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J Appl Physiol 85: 1070-1078, 1998;
8750-7587/98 $5.00
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Vol. 85, Issue 3, 1070-1078, September 1998

Regulation of the endogenous NO pathway by prolonged inhaled NO in rats

Deborah U. Frank1, Damian J. Horstman1, Geoffrey N. Morris2, Roger A. Johns2, and George F. Rich2

Departments of 1 Biomedical Engineering and 2 Anesthesiology, University of Virginia Health System, Charlottesville, Virginia 22906-0010

    ABSTRACT
Top
Abstract
Introduction
Methods
Results
Discussion
References

Nitric oxide (NO) modulates the endogenous NO-cGMP pathway. We determined whether prolonged inhaled NO downregulates the NO-cGMP pathway, which may explain clinically observed rebound pulmonary hypertension. Rats were placed in a normoxic (N; 21% O2) or hypoxic (H; 10% O2) environment with and without inhaled NO (20 parts/million) for 1 or 3 wk. Subsequently, nitric oxide synthase (NOS) and soluble guanylate cyclase (GC) activity and endothelial NOS (eNOS) protein levels were measured. Perfusate cGMP levels and endothelium-dependent and -independent vasodilation were determined in isolated lungs. eNOS protein levels and NOS activity were not altered by inhaled NO in N or H rats. GC activity was decreased by 60 ± 10 and 55 ± 11% in N and H rats, respectively, after 1 wk of inhaled NO but was not affected after 3 wk. Inhaled NO had no effect on perfusate cGMP in N lungs. Inhaled NO attenuated the increase in cGMP levels caused by 3 wk of H by 57 ± 11%, but there was no rebound in cGMP after 24 h of recovery. Endothelium-dependent vasodilation was not altered, and endothelium-independent vasodilation was not altered (N) or slightly increased (H, 10 ± 3%) by prolonged inhaled NO. In conclusion, inhaled NO did not alter the endogenous NO-cGMP pathway as determined by eNOS protein levels, NOS activity, or endothelium-dependent vasodilation under N and H conditions. GC activity was decreased after 1 wk; however, GC activity was not altered by 3 wk of inhaled NO and endothelium-independent vasodilation was not decreased.

nitric oxide; pulmonary hypertension; chronic hypoxia; nitric oxide synthase; guanylate cyclase; cyclic 3',5'-guanosine monophosphate; pulmonary vasodilation

    INTRODUCTION
Top
Abstract
Introduction
Methods
Results
Discussion
References

THE ENDOGENOUS NITRIC OXIDE (NO)-cGMP pathway plays an important role in modulating pulmonary vascular tone in normal and disease states (17, 22). Endogenous NO is produced by nitric oxide synthase (NOS) in basal concentrations and in response to shear stress or receptor-mediated increases in calcium (22). The NO produced in endothelial cells diffuses into underlying smooth muscle cells to stimulate soluble guanylate cyclase (GC), increase cGMP, and cause vasodilation (22). Inhibition of the endogenous NO-cGMP pathway has been demonstrated to increase pulmonary vascular resistance (31).

Endogenous and exogenous NO appear to negatively regulate all three NOS isoforms. Neuronal NOS (nNOS) is inhibited by NO in a concentration-dependent and reversible manner (29). Inducible NOS (iNOS) activity is also inhibited by NO donors (2). Endothelial NOS (eNOS) is inhibited by NO in its partially purified form (27, 28), in intact cells (27), and in a cellular bioassay of responses to bradykinin and increased shear stress (5). Endogenous and exogenous NO may also regulate GC. Several studies have found a desensitization of the GC in vascular smooth muscle made tolerant to NO donors (3, 26). However, other studies have demonstrated that tolerance is not occurring at the level of the GC (8). In contrast, a supersensitivity to nitrovasodilators and inhaled NO has been demonstrated with endothelium removal or inhibition of basal NO production (21).

Inhaled NO may be useful clinically as a selective pulmonary vasodilator (32). However, clinical reports indicate that discontinuation of inhaled NO after prolonged periods of administration may result in rebound pulmonary hypertension (14). Several studies have suggested that inhaled NO downregulates endogenous NO-cGMP, which may be an explanation for rebound pulmonary hypertension. We have previously demonstrated that 3 wk of NO inhalation decrease endothelium-dependent vasodilation in isolated rat lungs constricted with acute hypoxia (31). However, acute hypoxia is known to inhibit the NO-cGMP pathway and therefore may cause confusion in the interpretation of the results (30). Other groups have demonstrated that shorter periods (48 h) of inhaled NO decrease endothelium-dependent vasodilation in normoxic and acutely hypoxic rats (7, 25).

