1 Department of Internal Medicine, 2 Department of Physiology and Biophysics, and 3 Department of Surgery, University of Texas Medical Branch, Galveston, Texas 77555
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ABSTRACT |
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We determined the role of the multidrug resistance (MDR1) gene product, P-glycoprotein (PGP), in the secretion of aldosterone by the adrenal cell line NCI-H295. Aldosterone secretion is significantly decreased by the PGP inhibitors verapamil, cyclosporin A (CSA), PSC-833, and vinblastine. Aldosterone inhibits the efflux of the PGP substrate rhodamine 123 from NCI-H295 cells and from human mesangial cells (expressing PGP). CSA, verapamil, and the monoclonal antibody UIC2 significantly decreased the efflux of fluorescein-labeled (FL)-aldosterone microinjected into NCI-H295 cells. In MCF-7/VP cells, expressing multidrug resistance-associated protein (MRP) but not PGP, and in the parental cell line MCF7 (expressing no MRP and no PGP), the efflux of microinjected FL-aldosterone was slow. In BC19/3 cells (MCF7 cells transfected with MDR1), the efflux of FL-aldosterone was rapid and it was inhibited by verapamil, indicating that transfection with MDR1 cDNA confers the ability to transport FL-aldosterone. These results strongly indicate that PGP plays a role in the secretion of aldosterone by NCI-H295 cells and in other cells expressing MDR1, including normal adrenal cells.
transport; multidrug resistance; MDR1; P-glycoprotein; zona glomerulosa
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INTRODUCTION |
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STEROIDS WERE BELIEVED TO BE transported across plasma membranes by passive diffusion, a process favored by their relatively small size and high lipid solubility (31). However, active transport of cortisol and of the synthetic steroids dexamethasone and prednisolone has been described (11, 12, 31). Interactions between P-glycoprotein (PGP), the protein that confers multidrug resistance to cancer cells, and steroid transport have been reported by Ueda et al. (32). These authors transfected renal epithelial cells (LLC-PK1) with the multidrug resistance 1 (MDR1) cDNA isolated from human normal adrenal glands and observed that the cells acquired multidrug resistance and transported aldosterone from the basolateral to the apical side. Because PGP is expressed in the cortex of the adrenal gland, these results suggest the participation of PGP in the secretion of aldosterone (22, 25). In the mouse, PGP-dependent steroid resistance is gene and steroid specific, and the multidrug resistance provided by a given steroid is structure dependent. A keto oxygen at positions 3 and 20 as well as a 17-hydroxyl group enhance inhibition of drug transport (34). In addition, mutations in the PGP transmembrane domains 4-6 result in loss of the recognition of steroids with the 17-OH group and the 20-keto oxygen and loss of steroidal effect (34). Hyperreninemic hypoaldosteronism resistant to ANG II stimulation has been reported, in vivo, in rats treated with the PGP substrate cyclosporin A (CSA); the zona glomerulosa cells, isolated from the same animals, also failed to respond to ANG II stimulation (23, 28). In humans CSA produces hyperkalemic hyporeninism and a distal tubule defect in K+ excretion (16, 30). The aim of this study was to examine the hypothesis that aldosterone is a substrate for PGP, as a part of a larger research effort investigating the possible endogenous substrates of PGP. To this end, we studied the interaction of PGP and aldosterone in the human adrenal cell line NCI-H295, a cell line that exhibits ANG II-stimulated aldosterone secretion (4, 10, 14). We found that NCI-H295 cells express PGP and that ANG II-stimulated aldosterone secretion was inhibited by multidrug resistance modulators. In addition, aldosterone inhibits PGP-mediated transport of the prototype substrate rhodamine 123 (R123) in NCI-H295 cells as well as in human mesangial cells expressing PGP. Furthermore, BC19/3 cells expressing high levels of PGP have increased efflux of fluorescein-labeled aldosterone (FL-aldosterone). Inhibitors of PGP decrease the efflux of FL-aldosterone microinjected into NCI-H295 and BC19/3 cells (MCF7 cells transfected with MDR1). MCF-7/VP cells, which express multidrug resistance-associated protein (MRP) but not PGP (6, 27), and the parental cell line MCF7, exhibited a significantly slower efflux of microinjected FL-aldosterone than NCI-H295 and BC19/3 cells. These results strongly suggest that PGP plays a role in the secretion of aldosterone.
