help button home button
AJRCMB
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Published ahead of print on January 30, 2004, doi:10.1165/rcmb.2003-0287OC
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
2003-0287OCv1
30/6/908    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Hassoun, P. M.
Right arrow Articles by Bloch, K. D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Hassoun, P. M.
Right arrow Articles by Bloch, K. D.
American Journal of Respiratory Cell and Molecular Biology. Vol. 30, pp. 908-913, 2004
© 2004 American Thoracic Society
DOI: 10.1165/rcmb.2003-0287OC

Hypoxia Decreases Expression of Soluble Guanylate Cyclase in Cultured Rat Pulmonary Artery Smooth Muscle Cells

Paul M. Hassoun, Galina Filippov, Michael Fogel, Cameron Donaldson, Usamah S. Kayyali, Larissa A. Shimoda and Kenneth D. Bloch

Division of Pulmonary and Critical Care Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland; Pulmonary and Critical Care Division, Department of Medicine, Tufts University School of Medicine, New England Medical Center, Boston; and Cardiovascular Research Center, Massachusetts General Hospital, Charlestown, Massachusetts

Address correspondence to: Paul M. Hassoun, M.D., Associate Professor of Medicine, Johns Hopkins University School of Medicine, 5501 Hopkins Bayview Circle, Baltimore, MD 21224. E-mail: phassoun{at}jhmi.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Nitric oxide (NO) has an important role in modulating the pulmonary vascular tone. NO acts, in part, by stimulating soluble guanylate cyclase (sGC) to synthesize the intracellular second messenger cyclic GMP. In vascular smooth muscle cells, sGC is a heterodimer composed of {alpha}1 and ß1 subunits. The objective of this study was to test whether oxygen concentration regulates sGC expression in cultured rat pulmonary artery smooth muscle cells (rPaSMC). rPaSMC were exposed to 0, 3, and 20% oxygen for 1–48 h, and sGC subunit mRNA levels were measured. Compared with rPaSMC exposed to 20% oxygen, sGC {alpha}1 and ß1 subunit mRNA levels were markedly decreased in rPaSMC exposed to 0% and 3% oxygen. The decrease in sGC subunit mRNA levels in hypoxic rPaSMC was detected as early as 6 h of exposure. Compared with rPaSMC exposed to 20% oxygen, exposure of rPaSMC to 3% oxygen progressively decreased sGC subunit protein levels at 24 and 48 h. There was also a 30% and 50% decrease in sGC enzyme activity in cells exposed to hypoxia for 24 and 48 h (P < 0.05 and P < 0.001, respectively, as compared with cells maintained in normoxia). These results demonstrate that hypoxia decreases sGC expression in cultured pulmonary artery smooth muscle cells and suggest that, in hypoxic vascular smooth muscle, decreased cyclic GMP synthesis may limit the vasodilator response to NO.

Abbreviations: cyclic GMP, cGMP • nitric oxide, NO • particulate guanylate cyclase, pGC • rat pulmonary artery smooth muscle cells, rPaSMC • sodium dodecyl sulfate–polyacrylamide gel electrophoresis, SDS-PAGE • soluble guanylate cyclase, sGC


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
In contrast to the systemic vasculature, the pulmonary arterial vasculature is a low resistance system, which responds to decreased oxygen tension with vasoconstriction and vascular remodeling. The expression and release of several vasoactive substances, such as the vasoconstrictor endothelin or the vasodilator nitric oxide, appear to be altered in response to acute or chronic hypoxia (1–3). Endothelial-derived nitric oxide (NO), which results from the conversion of L-arginine to L-citrulline through the action of endothelial nitric oxide synthase 3 (NOS3), has a critical role in the regulation of pulmonary vascular tone (4).

NO acts, in part, through stimulation of soluble guanylate cyclase (sGC) to convert GTP to cyclic GMP (5). sGC is a heterodimer composed of {alpha} and ß subunits, which are both necessary for catalytic activity (6). Two isoforms of each subunit ({alpha}1, {alpha}2, ß1, and ß2) are encoded in the rat genome (7), with {alpha}1 and ß1 subunits being the predominant isoforms in the lung and vascular smooth muscle cells (8). Although the effects of oxygen tension on the expression of NOS have been extensively studied in vitro (2, 9) and in vivo (3, 10, 11), information related to the modulation of sGC by oxygen is scant and controversial. Crawley and colleagues reported that sGC function was impaired in pulmonary arteries from rats exposed to 10% oxygen, leading to decreased sodium nitroprusside-induced cyclic GMP (cGMP) accumulation and vasorelaxation (12). On the other hand, Li and coworkers recently reported that sGC levels were increased in lungs from rats exposed to 10% oxygen (13). The objective of this study was to investigate the regulation of sGC in cultured rat pulmonary artery smooth muscle cells (rPaSMC) in response to varying concentrations of oxygen. The results indicate that hypoxia decreases sGC expression in these cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
rPaSMC
Cultures of rPaSMC were prepared from explants of endothelium- and adventitia-stripped pulmonary arteries of adult Sprague-Dawley rats, as previously described by Yu and colleagues (14). The cells were maintained in RPMI supplemented with 10% NuSerum (Collaborative Biomedical Products, Bedford, MA), penicillin, and streptomycin and were studied between passages 3 and 10.

