Inhalation of nitric oxide (NO) selectively dilates pulmonary circulation and augments arterial oxygenation by enhancing the matching of perfusion and ventilation in patients with adult respiratory distress syndrome (ARDS) [1,2]. In approximately 30% of ARDS patients, minimal or no pulmonary vasodilatatory or oxygenation response to inhaled NO is observed . The mechanisms of hyporesponsiveness to inhaled NO are still unknown. There is some evidence that Gram-negative bacteraemia  and endotoxaemia  contribute to NO hyporesponsiveness. NO is a lipid-soluble free radical molecule with a short half-life in biological fluids. In the presence of oxygen, NO is produced from l-arginine by nitric oxide synthase (NOS) enzymes. NOS enzymes are divided into two classes: constitutive and inducible. The activities of the constitutive isoforms, neuronal NOS (NOS1) and endothelial NOS (NOS3), are dependent upon intracellular calcium levels. Inducible NOS (NOS2) expression is stimulated by endotoxin or cytokines  and inhibited by glucocorticoids . In pulmonary vascular smooth muscle cells, NO activates soluble guanylate cyclase, an enzyme responsible for the conversion of GTP to guanosine 3′,5′-cyclic monophosphate (cGMP) . cGMP activates cGMP-dependent protein kinases, leading to decreased pulmonary vascular smooth muscle tone.
We previously used an isolated-perfused rat lung model to investigate the mechanisms regulating pulmonary vascular responsiveness to inhaled NO . We observed that responsiveness to inhaled NO was decreased in isolated-perfused lungs from rats pretreated with endotoxin/lipopolysaccharide (LPS). In the same model, aminoguanidine (AG), a selective NOS2 inhibitor, improved responsiveness to inhaled NO and decreased the release of nitrate and nitrite into the perfusate . However, AG has also been demonstrated to scavenge peroxynitrite and to inhibit reactive oxygen species (ROS) formation, as well as lipid peroxidation and oxidant-induced apoptosis, which could have contributed to the development of hyporesponsiveness to inhaled NO [9,10]. In the present study, we hypothesized that the ability of NOS2 inhibitors to prevent hyporesponsiveness to inhaled NO in isolated-perfused lungs obtained from LPS-treated rats is dependent on their ability to reduce nitrite and nitrate levels in serum and released into the perfusate. To test this hypothesis, we employed increasing doses of two different specific inhibitors of NOS2 activity, AG and S-methylisothiourea (SMT), to induce pulmonary vasodilatation and decrease endogenous NO synthesis. We report here that AG and SMT restored hyporesponsiveness to inhaled NO which was dependent on their ability to reduce nitrite and nitrate levels in serum and pulmonary NO formation.
Isolated, perfused rat lungs
These investigations were approved by the Committee for Research Animal Studies for the University of Heidelberg, Germany. Lungs obtained from rats were isolated, perfused and ventilated as described previously . Briefly, adult Sprague-Dawley rats (Charles River Laboratories, Sulzfeld, Germany), weighing 400-450 g were killed by intraperitoneal injection of sodium pentothal (100 mg kg−1 body weight). Following a midline thoracotomy, the pulmonary artery and left atrium were cannulated. Lungs were perfused with Hank's balanced salt solution containing 5% dextran, 5% bovine serum and 30 μmol indomethacin using a roller pump (Abimed, Langenfeld, Germany) at a pulsatile flow of 0.03 L−1 kg−1 body weight−1 min−1 in a recirculating system at 37°C. The perfusate was continuously aerated with CO2 in the presence of NaHCO3 to adjust and maintain pH, PCO2 and PO2 between 7.35-7.40, 35-40 mmHg and 150-250 mmHg, respectively. Lungs were ventilated with air using a tidal volume of 0.03 L−1 kg−1 body weight−1.
Pulmonary artery pressure (PAP) and left atrial pressure were measured via small catheters (PE-50 tubing) placed within the lumens of the inflow and outflow perfusion catheters. Left atrial pressure was set at 4 cm H2O. The sensitivity of the PAP measurement in our preparation was 0.01 mmHg.
