Endogenous Nitric Oxide Can Act as Beneficial or Deleterious in the Hypoxic Lung Depending on the Reoxygenation Time.код для вставкиСкачать
THE ANATOMICAL RECORD 293:2193–2201 (2010) Endogenous Nitric Oxide Can Act as Beneficial or Deleterious in the Hypoxic Lung Depending on the Reoxygenation Time ALMA RUS, FRANCISCO MOLINA, M. ÁNGELES PEINADO, AND M. LUISA DEL MORAL* Department of Experimental Biology, University of Jaén, Jaén, Spain ABSTRACT Nitric oxide (NO) has been implicated in many pathophysiological situations in the lung, including hypoxia/reoxygenation. This work seeks to clarify the current controversy concerning the double protective/toxic role of endogenous NO under hypoxia/reoxygenation situations in the lung by using a nitric oxide synthase (NOS) inhibitor, in a novel approach to address the problems raised from assaults under such circumstances. A follow-up study was conducted in Wistar rats submitted to hypoxia/ reoxygenation (hypoxia for 30 min; reoxygenation of 0 h, 48 h, and 5 days), with or without prior treatment using the nonselective NOS inhibitor L-NAME (1.5 mM, in drinking water). Lipid peroxidation, apoptosis level, protein nitration, in situ NOS activity and NO production (NOx) were analyzed. This is the ﬁrst work to focus on the time-course effects of L-NAME in the adult rat lung submitted to hypoxia/reoxygenation. The results showed that after L-NAME administration, in situ NOS activity was almost completely eliminated and consequently, NOx levels fell. Lipid peroxidation and the percentage of apoptotic cells rose at the earliest reoxygenation time (0 h), but decreased in the later period (48 h and 5 days). Also nitrated protein expression decreased at 48 h and 5 days posthypoxia. These results suggest that NOS-derived NO exerts two different effects on lung hypoxia/reoxygenation injury depending on the reoxygenation time: NO has a beneﬁcial role just after the hypoxic stimulus and a deleterious effect in the later reoxygenation times. Moreover, we propose that this dual role of NO depends directly on the producer NOS isoform. C 2010 Wiley-Liss, Inc. Anat Rec, 293:2193–2201, 2010. V Key words: hypoxia; nitric oxide; nitric oxide synthase; L-NAME; apoptosis; lipid peroxidation; reoxygenation INTRODUCTION Hypoxia is one of the most frequently encountered types of stress in health and disease. To ﬁnd useful remedies that are capable of ameliorating its casualty is an essential effort. Although the underlying mechanisms of the hypoxia-induced injury and cell death are still not fully understood, it has been shown that hypoxia induces nitric oxide (NO) overproduction (Kiang and Tsen, 2006). In the lungs, endogenous NO plays an important role in regulating a number of physiological and pathological processes, including hypoxia. NO is produced by three isoenzymes of nitric oxide synthase (NOS): endothelial C 2010 WILEY-LISS, INC. V (eNOS or NOS III), inducible (iNOS or NOS II), and neuronal (nNOS or NOS I) (Vaughan et al., 2003). Grant sponsor: Ministry of Education and Sciences of Spain; Grant numbers: SAF-2003-04398-C02-02, BIO 2000-0405-P4-05. *Correspondence to: M. Luisa del Moral, Department of Experimental Biology, University of Jaén, Jaén, Spain. Fax: þ34953-211875. E-mail: firstname.lastname@example.org Received 3 March 2010; Accepted 1 June 2010 DOI 10.1002/ar.21229 Published online 23 August 2010 in Wiley Online Library (wileyonlinelibrary.com). 