We determined whether 1 and 3 wk of inhaled NO biochemically and functionally downregulate the endogenous NO-cGMP pathway in rats. Biochemically, we evaluated eNOS protein levels, total lung NOS activity, soluble GC activity, and cGMP production in rats. Functionally, we evaluated endothelium-dependent (bradykinin and calcium ionophore A-23187) and -independent (acute NO) vasodilation in saline-perfused isolated rat lungs constricted with the thromboxane analog U-46619. In the pulmonary circulation there are normally low levels of NOS activity (37). Therefore, we evaluated not only rats exposed to inhaled NO under normoxic conditions but also rats exposed to chronic hypoxia, which is a model of pulmonary hypertension in which eNOS is upregulated (33, 37).

    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

Chronic environment groups. This study was approved by the animal research committee at the University of Virginia. Male Sprague-Dawley rats (250-350 g) were placed in chambers that maintained different normobaric environments. Hypoxic (H) rats were maintained in 10% O2, whereas normoxic (N) rats were maintained in 21% O2. Rats exposed to inhaled NO were maintained with a NO level of 20 parts/million (ppm) and 10 (HNO) or 21% O2 (NNO) for 1 or 3 wk. In recovery groups, the rats were kept in a NO-free, hypoxic (NOS and GC studies), or room air (cGMP studies) environment for 24 h after the 1- or 3-wk exposure (NNOR, HNOR, or HR). For each study, rats (n = 8) were assessed in each of the six groups: N, NNO, NNOR, H, HNO, and HNOR.

NO was delivered to the chamber from a gas cylinder containing 1,600 or 800 ppm NO in N2 (BOC Gases, Port Allen, LA). NO and NO2 (NO2 < 1 ppm) levels were monitored with electrochemical sensors (Bedfont, UK). O2 levels were regulated by a Pro:ox model 350 unit (Reming Bioinstruments, Redfield, NY). A pump drew the chamber atmosphere through Drierite anhydrous calcium, BaraLyme barium hydroxide lime, and activated carbon to remove water vapor, CO2 and NO2 (<2 ppm), and ammonia, respectively.

NOS activity. Lungs were harvested by perfusion with ice-cold normal saline (30 ml) containing heparin (1 U/ml) via the pulmonary artery, snap-freezing in liquid N2, and storage at -80°C until homogenization. Total NOS activity was assessed in crude lung homogenate by minor modification of the method of Shaul et al. (33) of arginine-to-citrulline conversion measurement. Lung tissue was homogenized on ice in Tris buffer (50 mM, pH 7.5) containing EDTA (1.0 mM) and proteinase inhibitors (5 mM beta -mercaptoethanol, 10 µg/ml pepstatin A, 10 µg/ml leupeptin, and 90 µg/ml phenylmethylsulfonyl fluoride) with a Polytron homogenizer and centrifuged at 1,000 g at 4°C for 10 min. Homogenate (100 µl) was mixed with 25 µl of concentrated reaction mixture. The final reaction mixture was composed of 50 mM Tris (pH 7.4), 2 mM beta -NADPH, 2 µM BH4, 1.5 mM CaCl2, 15 nM calmodulin, 10 µM FAD, 10 µM riboflavin monophosphate, 10 µM L-arginine, and 10 µCi/ml L-[3H]arginine (Amersham, Arlington Heights, IL). After incubation for 30 min at 37°C, the reaction was stopped with 1 ml of ice-cold termination buffer [50 mM Tris (pH 5.5), 2 mM EDTA, and 2 mM EGTA]. Samples were applied to 1.25-ml columns of Dowex AG50WX-8 resin (sodium form) and eluted with 2 ml of termination buffer. The eluted sample was collected, and radioactivity was quantified in a scintillation counter. Enzyme activity was calculated as picomoles of arginine used per milligram of protein per minute. Protein concentration was determined in the lung homogenate by using the Bradford method (4). The assay was linear over time for ~50 min.

NOS protein quantitation: Western blot analysis. Lung protein was separated by SDS-PAGE by the method of Laemmli (13) in a 7.5% (wt/vol) polyacrylamide gel. Crude lung homogenate was prepared as described for NOS activity assay. One hundred and twenty micrograms of homogenate from each rat lung were loaded into separate wells of the gel and separated overnight with a constant current of 15-20 mA (Bio-Rad Laboratories, Hercules, CA). Proteins in the gel were then transferred to nitrocellulose paper, using an electrophoretic transfer cell (Bio-Rad Laboratories). Bovine pulmonary artery endothelial cell and lipopolysaccharide (LPS)-induced rat lung homogenates were included as control in Western analysis immunodetected with the eNOS and iNOS antibodies, respectively.

