NITRIC OXIDE: Biology and Chemistry Vol. 6, No. 2, pp. 153–159 (2002) doi:10.1006/niox.2001.0400, available online at http://www.idealibrary.com on
Nitric Oxide Gas Decreases Endothelin-1 mRNA in Cultured Pulmonary Artery Endothelial Cells Adrian P. L. Smith, 1 Eric A. G. Demoncheaux, and Tim W. Higenbottam 2 Section of Medicine and Pharmacology, Respiratory Medicine, Division of Clinical Sciences (South), School of Medicine and Biomedical Sciences, University of Sheffield, Sheffield, United Kingdom Received January 17, 2001, and in revised form August 16, 2001; published online December 12, 2001
Inhaled nitric oxide gas (iNO) vasodilates the pulmonary circulation. The effective “dose” of iNO for chronic treatment of pulmonary hypertension is unknown. Increased abundance of pulmonary mRNA for preproendothelin-1 (ppET-1) with its associated increase in endothelin-1 (ET-1) could contribute to the development of both clinical and experimental pulmonary hypertension. The benefit of iNO therapy may be from inhibition of ET-1 production. The present study was designed to compare the effects of two therapeutic concentrations of NO gas, 10 parts per million (p.p.m.) and 100 p.p.m. on the steady-state level of mRNA for ppET-1 and nitric oxide synthase (NOS III), in cultured bovine pulmonary artery endothelial cells. Uptake of NO gas was assessed by measurement of nitrite anions in the medium. The mRNA for ppET-1 and NOS III was determined by semiquantitative reverse-transcriptase polymerase chain reaction (RT-PCR). After 4 h exposure to 100 p.p.m. NO in air, nitrite anions levels were 1.6 M in the endothelial cell media as opposed to 0.23 M with 10 p.p.m. NO. The levels were 0.02 M in control cells exposed to air alone. Exposure to 100 p.p.m. NO reduced the steady state levels of mRNA for ppET-1, but not NOSIII mRNA levels. By comparison 10 p.p.m. NO did not affect levels of either mRNA. © 2001 Elsevier Science (USA) Key Words: endothelin-1; nitric oxide synthase; nitric oxide; endothelial cells; nitrite; von Willebrand factor; -actin; RT-PCR; chemiluminescence.
tension (PPH), chronic treatment with iNO may produce a sustained reduction in pulmonary vascular resistance (3) and long-term treatment is possible with pulsed delivery system where a fixed dose of iNO is delivered in each breath (4). However, unlike acute treatment where concentrations of 1 to 100 p.p.m. are effective, there is no consensus on the dose of iNO for chronic treatment. Some of the chronic therapeutic effects of iNO may result from the inhibition of the expression of preproendothelin-1 (ppET-1), the precursor of endothelin-1 (ET-1) as ET-1 expression appears elevated in all forms of primary and secondary pulmonary hypertension (5–7). Endothelin-1 is not only a powerful vasoconstrictor but is also a potent mitogen linked to remodelling of the pulmonary arteries in pulmonary hypertension (8). Nitric oxide donors are thought to inhibit the expression of preproendothelin-1 (9) and its own endogenous synthesis (10). We have questioned whether NO gas alters expression of endothelial nitric oxide synthase expression (NOS III) and ppET-1. We have compared the effects of a 4-h exposure to NO gas on the steady-state mRNA levels for ppET-1 and NOS III. Cultured bovine pulmonary endothelial cells were exposed to two concentrations, 10 and 100 p.p.m., of nitric oxide. EXPERIMENTAL PROCEDURES
Inhaled nitric oxide gas (iNO) 3 is a selective pulmonary vasodilator being used to treat neonatal pulmonary hypertension (1, 2). In primary pulmonary hyper1
Present address: Department of Molecular Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037. 2 To whom correspondence and reprint requests should be addressed. Fax: (44) (0)114 271 1711. E-mail:
[email protected]. 3 Abbreviations used: iNO, inhaled nitric oxide; PPH, primary pulmonary hypertension; ppET-1, preproendothelin-1; NOS III, endothelial nitric oxide synthase; vWF, von Willebrand factor. 1089-8603/01 $35.00 © 2001 Elsevier Science (USA) All rights reserved.
