Synergistic Stimulation of Aldosterone Production in Human Adrenocortical Carcinoma NCI-H295R Cells by Endothelin-1 and Angiotensin II
Chii-Whei Hu, Randy L. Webb, and Arco Y. Jeng
Abstract: Aldosterone has recently been implicated in the patho- genesis of heart failure. The purpose of the present study was to determine the effect of endothelin-1 (ET-1) and angiotensin II (Ang II), two potent vasoconstrictors that are also involved in heart failure, on aldosterone secretion by human adrenocortical carcinoma NCI- H295R cells grown in 96-well plates. Ang II stimulated the production of aldosterone dose-dependently in serum-free medium, and the presence of serum drastically decreased aldosterone secretion. In contrast, ET-1-stimulated aldosterone production absolutely required serum. Under optimal conditions, ET-1 was more effective than Ang II as an aldosterone secretagogue. In a suboptimal condition of 2.5% serum, ET-1 and Ang II at 1 µM produced 63 and 76 pmol aldosterone/mg protein, respectively, while 230 pmol aldosterone/mg protein was generated upon co- incubation with ET-1 and Ang II. The effect of ET-1 was inhibited dose-dependently by the selective ETA receptor antagonist BQ-123 with an IC50 of 23 nM, but the selective ETB receptor antagonist RES-701 had no effect up to 10 uM. These results suggest that ET-1 and Ang II stimulated aldosterone secretion synergistically in NCI- H295R cells and that the effect of ET-1 was mediated via the ETA receptor subtype.
Key Words: aldosterone, angiotensin II, endothelin, NCI-H295R cells, BQ-123 (J Cardiovasc Pharmaco!™ 2004;44(suppl 1):S289-S292)
F or decades since its discovery in 1953, the mineralo- corticoid hormone aldosterone has been thought to be produced exclusively in the adrenal gland, and functions primarily as a major regulator of fluid volume, sodium retention, and potassium excretion. However, recent studies have demonstrated that aldosterone can be generated extra- adrenally by endothelial and vascular smooth muscle cells in the heart and blood vessels, and that it causes a number of deleterious effects on the cardiovascular system, including myocardial necrosis and fibrosis.1-4 These results, together
with the clinical findings that treatment with aldosterone receptor antagonists such as spironolactone and eplerenone significantly reduced morbidity and mortality among heart failure patients already taking an angiotensin-converting enzyme inhibitor or a ß-blocker, have implicated aldosterone in the pathogenesis of heart failure.5,6
The biosynthesis of aldosterone is regulated by more than a dozen factors.7 Angiotensin II (Ang II), a potent vasoconstrictive peptide and mediator of heart failure,8 and potassium are well-documented aldosterone secretagogues in both animal and human studies. More recently, endothelin-1 (ET-1), another potent vasoconstrictor and potential mediator of heart failure,9 has also been shown to stimulate aldosterone secretion in primary human adrenocortical cells.10 The purpose of the present study was to evaluate the effect of ET-1 and Ang II on aldosterone secretion by human adrenocortical carcinoma NCI-H295R cells grown in 96-well plates. The subtype of the ET receptor mediating the effect of ET-1 on aldosterone production was also determined.
METHODS
Materials
The human adrenocortical carcinoma NCI-H295R cell line was obtained from American Type Culture Collection (Manassas, Virginia, U.S.A.). Insulin/transferrin/selenium-A supplement (100 x), Dulbecco’s modified Eagle’s medium/ Ham’s F-12 (DMEM/F-12), antibiotic/antimycotic (100 x), and fetal calf serum (FCS) were purchased from Gibco (Grand Island, New York, U.S.A.). The MTT cell proliferation kit and phosphate-buffered saline (PBS) were products of Roche Applied Science (Indianapolis, Indiana, U.S.A.). Anti-mouse PVT scintillation proximity assay beads and nonbinding surface 96-well plates were obtained from Amersham (Piscataway, New Jersey, U.S.A.) and Corning (Acton, Massachusetts, U.S.A.), respectively. Aldosterone and Ang II were purchased from Sigma (St Louis, Missouri, U.S.A.). D-[1,2,6,7-3H(N)]aldosterone was acquired from PerkinElmer (Boston, Massachusetts, U.S.A.). Nu-serum was a product of BD Biosciences (Franklin Lakes, New Jersey, U.S.A.). BQ-123, RES-701, ET-1, ET-2, and ET-3 were purchased from American Peptide (Sunnyvale, California, U.S.A.).
