This Article Abstract Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Chen, Y.-F. Articles by Claycomb, W. C. Search for Related Content PubMed PubMed Citation Articles by Chen, Y.-F. Articles by Claycomb, W. C. Pubmed/NCBI databases Compound via MeSH Substance via MeSH

(Hypertension. 1997;29:75.)
© 1997 American Heart Association, Inc.

Research Articles (Issue 1, Part 1)

Hypoxia Stimulates Atrial Natriuretic Peptide Gene Expression in Cultured Atrial Cardiocytes

Yiu-Fai Chen; Joan Durand; William C. Claycomb

the Vascular Biology and Hypertension Program, Division of Cardiovascular Disease, Department of Medicine, University of Alabama at Birmingham, and the Department of Biochemistry and Molecular Biology, Louisiana State University Medical Center, New Orleans (W.C.C.).

Correspondence to Yiu-Fai Chen, PhD, 1008 Zeigler Research Bldg, Vascular Biology and Hypertension Program, University of Alabama at Birmingham, Birmingham, AL 35294-0007.

	Abstract

Top Abstract Introduction Methods Results Discussion References

 
The current study tested the hypothesis that hypoxia stimulates atrial natriuretic peptide (ANP) gene expression and secretion in cultured atrial myocytes (AT-1 cells). AT-1 cells were obtained from a transplantable mouse atrial cardiomyocyte tumor lineage. Confluent AT-1 cells were exposed to hypoxia (1% oxygen) or normoxia (21% oxygen) as controls for 6 hours to 7 days. Medium ANP levels were measured by radioimmunoassay, and intracellular ANP gene transcripts were quantified by Northern and slot blot analyses. Exposure to hypoxia resulted in a significant increase in cellular ANP mRNA levels within 36 hours, which peaked (3.6-fold increase) at 2 days after hypoxic exposure, and produced a time-dependent increase in the release of ANP from AT-1 cells for 2 to 7 days. Transfection studies with recombinant DNA constructs that contained fragments of the -3003/+62 sequence of the ANP promoter and the luciferase reporter gene revealed that the regulatory sequences that mediate the hypoxia-induced increase in transcription are located within a region that extends from -638 to -518 bp to the transcriptional start site of the ANP gene. Gel mobility shift assays demonstrated that hypoxia-inducible nuclear proteins that bound to the 120-bp putative hypoxia-responsive elements of the ANP gene were produced during hypoxic exposure. We have thus defined a 120-bp region within the ANP gene promoter that contains hypoxia-responsive elements that might be responsible for the enhancement of ANP gene expression in atrial myocytes during hypoxic exposure.

Key Words: hypoxia • atrial natriuretic factor • gene expression

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
Atrial natriuretic peptide (ANP) is a peptide hormone that is synthesized, stored, and released primarily from adult mammalian atrial myocytes.¹ ANP is also found in normal ventricular tissue and a variety of extracardiac tissues, including aorta, lung, and brain.² ANP plays an important role in the regulation of cardiovascular functions, including blood pressure, sodium, and volume homeostasis. It exhibits natriuretic-diuretic and vasorelaxant properties through a direct action on membrane receptors in tissues and by a functional antagonism of the actions of angiotensin II, aldosterone, vasopressin, and endothelin.³ ANP is secreted into the circulation in response to a variety of stimuli, including atrial stretch, which generally occurs in response to volume loading in vivo⁴ ; acute hypoxia⁵ ; acetylcholine; epinephrine; dopamine⁶ ; arginine vasopressin⁷ ; and endothelin.⁴ Endogenous plasma ANP levels are elevated in a number of pathological states, including congestive heart failure and ischemic heart disease,⁸ chronic renal failure,⁸ and pulmonary hypertension, in humans and experimental animals.⁹

Studies from our laboratory and others have strongly suggested that ANP plays a role in regulating the pulmonary circulation and may be involved in the pathogenesis of hypoxia-induced pulmonary hypertension.^{10 11 12} In clinical studies, a significant positive correlation between plasma ANP level and pulmonary artery pressure has been observed in normal subjects and patients with heart disease.⁸ Patients with chronic pulmonary hypertension have elevated levels of circulating ANP in association with increased pulmonary artery pressure and pulmonary vascular resistance.¹³ Experiments performed in our laboratory have demonstrated that rats adapted to chronic hypoxia have significant pulmonary hypertension and right ventricular hypertrophy in association with an elevation in endogenous plasma ANP levels.^{10 11} Elevation of plasma ANP through chronic intravenous infusion of ANP or administration of an ANP clearance receptor antagonist attenuates the development of hypoxia-induced pulmonary hypertension and right ventricular hypertrophy in hypoxia-adapted rats.^{10 11} In addition, neutralization of endogenous ANP with monoclonal antibodies potentiates hypoxia-induced pulmonary hypertension.¹⁴ Thus, ANP appears to be an endogenous modulator of the pulmonary hypertensive response to chronic hypoxia.

Several studies have demonstrated that the main source of enhanced circulating ANP in hypoxia-adapted animals is the heart,^{12 15} but the mechanisms that regulate cardiac ANP synthesis and release during hypoxic exposure are not fully understood. It has been suggested that right atrial and ventricular distension resulting from pulmonary hypertension may be the major cause of ANP secretion during hypoxic exposure.^{12 16} However, there is increasing evidence that hypoxia per se is a potent stimulus for the ANP production and secretion from the heart of hypoxic animals.^{5 12 17 18 19}

In the present study, we use an in vitro system to test the hypothesis that hypoxic exposure can directly stimulate ANP gene expression and ANP release in cultured atrial myocytes (AT-1 cells). The AT-1 cells were isolated from a transplantable cardiac tumor lineage derived from transgenic mouse atria.^{20 21 22} This in vitro model system allowed us to study the ANP response to hypoxia in the absence of hemodynamic, neural, and hormonal influences. Our data demonstrated that hypoxia can act directly on cardiac myocytes to enhance ANP gene transcription and that there are positive cis-regulatory elements in the 5' promoter region of the ANP gene that mediate hypoxia-induced ANP gene expression, suggesting that ANP is a hypoxia response gene.

	Methods

Top Abstract Introduction Methods Results Discussion References

 
Cell Culture
AT-1 atrial myocytes were obtained from a transplantable mouse atrial cardiomyocyte tumor lineage derived from transgenic mouse atria.^{20 21 22} The transgenic mouse strain was first generated by Field.²¹ AT-1 cells were isolated and cultured as previously described, with minor modifications.^{20 22} Isolated AT-1 cells were plated at a density of 2.5x10⁶ cells per 5 mL culture medium per plate in 60-mm Falcon tissue culture plates precoated with fibronectin (5 µg/mL, Sigma Chemical Co) and gelatin (0.02%, Difco) in 5 mL Ex-Cell 320 medium (JRH Biosciences) supplemented with 10% fetal calf serum and antibiotics (100 U/mL penicillin and 100 µg/mL streptomycin). Cells were then cultured at 37°C, 95% air/5% CO₂, in a humidified incubator. Cells were allowed to attach for 18 to 24 hours before the medium was changed to fresh medium. Cells were cultured for 5 to 7 days until they reached 85% to 90% confluence before use in experimental protocols.