Materials All enzymes, molecular reagents, and cell culture materials were purchased from Gibco BRL (Paisley, UK) unless otherwise stated. Culture plastic-wares were obtained from Bibby Sterilin Ltd. (Stone, UK). General reagents, cytokines, and auto-radiographic materials were from Sigma Chemical Co. (Poole, UK). Fetal calf serum was bought as a reserved batch from Advanced Protein Products Ltd. (Brierly Hill, UK). Potassium iodide, potassium sulfate, sulfuric acid, and sodium nitrite were purchased from Aldricht (Gilling153
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Endothelial Cell Culture
FIG. 1. The culture chamber used to exposed endothelial cells to nitric oxide in air. Gas mixtures for control (as above) or containing NO (as above with 10 or 100 p.p.m.) were introduced from the source gas mixture by negative pressure. The gas was passed through water in an ante-chamber to humidify the gas before entering the culture chamber. At a stable plateau for NO levels in the box, the chamber was sealed.
ham, Dorset, UK). The 20% O 2 and 5% CO 2 gas mixture was obtained from Air Product (Basingstoke, UK). Certified low concentrations of NO gas in N 2 (9.9 and 100.0 p.p.m.) were purchased from BOC (Special Gases, Luton, UK). Exposure of Cultured Endothelial Cells to Nitric Oxide To expose the cells to NO gas we built a 4-liter volume perspex chamber (Fig. 1). In this chamber up to 20 culture flasks (T25 flasks) could be held at a controlled temperature of 37°C. All cultures were allowed to equilibrate for one hour under normoxic conditions (20% oxygen and 5% carbon dioxide, balanced with nitrogen). The control gas mixture as described above or the NO mixture was then drawn into the chamber at the rate of 750 ml/min using the inlet port of a commercially available NO analyzer (Model 42, Thermoelectron Ltd., Warrington, UK) through a water container to humidify the mixture from a Douglas bag (PK Morgan, Kent, UK). The contents of the chamber reached a steady gaseous concentration after approximately 20 min. The chamber was then sealed for 4 h. Nitric oxide, in concentrations of 10 or 100 p.p.m., was drawn into the chamber and its concentrations were determined at hourly intervals throughout the 4-h exposure in samples taken from the chamber gas space. Oxygen and carbon dioxide levels were verified by mass spectrometry (Centronic MG 200, Borough Green, UK).
Bovine pulmonary arteries were dissected from heart/lung plucks at a local abattoir and transported to the laboratory in ice cold phosphate buffered saline containing 100 U/ml penicillin, 100 g/ml streptomycin and 0.5 g/ml amphotericin B. Sections were cleaned of fat and connective tissue and were opened longitudinally, lumen down, in 1–2 ml of a 1 mg/ml solution of type II collagenase in PBS in a 150-mm diameter petri dish for 9 min. Endothelial cells were removed by gently scraping the lumen with a sterile scalpel blade (Swann Morton size 10). No area of the lumen was covered twice and after each stroke the cells were washed off into Dulbeco’s modified Eagle’s medium. The cells were pelleted (500g for 10 min at room temperature), resuspended in supplemented medium (see below), and seeded into 25-cm 2 culture flasks. All cultures were maintained until confluent at 37°C under 5% CO 2 in a humidified incubator in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 20 mM Hepes (pH 7.4), antibiotics/antimycotics (as above), and 10% fetal calf serum (FCS). Confluent cultures were passaged by washing twice in sterile PBS and mechanical displacement from the plastic culture flasks after a brief incubation with 0.25% (w/v) trypsin 0.2 mg/ml EDTA in PBS. The action of trypsin was terminated with the addition of supplemented medium and the cell suspension was seeded directly into new flasks (at a ratio 1:2). Culture medium pH, PO 2 and PCO 2 was tested using a blood gas analyzer (Model 1312; Instrumentation Laboratory, Milano, Italy). Parallel cultures of cells with identical morphology were tested for von Willebrand factor (vWF) expression by immunocytochemistry. Dividing cells were seeded onto a sterile cover slip. After 24 –36 h, slides were submerged in Hank’s balanced salt solution (BSS) for 1 min and the cells were fixed with freshly prepared paraformaldehyde at 2– 4% (w/v) in BSS for 10 min. The slides were washed three times in BSS and dehydrated in acetone at ⫺20°C. After a further three washes in BSS, slides were incubated with a 1:25 dilution of rabbit anti-vWF antibody (Dako Ltd, High Wycombe, UK) for 90 min at room temperature. Excess antibody was washed off with BSS and the cells were incubated with a 1:25 dilution of the secondary goatanti-rabbit IgG antibody for 90 min. Excess secondary antibody was washed off with BSS and the cover slips
© 2001 Elsevier Science (USA). All rights reserved.
