Introduction

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Endothelium-derived nitric oxide (NO) generated by the enzyme endothelial NO synthase (eNOS) is a potent vasodilator that is important in the modulation of pulmonary vasomotor tone. This is particularly evident during the normal cardiopulmonary transition from fetal to neonatal life when a variety of processes, including increasing oxygenation, rhythmic distension of the lungs, and shear stress, acutely enhance NO production by the pulmonary endothelium, causing an 8- to 10-fold increase in pulmonary blood flow (1). Under normal circumstances, the expression of eNOS in the lung is upregulated during late gestation to optimize the capacity for pulmonary NO production near term (4). In contrast, there is evidence that pulmonary eNOS expression is attenuated in certain conditions such as congenital diaphragmatic hernia and fetal pulmonary hypertension (6), potentially contributing to the pathogenesis of persistent pulmonary hypertension of the newborn (PPHN).

As a result of greater understanding of the biochemistry of the NO molecule and its role in the transitional pulmonary circulation over the past decade, inhaled NO therapy has recently been considered in the management of the patient with PPHN. The results reported are encouraging, as the majority of patients exhibit increased postductal oxygenation upon the initiation of NO therapy (9). However, some patients receive only transient benefit and others have prolonged requirements for NO administration (9, 11, 12). The latter clinical observations suggest that prolonged exposure to exogenous NO may have negative effects on the capacity for endogenous NO production. It has previously been demonstrated in experiments using the partially purified enzyme as well as intact endothelial cells from a variety of sources that NO acutely inhibits eNOS activity (15, 16). However, the effects of prolonged NO exposure on eNOS expression in intrapulmonary endothelium are not known.

To better understand the mechanisms regulating NO synthesis in the developing pulmonary circulation, the present investigation was designed to determine whether prolonged exposure to exogenous NO modulates eNOS expression in intrapulmonary artery endothelium. To avoid the potential effects of sustained changes in pulmonary blood flow that may result from inhaled NO administration in the intact animal, and to evaluate the direct effects of NO on the intrapulmonary endothelium, studies were performed with isolated, early-passage ovine fetal intrapulmonary artery endothelial cells (PAEC). This culture system has previously been used to investigate the modulation of pulmonary endothelial NO and prostacyclin production by oxygen; these studies revealed that many phenotypic characteristics related to the regulation of vasodilator synthesis are conserved in the early-passage PAEC (17- 19). On the basis of the clinical observations noted earlier (9, 11, 12), the hypothesis was raised that eNOS expression is attenuated by prolonged exposure to exogenous NO. In addition to testing this hypothesis, studies were performed to determine the rapidity of this process, to determine whether endogenous NO also modulates PAEC eNOS expression, and to begin to determine the mechanism(s) underlying NO modulation of eNOS expression.

	Materials and Methods

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Cell Culture and Treatment

PAEC were obtained from third-generation intrapulmonary arteries of mixed-breed fetal lambs at 125 to 135 d gestation, with term being 144 ± 4 d, using methods that we have previously described (20). The PAEC were propagated in RPMI media containing 10% iron-supplemented calf serum, 10% lamb serum, 1% L-glutamine, 1% antibiotic-antimycotic mixture, 0.15% Nystatin, 0.15% gentamycin, and 0.10% tylosin, in a humidified incubator with 5% CO₂ in air at 37°C. The identity of the cells was confirmed by phenotype (cobblestone appearance and contact inhibition), by immunofluorescence studies with antibody to factor VIII-related antigen, and by examinations of acetylated low-density lipoprotein uptake. Under quiescent conditions, the PAEC express eNOS and not the inducible isoform of NOS (iNOS) (20). The cells were studied at near-confluence at passages 4 to 6.