To examine the effects of hypoxia on ANP mRNA expression and release in cultured AT-1 cells, we transferred half of the plates with confluent cells into an air-tight hypoxic chamber (model 2710, Cell Culture Incubator, Queue Systems) containing 1% O₂/5% CO₂/94% N₂. The other half were used as normoxic controls (95% air/5% CO₂). After 6 hours or 1, 1.5, 2, 5, or 7 days of exposure to hypoxia or normoxia, 1 mL of medium was collected from each plate for determination of ANP by radioimmunoassay (RIA). The cells were then harvested for intracellular ANP mRNA measurement. For 5- and 7-day cultures, medium was changed once with preconditioned hypoxic or normoxic medium on day 3. PO₂ and PCO₂ in the media with cells reached 20 to 30 mm Hg and 38 to 40 mm Hg, respectively, pH 7.3 to 7.4, approximately 2 hours after the initiation of hypoxic exposure.

ANP Radioimmunoassay
ANP concentration in cultured media was measured by RIA with RIA kits (Peninsula Laboratories, Inc). Samples (1 mL) of media were collected in chilled polypropylene tubes containing EDTA (1 mg/mL) and aprotinin (500 kallikrein inhibitor units/mL) at 4°C. Medium was acidified with 0.1% trifluoroacetic acid (TFA, high-performance liquid chromatography [HPLC] grade), centrifuged at 3000g for 15 minutes at 4°C, and applied to a Sep-Pak C18 column (Waters Associates) that had been activated by washing with 60% acetonitrile (HPLC grade) in 0.1% TFA, 4x1 mL, followed by 0.1% TFA, 4x5 mL. ANP was eluted from the column with 60% acetonitrile in 0.1% TFA, 3x1 mL, into a polypropylene tube and evaporated to dryness in a centrifugal concentrator (SpeedVac, Savant Instruments, Inc). Samples were reconstituted in RIA buffer and subjected to RIA. The anti-ANP antiserum had 100% cross-reactivity with rat, human, and canine -ANP_1-28 and rat, rabbit, and mouse ANP_5-28; 5% cross-reactivity with rat, rabbit, and mouse atriopeptin II; and 0% cross-reactivity with rat, rabbit, and mouse atriopeptin I.

ANP mRNA Quantitation
RNA Isolation
Total RNA was extracted from cultured AT-1 cells by the method of Chomczynski and Sacchi.²³ Briefly, cells in each plate were dissolved with 0.5 mL of 4 mol/L guanidine isothiocyanate, 25 mmol/L sodium citrate (pH 7.0), 0.5% sodium-N-lauryl sarcosine, and 0.1 mol/L ß-mercaptoethanol. The protein in the cell extract was removed by phenol-chloroform extraction. The RNA was precipitated by isopropanol and washed with ethanol. The precipitated RNA was dissolved in water and measured by absorbance at 260/280 nm. Protein contamination was assessed by the ratio of optical absorbance at 260 and 280 nm. Samples with 260/280-nm optical density ratios less than 1.75 were subjected to further phenol-chloroform extraction. The ratio of absorbance at 260 and 280 nm in samples used for analysis in these studies was 1.8 to 2.0. To demonstrate that our rat ANP cDNA probe can detect mouse ANP mRNA in AT-1 cells, we collected right and left atria from an adult male Sprague-Dawley rat. Tissue was homogenized in 4 mol/L guanidine isothiocyanate solution and RNA extracted as described above.

ANP cDNA and 18S Ribosomal Oligonucleotide Probes
The cDNA probe used for identification and quantification of specific ANP mRNA was a 0.5-kb fragment of the rat ANP gene provided by Dr Wiegand at Monsanto (St Louis, Mo). For quantification of 18S rRNA, an oligonucleotide (5'-ACG-GTA-TCT-GAT-CGT-CTT-CGA-ACC-3') complementary to 18S rRNA was used. The ANP cDNA probe was radiolabeled with [-³²P]dCTP to a specific activity of 10⁸ to 10⁹ cpm/µg utilizing a random hexamer primer labeling system (Promega, Prime-a-Gene Labeling System). The oligonucleotide was end-labeled with [-³²P]ATP using T₄ polynucleotide kinase. The radiolabeled ANP cDNA probe was separated from unincorporated nucleotides using Quick Spin columns containing G-50 Sephadex (Boehringer Mannheim) and used for hybridization after denaturation.

Northern and Slot Blot Analyses
We used both Northern and slot blot analyses to identify and measure ANP mRNA. For Northern analysis, RNA isolated from cultured AT-1 cells and rat right and left atria was denatured in 50% formamide and 6% formaldehyde (in 22.5 mmol/L 3-[N-morpholino]propanesulfonic acid [MOPS] with 1.2 mmol/L EDTA); size-fractionated by electrophoresis through 1.5% agarose, 3% formaldehyde gels in 20 mmol/L MOPS, 5 mmol/L sodium acetate, and 1 mmol/L EDTA (pH 7.0); and blotted onto a Nytran membrane (0.45 µm, Schleicher & Schuell, Inc) in 20x SSC (1x SSC=0.15 mol/L NaCl, 15 mmol/L sodium citrate, pH 7.0). For slot blot analysis, two samples (5 and 1 µg) of total RNA from AT-1 cells were denatured in 6% formaldehyde in 8.3x SSC, incubated at 60°C for 20 minutes, and applied directly onto a Nytran membrane with a slot blot apparatus (Schleicher & Schuell). All Northern and slot blots were cross-linked by ultraviolet radiation for a total energy of 125 mJ in a GS Gene Linker (Bio-Rad). Blots were prehybridized in 50% deionized formamide, 5x Denhardt's solution (1x Denhardt's=0.02% Ficoll, 0.02% polyvinylpyrrolidone, 0.02% bovine serum albumin), 5x SSC, 1% sodium dodecyl sulfate (SDS), boiled salmon sperm DNA (200 µg/mL), and yeast tRNA (25 µg/mL) at 42°C for at least 2 hours and then hybridized in the same buffer with ³²P-labeled probes (10⁶ cpm/mL) at 55°C overnight. After hybridization, blots were washed twice in 500 mL of 2x SSC/0.1% SDS at room temperature for 15 minutes and three times in 0.2x SSC/0.1% SDS at 65°C for 30 minutes. Blots were dried and subsequently exposed to x-ray film (Kodak X-OMAT, Sigma). The density of autoradiographic signals was measured with an optical densitometer (model GS-670, Bio-Rad).

For quantitation of total RNA loaded onto slot blots, the ³²P-labeled ANP cDNA probe was stripped from the membrane after washing in 5 mmol/L Tris-HCl (pH 8.0), 0.2 mmol/L Na₂EDTA, 0.05% sodium pyrophosphate, 0.002% polyvinylpyrrolidone, 0.002% bovine serum albumin, and 0.002% Ficoll at 65°C for 2 hours. The membrane was then rehybridized with a ³²P-labeled 18S rRNA probe under the conditions specified above. Since the variability of gene expression of ß-actin and GAPDH (so-called housekeeping genes) in hypoxic conditions has recently been recognized²⁴ and rRNA (representing a fixed proportion of total RNA) has been proved to be unchanged by hypoxic or hypobaric exposure,²⁴ we elected to use 18S rRNA as a reference of the total cellular RNA loaded for each sample. To estimate cellular ANP mRNA steady-state levels, we determined the ratios of ANP mRNA to 18S rRNA by dividing the absorbance corresponding to ANP cDNA probe hybridization by the absorbance corresponding to 18S rRNA probe hybridization. This allows for correction for any misloading or overloading of the slot blots with RNA.