2194 RUS ET AL. Whereas eNOS and nNOS are mostly calcium/calmodulin-dependent and usually express constitutively (Palmer et al., 1987), iNOS is typically independent of intracellular calcium concentration and can be induced in a wide variety of cell types in response to some stimuli, such as hypoxia (Jung et al., 2000; Ricciardolo et al., 2004), or after stimulation with inﬂammatory mediators (Schulz et al., 1991; Kelly et al., 1996). Two main sites of NO production have been identiﬁed in the lung, the vasculature and the airways (Kobzik et al., 1993; Sherman et al., 1999). The main isoform in vascular endothelium is eNOS. In the airways, all three isoforms have been detected in the bronchial and bronchiolar epithelium (Kobzik et al., 1993; Shaul et al., 1995), iNOS being the major airway epithelial isoform (Guo et al., 1995). The reoxygenation of the hypoxic tissues is characterized by the formation of both reactive oxygen species (ROS) and reactive nitrogen species (RNS), resulting in widespread lipid peroxidation, protein oxidative and nitrosative modiﬁcations, alterations in DNA (Szabo, 1996) as well as apoptosis and necrosis (McCord et al., 1985). Peroxynitrite, formed by the reaction of NO and superoxide, is a ROS and RNS that can alter protein function by nitrating phenolic rings, including tyrosine, to create nitrotyrosines (Haddad et al., 1994). A number of works in the literature have shown controversial results reporting that NO can act both as a destructive as well as a protective agent in the pathogenesis of the hypoxia/reoxygenation injuries in the lung. In this sense, while several investigations have reported that NOS inhibitors exacerbate hypoxic injury in the lung (Naoki et al., 1999; Shirai et al., 2003), other studies using pharmacological inhibition of NOS showed protective effects against the same type of injuries (Baber et al., 2005; Barer et al., 2006). All this controversy exempliﬁes the need to provide a clearer view of the role of NOS-derived NO under hypoxia/ reoxygenation situations in the lung. For this, we studied parameters of cell and tissue damage (lipid peroxidation, apoptosis, and levels of nitrated proteins) before and after treatment with the NOS inhibitor NGnitro-L-arginine methyl ester (L-NAME) in the lung of Wistar rats submitted to hypoxia/reoxygenation. L-NAME is an analogue of L-arginine that acts as a nonselective inhibitor of the activity, but not of the expression, of the three NOS (Rees et al., 1990). Moreover, it bears mentioning that the literature dealing with this inhibitor in hypoxic lungs is very scant, and thus its possible effects on this organ remain completely unknown. This is the ﬁrst available study that describes the time-course effects of this NOS inhibitor in the adult rat lung submitted to hypoxia/reoxygenation. METHODS Animals The study was performed on mature adult (4–5 months old) male albino Wistar rats kept under standard conditions of light and temperature and allowed ad libitum access to food and water. All the experiments were conducted according to E.U. guidelines on the use of animals for biochemical research (86/609/EU). Experimental Protocol and NOS Inhibitor Administration The acute hypobaric hypoxia was carried out as previously published by our group (Lopez-Ramos et al., 2005; Martı́nez-Romero et al., 2006; Rus et al., 2010a). Brieﬂy, animals were placed in a chamber in which the air pressure was controlled by means of a continuous vacuum pump and an adjustable inﬂow valve. Hypoxia was induced by downregulating the environmental O2 pressure to a ﬁnal barometric pressure of 225 mmHg, resulting in a 48 mmHg O2 partial pressure (pO2). These conditions were maintained for 30 min. The ascent and descent speed was maintained below 1,000 feet/min. After the hypoxia period, animals were kept under normobaric normoxic conditions for different reoxygenation times (0, 48 h, and 5 days), and then were sacriﬁced. Control animals were maintained for 30 min in the chamber under normobaric normoxic conditions, before being sacriﬁced. The nonselective NOS inhibitor L-NAME or NG-nitroL-arginine methyl ester (Sigma) (1.5 mM) was dissolved in the drinking water of the animals for 2 weeks before hypoxia (Alonso et al., 2002). After the hypoxia period, animals were kept under normobaric normoxic conditions for different reoxygenation times (0, 48 h, and 5 days), and then were sacriﬁced. Control animals were maintained for 30 min in the chamber under normobaric normoxic conditions, before being sacriﬁced. The following experimental groups (n ¼ 5 rats per group) were studied: 1. Control: rats maintained for 30 min in the chamber under normobaric normoxic conditions. 