Measurement of pulmonary vasoreactivity to inhaled NO
In isolated-perfused lungs, the stable thromboxane analog U46619 (Cayman Chemical, Cedex, France) was administered to increase PAP by 6-8 mmHg. Next, the infusion rate was adjusted to infuse the minimum quantity required to maintain a stable elevated PAP. The lungs were ventilated with 0.4, 4 and 40 ppm NO in random order for periods of 3 min. After each period of NO ventilation, the PAP was allowed to increase to the elevated baseline. The variation to the former elevated PAP was within a 5% deviation range or less than 0.3 mmHg.
NO gas was obtained from AGA (Bottrop, Germany) as a mixture of 800 ppm NO in pure N2. Variable concentrations of NO were mixed with air just before entering the ventilator. NO levels were measured continuously by chemiluminescence analysis (Eco Physics CLD 77AM, Dürnten, Switzerland).
Pulmonary vasoreactivity to inhaled NO in rats treated with lipopolysaccharide, aminoguanidine and S-methylisothiourea
A total of 80 rats divided into 14 groups were studied. Animals were randomized using a computer-generated list of random numbers. About 16-18 h before the lung perfusion experiments, 38 rats received intraperitoneally 0.5 mg kg−1Escherichia coli 0111:B4 LPS (Difco Laboratories). To examine whether inhibition of NOS2-mediated NO production improves NO hyporesponsiveness, we administered AG, a selective inhibitor of NOS. In three groups of LPS-pretreated rats (n = 17) AG was administered in three different doses (3, 10 or 30 mg kg−1) intraperitoneally 4 h following LPS injection. To further examine whether a second inhibitor of NOS2 activity has the same impact on the development of hyporesponsiveness to inhaled NO in rats exposed to LPS, we used SMT. In three additional groups of LPS-pretreated rats (n = 14) SMT in three different doses (0.01, 0.1 or 1 mg kg−1) was intraperitoneally injected 4 h following LPS treatment.
Around 33 out of 80 rats were untreated controls that received intraperitoneal injections of either AG or SMT according to the protocol for LPS-treated rats.
One LPS group (n = 7) and one control group (n = 9) received neither AG nor SMT.
About 16-18 h after LPS injection, the lungs were isolated, perfused and ventilated. Then pulmonary vasoreactivity to inhaled NO was measured as described above and groups were divided into ‘responders' and ‘non-responders' depending on the dose or doses that caused pulmonary vasodilatation compared with lungs from rats treated with LPS alone.
Nitrite and nitrate levels in the serum and perfusate of isolated lungs from rats were treated with AG and SMT.
In 76 of the rats studied, serum samples were withdrawn during isolated lung preparation to measure nitrite/nitrate levels in serum. Perfusate samples for nitrite/nitrate measurements were taken 10 min after the beginning of lung perfusion. In additional rats (n = 24) only serum samples were withdrawn. Nitrite/nitrate levels were determined as described previously . In brief, 100 μL of the perfusate was diluted with 500 μL phosphate buffer (pH 7.5). Nitrate was reduced to nitrite using nitrate reductase (50 μL, 1 U mL−1) (Sigma, Deisenhofen, Germany) and NADPH (50 μL, 1.8 mmol; Sigma) was added. After 2 h of incubation, excess NADPH was oxidized by adding 50 μL phenazine methosulphate (80 μmol; Sigma). Then, 100 μL zinc acetate (0.5 mol; Sigma) and 100 μL NaOH (0.5 mol) were added for deproteinization. Nitrite was measured in the supernatant by Griess assay adding 250 μL sulphanilamide (0.1 mol in 1.5 mol phosphoric acid; Sigma) and 250 μL naphthyl ethylenediamine (8 mmol; Sigma). Colorimetric absorption at 540 nm correlated linearly with nitrite concentration.