Copyright @2004 by Lippincott Williams & Wilkins
Measurement of Aldosterone Production from NCI-H295R Cells
Human adrenocortical carcinoma NCI-H295R cells were seeded in 96-well plates at a density of 25,000 cells/well in 100 uL growth medium containing DMEM/F12 supplemented with 10% FCS, 2.5% Nu-serum, 1 µg insulin/ transferrin/selenium/mL, and 1 × antibiotic/antimycotic. The medium was changed after culturing for 3 days at 37℃ under an atmosphere of 5% CO2/95% air. On the following day, cells were rinsed with 100 uL DMEM/F12 and incubated with 100 µL treatment medium in quadruplicate wells at 37℃ for 1-3 days depending on the experiment. At the end of incubation, 50 uL medium was withdrawn from each well for measurement of aldosterone production by a radio- immunoassay using mouse anti-aldosterone monoclonal antibodies. The remaining cells were used either for protein determination or cell viability assay. For protein determination, cells were rinsed with 100 uL PBS and solubilized with 200 uL of 0.3 N NaOH containing 0.5% sodium dodecyl sulfate prior to assay. Cell viability was assessed according to manufacturer’s instructions.
Measurement of aldosterone was also performed using a 96-well plate format. Each test sample was incubated with 0.02 µCi of D-[1,2,6,7-3H(N)]aldosterone and 0.3 µg anti- aldosterone antibody in PBS containing 0.1% Triton X-100, 0.1% bovine serum albumin, and 12% glycerol in a total volume of 200 uL at room temperature for 1 hour. Anti- mouse PVT scintillation proximity assay beads (50 uL) were then added to each well and incubated overnight at room temperature prior to counting in a Microbeta plate counter. The amount of aldosterone in each sample was calculated by comparing with a standard curve generated using known quantities of the hormone.
Full concentration-response curves of an inhibitor of aldosterone production in NCI-H295R cells were performed at least three times. The 50% inhibitory concentration (IC50) values were derived using a non-linear least squares curve- fitting program from Microsoft XLfit.
RESULTS
Ang II stimulated the secretion of aldosterone by human adrenocortical carcinoma NCI-H295R cells in a time- dependent and dose-dependent manner. Figure 1 (top panel) shows that, upon incubation at 37ºC for 72 hours, Ang II increased the production of aldosterone dose-dependently in serum-free medium. The presence of fetal calf serum, even at 2.5%, drastically reduced the production of aldosterone. Similar results were obtained when the cells were incubated with Ang II for 24 and 48 hours, except that lower levels of aldosterone were generated (data not shown). ET-1 also stimulated the secretion of aldosterone by these cells in a time-dependent and dose-dependent manner. However, in contrast to the results obtained by Ang II, ET-1-stimulated
Aldosterone (pmol/mg protein)
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0% FCS
2.5% FCS
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Log[Angiotensin II] (M)
Aldosterone (pmol/mg protein)
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2.5% FCS
5% FCS
10% FCS
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Log[ET-1] (M)
aldosterone production absolutely required serum (Fig. 1, bottom panel). Under optimal conditions, ET-1 was more effective than Ang II as an aldosterone secretagogue. At 1 µM, ET-1 produced 870 pmol aldosterone/mg protein when incubated in 10% FCS for 72 hours, as compared with the 220 pmol aldosterone/mg protein for Ang II in serum-free medium (Fig. 1).