Transient Expression Assays
Plasmid Constructs and Transfection of AT-1 Cells
To identify the cis-sequences that mediate hypoxia-inducible expression of the rat ANP gene,²⁵ we subcloned a series of polymerase chain reaction (PCR)–generated rat ANP promoter fragments into a pSVOAL5' expression vector with a firefly luciferase reporter gene. The strategy and method used for the generation of ANP promoter–pSVOAL5' recombinant DNA constructs were modified from Knowlton et al.²⁶ The plasmid designation in this study is based on the most 5' and 3' nucleotides relative to the transcriptional start site in the native gene.²⁵ The constructed plasmids were transformed into library efficiency DH5 competent cells (GIBCO-BRL Life Technologies Inc), and the orientation and copy number of all ANP promoters inserted into pSVOAL5' were verified by double-stranded dideoxy chain termination sequencing. The plasmid DNA was amplified and extracted in large-scale preparation and purified by the CsCl₂ method before use in the transfection study. The plasmid DNAs (diluted to 1 µg/µL in water) from at least three separate preparations were used for transient transfection experiments.

Transient transfection of AT-1 cells with plasmids was performed by the lyposome-mediated transfection method. Cells were grown in 60-mm culture plates to 85% confluence and washed twice with serum-free medium. Each plate was then transfected with 1 µg of test plasmid mixed with 10 µL of LIPOFECTIN reagent (GIBCO-BRL) in 2 mL of serum-free Ex-Cell 320 medium. One microgram of pON249, a ß-galactosidase expression vector under the control of the human cytomegalovirus (CMV) promoter, was cotransfected and used as a control for transfection efficiency. Cells were incubated with the plasmid/LIPOFECTIN reagent mixture for 6 hours in 95% air/5% CO₂ at 37°C. The medium containing DNA was then replaced with fresh medium containing 10% fetal calf serum. One or 2 days after transfection, when the transfected cells reached 100% confluence, the plates were divided into two groups and incubated in either 1% or 21% oxygen for 48 hours before being harvested for intracellular luciferase and ß-galactosidase measurements.

Luciferase and ß-Galactosidase Assays
Transfected cells in each plate were washed twice with ice-cold phosphate-buffered saline (PBS) without Ca²⁺ and Mg²⁺ and then lysed on ice with 400 µL cell lysis buffer (0.1 mol/L KPO₄, 0.5% Triton X-100, 1 mmol/L dithiothreitol, pH 7.9) for 15 minutes. For luciferase assays, 25 µL cell extract was assayed in duplicate on an Optocomp I luminometer (MGM Instruments Inc) in 200 µL luciferase assay buffer containing KTME (100 mmol/L tricine, 10 mmol/L MgSO₄, 2 mmol/L EDTA, pH 7.8), 1 mmol/L dithiothreitol, 2.5 mmol/L ATP, and 75 µmol/L D-luciferin (Analytical Luminescence Laboratory). For ß-galactosidase assays, 50 µL cell extract was assayed in 250 µL ß-galactosidase assay buffer containing 0.1 mol/L Na₂HPO₄ (pH 7.3), 3.5 mmol/L o-nitrophenyl ß-D-galactopyranoside, 1 mmol/L MgCl₂, and 50 mmol/L ß-mercaptoethanol. Reactions were incubated at 37°C for 2 hours and stopped by the addition of 0.5 mL of 0.1 mol/L NaCO₃; absorbance was read at 410 nm with a spectrophotometer (Shimadzu UV-160). Relative luciferase activities were calculated as luciferase activities divided by their corresponding ß-galactosidase activities to correct for variations in transfection efficiency. These values were then normalized to the expression of -3003/+62 ANP-pSVOAL5' plasmid transfected into AT-1 cells that had been cultured in 21% oxygen. Mean data for all plasmids were derived from at least three independent transfection studies.

Gel Mobility Shift Assays
Preparation of Nuclear Extracts
After cells reached confluence, fresh complete medium was added to AT-1 cells in T-75 flasks, which were then incubated in 21% or 1% oxygen for 24 or 40 hours. Nuclear extracts were obtained by a modification of a standard method of Dignam et al.²⁷ In brief, cells were washed twice with ice-cold PBS, scraped into 5 mL PBS, and pelleted by centrifugation at 2000 rpm for 5 minutes at 4°C in a centrifuge (Beckman GS-6R). The cell pellet was washed with 5 packed-cell volumes of ice-cold buffer A (10 mmol/L HEPES [pH 7.9], 1.5 mmol/L MgCl₂, 10 mmol/L KCl, 0.4 mmol/L phenylmethylsulfonyl fluoride [PMSF], 0.5 mmol/L dithiothreitol, 2 µg/mL leupeptin, 2 µg/mL pepstatin, and 2 µg/mL aprotinin), resuspended in 5 packed-cell volumes of buffer A, and incubated on ice for 10 minutes. The cell suspension was homogenized with a B-type pestle glass homogenizer (10 strokes), and the nuclei were pelleted by centrifugation at 25 000g for 20 minutes at 4°C. The nuclear pellet was resuspended in 3 packed-cell volumes of buffer C (20 mmol/L HEPES [pH 7.9], 25% glycerol, 0.42 mol/L NaCl, 1.5 mmol/L MgCl₂, 0.2 mmol/L EDTA, 0.4 mmol/L PMSF, 0.5 mmol/L dithiothreitol, 2 µg/mL leupeptin, 2 µg/mL pepstatin, and 2 µg/mL aprotinin), homogenized with a B-type pestle glass homogenizer (10 strokes), and mixed gently on a rotator at 4°C for 30 minutes. Nuclear debris was pelleted by centrifugation at 25 000g for 30 minutes at 4°C. The supernatant was dialyzed against 50x volumes of buffer D (20 mmol/L HEPES [pH 7.9], 20% glycerol, 0.1 mol/L KCl, 0.2 mmol/L EDTA, 0.4 mmol/L PMSF, and 0.5 mmol/L dithiothreitol) for 5 hours at 4°C. The dialysate was centrifuged at 25 000g for 20 minutes at 4°C, and aliquots were frozen in liquid nitrogen and stored at -80°C. Protein concentrations were determined by a Bio-Rad assay.

Preparation of DNA Probe
A ³²P-labeled 120-bp DNA fragment that contains -638 to -518 bp of ANP 5'-flanking sequences was generated by PCR using the plasmid -3003/+62 ANP-pSVOAL5' as the template. Two oligonucleotides, 5'-AAT-TCT-TTA-GAG-CCT-GTA-TCA-TGT-TGG-CTT-3' and 5'-CCT-CGT-GGG-TGG-ACC-TCT-GGC-CCC-AGA-CAG-3', were 5' end-labeled with [-³²P]ATP and T₄ polynucleotide kinase and then used as primers for PCR. The labeled PCR probe was purified by 5% nondenaturing polyacrylamide gel electrophoresis. Probe was eluted from the excised gel in 500 µL TE buffer (10 mmol/L Tris-HCl [pH 7.6] and 1 mmol/L EDTA) for 16 hours at 23°C.