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MATERIALS AND METHODS |
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Cell Culture
NCI-H295 cells (10) were grown at 37°C with 5% CO2 in HyQ-CCM-1 medium (HyClone Laboratories, Logan, UT), a serum-free culture medium. Cells were seeded in custom-made glass-bottom perfusion chambers (3) for fluorescence experiments (R123 and FL-aldosterone effluxes), or in 12-well plates for aldosterone secretion experiments (see Aldosterone Secretion).The human breast cancer cells MCF7 (drug sensitive) and BC19/3 (MCF7 transfected with the human MDR1 cDNA) were used as negative and positive controls for the determination of the expression of MDR1 mRNA and for the study of the efflux of microinjected FL-aldosterone. These cells were grown at 37°C with 5% CO2 in Eagle's improved minimum essential medium (IMEM) supplemented with 10% fetal bovine serum (FBS) and in IMEM supplemented with 10% FBS and 0.01 µM adriamycin, respectively.
MCF-7/VP cells (MCF7 cells selected with etoposide, overexpressing MRP) were used as negative control for the efflux of microinjected FL-aldosterone; they were grown in IMEM supplemented with 10% FBS and 1% streptomycin/penicillin. Primary cultures of normal human mesangial cells were used to study the effect of aldosterone on the transport of R123, a prototype drug transported by PGP. The cells were grown as previously described (2). Briefly, normal pieces of human kidneys, surgically removed because of malignancies, or kidneys not used for transplantation were digested with collagenase. Glomeruli were isolated by sequential sieving (2). Decapsulated glomeruli were seeded in collagen-coated tissue culture flasks and grown in DMEM supplemented with 45 mM Na2HCO3, 25 mM HEPES, 10% FBS, 100 U/ml penicillin, and 10 µg/ml streptomycin (pH 7.4). Outgrowing mesangial cells were subcultured. Passages 1-6 were studied.
Downregulation of PGP by Antisense Oligonucleotide
Downregulation of PGP was achieved with the use of antisense oligonucleotide complementary to the 5' end of the human MDR1 cDNA. Control cells were incubated with sense oligonucleotide. NCI-H295 cells grown in custom-made perfusion chambers (efflux), on circular glass coverslips (immunofluorescence), were incubated for 3 days with growth medium containing 50 µg/ml of sense or antisense oligonucleotide. Incubation was started 24 h after plating of the cells, and the medium was renewed every 24 h. Sense and antisense oligonucleotide sequences were 5'GAGGTCGGGATGGAT3' and 5'ATCCATCCCGACCTC3', respectively (24). Oligonucleotides were custom synthesized by Genosys Biotechnologies (The Woodlands, TX).Expression of MDR1 mRNA and of PGP
Determination of the expression of MDR mRNA by RT-PCR. MDR mRNA expression was measured in NCI-H295 cells as described previously (2). Briefly, NCI-H295 cells were grown until subconfluent. Total RNA was prepared from 106 cells as previously described (2). Cells from a MDR1-transfected breast cancer cell line (BC19/3) and from the multidrug-sensitive parental cell line (MCF7) were used as positive and negative controls, respectively, for the expression of MDR1 mRNA. RT-PCR was performed on 500 ng of total RNA as described in Ref. 2. Isoform-specific PCR primers were as reported by Chin et al. (7). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) PCR primers (Stratagene, La Jolla, CA) were used for the amplification of internal control mRNA. PCR products were size fractionated by agarose electrophoresis and stained with ethidium bromide.