Exposure of Cells to Oxygen
Cells were placed in humidified airtight incubation chambers (Billups-Rothenberg, Del Mar, CA) and gassed with the desired concentration of oxygen (0, 3, and 20% oxygen), 5% CO2, and balance N2. The chambers were maintained in a New Brunswick incubator for the duration of exposure. The concentration of oxygen in the chambers was routinely checked using an oxygen analyzer (Beckman LB-2 O2 analyzer, Fullerton, CA) and was consistently within 2% of the desired level. The partial pressures of oxygen in cell culture media were typically 140–150 mm Hg for 20% oxygen exposure, 20–40 mm Hg for 3% oxygen, and 7–10 mm Hg for 0% oxygen. The concentration of CO2 in the chambers was also monitored (Beckman OM-11 CO2 analyzer) and was consistently 4–6%. Exposure of SMC to 0, 3, and 20% oxygen for up to 48 h did not cause cell toxicity as routinely determined by cell morphology under phase contrast microscopy and trypan blue exclusion.

RNA Blot Hybridization
RNA was extracted from rPaSMC in RNAzol according to the manufacturer's instructions. Total cellular RNA (10 µg) was fractionated in 1.3% agarose-formaldehyde gels containing ethidium bromide, transferred to nylon membranes (MAGNA CHARGE; Micron Separations, Westborough, MA), and hybridized with 32P-radiolabeled rat sGC {alpha}1 and ß1 subunit cDNA probes, as described previously (15, 16). Equal loading of RNA on gels was confirmed by staining 28S and 18S ribosomal RNA with ethidium bromide. All RNA blots shown are representative of at least three experiments. Films were scanned using a Color Imager Scanner (Seiko Epson Corp., Tokyo, Japan) and the National Institutes of Health Image 1.44 software. mRNA levels were estimated from the ratio of the absorbance on X-ray films corresponding to the sGC subunit mRNA divided by the absorbance on photographs corresponding to 28S ribosomal RNA.

Immunoblots for Rat sGC {alpha}1 and ß1 Subunits
rPaSMC were homogenized in buffer containing 50 mM Tris-HCl (pH 7.6), 1 mM EDTA, 1 mM dithiothreitol, and 2 mM phenylmethylsulfonyl fluoride. Extracts were centrifuged at 100,000 x g for 1 h at 4°C. Cell supernatants containing 30 µg of protein were fractionated by 8% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), transferred electrophoretically to nitrocellulose filters (Micron Separations Inc.), and incubated with polyclonal antisera directed against the rat sGC {alpha}1 or ß1 subunit, as described previously (16, 17). Bound antibodies were detected using horseradish peroxidase–protein A, enhanced chemiluminescence (Enhanced Chemiluminescence Kit; Amersham Life Sciences, Arlington Heights, IL) and exposure to X-ray film. Coomassie blue staining was performed on all SDS-polyacrylamide gels run in parallel to ensure equal protein loading. Films were scanned using a Color Imager Scanner (Seiko Epson Corp., Tokyo, Japan) and the National Institutes of Health Image 1.44 software. sGC subunit protein concentrations were estimated by measuring the absorbance on X-ray films corresponding to each sGC subunit.

Intracellular pH Measurements
rPaSMC were placed in a laminar flow cell chamber perfused with physiologic saline solution containing (in mM): 118.3 NaCl, 4.7 KCl, 1.2 MgSO4, 25 NaHCO3, 1.1 glucose, and 1.2 KH2PO4. pHi was measured in cells incubated with membrane permeant (acetoxymethyl ester) form of the pH-sensitive fluorescent dye 2',7'-bis(carboxyethyl)-5(6)-carboxyflourescein (BCECF-AM) for 60 min at 37°C under an atmosphere of 3% O2–5% CO2. Cells were then washed with physiologic saline solution for 10 min at 37°C to remove extracellular dye and allow complete de-esterification of cytosolic dye. Ratiometric measurement of fluorescence from the dyes was performed on a workstation (Intracellular Imaging Inc., Cincinnati, OH) consisting of a Nikon TSE 100 Ellipse inverted microscope with epifluorescence attachments (Nikon, Melville, NY). The light beam from a xenon arc lamp was filtered by interference filters at 490 and 440 nm and focused onto the individual rPaSMCS under examination via a x20 fluorescence objective (Super Fluor 20; Nikon). Light emitted from each cell at 530 nm was returned through the objective and detected by a cooled CCD imaging camera. An electronic shutter (Sutter Instruments, Novato, CA) was used to minimize photobleaching of dye. Protocols were executed and data collected on-line with InCyte software (Intracellular Imaging Inc.). Resting 490/440 ratio values were calculated for each cell as the mean of the values measured for 5 min. [H+]i and pHi were then estimated from in situ calibration for each cell after each experiment. Cells were perfused with a solution containing (in mM): 105 KCl, 1 MgCl2, 1.5 CaCl2, 10 glucose, 20 HEPES-Tris, and 0.01 nigericin to allow pHi to equilibrate to external pH. A two point calibration was created from fluorescence measured as pHi was adjusted with KOH from 6.5–7.5.

