1 Institut National pour la Santé et la Recherche Médicale, Unité 408, Faculté de Médecine Xavier Bichat, 75018 Paris, France; and 2 Eisai London Research Laboratories, University College London, London WC1E 6BT, United Kingdom
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ABSTRACT |
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Reactive oxygen species (ROS) increase the contractile response of airway smooth muscle (ASM). Heme oxygenase (HO) catabolizes heme to the powerful antioxidant bilirubin. Because HO is expressed in the airways, we investigated its effects on ASM contractility and ROS production in guinea pig trachea. HO expression was higher in the epithelium than in tracheal smooth muscle. Incubation of tracheal rings (TR) with the HO inhibitor tin protoporphyrin (SnPP IX) or the HO substrate hemin increased and decreased, respectively, ASM contractile response to carbamylcholine. The effect of hemin was reversed by SnPP and mimicked by the antioxidants superoxide dismutase (SOD) and catalase. Hemin significantly reduced the effect of carbamylcholine in rings treated with the guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ), compared with ODQ-treated rings without hemin incubation, suggesting that the CO-guanosine 3',5'-cyclic monophosphate pathway was not involved in the control of tracheal reactivity. SnPP and hemin increased and decreased ROS production by TR by 18 and 38%, respectively. Bilirubin (100 pM) significantly decreased TR contractility and ROS production. Hemin, bilirubin, and SOD/catalase decreased phosphorylation of the contractile protein myosin light chain, whereas SnPP significantly augmented it. These data suggest that modulation of the redox status by HO and, moreover, by bilirubin modulates ASM contractility by modulating levels of phosphorylated myosin light chain.
phosphorylation; myosin light chain; reactive oxygen species
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INTRODUCTION |
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REACTIVE OXYGEN SPECIES (ROS) are produced by all aerobic organisms as a consequence of oxygen consumption and cell respiration. They play a role of intracellular mediators at physiological concentrations. However, in stress situations, increasing production of ROS can lead to cellular injury (19).
One interesting biologically relevant effect of oxidants and of hydrogen peroxide and superoxide anion in particular is the regulation of cell functions by controlling the level of protein phosphorylation. This is modulated by activation of protein kinases or inactivation of protein phosphatases (4, 19, 22). In smooth muscle cells, levels of phosphorylated proteins are critical for cell contraction: phosphorylation of myosin light chain (MLC) allows the myosin ATPase to be activated by actin and thus the muscle to contract (36). Numerous experimental data have shown that airway reactivity was significantly increased by ROS, whereas it was decreased by different antioxidant molecules in different animal species such as guinea pigs (15), rats (45), rabbits (37), dogs (43), and humans (38). However, the role of ROS in the modulation of MLC phosphorylation is unknown.
The ubiquity of ROS production explains the need for strong antioxidant cellular systems. Heme oxygenase (HO), the enzyme responsible for heme degradation, is a powerful antioxidant and protective system (10, 31). Two main isoforms, products of different genes, have been identified: HO-1, the inducible form (also known as heat shock protein 32), and HO-2, the constitutive form (31). Degradation of the tetrapyrrolic ring produces carbon monoxide (CO) and biliverdin, reduced into bilirubin by the biliverdin reductase, one of the most important antioxidant molecules of the organism (33, 44). If the HO pathway has shown beneficial effects in different oxidative stress models (10, 14), its role in regulating cellular redox status in normal conditions has been poorly investigated.
We hypothesized that bilirubin, produced by HO, could modulate airway reactivity of guinea pig tracheal rings. If so, we wondered whether this effect could be related to the modulation of ROS production in the airways and how modulation of redox status could influence phosphorylation of the MLC.
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MATERIALS AND METHODS |
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Animals. Pathogen-free male Hartley guinea pigs (250-300 g body wt; Charles River) were housed in individual cages in climate-controlled animal quarters and were given water and food ad libitum. The experiments conducted in the present study were approved by the local institutional animal care and use committee, and the experimental protocol was in agreement with the recommendations related to animal studies under French law.
Western blot analysis of HO-1, HO-2, and phosphorylated MLC in
guinea pig trachea.
Guinea pigs were stunned by a blow to the head and exsanguinated. The
trachea was rapidly excised by open tracheotomy and placed in a Krebs
solution. Tracheal smooth muscle (TSM) was dissected under a binocular
microscope and cleaned of fat, blood, and connective tissue. In some
experiments, we removed the epithelial layer adjacent to the dissected
TSM by gently rubbing their mucosal surfaces with a cotton wool-coated
probe (35). Samples were then immediately frozen in liquid
nitrogen and stored at 80°C until use. Samples from each animal
were homogenized as described previously (42). We measured
protein concentration spectrophotometrically in 96-well plates using
the Bio-Rad protein reagent (Bio-Rad Laboratories, Richmond, CA) with
bovine serum albumin as standard.
Measurement of tracheal rings reactivity. Animals were killed as described in the previous section. The trachea was removed by open tracheotomy and cut into rings of ~2 mm in width. In some experiments, the epithelial layer was removed as described in the previous section.
We evaluated reactivity of tracheal rings by measuring isometric tension, as described earlier (35). The general protocol for measurement of tracheal ring reactivity was as follows: after readjustment of tension to 2 g, the preparation was contracted twice with 20 mM KCl, with intermediate washings. After 30 min of washing, we measured baseline tension, and the rings were incubated for 60 min in Krebs solution containing different pharmacological agents or their respective vehicles. Then, tension was measured again, and a cumulative concentration-response curve to carbamylcholine (carbachol) or histamine was performed (1 nM to 1 mM) as previously described (35).Measurement of superoxide anion release by tracheal rings. The release of superoxide anion by guinea pig tracheal rings was performed by measuring superoxide dismutase (SOD)-inhibitable lucigenin-dependent chemiluminescence (41). Guinea pig tracheal rings were obtained as described in Western blot analysis of HO-1, HO-2, and phosphorylated MLC in guinea pig trachea and placed in luminometer cuvettes containing 500 µM lucigenin, a chemiluminescence indicator, in a final volume of 0.5 ml of Krebs solution. Then, the rings were treated with different agents (see below), and chemiluminescence was measured every 15 min over a 60-min time period at 37°C. The integrated response was determined with a computer program supplied with the luminometer (AutoLumat LB 953; EG & G Instruments, Evry, France). Data are presented as the area under the curve over 60 min, normalized to the wet weight of the ring.
Reagents. Hemin chloride, tin protoporphyrin IX (SnPP), and zinc protoporphyrin IX (ZnPP IX) were purchased from Porphyrin Products (Logan, UT). Initial solutions were made in NaOH before dilution and pH adjustment in PBS. Bilirubin was purchased from Fluka (St Quentin-Fallavier, France) and diluted in NaOH and PBS. Reagents for SDS-PAGE and chemiluminescence substrates were from Bio-Rad Laboratories. All other chemicals were purchased from Sigma-Aldrich.
Statistical analysis. Values are given as means ± SE. We compared the effects of carbachol or histamine on tracheal ring contraction in the presence and absence of pharmacological agents by comparing the concentration-response curves by using two-way ANOVA for repeated measurements considering one "grouping" factor (i.e., factor group) and one "within" factor (i.e., factor concentration). Two-by-two comparisons between the concentration-response curves were made only when the overall comparison was significant. The other data were analyzed by one-way ANOVA; differences between means were analyzed with Fisher's protected least-significant difference, multiple-comparison test. Significance for all statistics was accepted at P < 0.05.
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RESULTS |
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Expression of HO-1 and HO-2 protein in epithelium and TSM. We first assessed HO-1 and HO-2 protein expression in TSM from epithelium-intact and epithelium-denuded tracheal rings.
Western blot analysis revealed HO-1 protein expression in both preparations; the molecular mass of HO-1 was identical to that of HO-1 expressed in rat spleen (Fig. 1A). Expression of HO-2 protein was also detected in both preparations, and the molecular mass of the band was identical to that of HO-2 expressed in rat testes (Fig. 1A).