The effects of other ET family peptides on aldosterone secretion were also examined. ET-1 and ET-2 were equipotent in stimulating aldosterone production by NCI- H295R cells (Fig. 2). In contrast, ET-3 did not have
1000
Aldosterone (pmol/mg protein)
ET-1
ET-2
800
ET-3
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Log[Peptide] (M)
significant effect at concentrations up to 10 nM. Upon incubation at 1 µM for 72 hours in 10% FCS, ET-3 produced about one-half of the amount of aldosterone as compared with those obtained from ET-1 or ET-2 (Fig. 2).
A suboptimal condition of 2.5% FCS was used to assess whether there were interactions between the ET-1 and Ang II signaling pathways. Under these conditions, ET-1 and Ang II at 1 µM produced 63 and 76 pmol aldosterone/mg protein, respectively, while 230 pmol aldosterone/mg protein was generated upon co-incubation with ET-1 and Ang II (Fig. 3). These results suggest that ET-1 and Ang II synergistically stimulated the secretion of aldosterone by NCI-H295R cells.
BQ-123, a specific ETA receptor antagonist, inhibited ET-1-stimulated secretion of aldosterone dose-dependently, with an IC50 of 23 ± 4 nM (mean ± standard error of the mean, n = 3) (Fig. 4). In contrast, the selective ETB receptor antagonist RES-701 had no significant effect at concentra- tions up to 10 µM.
DISCUSSION
The main observations in the present study are that the potent vasoconstrictors ET-1 and Ang II synergistically
Aldosterone (pmol/mg protein)
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Ang II
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Aldosterone (% control)
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KH
4
KH-OH
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D
80
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40
BQ-123
20
BQ-123 + RES-701
RES-701
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1
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[Compound] (nM)
stimulated the secretion of aldosterone from human adrenocortical carcinoma NCI-H295R cells and that the
effect of ET-1 was mediated by the ETA receptor subtype. In addition, methods were established to allow a rapid growth of NCI-H295R cells in 96-well plates for evaluation of the effect of aldosterone secretagogues. The methodology may be extended to measure the production of other steroids or hormones.
Ang II stimulated aldosterone production from NCI- H295R cells in a time-dependent and dose-dependent fashion, similar to the results reported previously.11 Interestingly, the presence of serum in culture medium markedly inhibited the Ang II-stimulated aldosterone secretion while serum was absolutely required for the ET-1- induced production of aldosterone. Further experiments are necessary to determine the serum factor(s) causing these effects.
In the present study, ET-1 and ET-2 were found to be equipotent as aldosterone secretagogues, while ET-3 was significantly less effective. These results, coupled with the findings that the ET-1-stimulated aldosterone secretion was inhibited dose-dependently by BQ-123 but not by RES-701, suggest the action of ET-1 was mediated through the ETA receptors. However, using primary human adrenocortical cells, Rossi et al. have reported that both ETA and ETB receptor subtypes are involved in ET-1-induced aldosterone secretion.10 Recently, the same group of researchers also showed that the aldosterone secretagogue effect of ET-1 was mediated via ETB receptors in patients with primary aldosteronism, whereas it was mediated via the ET_ receptor subtype in patients with high-to-normal renin hypertension.12 Thus, the ET receptor subtype involved in ET-1-induced aldosterone secretion remains to be clarified.
It has been postulated that, upon binding to its type I receptor, Ang II increases intracellular Ca2+ concentrations in human adrenocortical cells via a phospholipase C-dependent pathway and triggers phosphorylation of cAMP response element binding protein. This phosphorylated protein, in turn, binds to cAMP response element localized in the 5’ flanking region of aldosterone synthase, an enzyme responsible for the final step of aldosterone biosynthesis, thereby activating gene transcription of the enzyme. 13
Likewise, binding of ET-1 to its receptor also activates phospholipase C in various cell types.14 It is thus envisaged that ET-1 and Ang II could have an additive effect on the production of aldosterone in NCI-H295R cells. However, the mechanism by which ET-1 and Ang II exert the synergistic effect on aldosterone secretion in these cells observed in the present study is not clear. If this synergistic effect is confirmed in human studies, it would imply that a combined ET/renin-angiotensin system inhibition may provide a superior strategy for the treatment of heart failure.
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