Determination of the expression of MDR1 PGP by immunofluorescence on living cells. The fluorescence associated with PGP was measured in NCI-H295 cells that were incubated, in simultaneous experiments, with MDR1 sense or antisense oligonucleotides and detected with the monoclonal antibody MRK16. Immunofluorescence studies were performed as described previously (2). Briefly, cells growing on 25-mm-diameter no. 1 glass coverslips were washed once with HEPES-buffered solution containing (in mM) 125 NaCl, 5 KCl, 1 MgSO4, 1.36 Na2HPO4, 10 sodium acetate, 5 HEPES, 1.8 CaCl2, and 8 glucose, titrated to pH 7.4, and subsequently incubated for 1 h at room temperature in HEPES-buffered solution containing 10 µg/ml of MRK16 (Kamiya Biomedical, Thousand Oaks, CA). MRK16 is specific to the MDR1 isoform of PGP and recognizes an external epitope of the protein (26). The primary antibody was removed and the coverslips were washed with HEPES-buffered solution. Cells were incubated for 15 min at room temperature with HEPES-buffered solution containing 10 µg/ml FITC-labeled secondary antibody. After several washes with HEPES-buffered solution, fluorescence-labeled MDR1 PGP was observed by confocal microscopy (model Odissey; Noran Instruments, Middleton, WI). The membrane fluorescence was quantified as the relative fluorescence of membrane areas from eight fields containing 240 cells using a computer-imaging system (Quantex, Sunnyvale, CA). Briefly, A line-profile analysis program was utilized, and each field was divided by 10 horizontal lines out of 500 possible positions. The line-profile graphic was displayed in the screen, and the fluorescence peaks did coincide with the cell membranes; 555 peaks were read, each of them corresponding to the fluorescence at the contact points between two neighboring cells.
The expression of PGP in cell membranes of MCF7, NCI-H295, and BC19/3 cells was measured by fluorescence-activated cell sorting using FITC-4E3 monoclonal antibody against an external epitope of PGP (Signet Laboratory, Dedham, MA). Briefly, cells were detached from the cell culture flask by incubation in a trypsin-containing, Ca2+-free PBS. Trypsin digestion was stopped by addition of cold HEPES-buffered solution supplemented with 2% newborn bovine serum (NBS). Antibody was added per the manufacturer's recommendations. Samples were incubated at room temperature for 30 min, washed twice on PBS + 2% NBS, resuspended to a final volume of 500 µl, and kept in the dark until analyzed. Isotype-matched mouse IgG2a antibody was the negative control. Cell aliquots were placed in 12 × 75 Falcon tubes (Becton Dickinson, Lincoln Park, NJ.) Fluorescence measurements were made using a fluorescence-activated cell sorting scan (Becton Dickinson Immunocytometry Systems). In preliminary experiments, the expression of MRP was determined in MCF-7/VP and in NCI-295 cells treated with Tween 20 and fixed with 1% paraformaldehyde, using the human-specific QCRL-1 monoclonal antibody that recognizes an internal epitope of MRP (Signet Laboratory). The isotypic antibody was used as negative control (13). Although MCF-7/VP were positive for MRP, NCI-H295 cells were negative.Aldosterone Secretion
NCI-H295 cells were grown to confluence in 12-well culture plates. Growth medium was removed and cells were preincubated for 24 h at 37°C with 1 ml per well of control HyQ-CCM-1 medium or HyQ-CCM-1 medium containing 50 nM ANG II. The preincubation solution was removed and the cells were then incubated for 24 h at 37°C with 0.5 ml per well of control HyQ-CCM-1 medium or HyQ-CCM-1 medium containing 50 nM ANG II, in the absence or presence of 10 µM of one of several PGP inhibitors (verapamil, CSA, PSC-833, vinblastine). Supernatants were collected at the end of the incubation period and kept frozen before determining the aldosterone levels by radioimmunoassay, as previously described (14). Cells were seeded simultaneously in 96-well culture plates, and the effect of the various PGP inhibitors on cell growth was measured, as described previously (2), with the use of the colorimetric dye Alamar blue (Alamar Biosciences, Sacramento, CA).Efflux of R123 and FL-Aldosterone
Efflux of R123 from loaded cells.