NO GAS DECREASES ET-1 mRNA
were mounted and examined under a fluorescence microscope. mRNA Analysis Total cellular RNA was extracted and purified by the acid guanidinium isothiocyanate-phenol-chloroform method with RNAzol B (Biogenesis, Poole, UK) according to the manufacturer’s protocol. The integrity and quantity of total RNA was determined by spectrophotometric analysis and electrophoresis through a 1% agarose, 2.2 M formaldehyde gel. Primers were tested against 100 ng genomic DNA to determine if genomic amplification products interfered with the assay. The primers used were: for NOS III; (5⬘–3⬘, sense) GTG ACC CTC ACC GCT ACA AT and (antisense) CCG ACA TCT CCA TCA GGG; and for ppET-1, (sense) CCC TCC CCA GAA TGG ATT AT and (antisense) AGT TCT TTT CCT GCT TGG CA. The primers used for amplification of -actin were (sense) ATC ATG TTTG AGA CCT TCA ACA CCC CAG CC and (antisense) AAG AGA GCC TCG GGG CAT CGG AAC CGC TCA. The amplified cDNA fragments, 681, 400, and 421 bp, respectively. The cDNA was synthesised by 200 U murine Moloney-leukemia virus reverse transcriptase in the presence of 100 ⌴ random hexamer primers, 1 mM dNTPs, 250 ⌴ ribonuclease inhibitor and reaction buffer for 1 h at 37°C in a volume of 20 l. Samples (1 l) of these reaction solutions were used in the polymerase chain reaction (PCR) amplification using the oligonucleotide primers given above. Each reaction contained 2.5 U Taq DNA polymerase (Gibco), 1.0 ⌴ specific primers, 1.5 mM Mg 2⫹, 200 ⌴ dNTPs, 100 mM Tris–HCl (pH 8.2), and 50 mM KCl in a volume of 50 l. The identity of the PCR products were confirmed on 1% agarose gels by comparison with the expected size of the products and by automated dideoxy chain termination (Applied Biosystems Model 373A, Warrington, UK) as described elsewhere (11). Primer-dimer artefacts and unincorporated nucleotides and primers were removed by a centrifugation through Qiaquick spin-columns (Qiagen, Crawley, UK), according to the manufacturers protocol. Negative controls contained all PCR components excluding cDNA. All primers spanned introns and either failed to amplify a product or yielded products of greater size when tested against 100 ng of genomic DNA extracted from whole bovine blood (Nucleon II kit, Scot lab Bioscience, Lanarkshire, UK). There was no evidence of genomic contamination when PCR products amplified
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from cDNA preparations were separated on agarose gels after 35 cycles. The relative intensities on high density scans of autoradiographs were determined using the NIH Image 1.44 software (National Institute of Health, USA) as described previously (11). Semilogarithmic plots of densitometry vs cycle number were constructed. Before reaching plateau, and at equal PCR efficiencies, the ratio between the logarithm of the PCR products is equivalent to the ratio of the corresponding target mRNAs. In each of the three experiments, RNA extraction, reverse transcription, and PCR analysis was carried out on all samples simultaneously and in duplicate using common reagents. This allowed intragroup analysis but excluded intergroup comparison. Nitric Oxide and Nitrite Anion Analysis Gaseous NO was quantified by chemiluminescence (Model Mk2B, GlaxoSmithKline, Beckenham, UK and Model 42, Thermo ONIX, Warrington, UK). The analyzers were calibrated with known volumes of 9.9 or 100 p.p.m. NO in nitrogen. The