The data are expressed as absolute changes (means ± SEM) of elevated PAP (mmHg) in response to inhalation of NO. Multivariate analysis of variance (ANOVA) with repeated-measures techniques was used to examine the effects of LPS (controls vs. LPS-treated). Scheffé test was used to examine specific treatment (AG or SMT vs. none) and NO dose simultaneously. All data are expressed as mean ± standard deviation (SD). Differences between effective treatment vs. ineffective treatment were evaluated using pooled data followed by a simple t-test. Statistical significance level was reached when P < 0.05.
Aminoguanidine treatment improves responsiveness to inhaled NO in a reverse dose-dependent manner
Compared with untreated controls, administration of LPS reduced the response to 0.4, 4 and 40 ppm NO (P < 0.05, Fig. 1). All doses but 30 mg kg−1 at 0.4 ppm NO of AG improved responsiveness to inhaled NO in isolated-perfused lungs from LPS-treated rats compared with lungs obtained from rats treated with LPS alone (P < 0.05). Furthermore, doses of 3 and 10 mg kg−1 AG improved the ability of lungs from LPS-treated rats to vasodilatate in response to 0.4, 4 and 40 ppm NO to the same extent as that observed in lungs obtained from control rats (P = n.s.). In lungs obtained from control rats, treatment with AG had no effect on pulmonary vasoreactivity in response to 0.4, 4 and 40 ppm NO (data not shown).
Treatment with S-methylisothiourea improves responsiveness to inhaled NO in a dose-dependent manner
Compared with untreated controls, administration of LPS reduced the response to 0.4, 4 and 40 ppm NO (P < 0.05, Fig. 2). In lungs obtained from LPS-treated rats, administration of SMT at doses of 0.01 and 0.1 showed no improvement of responsiveness to 0.4, 4 and 40 ppm NO compared with the lungs of rats treated with LPS alone (P = n.s. for all doses). In contrast, in lungs obtained from rats treated with LPS and 1 mg kg−1 SMT, response to inhaled NO improved to the same extent as that observed in lungs of control rats (P = n.s. and P < 0.05 vs. LPS alone). In lungs obtained from control rats, SMT had no effect on responsiveness to inhaled NO (data not shown).
Nitrite/nitrate levels in the serum of rats treated with LPS and AG
To demonstrate that AG inhibits endogenous NO production in rats exposed to LPS, we measured nitrite/nitrate levels in serum and perfusate from rats with and without LPS pretreatment (Table 1a). Compared with rats treated with LPS alone, the total serum levels of nitrite/nitrate in rats treated with LPS and AG at doses of 10 and 30 mg kg−1 were decreased (P < 0.05). In LPS-treated rats that received AG at a dose of 3 mg kg−1, no difference of nitrite/nitrate serum levels compared with that of rats treated with LPS alone was observed (P = 0.06). Compared with control rats, serum levels of nitrite and nitrate were increased in LPS-treated groups with or without AG (P < 0.05 vs. LPS with/without AG). AG treatment did not affect nitrate and nitrite levels in serum from control rats (data not shown).
Nitrite/nitrate serum levels in rats exposed to LPS and S-methylisothiourea
To examine whether SMT would have the same effect as AG to inhibit endogenous NO production, we determined serum levels of nitrite/nitrate in LPS-treated rats with and without administration of SMT (Table 1b). Compared with LPS-treated rats without SMT, doses of 0.01 and 0.1 mg kg−1 did not decrease serum nitrite/nitrate levels in rats exposed to LPS and SMT (P = n.s.). In contrast, LPS-treated rats that received SMT at a dose of 1 mg kg−1 serum levels of nitrite/nitrate were decreased when compared with rats treated with LPS alone (P < 0.05). Compared with the control group, LPS-treated rats with or without SMT showed increased serum levels of nitrite and nitrate (P < 0.05 with/without SMT). In the control group, treatment with SMT had no effect on nitrite/nitrate levels in serum (data not shown).