Soluble Guanylate Cyclase Enzyme Activity
sGC enzyme activity was measured, as described by Mittal (18), in cell extracts containing 30 µg of protein incubated for 10 min at 37°C in a reaction mixture containing 50 mM Tris-HCl, pH 7.5, 4 mM MgCl2, 0.5 mM 1-methyl-3-isobutyl-xanthine, 7.5 mM creatinine phosphate, 0.2 mg/ml creatinine phosphokinase, 1 mM GTP, and 1 mM sodium nitroprusside. The reaction was terminated by addition of 0.9 ml 0.05 N HCl and boiling for 3 min. Newly synthesized cGMP was measured in the reaction mixture using a commercial RIA kit (Biomedical Technologies, Inc., Stoughton, MA). The enzyme activity was expressed as picomoles of cGMP produced per minute per milligram protein in the cell supernatant (mean ± SD). sGC enzyme activities were compared by a factorial model of ANOVA. When significant differences were detected, Fisher's analysis was used post hoc to compare groups. Significance was declared if P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Exposure of rPaSMC to Hypoxia Decreases sGC Subunit Gene Expression
To examine the effects of hypoxia on sGC expression, sGC subunit mRNA levels were compared in rPaSMC exposed to 3% and 20% O2 for various periods of time. To avoid differences in cell confluence between groups, all cells were collected at the same time after the desired oxygen and time exposure. As described previously (16), high levels of sGC subunit gene expression were detected in normoxic rPaSMC (Figure 1). Exposure of rPaSMC to 3% oxygen for 6 and 24 h significantly decreased sGC {alpha}1 and ß1 subunit mRNA levels (Figure 1). A further decrease in mRNA levels was observed when cells were exposed to 3% oxygen for 48 h (results not shown).




View larger version (95K):
[in this window]
[in a new window]
  		Figure 1. Exposure of rPaSMC to hypoxia decreases {alpha}1 and ß1 sGC subunit mRNA expression in a time-dependent fashion. (A) RNA extracted from rPaSMC incubated in normoxia (20% oxygen) or hypoxia (3% oxygen) for 1, 6, and 24 h was hybridized with sGC subunit cDNAs. A photograph of 28S ribosomal RNA in the ethidium-containing agarose gel is shown to confirm equal loading of RNA samples. There was a progressive decrease of both {alpha}1 and ß1 mRNA levels by hypoxia. (B) Densitometric ratios of sGC subunit mRNA over 28S ribosomal RNA (black and gray bars for {alpha}1 and ß1 sGC subunit, respectively). *P < 0.05 versus time 0.

 
To determine the effects of oxygen concentration on sGC subunit gene expression, confluent rPaSMC were exposed to 0, 3, and 20% oxygen for a period of 24 h. As shown in Figure 2, sGC subunit mRNA levels correlated with the oxygen concentration to which the cells were exposed, with a progressive decrease in sGC subunit {alpha}1 and ß1 mRNA levels in cells exposed to 3% and 0% oxygen, as compared with cells maintained in 20% oxygen. Exposure to 0, 3, and 20% oxygen for 24 h did not cause cell toxicity as determined by cell morphology under phase contrast microscopy or trypan blue exclusion. To further confirm that the decrease in sGC subunit mRNA levels in hypoxic RPaSMC was not attributable to cell toxicity, rPaSMC were exposed to hypoxia (3% oxygen) for 24 h and then returned to normoxia for another 24 h (total of 48 h exposure) before collection. sGC subunit mRNA levels did not differ in rPaSMC exposed to normoxia for 48 h from that in rPaSMC exposed to hypoxia for 24 h followed by normoxia for 24 h (not shown).



View larger version (89K):
[in this window]
[in a new window]
  		Figure 2. Effect of oxygen concentration on sGC subunit mRNA levels RNA extracted from rPaSMC that had been exposed to 0, 3, and 20% oxygen for 24 h was hybridized with sGC subunit cDNAs. A photograph of 28S ribosomal RNA in the ethidium-containing agarose gel is shown to confirm equal loading of RNA samples. sGC {alpha}1 and ß1 subunit mRNA levels correlated with the oxygen concentration to which the cells were exposed, such that the lowest levels were observed at 0% oxygen.

 
Hypoxia Decreases sGC Subunit Protein Levels and NO-Stimulated sGC Activity
To determine whether the hypoxia-induced decrease in sGC subunit gene expression was associated with a decrease in sGC subunit protein levels, cytosolic extracts of rPaSMC exposed to 3% oxygen for 24 and 48 h were fractionated by SDS-PAGE. Proteins were transferred to nitrocellulose membranes, which were then incubated with sGC subunit-specific antisera. Both sGC {alpha}1 and ß1 subunits were detected in normoxic rPaSMC (Figure 3A). Following exposure to 3% oxygen, there was a progressive decline in {alpha}1 and ß1 subunit protein levels at 24 and 48 h of exposure. At the end of the 48-h exposure to hypoxia, the residual protein expression for both subunits was 30% of control normoxic cells (Figure 3B).




View larger version (38K):
[in this window]
[in a new window]
  		Figure 3. Exposure of rPaSMC to hypoxia decreases {alpha}1 and ß1 sGC protein expression. (A) Immunoblots were prepared from extracts of rPaSMC incubated under hypoxic condition for 24 and 48 h. sGC subunit levels were measured using isoform-specific anti-sGC subunit antisera. Coomassie blue staining of the gels was used to ensure equal protein loading. Levels of sGC {alpha}1 and ß1 subunit protein were decreased at 24 h with a further decrease at 48 h. (B) Densitometric assessment of {alpha}1 (black bars) and ß1 (gray bars) subunit protein expression (n = 3 for each evaluation). Expression for both subunits at 48 h of hypoxic exposure was reduced to 30% of control (normoxic) expression. *P < 0.002 versus control. +P < 0.0002 versus control.