limit of detection of NO was 1 ppb (40 pM at 20°C) at an inlet flow rate of 130 ml/min with a response time of 1 s for Model Mk2B and 2 ppb at an inlet flow rate of 750 ml/min with a response time of 40 s for Model 42. Nitrite anions were quantified by acid-reduction to NO followed by chemiluminescent analysis of eluted gas. Briefly, a sealed 20-ml universal tube containing a mixture of 0.1 M sulfuric acid 0.1 M potassium iodide and 0.14 M potassium sulfate was continuously bubbled with NO-free nitrogen using a method adapted from Dunham et al. (12). Nitrite concentrations were calculated from the integral of the detected signal over time and compared to those of a series of standards. The assay was linear up to 500 M with a limit of detection of 0.2 M in 100 l samples. The analogue output of the chemiluminescent analysers was recorded digitally at 2 Hz on a personal computer (Macintosh SE30, Cupertino, CA) by analogue to digital conversion (MP100, Biopac Systems, Goleta, CA). The area under the curve was determined using the origin graphic software (MicroCal Inc., Northampton, MA). Statistical Analysis Nitrite anion levels are presented as the mean ⫾ SD. The mRNA levels were expressed as the ratio of target sequence levels to those of -actin and are presented as median (range). The average density of du-
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plicate dot blots was used for each data point. Significance was determined using nonparametric Wilcoxon Signed-Rank test. Values of P ⬍ 0.05 were considered statistically significant. RESULTS Endothelial Characterzation Early culture primary endothelial cells were used after 1st passage to provide matched control and test cultures. The pH of the culture medium varied between 7.24 –7.48. The PCO 2 of the medium ranged from 30.1– 42.2 mmHg and values of PO 2 varied between 128 and 146 mmHg. The endothelial cells exhibited a typical “cobblestone morphology” when viewed under phase contrast microscopy (Fig. 2A) and achieved confluence within 1 week of initial seeding. Contact inhibition was evident during the late stages of growth towards confluence. During the exponential growth phase the cells divided with a mean population doubling time of approximately 14 h. These cells also expressed the mRNA for ppET-1 and NOS III and stained positive for vWF with immunocytochemistry (Fig. 2B). After 4 h exposure to NO cells remained viable as determined by Trypan blue exclusion (data not shown). Preproendothelin-1 mRNA and Nitric Oxide Synthase III Semi-logarithmic plots of densitometry vs cycle number showed that amplification of all three RNA species was logarithmic over 17–31 cycles with evidence that reaction components became limiting at 35 cycles. Aliquots taken at 27 cycles demonstrated that amplification of cDNA derived from 0.3–3.0 g of total RNA were linear (r ⬎ 0.99) for all three RNA species. Exposure to 10 p.p.m. NO caused no significant change in ppET-1 (1.60 [1.37–3.71] vs 2.94 [0.80 – 4.17], exposure vs control, P ⫽ 0.24) nor in NOS III mRNA (2.81 [1.31– 4.74] vs 2.23 [1.67– 4.02], P ⫽ 0.25). Exposure to 100 p.p.m. caused a decrease in NOS III mRNA in 5 of the 7 matched cultures but did not reach statistical significance (3.20 [2.46 – 6.75] vs 4.94 [1.82–9.90], P ⫽ 0.14). However, all seven of the matched cultures showed a significant decrease in ppET-1 mRNA when compared to controls (0.73 [0.71–3.65] vs 3.45 [1.43– 7.36], P ⫽ 0.03). Parallel cultures of pulmonary endothelial cells treated for over 4 h with TGF  (10 ng/ml) showed increased ppET-1 mRNA (2.14 [1.15–5.34] vs 0.20 [0.10 – 0.75]). Cells treated with TNF␣ (1 ng/ml)
