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Departments of Clinical and Experimental Medicine, Pharmacology (S.B., L.T.), Division of Endocrinology (F.F.), Institutes of Microbiology (G.P.) and Anatomy (A.S.B., G.G.N.), University of Padua Medical School, Padova, Italy; and Max Planck Institute of Psychiatry, Clinical Institute (U.P.), Munich, Germany
Address all correspondence and requests for reprints to: Gian Paolo Rossi, M.D., F.A.C.C., Clinica Medica 1, Dipartimento di Medicina Clinica e Sperimentale, University Hospital via Giustiniani, 2, 35126 Padova, Italy. E-mail: gprossi{at}ipdunidx.unipd.it
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Introduction |
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A human adrenocortical tumor cell line (NCI-H295) is now available (9) that expresses angiotensin II AT1 receptor functionally coupled to phosphoinositidase C, secretes aldosterone in response to angiotensin II, and has been widely used for investigating the regulation of adrenal steroidogenesis in vitro (10, 11, 12, 13). Hence, we hypothesized that NCI-H295 cells can be useful for investigating the role of ET-1 in the regulation of human adrenocortical function.
This study was, therefore, designed to investigate whether NCI-H295 cells 1) express the genes for prepro-ET-1, human ET-1 converting enzyme-1 (hECE-1), and ETA and ETB receptors; 2) are endowed with a functional ET-1 biosynthetic pathway; 3) possess functional ETA and ETB receptor subtypes; and 4) regulate the expression of the aldosterone synthase (AS) gene in response to ET-1.
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Materials and Methods |
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Cell culture
Human NCI-H295 adrenal carcinoma cells were obtained from the
American Type Culture Collection (Rockville, MD; catalogue no. CRL
10296). Cells were initially maintained in RPMI 1640 medium (Life
Technologies, Eggenstein, Germany; catalogue no. 041–02404M)
supplemented with 2% FCS, insulin (0.005 mg/ml; Monotard, Novo Nordisk
Farmaceutici, Rome, Italy), transferrin (0.01 mg/ml), sodium selenite
(30 nM), and antibiotics (penicillin-streptomycin). Cells
were maintained and grown on 25-cm2 flasks at 37 C under an
atmosphere of 5% CO2-95% air. During the initial 2 months
of culture, only attached cells were retained when medium was changed.
Cells used for the experiments described were routinely maintained as
monolayer cultures. When the responses of cells to agonists were
studied, the medium was removed and replaced with serum-free medium and
0.02% BSA.
NCI-H295 cells were used for RNA extraction by the guanidium isothiocyanate method. RNA was precipitated by the addition of 500 µl ethanol and 20 µl 3 M sodium acetate and standing for 1 h at -80 C. It was recovered by centrifugation (15 min, 12,000 x g, 4 C), and the pellet was washed once in 75% ethanol (1.0 ml) before being dried under vacuum and dissolved in diethylpyrocarbonate-water. After isolation, total RNA was checked for integrity by 1.5% agarose gel electrophoresis and quantified by measurement of UV absorbance at 260 nm.
Reverse transcription-PCR (RT-PCR)
For use in the PCR, total RNA was reversely transcribed to
complementary DNA (cDNA) with random hexamers (2.5 µM),
as previously detailed (5). After incubation at 42 C for 15 min,
temperature was raised to 95 C for 5 min and then quickly decreased to
5 C for 5 min. For amplification of the resulting cDNA, 20 µl of the
reverse transcription mixture were used. The sample volume was
increased to 100 µl with a solution containing 50 mM KCl,
10 mM Tris (pH 8.3), 2 mM MgCl2,
and 0.2 µM of up- and downstream primers as well as 2.5 U
Taq polymerase (AmpliTaq DNA Polymerase, Perkin-Elmer/Cetus,
Norwalk, CT). Amplification of prepro-ET-1, ETA,
ETB, and hECE-1 cDNA was carried out with the primers and
the thermal profiles previously reported (5, 18), using a Delphi 1000
Thermal Cycler (MJ Research, Waterston, MA). The specificity of the
amplification products for the gene of interest was confirmed by
hybridization with the following cDNA-specific probes: hECE-1, 5'-TTG
GAC TTT GAG ACG GCA CTG GC-3'; ETA receptor, 5'-CCT CAA CCT
CTG CGC TCT TAG TGT-3'; and ETB receptor, 5'-TCC TGC CTT
GTG TTC GTG CTG GGG-3'. As a positive control, amplification of a
838-bp fragment of the human ß-actin gene was carried out in
parallel, as described previously (5). As false positive results of
RT-PCR for the ß-actin gene, due to amplification of
retro-pseudogenes, have been reported (19), in parallel experiments
ETA and ETB receptor PCR was carried out with
no prior RT to further rule out the possibility of amplifying genomic
DNA.
