(Hypertension. 1996;28:743-753.)
© 1996 American Heart Association, Inc.
Articles |
Upregulation of Nitric Oxide Synthase Correlates Temporally With Onset of Pulmonary Vascular Remodeling in the Hypoxic Rat
the Department of Anesthesiology, University of Virginia Health Sciences Center, Charlottesville.
Correspondence to Roger A. Johns, MD, Department of Anesthesiology, Box 238, University of Virginia Health Sciences Center, Charlottesville, VA 22908. E-mail raj2d@virginia.edu.
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Alterations in nitric oxide signaling have been hypothesized to have an etiologic role in the development of hypoxic pulmonary hypertension. However, changes in the expression of nitric oxide synthase (NOS) in hypoxic lungs remains controversial. In this study, we used (1) Northern and Western analyses to measure NOS mRNA and protein expressions, (2) lung histology together with measurements of lung and heart weights to monitor pulmonary vascular remodeling, and (3) immunohistochemistry to localize NOS proteins. The data demonstrated that endothelial NOS mRNA and protein were upregulated over 1 to 7 days of hypoxia that temporally correlated with and preceded the vascular remodeling that occurred in the course of the development of hypoxic pulmonary hypertension. Hypoxia also induced brain NOS in bronchial epithelium and inducible NOS in vascular smooth muscle but did not affect inducible NOS expression in macrophages or basal guanylyl cyclase activity in the lung. These findings showed that upregulation of endothelial NOS was tightly correlated with the vascular remodeling induced by hypoxia, suggesting a role for nitric oxide in the development of pulmonary hypertension.
Key Words: nitric oxide synthase • nitric oxide • hypertension, pulmonary • anoxia • endothelium, vascular • muscle, smooth, vascular • guanylate cyclase • cGMP
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Introduction |
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Pulmonary hypertension, a common complication of chronic lung disease, is characterized by elevated pulmonary blood pressure, abnormally thickened pulmonary arteries, and right ventricular hypertrophy.1 2 3 Although its pathogenesis is still elusive, chronic alveolar hypoxia has been considered to be a critical factor for the development of pulmonary hypertension in various respiratory and cardiac disorders.3 4 5 Recently, evidence has suggested that the alteration of the NO signaling pathway in the lung may play a role in the development of hypoxic pulmonary hypertension.6 However, studies of the effect of chronic hypoxia on the NO pathway in the lung have yielded various conclusions.
NO, a potent endogenous vasodilator,7 8 was first identified more than a decade ago as endothelium-derived relaxing factor (EDRF).9 It is synthesized from L-arginine in endothelial cells and diffuses to adjacent smooth muscle, where it activates the soluble guanylyl cyclase, with a resulting increase in cGMP, which leads to vascular dilation by relaxing the smooth muscle.7 Thus, the role of NO was hypothesized to be essential in the maintenance of low basal pulmonary artery pressure,10 11 12 and a loss of apparent EDRF/NO activity in the lungs was proposed to be involved in the development of pulmonary hypertension induced by chronic hypoxia6 13 or in lung diseases associated with pulmonary hypertension, such as cystic fibrosis,14 Eisenmenger syndrome,15 and chronic obstructive lung disease.16 17
However, other studies have not supported the above findings. NO has been shown not to be responsible for low basal pulmonary vascular tone,18 19 20 21 and a loss of EDRF/NO-dependent vasodilation failed to appear in hypobaric-induced chronic hypoxic pulmonary hypertension.19 In addition, chronic inhibition of NO production by feeding N
-nitro-L-arginine methyl ester (L-NAME) to rats did not result in structural or functional changes consistent with the development of pulmonary hypertension.19 Even in the same laboratory, a decrease in endothelial NO production was reported,22 but an enhanced eNOS (NOS being the enzyme for NO synthesis) was later demonstrated in the same hypoxic pulmonary hypertension model.23
We first demonstrated upregulation of NOS in the chronic hypoxic rat model24 coincident with the characteristic histological changes of pulmonary hypertension.24 25 We also demonstrated the lack of NOS in normal small pulmonary resistance vessels24 and no effect on basal pulmonary vascular resistance with NOS inhibition by L-NAME infusion.25 NOS mRNA, protein, and enzyme activity were all correspondingly increased in the chronic hypoxic lung,24 25 and the increased NOS was found to be distributed in the endothelium of hypertrophied small pulmonary vessels24 26 and vascular smooth muscle of all pulmonary vessels.24 NOS inhibitors increased endothelium-dependent pulmonary artery pressure to a greater degree in chronic hypoxic versus normoxic lungs, and the response to endothelium-dependent vasodilators was enhanced.25 These data support the hypothesis that chronic hypoxia-induced pulmonary hypertension results in both structural and physiological upregulation of NOS.
Recently, Giaid and Saleh27 observed reduced eNOS expression in the lungs of patients with pulmonary hypertension, whereas we have observed an upregulation of NOS in a group of similar patients.28 Given the above controversial data, we thoroughly studied the time course of changes in NOS expression and its correlation with the temporal development of vascular remodeling during the development of hypoxic pulmonary hypertension using (1) Northern and Western analyses to measure NOS mRNA and protein expressions, (2) lung histology together with measurements of lung and heart weights to monitor pulmonary vascular remodeling, and (3) immunohistochemistry to localize NOS proteins. The results illustrate a temporal correlation between the upregulation of NOS and development of hypoxic pulmonary hypertension, suggesting a role of NO in the development of hypoxic pulmonary hypertension.
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Methods |
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Hypoxic Rat Protocol and Tissue Preparations
The protocol for exposure of rats to hypoxia or normoxia was identical to that previously described.24 A total of 64 male Sprague-Dawley adult rats were placed in either a Plexiglas chamber maintained at 10% oxygen (hypoxic group, n=32) or a chamber open to room air (normoxic group, n=32) for 1, 3, 5, and 7 days; 8 hypoxic and 8 normoxic rats were studied each day. Rats were anesthetized and killed, and their lungs and hearts were perfused with 20 mL normal saline and rapidly removed and weighed. Four to five pieces were cut as 1- to 1.5-mm-thick sections from different lobes of each lung and processed as described previously24 29 30 for immunohistochemistry. Of the remaining lung tissue, approximately half was homogenized in Tri Reagent (50 to 100 mg tissue/mL Tri Reagent; Molecular Research Center) for RNA extraction, and the other half was frozen in liquid nitrogen. The tissues were stored at -70°C for Northern and Western preparations.
For separation of lung tissue into soluble and particulate fractions, the stored lung tissue was taken out, thawed, and homogenized in ice-cold 50 mmol/L Tris-HCl buffer (pH 7.4) containing 250 mmol/L sucrose and 0.2 mmol/L benzamidine. The homogenate was centrifuged at 1000g for 10 minutes at 4°C, and the pellet was discarded. The supernatant of the homogenate was centrifuged again at 105 000g for 60 minutes at 4°C. The supernatant was then harvested as the soluble (cytosolic) fraction, and the pellet was dissolved in the homogenization buffer, serving as the particulate (microsomal) fraction.
Northern Blot
The lung homogenate in Tri Reagent was thawed and centrifuged at 12 000g. RNA remained exclusively in the aqueous phase, whereas DNA and proteins were in the interphase and organic phases. Poly(A)+ mRNA was purified from total RNA with an oligo(dT)-cellulose affinity spin column following the manufacturer's instructions (5 Prime-3 Prime Inc). The yield and concentration of mRNA were determined by spectroscopic measurement of absorbance at 260 nm. Twenty micrograms poly(A)+ mRNA per hypoxic rat was loaded in the gel. Because less than 20 µg poly(A)+ mRNA was able to be collected from the half lung in many normoxic rats, we mixed the mRNA from four rats exposed to normoxia at 1, 3, 5, and 7 days and loaded 20 µg of the poly(A)+ mRNA in the gel. The NOS mRNA was analyzed by standard Northern blot and hybridization techniques with one of the following cDNA probes: 4091-bp EcoRI eNOS fragment (the eNOS cDNA clone was a kind gift from Dr W.C. Sessa) or 3934-bp HincII–Ssp I iNOS fragment (the iNOS clone was a kind gift from Drs Q.W. Xie and C. Nathan). The total loaded poly(A)+ mRNA was normalized with the blot hybridized with a ß-actin cDNA fragment. The cDNA probes were labeled with [32P]dCTP by random primed labeling, and the probe was purified through a G50 spin column (Boehringer Mannheim; protocol by the manufacturer). The cDNA probe was denatured by boiling and then hybridized to the blots overnight at 65°C. After the blots were washed at high stringency, a hybridized probe was detected and measured with Phosphorimager and Imagequant software (Molecular Dynamics). Autoradiographs were also obtained by exposure to film (Hyperfilm, Amersham). A molecular weight standard (RNA ladder of 9.5, 7.5, 4.4, 2.4, and 1.4 kb) was blotted along with the mRNA samples and then cut off the blot and stained with methylene blue.
Western Blot
Western blotting was performed by a method described previously,30 with minor modifications. Control tissues were homogenates from rat brain, cultured bovine aortic endothelial cells (microsomal preparation), and RAW 264.7 macrophages (stimulated with 50 U/mL interferon and 40 ng/mL lipopolysaccharide). The lung samples (crude homogenate and soluble or particulate fractions) were loaded (150 µg each) and separated on a 7.5% sodium dodecyl sulfate–polyacrylamide gel, followed by blotting of the proteins onto nitrocellulose (Bio-Rad). The blots were blocked with buffer (50 mmol/L Tris-HCl [pH 7.4], 0.15 mol/L NaCl, 2% bovine serum albumin, and 0.1% Tween 20) for 1 hour at room temperature. Then the blots were incubated with one of the three NOS antibodies: bNOS polyclonal antibody (dilution, 1:2000; Affinity BioReagents), iNOS polyclonal antibody (dilution, 1:1000; a kind gift from Drs V. Riveros-Moreno and S. Moncada), or eNOS monoclonal antibody (dilution, 1:500; Transduction Laboratories) for 1 hour at room temperature. The blots were washed six times with Tris-buffered saline (5 minutes) and then incubated for 1 hour at room temperature with anti-mouse IgG for monoclonal primary antibodies or anti-rabbit IgG for polyclonal primary antibodies, which were conjugated with horseradish peroxidase (Bio-Rad). The blots were washed six times with PBS (5 minutes) followed by detection of immunoreactive proteins with enhanced chemiluminescence (Amersham). The signal bands were measured by densitometry (Personal Densitometer/Imagequant, Molecular Dynamics).
Immunohistochemistry
The immunohistochemical method used was that previously described,24 29 30 with some modification. After preincubation with 20% horse serum (Sigma Chemical Co) for 15 minutes, tissue cryostat sections were washed (2x10 minutes in PBS) and incubated with one of the following NOS antibodies: (1) a polyclonal antibody against rat bNOS amino acid 724-739 fragment (dilution, 1:200; Affinity BioReagents), (2) a polyclonal antibody against mouse iNOS peptide 48-71 fragment (dilution, 1:250, a kind gift from Drs V. Riveros-Moreno and S. Moncada), or (3) a monoclonal antibody raised against a peptide fragment of amino acids 1030-1209 of human eNOS (dilution, 1:50; Transduction Laboratories) at 4°C overnight. For negative control studies, primary antibodies were omitted and slides were incubated with rabbit IgG (for polyclonal antibodies) or mouse IgG (for monoclonal antibodies). After unbound primary antibodies were washed off with PBS, the sections were incubated with biotinylated antibodies against mouse or rabbit (dilution, 1:250; Amersham Life Sciences, Inc) for 1 hour, followed by incubation in avidin/biotin/horseradish peroxidase complex (1:50 dilution for 45 minutes, Vector Laboratories, Inc). Peroxidase activity was visualized by a color reaction with diaminobenzidine (0.5 mg/mL) as the substrate (brown) or enhanced by cobalt chloride (dark blue, 0.2 mg/mL). The slides then were counterstained with hematoxylin (blue in nuclei), mounted, and examined under a bright-field microscope (Olympus Vanox AHBS3).
An immunofluorescent method24 29 30 was also used for eNOS detection by binding of the biotin-conjugated antibodies with streptavidin-conjugated Texas red (Vector Laboratories). These slides were examined under a fluorescence microscope (Olympus Vanox AHBS3).
Double-Labeling Immunohistochemistry for Pulmonary Vascular Histology
Three slides from each rat were treated with 5% horse serum in PBS for 15 minutes. After washing with PBS (2x10 minutes), the sections were incubated in a mixture of rabbit antiserum raised against von Willebrand factor (DAKO Corp; dilution, 1:200) and 
-actin monoclonal antibody (dilution, 1:500; Sigma) at 4°C overnight in a humidified chamber. Tissue sections were then thoroughly washed in PBS and incubated for 1 hour at room temperature with a mixture of goat antiserum raised against rabbit IgG conjugated with horseradish peroxidase (dilution, 1:100; Sigma) and horse antiserum raised against mouse IgG conjugated with alkaline phosphatase (dilution, 1:100; Sigma). After washing in PBS (3x10 minutes), the actin-phosphatase was stained red by fast red/naphthol AS-TR phosphate (Sigma), and the von Willebrand factor–peroxidase was colored black by fast diaminobenzidine/urea H2O2 with metal enhancer (Sigma) in the sections. Finally, the slides were counterstained with hematoxylin (blue in nuclei), mounted, and examined with the bright-field microscope.
The features of the pulmonary vascular histology were analyzed according to methods previously described.3 In addition, we used von Willebrand factor as an endothelial marker and -actin to show vascular smooth muscle. Approximately 50 arteries were randomly evaluated per rat. The small arteries (internal diameter <80 µm) were classified as nonmuscular, partially muscular, or muscular according to the -actin staining; the percentage of wall thickness was calculated by the formula 2xWall Thicknessx100/External Diameter3 25 for three sizes of vessels (
80, 81 to 149, and 
150 µm).24
Guanylyl Cyclase Activity Assay
Guanylyl cyclase activity was evaluated as described previously.31 Twenty micrograms of the lung crude homogenate, supernatant, or particulate fractions was added in the reaction solution at a final volume of 250 µL containing 50 mmol/L Tris-HCl (pH 7.4), 1 mmol/L isobutylmethylxanthine, 4 mmol/L MnCl2, 0.5 mmol/L ATP (disodium salt, grade I, from yeast), 1 mmol/L GTP (sodium salt, type III), 15 mmol/L creatine phosphate, and 100 µg creatine phosphokinase. The reaction was controlled at 37°C for 5 minutes and terminated by addition of 250 µL ice-cold 0.2N HCl. cGMP was quantified by automated radioimmunoassay as previously described.31 The time course and conditions for optimization of the guanylyl cyclase assays have been previously described.31
Analysis and Statistics
Values are expressed as mean±SE. Each value was calculated from the data collected from the experimental group (usually eight rats as a group, with two to four experiments performed). Comparisons of lung and heart weights, artery medial thickness, muscularization of small arteries, NOS mRNA, NOS protein, and guanylyl cyclase activity in normoxic versus hypoxic rat lungs were made by modified t test for two samples assuming equal or unequal variance. When more than two groups were compared, we used one-way ANOVA followed by Dunnett's test when indicated. A value of P<.05 was considered statistically significant.
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Histological Changes of Pulmonary Hypertension
The mean values of absolute heart weights (grams, normoxia/hypoxia) were 1.06±0.09/1.14±0.12 for day 1, 1.15±0.12/1.10±0.12 for day 3, 1.12±0.07/1.10±0.08 for day 5, and 1.12±0.06/1.16±0.07 for day 7. The mean values for absolute lung weights (grams, normoxia/hypoxia) were 1.68±0.18/1.61±0.35 for day 1, 1.76±0.20/2.20±0.22 for day 3, 1.71±0.11/2.09±0.26 for day 5, and 1.50±0.08/2.21±0.19 for day 7. Fig 1A and 1B
present heart and lung weights as a percentage of whole body weight in sets of 1- to 7-day hypoxic rats compared with normoxic rats. The absolute lung weights and lung weights as a percentage of whole body weight (Fig 1A) were first significantly increased on the third day of exposure to hypoxia, followed by heart weight as a percentage of whole body weight (Fig 1B) increasing significantly on the seventh day of exposure to hypoxia. No significant increase in absolute heart weights was seen in hypoxic rats.
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To histochemically characterize the development of vascular changes associated with pulmonary hypertension, we monitored the two major vascular components, endothelium and smooth muscle, in the lungs of rats exposed to an increasing duration of hypoxia using a double-immunolabeling technique: the endothelium was immunostained by von Willebrand factor antibody (black staining in Fig 2), and the smooth muscle was immunolabeled by -actin antibody (red staining in Fig 2). Fig 2 demonstrates that hypoxia caused a gradual increase (from normoxic lungs [Fig 2A] to hypoxic lungs at days 1 [Fig 2C and 2D], 3 [Fig 2E and 2F], and 7 [Fig 2G and 2H]) in the pulmonary vascular wall thickness of both large (Fig 2D, 2F, and 2H) and small (Fig 2A, 2C, 2E, and 2G) arteries. Furthermore, observations regarding the changes in vascular smooth muscle clearly showed that the increase in vascular wall thickness was due to vascular smooth muscle remodeling, with an increase in both the size and quantity of vascular smooth muscle cells in pulmonary vessels (Fig 2G and 2H). Fig 1C
further demonstrates quantitative analysis of the changes in vascular wall thickness, which were also found to be coordinated with the increase in the percentages of muscular and partially muscular small arteries (asterisks in Fig 1D). At the same time, the number of nonmuscular small arteries was decreased (Fig 1D). This increase in vascular wall thickness and artery muscularization was proportional to the duration of hypoxia. These changes in vascular histology were significant at the third day of exposure to hypoxia, corresponding with the timing of the increase in lung weight. Thus, this result suggests that the increase in lung weight is related to the vascular remodeling induced by hypoxia.
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eNOS Expression
Northern and Western blot data (Fig 3) showed that hypoxia resulted in a gradual increase in both eNOS mRNA (Fig 3A and 3A') and eNOS protein (Fig 3B and 3B'). The level of eNOS mRNA (normalized to ß-actin) was increased by 127.3±4.5%, 162.3±6.9%, 217.3±16.7%, and 258.8±10.4% after 1, 3, 5, and 7 days of exposure to hypoxia (Fig 3A), respectively. The level of eNOS protein was also shown to be increased in the lung by 135.0±13.5%, 168.5±15.6%, 199.7±17.9%, and 247.2±12.7% at 1, 3, 5, and 7 days of exposure to hypoxia, respectively (panel T in Fig 3B and 3B'). The increased eNOS primarily occurred in the particulate (microsomal) fraction (panel M). eNOS was unchanged in the cytosolic (soluble) fraction of the protein extract (panel C).
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In normoxic lungs, eNOS immunoreactivity was found to be distributed within endothelium among 95.8% (Table) of the large vessels (internal diameter >150 µm; arrows in Fig 4A and 4B) but only in 1.7% (Table) of the small vessels (internal diameter <80 µm; v in Fig 4A and 4B); in hypoxic lungs, eNOS immunoreactivity was frequently found in the endothelial cells of both large (arrows in Fig 4C) and small, thickened (Fig 4D and 4E) pulmonary vessels. The Table presents quantitative data on the distribution of eNOS immunoreactivity in the pulmonary vasculature of hypoxic versus normoxic lungs. The percentage of eNOS-positive vessels was found to increase in small and medium-sized pulmonary vessels of hypoxic lungs compared with vessels of normoxic lungs.
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bNOS and iNOS Expression
Although bNOS was found in the neurons surrounding respiratory bronchi (data not shown), the majority of bNOS immunoreactivity was distributed in bronchial epithelial cells (Fig 6A). Under hypoxic conditions, Western blot analysis showed bNOS protein to be gradually increased (Fig 5A).
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In normoxic lung, a 4.4-kb mRNA was detected by the iNOS cDNA probe (Fig 5B). When the lungs were exposed to hypoxia, an additional band (approximately 4.0 kb) was shown, and both bands were increased compared with normoxic lungs (Fig 5B). Also, the hypoxia-enhanced iNOS protein was at the 118-kD mass (Fig 5C), lower than the macrophage iNOS molecular mass (approximately 130 kD, see lane M in Fig 5C). Immunohistochemistry demonstrated increased iNOS protein located in the vascular smooth muscle of large (Fig 6C), medium-sized, and small (Fig 6D) pulmonary arteries. In contrast, very limited iNOS immunoreactivity was found in the pulmonary vasculature exposed to normoxia (arrows in Fig 6E).
Basal Guanylyl Cyclase Activity
Guanylyl cyclase activity of normoxic and hypoxic lungs is shown in Fig 7. No significant decrease or increase in cGMP level was seen in hypoxic lungs in crude homogenate, supernatant, or particulate preparations (Fig 7).
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In this study, we demonstrated that NOS expression is upregulated in the early course of development of hypoxic pulmonary hypertension and that this upregulation correlates temporally with the development of vascular remodeling associated with the pulmonary hypertension.
The observed effects of hypoxia on the pulmonary vasculature were a gradual increase in lung weight (Fig 1A), in the wall thickness of all sizes of pulmonary vessels (Figs 1C and 2), and in the percentage of small muscular arteries (asterisks in Fig 1D), whereas the number of small nonmuscular vessels was simultaneously decreased. Although no significant increase was seen in absolute heart weights, an increase in heart weight as a percentage of whole body weight (Fig 1B) was found on the seventh day of exposure to hypoxia. In addition, increased heart weights have been reported in rats exposed to hypoxia for 2 to 4 weeks.25 These data suggest that hypoxia first caused vascular remodeling and pulmonary hypertension, which secondarily resulted in cardiac muscle hypertrophy. This process is similar to that found in patients with chronic lung disease who first develop pulmonary hypertension but then eventually die of heart failure. Although the cellular mechanisms responsible for hypoxia-induced vascular remodeling are unclear, much evidence indicates that the endothelium releases vasoactive mediators that act locally in a paracrine manner to effect the proliferation of vascular smooth muscle cells.32 In the pulmonary vasculature, NO is well known as a vasodilator released from endothelium, as we have demonstrated24 25 28 33 (Fig 4). In the present study, a gradual increase in eNOS mRNA and protein was demonstrated (Fig 3A), which temporally correlated with the development of pulmonary vascular remodeling (Figs 1 and 2), supporting the concept that upregulated NOS may be associated with the cellular mechanism for the development of pulmonary hypertension.
NO was previously proposed as an antimitogen in repetitively subcultured cells.34 This report was a major basis for the previously held hypothesis that downregulation of NOS was an etiologic factor in the development of pulmonary hypertension. However, further study from the same laboratory showed recently that in primary cultured smooth muscle cells, NO was able to actually stimulate vascular smooth muscle mitogenesis and proliferation through selective amplification of fibroblast growth factor-2 and had no antimitogenic effects.35 Thus, NO, particularly at the higher concentrations present after hypoxic upregulation, may be more likely to be mitogenic rather than antimitogenic in the pulmonary vasculature exposed to hypoxia. NO has also been found to promote tumor growth36 and play an important role in angiogenesis in both fetal lung development30 and the wound tissue healing process.37 Furthermore, NO has been implicated in apoptosis,38 a critical component of vascular remodeling. In the present study, only 1.7% of normal small pulmonary vessels were immunostained by eNOS antibody (Fig 4 and Table), but 5.0%, 11.4%, 40.7%, and 44.7% of the same organ vessels stained positive for eNOS immunoreactivity when they were exposed to hypoxia at 1, 3, 5, and 7 days (Fig 4 and Table). The pattern of increased eNOS in small and medium-sized vessels was similar to that of eNOS mRNA and protein expression in the hypoxic lungs (Fig 3), suggesting that the upregulated eNOS was primarily due to the increase of eNOS expression in the endothelium of these smaller vessels, which demonstrated a temporal correlation with vascular remodeling (Figs 2 and 4). Interestingly, hypoxia increased eNOS expression in the pulmonary vasculature on the first day, preceding the significant increases in lung weight (Fig 1A), in vascular wall thickness (Figs 2 and 1C), and in the percentage of muscular arteries (Figs 2 and 1D). The temporal correlation of these parameters (Figs 1 and 4; Table) would be consistent with a role for NO acting as a mitogen or potentiating mitogenesis by other factors in the development of hypoxic vascular remodeling, leading to the subsequent development of pulmonary hypertension.
Hypoxia also upregulated iNOS mRNA and protein (Fig 5B and 5C). Interestingly, the species of iNOS mRNA induced by hypoxia migrated primarily at 4.0 kb, not at 4.4 kb, which is the reported range of the macrophage iNOS mRNA species (Fig 5B). Consistent with the smaller mRNA, the increased iNOS protein was at approximately 118 kD (hypoxia lanes 1, 3, 5, and 7 in Fig 5C), lower than the molecular mass (130 kD, lane M in Fig 5C) of macrophage iNOS protein. Immunohistochemistry further showed that iNOS immunoreactivity was increased in the vascular smooth muscle layers (Fig 6C and 6D) of hypoxic lung compared with normoxic lung (Fig 6E). Note that no iNOS immunoreactivity was seen in macrophages from hypoxic lungs (Fig 6C and 6D). We have previously reported iNOS expression in pulmonary macrophages, but it was seen in both lipopolysaccharide-induced lungs (C.X. and R.A.J., unpublished data, 1995) and lung from patients with chronic inflammatory diseases.39 These results are consistent with our previous data reported on chronic hypoxic lung,24 26 indicating that the mechanism for iNOS expression in the lungs is different between inflammatory and hypoxic stimuli.
Considering that the cDNA sequence of the iNOS isoform expressed in rat vascular smooth muscle was almost identical to that of the macrophage iNOS,40 we think that the difference between the hypoxia-induced iNOS from pulmonary smooth muscle and inflammation-induced iNOS from macrophages results from alternative transcription sites or different splicing of the primary RNA transcripts, as demonstrated by Chu et al.41 Two major mRNA species for human iNOS, which differed in size by approximately 0.2 kb, have also been demonstrated in several cultured lung epithelial cell lines.41 Cytokines enhanced the transcription of iNOS mRNA from both upstream and downstream of the TATA box, producing longer iNOS mRNA species. The shorter iNOS mRNA was shown to lack exon 1, which was associated with alternative splicing of the primary RNA transcript. These different transcription initiation sites on the iNOS gene may affect the rate of gene expression, because the 5' and 3' encoding regions of mRNAs were related to the efficiency of mRNA translation and degradation.41 The diversity of the transcriptional sites, together with their special promoters, may reflect different transcriptional responses to various environmental stimuli, such as inflammation and hypoxia. We demonstrated in this and previous studies that lipopolysaccharide primarily induced a 4.4-kb mRNA (lane M in Fig 5B) and 130-kD protein of iNOS in macrophages26 39 (lane M in Fig 5C), and hypoxia stimulated a 4.0-kb mRNA (lanes 1, 3, 5, and 7 in Fig 5B) and 118-kD protein (hypoxic lanes 1, 3, 5, and 7 in Fig 5C) of iNOS in smooth muscle cells (Fig 6C and 6D).
Although we found bNOS immunoreactivity in the neurons surrounding the large bronchi of the rat lung (data not shown), the majority of bNOS immunoreactivity was distributed in bronchial epithelial cells (Fig 6A), consistent with our previous observation.30 The increase in bNOS protein in hypoxic lungs revealed by Western blot analysis (Fig 5A) supports the role of NO in the regulation of bronchial tone as suggested previously.42 43 Note that although all three NOS isoforms are produced by three individual genes, our present results showing upregulation of all three NOS types by hypoxia indicate that all of these NOS isoforms bear regulatory components that respond to oxygen modulation.
The upregulation of NOS proteins induced by hypoxia in the pulmonary vasculature may be of physiological significance as a mechanism for pulmonary adaptation to hypoxic stress. For example, it may be a means of modulating the hypoxia-induced pulmonary hypertension seen in adult respiratory distress syndrome, chronic obstructive lung disease, and other pulmonary disease states and may be a physiological adaptation to such pulmonary vascular changes. However, Giaid and Saleh27 recently observed reduced eNOS expression in the lungs of patients with pulmonary hypertension. We have been unable to obtain the same results. Instead, we have seen an increase in eNOS in the pulmonary vasculature of patients with similar pulmonary diseases.28
It is also possible that hypoxia-induced pulmonary hypertension can induce eNOS expression through shear stress stimulation, because the eNOS gene contains a shear stress–responsive element in the promoter region.44 However, in a rat model with increased flow created by an aorta–pulmonary artery shunt, we found no significant increase in eNOS mRNA or protein expressions in the lungs (A. Everett, T.D. Le Cras, C.X, and R.A.J., unpublished data, 1995). Furthermore, in our hypoxic pulmonary hypertension model, the present study demonstrated that not only eNOS increased (Figs 3 and 4; Table) but iNOS (Figs 5B, 5C, 6C, and 6D) and bNOS (Fig 6A) also increased. As neither of these latter isoforms contains the shear stress–responsive element and bNOS was primarily located in the pulmonary epithelium (Fig 6A), it is unlikely that the increase in bNOS in the hypoxic lung model was directly caused by the pulmonary hypertension–related shear stress. A shear stress–unrelated upregulation of eNOS and bNOS was also seen in vascular endothelium and bronchial epithelium of normally hypoxic fetal lungs, in which there is no significant circulation to create shear stress, but was decreased in postnatal lungs when the pulmonary hypoxia was relieved.30 45 These data therefore suggest that the upregulation of the NOS isoforms in the lungs of the rat model may be primarily due to low oxygen tension. Further evidence for NOS regulation by oxygen tension has been produced by our own and other physiological studies showing that NOS activity was enhanced by chronic hypoxia20 24 46 and by two brain ischemia studies that show the upregulation of eNOS in small vessels after focal cerebral ischemia.47
We previously discussed the possibility of an alteration of the NO signaling pathway downstream at the level of guanylyl cyclase24 in order to explain the disparity between the increased NOS expression and an early physiological study that showed a loss of endothelium-dependent relaxant activity in the pulmonary circulation of rats exposed to chronic hypoxia.13 We tested this hypothesis in the present study using a biochemical assay to compare the basal guanylyl cyclase activity between normoxic and hypoxic lungs. Fig 7 illustrates that basal guanylyl cyclase activity did not differ significantly between normoxic and hypoxic lungs in total homogenate, cytosolic, or particulate fractions. We conclude that hypoxia did not affect both cytosolic and particulate basal guanylyl cyclase activities. This is consistent with subsequent physiological studies from our laboratory27 and others19 20 46 showing an enhanced NO response in the pulmonary vasculature of chronic hypoxic rats.
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Acknowledgments |
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This study was supported by National Institutes of Health grants RO1-HL-39706 and RO1-GM-49111 (R.A.J.) and by a research award from the American Heart Association, Virginia Affiliate (C.X.). The authors are grateful to Drs Zhiyi Zuo and Timothy D. Le Cras for advice regarding the guanylyl cyclase assay and Western and Northern blotting procedures. The authors wish to thank Dr Lan Xu for help in the microscopic counting of eNOS-positive vessels, measurements of pulmonary vascular histology, and preparation of figures. The authors also acknowledge the work of Nan Zhou and Linghong Da for technical assistance and Mary E. Davison for help in manuscript preparation.
Received April 3, 1996; first decision May 8, 1996; accepted July 5, 1996.
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References |
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