Department of Pharmacology and Toxicology, Medical College of Wisconsin, Milwaukee, Wisconsin 53226
Submitted 26 April 2004 ; accepted in final form 11 June 2004
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ABSTRACT |
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surface-enhanced Raman scattering
Inhibition of nitric oxide (NO) synthase with nitro-L-arginine methyl ester (L-NAME) resulted in an increase in cellular expression of ANG II receptors in adrenal glands and smooth muscle cells (3, 16, 19, 34, 38). Administration of L-NAME to rats increased plasma aldosterone and AT1 receptor mRNA and protein in adrenal homogenates (38). Chronic administration of L-NAME to normal rats also increased cardiac AT1 and AT2 receptor expression (19). Stimulation of vascular smooth muscle cells with S-nitroso-N-acetyl-DL-penicillamine, an NO donor, significantly decreased the receptor density (Bmax) but did not affect the affinity (Kd) of ANG II (3). Chronic treatment of rat vascular smooth muscle cells with NO decreased the binding of ANG II through a guanylyl cyclase/cGMP-independent mechanism. It is not known whether NO affects the expression and localization of ANG II receptors in ZG cells. We (9, 10) previously showed that acute treatment with NO inhibited the ANG II-, 25-hydroxycholesterol-, and pregnenolone-stimulated aldosterone synthesis in ZG cells, and this mechanism is guanylyl cyclase/cGMP independent. NO inhibited aldosterone biosynthesis by binding steroidogenic enzymes. The sensitivity to NO inhibition was increased at low oxygen concentrations (9). These observations led us to examine the effects of chronic exposure of ZG cells to NO on the expression of AT1 receptors and aldosterone synthesis.
Adrenal endothelial cells contain NO synthase and produce NO, whereas ZG cells do not (9). Thus endothelial cells are the major source of NO in the adrenal cortex. The adrenal gland is highly vascularized, so ZG cells are in close proximity to capillaries and endothelial cells (39). Thus ZG cells are chronically exposed to endothelial NO. ANG II stimulates endothelial NO release (11). In the presence of endothelial cells, ZG cells release less aldosterone in response to ANG II compared with ZG cells alone. This reduced aldosterone release can be reversed by NO synthase inhibitors. These studies indicate that endothelial NO can regulate aldosterone release from ZG cells.
In this study, the effects of chronic NO administration on the expression and localization of AT1 receptors and on aldosterone synthesis were investigated in cultured bovine adrenal ZG cells. AT1 receptor-overexpressing Chinese hamster ovary (CHO) cells were used as a positive control. Western blot analysis was used to determine the expression of AT1 receptors in ZG cells undergoing NO treatment at various times. The newly developed technique of confocal Raman microspectroscopy, using Raman labels and silver colloidal surface-enhanced Raman scattering (SERS) (26), and immunocytochemistry were used to detect AT1 receptors in single ZG cells. Radioligand binding was used to determine the Kd and Bmax of ANG II binding to the receptors. These multiple approaches all showed that chronic NO administration reduced AT1 receptor expression.
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MATERIALS AND METHODS |
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Culture of bovine adrenal ZG cells.
Bovine adrenal ZG cells were cultured as previously described (9, 10, 30, 31). Briefly, small bovine adrenal glands were obtained from a local abattoir and stored on ice before dissection. Glands were trimmed of fat and dipped sequentially in 70% ethanol and Earle's buffer containing 2% penicillin-streptomycin solution. Glands were then bisected and sectioned on a Stadie-Riggs microtome. Sections were digested in 50 mM HEPES buffer, pH 7.4, in the presence of a mixture of collagenase, dispase, hyluronidase, deoxyribonuclease, and 1 mg/ml BSA for 1 h at 37°C. Sections were then dispersed by repeated pipetting through a wide-bore 10-ml pipette. Suspended cells were pelleted by centrifugation and rinsed with Earle's buffer. Sections were then returned to the digestion solution for a total of two digestion cycles. Isolated cells were plated in a growth medium consisting of Ham's F-12 medium supplemented with 140 mM NaCl, 14 mM NaHCO3, 10% fetal bovine serum, 50 µM butylated hydroxyanisole, 1.2 µM -tocopherol, 100 µM sodium ascorbate, 50 nM sodium selenite, 0.15 µM glutathione, 20 nM insulin, 10 µg/ml transferrin, 5 µM metyrapone, 200 U/ml penicillin, 200 µg/ml streptomycin, 3 U/ml nystatin, 30 µg/ml gentamicin, and 2.5 µg/ml fungizone. Cells were plated in six-well plates at a density of 87,500 cells/well and maintained at 37°C in a humidified atmosphere of 5% CO2 in air. The medium was replaced with fresh growth medium containing 2% fetal bovine serum every 24 h. The cells were maintained in culture for 35 days and used at
50% confluence. The purity of ZG cells was determined by morphology as previously described and averaged 95% (29).