PAEC grown in 75-cm² flasks were placed in serum-free media for 8 to 48 h under four different conditions. The first two conditions, which were chosen to determine the effects of exogenous NO on eNOS expression, were media with L-arginine (1 mM) plus spermine (Sp) (1 µM), and media with L-arginine plus Sp NONOate (SpNO) (1 µM). SpNO is an NO donor compound that is stable in solution at alkaline pH, has a half-life of 39 min, releases NO when added to media with a normal pH, and requires no bioconversion to release NO; Sp is the parent compound (21). Sp and SpNO were prepared daily in phosphate-buffered saline (PBS) (100 mM NaCl, 25 mM NaH₂PO₄, and 80 mM Na₂HPO₄) at pH 8.5. Preliminary experiments revealed that Sp alone had no effect on eNOS expression. The second two conditions, which were selected to evaluate the effects of endogenous NO in eNOS expression, were media deficient in L-arginine (control) (Endothelial-SFM Growth Media; Life Technologies, Inc., Great Island, NY), and media deficient in L-arginine containing the competitive inhibitor of NOS, nitro-L-arginine-methyl ester (L-NAME) (2 mM). The pH was comparable in the control media and the media containing L-NAME. Cells were treated at the initiation of study and every 24 h thereafter.

To determine the optimal concentrations of Sp and SpNO for study, the effects of these agents on cell viability were evaluated in a quantitative colorimetric assay. We have previously used this method in studies of the effects of varying oxygen tension on PAEC eNOS expression (20). The assay is dependent on the reduction of the tetrazolium salt 3-(4,5-dimethylthazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT) by mitochondrial dehydrogenase in viable cells to form a blue formazan product. The procedure used was modified from that of Denizot and Lang (22). Cells grown in 24-well plates were exposed to Sp or SpNO at concentrations ranging from 10⁴ M to 10¹⁰ M for 24 h. After washing the cells with PBS, 100 µl of MTT solution (5 mg/ml) was added to 0.5 ml RPMI in each well. The cells were incubated at 37°C for 3 h and 0.5 ml of buffer containing 1:1 N,N-dimethyl formamide in distilled water with 0.2% sodium dodecyl sulfate (SDS) was added to each well. The cells were then placed in an incubator overnight. The supernatant was removed and absorbance was measured at dual wavelength (sample filter 540 nm, reference filter 690 nm) (Bio-Rad Model 2550 EIA Reader; Bio-Rad Laboratories, Inc., Melville, NY). Readings for cells exposed to Sp and SpNO were compared with control cells designated to have 100% viability, and with wells without cells reflecting 0% viability. Measured in this manner, the viability of the cells exposed to Sp was 0% at 10⁴ M, 77% at 10⁵ M, and 100% for 10⁶ M to 10¹⁰ M. Similar results were noted for viability of cells exposed to SpNO (0% at 10⁴ M, 66% at 10⁵ M, and 100% for 10⁶ M to 10¹⁰ M). Except where stated otherwise, the greatest concentration of Sp and SpNO that did not affect cell viability (10⁶ M) was used.

To confirm that L-NAME under the conditions employed inhibits NOS activity in intact PAEC, basal NOS activity was determined in intact cells by measuring ³H-L-arginine conversion to ³H-L-citrulline over 60 min using methods we have described previously (23). L-NAME (2 mM) inhibited NOS activity by 89%, and the inhibition was fully reversed by the provision of excess L-arginine (100 mM) but not D-arginine (100 mM). Thus, nearly complete NOS inhibition was achieved with exposure of the intact cells to the enzyme antagonist, allowing for the study of the role of endogenous NO in eNOS expression.

Immunoblot Analysis

The methods used for immunoblot analysis generally followed those we have reported previously (20). After exposure to Sp or SpNO, or control conditions versus L-NAME, PAEC were harvested in ice-cold PBS, pH 7.4, containing 120 mM NaCl, 4.2 mM KCl, 2.5 mM CaCl₂, 1.3 mM MgSO₄, 7.5 mM glucose, 10 mM N-2-hydroxyethylpiperazine-N'-2-ethane sulfonic acid (Hepes), 1.2 mM Na₂HPO₄, and 0.37 mM KH₂PO₄. After pelleting and resuspension in 50 mM KH₂PO₄ buffer (pH 7.8) containing 250 mM mannitol, 5 mM disodium ethylenediaminetetraacetic acid (EDTA), 0.1 mM diethyldithiocarbamate, 0.1 mM indomethacin, and 1% Tween-20, the cells were ultrasonically disrupted (Branson Ultrasonics, Chicago, IL). The protein content of the preparation was determined, SDS/polyacrylamide gel electrophoresis was performed on 25 µg protein with 7% acrylamide, and the proteins were electrophoretically transferred to nitrocellulose filters. The filters were blocked for 1.5 h in buffer containing 150 mM NaCl and 10 mM Tris (pH 7.5) with 0.5% Tween-20 and 5% dried milk, and incubated overnight at 4°C with a 1:2,000 dilution of primary antiserum generated to the unique midmolecule peptide PYNSSPRPEQHKSYK of eNOS, which corresponds to a conserved epitope identical in sequence between bovine and human (24, 25). After incubation with primary antiserum, the nitrocellulose filters were washed with the 150-mM NaCl buffer with Tween-20 and incubated for 1.5 h with a 1:5,000 dilution of a donkey antirabbit immunoglobulin antibody-horseradish peroxidase conjugate (Amersham, Bucks, UK). The filters were washed in the 150-mM NaCl buffer with Tween-20 and the bands for eNOS were visualized by chemiluminescence (ECL Western Blotting Analysis System; Amersham) and quantitated densitometrically. Standardization of densitometry for eNOS protein detection by these methods has been demonstrated previously using 10 to 75 µg of PAEC protein. A linear relationship was seen between the amount of protein and the densitometric value obtained (r = 0.96-0.98) (20). The antiserum to eNOS was the kind gift of Dr. Thomas Michel (Cardiovascular Division, Brigham and Women's Hospital, Harvard Medical School, Boston, MA).

NOS Activity in Cell Lysates

To assess differences in eNOS protein expression after treatment with SpNO or the parent compound Sp by a method other than immunoblot analysis, the abundance of NOS enzymatic activity was evaluated in whole cell lysates. Treated PAEC were washed with ice-cold PBS, pelleted, and resuspended in ice-cold 50 mM Tris buffer (pH 7.4) containing 1.0 mM EDTA, 5 mM mercaptoethanol, 10 µg/ml pepstatin A, 10 µg/ml leupeptin, 90 µg/ml phenylmethylsulfonyl fluoride, and 1.0 µM tetrahydrobiopterin. The cells were disrupted by freeze-thawing in liquid nitrogen, and NOS activity was determined in the cell lysate by measuring the conversion of ³H-L-arginine to ³H-L-citrulline (26). A total of 50 µl of cell lysate was added to 50 µl of buffer, yielding final concentrations of reagents as follows: 2 mM -nicotinamide adenine dinucleotide phosphate, 2 µM tetrahydrobiopterin, 10 µM flavin adenine dinucleotide, 10 µM flavin mononucleotide, 0.5 mM CaCl₂ in excess of EDTA, 15 nM calmodulin, 2 µM cold L-arginine, and 2.0 µCi/ml ³H-L-arginine. After incubation at 37°C for 30 min the assay was terminated by the addition of 400 µl of 40-mM Hepes buffer, pH 5.5, with 2 mM EDTA and 2 mM ethyleneglycol-bis-(-aminoethyl ether)- N,N'-tetraacetic acid. The terminated reactions were applied to 1-ml columns of Dowex AG50WX-8 (Tris form) and eluted with 1 ml of the 40-mM Hepes buffer. ³H-citrulline was collected in scintillation vials and quantified by liquid scintillation spectroscopy. NOS activity in cell lysates was linear with time for up to 1 h, and it was fully inhibited by 2.0 mM L-NAME. We have previously noted excellent correlation between changes in eNOS protein expression evaluated by immunoblot analysis and changes in NOS enzymatic activity in cell lysates (20).

Reverse Transcription/Polymerase Chain Reaction Assays

A semiquantitative reverse transcription/polymerase chain reaction (RT-PCR) assay was established to evaluate eNOS messenger RNA (mRNA) abundance in PAEC because detection of the mRNA by Northern analysis requires the use of poly(A)⁺ RNA from a large volume of cells. Total cellular RNA was obtained from the cells, and RT was performed using 5 µg total RNA. Briefly, complementary DNA (cDNA) synthesis was carried out using 200 U murine MMLV reverse transcriptase, 5 uM oligo-dT, 1 mM deoxynucleotide triphosphates (dNTPs) and 3 mM Mg²⁺ in a volume of 20 µl. In selected tubes the reverse transcriptase was omitted to control for amplification from contaminating cDNA or genomic DNA. The temperature profile was: (1) annealing at room temperature for 5 min, (2) extension at 42°C for 60 min, and (3) termination at 99°C for 5 min. PCR was performed on the resulting RT products using specific oligonucleotide primers for ovine eNOS (8). The sequence of the sense primer was 5'-AGCTCGAGACCCTCAGTCAGGA-3' and the antisense primer 5'-GTCTCCAGTCTTGAGTTGGC-3'. The PCR reactions contained 1.5 mM Mg²⁺, 1 µM primers, 200 µM dNTPs, reaction buffer, and 5 µl cDNA in a final volume of 45 µl. To minimize nonspecific amplification, a "hot start" procedure was employed in which the PCR reaction tubes were placed in a thermal cycler (Perkin-Elmer model 480) prewarmed to 94°C. After 2 min, each tube was opened sequentially and 2.5 U (in 2 µl) of TaqDNA polymerase was added. The PCR temperature profile consisted of 35 cycles of 94°C for 45 s (denaturation), 60°C for 45 s (annealing), and 72°C for 1.25 min. (extension), followed by an additional 5 min final extension at 72°C. Preliminary studies with varying cycle numbers revealed that 35 cycles was within the linear component of the reaction. The primer location, primer concentration, Mg²⁺ concentration, and annealing temperature were optimized to produce the greatest amount of a single PCR product.

The PCR products were size-fractionated by agarose gel electrophoresis. The identity of the PCR products was confirmed and they were quantitated by transferring the DNA to nylon filters, probing with a ³²P end-labeled internal oligonucleotide primer specific for ovine eNOS, and performing densitometric analysis of the resulting autoradiographs. PCR product identity was also confirmed by direct double-stranded sequencing (Fmol DNA Sequencing Kit; Promega Corp., Madison, WI). To control for the RT step, RT-PCR was also done for the housekeeping gene malate dehydrogenase (MDH) (8). The PCR temperature profile for MDH was identical to that described earlier except for an annealing temperature of 56°C.

Preliminary experiments were performed to evaluate the relationship between the quantity of total RNA subjected to RT-PCR and the amount of PCR product generated. Linear regression analysis showed high correlations between densitometry values and the quantity of RNA used for eNOS RT-PCR (Figure 1, r = 0.97-0.99, n = 3 experiments) and for MDH RT-PCR (r = 0.96-0.99, n = 3) (27). We have previously employed RT-PCR assays performed in this semiquantitative manner in studies of the ontogeny of eNOS and neuronal NOS (nNOS) mRNA expression in fetal and newborn rat lung, and in studies of changes in their expression in lungs from fetal rats with congenital diaphragmatic hernia (CDH) and lungs from adult rats subjected to prolonged hypoxia (4, 7, 28). In all investigations in which we have performed Northern analyses in parallel with RT-PCR assays, identical results have been obtained with the two techniques (4, 28).

View larger version (18K): [in this window] [in a new window]   Figure 1.   Relationship between quantity of RNA subjected to RT-PCR and resulting PCR products for eNOS. Varying amounts of total RNA from PAEC were subjected to RT-PCR using primers specific to ovine eNOS, and cDNA generated was separated by electrophoresis and probed with an internal oligonucleotide unique to ovine eNOS. (A) Representative Southern analysis. Quantitative densitometry was performed, and abundance of RT-PCR product for 16 µg RNA was given an arbitrary value of 100. (B) Plot of PCR product abundance versus quantity of RNA used.

Statistical Analysis

Data for the immunoblot analyses and RT-PCR assays are presented as the percent of the value observed under control conditions. Standard errors were calculated for the experimental values for graphic display of the variability of results between studies. Significance was determined using two-tailed Student's t tests for single comparisons, and analysis of variance and post hoc Neuman-Keuls testing for multiple comparisons (29). Significance was accepted at the 0.05 level of probability.

	Results

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Effect of NO on eNOS Protein Expression

To first determine whether exogenous NO modulates eNOS protein expression, the effect of NO from the donor agent SpNO (10⁶ M) was assessed over 24 h (Figure 2). Signal for eNOS protein was detected at 135 kD. In the representative immunoblot shown (Figure 2A), there was enhanced eNOS protein expression in cells exposed to exogenous NO from SpNO compared with Sp alone. Quantitative densitometry for six separate experiments is shown in Figure 2B. There was a 52% increase in eNOS protein abundance in PAEC treated with exogenous NO. The rise in eNOS expression was similar with exposure to 10⁶ M and 10⁸ M SpNO (152 ± 20% and 152 ± 18% of control, respectively; n = 4).

View larger version (20K): [in this window] [in a new window]   Figure 2.   Effect of exogenous NO on eNOS protein expression. PAEC were exposed to the NO donor SpNO (106 M) or to the parent compound Sp (106 M) for 24 h, and eNOS protein abundance was assessed by immunoblot analysis (A). Signal for eNOS was evident at 135 kD. These results are representative of six independent experiments. (B) Summary data for quantitative densitometry for six experiments. Values are means ± standard error of the mean (SEM) for protein abundance expressed as a percent of eNOS for control conditions (Sp). *P < 0.05 versus Sp.

Having demonstrated a change in eNOS expression within 24 h of varying exogenous NO exposure, the time course of the effect was then evaluated in studies performed over 8 to 48 h (Figure 3). Immunoblot analysis revealed a detectable increase in eNOS protein with SpNO treatment to levels that were 223% of control values (Sp alone) by 16 h. The increase in eNOS expression in response to exogenous NO persisted for at least 48 h.

View larger version (17K): [in this window] [in a new window]   Figure 3.   Time course of the effect of exogenous NO on eNOS protein expression. PAEC were exposed to the NO donor SpNO (106 M) or to the parent compound Sp (106 M) for 8 to 48 h. eNOS protein abundance was assessed by immunoblot analysis, and quantitative densitometry was performed. Values are means ± SEM for protein abundance in SpNO-treated cells, expressed as a percent of eNOS for control conditions (Sp alone), n = 4 experiments. *P < 0.05 versus control.

To determine whether endogenous NO also modulates eNOS expression, the effect of NOS inhibition on eNOS protein abundance was evaluated over 24 h (Figure 4). The representative immunoblot reveals a decrease in eNOS protein expression in cells treated with L-NAME (Figure 4A). Quantitative densitometry for six independent experiments (Figure 4B) confirmed these results, showing a 43% decrease in eNOS protein with the inhibition of endogenous NO production.

View larger version (21K): [in this window] [in a new window]   Figure 4.   Effect of endogenous NO on eNOS protein expression. PAEC were exposed to NOS inhibition with L-NAME (2.0 mM) or to control conditions (C) for 24 h, and eNOS protein abundance was assessed by immunoblot analysis (A). Signal for eNOS was evident at 135 kD. These results are representative of six independent experiments. (B) Summary data for quantitative densitometry for six experiments. Values are means ± SEM for protein abundance expressed as a percent of eNOS for control conditions. *P < 0.05 versus control.

The time course of the effect of endogenous NO inhibition on eNOS expression was then evaluated in studies performed over 8 to 48 h (Figure 5). Immunoblot analysis revealed a decrease in eNOS protein to levels that were 41% of control values within 8 h, and eNOS protein abundance was down to 33% of control levels after 16 h. The decline in eNOS expression in response to endogenous NO inhibition persisted for 24 h, but was no longer apparent after 48 h.

View larger version (16K): [in this window] [in a new window]   Figure 5.   Time course of the effect of endogenous NO on eNOS protein expression. PAEC were exposed to NOS inhibition with L-NAME (2.0 mM) or to control conditions for 8 to 48 h. eNOS protein abundance was assessed by immunoblot analysis, and quantitative densitometry was performed. Values are means ± SEM for protein abundance in L-NAME-treated cells, expressed as a percent of eNOS for control conditions, n = 4 experiments. *P < 0.05 versus control.

Effect of NO on eNOS Enzymatic Activity

To confirm the enhancement in eNOS protein expression seen with exposure to exogenous NO, NOS enzymatic activity was quantitated in lysates of PAEC exposed to either Sp or SpNO (10⁶ M for 24 h) by measuring the conversion of ³H-L-arginine to ³H-L-citrulline (Figure 6). Paralleling the rise in eNOS protein expression, NOS activity was increased by 120% in PAEC exposed to exogenous NO.

View larger version (11K): [in this window] [in a new window]   Figure 6.   Effect of exogenous NO on NOS activity. PAEC were exposed to the NO donor SpNO (106 M) or to the parent compound Sp (106 M) for 24 h and NOS enzymatic activity was assessed in the cell lysates by measuring 3H-L-arginine conversion to 3H-L-citrulline. Values are means ± SEM, n = 4. *P < 0.05 versus Sp.

Effect of NO on eNOS mRNA Expression

To determine the basis for the increase in eNOS protein expression and NOS enzymatic activity with exposure to exogenous NO, eNOS mRNA abundance was evaluated by RT-PCR in cells treated with the parent compound Sp or the NO donor SpNO for 16 h. A single PCR product was obtained for eNOS at the expected size of 281 base pairs (bp) (Figure 7A, upper panel). The representative Southern blot reveals an increase in eNOS mRNA as determined by RT-PCR in cells exposed to exogenous NO, paralleling the findings for eNOS protein and NOS enzymatic activity. PCR was also performed for MDH to serve as a control for the RT step, yielding a single PCR product at the expected size of 396 bp (Figure 7A, lower panel). These results were confirmed in four independent experiments. Summary data are shown in Figure 7B, revealing a 2.8-fold increase in eNOS mRNA at 16 h.

View larger version (15K): [in this window] [in a new window]   Figure 7.   Effect of exogenous NO on eNOS mRNA expression. (A) Southern analysis of RT-PCR products for eNOS (top) and MDH (bottom) in PAEC exposed to the NO donor SpNO (106 M) or the parent compound Sp (106 M) for 16 h. Band sizes were as follows: eNOS, 281 bp; MDH, 396 bp. Data are representative of four independent experiments. (B) Summary data for four experiments. eNOS densitometry values corrected for MDH are expressed as percent in control, Sp-treated cells (means ± SEM). *P < 0.05 versus control.

To assess the mechanism underlying the decrease in eNOS protein expression with inhibition of endogenous NO, eNOS mRNA abundance was also determined by RT-PCR in control cells and cells treated with L-NAME for 16 h. The representative Southern blot for eNOS (Figure 8A, upper panel) reveals a decrease in the mRNA as determined by RT-PCR in cells undergoing NOS inhibition, paralleling the findings for eNOS protein. PCR product abundance for MDH was similar in control and L-NAME-treated cells (Figure 8A, lower panel). These results were confirmed in four independent experiments. Summary data are shown in Figure 8B, revealing a 57% decrease in eNOS mRNA at 16 h.

View larger version (17K): [in this window] [in a new window]   Figure 8.   Effect of endogenous NO on eNOS mRNA expression. (A) Southern analysis of RT-PCR products for eNOS (top) and MDH (bottom) in PAEC exposed to NOS inhibition with L-NAME (2.0 mM) or to control conditions (C) for 16 h. Band sizes were as follows: eNOS, 281 bp; MDH, 396 bp. Data are representative of four independent experiments. (B) Summary data for four experiments. eNOS densitometry values corrected for MDH are expressed as percent in control cells (means ± SEM). *P < 0.05 versus control.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

In the present study, we have determined whether prolonged exposure to exogenous NO modulates eNOS expression in intrapulmonary artery endothelium. Contrary to the hypothesis raised, we have shown that there is an increase in eNOS protein expression in PAEC exposed to exogenous NO, resulting in a parallel increase in NOS enzymatic activity. These findings indicate that prolonged exposure to exogenous NO does not have a negative effect on the abundance of eNOS, but rather that exogenous NO upregulates the expression of the enzyme.

In addition to demonstrating that exogenous NO upregulates eNOS protein expression, we have evaluated the role of endogenous NO. Consistent with the observed upregulation in response to exogenous NO, the prolonged inhibition of endogenous NO production with L-NAME caused a decline in PAEC eNOS expression. This finding indicates that enzymatically generated NO may play an important positive-feedback regulatory role in eNOS expression in PAEC, thereby affecting pulmonary vascular endothelial function in an autocrine manner.

Along with showing that either exogenous or endogenous NO upregulates eNOS expression in the fetal intrapulmonary artery endothelium, we have evaluated the time courses of these effects. Changes in eNOS protein were readily detectable within 16 h of increasing exogenous NO levels with SpNO, and within 8 h of decreasing endogenous NO with L-NAME. For both interventions, the maximal effects were evident by 16 h and effects then declined in a relative manner beyond 16 h. The resulting mirror images of the time courses displayed in Figures 3 and 5 suggest that the underlying mechanisms are similar for exogenous and endogenous NO. The NO-induced changes in eNOS expression in the fetal intrapulmonary artery endothelium were considerably more rapid than those we have previously demonstrated with varying levels of oxygenation or with estrogen exposure, which were not evident before 24 to 48 h. However, the maximal effects of exogenous or endogenous NO (2- to 3-fold changes at 16 h) are comparable to those observed with varying oxygenation or extrogen exposure (20, 30).

To determine the basis for NO modulation of eNOS protein expression in the fetal intrapulmonary artery endothelium, RT-PCR assays were established to evaluate eNOS mRNA levels in cells exposed either to exogenous NO or to NOS inhibition with L-NAME. Paralleling the observed changes in eNOS protein abundance, steady-state eNOS mRNA levels were upregulated by exogenous NO donated by SpNO and they were downregulated by L-NAME. This indicates that the mechanism(s) by which NO modulates eNOS expression involves processes at the level of gene transcription or mRNA stability. With the exception of tumor necrosis factor-, which principally alters eNOS mRNA stability (31), studies of both humoral and physical modulators of eNOS indicate that levels of expression are primarily under transcriptional control (32, 33). Consistent with the present observations, it has been shown that exogenous cyclic guanosine monophosphate (cGMP), the signaling molecule through which NO has its principal effects, causes upregulation of eNOS expression in endothelial cells grown from bovine main pulmonary artery, and that this most likely involves transcriptional events (34). These cumulative observations suggest that there is positive-feedback control of eNOS gene expression by the physiologically relevant product of the enzyme NO, and that this is mediated by the second messenger, cGMP.

The current observation that both exogenous and endogenous NO upregulate eNOS in the fetal intrapulmonary artery endothelium contrasts with recent findings in main pulmonary arterial endothelial cells from fetal lambs. Studies in the latter model revealed no change in eNOS protein or mRNA abundance after exposure to 1 mM sodium nitroprusside for up to 24 h (16). This disparity may be explained by the difference in cell types studied because the intrapulmonary vasculature and the main pulmonary artery originate from unique embryonic sources (35). Alternatively, it may be related to the requirement for bioconversion for NO release from nitroprusside, whereas bioconversion is not necessary for NO generation by SpNO (21).

To our knowledge, the present investigation is the first to demonstrate that NO modulates eNOS gene expression. However, effects of NO on iNOS expression have already been described. In studies of renal mesangial cells, NO donor compounds have been found to potently augment cytokine-induced expression of macrophage-type iNOS at the transcriptional level (36). In contrast, investigations of macrophage-type iNOS induction in central nervous system glial cells indicate that NO inhibits iNOS gene transcription (37). Further, studies of hepatic iNOS induction in a model of chronic liver inflammation induced by Corynebacterium parvum have revealed that NO downregulates iNOS mRNA and protein expression (38). Thus, the effects of NO on NOS gene expression appear to be cell- and isoform-specific.

The present observation that endogenous and exogenous NO cause upregulation of eNOS expression contrasts with the acute effects of the molecule on NOS activity. It has been demonstrated previously that NO acutely inhibits NOS activity, and that eNOS and nNOS are more sensitive than iNOS. In addition, not only exogenously added NO but also enzymatically generated NO acutely inhibits the activity of eNOS and nNOS (15, 16). Oxyhemoglobin partially prevents the inhibition of NOS activity by NO, suggesting that the inhibition involves a direct interaction of NO with NOS and not an indirect mechanism such as limitations in cofactor or oxygen availability (39). The mechanism appears to involve the heme iron prosthetic group of NOS. The oxidation state of the heme iron is critical in determining the magnitude of inhibition of NOS by NO, and one of the cofactor roles of tetrahydrobiopterin may be to reduce the negative feedback effect of NO on NOS activity by favoring the formation of the ferrous heme state in NOS (15). Thus, we now have evidence of both short-term negative feedback and long-term positive feedback regulation of eNOS expression and enzymatic activity.

Keeping in mind the potential limitations of extrapolating the present findings in cultured cells to processes in the intact lung, there are important ramifications of NO modulation of intrapulmonary artery eNOS expression that are relevant to the perinatal period. For the past several years, patients with PPHN and other forms of pulmonary hypertension have been exposed to exogenous NO gas in studies of its therapeutic efficacy as a selective pulmonary vasodilator. Although a large proportion of the patients have sustained improvements in systemic oxygenation with inhaled NO, some patients receive only temporary benefit from the gas and others have prolonged requirements for the therapy (9, 11, 12). The findings of the present study in the cultured cell model suggest that these clinical observations are most likely not related to downregulation of eNOS expression. Alternatively, difficulties may arise in the delivery of the gas to the level of the alveolus, or there may be negative effects of prolonged NO therapy on the responsiveness of the vascular smooth muscle to the vasodilator. Consistent with the latter possibility, it has been demonstrated that NO causes homologous and heterologous desensitization of the target for NO action, soluble guanylyl cyclase, in certain cell types (40).

There are also important physiologic and pathophysiologic implications of the present findings. Under normal circumstances, the increasing capacity for NO production that has been demonstrated to occur during late fetal life may play a role in the upregulation in eNOS that optimizes NO-mediated pulmonary vasodilation during transition at birth (4, 41). In contrast, when the capacity for fetal pulmonary endothelial NO production is attenuatedsuch as with congenital diaphragmatic hernia or fetal pulmonary hypertension (6)or when fetal pulmonary endothelial NO production is decreased due to fetal hypoxemia (18, 19), this may blunt the developmental upregulation of the enzyme that normally occurs during late gestation, thereby contributing to the pathophysiology of pulmonary hypertension in the newborn period. Further studies of the basis for NO-mediated regulation of intrapulmonary endothelial cell gene expression will increase our understanding of the role of this molecule in the developing lung and also its application as a therapeutic agent.