showed decreased NOS III mRNA (0.10 [0.04 – 0.15] vs 0.57 [0.42–1.62]). Nitric Oxide and Nitrite Anion Accumulation The concentration of nitrite anions rose to 1,670 ⫾ 158 nM (n ⫽ 7) compared to 18 ⫾ 5 nM (n ⫽ 7) in the control cells, with 100 p.p.m. after 4 h exposure. With 10 p.p.m. NO, the medium contained 243 ⫾ 8 nM (n ⫽ 5) nitrite anions compared to that of 15 ⫾ 7 nM (n ⫽ 5) in controls after 4 h. DISCUSSION In this study, we exposed first passage primary cultures of pulmonary artery endothelial cells to two concentrations of NO gas, 10 and 100 p.p.m. The medium nitrite anions content was 0.02 M with a control atmosphere, 0.2 M with 10 p.p.m. and 1.6 M with 100 p.p.m. NO after 4 h static exposure at 37°C. Under these conditions, we observed a decrease in steady state ppET-1 mRNA levels in cells exposed to 100 p.p.m. NO, but not with 10 p.p.m. No change in levels of mRNA for NOS III were observed with either concentration. Nitric oxide can be delivered in a number of ways in biological solutions. A “transient” dose of NO can be delivered as a bolus injection of a de-aerated solution containing a known dissolved concentration of the gas. The chemistry of nitrogen oxides in biological fluids is complex (13). As a result of the cell culture medium containing a variety of chemicals, which enhance “oxidation” of dissolved NO (14), the exact dose of NO reaching the cultured cells remains unknown. A NO donor such as nitroprusside, may offer a more precise means of NO “dosing,” but they release small organic or inorganic fragments after releasing NO (15, 16), which are likely to have direct effects on the phenotype of the cultured cells. We chose to use mixtures of NO in air and to measure nitrite anions, after a fixed exposure period in cell culture medium bathing the cells as it seemed a more physiological approach to the delivery of nitric oxide. Our technique for measuring mRNA levels is sensitive and therefore allows small differences to be detected with reproducibility within 19% (11). We used a commonly used housekeeping gene,  actin, as an internal mRNA standard to allow for correction of the mRNA quantification to the original amounts of extracted mRNA. Validation of amplification kinetics confirmed that there was an exponential increase in PCR products over the range of PCR cycles chosen (11).
© 2001 Elsevier Science (USA). All rights reserved.
NO GAS DECREASES ET-1 mRNA
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FIG. 2. Photomicrograph of confluent cultured bovine pulmonary artery endothelial cells viewed under phase contrast (A, ⫻40). Fluorescence photomicrograph of subconfluent endothelial cells stained with anti-von Willebrand IgG (B, ⫻400).
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FIG. 3. mRNA levels for ppET-1 and NOS III in bovine pulmonary artery endothelial cells cultured in 20% O 2, 5% CO 2 (controls), and containing 10 or 100 p.p.m. nitric oxide. Data are presented as a ratio of the image density for ppET-1 to that of -actin in arbitrary units. Each point represents an average of duplicate assay from each sample taken at 27 cycles. * P ⬍ 0.05 (Wilcoxon Sign-Rank test).