2. Hypoxia/reoxygenation: rats submitted to 30 min of hypoxia followed by 0 h, 48 h, and 5 days of reoxygenation. 3. Control þ L-NAME: control rats treated with L-NAME. 4. Hypoxia/reoxygenation þ L-NAME: rats submitted to the same procedure as the second group but treated with L-NAME. A total of 40 albino Wistar rats were used for the biochemical experiments (ﬁve animals per experimental group). After the corresponding reoxygenation times, the rats were killed by cervical dislocation and the lungs were immediately removed, rinsed in saline solution, and stored at 80 C until used. Another 40 rats were used for histochemistry and immunohistochemistry (ﬁve animals per experimental group). The rats were anaesthetized with Ketolar (Parke Davis, 1 mL/250 g weight) by intraperitoneal injection and perfused at each reoxygenation time. Then, the lungs were removed, rinsed in saline solution, and ﬁxed. NADPH-Diaphorase Histochemistry NOS have been shown to have NADPH-diaphorase activity, as evidenced by colocalization and coprecipitation of NADPH-diaphorase and NOS activity (Snyder, 1992). In fact, NADPH-diaphorase histochemistry has been widely used as an indirect way to determine in situ NOS activity (Kugler et al., 1994; Roufail et al., 1995; Moraes et al., 2001). Lung sections, 40-lm thick, were cut using DUAL ROLES OF NO IN THE HYPOXIC LUNG a cryostat (2800 Frigocut E, Reichert-Jung Vienna, Austria). Free-ﬂoating sections were incubated for 4 h in PBS containing 0.1% Triton X-100. After several washes in 0.1 M Tris-HCl pH 7.4 buffer, they were incubated in the dark for 45 min at 37 C in 0.1 M Tris-HCl pH 7.4 containing 1 mM b-NADPH and 2 mM NBT (in 70% dimethylformamide). The sections were then washed twice with 0.1 M Tris-HCl pH 7.4, quickly dehydrated in a graded ethanol series, cleared, and mounted in DPX (Fluka, Madrid, Spain). NO Measurement The reaction of NO with ozone results in the emission of light, and this light (emitted in proportion to the NO concentration) is the basis for one of the most accurate NO assays available (Fontijn et al., 1997; Laitinen et al., 1993). NO production was indirectly quantiﬁed by measuring nitrate/nitrite and S-nitrose compounds (NOx) using an ozone chemiluminescence-based method. For this technique, lungs were homogenized in PBS with protease inhibitors. Homogenates were then sonicated, centrifuged, and deproteinized with NaOH 0.8 N and ZnSO4 16% solutions. The total amount of NOx was determined by a modiﬁcation (Lopez-Ramos et al., 2005) of the procedure described by Braman and Hendrix (1989) using a NO analyzer (NOATM 280i Sievers Instruments). A saturated solution of vanadium chloride (VCl3) in 1 M HCl was added to the nitrogen-bubbled purge vessel ﬁtted with a cold water condenser and a water jacket to heat the reagent to 90 C using a circulating bath. HCl vapors were removed by a gas bubbler containing 1 M NaOH. The gas-ﬂow rate into the detector was controlled by a needle valve adjusted to yield a constant pressure. Once the detector signal was stabilized, samples were injected into the purge vessel to react with the reagent, converting NOx to NO, which was then detected by ozone-induced chemiluminescence. NOx concentrations were calculated by comparison with standard solutions of sodium nitrate. Final NOx values were referred to the total protein concentration in the initial extracts. Thiobarbituric Acid Reactive Substances Thiobarbituric acid reactive substances (TBARS) were determined in lung homogenates as described by Buege and Aust (1978). Brieﬂy, lungs were homogenized in PBS, and then sonicated and centrifuged. In the supernatant, the amount of proteins was determined using the Bradford assay (1976). After 700 lL of thiobarbituric acid reagent (15% TCA or trichloroacetic acid, 0.38% TBA or 2 