 
To examine whether the decrease in sGC subunit mRNA and protein levels in hypoxic cells caused a similar decrease in sGC enzyme activity, rPaSMC were exposed to normoxia (20% oxygen) or hypoxia (3% O2) for 24 and 48 h. After exposure, cytosolic extracts were prepared and NO-stimulated sGC activity was measured. As shown in Figure 4, exposure to 3% oxygen for 24 and 48 h decreased NO-stimulated sGC enzyme activity by 30% (P < 0.05 versus control normoxic cells) and 50% (P < 0.001 versus control normoxic cells), respectively. However, exposure to 3% O2 for 24 or 48 h did not alter intracellular H+ anion concentration or pH (Figure 5), suggesting that, unlike changes previously reported from cardiomyocytes (19), the decrease in cGMP synthesis in response to hypoxia was not due to pH alteration.



View larger version (15K):
[in this window]
[in a new window]
  		Figure 4. Exposure of rPaSMC to hypoxia significantly decreases sGC enzymatic activity NO-stimulated sGC enzyme activity was measured in extracts of rPaSMC incubated in normoxia (control) and hypoxia for 24 and 48 h. NO-stimulated sGC enzyme activity decreased in hypoxic rPaSMC by 30% and 50% at 24 and 48 h, respectively. *P < 0.05 versus control. +P < 0.001 versus control.

 


View larger version (20K):
[in this window]
[in a new window]
  		Figure 5. Effect of hypoxia exposure on intracellular [H+] concentration and pH. Exposure of SMC to 3% oxygen for 24 and 48 h did not alter intracellular [H+] and pH. Values represent means ± SD of measurements obtained from at least 40 different cells for each time point and oxygen exposure.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Endothelial-derived NO and its major target in smooth muscle cells, sGC, are thought to play an important role in maintaining a low resistance in the pulmonary vascularature. Through stimulation by NO, sGC converts GTP to cGMP, an intracellular second messenger that activates cGMP-dependent protein kinase as well as other cGMP receptor proteins, resulting in relaxation of smooth muscle cells among other phenomena. The distribution of sGC throughout the pulmonary vasculature is uneven, with greater expression reported to be present in venules and capillaries as compared with proximal pulmonary arteries (13, 20). There are also significant differences in sGC expression between the fetal and adult lungs. sGC is thought to play a significant vasodilatory role in the developing lung as evidenced by the abundant distribution of this enzyme in resistance vessels (small arteries and preacinar veins) of the fetal pulmonary circulation (21). Although abundant pulmonary sGC remains present in the perinatal period, presumably contributing to the profound NO-mediated pulmonary vasodilation characteristic of that period (15), there appears to be comparatively less staining in the adult circulation, in particular in large and medium-sized pulmonary arteries (13). However, the importance of the sGC system in the control of pulmonary vascular tone in the adult lung is illustrated by the fact that inhibitors of cGMP-specific phosphodiesterases can significantly vasodilate the pulmonary circulation (22) and enhance the effect of NO (23). Therefore, changes in sGC expression are likely to affect physiologic responses to NO, such as vasorelaxation, and cell functions regulated by activation of cGMP-dependent protein kinases and other cGMP receptor proteins (24). In this report, we observed that sGC subunit mRNA and protein levels, as well as NO-stimulated sGC activity, were significantly decreased in pulmonary artery smooth muscle cells exposed to low oxygen concentrations for up to 48 h.

Several factors are known to regulate smooth muscle cell sGC expression. In rPASMC, NO and NO donors decrease sGC subunit mRNA stability by transcription- and translation-dependent mechanisms (17) associated with a decrease sGC protein expression and enzymatic activity (17, 25–27). Analogues of cAMP decrease sGC subunit mRNA and enzyme activity in rat fetal lung fibroblasts (28), aortic smooth muscle cells (29), and pheochromocytoma (PC12) cells (30). Similarly, agents such as sodium nitroprusside and SIN-1, which increase intracellular cGMP, also decrease sGC subunit mRNA levels and enzymatic activity in rat medullary interstitial cells (27). Nerve growth factor has been shown to decrease sGC subunit mRNA levels (via mechanisms dependent on protein tyrosine phosphorylation and Ras activation) and enzymatic activity in rat pheochromocytoma PC12 cells (30). More recently, Takata and coworkers have demonstrated that cytokines, such as interleukin-1{alpha} and tumor necrosis factor-{alpha}, decrease sGC through NO-dependent and -independent mechanisms (16).

The effect of hypoxia on sGC expression has been a source of controversy. Using rats exposed to hypoxia for 2 and 7 d, Crawley and colleagues (12) demonstrated that acetylcholine and sodium nitroprusside relaxed rings obtained from main pulmonary arteries of normoxic animals by elevating cGMP, but failed to do so in rings obtained from hypoxic animals. The authors concluded from these indirect studies that chronic hypoxic exposure results in a selective impairment of sGC activity in rat pulmonary arteries (12).

