2010), may exert additional cardiovascular actions, such as an anti-atherosclerotic action, through holding up the age- associated declines in adrenal androgen production in the aged patients with hypertension. Our present results showed that, at least, efonidipine and R(-)-efonidipine enhanced the dbcAMP-induced production of DHEA-S which reflects increased cholesterol transport to mitochon- dria due to increased expression of StAR because other ste- roidogenic enzymes are not siginificantly changed by dbcAMP with efonidipine or R(-)-efonidipine. On the con- trary, angiotensin II decreased the production of DHEA-S, and CCBs enhanced the decrease of DHEA-S induced by angiotensin II, possibly due to enhancement of the expres- sion of the steroidogenic enzymes, such as HSD3B2 and CYP21A2. The detailed mechanism of enhancement by CCBs on angiotensin II is not clarified and the further stud- ies should be required. In conclusion, the present results indicate that efoni- dipine may enhance the production of adrenal DHEA-S via the modulation of StAR independent blockade of Ca2+ influx through Ca2+ channels. Therefore, although the detailed mechanism still remains to be clarified, efonidipine <!-- PageBreak --> <!-- PageNumber="269" --> <!-- PageHeader="Modulation of Steroidogenesis by Dihydropyridines" --> <figure> <figcaption>Fig. 5. The effects of dihydropyridine CCBs on the expression of dbcAMP- and angiotensin II-induced steroidogenic enzymes in NCI-H295R adrenocortical carcinoma cells. CCBs enhanced angiotensin II-induced expression of these enzymes, whereas CCBs did not exert the significant action on the protein expression of these enzymes. A) Image of western blot analysis of CYP11A1, HSD3B2, and CYP21A2. B)-D) Quantification of the protein expression of CYP11A1, HSD3B2, and CYP21A2. D: DMSO (vehicle, 0.1%), Aml: amlodipine 10-6 mol/l, Nif: nifedipine 10 6 mol/l, Efo: efonidipine 10 6 mol/l, Aze: azelnidipine 10-6 mol/l, R(-)- Efo: R(-)-efonidipine 10-6 mol/l.</figcaption> A dbcAMP Ang II R(-)- R(-)- R(-)- D Aml Efo Nif Aze Efa Aml Efo Nif Aze Efo Aml Efo Nif Aze Efo CYP11A1 HSD3B2 CYP21A2 B-actin Protein Expression (CYP11A1, folds of DMSO) W 25 20 Relative 15 10 5 0 D Aml Efo Nif Aze R(-)-Efo Aml Efo Nif Aze R(-)-Efo Aml Efo Nif Aze R(-)-Efo C Protein Expression (HSD3B2, folds of DMSO) dbcAMP Ang II 6 Relative 4 2 0 D Aml Efo Nif Aze R(-)-Efo Aml Efo Nif Aze R(-)-Efo Aml Efo Nif Aze R(-)-Efo Protein Expression (CYP21A2, folds of DMSO) 6 dbcAMP Ang II 15 12 9 6 3 0 D Aml Efo Nif Aze R(-)-Efo Aml Efo Nif Aze R(-)-Efo Aml Efo Nif Aze R(-)-Efo dbcAMP Ang II </figure> <!-- PageBreak --> <!-- PageNumber="270" --> <!-- PageHeader="K. Ikeda et al." --> <figure> <figcaption>Fig. 6. Effects of CCBs (10-6 mol/l) on the production of DHEA-S in NCI-H295R adrenocortical carcinoma cells. A) Efonidipine and R(-)-efonidipine, but not azelnidipine, amlodipine, and nifedipine, enhanced the dbcAMP-induced production of DHEA-S. B) CCBs further decreased the angiotensin II-induced suppression on the production of DHEA-S. C) and D) DbcAMP- and angiotensin II-induced production of cortisol in NCI-H295R adrenocortical carci- noma cells was significantly decreased by CCBs, confirming the effects of CCBs on NCI-H295R adrenocortical carci- noma cells. The data were reproduced in at least two independent experiments. D: DMSO (vehicle, 0.1%), Aze: azelni- dipine 106 mol/l, Aml: amlodipine 10-6 mol/l, Nif: nifedipine 10-6 mol/l, Efo: efonidipine 10-6 mol/l, R(-)-Efo: R(-)- efonidipine 10 6 mol/l.</figcaption> A \* \* B 20 \# $ & 12 DHEA-S production (nmol/mg protein) \# DHEA-S production (nmol/mg protein) ** T \- \* T \* 15 \* \# LO \* ₩ KA T T \## T 10 2 T 6 T T T T 5 3 0 0 0 Aze Aml Nif Efo R(-)-Efo Aze Aml Nif Efo R(-)-Efo D Aze Aml Nif Efo R(-)-Efo Aze Aml Nif Efo R(-)-Efo C dbcAMP D Ang II 2500 \* \# \* 1600 \* A \# \* Cortisol production (nmol/mg protein) O 2000 Cortisol production (nmol/mg protein) ,# A 1 1200 \* \# \* $ * A 1500 S A T 800 1000 500 400 0 0 D Aze Aml Nif Efo R(-)-Efo Aze Ami Nif Efo R(-)-Efo D Aze Aml Nif Efo R(-)-Efo Aze Aml Nif Efo R(-)-Efo dbcAMP Ang II </figure> may exert an additional action beyond anti-hypertensive actions via slowing the age-associated decline of adrenal androgen production in aged patients with hypertension. ## Acknowledgments The authors wish to thank Yuri Inada, and Yuko Takada for their excellent technical assistance. 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