Binding Reaction and Gel Retardation Assay
Binding reactions were carried out in a total volume of 20 µL containing 5 or 10 µg nuclear extract, 2 µg poly(dI-dC), and 6 µg bovine serum albumin. After preincubation for 5 minutes at room temperature and addition of 10 000 cpm ³²P-labeled DNA probe, the incubation was continued for an additional 15 minutes, after which the reaction mixtures were loaded onto 5% nondenaturing polyacrylamide gels. Electrophoresis was performed at 175 V at 4°C. Gels were vacuum-dried and autoradiographed at -80°C for 24 hours. Competitor DNA (120-bp fragment generated by the same PCR but with unlabeled primers) was preincubated with nuclear extract for 5 minutes before addition of labeled probe.

Statistical Analysis
Results are expressed as mean±SE. Data were analyzed with the CRUNCH statistical package on an IBM-compatible computer. Statistical comparisons of medium ANP and cellular ANP mRNA levels over time and of activities of ANP promoters of different sizes and sequences in the transfection experiments were performed with one- and two-way ANOVAs with a post hoc Newman-Keuls test. Differences were considered significant at a value of P<.05.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Cell Viability
Primary cultures of mouse AT-1 cells were exposed to a hypoxic atmosphere with 1% O₂/5% CO₂ (PO₂=20 to 30 mm Hg in media) or to a normoxic atmosphere with 21% O₂/5% CO₂ (PO₂=140 mm Hg in media). After hypoxic exposures for 1, 1.5, 2, 5, and 7 days, the AT-1 cells were assessed for viability by trypan blue exclusion (0.4% trypan blue solution, Sigma) and cell confluence. No significant cell death (cell confluence >98% and no floating cells, n=5 plates for each time point) and differences in trypan blue exclusion (exclusion >95%, n=5 plates for each time point) between cells exposed to hypoxia and normoxia were seen at any time. The general light microscopic characteristics (shape and size) of the cultured AT-1 cells were similar in hypoxic and normoxic groups and were the same as reported previously.²⁰

Effect of Hypoxia on ANP Secretion
During normoxic exposure, the ANP levels in the medium did not change significantly, indicating a balanced secretion and reuptake/hydrolysis of ANP by the cultured AT-1 cells (Fig 1). Exposure of AT-1 cells to 1% oxygen caused a time-dependent increase in the ANP levels in the culture media (Fig 1). At the initial time points studied (6 hours and 1 and 1.5 days), ANP release into the media by AT-1 cells exposed to normoxia or hypoxia did not differ significantly. However, by 2 days, the amount of ANP detected in the media from hypoxic cells (37.6±2.9 pg/mL, n=10) was significantly higher than the amount in the media from corresponding normoxic cells (21.5±2.4 pg/mL, n=11). Levels of ANP in the media from hypoxic cells were approximately 3.5-fold of those in media from normoxic control cells at 5 days of hypoxic exposure (Fig 1). The delay in the increase in media ANP levels suggests that hypoxia affects transcription, translation, or some other parameter of peptide synthesis rather than ANP secretion from AT-1 cells.

View larger version (18K): [in this window] [in a new window]   Figure 1. Time course of increase in atrial natriuretic peptide (ANP) levels in culture medium of atrial myocytes (AT-1 cells) during exposure to hypoxia (1% oxygen) compared with normoxic (21% oxygen) control cultures. Medium ANP was extracted on Sep-Pak C18 columns and measured by radioimmunoassay. Results are expressed as mean±SE; n indicates number of plates from at least three separate experiments. **P<.01, *P<.05, compared with respective normoxic groups.

Effect of Hypoxia on Cellular ANP mRNA Levels
Hybridization of RNA from the AT-1 cells with the labeled ANP cDNA probe detected a single transcript of the expected size, 0.9 kb, which was identical to that of authentic ANP mRNA in rat atrial tissues (Fig 2) as described previously.²⁸ To determine whether the increased ANP release during hypoxic exposure was accompanied by an induction of ANP mRNA, we harvested cells from the same plates for measurement of steady-state ANP mRNA levels. Quantitative slot blot analysis indicated that hypoxic exposure resulted in a 1.7-fold increase in ANP mRNA levels within 36 hours (Figs 2 and 3), and the increase peaked at 2 days after hypoxic exposure (Fig 3). ANP mRNA levels dropped to normoxic control levels after 7 days of hypoxic exposure. The ANP mRNA levels of the normoxic control group did not change significantly during the course of the experiment (Fig 3). The increase in ANP mRNA before the secretory response (Fig 1) indicates that the increase in ANP in culture media of AT-1 cells during hypoxic exposure is due to increased synthesis of the peptide, most likely related to increased gene transcription. The increase in medium ANP plateaued at 5 to 7 days after hypoxic exposure, whereas ANP mRNA gradually returned to baseline levels, suggesting that medium ANP may have a more prolonged half-life than that of cellular ANP mRNA.

View larger version (42K): [in this window] [in a new window]   Figure 2. Northern analysis of atrial natriuretic peptide (ANP) mRNA in rat atrium (lane 1) and ventricle (lane 2) and in mouse atrial myocytes (AT-1 cells) cultured in 21% oxygen (lanes 3 and 4) or 1% oxygen (lanes 5 and 6) conditions. Total RNA was extracted from left atrium and ventricle of a Sprague-Dawley rat exposed to hypoxia (10% oxygen) for 2 weeks and from AT-1 cells cultured in hypoxic (1% oxygen) or normoxic (21% oxygen) conditions for 36 hours. Total RNA (10 µg) was loaded onto each lane. Hybridization with 32P-labeled ANP cDNA detected a single 0.9-kb band with both mouse AT-1 cell RNA and rat atrial and ventricular RNAs. An oligonucleotide (24 bases) specific for 18S rRNA was used to measure amount of total RNA loaded (bottom lanes).

View larger version (19K): [in this window] [in a new window]   Figure 3. Time course of increase in steady-state atrial natriuretic peptide (ANP) mRNA levels in cultured atrial myocytes (AT-1 cells) after exposure to hypoxia (1% oxygen) or normoxia (21% oxygen). Slot blot analysis was carried out with 1 and 5 µg total RNA extracted from cultured AT-1 cells and probed with radiolabeled ANP cDNA probe. Data were normalized to allow for variations in RNA loading using a 32P-labeled 18S rRNA probe and expressed as percentage of normoxic controls. Results are expressed as mean±SE; n indicates number of plates from at least three separate experiments. **P<.01, *P<.05, compared with respective normoxic groups.