thiobarbituric acid and 2% HCl or chloride acid) was added to 300 lL of the supernatant, the solution was heated at 95 C for 15 min. After heating, the tubes were cooled in a water bath and centrifuged. The absorbance of the supernatant was read at 535 nm. TUNEL Assay for Assessment of Apoptotic Cell Death and Image Processing Terminal deoxynucleotidyl transferase (TdT)-mediated desoxyuridinetriphosphate (dUTP) nick end-labelling (TUNEL) is a technique to estimate apoptosis in tissue sections. The protocol was performed in sections 2195 obtained from lungs embedded in parafﬁn according to the manufacturer’s recommendations (TdT-FragELTM DNA Fragmentation Detection Kit, Calbiochem). Deionized water was replaced by TdT enzyme as a negative control. Apoptotic bodies were stained brown. Ten similar microphotographs per rat were digitally captured with a light microscope (Olympus, Hamburg, Germany). They were then analyzed, after background subtraction (minimal particle size 10 pixels), in two different color channels using ImageJ (an NIH image analysis and processing software downloaded free from http://rsbweb.nih.gov/ij/). The image derived from the green channel was used to determine the number of living cells while the image acquired from the red one was used to determine the apoptotic cells. The percentage of area of apoptotic cells in each microphotograph was quantiﬁed by computer-assisted image analysis using the same software. Western Blot Analysis for Nitrotyrosine Expression For Western blot analysis, equal amounts of denatured lung total-protein extracts were loaded and separated in 7.5% SDS-polyacrylamide gel. Proteins in the gel were transferred to a PVDF membrane (Amersham Pharmacia Biotech, Buckinghamshire, UK) and then blocked. Polyclonal rabbit anti-3-nitro-L-tyrosine A4 antibody (1:3000, gift from Professor J. Rodrigo from CSIC Cajal Institute of Madrid) was used to detect nitrated proteins, and monoclonal antibody to a-tubulin (Sigma) was used as the internal control. Antibody reaction was revealed with chemiluminescence detection following the manufacturer’s recommendations (ECL kit, Amersham Corp., Buckinghamshire, UK). Nitrotyrosine Immunohistochemistry Lung samples were embedded in parafﬁn (Paraplast Extra, Tyco). Sections were incubated with 10% goat serum during 30 min. Afterward, they were ﬁrst incubated with diluted polyclonal rabbit anti-3-nitro-L-tyrosine A4 antibody (1:500, gift from Professor J. Rodrigo from CSIC Cajal Institute of Madrid), used to detect nitrated proteins, in PBS overnight at 4 C, and later with a goat anti-rabbit biotinylated secondary antibody (Pierce) followed by peroxidase-linked ABC. The peroxidase activity was demonstrated following the nickel-enhanced diamino-benzidine procedure (Shu et al., 1988). Sections were mounted on slides, dehydrated, and covered using DPX. Controls for background staining were performed by replacing the primary antibody with PBS. Statistical Analysis Data were expressed as mean standard deviation (SD). The statistical treatment to evaluate signiﬁcant differences between groups was performed with SPSS 15.0 software. The data followed a normal distribution (tested with Kolmogorov–Smirnov test) and the principle of homoscedasticity of variances (tested with Levene test), and were tested by a two-way ANOVA. The statistical signiﬁcance was established by applying an unpaired Student’s t-test to compare differences between means. The statistically signiﬁcant differences regarding 2196 RUS ET AL. Fig. 2. Inﬂuence of hypoxia/reoxygenation on nitrate, nitrite, and other nitrose compounds (NOx) in rat lung (lmol/mg protein). Experimental groups: Control and 0 h, 48 h, and 5 days posthypoxia; control L-NAME and 0 h L-NAME, 48 h L-NAME, and 5 days L-NAME posthypoxia. Results are mean values of three independent experiments with ﬁve animals per group. The statistically signiﬁcant differences regarding the control group in the untreated groups were expressed