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Role of HO on tracheal ring contractile response. Having demonstrated that the two HO isoforms are expressed both in TSM and epithelium, we evaluated the effect of HO activity modulation on the contractile response of epithelium-intact preparations.
Incubation of tracheal rings for 60 min with the HO inhibitor SnPP (10 µM) significantly increased the response to carbachol compared with its vehicle (P < 0.05, Fig. 2). Similar results were observed when the tracheal ring was contracted with histamine (data not shown). For the maximal concentration of carbachol and histamine, the increase averaged 20 and 30% compared with vehicle-incubated preparations. Another HO inhibitor, ZnPP IX (10 µM), had an effect similar to SnPP (data not shown).
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Role of epithelial HO in modulating smooth muscle contractility. To assess whether epithelial or muscular HO could be involved in the effect of HO modulation on the contractile response of tracheal rings, we repeated the experiments using epithelium-denuded tracheal rings.
As previously described (24), contractile response to carbachol was significantly increased in epithelium-denuded compared with epithelium-intact rings (P < 0.05, Fig. 2). This response was modulated neither by SnPP nor by hemin (Fig. 2), suggesting that airway smooth muscle HO did not modulate the contractile response of the muscle. Therefore, further experiments were performed in tracheal rings with intact epithelia.Role of the CO-guanosine 3',5'-cyclic monophosphate pathway. As the CO-guanosine 3',5'-cyclic monophosphate (cGMP) pathway has been involved in the modulation of bronchial tone (8), we evaluated the effects of the selective inhibitor of soluble guanylate cyclase (sGC) 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ, 10 µM) (25) on the contractile response to carbachol of hemin- or vehicle-treated tracheal rings. The rationale for this experiment was that, if CO is involved in the effects of hemin, inhibition of sGC by ODQ should inhibit the effect of hemin. Conversely, if the effect of hemin is independent of CO, hemin should be able to reduce carbachol-induced contractions, even in the presence of ODQ.
The results of the experiments showed that 1) as previously described (25), ODQ significantly increased the contractile response to carbachol compared with control rings (P < 0.05, Fig. 3) and that 2) hemin significantly reduced the effect of carbachol in ODQ-treated rings compared with ODQ-treated rings without hemin incubation (P < 0.05, Fig. 3). These results suggest that inhibition of the sGC did not impair the effect of hemin.
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Role of ROS on HO effects on tracheal rings. Because HO is known as a powerful antioxidant system (10, 31), we next investigated the role of ROS in mediating the effects of HO.
First, we assessed whether HO could modulate ROS production in our model. Rings incubated with the vehicles for SnPP, hemin, or the intracellular antioxidant mixture polyethylenglycol-SOD (PEG-SOD) plus PEG-catalase showed no difference compared with rings incubated with Krebs solution alone. Therefore, the results of these experiments were pooled and presented as "control" rings. Control rings released a small, but significant amount of superoxide anion, which was significantly decreased by the intracellular antioxidants PEG-SOD plus PEG-catalase (250 and 500 U/ml respectively, P < 0.05, Fig. 4). Superoxide anion production was modified neither by carbachol nor by histamine (data not shown); however, it was significantly increased by SnPP (P < 0.05). The effect of SnPP was suppressed by PEG-SOD plus PEG-catalase (P < 0.005, Fig. 4). Superoxide anion production was significantly reduced by hemin (P < 0.01, Fig. 4), and this effect was reversed by SnPP.
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Role of bilirubin.
Because bilirubin, the end-step product of heme degradation, is one of
the most powerful endogenous antioxidant (44), we evaluated its involvement in the antioxidant effect of HO in the present experimental model. Superoxide anion production by control and
SnPP-treated rings was significantly reduced by bilirubin at 100 pM
(P < 0.05, Fig. 6).
However, no modification was observed when bilirubin was used at 10 nM
(Fig. 6). Bilirubin, at a concentration of 100 pM, significantly
reduced the contractile response of vehicle-treated rings
(P < 0.05, data not shown) and abrogated the
potentiation induced by SnPP (P < 0.05, Fig.
7). These effects were not observed when
bilirubin was used at 10 nM (Fig. 7).
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Role of HO on the level of phosphorylated MLC. Finally, we evaluated the effect of HO on MLC phosphorylation in TSM obtained from epithelium-intact rings.
Western blot analysis of TSM incubated with 1 mM carbachol showed a basal level of phosphorylated MLC (Fig. 8). This level was significantly increased by SnPP (P < 0.05) and significantly reduced by PEG-SOD plus PEG-catalase, hemin, or bilirubin at the aforementioned concentrations (100 pM for bilirubin, P < 0.05, Fig. 8, A and B). Furthermore, PEG-SOD plus PEG-catalase and bilirubin abrogated the increase in phosphorylated MLC induced by SnPP (P < 0.05 respectively, Fig. 8B).
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DISCUSSION |
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The main results of this study are as follows. 1) Hemin, the HO substrate and inductor, decreased and SnPP IX, a HO inhibitor, increased tracheal rings' response to carbachol. 2) Epithelial HO was involved in modulating tracheal ring reactivity, whereas muscular HO does not seem to be involved in this phenomenon. 3) Hemin decreased production of superoxide anion by tracheal rings, whereas SnPP significantly increased it. The antioxidants PEG-SOD plus PEG-catalase reduced SnPP-induced superoxide production. Moreover, they abolished the contractile response to SnPP. 4) Bilirubin, the end-step product of HO activity, reversed the effect of SnPP on ROS production and tracheal ring reactivity. 5) Hemin decreased phosphorylation of the MLC. Bilirubin and PEG-SOD plus PEG-catalase mimicked this effect, whereas SnPP significantly increased the amount of the phosphorylated protein. These data would suggest that modulation of the redox status by HO and, moreover, by its end-step product bilirubin regulates airway smooth muscle contractility by modulating the levels of phosphorylated MLC.
Expression and role of HO in the airways. HO is strongly expressed in normal and pathological human airways, especially in epithelial cells (16, 28) but also in smooth muscle cells, parasympathetic ganglia, endothelium, and macrophages. The HO pathway has been shown to be involved in controlling both airway inflammation and reactivity in animal models of allergyinduced and ozone-induced airway hyperresponsiveness (23, 26). HO is able to induce relaxation (8, 34) and/or to decrease contractile response in smooth muscle from different tissues (1, 7, 9, 18, 20). For example, increasing HO-1 activity in myometrium strips by short-term incubation with hemin, in an experimental protocol quite similar to ours, is able to inhibit contractile response to KCl, whereas SnPP has the opposite effect (1). This effect is often related to the binding of CO to the heme moiety of sGC and the resultant formation of the second messenger cGMP (34). However, Canning and Fischer (7) found that neither ZnPP IX nor the sGC inhibitor ODQ has marked effects on vagally mediated bronchial contractions, suggesting that a CO-independent mechanism could be involved in modulation of contractile response by the HO pathway in the airway. Our study is the first to provide evidence that the HO pathway modulates contractile response to carbachol and histamine in airway smooth muscle. Moreover, our data present a new and original mechanism to explain the role of HO, linking for the first time its antioxidant properties with the decrease in muscle contractility. In fact, our data obtained with ODQ makes unlikely a role for CO-cGMP in the decreased contractility induced by hemin. However, if we exclude the participation of a CO-related production of cGMP in airway smooth muscle contraction, we do not exclude the possibility that CO can modulate airway reactivity in the present experimental model by a cGMP-independent mechanism, such as modulation of potassium channels (29, 48) or kinase activity (2, 6), as demonstrated in other tissues.
The present results show that epithelial HO, and not muscular HO, modulates TSM contractility. SnPP actually failed to increase the response to carbachol when the epithelium was removed, whereas it increased it in intact rings. A similar lack of effect was observed with hemin. This phenomenon could be related to the low level of expression of HO-1 and HO-2 in TSM compared with the epithelium. Airway epithelium not only is exposed to exogenous oxidative damage but also is an important source of oxygen and nitrogen species (32), thus explaining the need for a strong local antioxidant system (49). Catalase, for example, is more strongly expressed in epithelial than in smooth muscle cells (3). Our results are in agreement with this data, thus confirming the protective role of epithelia against oxidant-induced contraction.Role of bilirubin. Our results show that bilirubin decreases TSM contractile response to carbachol. Previous works have shown that bilirubin decreases vascular or gastric smooth muscle contractility (5, 13, 46, 47). However, in one of these studies, the authors showed that this effect was highly dependent on bilirubin concentration: relaxation with the lowest doses (about 10 µM) and contraction with the highest (about 200 µM) (5). We observed such a dose-related effect in our experiments: bilirubin, at a concentration of 100 pM, decreased oxidant production and TSM contractility, whereas this effect was not observed at 10 nM. A similar disappearance of the protective effect of bilirubin at such concentrations was observed by Doré and coworkers (17) in neuronal cultures and was related to the toxicity of bilirubin. Favorable antioxidant effect of bilirubin has been demonstrated also in vivo (11) and ex vivo in ischemic hearts at nanomolar concentrations (12, 17). In the present study, the simultaneous decrease in oxidant production and TSM contractility observed with 100 pM of bilirubin and the fact that this latter effect was mimicked by antioxidants strongly suggest a major role of the antioxidant properties of bilirubin in the decreased contractility. Indeed, different studies have shown that airway reactivity is significantly increased by ROS (38, 40). Our results agree with data published by Colpaert and Lefebvre (13), showing that bilirubin potentiated the relaxant effect of nitric oxide in gastric smooth muscle with a potency similar to other antioxidants, such as urate or glutathione. In fact, bilirubin has been known for a long time to possess strong antioxidant properties: it is able to scavenge different oxygen and nitrogen species (33) and to inhibit production of superoxide by NADPH oxidase (27).
However, if bilirubin antioxidant properties appear to be an important mechanism explaining the decrease in TSM contractility, we cannot exclude other mechanisms. Bilirubin actually modulates calcium uptake by vascular smooth muscle cells (47) and inhibits protein phosphorylation, especially in the MLC, probably by interacting with different domains of the kinase (21). The relative importance of these pathways in the decreased of ASM contractility warrants further investigations. Several mechanisms can be involved in the modulation of smooth muscle contractility by ROS: ROS are considered as intracellular messengers involved in control of calcium channel sensitivity, mitochondrial respiration or activation of protein-tyrosine kinase, and inhibition of protein-tyrosine phosphatase (reviewed in Ref. 19), all features involved in muscular contraction. Among those mechanisms, phosphorylation of the MLC is actually the key event for initiating smooth muscle contraction by modifying the conformation of myosin, and regulation of the amount of phosphorylated/ dephosphorylated protein is critical for the cellular physiology. We show in this study that increasing ROS production by SnPP increases both contractile responses to carbachol and the amount of phosphorylated MLC, whereas hemin and antioxidants have the opposite effect. These results are in line with data published by Lopez-Ongil and coworkers (30), showing that exogenously added hydrogen peroxide increased both the amount of phosphorylated MLC and contraction of endothelial cells. Bilirubin was able to reverse the three effects: ROS production, contractile response, and MLC phosphorylation. These data suggest a link between anticonstrictive effect of bilirubin and its ability to decrease ROS production by tracheal rings, emphasizing the role of redox status in modulating the amount of phosphorylated MLC in TSM. In conclusion, our data provide evidence that the HO pathway, and especially bilirubin, controls tracheal reactivity by decreasing ROS-dependent phosphorylation of MLC. This would argue for the redox modulation of airway contractility by bilirubin and support the interest for antioxidant therapy in airway inflammatory disease. ![]() |
FOOTNOTES |
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* A. Samb and C. Taillé contributed equally to this work.
Address for reprint requests and other correspondence: C. Taillé, INSERM U408, Faculté Xavier Bichat, 16 rue Henri Huchard, 75018 Paris, France (E-mail: c-taille{at}bichat.inserm.fr).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
April 5, 2002;10.1152/ajplung.00446.2001
Received 3 December 2001; accepted in final form 3 April 2002.
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