Culture of CHO cells. CHO cells, stably transfected with the AT1B receptor (27), were fed daily with Ham's F-12 medium supplemented with 10% fetal calf serum and geneticin (G418; 250 mg/l) as previously described (8). After treatment with DETA NONOate (104 M) for 0, 12, 24, 48, 72, and 96 h (DETA NONOate was replaced daily), cells were washed three times with 25 mM HEPES buffer, pH 7.4, containing (in mM) 150 NaCl, 5 KCl, 1.8 CaCl2, 1 MgCl2, and 6 glucose. Then, 0.5 ml of lysis buffer containing 50 mM HEPES, 150 mM NaCl, 1.5 mM MgCl2, 1 mM EGTA, 10% glycerol, 1% Triton X-100, and Complete protease inhibitor mixture was added to the flask and incubated at 20°C for 10 min. After the incubation, the cells were scraped and run five times through a 22-gauge needle. The lysates were then aliquotted and frozen at 80°C. The protein concentration was determined by Bio-Rad protein assay kit.
Western blot analysis of AT1 receptors in CHO cells.
Protein (10 µg) was added to an equal volume of 5x sample buffer (containing 4.8 ml of glycerol, 2.4 ml of -mercaptoethanol, 9.6 ml of 10% SDS, 1.2 ml of 0.1% bromophenol blue, 6.0 ml of 0.5 M Tris·HCl buffer, pH 6.8, and 24 ml of H2O). Samples were heated at 65 or 95°C for 5 min, cooled on ice, and loaded onto the gel. These two temperatures for protein denaturing gave identical results for AT1 receptors on Western blot analysis. The proteins were separated electrophoretically on a Bio-Rad 10% polyacrylamide Redi-Gel at 150 V for 1 h. The separated protein bands were transferred to a nitrocellulose membrane with 100 V for 1 h. The membranes were incubated in Tris-buffered saline (TBS) containing 0.1% Tween-20 and 5% milk at room temperature for 1 h.
The membrane was washed five times with cold TBS and then incubated overnight at 4°C with a 1:1,000 dilution of rabbit polyclonal anti-AT1 receptor antibody in TBS blocking solution. Anti-AT1 receptor antibodies from Santa Cruz and Alpha Diagnostic International were used and gave identical results. Then, the membrane was washed twice with cold TBS containing 0.1% Tween-20 and three more times with cold TBS. Goat anti-rabbit IgG-horseradish peroxidase-conjugated secondary antibody at 1:5,000 in TBS containing 5% nonfat dry milk was added and incubated at room temperature for 1 h. The membrane was again washed twice with cold TBS containing 0.1% Tween-20 and three more times with cold TBS.
The immunoreactive bands were detected by chemiluminescence. The membranes were incubated with 4 ml of ECL solution for 2 min, the excess solution was discarded, and the membranes were wrapped in a plastic wrap. The membranes were exposed to Kodak Biomax-ML film for 515 s, and the photographic films were developed in an automated film processor.
Western blot analysis of AT1 receptors and cholesterol side-chain cleavage enzyme in bovine adrenal ZG cells.