as aP < 0.05; bP < 0.02; cP < 0.01; and dP < 0.001. The statistically signiﬁcant differences between the treated groups and the corresponding untreated groups were expressed as wP < 0.05; xP < 0.02; yP < 0.01; and zP < 0.001. Fig. 1. NADPH-diaphorase staining. (A–H): Micrographs showing in situ NOS activity in rat lung sections in endothelial cells (arrows) and bronchiolar epithelial cells (arrow heads). Experimental groups: Control (C) and 0 h, 48 h, and 5 days (5d) posthypoxia; control (C) L-NAME and 0 h L-NAME, 48 h L-NAME, and 5 days (5d) L-NAME posthypoxia. Micrographs (C), (E), and (G) show the presence of slightly more intense NADPH-diaphorase staining in both types of cells when compared with the control group in the untreated groups. Micrographs (B), (D), (F), and (H) do not show NADPH-diaphorase staining in relation to the untreated groups. Scale bars ¼ 200 lm. the control group in the untreated groups were expressed as aP < 0.05; bP < 0.02; cP < 0.01; and dP < 0.001. The statistically signiﬁcant differences between the treated groups and the corresponding untreated groups were expressed as wP < 0.05; xP < 0.02; yP < 0.01; and zP < 0.001. RESULTS NADPH-Diaphorase Histochemistry In the microphotographs (Fig. 1, microphotographs A, C, E, and G taken from Rus et al., 2010b), the NADPHdiaphorase staining, representative of in situ NOS activity, was found in all the experimental groups in endothelial cells and bronchiolar epithelial cells. An increase in NADPH-diaphorase staining was detected at 0 h, 48 h, and 5 days posthypoxia in those cell types in the untreated groups. On the other hand, after the treat- ment with the nonselective NOS inhibitor L-NAME, the NADPH-diaphorase staining was almost completely eliminated in all the experimental groups. NO Production Figure 2 shows the determinations of nitrate/nitrite and other S-nitrose compounds (NOx) in the different experimental groups. NOx levels rose early, at 0 h of reoxygenation (P < 0.001), in the untreated groups. However, the administration of this NOS inhibitor signiﬁcantly lowered NOx levels in control (P < 0.001) and throughout all the reoxygenation periods: 0 h (P < 0.001), 48 h (P < 0.05), and 5 days (P < 0.05). Thiobarbituric Acid Reactive Substances Malondialdehyde and other lipid peroxidation products, which react with thiobarbituric acid, are good indicators of oxidative stress (Drapper et al., 1993). Figure 3 shows that hypoxia/reoxygenation raised the TBARS level at 48 h (P < 0.001), and 5 days (P < 0.001) posthypoxia in the untreated groups. Moreover, in the groups treated with L-NAME, TBARS increased at 0 h (P < 0.05), but decreased in the later reoxygenation periods (48 h: P < 0.02; 5 days: P < 0.001). TUNEL Assay The TUNEL assay, which identiﬁes apoptotic cells, showed that hypoxia/reoxygenation signiﬁcantly raised DUAL ROLES OF NO IN THE HYPOXIC LUNG 2197 Fig. 3. Inﬂuence of hypoxia/reoxygenation on lipid peroxidation in rat lung. Results were expressed as absorbance at 535 nm. Experimental groups: control and 0 h, 48 h, and 5 days posthypoxia; control L-NAME and 0 h L-NAME, 48 h L-NAME, and 5 days L-NAME posthypoxia. Results are mean values of three independent experiments with ﬁve animals per group. The statistically signiﬁcant differences regarding the control group in the untreated groups were expressed as aP < 0.05; bP < 0.02; cP < 0.01; and dP < 0.001. The statistically signiﬁcant differences between the treated groups and the corresponding untreated groups were expressed as wP < 0.05; xP < 0.02; yP < 0.01; and zP < 0.001. TABLE 1. Quantitative data from image analysis of histological sections of rat lungs stained for TUNEL assay Group Untreated groups Control 0h 48 h 5 days L-NAME-treated groups Control 0h 48 h 5 days Percentage of area of apoptotic cells (%) 3.31 3.80 20.18 34.67 1.10 1.00 2.52d 2.47d 5.12 22.84 9.46 7.13 2.07 1.71z 2.20z 2.63z Experimental groups: control and 0 h, 48 h, and 5 days posthypoxia; control L-NAME