Immunocytochemistry
NCI-H295 cells were cultured in double slide flasks at a
concentration of 20,000 cells/well in RPMI 1640 with all ingredients
and 2% FCS for 4 days, then washed in PBS and cultured for 24 h
in RPMI supplemented only with antibiotics, glutamine, selenium, and
transferrin. Immunocytochemistry was performed with minor modifications
as previously described (20). Briefly, two antibodies for ET-1 were
used: a rabbit polyclonal (Peninsula, Heidelberg, Germany) and a mouse
monoclonal (Dianova, Hamburg, Germany; cross-reactivity for ET-3, 7%
and 2%, respectively). Rabbit and swine serum and biotinylated swine
antirabbit and antimouse IgGs were obtained from Dako (Hamburg,
Germany). After medium removal and several washes in cold PBS, the cell
monolayer was rapidly dried and fixed in 4% paraformaldehyde, and
endogenous peroxidase was blocked with 1%
H2O2. The sections were then preincubated in
rabbit or swine serum (1:10), followed by application of polyclonal or
monoclonal ET-1 antibody (1:1500 and 1:125) overnight at 4 C. After
incubation with the respective biotinylated IgGs (1:300) and the
streptavidin-biotin-horseradish peroxidase complex (1:100) for 1 h
at room temperature each, the enzymatic reaction was performed for
5–10 min at room temperature using 0.1% 3,3-diaminobenzidine
tetrahydrochloride (diluted in PBS with 0.05% hydrogen peroxide) as
chromogen. After immunostaining, the sections were inserted for
dehydration in a series with increasing concentrations of
ethanol-xylol, and the coverslips were attached with Eukitt (Riedel de
Haen, Seelze, Germany). For immunocytochemical detection, negative
controls with omission of the primary antibody were run in
parallel.
Autoradiography
Cell monolayers were immediately frozen at -30 C by immersion
in isopentane and stored at -80 C. They were processed according to
the method of Kuhar (21) with minor modifications (6). ET-1-binding
sites were labeled in vitro by incubation for 120 min with
100 pM [125I]ET-1; nonspecific binding was
determined by adding 1 µM unlabeled ET-1. Selective
[125I]ET-1 binding to ETA and ETB
was studied by adding 100 nM BQ-123 and sarafotoxin 6C,
respectively. The reaction was stopped by washing the samples three
times in 50 nM Tris-HCl buffer. After rinsing, the sections
were rapidly dried, fixed in paraformaldehyde vapors at 80 C for 120
min, and coated with NTB2 Kodak nuclear emulsion (Eastman Kodak,
Rochester, NY). The autoradiograms were exposed for 2 weeks at 4 C and
then developed with undiluted D19 Kodak developer. They were stained
with hematoxylin-eosin, and observed and photographed with a Leitz
Laborlux microscope (Leitz, Rockleigh, NJ). For the purpose of
quantitation, autoradiograms were analyzed by computer-assisted
densitometry using a camera-connected microscope and a personal
computer equipped with software specifically written for this purpose
(Studio Casti Imaging, Venice, Italy). Given the uneven distribution of
cells on monolayers, only areas corresponding to identifiable cells
were outlined and measured. For each autoradiogram (n = 4), 10–18
areas of about 55,000 µm2 (79,500 pixels) were analyzed.
The density of the ETA subtype was assessed in the presence
of the ETB weak agonist sarafotoxin 6C (500
nM), and that of ETB was determined in the
presence of BQ-123 (500 nM).
Gene expression
NCI-H295 cells (5 x 105 cells/well) were
incubated for 24 h at 37 C with ET-1 (10-8
M) or IRL-1620 (10-7 M) and with
ET-1 (10-8 M) plus BQ-123 or BQ-788
(10-6 M). At the end of the incubation period,
cells were harvested, and RNA was extracted and reversed transcribed
(see above). PCR amplification was performed (see above) with 21-mer
5'-ACATTGGTACAGGTTTTCCTC-3' (sense) and 5'-CAGATGCAAGACTAGTTAATC-3'
(antisense) (22). To evaluate the kinetics of the amplification
reaction, 5-µl aliquots of the amplification mixture were collected
after every two cycles starting from the 20th up to the 38th cycle.