Transfection Study
To determine whether ANP gene transcription is increased during hypoxic exposure of AT-1 cells by activation of cis-elements within the 5'-flanking promoter region of the gene, we constructed a series of ANP promoter–luciferase reporter fusion genes by inserting various fragments of the 5'-flanking region of the ANP gene upstream of the luciferase reporter gene.²⁶ Each of these recombinant plasmids was cotransfected into AT-1 cells with a CMV–ß-galactosidase vector, pON249, a plasmid in which the transcription of bacterial ß-galactosidase coding sequences is driven by the human CMV promoter. In all experiments, transfection efficiency was monitored by the expression of ß-galactosidase activity, and relative luciferase expression was normalized by calculation of the ratio of luciferase to ß-galactosidase activity. To test the response to hypoxia, we cultured two sets of transfected AT-1 cells in a 95% air/5% CO₂ (normoxia) incubator or in a sealed chamber containing 1% O₂/5% CO₂/94% N₂ for 48 hours. In the normoxic cells, there were two significant reductions (P<.05, one-way ANOVA) in luciferase activity, between the -3003/+62 and -638/+62 and between the -188/+62 and -95/+62 pSVOAL5' plasmids, indicating that regions within these sequences contain essential elements that control basic ANP promoter activity. During hypoxic exposure, expression of luciferase activity of the -638/+62 fusion gene was 3.1-fold greater in hypoxic AT-1 cells than in normoxic AT-1 cells (Fig 4). Hypoxic exposure resulted in a modest (approximately 30% to 60%) but significant induction of luciferase activity in the other ANP promoter–pSVOAL5' plasmids that disappeared when the -95/+62 pSVOAL5' plasmid was used (Fig 4). Since a significantly greater increase (P<.01, two-way ANOVA) in hypoxia-inducible expression was observed between the constructs containing -638 and -518 bp of ANP 5'-flanking sequences, these experiments suggest a role for this intervening sequence in the hypoxia-inducible expression of the ANP gene. As a negative control, AT-1 cells were transfected with a promoterless luciferase plasmid pSVOAL5', which displayed negligible basal luciferase activity and was not induced after hypoxic exposure, indicating no potential contribution of cryptic promoter activity by the expression vector itself in these transfection assays (Fig 4).

View larger version (20K): [in this window] [in a new window]   Figure 4. Transcriptional analysis after transfection of AT-1 cells with recombinant atrial natriuretic peptide (ANP) promoter–luciferase (Luc) DNA constructs. Eight different polymerase chain reaction–generated rat ANP promoter fragments were subcloned 5' to the Luc reporter gene in a pSVOAL5' vector. The constructed plasmid was transfected into AT-1 cells by the lipofection method. Lengths of the polymerase chain reaction fragments (relative to the transcriptional start site) tested in the transfection assay are indicated on the y axis. A cytomegalovirus promoter–driven ß-galactosidase (B-Gal) gene was cotransfected for assessment of transfection efficiency. Transfected AT-1 cells were placed in normoxic (21% O2/5% CO2) or hypoxic (1% O2/5% CO2) conditions for 48 hours. ANP promoter activities were expressed as ratio of Luc to B-Gal activity in cell extracts as a percentage of the value of -3003/+62 normoxic group. Results are expressed as mean±SE; numbers in parentheses indicate number of plates from three to six independent transfection experiments. **P<.01, *P<.05, compared with respective normoxic groups.

Gel Mobility Shift Assays
To test the hypothesis that hypoxia-stimulated ANP gene expression requires the induction of nuclear transcription regulatory factors that bind ANP cis-regulatory sequences, we used an electrophoretic mobility shift assay to analyze the complexes formed between nuclear proteins of AT-1 cells and the ANP 5'-flanking sequences from -638 to -518 bp. Nuclear proteins extracted from AT-1 cells exposed to 1% oxygen for 40 hours caused an upward mobility shift of the complexes (Fig 5, lanes 4 and 7), indicating the presence of trans-acting proteins after hypoxic exposure. The specificity of the complexes formed was accessed by competition with an excessive amount of unlabeled ANP (-638 to -518) DNA fragment. We found that at least two specific DNA-protein complexes were formed when a large amount of nuclear extracts (10 µg, Fig 5, lane 4) was added to the reaction, indicating that multiple nuclear factors that interacted with the ANP promoter sequences from -638 to -518 bp were induced by hypoxia. A similar gel shift pattern was not found when nuclear extracts of AT-1 cells exposed to 24 hours of hypoxia were used in the reaction, indicating that these hypoxia-inducible transcription regulatory factors were not generated during the first 24 hours of hypoxic exposure.

View larger version (84K): [in this window] [in a new window]   Figure 5. Interaction of nuclear extracts from AT-1 cells exposed to 1% oxygen for 24 (lanes 8 and 9) or 40 (lanes 4 through 7) hours with the polymerase chain reaction–generated, 32P-labeled, 120-bp DNA fragment that contains -638 to -518 bp of ANP 5'-flanking sequences. Specificity of the complexes formed was assessed by competition with x20 or x100 unlabeled ANP (-638 to -518) fragment (lanes 3, 5, 6, and 9). Nuclear extracts from AT-1 cells exposed to normoxia (21% oxygen) were used as controls (lanes 2 and 3). Arrowheads indicate the position of the probe specifically associated with nuclear proteins extracted from AT-1 cells exposed to hypoxia.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
The current data provide the first direct evidence that ANP gene expression is enhanced in cardiomyocytes in response to reduced oxygen tension (hypoxia). ANP secretion from cultured AT-1 atrial myocytes was increased within 48 hours of hypoxic exposure; the stimulatory effect lasted for at least 7 days. Steady-state ANP mRNA levels were also significantly increased in cultured AT-1 cells in response to 48 hours of hypoxic exposure, suggesting that the hypoxia-induced alterations in ANP activity were determined at the transcriptional level. A transfection study using ANP promoter–luciferase DNA constructs demonstrated that a segment of the 5'-flanking region of the ANP gene that extends from -638 to -518 bp relative to the transcriptional start site contains positive cis-regulatory elements that mediate hypoxia-induced ANP gene expression. These results support our previous in vivo findings which showed that enhanced expression of the ANP gene in atria of hypoxia-adapted rats is a function of hypoxia per se rather than of increased atrial pressure, cardiac hypertrophy, or neurohumoral factors activated by hypoxia.

In the current study, we used AT-1 atrial myocytes as a model system to study the regulation of ANP gene expression during hypoxia. The AT-1 cells are a differentiated transplantable tumor line derived from transgenic mice expressing an ANP–simian virus 40 T antigen fusion gene^{20 21} and are the only in vitro model of adult atrial myocyte currently available. Even AT-1 cells are not "natural" atrial myocytes; the cultured AT-1 cells are a useful model system for studies of ANP expression and secretion and the expression of adult cardiac-specific genes because they possess properties similar to those of adult atrial myocytes.^{20 22} Cultured AT-1 cells express adult contractile cardiac protein isoforms,²² retain ultrastructural features characteristic of cardiomyocytes, and display spontaneous electrical and contractile activity.²⁰ In addition, cultured AT-1 cells synthesize, store, and secrete ANP in a manner similar to that of adult atrial myocytes in vivo.^{22 29} In the current study, we demonstrated that 1% oxygen has no adverse effects on AT-1 cell viability for at least 7 days in culture. This in vitro model allowed us to dissociate hypoxia from increased pressure and stretch and from cardiac hypertrophy as possible stimuli for ANP synthesis and release under hypoxic conditions. Furthermore, experiments in which ANP promoter–DNA constructs were transfected into AT-1 cells defined positive cis-regulatory elements that mediate hypoxia-induced ANP gene expression.