For detection of eNOS protein, the blot was incubated with a monoclonal antibody specific to the eNOS protein (Transduction Laboratories, Lexington, KY). For detection of iNOS protein, the blot was incubated with a polyclonal antibody specific to the iNOS protein (Santa Cruz Biotech, Santa Cruz, CA). Protein bands recognized by the antibodies were detected on film by enhanced chemiluminescence (Amersham) and quantified by using a laser densitometer and Imagequant software (Molecular Dynamics, Sunnyvale, CA). The band density was normalized to the level of the normoxic samples. The density of eNOS protein bands detected by Western blot analysis was linearly related to the amount of protein loaded onto the polyacrylamide gel.

GC activity. GC enzyme activity was measured in lung homogenates that were separated into soluble and particulate fractions by ultracentrifugation. The method used was a slight modification of the method of Zuo and Johns (38). Basal and sodium nitroprusside (SNP)-stimulated enzyme activity were measured in the soluble fraction. Basal activity was measured in the particulate fraction. Lung tissue was homogenized in buffer [50 mM Tris · HCl (pH 7.4), 250 mM sucrose, 0.2 mM benzamidine] with a Polytron blender and centrifuged at 1,000 g for 10 min at 2°C. The resultant supernatant was centrifuged at 105,000 g for 60 min at 4°C. The supernatant (soluble fraction) was pipetted off, and the pellet (particulate fraction) was washed and resuspended. Protein concentrations were determined by the Bradford method (4).

The GC reaction mixture contained (in mM) 50 Tris · HCl (pH 7.4), 1 IBMX, 4 MnCl2, 0.5 ATP (disodium salt, grade I: from yeast), 1 GTP (sodium salt, type III), and 15 creatine phosphate as well as 100 µg/100 µl creatine phosphokinase. The protein sample (20 µg in <10 µl) was added to the reaction mixture (100 µl), and the reaction was allowed to proceed for 5 min and then was terminated by addition of ice-cold 0.2 N HCl (100 µl). For stimulated activity, 10 µl of SNP were added immediately after protein (final concentration 10-2 M). Samples were assayed for cGMP content by radioimmunoassay. The assay of soluble basal activity was linear for at least 10 min, and 10-2 M SNP gave up to a 12-fold stimulation in activity.

Isolated rat lung preparation. Experiments were initiated within 30 min after removal from the chamber. Rats were anesthetized with halothane and injected with pentobarbital sodium (5 mg/100 g ip). A 17-gauge metal cannula was inserted into the trachea, and the lungs were ventilated with warmed gas with a tidal volume of 1 ml/100 g body weight and a frequency of 60 breaths/min. The heart and lungs were exposed, and then the rat was heparinized (100 U) and partially exsanguinated. Via an incision in the right ventricle, a 13-gauge steel cannula, connected to the primed perfusion system, was inserted through the pulmonic valve into the main pulmonary artery. A 3.5-mm-OD plastic cannula was secured in the left ventricle. Perfusate drained from the left ventricle to a warmed glass reservoir and returned to the pulmonary artery by a roller pump. The initial 30 ml of perfusate were discarded to ensure all blood was flushed from the lungs. Thirty milliliters of perfusate were then recirculated for the duration of the experiment.

Perfusate was a modified physiological salt solution containing (in g/l) 0.265 CaCl2 · 2H2O, 0.098 MgSO4, 0.4 KCl, 6.8 NaCl, 0.122 NaH3PO4, and 1.0 D-glucose. Additional inclusions were Ficoll (4 g/100 ml, type 70) to act as a colloid, sodium meclofenamate (3.1 µM) to inhibit cyclooxygenase, and sodium bicarbonate (1.6 g/l) to maintain pH within the physiological range (4). Perfusate pH was continuously monitored and maintained between 7.35 and 7.45 by addition of HCl or NaHCO3 as necessary.

The pulmonary arterial pressure (Ppa), left atrial pressure, and airway pressure of the isolated lungs were measured by using pressure transducers and a Datascope 2001A monitor. A Gould 2400S strip-chart recorder and a personal computer enabled analog and digital recording. The ventilatory gases were administered through separate flowmeters to the ventilator. Inspired O2 and NO (Exidyne Instrumentation Technologies, Exton, PA) concentrations were monitored near the tracheal tube. NO for acute inhalation was a mixture of 254 ppm NO in N2 (Roberts Oxygen, Baltimore, MD).

Once the pulmonary circulation was isolated, it was ventilated with 95% air-5% CO2, and the perfusate flow rate was increased to 16 ml/min. The height of the reservoir was adjusted such that left atrial pressure was greater than the average airway pressure (~4 mmHg). Twenty minutes after the final adjustment, the experimental protocol began. All vasodilators and constrictors were obtained from Sigma Chemical (St. Louis, MO).

Responses to endothelium-dependent vasodilators. A Ppa 10 mmHg above baseline was established by constricting the pulmonary circulation with U-46619, a thromboxane A2 mimetic. Endothelium-dependent vasodilation was assessed with injections of 3 µg of bradykinin (in 0.1 ml saline) followed by 1 µg of the calcium ionophore A-23187 (in 0.5% DMSO in saline). A control 0.1-ml injection of the A-23187 vehicle (0.5% DMSO in saline) had no effect on Ppa.