However, a report by Li and associates (13) suggests that exposure of rats to hypoxia (1–21 d) significantly upregulates pulmonary sGC expression as assessed by immunoblot analysis, immunohistochemistry, and sGC enzyme activity. The authors demonstrated that, in lungs of rats exposed to hypoxia, pulmonary sGC {alpha}1-subunit protein levels increased 2-fold (at 3, 5, and 21 d of hypoxia), and increased expression of sGC was detected in the smooth muscle cells of the pulmonary arteries and arterioles (13). The discrepancy between the latter study showing increased expression of sGC in response to hypoxia, and the present study showing decreased expression of sGC in response to hypoxia, is difficult to reconcile at the present time. Both studies examined expression of sGC in response to hypoxia in the same species. However, the study by Li and coworkers examined sGC expression in distal pulmonary artery smooth muscle cells, and presumably newly muscularized arterioles, whereas the present study, as well as the study by Crawley and colleagues, studied the impact of hypoxia on smooth muscle cells from the proximal pulmonary arteries. Other obvious differences between the study by Li and coworkers and the present study include length of exposure to hypoxia (days versus hours of hypoxia exposure) and in vivo versus in vitro experiments, respectively.

There have been previous examples of discrepancy in findings between in vivo and in vitro studies, in particular when they relate to hypoxia. A relevant example is the response of endothelial NOS (eNOS) expression to a hypoxic stimulus. Reports by McQuillan and associates (9) and Liao and colleagues (2), both done on cultured endothelial cells, showed decreased eNOS gene expression in response to acute hypoxia (i.e., 24 h). Conversely, studies by Shaul and coworkers (31) and Le Cras and associates (10), both using chronic exposure of rats to hypoxia (i.e., 7 d and 3 wk, respectively), found increased pulmonary eNOS expression. Although such divergence in the NO/cGMP expression between in vivo and in vitro systems has not been adequately explained, several reasons could be invoked. Cell–cell interaction and signaling are phenomena likely to significantly alter individual gene expression in vivo. For instance, decreased or increased endothelial NOS expression may directly affect smooth muscle cell cGC expression. For example, the increase in pulmonary sGC expression observed in vivo in response to chronic hypoxia (13) appears to be related to endothelial NOS expression or eNOS-derived NO, because congenital disruption of eNOS is associated with lower concentrations of lung cGMP and no change in sGC protein levels as compared with wild-type (eNOS+/+) animals exposed to normoxic conditions or during the development of hypoxia-induced pulmonary hypertension (32). Shear stress, which is operative in vivo but not in usual in vitro conditions, is quite likely to alter both NOS and sGC expression. Shear stress is known to upregulate eNOS expression (33), and there is evidence that it might also stimulate the sGC/cGMP system. For example, shear stress has been reported, at least in cultured endothelial cells, to elevate cGMP via an NO-dependent mechanism. This effect appears to be mediated by a unique signal transduction pathway that is coupled to a pertussis toxin–sensitive G protein and which requires the activity of an endothelial potassium channel (34). Stimulation of the cGMP system by shear stress was also demonstrated in vivo by Qiu and coworkers (35). Flow-induced release of cGMP was determined to be significantly greater in spontaneously hypertensive rats as compared with control Wistar Kyoto rats. The authors concluded that upregulation of the NO/cGMP pathway occurred to compensate for the increased vascular tone in spontaneously hypertensive rats (5). Therefore, one might speculate that increased expression of sGC observed in vivo may be related, at least in part, to the observed elevation in pulmonary artery pressure in the hypoxic animals (13) as well as shear stress involving the pulmonary circulation.

Our findings of decreased cellular cGMP synthesis in response to hypoxia are consistent with a recent report by Agullo and colleagues describing a significant decrease in cGMP synthesis in cardiomyocytes in response to ischemia (19). However, significant differences between the two studies are worth noting. Although Agullo and coworkers observed a > 50% decrease in particulate GC (pGC)-dependent synthesis, they reported no change in sGC-dependent cGMP synthesis. In addition, the decrease in pGC-mediated synthesis was related to intracellular acidosis. In the present study, intracellular pH was unaltered by hypoxia and, therefore, could not account for changes in GC. Discrepancies in findings related to sGC may be related to differences in hypoxia exposure time (2 h in the study by Agullo and associates versus several hours in the present study) and differences in cell types (cardiomyocytes versus SMC). In addition, whereas our studies examined the effects of hypoxia on sGC gene expression and activity, conclusions by Agullo and coworkers inferring changes in GC were reached indirectly (i.e., assessment of cGMP synthesis in response to NO donors and natriuretic peptides for sGC- and pGC-mediated cGMP changes, respectively).

Finally, we demonstrated that both sGC gene expression and activity were significantly decreased by exposure to hypoxia and, therefore, postulated that these changes would result in decreased cellular cGMP. However, decreased cGMP may also be the result of increased phosphodiesterase activity as recently reported by Jernigan and Resta (36). In that study, the authors demonstrated that altered vasodilator reactivity to NO in rats exposed to chronic hypoxia for 4 wk is related to increased degradation of cGMP by the cGMP-specific phosphodiesterase 5, as well as decreased pulmonary vascular smooth muscle sensitivity to cGMP (36). Therefore, decreased cGMP synthesis and/or availability in response to hypoxia may be multifactorial.

The present study was limited to the expression and activity of sGC in response to hypoxia. However, cGMP can also be synthesized by the natriuretic pathway which operates through pGC. This pathway appears to be the primary source of cGMP synthesis in hypoxia-induced pulmonary hypertension (37). In addition, exposure of animals to hypoxia elevates both atrial and brain natriuretic peptides (ANP and BNP), which act through cGMP to limit the rise in pulmonary vascular resistance and right ventricular load in response to chronic hypoxic exposure (38). The role of the natriuretic pathway in chronic hypoxia is further demonstrated by the higher basal right ventricular systolic pressure in mice lacking the guanylyl-cyclase-linked natriuretic peptide receptor NPR-A (NPR-A–/–), as compared with wild-type mice (NPR-A+/+) (39). Taken together, these studies suggest that, as opposed to sGC which is downregulated by hypoxia, pulmonary vascular pGC may respond to increased ANP and BNP levels to modulate vasoconstriction in response to hypoxia.

In summary, this report suggests that hypoxia downregulates sGC subunit gene and protein expression as well as enzyme activity in cultured rPaSMC. We speculate that mechanisms other than the direct effects of hypoxia (e.g., shear stress, eNOS-derived NO) on sGC subunit gene regulation might be responsible for the upregulation of lung sGC previously observed in vivo (13).


   Acknowledgments
 
The authors thank Ms. Rebecca Nowak for technical assistance. This work was supported by grants from NHLBI, HL49441 (P.M.H.), HL07208 (G.F.), and HL 55377 (K.D.B.), the American Lung Association (U.S.K.), and the Massachusetts Tobacco Control Program (U.S.K.). During the course of these studies K.D.B. was supported by an Established Investigator Award from the American Heart Association.

Received in original form July 31, 2003

Received in final form December 18, 2003


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Kourembanas, S., P. A. Marsden, L. P. McQuillan, and D. V. Faller. 1991. Hypoxia induces endothelin gene expression and secretion in cultured human endothelium. J. Clin. Invest. 88:1054–1057.
  2. Liao, J. K., J. J. Zulueta, F. S. Yu, H. B. Peng, C. G. Cote, and P. M. Hassoun. 1995. Regulation of bovine endothelial constitutive nitric oxide synthase by oxygen. J. Clin. Invest. 96:2661–2666.
  3. Xue, C., A. Rengasamy, T. D. Le Cras, P. A. Koberna, G. C. Dailey, and R. A. Johns. 1994. Distribution of NOS in normoxic vs. hypoxic rat lung: upregulation of NOS by chronic hypoxia. Am. J. Physiol. 267:L667–L678.
  4. Moncada, S., and A. Higgs. 1993. The L-arginine-nitric oxide pathway. N. Engl. J. Med. 329:2002–2012.[Free Full Text]
  5. Schmidt, H. H., and U. Walter. 1994. NO at work. Cell 78:919–925.[CrossRef][Medline]
  6. Buechler, W. A., M. Nakane, and F. Murad. 1991. Expression of soluble guanylate cyclase activity requires both enzyme subunits. Biochem. Biophys. Res. Commun. 174:351–357.[CrossRef][Medline]
  7. Wong, S. K., and D. L. Garbers. 1992. Receptor guanylyl cyclases. J. Clin. Invest. 90:299–305.
  8. Nakane, M., K. Arai, S. Saheki, T. Kuno, W. Buechler, and F. Murad. 1990. Molecular cloning and expression of cDNAs coding for soluble guanylate cyclase from rat lung. J. Biol. Chem. 265:16841–16845.[Abstract/Free Full Text]
  9. McQuillan, L. P., G. K. Leung, P. A. Marsden, S. K. Kostyk, and S. Kourembanas. 1994. Hypoxia inhibits expression of eNOS via transcriptional and posttranscriptional mechanisms. Am. J. Physiol. 267:H1921–H1927.
  10. Le Cras, T. D., C. Xue, A. Rengasamy, and R. A. Johns. 1996. Chronic hypoxia upregulates endothelial and inducible NO synthase gene and protein expression in rat lung. Am. J. Physiol. 270:L164–L170.
  11. Xue, C., and R. A. Johns. 1996. Upregulation of nitric oxide synthase correlates temporally with onset of pulmonary vascular remodeling in the hypoxic rat. Hypertension 28:743–753.[Abstract/Free Full Text]
  12. Crawley, D. E., L. Zhao, M. A. Giembycz, S. Liu, P. J. Barnes, R. J. Winter, and T. W. Evans. 1992. Chronic hypoxia impairs soluble guanylyl cyclase-mediated pulmonary arterial relaxation in the rat. Am. J. Physiol. 263:L325–L332.
  13. Li, D., N. Zhou, and R. A. Johns. 1999. Soluble guanylate cyclase gene expression and localization in rat lung after exposure to hypoxia. Am. J. Physiol. 277:L841–L847.
  14. Yu, F. S., S. L. Lee, J. J. Lanzillo, and B. L. Fanburg. 1989. Endothelial cell inhibition of hypoxia-induced stimulation of serotonin uptake by vascular smooth muscle cells. Am. Rev. Respir. Dis. 139:1144–1148.[Medline]
  15. Bloch, K. D., G. Filippov, L. S. Sanchez, M. Nakane, and S. M. de la Monte. 1997. Pulmonary soluble guanylate cyclase, a nitric oxide receptor, is increased during the perinatal period. Am. J. Physiol. 272:L400–L406.
  16. Takata, M., G. Filippov, H. Liu, F. Ichinose, S. Janssens, D. B. Bloch, and K. D. Bloch. 2001. Cytokines decrease sGC in pulmonary artery smooth muscle cells via NO-dependent and NO-independent mechanisms. Am. J. Physiol. Lung Cell. Mol. Physiol. 280:L272–L278.[Abstract/Free Full Text]
  17. Filippov, G., D. B. Bloch, and K. D. Bloch. 1997. Nitric oxide decreases stability of mRNAs encoding soluble guanylate cyclase subunits in rat pulmonary artery smooth muscle cells. J. Clin. Invest. 100:942–948.[Medline]
  18. Mittal, C. K. 1986. Determination of adenylate cyclase and guanylate cyclase activities in cells of the immune system. Methods Enzymol. 132:422–428.[Medline]
  19. Agullo, L., D. Garcia-Dorado, N. Escalona, M. Ruiz-Meana, J. Inserte, and J. Soler-Soler. 2003. Effect of ischemia on soluble and particulate guanylyl cyclase-mediated cGMP synthesis in cardiomyocytes. Am. J. Physiol. Heart Circ. Physiol. 284:H2170–H2176.[Abstract/Free Full Text]
  20. Bina, S., J. L. Hart, Y. Sei, and S. M. Muldoon. 1998. Factors contributing to differences in the regulation of cGMP in isolated porcine pulmonary vessels. Eur. J. Pharmacol. 351:253–260.[CrossRef][Medline]
  21. D'Angelis, C. A., P. A. Nickerson, R. H. Steinhorn, and F. C. Morin, III. 1998. Heterogeneous distribution of soluble guanylate cyclase in the pulmonary vasculature of the fetal lamb. Anat. Rec. 250:62–69.[CrossRef][Medline]
  22. Dukarm, R. C., J. A. Russell, F. C. Morin, III, B. J. Perry, and R. H. Steinhorn. 1999. The cGMP-specific phosphodiesterase inhibitor E4021 dilates the pulmonary circulation. Am. J. Respir. Crit. Care Med. 160:858–865.[Abstract/Free Full Text]
  23. Steinhorn, R. H., J. B. Gordon, and M. L. Tod. 2000. Site-specific effect of guanosine 3',5'-cyclic monophosphate phosphodiesterase inhibition in isolated lamb lungs. Crit. Care Med. 28:490–495.[CrossRef][Medline]
  24. Lincoln, T. M., and T. L. Cornwell. 1993. Intracellular cyclic GMP receptor proteins. FASEB J. 7:328–338.[Abstract]
  25. Papapetropoulos, A., C. Y. Go, F. Murad, and J. D. Catravas. 1996. Mechanisms of tolerance to sodium nitroprusside in rat cultured aortic smooth muscle cells. Br. J. Pharmacol. 117:147–155.[Medline]
  26. Scott, W. S., and D. K. Nakayama. 1998. Sustained nitric oxide exposure decreases soluble guanylate cyclase mRNA and enzyme activity in pulmonary artery smooth muscle. J. Surg. Res. 79:66–70.[CrossRef][Medline]
  27. Ujiie, K., L. Hogarth, R. Danziger, J. G. Drewett, P. S. Yuen, I. H. Pang, and R. A. Star. 1994. Homologous and heterologous desensitization of a guanylyl cyclase-linked nitric oxide receptor in cultured rat medullary interstitial cells. J. Pharmacol. Exp. Ther. 270:761–767.[Abstract/Free Full Text]
  28. Shimouchi, A., S. P. Janssens, D. B. Bloch, W. M. Zapol, and K. D. Bloch. 1993. cAMP regulates soluble guanylate cyclase beta 1-subunit gene expression in RFL-6 rat fetal lung fibroblasts. Am. J. Physiol. 265:L456–L461.
  29. Papapetropoulos, A., N. Marczin, G. Mora, A. Milici, F. Murad, and J. D. Catravas. 1995. Regulation of vascular smooth muscle soluble guanylate cyclase activity, mRNA, and protein levels by cAMP-elevating agents. Hypertension 26:696–704.[Abstract/Free Full Text]
  30. Liu, H., T. Force, and K. D. Bloch. 1997. Nerve growth factor decreases soluble guanylate cyclase in rat pheochromocytoma PC12 cells. J. Biol. Chem. 272:6038–6043.[Abstract/Free Full Text]
  31. Shaul, P. W., A. J. North, T. S. Brannon, K. Ujiie, L. B. Wells, P. A. Nisen, C. J. Lowenstein, S. H. Snyder, and R. A. Star. 1995. Prolonged in vivo hypoxia enhances nitric oxide synthase type I and type III gene expression in adult rat lung. Am. J. Respir. Cell Mol. Biol. 13:167–174.[Abstract]
  32. Li, D., V. E. Laubach, and R. A. Johns. 2001. Upregulation of lung soluble guanylate cyclase during chronic hypoxia is prevented by deletion of eNOS. Am. J. Physiol. Lung Cell. Mol. Physiol. 281:L369–L376.[Abstract/Free Full Text]
  33. Nishida, K., D. G. Harrison, J. P. Navas, A. A. Fisher, S. P. Dockery, M. Uematsu, R. M. Nerem, R. W. Alexander, and T. J. Murphy. 1992. Molecular cloning and characterization of the constitutive bovine aortic endothelial cell nitric oxide synthase. J. Clin. Invest. 90:2092–2096.
  34. Ohno, M., G. H. Gibbons, V. J. Dzau, and J. P. Cooke. 1993. Shear stress elevates endothelial cGMP. Role of a potassium channel and G protein coupling. Circulation 88:193–197.[Abstract/Free Full Text]
  35. Qiu, H. Y., D. Henrion, J. Benessiano, C. Heymes, B. Tournier, and B. I. Levy. 1998. Decreased flow-induced dilation and increased production of cGMP in spontaneously hypertensive rats. Hypertension 32:1098–1103.[Abstract/Free Full Text]
  36. Jernigan, N. L., and T. C. Resta. 2002. Chronic hypoxia attenuates cGMP-dependent pulmonary vasodilation. Am. J. Physiol. Lung Cell. Mol. Physiol. 282:L1366–L1375.[Abstract/Free Full Text]
  37. Muramatsu, M., R. C. Tyler, J. Gutkowska, J. R. Klinger, N. S. Hill, D. M. Rodman, and I. F. McMurtry. 1997. Atrial natriuretic peptide accounts for increased cGMP in hypoxia-induced hypertensive rat lungs. Am. J. Physiol. 272:L1126–L1132.
  38. Zhao, L., R. J. Winter, T. Krausz, and J. M. Hughes. 1991. Effects of continuous infusion of atrial natriuretic peptide on the pulmonary hypertension induced by chronic hypoxia in rats. Clin. Sci. (Lond.) 81:379–385.[Medline]
  39. Zhao, L., N. A. Mason, J. W. Strange, H. Walker, and M. R. Wilkins. 2003. Beneficial effects of phosphodiesterase 5 inhibition in pulmonary hypertension are influenced by natriuretic peptide activity. Circulation 107:234–237.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 J. Appl. Physiol.Home page
Q. Xue, C. A. Ducsay, L. D. Longo, and L. Zhang
Effect of long-term high-altitude hypoxia on fetal pulmonary vascular contractility
J Appl Physiol, June 1, 2008; 104(6): 1786 - 1792.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
C. J. Mingone, M. Ahmad, S. A. Gupte, J. L. Chow, and M. S. Wolin
Heme oxygenase-1 induction depletes heme and attenuates pulmonary artery relaxation and guanylate cyclase activation by nitric oxide
Am J Physiol Heart Circ Physiol, March 1, 2008; 294(3): H1244 - H1250.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Lung Cell. Mol. Physiol.Home page
L. He, J. Chen, X. Liu, B. Dinger, and S. Fidone
Enhanced nitric oxide-mediated chemoreceptor inhibition and altered cyclic GMP signaling in rat carotid body following chronic hypoxia
Am J Physiol Lung Cell Mol Physiol, December 1, 2007; 293(6): L1463 - L1468.
[Abstract] [Full Text] [PDF]

Home page
 Arterioscler. Thromb. Vasc. Bio.Home page
M. P. Coggins and K. D. Bloch
Nitric Oxide in the Pulmonary Vasculature
Arterioscler. Thromb. Vasc. Biol., September 1, 2007; 27(9): 1877 - 1885.
[Abstract] [Full Text] [PDF]

Home page
 Cardiovasc ResHome page
M. Oka, V. Karoor, N. Homma, T. Nagaoka, E. Sakao, S. M. Golembeski, J. Limbird, M. Imamura, S. A. Gebb, K. A. Fagan, et al.
Dehydroepiandrosterone upregulates soluble guanylate cyclase and inhibits hypoxic pulmonary hypertension
Cardiovasc Res, June 1, 2007; 74(3): 377 - 387.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Lung Cell. Mol. Physiol.Home page
C. J. Mingone, S. A. Gupte, J. L. Chow, M. Ahmad, N. G. Abraham, and M. S. Wolin
Protoporphyrin IX generation from {delta}-aminolevulinic acid elicits pulmonary artery relaxation and soluble guanylate cyclase activation.
Am J Physiol Lung Cell Mol Physiol, September 1, 2006; 291(3): L337 - L344.
[Abstract] [Full Text] [PDF]

Home page
 J. Appl. Physiol.Home page
J. M. Williams, C. R. White, M. M. Chang, E. R. Injeti, L. Zhang, and W. J. Pearce
Chronic hypoxic decreases in soluble guanylate cyclase protein and enzyme activity are age dependent in fetal and adult ovine carotid arteries
J Appl Physiol, June 1, 2006; 100(6): 1857 - 1866.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Lung Cell. Mol. Physiol.Home page
C. J. Mingone, S. A. Gupte, N. Ali, R. A. Oeckler, and M. S. Wolin
Thiol oxidation inhibits nitric oxide-mediated pulmonary artery relaxation and guanylate cyclase stimulation
Am J Physiol Lung Cell Mol Physiol, March 1, 2006; 290(3): L549 - L557.
[Abstract] [Full Text] [PDF]

Home page
 CirculationHome page
R. Dumitrascu, N. Weissmann, H. A. Ghofrani, E. Dony, K. Beuerlein, H. Schmidt, J.-P. Stasch, M. J. Gnoth, W. Seeger, F. Grimminger, et al.
Activation of Soluble Guanylate Cyclase Reverses Experimental Pulmonary Hypertension and Vascular Remodeling
Circulation, January 17, 2006; 113(2): 286 - 295.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
2003-0287OCv1
30/6/908    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Hassoun, P. M.
Right arrow Articles by Bloch, K. D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Hassoun, P. M.
Right arrow Articles by Bloch, K. D.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Proc. Am. Thorac. Soc. Am. J. Respir. Crit. Care Med.
Copyright © 2004 American Thoracic Society.