A growing number of mammalian genes whose expression is inducible by hypoxia have been identified. These include erythropoietin,³⁰ the B chain of platelet-derived growth factor (PDGF-B),³¹ endothelin-1,³² vascular endothelial grow factor,²⁴ tyrosine hydroxylase,³³ and the glycolytic enzymes aldolase A, phosphoglycerate kinase-1, and pyruvate kinase M.³⁰ It also has been demonstrated that ANP plays an important role in the adaptation to hypoxia. Acute hypoxia stimulates the release of ANP from isolated rat and rabbit hearts and increases circulating ANP levels in anesthetized rabbits.⁵ Animals exposed to chronic hypoxia (10% oxygen at atmospheric pressure) exhibit increased pulmonary arterial pressure, plasma ANP levels, and cardiac ANP gene expression.^{10 11 12} In our previous in vivo study, we demonstrated that the PO₂ was 45±2 and 34±2 mm Hg (n=6) in arterial and venous blood, respectively, in male Sprague-Dawley rats exposed to 10% oxygen for 24 hours. In the current in vitro study, PO₂ was 20 to 30 mm Hg in the media during hypoxic (1% oxygen) exposure, which may be comparable to the tissue oxygen concentration in rats exposed to 10% oxygen. Studies from our laboratory demonstrate that hypoxia-induced ANP secretion provides a compensatory mechanism, through its vasodilator and antiproliferative effects, for modulation of the development of hypoxia-induced pulmonary hypertension and vascular remodeling in rats exposed to chronic hypoxia.^{10 11} Despite the conventional interpretation that ANP secretion responds to the distension of the atrial wall resulting from pulmonary hypertension during hypoxic exposure, there is increasing evidence that ANP responds directly to hypoxia. Acute hypoxia causes a greater release of ANP than volume loading in anesthetized rabbits⁵ and conscious lambs.¹⁷ Further hypoxia stimulates the release of ANP from isolated rat and rabbit hearts in vitro in the absence of atrial distension and changes in heart rate.¹⁸ In addition, Winter et al¹⁹ demonstrated that elevated plasma ANP levels in male Wistar rats exposed to 7 days of hypoxia (10% oxygen at atmospheric pressure) return to baseline within 24 hours of reexposure to normoxia. Stockmann et al¹² also demonstrated that chronic hypoxic exposure (10% oxygen at atmospheric pressure) results in a significant increase in ANP mRNA and peptide contents in the right and left ventricles in male Sprague-Dawley rats. The upregulation in right ventricular ANP expression induced by chronic hypoxia decreases within days of normoxic recovery, at a time when elevated right ventricular pressure and right ventricular hypertrophy are still present. Furthermore, our previous studies demonstrated that ANP mRNA expression was increased in the left atrium of male Sprague-Dawley rats during acute (<24 hours) and chronic (4 weeks) hypoxic exposure (10% oxygen at atmospheric pressure)^{34 35} as well as in both ventricles exposed to hypoxia for 4 weeks.³⁵ Since only the right ventricle was hypertrophied and only right-sided cardiac pressures were elevated in this model, our results suggest that enhanced expression of the ANP gene in the left ventricle of rats during hypoxic exposure may be a function of hypoxia per se rather than of increased pressure or a consequence of cardiac hypertrophy. These findings suggest that a reduction in oxygen tension plays a role in upregulating cardiac ANP synthesis and raising plasma ANP levels that is independent of changes in pulmonary artery pressure and cardiac tissue remodeling. Our current findings that hypoxic exposure increased steady-state ANP mRNA levels and stimulated ANP release from cultured AT-1 cells provide strong evidence in support of this conclusion.

Transfection of primary cultured AT-1 cells with ANP-luciferase hybrid genes permits assessment of cis-regulatory nucleotide fragments responsible for the regulation of ANP gene expression and modulation by hypoxia. Background luciferase expression of the pSVOAL5' vector is negligible in this system. In cells exposed to normoxia, the minimal expression of luciferase by -95/+62 ANP-pSVOAL5' hybrid genes suggests that sequences located immediately proximal to the ANP gene are insufficient to direct ANP expression. Transfection with ANP-pSVOAL5' constructs containing 188 bp of 5' ANP promoter region, including the cap site and TATA box, promoted basal levels of luciferase expression, suggesting that regulatory sequences located within this region are essential for ANP expression in atrial myocytes. A 3-kb region on the 5' side of the ANP gene (-3003/+62 ANP-pSVOAL5') promoted significantly greater luciferase expression than the shorter ANP-pSVOAL5' (-638/+62 and shorter) constructs in AT-1 cells, suggesting that a signal required for high-level atrial ANP gene expression lies in a region from -3003 to -638 bp on the 5' side of the ANP gene. These results are consistent with a previous report by Rosenzweig et al³⁶ using primary cultured neonatal rat atrial cells as a functional assay in vitro. These investigators demonstrated that at least three cis-acting regulatory elements are required for expression of the rat ANP gene in atrial myocytes. One distal cis-acting regulatory element lies more than 640 bp from the transcription initiation site, and two other elements (activator protein-1 and CRE-like [cyclic AMP response element] sites) lie within a 213-bp region located 609 bp upstream of the transcriptional initiation site. Wu et al³⁷ have demonstrated that basal human ANP promoter activity in cultured neonatal rat atrial cells decreases significantly between the -1150- and -410-bp region and drops to minimal levels when the ANP promoter is truncated to the -130-bp region. This report is consistent with our current findings in AT-1 cells exposed to normoxia. In cells exposed to hypoxia, the same modest degree of induction (range, 30% to 59%) of luciferase activity has been found in hypoxic cells transfected with all ANP-pSVOAL5' hybrid genes except -638/+62 and -95/+62 constructs, suggesting that a common signal is involved in regulating expression of these genes during hypoxic exposure. The mechanism of this common signal for the modest induction of ANP gene transcription during hypoxic exposure is not understood and needs further investigation. Since hypoxia induced a significantly greater increase (>threefold, P<.01, two-way ANOVA) in luciferase activity only in cells transfected with the -638/+62 gene construct, we suggest that putative positive hypoxia-inducible elements are contained within a region that extends from -638 to -518 bp in the 5'-flanking sequence of the ANP gene. Our results also indicated that the luciferase activity of the -638/+62 fusion gene is significantly higher than that of the -3003/+62 gene in hypoxia-exposed cells. We postulate that ANP gene expression is regulated by multiple factors that can act on different positive and negative cis-elements in the promoter region of the ANP gene. The presence of other negative regulatory elements may be responsible for the lower inducibility of the 3003-bp ANP promoter fragment in our transfection studies. In future studies, we will further dissect the ANP promoter to examine in detail the regulation of ANP gene expression.

Results obtained from electrophoretic mobility shift assays complemented the results of functional assays. Nuclear extracts prepared from AT-1 cells exposed to hypoxia for 40 hours exhibited a binding pattern different from that in AT-1 cells exposed to normoxia, indicating that specific nuclear proteins that bound to the 120-bp putative hypoxia-inducible elements of the ANP gene were induced or activated (eg, by posttranslational modification) during hypoxic exposure. The data that at least two specific DNA-protein complexes were formed when a large amount of nuclear extract of hypoxic AT-1 cells was added to the binding reaction suggest that multiple proteins might be induced or activated during hypoxic exposure and indicate the complexity of transcriptional regulation of ANP gene expression during hypoxic exposure. The observation that this specific binding pattern was absent when nuclear extracts from cells exposed to 24 hours of hypoxia were used in the reaction suggests that the induction of these hypoxia-inducible nuclear proteins was not accomplished during the first 24 hours of hypoxic exposure. This delayed response to hypoxia could explain the late onset of the increases in cellular ANP mRNA and secretion of ANP from AT-1 cells during hypoxic exposure (Figs 1 and 3).