Hyporesponsiveness to inhaled NO in correlation to serum and perfusate nitrite/nitrate levels
Because LPS-treated rats injected with 3, 10 or 30 mg kg−1 AG and 1 mg kg−1 SMT showed improved responsiveness to inhaled NO, they were summarized as ‘responders' (Table 2). Rats treated with LPS alone and LPS-pretreated rats treated with 0.01 and 0.1 mg kg−1 SMT were called ‘non-responders' because of hyporesponsiveness to inhaled NO. Compared with responders, non-responders had higher levels of serum as well as peak perfusate levels of nitrite and nitrate (P < 0.05).
To investigate whether responsiveness to inhaled NO was associated with inhibition of endogenous NOS2-mediated NO production, we plotted the decrease of PAP in response to 40 ppm NO of both responder and non-responder groups against nitrite/nitrate levels in serum and perfusate (Fig. 3a,b). The improved response to inhaled NO in the responder group was associated with a lower level of nitrite/nitrate in serum and perfusate (P < 0.001 vs. non-responder). The observation that the decreased endogenous NO formation was associated with the inhalation of 40 ppm was also seen when compared with 0.4 and 4 ppm NO (data not shown).
Inhalation of NO selectively dilates pulmonary circulation and improves arterial oxygenation in patients with ARDS [2,12]. However, up to 60% of septic ARDS patients do not respond or respond only minimally to inhaled NO with improved oxygenation . We observed that in isolated-perfused lungs from rats exposed to LPS, the ability of inhaled NO to induce pulmonary vasodilatation is impaired . Systemic administration of LPS stimulates NOS2 synthesis in a variety of cell types [6,13], including pulmonary vascular cells . Because sepsis and endotoxaemia, the most common causes of ARDS, promote increased NO production by NOS2 [6,14,15], we studied responsiveness to inhaled NO in isolated, perfused and ventilated lungs from rats exposed to LPS. We demonstrated that inhibition of endogenous NO production or NOS2 gene expression prevented the development of hyporesponsiveness to inhaled NO in rats exposed to LPS . Because AG affects oxygen species formation, which could have contributed to the development of hyporesponsiveness to inhaled NO, we employed AG and a second NOS2 inhibitor to demonstrate that the improvement of NO responsiveness is associated with decreased NOS2-mediated NO production.
A number of studies have confirmed that AG selectively inhibits NOS2 in vivo [16,17] and in vitro  without an effect on NOS3 activity. In rats, pulmonary NOS2 activity and serum nitrite and nitrate levels were increased by an intraperitoneal injection of 15 mg kg−1 endotoxin . Treatment with 20 mg kg−1 AG 2 h after LPS injection decreased NOS2 activity in the lungs by approximately 80% and serum nitrate/nitrate levels by approximately 70%. In a recent study, Hayashi and colleagues could demonstrate that LPS-induced increase in NO in plasma strongly correlated with NOS activity in lungs, depending on NOS2 protein expression . In our model, administration of a single dose of AG 4 h after LPS administration restored pulmonary vascular responsiveness to inhaled NO (Fig. 1) and decreased serum levels of nitrite and nitrate 16-18 h after LPS treatment (Table 1). These results suggest that enhanced endogenous NO production by NOS2 is required for the development of hyporesponsiveness to inhaled NO in LPS-treated rats. These observations are supported by a study of Weimann and colleagues using NOS2-deficient mice in which administration of LPS decreased responsiveness to inhaled NO in isolated-perfused lungs of NOS2-deficient mice after breathing 20 ppm NO for 16 h, but not with LPS-treatment alone . However, it is uncertain whether increased pulmonary NO concentrations alone are sufficient to produce hyporesponsiveness to inhaled NO. Combs and colleagues reported that in isolated-perfused lungs from rats breathing 30 ppm NO for 48 h under normoxic conditions, vasodilatation to sodium nitroprusside was preserved . Frank and colleagues reported that responsiveness to inhaled NO was not altered in isolated-perfused lungs from rats exposed to 20 ppm NO for up to 3 weeks . In contrast, we observed that in isolated-perfused lungs obtained from rats breathing 80 ppm NO for 20 h under normoxic conditions, the response to acute inhalation of 4 ppm NO was decreased by 15% compared with untreated rats . Furthermore, Roos and colleagues demonstrated that in isolated-perfused lungs obtained from rats exposed to 10% normobaric oxygen in combination with 20 ppm NO for 3 weeks, response to acute inhalation of 40 ppm NO was diminished compared with chronic hypoxic or normoxic rats .