Endothelium-independent vasodilation: acute inhaled NO responses. After determination of endothelium-dependent vasodilation, nitro-L-arginine methyl ester (L-NAME; 100 µM) was added to the perfusate. Twenty minutes were allowed to ensure effective inhibition of NOS. When necessary, perfusion Ppa was reestablished 10 mmHg above the original baseline with additional U-46619. Endothelium-independent vasodilation was determined with acute inhalation of NO in concentrations of 6 and 40 ppm.

Measurement of lung production of cGMP. Samples of perfusate were collected at 20-min intervals for measurement of cGMP. To ensure adequate substrate for the NOS enzyme, 100 µM L-arginine were included in the perfusate. Samples were collected for 1 h while the lungs were ventilated with a normoxic gas mixture (95% air-5% CO2) and an additional hour while lungs were alternately ventilated with the normoxic mixture and a hypoxic gas mixture (95% N2-5% CO2).

Three additional rats in the N group and H groups were studied under this protocol with the addition of 580 µM L-NAME to the perfusate. This was done to determine the relative contribution of soluble and particulate GC to the cGMP accumulating in the perfusate.

Analysis and statistics. NOS protein and GC activity levels were normalized to the mean level of the N group. Data from the enzyme activity studies and Western blots, for which one value was obtained for each rat, were analyzed by two-factor ANOVA. cGMP data were analyzed by using a two-way repeated-measures ANOVA. The bradykinin, A-23187, and acute NO vasodilation responses were calculated as the maximal percent drop in Ppa, with 100% being the pressure immediately before drug dose minus the baseline pressure. Data were analyzed by ANOVA. Individual comparisons among group means were made by using the Student-Newman-Keuls post hoc test. A P value <0.05 was considered significant. Data are reported as means ± SE.

    RESULTS
Top
Abstract
Introduction
Methods
Results
Discussion
References

General results. Inhaled NO significantly attenuated the increase in right ventricular hypertrophy (right ventricle wt-to-left ventricle plus septum wt ratio) in rats exposed to H for 3 wk but did not have a significant effect in the 1-wk H rats or N rats (Table 1). Inhaled NO did not alter the hypoxia-induced decrease in weight gain, increase in hematocrit, or increase in dry lung weight-to-body weight ratio. The Ppa measured in the absence of NO in saline-perfused isolated lungs under normoxic conditions was not different in rats exposed to prolonged NO at 1 wk (N = 9.1 ± 0.3, H = 13.2 ± 0.4 mmHg) or 3 wk (N = 9.0 ± 0.2, H = 18.5 ± 0.7 mmHg) compared with rats not exposed to NO at 1 wk (N = 8.9 ± 0.2, H = 13.0 ± 0.4 mmHg) and 3 wk (N = 8.9 ± 0.3, H = 17.0 ± 0.4 mmHg).

                              
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  		Table 1.   General results

NOS: protein levels and enzyme activity. The monoclonal antibody to eNOS detected eNOS protein on the Western blots of rat lung homogenate at a molecular mass of ~135 kDa. Prolonged inhaled NO did not affect eNOS protein in either H or N rats, whereas 1 and 3 wk of H significantly increased eNOS levels compared with N levels (Figs. 1 and 2). Twenty-four hours of recovery from inhaled NO also did not affect eNOS protein levels. There was no difference in total lung NOS activity due to prolonged inhaled NO, H, or recovery in either the 1- or 3-wk lungs compared with N lungs (Fig. 3).


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  		Fig. 1.   Representative Western blot of bovine endothelium control (E) and endothelial nitric oxide synthase (eNOS) protein for rats exposed to normoxia (N), N+inhaled NO (NNO), hypoxia (H), or H+NO (HNO) for 1 (A) and 3 wk (B). Recovery rats were maintained in N (NNOR) or H (HNOR) without NO for 24 h. eNOS was increased by 1 and 3 wk of hypoxia but was unaffected by inhaled NO or recovery.


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  		Fig. 2.   eNOS protein levels normalized to N for rats exposed to N, NNO, H, or HNO for 1 (A) and 3 wk (B). NNOR and HNOR rats were maintained without NO for 24 h. eNOS was unaffected by inhaled NO. Values are means ± SE. * Significant increase in eNOS in H, HNO, and HNOR groups compared with N group, P < 0.05.


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  		Fig. 3.   Total lung NOS activity normalized to N from crude lung homogenate from rats exposed to N, NNO, H, or HNO for 1 (A) and 3 wk (B). NNOR and HNOR rats were maintained without NO for 24 h. NOS activity was unaltered by inhaled NO or H. Values are means ± SE.

The polyclonal antibody to iNOS detected iNOS protein on the Western blots only in the LPS-induced rat lung homogenate that was included as a positive control. The iNOS band detected was at a molecular mass of ~118 kDa. No iNOS was detected in the N or H lungs from experimental rats.

GC: enzyme activity. The stimulated activity of the GC enzyme was decreased by 1 wk of inhaled NO (P < 0.001), and it remained lower than the normoxic or hypoxic levels 24 h after removal from the NO environment (NNOR and HNOR groups) (Fig. 4). A decrease in enzyme activity by NO was suggested by the levels for basal activity, but the results did not reach statistical significance (P = 0.10). There was no significant effect of prolonged inhaled NO on basal or stimulated GC activity after 3 wk. One week of hypoxia did not affect either the basal or stimulated soluble GC enzyme activity, whereas 3 wk of hypoxia increased both the basal (P < 0.001) and the stimulated (P = 0.04) activity compared with normoxic levels.


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  		Fig. 4.   Basal and stimulated [sodium nitroprusside (SNP; 10-2 M)] soluble guanylate cyclase (GC) activity normalized to N in rat lung homogenate from rats exposed to N, NNO, H, or HNO for 1 (A) and 3 wk (B). NNOR and HNOR rats were maintained without NO for 24 h. H group values were not different from N group values. Open bars, basal GC activity; hatched bars, 10-2 M SNP-stimulated GC activity. Values are means ± SE. # Decrease in stimulated GC activity by inhaled NO (P < 0.001). This inhibition was not reversed after 24 h of recovery. * Significant increase in both basal (P < 0.001) and stimulated (P = 0.04) GC activity in H groups vs. N groups. There was no effect of inhaled NO.

Particulate GC enzyme activity was not altered by prolonged inhaled NO in 1- or 3-wk rats. Particulate GC activity was decreased by 1 wk of hypoxia compared with normoxia (0.83 ± 0.02 vs. 0.99 ± 0.04, P = 0.002). After 3 wk, there were no effects of hypoxia on the particulate activity (1.06 ± 0.07 vs. 1.07 ± 0.07).

cGMP in lung perfusate. Perfusate cGMP levels (measured at 140 min) were not altered by inhaled NO in N lungs after 1 wk (N = 174 ± 16, NNO = 170 ± 18 pmol/ml) or 3 wk (N = 192 ± 17, NNO = 190 ± 16 pmol/ml). The perfusate cGMP levels were increased by chronic H (3 wk > 1 wk, 1,450 ± 110 vs. 905 ± 116 pmol/ml) compared with the N group (Fig. 5). Prolonged NO exposure attenuated the increase in cGMP release, reaching statistical significance in the 3-wk group (610 ± 80 pmol/ml) but not the 1-wk group (426 ± 78 pmol/ml).


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  		Fig. 5.   cGMP accumulation in isolated lung perfusate with time. Rats were exposed to N, H, or HNO for 3 wk. H (HR) and HNOR groups were allowed to recover in air for 24 h. Values are means ± SE. cGMP levels (pmol/ml) increased with time (P < 0.001). * Significant increase in 3-wk H cGMP levels over levels in HNO, HR, HNOR, and N groups (P < 0.05). # No difference in values in HNO, HR, and HNOR rats, but values are increased compared with N rats (P < 0.05).

To determine whether differences in cGMP release were caused by downregulation secondary to inhaled NO or attenuation of hypoxic-induced upregulation, additional 3-wk H and HNO animals were studied after a 24-h recovery in room air (HR and HNOR groups). cGMP levels were decreased in the HR group (605 ± 92 pmol/ml) compared with the 3-wk H group but were unchanged in the HNOR group (696 ± 110 pmol/ml, Fig. 5). There was no difference among the HNO, HNOR, and HR groups.

In rats studied to determine whether cGMP released into the perfusate was produced by soluble or particulate GC, L-NAME significantly decreased cGMP. L-NAME decreased cGMP levels by 82 ± 4% in the H rats (98% relative to the N levels). The cGMP levels in N lungs did not change with the addition of L-NAME.

Endothelium-dependent vasodilation. In U-46619-constricted N lungs, prolonged inhaled NO did not affect the vasodilation to either bradykinin or the calcium ionophore A-23187 in the N groups, the 3-wk H group, nor did it affect the calcium ionophore A-23187 vasodilation in the 1-wk H group (Fig. 6). There was less bradykinin vasodilation in the 1-wk HNO group compared with the 1-wk H group.


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  		Fig. 6.   Endothelium-dependent vasodilation (calcium ionophore A-23187 and bradykinin) by using normoxic isolated lung constricted with U-46619. Rats were previously exposed to N, H, NNO, or HNO for 1 or 3 wk. Values are means ± SE. Percent change in pulmonary arterial pressure (Ppa) due to vasodilation vs. rat group is shown. * Significantly greater 1-wk H bradykinin vasodilation than N vasodilation (P < 0.05). # Bradykinin vasodilation in 1-wk HNO group was less than in H group (P < 0.05). ** Significantly greater bradykinin and A-23187 vasodilations in 3-wk H group than in N and 1-wk H groups (P < 0.05). Inhaled NO did not alter endothelium-dependent vasodilation in N or H lungs.

Bradykinin vasodilation was greater after H (3 wk > 1 wk) compared with the N group. The calcium ionophore A-23187-induced vasodilation was greater in the 3-wk H group but not in the 1-wk H group compared with the N group.

Endothelium-independent vasodilation: acute inhaled NO. In U-46619-constricted lungs, the vasodilation secondary to acute inhaled NO was greater with 40 ppm than with 6 ppm in all groups (P < 0.001). Prolonged NO did not affect endothelium-independent vasodilation with either acute NO concentration in the N groups (Fig. 7). Prolonged NO did not affect the acute response to 6 ppm NO in the H groups, but with 40 ppm the vasodilation was increased in the 1- and 3-wk groups.


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  		Fig. 7.   Endothelium-independent vasodilation (acute 6 and 40 ppm NO) by using normoxic isolated lung constricted with U-46619. Rats were previously exposed to N, H, NNO, or HNO for 1 or 3 wk. Percent change in Ppa due to vasodilation vs. rat group is shown. Values are means ± SE. Vasodilation to 40 ppm NO is greater than vasodilation to 6 ppm NO for all groups studied (P < 0.001). * Significantly greater 6 ppm NO vasodilation in 1-wk H rats than N response (P < 0.05). ** Significantly greater 6 and 40 ppm NO vasodilations in 3-wk H rats than N and 1-wk H responses (P < 0.05). # Significantly greater 40 ppm NO vasodilation in 1- and 3-wk HNO groups than in corresponding H groups (P < 0.05). Inhaled NO had no effect on endothelium-independent vasodilation in N lungs.

Vasodilation to 6 ppm but not 40 ppm NO was greater after 1 wk of H compared with N. Vasodilation to both concentrations of NO was greater in the 3-wk H group compared with the corresponding concentrations in the N and 1-wk H groups.

The U-46619 dose required to raise the Ppa by 10 mmHg was decreased by 1 and 3 wk of H (0.8 ± 0.1 and 0.5 ± 0.0 µg, respectively) compared with the N groups (4.0 ± 0.5 µg). Prolonged inhaled NO did not alter this required dose of U-46619 in N or H rats.

    DISCUSSION
Top
Abstract
Introduction
Methods
Results
Discussion
References

We determined whether exposure to inhaled NO for 1 or 3 wk downregulates the endogenous NO-cGMP pathway by examining lung NOS protein levels, NOS activity, GC activity, cGMP levels, and endothelium-dependent vasodilation. On the basis of these studies, it appears that NOS protein levels and activity are not altered but that GC activity and cGMP levels may be affected by prolonged inhaled NO. Endothelium-dependent and -independent vasodilation are not decreased by prolonged inhaled NO under normoxic and chronically hypoxic conditions.

Rat lung eNOS protein levels were not altered by 1 or 3 wk of inhaled NO in N or H rats. The increase in lung eNOS protein observed with prolonged hypoxia is consistent with previous rat studies (33, 37); however, the effect of exogenous NO on eNOS protein has not been previously studied. Shorter periods of inhaled NO (48 h) have been shown to not alter mRNA levels for eNOS (25). NO donors have been shown to regulate eNOS gene and protein expression in vitro, but an increase rather than a decrease in both mRNA and protein has been demonstrated (20). It is unlikely that iNOS is significantly regulated by inhaled NO in our experiments because iNOS protein was undetectable. However, we cannot completely rule out the effects of inhaled NO on iNOS because mRNA and protein have been demonstrated by others in the vascular smooth muscle of H rat lungs, although they have been shown to be present minimally or not at all in N lungs (6, 37).

Total lung NOS activity per milligram of lung protein was not altered by inhaled NO in N or H rats. Inhibition of iNOS activity has been demonstrated in vascular rings with 5 ppm NO after treatment with LPS (10). In contrast, after a 48-h LPS induction, 100 ppm NO did not alter iNOS activity or induction in rat lungs (11). In vitro inhibition of eNOS activity by NO has been shown to be a reversible effect, which may result from the NO-induced formation of intra- or intermolecular disulfide bonds (27) or from interaction with the heme moiety (9, 28). In our study the NOS enzymes were fully reduced because of the presence of beta -mercaptoethanol in the buffer, which may prevent detection of inhibition by disulfide formation (27). The oxidation state of the heme moiety also affects the inhibition of NOS by NO as tetrahydrobiopterin diminishes and heme oxidants enhance NO inhibition of purified nNOS (9). Despite the presence of reducing agents, Ravichandran et al. (28) demonstrated that NO inhibits NOS activity; however, inhibition of NOS activity was demonstrated with higher concentrations of NO (10-200 µM) than those used to inhibit the nonreduced eNOS enzyme (0.2-1.0 µM) (27) or purified nNOS (0.1-10 µM) (9). It is possible that higher NO levels might be necessary to inhibit NOS in the presence of reducing agents. The calculated tissue concentration of NO during inhalation of 100 ppm is 0.16 µM (11). At such levels of NO, cellular antioxidants may be able to maintain NOS in a reduced state and prevent inhibition by prolonged inhaled NO. Although total lung NOS activity does not appear to be altered by inhaled NO, we cannot rule out effects of inhaled NO on eNOS activity because total lung NOS activity is not limited to the pulmonary vascular endothelium (37).

The NOS activity was not altered by chronic hypoxia, which is in contrast to previous studies demonstrating that NOS activity is increased in H lungs (33, 37). This discrepancy may partially be due to expression of different NOS isoforms. Xue et al. (37) demonstrated an increased iNOS but not eNOS activity. In our study, iNOS protein was not detected, whereas Xue et al. found iNOS in N lungs and increased levels in H lungs. Shaul et al. (33) reported an increase in total lung NOS activity after 3 wk of hypoxia. Increased mRNA for eNOS and nNOS was found, but the iNOS was not examined.

Stimulated GC activity was decreased by 1 wk of inhaled NO. The stimulated GC activity was decreased in N and H rats, indicating that the regulation is independent of the hypoxic effects on GC. This decrease in GC is consistent with reports using NO donors (3, 26). NO binding to the heme moiety of GC is the likely method of activation of the enzyme (24), but the GC enzyme also contains regulatory vicinal thiols that may be regulated by NO and oxidative-related species (19). These regulatory sites suggest that NO could cause a reversible inhibition of GC (19). Basal GC enzyme activity, which does not depend on the heme prosthetic group (24), was not significantly affected by inhaled NO. The inhibition of stimulated but not basal activity might support a reversible inhibition of the enzyme. Although GC activity did not recover after 24 h, the inhibition by inhaled NO was no longer present after 3 wk. This indicates that the inhibition observed at 1 wk is not irreversible and that a slow compensatory mechanism exists to restore GC activity or protein levels. It is possible that direct NO interaction with gene transcription factors regulates gene expression (35). Additionally, NO-induced increases in cGMP levels can increase cAMP levels. cAMP activates protein kinase A, which has been shown to regulate gene expression (26).

Neither basal nor stimulated GC activity was altered by 1 wk of hypoxia; however, 3 wk of hypoxia increased both basal and stimulated GC activity. This increase in GC activity is consistent with a preliminary report by LeCras et al. (15), which demonstrated an increase in the GC mRNA after 3 wk of hypoxia. However, whether protein expression is upregulated after 3 wk of hypoxia remains unknown.

Perfusate cGMP accumulation was not affected by inhaled NO in N lungs. cGMP was increased by chronic hypoxia, whereas inhaled NO attenuated this increase in the 3-wk H group. The 24-h recovery studies suggest that prolonged inhaled NO attenuated the upregulation of cGMP secondary to hypoxia rather than downregulating the production of cGMP. This is because after recovery the H group showed a large decrease in cGMP, whereas the HNO group's level did not change. If downregulation were occurring, an increase in cGMP in the HNO group and no change in the H group should have been evident. The observation that the HNO group's cGMP level did not change after recovery and the fact that cGMP was not altered in N lungs suggest that discontinuation of inhaled NO does not cause a rebound effect on the endogenous NO-cGMP pathway.

The mechanisms by which inhaled NO attenuates the increase in perfusate cGMP caused by prolonged hypoxia are unclear. Most importantly, possible mechanisms may include an inhibitory effect of NO on the lung NOS and/or GC enzymes. However, neither NOS protein nor activity was affected by prolonged inhaled NO. Although 1-wk GC activity was decreased by inhaled NO, the 3-wk GC activity was not altered; therefore, it is unlikely that the attenuation of perfusate cGMP release was solely due to an inhibition of GC. Alternatively, the decreased perfusate cGMP with inhaled NO may involve effects on hypoxic pulmonary vascular smooth muscle remodeling. Inhaled NO attenuates the hypoxic increases in muscularization of vessels (31); and a decrease in muscle mass may account for the decrease in perfusate cGMP. However, hypoxic remodeling does not resolve in 24 h (1); therefore, if cGMP release were related solely to vascular smooth muscle mass, the cGMP accumulation would not be expected to decrease significantly after 24 h. A NO-induced inhibition of cGMP transport or a decreased half-life of tissue NO (12) after prolonged inhaled NO could also account for the changes in cGMP release; however, these mechanisms should have also decreased cGMP in N lungs. Particulate GC did not contribute significantly to the cGMP accumulation in our study because L-NAME completely prevented the hypoxic increase in perfusate cGMP. This is in agreement with Kurrek et al. (11); however, other investigators have suggested that ANP-stimulated, particulate GC is a source of the cGMP (18) and that plasma ANP levels are increased in H rats (36).

Prolonged NO inhalation did not affect endothelium-dependent vasodilation in N rats, nor did it alter vasodilation with calcium ionophore A-23187 after 1 or 3 wk or with bradykinin after 3 wk of H. These results indicate that the NO-cGMP pathway is functionally intact after prolonged inhaled NO. The 1-wk HNO response to bradykinin was decreased, suggesting that bradykinin receptors may be altered by prolonged inhaled NO. However, this is inconclusive because bradykinin-induced vasodilation was not altered after 3 wk of inhaled NO. Bradykinin-induced vasodilation may also be difficult to interpret because bradykinin is inactivated by angiotensin-converting enzyme activity, which is altered in chronically hypoxic rat lungs (23). The observation that prolonged inhaled NO does not alter endothelium-dependent vasodilation is consistent with the absence of effects of inhaled NO on NOS protein and activity.

Our observation that prolonged inhaled NO does not alter endothelium-dependent vasodilation contrasts with previous studies by Oka et al. (25) and Combes et al. (7), who demonstrated that endothelium-dependent vasodilation was decreased after 48 h of inhaled NO. Roos et al. (31) also detected a decrease in bradykinin-induced vasodilation after 3 wk of inhaled NO in H rats. There are important study differences that may account for the discrepancy in results. First, the studies by Roos et al. and Oka et al. evaluated bradykinin-induced vasodilation in isolated rat lungs constricted with acute hypoxia, whereas in this study the lungs were constricted with the thromboxane analog U-46619. O2 is a required substrate for NO production, and its availability can limit NOS activity in vitro (19). Acute hypoxia also reduces angiotensin-converting enzyme activity, which may alter the availability of bradykinin (34). This cannot entirely explain the differences because Combes et al. observed a decrease in arginine vasopressin induced endothelium-dependent vasodilation in U-46619-constricted lungs. However, in contrast to Combes et al., who administered inhaled NO to N rats for 48 h, we administered NO for 1 and 3 wk. It is possible that decreased endothelium-dependent vasodilation occurs after 48 h of inhaled NO but the NO-cGMP pathway recovers by 1 wk.

Prolonged inhaled NO did not alter endothelium-independent vasodilation in N rats or vasodilation to 6 ppm NO in H rats, whereas there was a slight increase in vasodilation with 40 ppm NO in H rats. These results agree with previous studies by Oka et al. (25) and Combes et al. (7) and demonstrate that the distal portion of the NO-cGMP pathway is not downregulated by prolonged inhaled NO. Importantly, the decreased GC activity that occurred after 1 wk of inhaled NO does not appear to result in functional downregulation, as measured by endothelium-independent vasodilation. Furthermore, clinical tolerance to inhaled NO, which may be consistent with decreased GC activity, does not appear to occur (32).

The increased endothelium-dependent vasodilation with prolonged hypoxia agrees with most other reports in isolated lungs (17, 31) and may be secondary to an increase in eNOS protein levels or an increase in NOS activity (16, 33). Prolonged hypoxia also increased endothelium-independent vasodilation, which may be secondary to increases in GC activity (which we demonstrated in the 3-wk studies), or a protein involved in vasodilation such as cGMP-dependent protein kinase, which is distal to GC. There is also evidence that, in the H rat, mRNA for GC is increased (15).

In conclusion, inhaled NO does not appear to alter NOS activity or eNOS protein levels. GC activity is decreased after 1 wk of inhaled NO but not altered after 3 wk. Inhaled NO does not alter cGMP levels during normoxic conditions but attenuates the upregulation of cGMP production caused by prolonged hypoxia. Finally, 1 and 3 wk of 20 ppm inhaled NO has no effect on endothelium-dependent vasodilation in N or H rats, suggesting that the endogenous NO-cGMP pathway is not functionally altered.

    ACKNOWLEDGEMENTS

This work was supported by a grant from Ohmeda Pharmaceutical (G. F. Rich).

    FOOTNOTES

Address for reprint requests: G. F. Rich, Dept. of Anesthesiology, PO Box 10010, Charlottesville, VA 22906-0010 (E-mail: gfr2f{at}virginia.edu).

Received 14 October 1997; accepted in final form 13 May 1998.

    REFERENCES
Top
Abstract
Introduction
Methods
Results
Discussion
References

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