and 0 h L-NAME, 48 h LNAME, and 5 days L-NAME posthypoxia. Results are mean values of 50 microphotographs (10 microphotographs per animal and ﬁve animals per group) SD. The statistically signiﬁcant differences regarding the control group in the untreated groups were expressed as aP < 0.05; bP < 0.02; c P < 0.01. d P < 0.001. The statistically signiﬁcant differences between the treated groups and the corresponding untreated groups were expressed as wP < 0.05; xP < 0.02; yP < 0.01; and. z P < 0.001. Fig. 4. (A–H): Micrographs showing histological sections of rat lung stained for TUNEL assay. Experimental groups: control (C) and 0 h, 48 h, and 5 days (5d) posthypoxia; control (C) L-NAME and 0 h L-NAME, 48 h L-NAME, and 5 days (5d) L-NAME posthypoxia. Apoptotic, TUNEL-positive cells are indicated by the brown nuclear staining (arrows). Micrographs (E) and (G) show higher apoptosis levels when compared with the control group in the untreated groups. Micrograph (D) shows higher apoptosis levels in relation to the corresponding untreated group. Micrographs (F) and (H) show lower apoptosis levels in relation to the corresponding untreated groups. Scale bars ¼ 50 lm. the percentage of apoptotic cells at the late posthypoxia times (48 h: P < 0.001, 5 days: P < 0.001) in the untreated groups (Table 1). However, after the treatment with L-NAME, this percentage rose at the earliest reoxygenation period (0 h: P < 0.001), but decreased in the later posthypoxia times (48 h: P < 0.001, 5 days: P < 0.001) when compared with the corresponding untreated groups (Fig. 4, microphotographs A, C, E, and G taken from Rus et al., 2010b). Nitrotyrosine Expression Three nitrotyrosine immunoreactive bands, corresponding to proteins of 126, 112, and 72 kDa, were detected in all the experimental groups (Fig. 5, right panel). The quantitative evaluation of the bulk-nitrated proteins increased signiﬁcantly from 48 h (P < 0.001) to 5 days (P < 0.001) posthypoxia in the untreated groups 2198 RUS ET AL. Fig. 5. Inﬂuence of hypoxia/reoxygenation on nitrotyrosine-modiﬁed protein expression in rat lung. Left panel: densitometric quantiﬁcation of nitrotyrosine-modiﬁed proteins in the experimental groups: Control (C) and 0 h, 48 h, and 5 days (5d) posthypoxia; control (C) L-NAME and 0 h L-NAME, 48 h L-NAME, and 5 days (5d) L-NAME posthypoxia. Results were expressed as arbitrary units (A.U.). Results are mean values of three independent experiments with ﬁve animals per group. Right panel: representative autoradiography of the nitrotyrosine-modiﬁed protein bands; a-tubulin immunodetection was also included as a proteinloading control. The statistically signiﬁcant differences regarding the control group in the untreated groups were expressed as aP < 0.05; bP < 0.02; cP < 0.01; and dP < 0.001. The statistically signiﬁcant differences between the treated groups and the corresponding untreated groups were expressed as wP < 0.05; xP < 0.02; yP < 0.01; and zP < 0.001. (Fig. 5, left panel). However, the administration of this nonselective inhibitor decreased nitrated protein expression at 48 h (P < 0.05), and 5 days (P < 0.02) of reoxygenation. nation situations in the lung. The results indicate that our model of hypoxia/reoxygenation increased the NADPH-diaphorase staining, indicative of in situ NOS activity, in endothelial and bronchiolar epithelial cells throughout the reoxygenation periods (0 h, 48 h, and 5 days) in the untreated groups. However, this staining was almost completely eliminated in those cell types after L-NAME administration, as expected. On the other hand, we have also shown that NO levels, indirectly quantiﬁed by nitrate/nitrite and S-nitrose compounds (NOx), signiﬁcantly rose at 0 h posthypoxia in the untreated groups when compared with the normobaric normoxic control. Nonetheless, the administration of this nonselective NOS inhibitor signiﬁcantly lowered NOx levels in all the experimental groups. Because it is well known that L-NAME is a NOS