Cells were grown on collagen-coated glass coverslips forming the bottom
of custom-made chambers (3). PGP-mediated transport was assessed by
measuring the efflux of the fluorescent substrate R123 (2). Cells were
loaded for 1 h at 37°C by exposure to 10 µM R123 in
HEPES-buffered solution (see Determination of the expression of
MDR1 PGP by immunofluorescence on living cells). At
time zero, the dye was rapidly removed by superfusion with HEPES-buffered solution. The fluorescence of R123 was measured in
individual cells by epifluorescence with a Nikon microscope connected
to an imaging system (Quantex, Sonnyvale, CA). Rate constants for
fluorescence decay (FR123, k) were estimated from fits of Eq. 1 to the data, as previously described (2), using SigmaPlot software (SigmaPlot; SSPS, Chicago, IL)
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(1) |
Efflux of R123 and of FL-aldosterone from microinjected cells. NCI-H295 cells were grown on collagen-coated glass coverslips. At confluence, the coverslips were mounted in a custom-made chamber on the stage of a Nikon inverted microscope. R123 or FL-aldosterone were diluted in a PBS containing 53 meq of K+ and 8 meq of Na+, pH 7.3. Approximately 10 fl of R123 or FL-aldosterone were microinjected into cells using a Femtotip Eppendorf (Eppendorf North America, Madison, WI) mounted on an Eppendorf microinjector (model 5246) attached to an Eppendorf micromanipulator (model 5171). Final intracellular concentrations were estimated to be 0.1-0.5 µM. FL-aldosterone was custom synthesized by Molecular Probes (Eugene, OR) by carbodiimide coupling of the 21-OH group of aldosterone to 5-carboxyfluorescein. The efflux of R123 or FL-aldosterone was monitored using a Deltascan imaging system [Photon Technology International (PTI), South Brunswick, NJ] and a charge-coupled device camera. Excitation and emission wavelengths were 495 and 535 nm, respectively, for R123 and 490 and 520 nm, respectively, for FL-aldosterone. Images were processed using the Image Master software (PTI). MCF7 (the parental cell line expressing no PGP), MCF-7/VP cells (expressing MRP but not PGP), and BC19/3 (MCF7 cells transfected with MDR1) were microinjected using the same protocol, and the efflux of FL-aldosterone was monitored as described above.
Statistics
Values are means ± SE. Population analysis (36) and paired or unpaired Student's t-tests were used as required by the experimental design (35). P < 0.05 was taken as statistically significant. ![]() |
RESULTS |
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MDR mRNA is Expressed in NCI-H295 Cells
Figure 1 shows the results of RT-PCR experiments designed to demonstrate the presence of MDR mRNA in NCI-H295 cells. RT-PCR products stained with ethidium bromide were observed in the lanes corresponding to MDR1-specific amplification of mRNA from the positive-control MDR1-transfected breast cancer cells BC19/3 and from the human adrenal cancer cells NCI-H295 (two different cultures H295-A, H295-B). The detected products were of the expected 523 bp size. No RT-PCR product (expected size 771 bp) was amplified when MDR2-specific primers were used. GAPDH, which was used as an internal control for mRNA integrity, was amplified in all lanes (600-bp RT-PCR product). In the negative-control MCF7 cells (multidrug-sensitive breast cancer cells expressing no detectable PGPs), multidrug resistance-derived PCR products were absent. Thus the only multidrug resistance mRNA isoform expressed in NCI-H295 cells was MDR1. The expression of MDR1 mRNA in NCI-H295 cells, while clearly detectable, was lower than in the MDR1-transfected cells.
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MDR1 PGP Is Expressed in Living NCI-H295 Cells
MDR1 PGP expressed in living NCI-H295 cells was detected using MRK16, a monoclonal antibody specific for an extracellular epitope of the MDR1 isoform of human PGPs. NCI-H295 cells were incubated in the presence of MDR1 antisense oligonucleotide in an attempt to downregulate the production of PGP and thus its membrane insertion. In parallel, control cells were treated with MDR1 sense oligonucleotide. Figure 2A, left, depicts specific fluorescence localized at the plasma membrane of NCI-H295 cells treated with sense oligonucleotide, thus demonstrating the presence of MDR1 PGP in these cells. It can be appreciated that the fluorescence is not homogeneous, indicating that cells from the same culture express different levels of PGP. A decrease in membrane fluorescence (by ~55% of that measured in sense oligonucleotide-treated cells) was noted in cells treated with antisense oligonucleotide, as demonstrated in Fig. 2A, right, thus indicating a decrease in membrane PGP. The fluorescence in membranes of cells treated with sense oligonucleotide was 130 ± 5 (in arbitrary units), while the fluorescence in membranes of cells treated with antisense oligonucleotide was 59 ± 3 (P < 0.0001). The decrease in membrane fluorescence measured after incubation of cells with MDR1 antisense oligonucleotide constitutes further evidence of the expression of MDR1 PGP in NCI-H295 cells.
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To determine the relative levels of PGP in living MCF7, NCI-H295, and BC19/3 cells, each cell type was immunolabeled with the monoclonal antibody FITC-4E3, which recognizes an external epitope of MDR1 PGP, and was analyzed by flow cytometry. MCF7 cells exhibited minimal fluorescence (mean 7.1 ± 0.8 arbitrary units, see Fig. 2B), not different from the fluorescence of untagged cells or cells tagged with the isotype-matched negative-control antibody (data not shown). By comparison, cells expressing PGP exhibited higher levels of fluorescence. As expected, BC19/3 cells exhibited the highest fluorescence and NCI-H295 cells exhibited fluorescence at an intermediate level (903.0 ± 7.9 and 30.1 ± 0.8 arbitrary units, respectively).
ANG II Stimulates Aldosterone Secretion by NCI-H295 Cells
Figure 3 shows the results of experiments designed to demonstrate the secretion of aldosterone by NCI-H295 cells and to determine the effect of the MDR1 PGP blockers verapamil, CSA, PSC-833, and vinblastine on aldosterone secretion. In preliminary experiments cell growth was evaluated by measurement of the metabolic activity using the colorimetric dye Alamar blue (Almar Biosciences) (2, 8) to ascertain that the drugs used in this study had no inhibitory effect on NCI-H295 cell growth. Over the 24-h incubation period, 10 µM of verapamil, CSA, PSC-833, or vinblastine did not modify NCI-H295 metabolic activity (data not shown). Levels of aldosterone secreted into the culture medium were measured under control conditions as well as in the presence of ANG II. The baseline secretion of aldosterone was very low, ranging from undetectable to 0.2 pmol · mg protein
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Aldosterone Inhibits Transport of Other PGP Substrates
We have previously demonstrated that human mesangial cells express PGP, that the isoform expressed is MDR1, and that it transports xenobiotics (2). We assessed the function of PGP expressed in human mesangial cells in culture from the efflux of R123 (see Fig. 4A). Under control conditions, the rate constant of R123 efflux was 0.984 ± 0.004 min
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Downregulation of PGP by Antisense Oligonucleotide Leads to a Reduction of R123 Efflux in NCI-H295 Cells
The efflux of the fluorescent substrate R123 was measured in NCI-H295 cells loaded for 1 h, and the rate constant was calculated. The rate constant was 2.071 ± 0.151 minFigure 5 depicts an example of R123 efflux
under control conditions and after cells were incubated with MDR1 sense
or antisense oligonucleotide.
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Blockers of PGP Reduce the Efflux of FL-Aldosterone Microinjected Into NCI-H295 Cells
Preliminary experiments were performed to compare the load and efflux rates of [3H]aldosterone and FL-aldosterone. Both molecules entered the cells, but the loading was low. The effluxes of [3H]aldosterone and FL-aldosterone were completed in 90 and 60 s, respectively. The small influx and rapid efflux prevented meaningful kinetic studies. Nevertheless, these experiments demonstrate that fluorescent-tagged aldosterone and [3H]aldosterone have similar loading and efflux characteristics, allowing for the study of the efflux of FL-aldosterone in cells loaded by microinjection. NCI-H295 cells were microinjected with fluorescent aldosterone as described in MATERIALS AND METHODS, and the rate constant of efflux was calculated from the decay of intracellular fluorescence. Figure 6A shows the efflux of FL-aldosterone microinjected into NCI-H295 cells in the presence or absence of the inhibitors verapamil and CSA. In the absence of inhibitor, the rate constant of efflux of FL-aldosterone was 0.112 ± 0.014 min
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The efflux of microinjected R123 was used as a control of the quality
of FL-aldosterone injections into NCI-H295 cells in the absence and the
presence of verapamil (see Fig. 6B). Under control conditions,
the rate constant of the efflux of R123 was 0.096 ± 0.013 min1 (n = 12). In the presence of verapamil,
the rate constant was 0.043 ± 0.011 min
1,
(n = 7, P = 0.025). There was no significant difference
between the efflux of R123 and FL-aldosterone under control conditions. These experiments indicate that FL-aldosterone and R123 have similar efflux rates and that both fluxes are partially inhibitable by PGP
blockers in NCI-H295 cells.
The PGP Inhibitory Antibody UIC2 Lowers FL-Aldosterone Efflux From NCI-H295 Cells
The effects of UIC2, a MDR1-specific, nontoxic, inhibitory antibody (22), and of the isotypic noninhibitory antibody UPC10 were studied in cells microinjected with FL-aldosterone (see Fig. 6C).The rate constant of FL-aldosterone efflux was 0.112 ± 0.014 min1 under control conditions and remained
unchanged (0.114 ± 0.012 min
1; n = 20, P = NS, experimental vs. control) in the presence of the
isotypic UPC10 monoclonal antibody used as a control antibody (see Fig.
6C). In the presence of UIC2, the FL-aldosterone efflux rate
constant was decreased to 0.072 ± 0.007 min
1 (n = 18, P = 0.015).
The UIC2 antibody also decreased the efflux of R123 microinjected into
NCI-H295 cells. The rate constant of R123 efflux in the presence of the
antibody was 0.017 ± 0.008 min1 (n = 9)
compared with rate constant of 0.093 ± 0.016 min
1
(n = 12) for cells microinjected with R123 in the absence of the inhibitory antibody (P = 0.0015). In cells
microinjected with R123 in the presence of UPC10 (non inhibitory
antibody), the rate constant was 0.168 ± 0.007 min
1, a value similar to the rate constant in
control cells in the absence of antibody. The inhibition by UIC2
antibody was smaller for FL-aldosterone than for R123, albeit the
control efflux time was similar. This indicates that, most likely, MDR1
does play a role in aldosterone secretion but that it is only
responsible for part of the efflux, with another mechanism operating in
parallel (see DISCUSSION).
Efflux of FL-Aldosterone From MCF7, MCF-7/VP, and BC19/3 Cells
The efflux of microinjected FL-aldosterone from MCF7 cells (the parental cell line that does not express PGP) and from BC19/3 cells (MCF7 cells transfected with MDR1) was also studied (see Fig. 7). As expected, the efflux of FL-aldosterone was higher in BC19/3 cells (expressing a high level of PGP, see also Fig. 4B) with a rate constant k of 0.532 ± 0.039 (n = 9) that was reduced by verapamil (20 µM) to 0.268 ± 0.087 (n = 12, P < 0.03). In MCF7 cells, k was 0.059 ± 0.001 (n = 17) and 0.050 ± 0.002 (n = 14) without and with verapamil (20 µM) in the bath, respectively. The rate constant in MCF7 cells was significantly lower than in BC19/3 cells (P < 0.0001). This experiment demonstrates that the transfection of cells with MDR1 cDNA elicits the capacity to transport FL-aldosterone from the cytosol to the extracellular medium.
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To rule out the participation of other multidrug transporters in the
efflux of FL-aldosterone, MCF-7/VP cells (expressing MRP but not PGP)
were microinjected as described for NCI-295 cells, and the efflux of
FL-aldosterone was measured following the same protocol. The rate
constant of efflux of FL-aldosterone from MCF-7/VP cells was 0.037 ± 0.001 min1 (n = 40). This rate constant is
significantly smaller than the rate constant of FL-aldosterone measured
in NCI-H295 cells, expressing PGP but not MRP (0.112 ± 0.014 min
1, P < 0.0001). It is comparable to the
rate constant of efflux of FL-aldosterone measured in MCF7 cells, and
in NCI-H295 cells in the presence of PGP inhibitors.
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DISCUSSION |
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PGP mediation of aldosterone secretion was suggested by the demonstration by Ueda et al. (32) that kidney cells transfected with MDR1 actively transport aldosterone from the basolateral to the apical side, together with the demonstration of MDR1 expression in the zona glomerulosa of the adrenal gland (25, 29). Aldosterone is a hydrophobic small molecule and its efflux from the adrenal cells was expected to occur by simple diffusion. Hypothetically, the presence in parallel of an active, presumably high-affinity secretory mechanism would result in rapid aldosterone export, even at very low intracellular concentrations. This would keep the intracellular concentration of the hormone low, facilitating (theoretically) its continuous synthesis.
In most of the experiments we used NCI-H295 cells, a human adrenocarcinoma cell line that expresses the major pathways of adrenal steroidogenesis (10) and in which ANG II stimulates aldosterone secretion (14). We confirmed that these cells have ANG II-stimulated aldosterone secretion in our culture conditions. In addition, we demonstrated that they express MDR1 PGP on the plasma membrane, that PGP transports xenobiotics, and that this process is predictably inhibited by PGP substrates and modulators. Furthermore, the efflux of the PGP substrate R123 decreased following preincubation with MDR1 antisense oligonucleotide, a correlation that has been demonstrated in other cells (1). For these reasons NCI-H295 cells are a good model to study the role of PGP in aldosterone secretion.
Aldosterone secretion was inhibited by the PGP substrates CSA, PSC-833 (a nonimmunosuppressive CSA analog), and vinblastine. These observations, with three agents of very different chemical structures and cellular targets, suggest that their effects on aldosterone secretion are exerted via PGP and not by other mechanisms. Similar experimental approaches are used to establish whether or not a given drug is transported by PGP (18). The lack of a specific inhibitor of PGP transport mandates the use of several inhibitors, which exhibit other cellular effects in addition to blocking PGP-mediated transport.2 This is particularly relevant to the experiments reported here because verapamil is a Ca2+ channel blocker, and ANG II-stimulated aldosterone secretion is mediated by increases in intracellular Ca2+ that, at least in part, are sensitive to removal of external Ca2+ or to Ca2+ channel blockers (4, 5). Thus a decrease in aldosterone secretion with verapamil could result from diminished aldosterone production, instead of from inhibition of PGP-mediated transport. In the experiments reported here, verapamil weakly decreased aldosterone secretion, an effect that could be due to inhibition of production instead of secretion. On the other hand, CSA has been reported to increase cytosolic Ca2+ by increasing Ca2+ influx in renal cells (15, 19), and if the same effect occurs in NCI-H295 cells, we would expect an increase in aldosterone production. In contrast, the ANG II-dependent secretion of aldosterone was decreased by CSA, suggesting an effect on PGP-mediated secretion. In addition, two other modulators of PGP also decreased aldosterone secretion. Finally, verapamil and CSA were also blockers of the efflux of microinjected FL-aldosterone, an experimental condition in which aldosterone production is bypassed. Although individually these experiments are inconclusive, together they suggest that the transport of aldosterone occurs via PGP.
The most compelling evidence for a role of PGP in aldosterone transport is provided by the FL-aldosterone microinjection experiments. These experiments demonstrate inhibition of efflux by PGP blockers. It can be argued that the molecule modification, resulting from incorporation of the fluorophore, may have altered its transport properties. However, we found that incubation of NCI-H295 cells in medium containing FL-aldosterone resulted in cell loading and efflux, which were not different from those observed with [3H]aldosterone (data not shown). Because influx cannot be via PGP, because the pump only extrudes lipophilic substrates, it is likely that it occurs entirely by diffusion. The magnitude of the load in both cases was small enough to prevent kinetic studies, so we resorted to intracellular microinjections.
The comparison between the effluxes of microinjected FL-aldosterone and R123 revealed similar rate constants, indicating rapid extrusion of both molecules under control conditions. R123 is a good substrate for PGP and a poor one for the MRP pump. In addition we were unable to demonstrate, by immunofluorescence, the presence of MRP in NCI-H295 cells, and the efflux of microinjected FL-aldosterone from cells expressing MRP but not PGP (MCF-7/VP) was significantly slower than in cells expressing PGP. Cells that do not express PGP (MCF7) have very slow efflux of FL-aldosterone, but after transfection with MDR1 (BC19/3 cells) they exhibit a 10-fold increase in the rate constant for the efflux of FL-aldosterone. Finally, the MDR1-specific blocking antibody UIC2, a good blocker of R123 efflux, significantly inhibited FL-aldosterone efflux, suggesting that, under these experimental conditions, PGP is responsible for at least a fraction of the FL-aldosterone efflux.
Our results on MCF7 and BC19/3 cells are in agreement with the experiments by Ueda et al. (32), who demonstrated that LLC-PK1 epithelial cells transfected with the human MDR1 cDNA from normal adrenal gland acquired multidrug resistance and the ability to translocate aldosterone from the basolateral to the apical side. The interaction between multidrug resistance proteins and steroids has been studied in several cell lines, and some experiments suggested that neither the corticosteroids dexamethasone and hydrocortisone nor aldosterone are substrates of PGP (9, 20, 33). However, the experiments conducted by Fojo et al. (9) were performed in cell lines expressing a mutated PGP that has been reported to modify both multidrug efflux and photoaffinity binding of PGP substrates (17, 31). The negative results from van Kalken et al. (33) could be due to differences in PGP structure or in the experimental methodology. These authors did not demonstrate cell loading with the polar steroids progesterone and aldosterone in either drug-sensitive or drug-resistant cells; in the present studies we had to resort to intracellular microinjections for the substrate-efflux experiments. As quoted in the Introduction, evidence obtained by Vo and Gruol (34) in mice PGP supports the notion that steroid transport depends on both the chemical structure of the substrate and the primary structure of PGP.
In summary, we have demonstrated that aldosterone and FL-aldosterone are substrates of PGP, a membrane protein expressed in the adrenal cell line NCI-H295 as well as in other cells. These findings suggest that PGP participates in aldosterone secretion in cells from the zona glomerulosa of the adrenal gland.
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ACKNOWLEDGEMENTS |
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We are indebted to Dr. L. Reuss for valuable comments during the preparation of this manuscript. We also thank Dr. Eugene B. Mechetner for donation of the UIC2 monoclonal antibody and K. L. Ives for the technical assistance with microinjections. The technical help of R. Zabriskie and the secretarial help of Y. Trevino are gratefully acknowledged.
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FOOTNOTES |
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This work was supported in part by American Heart Association Grant 96G-643 and institutional funds from the Department of Internal Medicine, University of Texas Medical Branch, Galveston, TX.
Present addresses: S. Ernest, Institut Pasteur, Unité de Génétique des Déficits Sensoriels, CNRS URA 1968, 25 rue du Dr. Roux, 75724 Paris Cedex 15, France; B. Holland, Central Texas Medical Foundation, 601 East 15th St., Austin, TX 78701.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
1
Baseline aldosterone levels in cells cultured
with HyQCCM-1 serum free medium are lower that the ones measured in the
same laboratory using cells grown in enriched medium or medium
containing progesterone (Ref. 14 and our unpublished observations).
Nevertheless, the highest levels obtained after ANG II stimulation in
the cells grown in HyQCCM-1 medium are close to the mean increases in
aldosterone detected after stimulation with 1010 M
ANG II in cells growing in enriched medium.
2 Verapamil does not modify the influx or efflux of NH+4/NH3 in NCI-H295 cells. The intracellular pH changes during NH4Cl exposure and removal were the same in the absence and in the presence of verapamil.
Address for reprint requests and other correspondence: E. Bello-Reuss, Dept. of Internal Medicine, Div. of Nephrology, Univ. of Texas Medical Branch, 4.200 John Sealy Annex, 301 University Blvd., Galveston, TX 77555-0562.
Received 30 March 1999; accepted in final form 12 January 2000.
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