Semenza et al³⁰ have identified a hypoxia-responsive transcriptional enhancer in the 3'-flanking region of the human erythropoietin gene that can induce the transcription of erythropoietin in response to hypoxia in cultured hepatocytes. In later studies, Wang and Semenza³⁸ purified and characterized hypoxia-inducible factor-1 (HIF-1), a nuclear protein whose DNA binding activity is induced by hypoxia. HIF-1 binds to the hypoxia enhancer element in the erythropoietin gene and acts as a physiological regulator of erythropoietin gene expression in response to changes in cellular oxygen tension. The enhancer sequence 5'-(C/G/T)ACGTGC(G/T)-3', which has been shown to bind to HIF-1, is found in genes encoding erythropoietin and the glycolytic enzymes³⁰ but is not found in the rat ANP gene.^{25 35} Further study is needed to identify precisely the hypoxia-responsive transcriptional modifier in the ANP gene sequence and to isolate and characterize protein factors that direct the transcriptional response of the ANP gene to hypoxia.

It is important to note that the time course for the increases in transcription and release of ANP in AT-1 cells (onset at 36 to 48 hours) during hypoxia is longer than previously described for the other hypoxia-inducible genes. Hypoxia-induced increases in steady-state levels of mRNA for tyrosine hydroxylase occur within 1 to 3 hours³³ ; for erythropoietin, within 2 to 6 hours³⁰ ; for endothelin-1, within 1 hour and peak at 48 hours³² ; and for PDGF-B, at more than 24 hours.³¹ We speculate that the family of hypoxia-inducible HIF-1 proteins is responsible for the early phase of hypoxia-induced gene expression (eg, erythropoietin and endothelin-1) but that factors other than HIF-1 may act on different hypoxia-regulatory elements that are responsible for the late response (eg, ANP and PDGF-B genes) during hypoxic exposures.

In summary, our present results clearly indicate that hypoxia is a powerful stimulus for ANP secretion from the isolated atrial myocyte. Our data show that hypoxic exposure activates the transcription of the ANP gene, which likely accounts for the increases in its respective mRNA and protein levels in this model system. Transfection of primary cultured AT-1 cells with recombinant ANP-luciferase genes has delineated a 120-bp region of 5'-flanking sequence that modulates ANP gene expression during hypoxic exposure. Electrophoretic mobility shift assays demonstrated that hypoxia-inducible nuclear proteins that bound to the 120-bp putative hypoxia-responsive elements of the ANP gene were produced during hypoxic exposure. Future study is needed to define precisely the hypoxia-responsive regulatory elements in this region of the ANP gene and to determine whether sequences identified by these studies are effective in enhancing ANP gene expression in intact hypoxia-adapted animals.

	Acknowledgments

 
This work was supported in part by grants HL-44195, HL-47081, HL-07457, HL-50147, HL-43124, and HL-45453 from the National Heart, Lung, and Blood Institute, National Institutes of Health, and Grant-in-Aid No. 93014260 from the American Heart Association. The authors thank Nancy Penney for her assistance in the preparation of this manuscript and Beverly Stallworth for her excellent technical assistance.

Received August 9, 1996; first decision August 9, 1996; accepted August 9, 1996.

	References

Top Abstract Introduction Methods Results Discussion References

 