inhibitor (Viñas et al., 2006; Bertuglia, 2008), the decrease reported here in NOx levels and in situ NOS activity should be attributed to the inhibition of NOS isoforms. Our model of hypoxia/reoxygenation signiﬁcantly raised the lipid peroxidation level, determined by TBARS, at 48 h, and 5 days posthypoxia in the untreated groups, proposing that changes consistent with oxidative processes occur in the lung in response to hypoxia/reoxygenation. On the other hand, once L-NAME was administrated, the TBARS levels signiﬁcantly increased during the reoxygenation period at 0 h, but decreased in the later posthypoxia times (48 h, 5 days). Our results have also shown that our hypoxia/reoxygenation model provokes cell damage, indicated by the use of the TUNEL assay, which measures DNA damage and is an indicator of apoptosis. In this sense, an increased percentage of apoptotic cells were detected at 48 h and 5 days posthypoxia in the untreated groups. Nevertheless, the L-NAME treatment produced similar effects on Nitrotyrosine Immunohistochemistry In all the experimental groups, the nitrotyrosine immunoreactivity was detected in bronchiolar epithelial cells and vascular endothelial cells (Fig. 6, microphotographs A, C, E, and G taken from Rus et al., 2010b). Corroborating the previous results, the nitrotyrosinepositive staining was detected more intense from 48 h to 5 days posthypoxia in these cell types in the untreated groups and less intense during those times in the groups treated with the NOS inhibitor L-NAME. DISCUSSION Hypoxia-associated pathophysiology is complicated and elusive but noticeably signiﬁcant. Many adverse effects of hypoxia are commonly observed under conditions generated by ischemia. Like reperfusion, reoxygenation eventually does not completely reverse the hypoxia-induced changes. The complexity of the cell response to hypoxia complicates efforts to design approaches to treat or prevent injury resulting from reoxygenation. As mentioned in the Introduction section, there is a remarkable dearth of literature dealing with the inhibitor L-NAME in lungs submitted to hypoxia/reoxygenation, making its possible effects on this organ completely unknown. To the best of our knowledge, this is the ﬁrst study that describes the time-course effects of this nonselective NOS inhibitor in the hypoxic adult rat lung. Alterations in the production of NOS-derived NO are critical in the injury that occurs during hypoxia/reoxyge- DUAL ROLES OF NO IN THE HYPOXIC LUNG Fig. 6. (A–H): Micrographs showing nitrotyrosine immunoreactivity in rat lung sections in endothelial cells (arrows) and bronchiolar epithelial cells (arrow heads). Experimental groups: control (C) and 0 h, 48 h, and 5 days (5d) posthypoxia; control (C) L-NAME and 0 h L-NAME, 48 h L-NAME, and 5 days (5d) L-NAME posthypoxia. Micrographs (E) and (G) show higher nitrotyrosine staining intensity when compared with the control group in the untreated groups. Micrographs (F) and (H) show less nitrotyrosine staining intensity in relation to the corresponding untreated groups. apoptosis as on the lipid peroxidation level in the rat lung: the percentage of apoptotic cells raised at 0 h of reoxygenation, but decreased at 48 h and 5 days. We have previously reported that eNOS increased in the hypoxic rat lung at the earliest reoxygenation time (0 h), while the inducible isoform augmented from 48 h to 5 days posthypoxia (Rus et al., 2010b). All these ﬁndings together suggest that eNOS-derived NO may exert a beneﬁcial role against the lipid peroxidation and apoptosis processes, which occur during hypoxia/reoxygenation in the lung, while iNOS-derived NO appears to be involved in both of these harmful phenomena. As stated above, currently there are only scarce and controversial studies dealing with NOS inhibitors in the hypoxic lung, and they mostly are performed in cultured cells, not in vivo, as this work. In this sense, while some authors showed that inhibition of NO synthesis