ZG cells were grown in 60-mm dishes at 200,000 cells per dish and incubated with DETA NONOate (104 M) for 0, 12, 24, 48, 72, and 96 h (DETA NONOate was replaced daily). DETA NONOate was chosen as the NO donor for these chronic studies because of its long, 56-h half-life (15). This assures the consistent release of a low concentration of NO over the duration of the study. Furthermore, DETA NONOate was replaced daily in these treatments. This concentration of DETA NONOate gave an optimal inhibition of ANG II-induced aldosterone synthesis in ZG cells acutely treated with DETA NONOate (11). In preliminary studies, ZG cells were treated with various concentrations of DETA NONOate (107 to 103 M) for 96 h. The 103 M concentration was toxic to the cells; lower concentrations did not affect ZG cell viability. Maximal inhibition of AT1 receptor expression and maximal inhibition of ANG II-induced aldosterone release occurred with 104 M DETA NONOate. Thus this concentration was used for subsequent studies. DETA NONOate at 1 mM gives a steady-state concentration of NO of 40 nM (10), and the steady-state release of NO from bovine aortic endothelial cells was 12.7 ± 0.7 nM (41). Thus the concentration used in this study (104 M) is in the range of NO released by endothelial cells. Control cells were incubated in the absence of DETA NONOate. Cells were washed three times with 25 mM HEPES buffer. Then, 0.5 ml of lysis buffer was added to the flask and kept on ice for 5 min. Lysed cells were scraped, transferred to a 1.5-ml centrifuge tube, and centrifuged at 15,000 g at 4°C for 15 min. Supernatant was immediately frozen and stored at 80°C until analysis. The protein concentration was determined by Bio-Rad protein assay kit. For AT1 receptors, the Western blot protocol is the same as for AT1 receptors in CHO cells described above.
For cholesterol side-chain cleavage, the membrane was washed as above and then incubated overnight at 4°C with a 1:1,000 dilution of rabbit polyclonal anti-cholesterol side-chain cleavage antibody in TBS blocking solution. Then, the membrane was washed twice with cold TBS containing 0.1% Tween-20 and three more times with cold TBS. Goat anti-rabbit IgG-horseradish peroxidase-conjugated secondary antibody at 1:5,000 in TBS containing 5% nonfat dry milk was added and incubated at room temperature for 1 h. The membrane was again washed twice with cold TBS containing 0.1% Tween-20 and three more times with cold TBS. The immunoreactive bands were detected by chemiluminescence as described above.
ANG II radioligand-binding assay. A previous reported radioligand-binding assay (40) was used with slight modifications. ZG cells at 2.0 x 106 cells were grown in a T225-cm2 flask in the absence or presence of DETA NONOate (104 M) for 0 and 96 h. DETA NONOate was replaced daily. ZG cells were washed three times with PBS buffer, pH 7.4. Then, 2 ml of 20 mM NaHCO3, pH 7.4, were added to lyse the cells. The cells were then scraped into a 15-ml tube and immediately placed on ice. The cells were homogenized using a Polytron. Cellular homogenate was centrifuged at 600 g at 4°C for 15 min. Supernatant was decanted into a centrifuge tube and centrifuged at 30,000 g at 4°C for 15 min to separate the nuclear and plasma membrane fractions from the ZG cell homogenate. The supernatant was discarded, and the membrane fraction was then suspended in binding buffer consisting of 50 mM Tris·HCl buffer, pH 7.4, 125 mM NaCl, 6.5 mM MgCl2, 1 mM EDTA, 2 mg/ml BSA, and complete protease inhibitor mixture. The membrane fraction was then again homogenized. The protein content in the membrane fraction was determined using the Bradford protein assay in the absence of BSA in the buffer. The binding volume of 250 µl consisted of 200 µl of the membrane fraction in binding buffer, 25 µl of 0.15 nM of 125I-labeled ANG II, and 25 µl of ANG II at various concentrations. The solution was incubated at room temperature for 60 min. Then, 2 ml of ice-cold PBS were added to each tube. Bound and free ligands were separated by filtering through a no. 2 glass filter by use of the Brandel M-24 R cell filter unit. Nonspecific binding was determined in the presence of 106 M ANG II. Bmax and Kd were determined by plotting the total ANG II vs. specifically bound ANG II, which was converted to a Scatchard plot with the Prism software program. The results were averaged from four different experiments of six concentrations of ANG II (with triplicate samples at each concentration).
Immunofluorescence detection of AT1 receptors in bovine adrenal ZG cells.
Cells were grown in 12-well plates to 40% confluence, treated daily with or without DETA NONOate (104 M) for 0, 24, and 96 h, and washed three times with phosphate-buffered saline (PBS), pH 7.4. Then, they were fixed with ice-cold methanol-ethanol (1:1) for 15 min at 4°C. Cells were again washed three times with ice-cold PBS and stored at 4°C. These fixed cells were ready for use for detection of AT1 receptors.
Fixed ZG cells were incubated with 5% normal goat serum in PBS buffer containing 0.05% Pluronic F-127 for 30 min at room temperature. Cells were washed once with ice-cold PBS and then incubated for 30 min with a 1:1,000 dilution of rabbit polyclonal anti-AT1 receptor primary antibody in PBS buffer containing 0.05% Pluronic F-127. Cells were washed again with PBS and incubated with a 1:500 dilution of Alexa-594-goat anti-rabbit IgG secondary antibody in PBS buffer containing 0.05% Pluronic F-127 for 30 min at room temperature. Cells were washed three times with ice-cold PBS buffer and kept on ice until detection. Control cells (or blank cells) were incubated the same way in the absence of primary antibody. The fluorescence in the cells was detected with a fluorescence microscope with excitation/emission wavelengths of 590/615 nm. The images were captured with a fluorescence microscope (Nikon Eclipse E600, Diagnostics Instruments).
Expression and localization of AT1 receptors by confocal Raman microspectroscopy. Expression and localization of AT1 receptors in ZG cells by confocal Raman microspectroscopy was previously reported (26). Briefly, fixed ZG cells were incubated with 5% normal goat serum, washed three times with ice-cold PBS buffer, and then incubated for 30 min with a 1:500 dilution of rabbit polyclonal anti-AT1 receptor antibody in PBS buffer containing 0.05% Pluronic F-127. Cells were again washed with PBS buffer once and incubated with a 1:500 dilution of biotinylated goat anti-rabbit in PBS buffer containing 0.05% Pluronic F-127 for 30 min at room temperature. Cells were washed and incubated with a 1:1,000 dilution of Neutravidin in PBS buffer containing 0.05% Pluronic F-127 for 15 min at room temperature. Then, a 1:500 dilution of biotin-CV in PBS buffer containing 0.05% Pluronic F-127 was added and incubated for 15 min. Cells were washed three times with ice-cold PBS buffer and readied for detection. Control cells were treated the same way but in the absence of primary AT1 antibody.
For SERS detection of AT1 receptors, a cold, diluted silver colloidal solution of 448 µl was aggregated by adding 22 µl of 1 M NaCl and mixing thoroughly by inversion. PBS buffer was aspirated from the wells, and then the aggregated colloids were added to the wells. The most consistent and reproducible spectra were obtained between 3 and 30 min after aggregation. The culture plate was then placed on the microscope stage, and single cells were visualized on a video monitor. SERS was measured by the Explorer Series 1 Raman spectrometer (JYHoriba, Edison, NJ) with an Olympus BH-2 microscope and x80 (NA 0.75) objective. The 12-mW HeNe laser with an exciting light at 632.8 nm was used. Rayleigh scatter was eliminated with a holographic notch filter (Kaiser Optical Systems, Ann Arbor, MI). Spatial resolution of the Raman scatter from the object was obtained by use of a 200-µm confocal pinhole. Spectral resolution of the Raman scatter of 6 cm1 was obtained using a 600 grooves/mm grating. Raman scatter was detected by use of a thermoelectrically cooled 1,024 x 256-pixel charge-coupled device camera. Peak frequencies were calibrated with the silicon phonon line at 520 cm1. Spectral data were visualized on a microcomputer and the Spectramax version 1.1d software program.
SERS spectra were acquired for 10 s each, and spectra were stored on the computer. For detection of spectra in z plane, the microscope stage was moved up or down in 2-µm increments while its x and y planes were maintained. This vertical movement of the stage places the laser focus at many different levels in the z plane. For the stage movement in the x or y direction, the movement was made in 2-µm increments while the other two planes were kept constant.
Measurement of aldosterone synthesis in bovine adrenal ZG cells. ZG cells were treated with DETA NONOate (104 M) for 96 h in the incubator (DETA NONOate was replaced daily). Then, ZG cells were washed twice with 0.5 ml of SM1 buffer, consisting of Ham's F-12 medium, 140 mM NaCl, 14 mM NaHCO3, and 1 mg/ml BSA. Cells were then incubated with SM1 buffer for 2 h at 37°C, and the buffer was removed and replaced with 0.5 ml of SM2 buffer, consisting of Ham's F-12 medium, 140 mM NaCl, 14 mM NaHCO3, 1.8 mM CaCl2 and 2 mg/ml BSA. Then, ANG II or 25-hydroxycholesterol at various concentrations was added and incubated for 1 h at 37°C. The incubation was stopped by transferring the SM2 buffer to plastic tubes and freezing at 40°C for subsequent analysis of aldosterone. It should be emphasized that no DETA NONOate was added to the SM1 washes or SM2 incubation. Thus the effects of chronic administration of NO, not acute NO, are studied.
Aldosterone was measured by enzyme-linked immunosorbant assay (ELISA) using a mouse anti-aldosterone monoclonal primary antibody, aldosterone-horseradish peroxidase conjugate and a goat anti-mouse, Fc fragment specific secondary antibody (9, 10). ELISA 96-well plates were precoated with the secondary antibody by incubating 300 µl of 3.3. µg/ml of goat anti-mouse IgG in 0.1 M Na2CO3, pH 9.6, for 18 h at 4 °C. The plates were then washed 3 times with 300 µl of washing buffer containing 135 mM NaCl, 20 mM NaH2PO3, 0.01% thimerosal, and 0.2% Tween 80 (wash buffer) using Biotek automatic plate washer (Model EL402, Winooski, VT). Coated ELISA plates were stored in 10 mM PBS containing 138 mM NaCl and 2.7 nM KCl at 4 °C until used. The aldosterone-horseradish peroxidase conjugate and anti-aldosterone antibody were each diluted 1:6,000 in the assay buffer containing 150 mM NaCl, 100 mM NaH2PO4, 0.1% Tween 80, 0.01% thimerosal, and 0.5% bovine serum albumin. The assay buffer (250 µl) was added to 50 µl of the standard or sample in each well. The assay was then allowed to equilibrate overnight at 4 °C. The plates were washed 6 times with 300 µl/well with wash buffer on an automatic plate washer with a 1-min agitation on an orbital shaker after the third wash. The assay was developed by the addition of 0.01% urea peroxide in 100 mM citric acid and 40 mM 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) as a color reagent. Aldosterone was quantified by colorimetric measurement using a Biotek Model EL309 automated plate reader with a 490-nm filter. ACTH-stimulated aldosterone synthesis was included as a positive control in each experiment.
Statistical analysis. All values are expressed as means ± SE. The significance of difference among groups was calculated by InSat 3 software (GraphPad Software, San Diego, CA) using unpaired t-test for two-tailed P value. P < 0.05 is considered significantly different.
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RESULTS |
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ANG II receptor binding in chronic NO-treated ZG cells. The effect of chronic treatment of ZG cells with NO on the binding of 125I-ANG II to its receptors was determined by radioligand-binding assay (Fig. 2). ANG II demonstrated saturatable, specific binding in the control and DETA NONOate-treated ZG cells. ZG cells treated with DETA NONOate (104 M) for 96 h resulted in a decrease in Bmax (1,864 ± 129 vs. 3,157 ± 220 fmol/mg protein for control). However, Kd was not significantly different between DETA NONOate-treated and control ZG cells (1.95 ± 0.22 vs. 1.88 ± 0.21 nM). These results suggest that chronic NO treatment of ZG cells decreases the receptor number or downregulates the expression of AT receptors.
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DISCUSSION |
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The Western immunoblot analysis of the cell lysates of ZG cells treated with NO shows a significant decrease in the intensity of the immunoreactive bands of AT1 receptors at 24, 48, 72, and 96 h. The chronic NO treatment of ZG cells also resulted in a significant decrease in number of binding sites but not the affinity for the binding of ANG II to its receptors compared with control cells. These data suggest that the number of AT (AT1 and/or AT2) receptors decreased with exposure of the cells to NO. Confocal Raman microspectroscopy revealed that AT1 receptors were densely located on the plasma membranes and to a much lesser extent on the nuclear envelopes of the ZG cells. It has been shown that AT1 receptors are mainly on the plasma membranes and can translocate to the nucleus upon the binding of ANG II to the receptors (2, 36). Furthermore, a decrease in the SERS intensity at 592 cm1 indicates a marked decrease in the number of AT1 receptors after 96 h of treatment of ZG cells with NO. The decrease of AT1 receptors with NO administration to ZG cells was also observed with fluorescence immunocytochemistry. With a variety of methods, these studies indicate that chronic NO administration reduces AT1 receptors in ZG cells. In contrast, inhibition of NO synthesis with L-NAME increased AT1 but not AT2 receptors in rat adrenal glands (38). Not all receptors are susceptible to inhibition by NO. Treatment of endothelial cells with NO did not change bradykinin B2 receptor number or receptor protein expression (22).
ANG II-stimulated aldosterone release from ZG cells was inhibited by chronic NO exposure. In these studies, DETA NONOate was added to the ZG cells chronically but was not present in the wash buffer or the incubation buffer with ANG II. Thus these data represent the chronic, not acute, effect of NO administration. This decrease in the ANG II-stimulated aldosterone synthesis could be due to the decrease in the number of AT receptors. However, the stimulation of aldosterone synthesis by 25-hydroxycholesterol was also inhibited by chronic NO administration. Because 25-hydroxycholesterol bypasses the AT receptors and promotes aldosterone biosynthesis by acting directly as a precursor in aldosterone synthesis, chronic NO administration must inhibit aldosterone biosynthesis by another mechanism. The Western immunoblot of cholesterol side-chain cleavage enzyme, an initial step in aldosterone biosynthesis, indicates that the chronic NO administration downregulates the enzyme expression and may represent the mechanism for inhibition of 25-hydroxycholesterol-stimulated aldosterone synthesis. Thus, although acute NO binds to cholesterol side-chain cleavage enzyme and blocks the conversion of 25-hydroxycholesterol to pregnenolone (10), chronic NO administration decreases the expression of this key enzyme as well. This enzyme is critical in the synthesis of other steroid hormones, including cortisol, estrogen, testosterone, and progesterone (1, 21). This finding raises the possibility that chronic NO exposure regulates steroidogenesis in other endocrine tissues besides the adrenal cortex.
The mechanism(s) responsible for the reduction in AT1 receptors and cholesterol side-chain cleavage enzyme by chronic NO administration was not determined. Previous studies in vascular smooth muscle cells indicated that an increase in cGMP was not responsible for an NO reduction in AT receptors (3). This may involve a DNA-binding protein that binds the proximal promoter region of the AT1 receptor gene. In previous studies (10), we found that acutely administered NO increased cGMP even though this was not responsible for the inhibition of aldosterone release. It is not known whether chronic NO administration will maintain elevated concentration of cGMP. Furthermore, it is not known whether elevation in cellular cGMP will reduce the expression of cholesterol side-chain cleavage enzyme.
Inhibition of AT1 receptor and cholesterol side-chain cleavage enzyme expression by chronic exposure to NO may represent a negative feedback mechanism in hypertension. Elevated perfusion pressure and shear stress is a physiological stimulus to NO release from endothelial cells (13, 20, 37). In hypertension, ZG cells may be chronically exposed to endothelial NO, which would decrease aldosterone release by downregulating AT1 receptors and cholesterol side-chain cleavage enzyme. The reduction in aldosterone release would promote sodium excretion, reduce the extracellular volume, and reduce the blood pressure toward normal.
This study demonstrates the effects of chronic NO administration on the expressions of AT1 receptors and cholesterol side-chain cleavage enzyme and, hence, the inhibition of aldosterone synthesis from the ANG II-stimulated and 25-hydroxycholesterol-stimulated pathways, respectively. The results suggest that NO produced locally by the adrenal vascular endothelium can play an important role in the regulation of steroidogenesis in adrenal gland and may lower blood pressure from either direct vasodilation effect or inhibition of aldosterone synthesis.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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. Section 1734 solely to indicate this fact.
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REFERENCES |
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