Aliquots of the PCR products were transferred to a nylon membrane
(Immobilon-S, Millipore, Milan, Italy) by a slot blot apparatus
(Milliblot, Millipore) and UV cross-linked (Stratagene UV-Crosslinker
1800, Stratagene-Duotech, Milan, Italy). Detection of the
digoxigenin-labeled amplification products on the nylon membrane was
carried out by high affinity antidigoxigenin antibody Fab fragments
conjugated to alkaline phosphatase using a chemiluminescent detection
kit (DIG Luminescent Detection Kit, Boehringer Mannheim, Mannheim,
Germany). Light generated via dephosphorylation of the chemiluminescent
substrate (CSPD, Boehringer Mannheim) was used to impress x-ray films
(BioMax MR-1 film, Sigma) with a 20-min exposure, as in an
autoradiographic procedure (5). Quantification of the PCR products was
carried out by measuring the integrated optical density of the
autoradiographies with an image analyzer IBAS 2000 (Zeiss, Unterkochen,
Germany). Plots of the integrated optical density vs. the
number of cycles were then elaborated; the cycle number
(N50) that corresponded to the half-maximal PCR products
was also calculated as an estimate of the amount of initial amplifiable
template of the gene (5, 23). Comparison of the N50 of
cells exposed to the different agonists and antagonists was carried out
by one-way ANOVA followed by Bonferroni post-hoc test; the
significance level was set at 0.05.
Measurement of intracellular Ca2+concentration
([Ca2+]i)
[Ca2+]i was measured by fluorometry in
a double wavelength mode in a Perkin-Elmer LS-50B spectrofluorometer
equipped with a magnetic stirrer and a temperature regulator
(Perkin-Elmer, Norwalk, CT). Cell monolayers were loaded with
fura-2/acetoxymethyl ester (fura-2/AM; Molecular Probes, Eugene, OR) as
described by Bird et al. (24). Briefly, NCI-H295 cells were
plated on 8 x 30-mm glass coverslips and cultured in growth
medium, as described above, for 7 days. For cell loading, coverslips
were incubated in a buffer (130 mM NaCl, 4.8 mM
KCl, 1 mM MgCl2, 1.5 mM
CaCl2, 1 mM NaH2PO4, 15
mM glucose, 1 mg/ml BSA, and 10 mM HEPES, pH
7.4) containing 5 µM fura-2/AM for 45 min at 37 C in a
5% CO2-95% O2 atmosphere. Coverslips were
then transferred to a customized holder, inserted into a quartz
cuvette, and placed into the spectrofluorometer, where they were
superfused at a rate of 5 ml/min with the same buffer without BSA (25).
The cells were left to recover for 20 min before starting the
experiment. [Ca2+]i was calculated by the
formula of Grynkiewicz et al. (26):
[Ca2+]i = {[(R -
Rmin)/(Rmax - R)] x
(Sf/Sb) x Kd}, where
Kd is the dissociation constant of fura-2 and was assumed
to be 225 nM, R is the 340/380 nm ratio of fura-2
fluorescence measured in the cells, Rmax is the 340/380 nm
ratio in the presence of a saturating calcium concentration,
Rmin is the 340/380 nm ratio of fura-2 fluorescence in a
Ca2+-free solution containing EGTA, and
Sf/Sb is the ratio of Ca2+-free to
Ca2+-bound fura-2 fluorescence at 380 nm. Calibration was
performed by using fura free acid. Rmin was determined in a
solution containing 115 mM KCl, 10 mM NaCl, 2
mM MgSO4, 10 mM
K2H2-EGTA, and 10 mM
K2-3-(N-morpholino)propanesulfonic acid (MOPS)
at pH 7.2. For the determination of Rmax, 2 mM
CaCl2 was added to the medium, and CaK2-EGTA
was substituted for K2H2-EGTA.
Rmax, Rmin, and Sf/Sb
values were 17.36, 1, and 7.81, respectively.
The effect of ET-1 (10 nM) and the ETB agonist IRL 1620 (100 nM) on [Ca2+]i was assessed with and without 10-min pretreatment with BQ-788 (0.8 mM) and/or BQ-123 (1 mM). As exposure to ET-1 was reported to induce homologous down-regulation (27), each experiment was carried out on a different preparation of NCI-H295 cells. Each set of experiments was carried out four times on different cell preparations with consistent results.
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Results |
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, A and B). The
specificity of the amplification products obtained was confirmed by 1)
hybridization with cDNA-specific probes, 2) size identity, and 3) lack
of amplification of each cDNA when diethylpyrocarbonate-water was
used instead of mRNA as the template for RT-PCR.
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),
whereas no staining was seen when the primary antibody was omitted
(Fig. 2B).
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) and
binding in the presence of excess cold ET-1 (Fig. 3B). The binding was
partially displaced by both the ETA antagonist BQ-123 (Fig. 3C) and the ETB antagonists BQ-788 (Fig. 3D). Quantitative
densitometric analysis of the autoradiograms revealed that the
ETB subtype accounted for the majority (mean ±
SEM, 72.6 ± 9%) and the ETA accounted
for the minority (37.4 ± 5%) of the total
[125I]ET-1-specific binding.
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, where the amount of amplified DNA (log
integrated optical density units) is plotted vs. the
number of PCR cycles. The curve corresponding to the ET-1-treated cells
cDNA was shifted to the left compared with that in the controls (Fig. 4), thereby indicating a higher initial abundance of AS mRNA
(i.e. an enhanced expression of the AS gene) (23); however,
the slopes of all curves were similar, suggesting a similar efficiency
of amplification. The specific ETB agonist IRL-1620
displayed a similar shift to the left compared with the control curve.
The concomitant exposure to ET-1 and either BQ-123 or BQ-788 did not
abolish the stimulatory effect of ET-1. The N50,
i.e. the number of cycles at which half-maximal
amplification was attained, was significantly lower for ET-1
(25.00 ± 0.24 SEM; P = 0.006) and
IRL-1620 (26.18 ± 0.59; P < 0.05) than for the
controls (28.90 ± 0.20). Similarly, the N50 of cells
treated with both ET-1 plus BQ-788 (25.61 ± 0.84) and ET-1 plus
BQ-123 (26.04 ± 0.70) was lower than the control value, although
not significantly so, due to the larger dispersion of the data.
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). This effect rapidly disappeared with
repeated exposure to ET-1 and was abolished by 10-min pretreatment with
the BQ-788 (Fig. 5B), whereas it was not affected by pretreatment with
BQ-123 (Fig. 5C). Either BQ alone did not modify resting
[Ca2+]i. The exposure of the cells to IRL
1620 induced a 91% increase in [Ca2+]i (from
65 ± 7 to 124 ± 22; n = 3; Fig. 5D). This effect was
prevented by 10-min pretreatment with BQ-788 (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Our coupled cell biology, immunocytochemical, and autoradiographic study shows that NCI-H295 cells not only express the mRNAs of prepro-ET-1 and hECE-1 and can synthesize immunoreactive ET-1, a finding in partial agreement with the results of Li et al. (28) in adrenocortical carcinomas, but also that they can express and translate into functional proteins both ETA and ETB receptor subtypes. Thus, these results strongly suggest that ET-1 may act as an autocrine-paracrine factor in the regulation of NCI-H295 cell function.
This contention is supported by the demonstration that ET-1 is able to enhance the expression of the AS gene through a mechanism that is likely to mainly involve the ETB receptor subtype. In fact, our findings indicate that ET-1 exposure induces a higher initial abundance of AS mRNA in the NCI-H295 cells, and the ETB agonist IRL-1620 is equipotent to ET-1 as far as this effect is concerned. It must be pointed out, however, that exposure to either the specific ETA antagonist BQ-123 or the specific ETB antagonist BQ-788 alone could not abolish the stimulatory effect of ET-1, thereby suggesting that both receptor subtypes are somehow involved in the mediation of this effect of ET-1.
As concerns the intracellular event brought about by ET receptor activation, we found that activation of the ETB receptor subtype by either ET-1 or IRL 1620 leads to a clear-cut increase in [Ca2+]i, which vanished with repeated ET-1 stimulation, probably due to homologous desensitization (27). This effect was abolished by pretreating the cells with the specific ETB antagonist BQ-788 alone or in combination with BQ-123, but not by treatment with the specific ETA antagonist BQ-123 alone, thereby confirming that the ET-1-induced increase in [Ca2+]i is an ETB receptor-mediated effect. Calcium mobilization is deemed to play an important role in the control of steroidogenesis in the adrenal cortex (29); accordingly, the bulk of the evidence indicates that activation of the ETB receptor subtype mediates the aldosterone secretagogue effect of ET-1 (for review, see 8 .
Hence, the question arises of what are the cellular events associated with ETA receptor subtype activation and ensuing stimulation of AS gene expression. The fact that no evident increase in [Ca2+]i and aldosterone secretion was elicited by ET-1 activation of the ETA subtype is intriguing and obviously worthy of further investigation.
In conclusion, our results provide evidence for the concomitant expression AS and the prepro-ET-1, hECE-1, ETA, and ETB receptor genes in NCI-H295 adrenocortical carcinoma-derived cells. Furthermore, they demonstrate that NCI-H295 cells synthesize mature ET-1 and express both the ETA and ETB receptor subtypes at the protein level. Both ET receptors are able to bind [125I]ET-1 and can mediate the secretagogue effect of ET-1 on aldosterone by acting in an autocrine-paracrine fashion at the transcriptional level of AS; however, only ETB activation was found to be associated with a sizable increase in [Ca2+]i. Thus, these findings indicate that NCI-H295 cells may be a useful model for investigating the role of ET-1 and its receptor subtypes in the regulation of adrenocortical function in humans as well as the potential autocrine-paracrine role of ETs in the pathogenesis of adrenal tumors.
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Acknowledgments |
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Footnotes |
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Received April 23, 1997.
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