by L-NAME decreased greatly the production of lung TBARS after ischemia/reperfusion (Ischiropoulos et al., 2199 1995), others found that the same NOS inhibitor increased the concentrations of TBARS after intermittent hypoxia in hamster cheek pouch microcirculation (Bertuglia, 2008). Regarding apoptosis, Maejima et al. (2003) reported that L-NAME greatly increased the apoptosis rate in neonatal rat cardiomyocytes under ischemia/reperfusion, indicating that the NO released during these situations exerts an antiapoptotic effect. By contrast, Yang et al. (2005) found that hypoxia/reoxygenation-induced apoptosis was signiﬁcantly decreased by L-NAME in liver sinusoidal endothelial cells, and Viñas et al. (2006) showed that the number of TUNEL-positive cells, increased by ischemia/reperfusion, decreased with L-NAME in rat kidney, suggesting a role for NO in the development of apoptosis. In fact, it has been previously reported that NO can exert both pro- and antiapoptotic effects. In this way, NO can act as a second messenger, activating a number of cytokines that induce apoptosis (Saugstad, 2000), or it can inhibit aconitase in the tricarboxylic acid cycle, leading to a glycolysis inhibition (Stadler et al., 1991). On the contrary, NO can also inhibit apoptosis by nitrosylating active sites of cysteine residues in caspases, essential enzymes for the apoptotic process (Leist et al., 1999). Finally, our results have also shown that our model of hypoxia/reoxygenation signiﬁcantly augmented nitrotyrosine-modiﬁed protein expression at 48 h, and 5 days posthypoxia in the untreated groups. However, the treatment with the NOS inhibitor L-NAME signiﬁcantly decreased nitrated proteins at those same times, suggesting an involvement of NOS-derived NO in nitrotyrosine formation in the hypoxic lung. This decrease in nitrated protein expression is consistent, since L-NAME reduces NOx levels, and as a result, the formation of peroxinitrite, diminishing the nitrotyrosine-modiﬁed proteins formation. These results were corroborated by the nitrated protein location data, found in vascular endothelial cells and bronchiolar epithelium. There are, nonetheless, controversial results around the effects of this nonselective NOS inhibitor on nitrotyrosine levels in other organs and models. In this sense, Ischiropoulos et al. (1995) reported that the treatment with L-NAME counteracted the rise in nitrotyrosine levels after ischemia/reperfusion in the lung, and Serrano et al. (2006) showed that the administration of the same NOS inhibitor prevented the increase in nitrotyrosine immunoreactivity provoked by hypobaric hypoxia in rat cerebral cortex. On the contrary, other authors demonstrated that nitrotyrosine increased in the groups treated with L-NAME in the acutely hypoxic brain (Litt et al., 1999). In summary, the results of this study demonstrate that a treatment with the nonselective NOS inhibitor L-NAME in hypoxic lungs exacerbates lipid peroxidation and apoptosis just after the hypoxic stimulus, but decreases them in the later posthypoxia times. These ﬁndings suggest that NOSderived NO exerts two different effects on lung hypoxia/ reoxygenation-induced damage depending on the reoxygenation time: NO may have a beneﬁcial role at the earliest posthypoxia period and a deleterious role in the later reoxygenation times. Moreover, this dual role of NO may depend directly on the producer NOS isoform: while eNOSderived NO would exert a protective effect against the injuries which occur during hypoxia/reoxygenation in the lung, iNOS-derived NO appears to have a detrimental role. We suggest that the administration of L-NAME would be 2200 RUS ET AL. beneﬁcial for the treatment of lung hypoxia/reoxygenation injuries after 48 h posthypoxia. ACKNOWLEDGMENTS The authors thank Rafael Lomas for his statistic assistance, and David Nesbitt for his editorial help with the English version of the manuscript. 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