It is likely that other sepsis-induced factors may act together with NO leading to the development of NO hyporesponsiveness. One such a sepsis-induced factor could be superoxide, which can react with NO to form the potentially toxic metabolite peroxynitrite . In cultured macrophages, AG inhibited peroxynitrite-induced benzoate hydroxylation and 4-hydroxyphenylacetic acid nitration . Furthermore, AG inhibits H2O2-induced intracellular ROS production, lipid peroxidation and oxidant-induced apoptosis in rat retinal Muller cells . Therefore, prevention of tissue damage associated with ROS production by the antioxidant effects of AG could have preserved pulmonary smooth muscle cells function in our model and therefore be at least in part attributable for improvement of NO responsiveness.
Because AG has been shown to inhibit other enzymes systems, we employed a second NOS2 inhibitor to confirm our observations. Furthermore, between substituted aminoguanidines and aminoisothioureas, selectivity to NOS2 may vary . We therefore used SMT to verify that amelioration of pulmonary responsiveness to inhaled NO in LPS-treated rats is due to NOS2 inhibition . Jang and colleagues observed in cultured bovine chondrocytes that SMT is 5-10 times more potent in blocking NOS2 activity than AG . SMT has been used to block NOS2 activity in a variety of models [19,29,30]. Arkovitz and colleagues demonstrated that in rats a bolus administration of 5 mg kg−1 SMT given 2 h after intraperitoneal LPS injection reduced LPS-mediated increase of pulmonary NOS2 activity and serum nitrate/nitrite levels 6 h later . However, in contrast to AG , SMT has also been demonstrated to block NOS2 expression in the tail arteries of rats 3 days after coronary artery ligation and in liver and heart 24 h after LPS treatment [31-33]. In our model, SMT only at a dose of 1 mg kg−1 given 4 h after LPS-injection prevented the development of hyporesponsiveness to inhaled NO (Fig. 2) and decreased serum nitrite/nitrate levels (Table 1) 16-18 h after LPS treatment. These results suggest that inhibition of NOS2 activity contributes to the development of hyporesponsiveness to inhaled NO in rats treated with LPS.
To further evaluate the contribution of NOS2-mediated NO formation to the development of hyporesponsiveness to inhaled NO in LPS-treated rats, groups were divided into responders and non-responders (Fig. 3a,b). In responders, improved responsiveness to inhaled NO was associated with low levels of nitrite and nitrate in serum and release into the perfusate. In contrast, in non-responders high levels of nitrite and nitrate in serum and increased release into the perfusate contributed to hyporesponsiveness to inhaled NO. Because in rats LPS-induced increase of plasma NO is dependent on pulmonary NOS activity, which mainly derives from NOS2 , we suggest that increase of nitrite and nitrate in serum in our model was mediated by pulmonary NOS2. Furthermore, decreased NO levels into the perfusate indicate a decrease in pulmonary NO synthesis. These results suggest that direct inhibition of pulmonary NOS2 following LPS by AG or SMT contributes to the improved pulmonary vasodilatation in response to inhaled NO.
In summary, our observations demonstrate that a single injection of NOS2 inhibitor AG or SMT prevented the development of hyporesponsiveness to inhaled NO and decreased endogenous NO synthesis in rats exposed to LPS. Decreased nitrite and nitrate levels in serum and perfusate following NOS2 inhibition suggests that increased pulmonary NO production contributes to the development of NO hyporesponsiveness. It is possible that this mechanism is at least in part responsible for the development of hyporesponsiveness to inhaled NO in patients with ARDS associated with sepsis.
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