Variability in the baseline levels of ET-1 and NOS III mRNAs may reflect differences in the harvest site within the pulmonary artery tree or small variations in the exact number of population doublings since seeding. These most likely reflect real variation since assay reproducibility was within 19% (11). The observed reduction of ppET-1 mRNA with 100 p.p.m. gaseous NO is the same as that seen with NO donors and consistent with the 15 min half life of ppET-1 mRNA (17). As expected, TGF  increased ppET-1 mRNA, indicating that our bovine pulmonary artery endothelial cells can change their phenotype in a predictable fashion and consistent with the established doctrine that transcription is the primary level of ET-1 regulation (18). In contrast, the half-life of NOS III mRNA was not
changed. Failure to change NOS III mRNA with NO gas agrees with the observed failure of NOS III mRNA to fall in the lungs of rats exposed to gaseous NO over 1–3 weeks (19). However, the normally long half-life of NOS III was dramatically decreased by TNF ␣ over 4 h exposure, which is known to regulate the transcript stability (20). Therefore, we can conclude that NO gas is unlikely to act on NOS III through a dramatic decrease in mRNA stability. Whilst our data does not provide information on the enzymatic activity of NOS III nor the peptide levels for ET-1 they do, however, show that NO gas alters the phenotype of endothelial cells under these special conditions. Furthermore, a recent study by Lin et al. (21) indicates a correlation between transcription and ET-1 peptide expression in vitro. In vivo, there does appear
© 2001 Elsevier Science (USA). All rights reserved.
NO GAS DECREASES ET-1 mRNA
to be an interplay between NO and ET-1 production (22, 23) and exposure to short-term NO gas may reduce constitutive NOS activity, including NOS III (23). The down regulation in mRNA transcription for preproendothelin-1 without an effect on NOS III mRNA could be considered valuable in the treatment of pulmonary hypertension. We cannot describe the mechanism of action of NO on the regulation of expression of ppET-1 but it has been argued to result from both a cGMP dependent mechanism and one sensitive to the cell redox potential (9). In the lung, we might speculate that concentrations greater than 10 p.p.m. NO gas may be required to suppress the overexpression of ET-1 in pulmonary hypertension. In conclusion, 100 p.p.m. NO gas suppresses ppET-1 mRNA levels in cultured pulmonary artery endothelial cells over a 4-h period without significantly altering NOS III mRNA levels. REFERENCES 1. Pepke-Zaba, J., Higenbottam, T. W., Dinh-Xuan, A. T., Stone, D., and Wallwork, J. (1991). Inhaled nitric oxide as a cause of selective pulmonary vasodilation in pulmonary hypertension. Lancet 338, 1173–1174. 2. Kinsella, J. P., Neish, S. R., Shaffer, E., and Abman, S. H. (1992). Low-dose inhalation nitric oxide in persistent pulmonary hypertension of the newborn. Lancet 340, 819 – 820. 3. Channick, R. N., Newhart, J. W., Johnson, F. W., Williams, P. J., Auger, W. R., Fedullo, P. F., and Moser, K. M. (1996). Pulsed delivery of inhaled nitric oxide to patients with primary pulmonary hypertension—An ambulatory delivery system and initial clinical tests. Chest 109, 1545–1549. 4. Katayama, Y., Higenbottam, T. W., Cremona, G., Akamine, S., Demoncheaux, E. A. G., Smith, A. P. L., and Siddons, T. E. (1998). Minimising the inhaled dose of NO with breath-by-breath delivery of spikes of concentrated gas. Circulation 98, 2429 – 2432. 5. Stewart, D. J., Levy, R. D., Cernacek, P., and Langleben D. (1991). Increased plasma endothelin-1 in pulmonary hypertension-marker or mediator of disease. Ann. Intern. Med. 114, 464 – 469. 6. Ferri, C., Bellini, C., Deangelis, C., Desiati, L., Perrone, A., Properzi, G., and Santucci, A. (1995). Circulating endothelin-1 concentrations in patients with chronic hypoxia. J. Clin. Pathol. 48, 519 –524. 7. Saleh, D., Furukawa, K., Tsao, M. S., Maghazachi, A., Corrin, B., Yanagisawa, M., Barnes, P. J., and Giaid, A. (1997). Elevated expression of endothelin-1 and endothelin-converting enzyme-1 in idiopathic pulmonary fibrosis: Possible involvement of proinflammatory cytokines. Am. J. Resp. Cell Mol. 16, 187–193. 8. Chen, S. J., Chen, Y. F., Meng, Q. C., Durand, J., Dicarlo, V. S., and Oparil, S. (1995). Endothelin-receptor antagonist bosentan
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