1. de Bold AJ. Atrial natriuretic factor: a hormone produced by the heart. Science. 1985;230:767-770.[Abstract/Free Full Text]
2. Matsuo H, Kangawa K, Miyata A. Atrial natriuretic polypeptide (ANP) molecular forms and distribution in mammalian tissues and plasma. In: Deber CM, Hruby VJ, Kopple KD, eds. Peptides: Structure and Function. Rockford, Ill: Pierce Chemical Co; 1985:932-952.
3. Brenner BMB, Ballermann BJ, Gunning ME, Zeidel ML. Diverse biological actions of atrial natriuretic peptide. Physiol Rev. 1990;70:665-699.[Free Full Text]
4. Fyhrquist F, Sirvio ML, Helin K, Saijonmaa O, Metsarinne K, Paakkari I, Jarvinen A, Tikkanen I. Endothelin antiserum decreases volume-stimulated and basal plasma concentration of atrial natriuretic peptide. Circulation. 1993;88:1172-1176.[Abstract/Free Full Text]
5. Baertschi AJ, Adams JM, Sullivan MP. Acute hypoxiemia stimulates atrial natriuretic factor secretion in vivo. Am J Physiol. 1988;255:H295-H300.[Medline] [Order article via Infotrieve]
6. Chen YF, Jin H, Paul R, Nagahama S. Blunted pressor responsiveness to quinpirole, a specific dopamine D2 receptor agonist, in conscious DOCA/NaCl hypertensive rats is related to ANP release. J Pharmacol Exp Ther. 1988;246:485-493.[Abstract/Free Full Text]
7. Jin H, Chen YF, Yang RH, McKenna TM, Jackson RM, Oparil S. Vasopressin lowers pulmonary artery pressure in hypoxic rats by releasing atrial natriuretic peptide. Am J Med Sci. 1989;298:227-236.[Medline] [Order article via Infotrieve]
8. Naruse M, Takeyama Y, Tanabe A, Hiroshige J, Naruse K, Yoshimoto T, Tanaka M, Katagiri T, Demura H. Atrial and brain natriuretic peptides in cardiovascular diseases. Hypertension. 1994;23(suppl I):I-231-I-234.
9. Hirata Y, Suzuki E, Hayakawa H, Matsuoka H, Sugimoto T, Kojima M, Kangawa K, Matsuo H. Role of endogenous ANP in sodium excretion in rats with experimental pulmonary hypertension. Am J Physiol. 1992;262:H1684-H1689.[Medline] [Order article via Infotrieve]
10. Jin H, Chen YF, Yang RH, Jackson RM, Oparil S. Atrial natriuretic peptide clearance receptor agonist lowers pulmonary pressure in hypoxic rats. J Appl Physiol. 1990;68:2413-2418.[Abstract/Free Full Text]
11. Jin H, Yang RH, Chen YF, Jackson RM, Oparil S. Atrial natriuretic peptide attenuates the development of pulmonary hypertension in rats adapted to chronic hypoxia. J Clin Invest. 1990;85:115-120.[Medline] [Order article via Infotrieve]
12. Stockmann PT, Will DH, Sides SD, Brunnert SR, Wilner GD, Leahy KM, Wiegand RC, Needleman P. Reversible induction of right ventricular atriopeptin synthesis in hypertrophy due to hypoxia. Circ Res. 1988;63:207-213.[Abstract/Free Full Text]
13. Brun-Buisson C, Gutkowska Y, Braquet P. Atrial natriuretic peptide concentrations and pulmonary hemodynamics in patients with pulmonary artery hypertension. Am Rev Respir Dis. 1987;136:951-956.[Medline] [Order article via Infotrieve]
14. Jin H, Yang RH, Chen YF, Jackson RM, Itoh H, Mukoyama M, Nakao K, Imura H, Oparil S. Atrial natriuretic peptide in acute hypoxia-induced pulmonary hypertension in rats. J Appl Physiol. 1991;71:807-814.[Abstract/Free Full Text]
15. Oehlenschlager WF, Kurtz DT, Baron DA, Currie MG. Enhanced activity of the cardiac endocrine system during right ventricular hypertrophy. Mol Cell Endocrinol. 1989;62:243-251.[Medline] [Order article via Infotrieve]
16. Dietz JR. Release of atrial natriuretic factor from rat heart-lung preparation by atrial distension. Am J Physiol. 1984;247:R1093-R1096.[Medline] [Order article via Infotrieve]
17. Baertschi AJ, Teague WG. Alveolar hypoxia is powerful stimulus for ANF release in conscious lambs. Am J Physiol. 1989;256:H990-H998.[Medline] [Order article via Infotrieve]
18. Lew RA, Baertschi AJ. Mechanisms of hypoxia-induced atrial natriuretic factor release from rat hearts. Am J Physiol. 1988;255:H295-H300.[Medline] [Order article via Infotrieve]
19. Winter RJD, Meleagros L, Pervez S, Jamal H, Krausz T, Polak JM, Bloom SR. Atrial natriuretic peptide levels in plasma and in cardiac tissues after chronic hypoxia in rats. Clin Sci. 1989;76:95-101.[Medline] [Order article via Infotrieve]
20. Delcarpio JB, Lanson NA, Field LJ, Claycomb WC. Morphological characterization of cardiomyocytes isolated from a transplantable cardiac tumor derived from transgenic mouse atria (AT-1 cells). Circ Res. 1991;69:1591-1600.[Abstract/Free Full Text]
21. Field LJ. Atrial natriuretic factor-SV40 T antigen transgenes produce atrial tumors and cardiac arrhythmias in mice. Science. 1988;239:1029-1033.[Abstract/Free Full Text]
22. Lanson NA, Glembotski CC, Steinhelper ME, Field LJ, Claycomb WC. Gene expression and atrial natriuretic factor processing and secretion in cultured AT-1 cardiac myocytes. Circulation. 1992;85:1835-1841.[Abstract/Free Full Text]
23. Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987;162:156-159.[Medline] [Order article via Infotrieve]
24. Tuder RM, Flook BE, Voelkel NF. Increased gene expression for VEGF and the VEGF receptors KDR/Flk and Flt in lungs exposed to acute or chronic hypoxia. J Clin Invest. 1995;95:1798-1807.[Medline] [Order article via Infotrieve]
25. Argentin S, Nemer M, Drouin J, Scott GK, Kennedy BP, Davies PL. The gene for rat atrial natriuretic factor. J Biol Chem. 1985;260:4568-4571.[Abstract/Free Full Text]
26. Knowlton KU, Baracchin E, Ross RS, Harris AN, Henderson SA, Evans SM, Glembotski CC, Chien KR. Co-regulation of the atrial natriuretic factor and cardiac myosin light chain-2 genes during -adrenergic stimulation of neonatal rat ventricular cells. J Biol Chem. 1991;266:7759-7768.[Abstract/Free Full Text]
27. Dignam JD, Lebovitz RM, Roeder RG. Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids Res. 1983;11:1475-1489.[Abstract/Free Full Text]
28. Gardner DG, Hane S, Irachewsky D, Schenk D, Baxter JD. Atrial natriuretic peptide mRNA is regulated by glucocorticoids in vivo. Biochem Biophys Res Commun. 1986;139:1047-1054.[Medline] [Order article via Infotrieve]
29. Thibault G, Lazure C, Shiffrin EL, Gukowska J, Chartier L, Garcia R, Seidah NG, Chretien M, Genest J, Cantin M. Identification of a biologically active circulating form of atrial natriuretic factor. Biochem Biophys Res Commun. 1985;130:981-986.[Medline] [Order article via Infotrieve]
30. Semenza GL, Roth PH, Fang HM, Wang GL. Transcriptional regulation of genes encoding glycolytic enzymes by hypoxia-inducible factor 1. J Biol Chem. 1994;269:23757-23763.[Abstract/Free Full Text]
31. Kourembanas S, Hannan RL, Faller DV. Oxygen tension regulates the expression of the platelet-derived growth factor-B chain gene in human endothelial cells. J Clin Invest. 1990;86:670-674.[Medline] [Order article via Infotrieve]
32. Li HB, Chen SJ, Chen YF, Meng QC, Durand J, Oparil S, Elton T. Enhanced endothelin-1 and endothelin receptor gene expression in chronic hypoxia. J Appl Physiol. 1994;73:1451-1459.
33. Czyzyk-Krzeska MF, Fumari BA, Lawson EE, Millhorn DE. Hypoxia increases rate of transcription and stability of tyrosine hydroxylase mRNA in pheochromocytoma (PC12) cells. J Biol Chem. 1994;269:760-764.[Abstract/Free Full Text]
34. Chen YF, Elton TS, Bounelis P, Jin H, Oparil S. Acute hypoxia increases atrial natriuretic peptide (ANP) gene expression in left atrium of the rat. Clin Res. 1993;38:18. Abstract.
35. Chen YF, Elton TS, Bounelis P, Jin H, Oparil S. Altered atrial natriuretic peptide gene expression in atria of rats adapted to chronic normobaric hypoxia. Am J Hypertens. 1989;2:45. Abstract.
36. Rosenzweig A, Halazonetis TD, Seidman JG, Seidman CE. Proximal regulatory domains of rat atrial natriuretic factor gene. Circulation. 1991;84:1256-1265.[Abstract/Free Full Text]
37. Wu JP, Kovacic-Milivojevic B, Lapointe MC, Nakamura K, Gardner DG. CIS-Active determinants of cardiac-specific expression in the human atrial natriuretic peptide gene. Mol Endocrinol. 1991;5:1311-1322.[Abstract]
38. Wang GL, Semenza GL. Characterization of hypoxia-inducible factor 1 and regulation of DNA binding activity by hypoxia. J Biol Chem. 1993;268:21513-21518.[Abstract/Free Full Text]

This article has been cited by other articles:

				Y. Luo, C. Jiang, A. J. Belanger, G. Y. Akita, S. C. Wadsworth, R. J. Gregory, and K. A. Vincent A Constitutively Active Hypoxia-Inducible Factor-1{alpha}/VP16 Hybrid Factor Activates Expression of the Human B-Type Natriuretic Peptide Gene Mol. Pharmacol., June 1, 2006; 69(6): 1953 - 1962. [Abstract] [Full Text] [PDF]

				W. E. Hopkins, Z. Chen, N. K. Fukagawa, C. Hall, H. J. Knot, and M. M. LeWinter Increased Atrial and Brain Natriuretic Peptides in Adults With Cyanotic Congenital Heart Disease: Enhanced Understanding of the Relationship Between Hypoxia and Natriuretic Peptide Secretion Circulation, June 15, 2004; 109(23): 2872 - 2877. [Abstract] [Full Text] [PDF]

				I. E. Konstantinov, J. G. Coles, C. Boscarino, M. Takahashi, J. Goncalves, J. Ritter, and G. S. Van Arsdell Gene expression profiles in children undergoing cardiac surgery for right heart obstructive lesions J. Thorac. Cardiovasc. Surg., March 1, 2004; 127(3): 746 - 754. [Abstract] [Full Text] [PDF]

				J. R. Klinger, L. Pietras, R. Warburton, and N. S. Hill Reduced Oxygen Tension Increases Atrial Natriuretic Peptide Release from Atrial Cardiocytes Experimental Biology and Medicine, October 1, 2001; 226(9): 847 - 853. [Abstract] [Full Text] [PDF]

				B. C Kone Molecular biology of natriuretic peptides and nitric oxide synthases Cardiovasc Res, August 15, 2001; 51(3): 429 - 441. [Abstract] [Full Text] [PDF]

This Article Abstract Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar