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A new nitrosyl ruthenium complex nitric oxide donor presents higher efficacy than sodium nitroprusside on relaxation of airway smooth muscle
European Journal of Pharmaceutical Sciences 43 (2011) 370–377
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European Journal of Pharmaceutical Sciences journal homepage: www.elsevier.com/locate/ejps
A new nitrosyl ruthenium complex nitric oxide donor presents higher efficacy than sodium nitroprusside on relaxation of airway smooth muscle Patrícia F.S. Castro a, Amanda de C. Pereira b, Gerson J. Rogrigues b, Aline C. Batista c, Roberto S. da Silva b, Lusiane M. Bendhack b, Matheus L. Rocha a,⇑ a b c
Faculty of Pharmacy, Federal University of Goias, Av. Universitária s/n, 74605-220 Goiânia, GO, Brazil Faculty of Pharmaceutical Sciences, University of São Paulo, Av. do Café s/n, 14040-903 Ribeirão Preto, SP, Brazil Faculty of Odontology, Federal University of Goias, Av. Universitária s/n, 74605-220 Goiânia, GO, Brazil
a r t i c l e
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Article history: Received 17 February 2011 Received in revised form 2 May 2011 Accepted 8 May 2011 Available online 14 May 2011 Keywords: Nitric oxide Airway smooth muscle Guanylyl cyclase K+ channels Trachea
a b s t r a c t Nitric oxide (NO) has been demonstrated to be the primary agent in relaxing airways in humans and animals. We investigated the mechanisms involved in the relaxation induced by NO-donors, ruthenium complex [Ru(terpy)(bdq)NO+]3+ (TERPY) and sodium nitroprusside (SNP) in isolated trachea of rats contracted with carbachol in an isolated organs chamber. For instance, we verified the contribution of K+ channels, the importance of sGC/cGMP pathway, the influence of the extra and intracellular Ca2+ sources and the contribution of the epithelium on the relaxing response. Additionally, we have used confocal microscopy in order to analyze the action of the NO-donors on cytosolic Ca2+ concentration. The results demonstrated that both compounds led to the relaxation of trachea in a dependent-concentration way. However, the maximum effect (Emax) of TERPY is higher than the SNP. The relaxation induced by SNP (but not TERPY) was significantly reduced by pretreatment with ODQ (sGC inhibitor). Only TERPYinduced relaxation was reduced by tetraethylammonium (K+ channels blocker) and by pre-contraction with 75 mM KCl (membrane depolarization). The response to both NO-donors was not altered by the presence of thapsigargin (sarcoplasmic reticulum Ca2+-ATPase inhibitor). The epithelium removal has reduced the relaxation only to SNP, and it has no effect on TERPY. The both NO-donors reduced the contraction evoked by Ca2+ influx, while TERPY have shown a higher inhibitory effect on contraction. Moreover, the TERPY was more effective than SNP in reducing the cytosolic Ca2+ concentration measured by confocal microscopy. In conclusion, these results show that TERPY induces airway smooth muscle relaxation by cGMP-independent mechanisms, it involves the fluxes of Ca2+ and K+ across the membrane, it is more effective in reducing cytosolic Ca2+ concentration and inducing relaxation in the rat trachea than the standard drug, SNP. Ó 2011 Elsevier B.V. All rights reserved.
1. Introduction Airway smooth muscle tone is maintained by equilibrium between bronchoconstriction and bronchodilation. The aim of clinical treatment for pathological conditions, such as allergy, asthma and inflammation is to avoid excessive constriction, and to reestablish a balance by producing dilation. In this context, nitric oxide (NO) has been demonstrated to be the primary agent in relaxing airways in humans and animals (Ellis, 1997; Ellis and Undem, 1992; Gaston et al., 1993). NO is released in the epithelial cells of the airways in basal conditions causing relaxation in vivo and in vitro, through its action on the smooth muscle (Arnold et al., 1984). This relaxation is
⇑ Corresponding author. Address: UFG – Faculdade de Farmácia, Av. Universitária com 1ª Avenida s/n, Setor Universitário CEP, 74605-220 Goiânia, GO, Brazil. Tel.: +55 62 3209 6440; fax: +55 62 3209 6037. E-mail address: [email protected] (M.L. Rocha). 0928-0987/$ - see front matter Ó 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.ejps.2011.05.004
mediated by endogenous NO produced mainly by epithelial cells and nerves, which diffuses to the underlying smooth muscle (Barnes and Belvisi, 1993). Thus, NO-donors may be useful pharmacological tools to induce relaxing response in airways. Studies in bronchial and tracheal smooth muscle have shown that a major target of NO is the enzyme soluble guanylyl cyclase (sGC) (Ellis, 1997; Hwang et al., 1998). The activation of sGC by NO increases intracellular levels of cGMP which, in turn, amplifies the cellular response (Arnold et al., 1977). The main target of cGMP is the family of cGMP-dependent protein kinases (PKG). Activation of PKG and subsequent phosphorylation of various proteins constitute a cascade of reactions that lead to the reduction of cytosolic Ca2+ concentration, leading to relaxation (McDaniel et al., 1992). However, not all studies support the role of sGC/cGMP pathway in mediating the relaxant effect of NO, and cGMP-independent pathways are increasingly being recognized (Redington, 2006; Stuart-Smith et al., 1994). The controversial results of these studies may be due to different species and NO-donors used.
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system and WinDaq Resource (DATAQ Instruments, Akron, OH, USA, data acquisition unit) to measure tension in the preparations. The tracheal rings were placed in a 10 ml organ chamber containing Krebs solution with the following composition (mM): NaCl 130, KCl 4.7, KH2PO4 1.2, MgSO4 1.2, NaHCO3 14.9, glucose 5.5, CaCl2 1.6. The solution was maintained at pH 7.4, and gassed with 95% O2 and 5% CO2 at 37 °C. The rings were initially stretched to a basal tension of 1.0 g (optimal basal tone, previously determined by length–tension relationship experiments) before allowing them to equilibrate in the bathing medium. 2.2. Experimental protocols
Fig. 1. Struture of [Ru(terpy)(bdq)NO+]3+.
The main prototypes of NO-donors traditionally used, such as sodium nitroprusside (SNP) and nitroglycerine, have severe wellknown adverse effects, such as sharp tachycardia, high toxicity and induce rapid tolerance (Yakazu et al., 2001). In order to avoid these unwanted effects, some metallonitrosyl complexes have been used as NO deliver agents. NO coupled to nitrosyl ruthenium complexes is attractive because of its thermal stability, stability in physiological pH and is practically nontoxic (de Lima et al., 2006; Oliveira et al., 2007; Sauaia et al., 2003; Wang et al., 2000). Studies have shown that these compounds exert a NO type biological activity, such as relaxation of vascular smooth muscle, inhibition of platelet aggregation and increasing levels of cGMP (Ignarro et al., 1980; Kowaluk and Fung, 1990; Radomski et al., 1992; Rotta et al., 2003). The Nitrosyl–terpyridine–phenylene–diamine ruthenium (II) ion complex [Ru(terpy)(bdq)NO+]3+ (TERPY, Fig. 1) is a NO-donor that induces relaxation in arteries of rats by acting on both K+ channels and sGC–cGMP pathway (Bonaventura et al., 2007). An increasing knowledge of the involvement of NO in numerous physiological pathways has not only expanded new therapeutic avenues for NO-related compounds but also led to an increased use of such compounds in pharmacological studies. Therefore, this study was dedicated to investigate the effects of a ruthenium complex NO-donor in tracheal smooth muscle of rats and to evaluate the advantages of its relaxing properties (in vitro) when compared with SNP. 2. Methods and materials 2.1. Isolated tracheal preparation Male Wistar rats were anaesthetized and killed by aortic exsanguinations. All the procedures were carried out in accordance with the Animal Research Ethical Committee of the Federal University of Goiás, Goiânia, Goiás, Brazil. The trachea was quickly removed, dissected, cleaned of adhering fat and connective tissue, and cut into 4-mm-long rings. The tracheal rings were placed between two stainless-steel stirrups and connected to an isometric force transducer. The responses were recorded using a computerized
2.2.1. Isometric tension recording in tracheal rings After 60 min of equilibration, each tracheal ring was exposed to KCl 75 mM to attain its maximum contractility. Each ring was sequentially washed and re-equilibrated and was allowed to relax to baseline. After 30 min, cumulative concentration–response curves for SNP (10 nM to 100 lM) and TERPY (10 nM to 100 lM) were carried out in preparations pre-contracted with cholinergic agonist carbachol (0.5 lM, EC50 previously determined in our laboratory) or after contraction induced by membrane depolarization with KCl 75 mM. To investigate some mechanism(s) responsible for SNP- or TERPY-induced relaxation, tracheal rings were contracted with carbachol (0.5 lM) 30 min after being incubated with one the following drugs: guanylyl cyclase inhibitor 1H-[1,2,4] oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, 1 lM), non-selective K+ channel blockers tetraethylammonium (TEA, 5 mM), a specific sarcoplasmic reticulum Ca2+-ATPase (SERCA) inhibitor (Thapsigargin, 1 lM). 2.2.2. Effect of TERPY and SNP on Ca2+ influx To further analyze the relative contribution of the TERPY or SNP in inhibit the contraction evoked by Ca2+ influx, the role of extracellular Ca2+ mobilization stimulated by contractile agonist was investigated by CaCl2-induced contraction in the presence of carbachol. Tracheal rings were first contracted with carbachol (EC50, 0.5 lM) to deplete the intracellular Ca2+ stores in Ca2+-free solution until the disappearance of any contractile response (approximately 60 min) and then rinsed in Ca2+-free solution containing carbachol (0.5 lM). After that, the cumulative concentration–response curves for CaCl2 (0.0– 1.6 mM) were obtained in the absence (control group) or after a 20 min incubation period with SNP or TERPY (100 lM). 2.3. Influence of the epithelial cells in the relaxation induced by NOdonors In some experiments, the epithelial cells were removed mechanically by rubbing the internal tracheal surface with a fine metallic wire (200 lm in diameter) as described previously (Wu et al., 2004). This procedure allowed investigating the influence of functional epithelium in the relaxation induced by NO-donors. After removal of the epithelium, cumulative concentration-effect curves to SNP or TERPY (10 nM to 100 lM) were performed under precontraction with carbachol (0.5 lM). The removal of the epithelial layer was confirmed by histological examination, with the observation of trachea tissue stained with hematoxylin and eosin by optical microscopy. 2.4. Measurements of intracellular Ca2+ by confocal microscopy and image analysis Calcium levels were detected as previously described (Lunardi et al., 2006; Rodrigues et al., 2008) with some adaptations to the tracheal smooth muscle. Trachea slices (150 lm thick) were placed in a glass coverslip covered with poly-L-lysine (50%) in Hanks
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solution (composition in mM: NaCl 145.0; KCl 5.0; MgSO4; 7H2O 1.0; CaCl2 1.6; NaH2PO4 0.5; glucose 10.0; Hepes 10.0) at pH 7.4 and 37 °C. Then, tracheal slices were loaded with the membranepermeating indicator fluorescent Ca2+ dye Fluo 3-AM (10 lM, Sigma Probes) for 30 min at room temperature. Excess dye was removed by washing out the dye with a bath solution. After washing with Hanks solution, coverslips were placed on a chamber (1.0 ml in volume) and placed in a confocal microscope (Leica TCS SP5). Ca2+-images of the distinct slices of tracheal rings were taken sequentially, in Hanks buffer and assessed through a water immersion objective (63). Fluo 3-AM was excited with the 488 nm line of an argon ion laser, and the emitted fluorescence was measured at 510 nm. Time course software was used to capture images of the rings 4 min after TERPY or SNP (100 lM) addition, at intervals of 2.5 s (xyt mode), 512 512 pixel at 700 Hz. The computer software Leica Microsystem LAS AF Lite, was used to measure the intensities of the intracellular maximum or minimum fluorescence in regions of interest (smooth muscle layer) in the ring. From these data, the initial fluorescence value (F0) was used and the final fluorescence value was denoted as F. The difference in fluorescence intensity (DFI) was obtained for each protocol: % DFI = (F F0)/ F0 100.
The maximal response (Emax) of TERPY (97.17 ± 2.82%) was approximately 43% higher than SNP (55.14 ± 5.5%, p < 0.001). Furthermore, SNP and TERPY have presented different potency (pEC50: 6.07 ± 0.03 and 5.16 ± 0.14, respectively, p < 0.01). 3.2. Participation of sGC and K+ channels on the tracheal relaxation induced by TERPY and SNP As shown in Fig. 3A, the sGC inhibition decreased the potency (p < 0.01) and maximal effect (p < 0.05) of SNP in inducing trachea
2.5. Chemicals The drugs used in this study included carbachol, ODQ, TEA, SNP, thapsigargin, Fluo 3-AM, which were purchased from Sigma Chemical Co. (St. Louis, MO, USA). All other chemicals used in the present study were commercially available and of reagent grade. ODQ and thapsigargin were dissolved in DMSO. The other drugs were dissolved in distilled water. The concentration of DMSO in the organ chamber did not exceed 0.01% and had no effect on tension generation in this preparation. The TERPY complex was synthesized as previously described (de Lima et al., 2006). TERPY synthesis was conducted at the Laboratory of Analytical Chemistry by Dr. Roberto Santana da Silva, Faculty of Pharmaceutical Sciences, University of São Paulo, Brazil.
Fig. 2. Relaxant concentration–response curves of SNP and TERPY in the carbacholcontracted rat trachea. Each point represents the mean ± SEM and is expressed as percentage of relaxation to SNP (n = 5) or TERPY (n = 7). Statistic ⁄⁄⁄p < 0.001.
2.6. Statistical analysis In the graphics, the data are presented as means ± SEM. In each set of experiments, n indicates the number of rats studied. The values for reactivity and responses to SNP or TERPY are expressed as percentage of the preceding contraction. The concentration of the compound producing a half-maximal response (EC50) was determined after logarithmic transformation of the normalized concentration– response curves and is reported as the negative logarithm (log EC50 = pEC50 values) of the mean of individual values for each tissue, using GraphPad Prism version 5.0 (GraphPad Software Inc., San Diego, CA). The maximum relaxant effect (Emax) was considered as the maximal amplitude response reached in concentration–effect curves for relaxant agents. Statistical analysis was performed using the GraphPad Prism version 5.0 (GraphPad Software Corporation, San Diego, CA). Comparisons among groups were performed using one-way ANOVA (post-test: Newman–Keuls) and Student’s t test, and values of p < 0.05 were considered to be significant. 3. Results 3.1. Relaxant effect of TERPY and SNP in the rat tracheal smooth muscle TERPY or SNP (10 nM to 100 lM) added cumulatively to the bath solution during the sustained contraction induced by carbachol (0.5 lM) have evoked concentration-dependent relaxation (Fig. 2).
Fig. 3. Relaxant concentration–response curves of SNP or TERPY in presence ODQ in carbachol-contracted rat trachea. (A) Relaxation SNP in absence (control, n = 5) and presence (20 min) of the ODQ (1 lM, n = 7). (B) Relaxation TERPY in absence (control, n = 7) and presence (20 min) of the ODQ (1 lM, n = 5). Each point represents the mean ± SEM and is expressed as percentage of relaxation. Statistic ⁄ p < 0.05.
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relaxation (Emax 35.0 ± 7.7% and pEC50 5.19 ± 0.21), compared to control (Emax 55.14 ± 5.5% and pEC50 6.07 ± 0.03). To TERPY (Fig. 3B), no differences were observed in the maximum effect and potency in the presence of ODQ when compared to the control group (Emax 98.7 ± 1.26%, pEC50 5.22 ± 0.11). These results suggest that the pathway sGC/cGMP is involved only on relaxation effect induced by SNP. Conversely, as showed in Fig. 4A and B, the K+ channels blocker TEA altered the Emax for SNP (from 55.14 ± 5.5% to 82.4 ± 5.8%), leading to an increase of about 49% (p < 0.05) and for TERPY (from 97.17 ± 2.82% to 66.14 ± 7.7%), a decrease of about 32% (p < 0.01) compared with the controls groups. In the presence of TEA no differences were observed in the potency compared with the control group (NPS pEC50: 6.86 ± 0.39; TERPY pEC50: 4.30 ± 0.40). In preparations contracted with 75 mM KCl, a concentration that induces depolarization of the cell membrane, no difference was observed in the relaxation induced by SNP when compared with contraction with EC50 carbachol (Fig. 4C, Emax 61.19 ± 6.03%, pEC50 6.24 ± 0.08, n = 5). On the other hand, the relaxation induced by TERPY was significantly reduced by 49% (Fig. 4D, p < 0.001). Emax was reduced from 97.17 ± 2.82% to 49.54 ± 6.97%, n = 5 and pEC50 from 5.16 ± 0.14 to 4.94 ± 0.16, n = 5. These results suggest that the K+ channels play an important role in the relaxation induced by TERPY.
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45.4 ± 3.94, pEC50 6.05 ± 0.11) or TERPY (Fig. 5B, Emax 99.4 ± 2.97%, pEC50 5.34 ± 0.17) compared with the controls groups. The results with thapsigargin suggest that calcium stored in sarcoplasmic reticulum does not have significant importance for relaxing effect both SNP and TERPY in tracheal smooth muscle. 3.4. Effect of TERPY and SNP on CaCl2-induced contractions Tracheal ring was used to investigate contractile responses dependent on extracellular Ca2+ influx (Fig. 6). Cumulative additions of CaCl2 produce concentration-dependent tracheal smooth muscle contractions in the presence carbachol. Contraction responses are expressed as a percentage of the response to KCl 75 mM. The group incubated with SNP (100 lM for 20 min) significant decreased (p < 0.001) contraction in 34.8% compared with the control. Emax of group incubated with SNP was 76.87 ± 9.59% and control group was 117.9 ± 5.98%. The group incubated with TERPY (100 lM for 20 min) also significantly reduced the contraction (p < 0.001) in 74.7% compared with the control. Emax of group incubated with TERPY was 27.42 ± 4.08% and control group was 117.9 ± 5.98%. Comparing the incubation to TERPY with SNP, they are different in 64% (p < 0.001) (Fig. 6). These results suggest that the inhibition of Ca2+ influx is an important mechanism to bring about relaxation the tracheal smooth muscle for the two NO-donors.
3.3. Effects of the SERCA inhibitor on the relaxation induced by TERPY and SNP
3.5. Role of epithelium in relaxation rat tracheal smooth muscle
Our data showed that thapsigargin (1 lM, SERCA inhibitor), did not alter the relaxation and potency induced by SNP (Fig. 5A, Emax
Fig. 7 shows rat trachea with and without epithelium and its effect on the relaxation of smooth muscle. Fig. 7A and B is
Fig. 4. Relaxant concentration–response curves of SNP or TERPY in presence TEA in carbachol-contracted trachea. (A) Relaxation SNP in absence (control, n = 5) and presence (20 min) of the TEA (5 mM, n = 8). (B) Relaxation TERPY in absence (control, n = 7) and presence (20 min) of the TEA (5 mM, n = 11). (C) Relaxant concentration–response curves of SNP in carbachol- and KCl (75 mM)-contracted trachea. (D) Relaxant concentration–response curves of TERPY in carbachol- and KCl (75 mM)-contracted trachea. Each point represents the mean ± SEM and is expressed as percentage of relaxation. Statistic ⁄p < 0.05, ⁄⁄p < 0.01, ⁄⁄⁄p < 0.001.
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Fig. 7. Hematoxylin- and eosin-stained histological (400) sections of rat trachea displayed before (A) and after (B) removal of the epithelial layer. E, epithelium; SM, smooth muscle. (C) Graphic in bar of maximal response (Emax) represent the mean ± SEM and are expressed as percentage of relaxation of SNP with (SNP E+, n = 5) and without (SNP E, n = 4) epithelium or TERPY with (TERPY E+, n = 7) and without (TERPY E, n = 4) epithelium in the carbachol-contracted rat trachea. Statistic ⁄⁄⁄p < 0.001. Fig. 5. Relaxant concentration–response curves of SNP or TERPY in presence thapsigargin in the carbachol-contracted rat trachea. Each point represents the mean ± SEM and is expressed as percentage of relaxation. (A) Relaxation SNP in absence (control, n = 5) and presence (20 min) of the thapsigargin (1 lM, n = 5). (B) Relaxation TERPY in absence (control, n = 7) and presence (20 min) of the thapsigargin (1 lM, n = 6).
The Emax induced by TERPY in trachea E+ was 97.17 ± 2.82%, and for trachea E 96.62 ± 2.16%, no significant difference was observed (Fig. 7C). These results suggest that the epithelium is important in mechanism of the relaxation induced by SNP. 3.6. Ca2+ measurements Using the confocal microscope, we examined the effect of SNP and TERPY (100 lM) on cytosolic Ca2+ concentration in rat tracheal smooth muscle, pre-loaded with Fluo 3-AM. Fig. 8 shows decrease in the fluorescence of Fluo 3 after the addition of TERPY or SNP. The decrease in cytosolic Ca2+ concentration in response to TERPY is sharper than SNP and this 61.7% reduction is significant (p < 0.05). The percentage of reduction in fluorescence intensity (% DFI) caused by SNP was 18.06 ± 3.09, n = 4 and the by the TERPY was 47.24 ± 9.97, n = 4. 4. Discussion
Fig. 6. Concentration–response curves to extracellular calcium (CaCl2) in the absence (control, n = 5) and presence (20 min) of the 100 lM of SNP (n = 5) or TERPY (n = 4) in rat tracheal preparations stimulated by carbachol. Each point represents the mean ± SEM and is expressed as maximal change from the contraction produced by KCl (75 mM), which was taken as 100%. Statistic ⁄⁄⁄ p < 0.001 between control and treated with SNP (100 lM) or TERPY (100 lM) and between these.
transverse histological section of the trachea of rats. Noteworthy was the presence of epithelium (E) and smooth muscle (SM) (Fig. 7A) and absence of epithelium in Fig. 7B. Thus we could confirm the absence of epithelium in the appropriate experiments. Tracheal rings with epithelium (E+) was pre-contracted by carbachol (0.5 lM), the response maximal induced by SNP was 55.14 ± 5.5% (Fig. 5C.) and for trachea without epithelium (E) 30.98 ± 3.81%. This reduction of 43.8% in the maximum response is significant (p < 0.001).
Nitric oxide is an important factor in the regulation of several physiological and pathological processes including airway smooth muscle tone. Nitroglycerin and SNP have been used as pharmacological tools to study the NO effects. In the respiratory system, NO-donors are known for many years to induce bronchial relaxation. Further more, the administration of exogenous nitric oxide is known to attenuate the response to bronchoconstricting agents in both laboratory animals and humans (Janssen et al., 2000; Elmedal et al., 2005; Toque et al., 2010). In this study, the effect of the new NO-donor TERPY is demonstrated to decreases intracellular Ca2+ concentration and dilates the tracheal smooth muscle. The most interesting findings in this study are that this ruthenium complex shown higher efficacy than SNP. Moreover, the mechanism by which TERPY induces airway relaxation is different from SNP, and seems to act on Ca2+ and K+ currents, leading to decreasing in cytosolic Ca2+ concentration.
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Fig. 8. Decrease of cytosolic Ca2+ concentration ([Ca2+]c) induced by TERPY and SNP (100 lM) in rat tracheal smooth muscle. A serial Ca2+ images of Fluo 3-AM fluorescence in slices of rat trachea were recorded at t = 0 (before addition of NO-donor), 90 and 180 s after addition of TERPY (A, B, C, respectively) or SNP (D, E, F, respectively). (G) % DFI indicates the average of % [Ca2+]c reduction after 180 s. Statistic ⁄p < 0.05.
Since the discovery that NO containing substances, such as SNP induce relaxation of the isolated airways, the ability of exogenous NO to provoke a bronchodilator effect in animals and man has been studied. In the present study, SNP relaxed contracted rat trachea in a concentration-dependent way, however, in a lesser proportion than the new NO-donor TERPY, which induced full relaxation. Our results with SNP corroborates with previous study in which this drug only induced a half-relaxation in airways (Corompt et al., 1998; Hjoberg et al., 1999; Vaali et al., 2000; Toque et al., 2010). On the other hand, our experiments have shown that TERPY induce 100% relaxation in pre-contracted rat trachea, and this can be a major advantage in relation to the other NO-correlated drugs. Although their mechanism of action is controversial, it is believed that NO (which in the airway is produced largely by epithelial cells) activates sGC and increases intracellular cGMP concentration, leading to relaxation of airway smooth muscles (Belik et al., 2007; Laursen et al., 2006; Toque et al., 2010). In fact, our results obtained with SNP corroborates with this authors, since that relaxation induced by SNP in rat trachea was decreased after inhibition of sCG by the selective inhibitor ODQ. However, in this tissue, the role of cGMP in mediating this relaxation is not clear and not all studies support the role of sGC/cGMP pathway in mediating the relaxant effects stimulated by NO, suggesting participation of other cellular pathways. In canine airway, both
relaxation and the increase in intracellular cGMP induced by NOdonors are inhibited by the putative sGC inhibitor methylene blue (Zhou and Torphy, 1991; Jones et al., 1994). In contrast, relaxation of human bronchi is unaffected by methylene blue, although increases in intracellular cGMP are attenuated (Gaston et al., 1994). Stuart-Smith et al. (1994), have shown that the relaxation induced by three different NO-donors in tracheal preparations may occur without a concomitant increase in cGMP, and whereas relaxation induced by some NO-donors may increase intracellular cGMP, the relaxation is not completely dependent upon it, since that sGC inhibition did not alter the relaxation. Our data suggest that the relaxation produced by TERPY do not involves sCG/cGMP pathway, since that ODQ did not modify the response, unlike the standard drug SNP. Several types of K+ channels have been identified in plasma membrane of airway smooth muscle. Opening of K+ channels will cause plasma membrane hyperpolarization by increasing K+ conductance (Standen and Quayle, 1998). Membrane hyperpolarization would reduce cell excitability, thereby promoting smooth muscle relaxation (Quast, 1993; Malerba et al., 2010). To investigate whether K+ channels are also involved in the trachea relaxation stimulated by SNP and TERPY, the preparation was pretreated with TEA, a non-selective K+ channel blocker. TEA was able to reduce significantly the relaxant effect of TERPY. Thus, K+
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channels appear to be a molecular target for the relaxant activity of TERPY. Moreover, the relaxant effect of TERPY was significantly inhibited in the muscle pre-contracted by KCl 75 mM, which indirectly shows the dependence of K+ channels in the relaxant response of TERPY. Differently from this, pretreatment with TEA augmented the relaxant effect of SNP; and the pre-contraction with KCl 75 mM did not alter the relaxant response to SNP, which indirectly shows the independence of K+ channels to the effects of SNP. Take together, these results suggest that the relaxant effect of the TERPY is in part mediated via K+ channels. Contrary to this, these channels do not seem to be important for SNP-induced relaxation. Is difficult explain (and is not the objective of this study) the unexpected effect of TEA on SNP-induced relaxation. TEA is also a know activator of gap junctions communications in smooth muscle cells, including trachea (Kannan and Daniel, 1978; Watts et al., 1994). Direct cell-to-cell communication along the muscular tissue is governed by gap junctions, enabling the production of coordinated multicellular responses (Christ, 1995; Rocha et al., 2008). Some studies also have shown that NO, through the cGMP/PKG pathway, regulates and further increases gap junctions communications (Lampe and Lau, 2004; Gönczi et al., 2009). Since gap junctions mediates the spread of ions and second messengers, such as cGMP, from one cell to another (Kumar and Gilula, 1996; Kanaporis et al., 2008), the increase in gap junction coupling should enhance the diffusion potential, leading to direct activation of a greater fraction of smooth muscle cells in the preparation (Kenakin, 1980; Christ, 1995). Christ et al. (1994) have proposed that NO-induced dilation in peripheral tissues is dependent on the cell-to-cell diffusion of cGMP through the gap junction. The effect of TEA potentiating NO-induced relaxation did not was seen to TERPY, once for TERPY, the cGMP/PKG pathway is not being recruited, unlike to SNP, which appears to activate the cGMP pathway. Although we think these hypotheses are reasonable, we cannot state here the real mechanism by which TEA increases the relaxant effect of SNP. Previously, it has been reported that calcium uptake into intracellular stores by SERCA is normally involved in the smooth muscle relaxation induced by NO. In the current study, we have shown that thapsigargin did not modify the relaxant response to both the SNP and for TERPY, suggesting that the relaxation to both NO-donors does not rely on SERCA. These findings are in agreement to previous results indicating that, although Ca2+ pumping back by SERCA seems to be active, it is not involved in intracellular Ca2+ decrease in airway myocytes (Roux and Marhl, 2004). In biological systems, the main mechanism for NO release from SNP is enzyme-dependent (Harrison and Bates, 1993). A membrane-bound enzyme may be involved in the generation of NO from SNP in biological tissues, and either NADH or NADPH appears to be required as a cofactor (Bates et al., 1991; Kowaluk et al., 1992). The same do not occur for NO-related ruthenium compounds. For those complexes, the NO release has been achieved by chemical or electrochemical reduction (Toledo et al., 2002; Sauaia et al., 2005). In this context, Bonaventura et al. (2008) verified in rat aortas that the relaxation induced by SNP is potentiated by the presence of vascular endothelium. The modulation of the endothelium on the vasorelaxation evoked by SNP is due to the activation of constitutive enzyme systems present in endothelial cells, particularly NO-synthase, which in turn could culminate in the generation of endogenous NO (Bonaventura et al., 2008). Moreover, human airway epithelium expresses endothelial NO-synthase (Shaul et al., 1993) and inducible NO-synthase, which continuously synthesizes NO in vivo (Guo et al., 1995). Therefore, in this study we have analyzed the epithelial modulation on the relaxant responses to NO-donors SNP and TERPY in isolated rat trachea. The results confirmed that epithelial cells modulate SNP-induced relaxation, since the relaxation induced by SNP after epithelium
removal was lesser to that observed in the presence of epithelial cells, similarly as observed in vascular bed (Bonaventura et al., 2008). However, the relaxation induced by TERPY was not affected by the epithelial layer removal, maybe because it does not need be metabolized to release NO (Toledo et al., 2002; Sauaia et al., 2005). In airway smooth muscle, extracellular Ca2+ influx is a very important determinant of intracellular Ca2+ elevation during the contraction phase and can to be stimulated by muscarinic agonist (Du et al., 2005; Tazzeo et al., 2008). Several different mechanisms for NO-induced inhibition of Ca2+ influx through of the plasma membrane have been proposed, including their inhibition by cGMP-dependent mechanisms (Blatter and Wier, 1994) or by membrane hyperpolarization due to direct (Bolotina et al., 1994) or indirect cGMP-dependent activation of K+ channels (Robertson et al., 1993; Ay et al., 2006). Our experiments also revealed the importance of extracellular Ca2+ influx inhibition induced by NO. We found that the both SNP and TERPY are able to inhibit the contraction evoked by extracellular Ca2+ influx stimulated by agonist, which can contribute to relaxant effect of these drugs. Moreover, TERPY was more effective in promoting this inhibition than SNP. Based on these results, we can suggest that TERPY mediates the relaxation by mechanisms that alter the mobilization or sensitivity to extracellular Ca2+. In confocal microscope, membrane permeable Fluo 3-AM readily enters the cells and is subsequently hydrolyzed by cytosolic esterases releasing free Fluo 3, which does not leak into the medium. At physiological pH, Fluo 3 is relatively non-fluorescent; however, in the presence of Ca2+, it forms a fluorescent product. This approach allows the direct visualization and semi-quantitative analysis of the basal Ca2+ availability at the cell level. The decrease in cytosolic Ca2+ concentrations in response to TERPY is sharper than SNP, these results support the tracheal reactivity study shows in Fig. 2. In summary, the new NO-donor TERPY is more effective in reducing cytosolic Ca2+ concentration and inducing relaxation in rat trachea than the SNP. Different of SNP, NO released from the ruthenium complex, induces airway smooth muscle relaxation by cGMP-independent mechanisms, appears to involve Ca2+ and K+ fluxes across the membrane, leading to decreased cytosolic Ca2+ concentrations. Conflict of interest None declare. Acknowledgments This study was supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES). References Arnold, W.P., Longnecker, D.E., Epstein, R.M., 1984. Photodegradation of sodium nitroprusside: biologic activity and cyanide release. Anesthesiology 61, 254– 260. Arnold, W.P., Mittal, C.K., Katsuki, S., Murad, F., 1977. Nitric oxide activates guanylate cyclase and increases guanosine 30 :50 -cyclic monophosphate levels in various tissue preparations. Proc. Natl. Acad. Sci. USA 74 (8), 3203–3207. Ay, B., Iyanoye, A., Sieck, G.C., Prakash, Y.S., Pabelick, C.M., 2006. Cyclic nucleotide regulation of store-operated Ca2+ influx in airway smooth muscle. Am. J. Physiol.: Lung Cell. Mol. Physiol. 290, L278–L283. Barnes, P.J., Belvisi, M.G., 1993. Nitric oxide and lung disease. Thorax 48, 1034– 1043. Bates, J.N., Baker, M.T., Guerra, R.J.R., Harrison, D.G., 1991. Nitric oxide generation from nitroprusside by vascular tissue: evidence that reduction of the nitroprusside anion and cyanide loss are required. Biochem. Pharmacol. 42, S157–S165. Belik, J., Hehne, N., Pan, J., Behrends, S., 2007. Soluble guanylate cyclase-dependent relaxation is reduced in the adult rat bronchial smooth muscle. Am. J. Physiol. 292 (3), L699–L703.
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European Journal of Pharmaceutical Sciences journal homepage: www.elsevier.com/locate/ejps
A new nitrosyl ruthenium complex nitric oxide donor presents higher efficacy than sodium nitroprusside on relaxation of airway smooth muscle Patrícia F.S. Castro a, Amanda de C. Pereira b, Gerson J. Rogrigues b, Aline C. Batista c, Roberto S. da Silva b, Lusiane M. Bendhack b, Matheus L. Rocha a,⇑ a b c
Faculty of Pharmacy, Federal University of Goias, Av. Universitária s/n, 74605-220 Goiânia, GO, Brazil Faculty of Pharmaceutical Sciences, University of São Paulo, Av. do Café s/n, 14040-903 Ribeirão Preto, SP, Brazil Faculty of Odontology, Federal University of Goias, Av. Universitária s/n, 74605-220 Goiânia, GO, Brazil
a r t i c l e
i n f o
Article history: Received 17 February 2011 Received in revised form 2 May 2011 Accepted 8 May 2011 Available online 14 May 2011 Keywords: Nitric oxide Airway smooth muscle Guanylyl cyclase K+ channels Trachea
a b s t r a c t Nitric oxide (NO) has been demonstrated to be the primary agent in relaxing airways in humans and animals. We investigated the mechanisms involved in the relaxation induced by NO-donors, ruthenium complex [Ru(terpy)(bdq)NO+]3+ (TERPY) and sodium nitroprusside (SNP) in isolated trachea of rats contracted with carbachol in an isolated organs chamber. For instance, we verified the contribution of K+ channels, the importance of sGC/cGMP pathway, the influence of the extra and intracellular Ca2+ sources and the contribution of the epithelium on the relaxing response. Additionally, we have used confocal microscopy in order to analyze the action of the NO-donors on cytosolic Ca2+ concentration. The results demonstrated that both compounds led to the relaxation of trachea in a dependent-concentration way. However, the maximum effect (Emax) of TERPY is higher than the SNP. The relaxation induced by SNP (but not TERPY) was significantly reduced by pretreatment with ODQ (sGC inhibitor). Only TERPYinduced relaxation was reduced by tetraethylammonium (K+ channels blocker) and by pre-contraction with 75 mM KCl (membrane depolarization). The response to both NO-donors was not altered by the presence of thapsigargin (sarcoplasmic reticulum Ca2+-ATPase inhibitor). The epithelium removal has reduced the relaxation only to SNP, and it has no effect on TERPY. The both NO-donors reduced the contraction evoked by Ca2+ influx, while TERPY have shown a higher inhibitory effect on contraction. Moreover, the TERPY was more effective than SNP in reducing the cytosolic Ca2+ concentration measured by confocal microscopy. In conclusion, these results show that TERPY induces airway smooth muscle relaxation by cGMP-independent mechanisms, it involves the fluxes of Ca2+ and K+ across the membrane, it is more effective in reducing cytosolic Ca2+ concentration and inducing relaxation in the rat trachea than the standard drug, SNP. Ó 2011 Elsevier B.V. All rights reserved.
1. Introduction Airway smooth muscle tone is maintained by equilibrium between bronchoconstriction and bronchodilation. The aim of clinical treatment for pathological conditions, such as allergy, asthma and inflammation is to avoid excessive constriction, and to reestablish a balance by producing dilation. In this context, nitric oxide (NO) has been demonstrated to be the primary agent in relaxing airways in humans and animals (Ellis, 1997; Ellis and Undem, 1992; Gaston et al., 1993). NO is released in the epithelial cells of the airways in basal conditions causing relaxation in vivo and in vitro, through its action on the smooth muscle (Arnold et al., 1984). This relaxation is
⇑ Corresponding author. Address: UFG – Faculdade de Farmácia, Av. Universitária com 1ª Avenida s/n, Setor Universitário CEP, 74605-220 Goiânia, GO, Brazil. Tel.: +55 62 3209 6440; fax: +55 62 3209 6037. E-mail address: [email protected] (M.L. Rocha). 0928-0987/$ - see front matter Ó 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.ejps.2011.05.004
mediated by endogenous NO produced mainly by epithelial cells and nerves, which diffuses to the underlying smooth muscle (Barnes and Belvisi, 1993). Thus, NO-donors may be useful pharmacological tools to induce relaxing response in airways. Studies in bronchial and tracheal smooth muscle have shown that a major target of NO is the enzyme soluble guanylyl cyclase (sGC) (Ellis, 1997; Hwang et al., 1998). The activation of sGC by NO increases intracellular levels of cGMP which, in turn, amplifies the cellular response (Arnold et al., 1977). The main target of cGMP is the family of cGMP-dependent protein kinases (PKG). Activation of PKG and subsequent phosphorylation of various proteins constitute a cascade of reactions that lead to the reduction of cytosolic Ca2+ concentration, leading to relaxation (McDaniel et al., 1992). However, not all studies support the role of sGC/cGMP pathway in mediating the relaxant effect of NO, and cGMP-independent pathways are increasingly being recognized (Redington, 2006; Stuart-Smith et al., 1994). The controversial results of these studies may be due to different species and NO-donors used.
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system and WinDaq Resource (DATAQ Instruments, Akron, OH, USA, data acquisition unit) to measure tension in the preparations. The tracheal rings were placed in a 10 ml organ chamber containing Krebs solution with the following composition (mM): NaCl 130, KCl 4.7, KH2PO4 1.2, MgSO4 1.2, NaHCO3 14.9, glucose 5.5, CaCl2 1.6. The solution was maintained at pH 7.4, and gassed with 95% O2 and 5% CO2 at 37 °C. The rings were initially stretched to a basal tension of 1.0 g (optimal basal tone, previously determined by length–tension relationship experiments) before allowing them to equilibrate in the bathing medium. 2.2. Experimental protocols
Fig. 1. Struture of [Ru(terpy)(bdq)NO+]3+.
The main prototypes of NO-donors traditionally used, such as sodium nitroprusside (SNP) and nitroglycerine, have severe wellknown adverse effects, such as sharp tachycardia, high toxicity and induce rapid tolerance (Yakazu et al., 2001). In order to avoid these unwanted effects, some metallonitrosyl complexes have been used as NO deliver agents. NO coupled to nitrosyl ruthenium complexes is attractive because of its thermal stability, stability in physiological pH and is practically nontoxic (de Lima et al., 2006; Oliveira et al., 2007; Sauaia et al., 2003; Wang et al., 2000). Studies have shown that these compounds exert a NO type biological activity, such as relaxation of vascular smooth muscle, inhibition of platelet aggregation and increasing levels of cGMP (Ignarro et al., 1980; Kowaluk and Fung, 1990; Radomski et al., 1992; Rotta et al., 2003). The Nitrosyl–terpyridine–phenylene–diamine ruthenium (II) ion complex [Ru(terpy)(bdq)NO+]3+ (TERPY, Fig. 1) is a NO-donor that induces relaxation in arteries of rats by acting on both K+ channels and sGC–cGMP pathway (Bonaventura et al., 2007). An increasing knowledge of the involvement of NO in numerous physiological pathways has not only expanded new therapeutic avenues for NO-related compounds but also led to an increased use of such compounds in pharmacological studies. Therefore, this study was dedicated to investigate the effects of a ruthenium complex NO-donor in tracheal smooth muscle of rats and to evaluate the advantages of its relaxing properties (in vitro) when compared with SNP. 2. Methods and materials 2.1. Isolated tracheal preparation Male Wistar rats were anaesthetized and killed by aortic exsanguinations. All the procedures were carried out in accordance with the Animal Research Ethical Committee of the Federal University of Goiás, Goiânia, Goiás, Brazil. The trachea was quickly removed, dissected, cleaned of adhering fat and connective tissue, and cut into 4-mm-long rings. The tracheal rings were placed between two stainless-steel stirrups and connected to an isometric force transducer. The responses were recorded using a computerized
2.2.1. Isometric tension recording in tracheal rings After 60 min of equilibration, each tracheal ring was exposed to KCl 75 mM to attain its maximum contractility. Each ring was sequentially washed and re-equilibrated and was allowed to relax to baseline. After 30 min, cumulative concentration–response curves for SNP (10 nM to 100 lM) and TERPY (10 nM to 100 lM) were carried out in preparations pre-contracted with cholinergic agonist carbachol (0.5 lM, EC50 previously determined in our laboratory) or after contraction induced by membrane depolarization with KCl 75 mM. To investigate some mechanism(s) responsible for SNP- or TERPY-induced relaxation, tracheal rings were contracted with carbachol (0.5 lM) 30 min after being incubated with one the following drugs: guanylyl cyclase inhibitor 1H-[1,2,4] oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, 1 lM), non-selective K+ channel blockers tetraethylammonium (TEA, 5 mM), a specific sarcoplasmic reticulum Ca2+-ATPase (SERCA) inhibitor (Thapsigargin, 1 lM). 2.2.2. Effect of TERPY and SNP on Ca2+ influx To further analyze the relative contribution of the TERPY or SNP in inhibit the contraction evoked by Ca2+ influx, the role of extracellular Ca2+ mobilization stimulated by contractile agonist was investigated by CaCl2-induced contraction in the presence of carbachol. Tracheal rings were first contracted with carbachol (EC50, 0.5 lM) to deplete the intracellular Ca2+ stores in Ca2+-free solution until the disappearance of any contractile response (approximately 60 min) and then rinsed in Ca2+-free solution containing carbachol (0.5 lM). After that, the cumulative concentration–response curves for CaCl2 (0.0– 1.6 mM) were obtained in the absence (control group) or after a 20 min incubation period with SNP or TERPY (100 lM). 2.3. Influence of the epithelial cells in the relaxation induced by NOdonors In some experiments, the epithelial cells were removed mechanically by rubbing the internal tracheal surface with a fine metallic wire (200 lm in diameter) as described previously (Wu et al., 2004). This procedure allowed investigating the influence of functional epithelium in the relaxation induced by NO-donors. After removal of the epithelium, cumulative concentration-effect curves to SNP or TERPY (10 nM to 100 lM) were performed under precontraction with carbachol (0.5 lM). The removal of the epithelial layer was confirmed by histological examination, with the observation of trachea tissue stained with hematoxylin and eosin by optical microscopy. 2.4. Measurements of intracellular Ca2+ by confocal microscopy and image analysis Calcium levels were detected as previously described (Lunardi et al., 2006; Rodrigues et al., 2008) with some adaptations to the tracheal smooth muscle. Trachea slices (150 lm thick) were placed in a glass coverslip covered with poly-L-lysine (50%) in Hanks
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solution (composition in mM: NaCl 145.0; KCl 5.0; MgSO4; 7H2O 1.0; CaCl2 1.6; NaH2PO4 0.5; glucose 10.0; Hepes 10.0) at pH 7.4 and 37 °C. Then, tracheal slices were loaded with the membranepermeating indicator fluorescent Ca2+ dye Fluo 3-AM (10 lM, Sigma Probes) for 30 min at room temperature. Excess dye was removed by washing out the dye with a bath solution. After washing with Hanks solution, coverslips were placed on a chamber (1.0 ml in volume) and placed in a confocal microscope (Leica TCS SP5). Ca2+-images of the distinct slices of tracheal rings were taken sequentially, in Hanks buffer and assessed through a water immersion objective (63). Fluo 3-AM was excited with the 488 nm line of an argon ion laser, and the emitted fluorescence was measured at 510 nm. Time course software was used to capture images of the rings 4 min after TERPY or SNP (100 lM) addition, at intervals of 2.5 s (xyt mode), 512 512 pixel at 700 Hz. The computer software Leica Microsystem LAS AF Lite, was used to measure the intensities of the intracellular maximum or minimum fluorescence in regions of interest (smooth muscle layer) in the ring. From these data, the initial fluorescence value (F0) was used and the final fluorescence value was denoted as F. The difference in fluorescence intensity (DFI) was obtained for each protocol: % DFI = (F F0)/ F0 100.
The maximal response (Emax) of TERPY (97.17 ± 2.82%) was approximately 43% higher than SNP (55.14 ± 5.5%, p < 0.001). Furthermore, SNP and TERPY have presented different potency (pEC50: 6.07 ± 0.03 and 5.16 ± 0.14, respectively, p < 0.01). 3.2. Participation of sGC and K+ channels on the tracheal relaxation induced by TERPY and SNP As shown in Fig. 3A, the sGC inhibition decreased the potency (p < 0.01) and maximal effect (p < 0.05) of SNP in inducing trachea
2.5. Chemicals The drugs used in this study included carbachol, ODQ, TEA, SNP, thapsigargin, Fluo 3-AM, which were purchased from Sigma Chemical Co. (St. Louis, MO, USA). All other chemicals used in the present study were commercially available and of reagent grade. ODQ and thapsigargin were dissolved in DMSO. The other drugs were dissolved in distilled water. The concentration of DMSO in the organ chamber did not exceed 0.01% and had no effect on tension generation in this preparation. The TERPY complex was synthesized as previously described (de Lima et al., 2006). TERPY synthesis was conducted at the Laboratory of Analytical Chemistry by Dr. Roberto Santana da Silva, Faculty of Pharmaceutical Sciences, University of São Paulo, Brazil.
Fig. 2. Relaxant concentration–response curves of SNP and TERPY in the carbacholcontracted rat trachea. Each point represents the mean ± SEM and is expressed as percentage of relaxation to SNP (n = 5) or TERPY (n = 7). Statistic ⁄⁄⁄p < 0.001.
2.6. Statistical analysis In the graphics, the data are presented as means ± SEM. In each set of experiments, n indicates the number of rats studied. The values for reactivity and responses to SNP or TERPY are expressed as percentage of the preceding contraction. The concentration of the compound producing a half-maximal response (EC50) was determined after logarithmic transformation of the normalized concentration– response curves and is reported as the negative logarithm (log EC50 = pEC50 values) of the mean of individual values for each tissue, using GraphPad Prism version 5.0 (GraphPad Software Inc., San Diego, CA). The maximum relaxant effect (Emax) was considered as the maximal amplitude response reached in concentration–effect curves for relaxant agents. Statistical analysis was performed using the GraphPad Prism version 5.0 (GraphPad Software Corporation, San Diego, CA). Comparisons among groups were performed using one-way ANOVA (post-test: Newman–Keuls) and Student’s t test, and values of p < 0.05 were considered to be significant. 3. Results 3.1. Relaxant effect of TERPY and SNP in the rat tracheal smooth muscle TERPY or SNP (10 nM to 100 lM) added cumulatively to the bath solution during the sustained contraction induced by carbachol (0.5 lM) have evoked concentration-dependent relaxation (Fig. 2).
Fig. 3. Relaxant concentration–response curves of SNP or TERPY in presence ODQ in carbachol-contracted rat trachea. (A) Relaxation SNP in absence (control, n = 5) and presence (20 min) of the ODQ (1 lM, n = 7). (B) Relaxation TERPY in absence (control, n = 7) and presence (20 min) of the ODQ (1 lM, n = 5). Each point represents the mean ± SEM and is expressed as percentage of relaxation. Statistic ⁄ p < 0.05.
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relaxation (Emax 35.0 ± 7.7% and pEC50 5.19 ± 0.21), compared to control (Emax 55.14 ± 5.5% and pEC50 6.07 ± 0.03). To TERPY (Fig. 3B), no differences were observed in the maximum effect and potency in the presence of ODQ when compared to the control group (Emax 98.7 ± 1.26%, pEC50 5.22 ± 0.11). These results suggest that the pathway sGC/cGMP is involved only on relaxation effect induced by SNP. Conversely, as showed in Fig. 4A and B, the K+ channels blocker TEA altered the Emax for SNP (from 55.14 ± 5.5% to 82.4 ± 5.8%), leading to an increase of about 49% (p < 0.05) and for TERPY (from 97.17 ± 2.82% to 66.14 ± 7.7%), a decrease of about 32% (p < 0.01) compared with the controls groups. In the presence of TEA no differences were observed in the potency compared with the control group (NPS pEC50: 6.86 ± 0.39; TERPY pEC50: 4.30 ± 0.40). In preparations contracted with 75 mM KCl, a concentration that induces depolarization of the cell membrane, no difference was observed in the relaxation induced by SNP when compared with contraction with EC50 carbachol (Fig. 4C, Emax 61.19 ± 6.03%, pEC50 6.24 ± 0.08, n = 5). On the other hand, the relaxation induced by TERPY was significantly reduced by 49% (Fig. 4D, p < 0.001). Emax was reduced from 97.17 ± 2.82% to 49.54 ± 6.97%, n = 5 and pEC50 from 5.16 ± 0.14 to 4.94 ± 0.16, n = 5. These results suggest that the K+ channels play an important role in the relaxation induced by TERPY.
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45.4 ± 3.94, pEC50 6.05 ± 0.11) or TERPY (Fig. 5B, Emax 99.4 ± 2.97%, pEC50 5.34 ± 0.17) compared with the controls groups. The results with thapsigargin suggest that calcium stored in sarcoplasmic reticulum does not have significant importance for relaxing effect both SNP and TERPY in tracheal smooth muscle. 3.4. Effect of TERPY and SNP on CaCl2-induced contractions Tracheal ring was used to investigate contractile responses dependent on extracellular Ca2+ influx (Fig. 6). Cumulative additions of CaCl2 produce concentration-dependent tracheal smooth muscle contractions in the presence carbachol. Contraction responses are expressed as a percentage of the response to KCl 75 mM. The group incubated with SNP (100 lM for 20 min) significant decreased (p < 0.001) contraction in 34.8% compared with the control. Emax of group incubated with SNP was 76.87 ± 9.59% and control group was 117.9 ± 5.98%. The group incubated with TERPY (100 lM for 20 min) also significantly reduced the contraction (p < 0.001) in 74.7% compared with the control. Emax of group incubated with TERPY was 27.42 ± 4.08% and control group was 117.9 ± 5.98%. Comparing the incubation to TERPY with SNP, they are different in 64% (p < 0.001) (Fig. 6). These results suggest that the inhibition of Ca2+ influx is an important mechanism to bring about relaxation the tracheal smooth muscle for the two NO-donors.
3.3. Effects of the SERCA inhibitor on the relaxation induced by TERPY and SNP
3.5. Role of epithelium in relaxation rat tracheal smooth muscle
Our data showed that thapsigargin (1 lM, SERCA inhibitor), did not alter the relaxation and potency induced by SNP (Fig. 5A, Emax
Fig. 7 shows rat trachea with and without epithelium and its effect on the relaxation of smooth muscle. Fig. 7A and B is
Fig. 4. Relaxant concentration–response curves of SNP or TERPY in presence TEA in carbachol-contracted trachea. (A) Relaxation SNP in absence (control, n = 5) and presence (20 min) of the TEA (5 mM, n = 8). (B) Relaxation TERPY in absence (control, n = 7) and presence (20 min) of the TEA (5 mM, n = 11). (C) Relaxant concentration–response curves of SNP in carbachol- and KCl (75 mM)-contracted trachea. (D) Relaxant concentration–response curves of TERPY in carbachol- and KCl (75 mM)-contracted trachea. Each point represents the mean ± SEM and is expressed as percentage of relaxation. Statistic ⁄p < 0.05, ⁄⁄p < 0.01, ⁄⁄⁄p < 0.001.
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Fig. 7. Hematoxylin- and eosin-stained histological (400) sections of rat trachea displayed before (A) and after (B) removal of the epithelial layer. E, epithelium; SM, smooth muscle. (C) Graphic in bar of maximal response (Emax) represent the mean ± SEM and are expressed as percentage of relaxation of SNP with (SNP E+, n = 5) and without (SNP E, n = 4) epithelium or TERPY with (TERPY E+, n = 7) and without (TERPY E, n = 4) epithelium in the carbachol-contracted rat trachea. Statistic ⁄⁄⁄p < 0.001. Fig. 5. Relaxant concentration–response curves of SNP or TERPY in presence thapsigargin in the carbachol-contracted rat trachea. Each point represents the mean ± SEM and is expressed as percentage of relaxation. (A) Relaxation SNP in absence (control, n = 5) and presence (20 min) of the thapsigargin (1 lM, n = 5). (B) Relaxation TERPY in absence (control, n = 7) and presence (20 min) of the thapsigargin (1 lM, n = 6).
The Emax induced by TERPY in trachea E+ was 97.17 ± 2.82%, and for trachea E 96.62 ± 2.16%, no significant difference was observed (Fig. 7C). These results suggest that the epithelium is important in mechanism of the relaxation induced by SNP. 3.6. Ca2+ measurements Using the confocal microscope, we examined the effect of SNP and TERPY (100 lM) on cytosolic Ca2+ concentration in rat tracheal smooth muscle, pre-loaded with Fluo 3-AM. Fig. 8 shows decrease in the fluorescence of Fluo 3 after the addition of TERPY or SNP. The decrease in cytosolic Ca2+ concentration in response to TERPY is sharper than SNP and this 61.7% reduction is significant (p < 0.05). The percentage of reduction in fluorescence intensity (% DFI) caused by SNP was 18.06 ± 3.09, n = 4 and the by the TERPY was 47.24 ± 9.97, n = 4. 4. Discussion
Fig. 6. Concentration–response curves to extracellular calcium (CaCl2) in the absence (control, n = 5) and presence (20 min) of the 100 lM of SNP (n = 5) or TERPY (n = 4) in rat tracheal preparations stimulated by carbachol. Each point represents the mean ± SEM and is expressed as maximal change from the contraction produced by KCl (75 mM), which was taken as 100%. Statistic ⁄⁄⁄ p < 0.001 between control and treated with SNP (100 lM) or TERPY (100 lM) and between these.
transverse histological section of the trachea of rats. Noteworthy was the presence of epithelium (E) and smooth muscle (SM) (Fig. 7A) and absence of epithelium in Fig. 7B. Thus we could confirm the absence of epithelium in the appropriate experiments. Tracheal rings with epithelium (E+) was pre-contracted by carbachol (0.5 lM), the response maximal induced by SNP was 55.14 ± 5.5% (Fig. 5C.) and for trachea without epithelium (E) 30.98 ± 3.81%. This reduction of 43.8% in the maximum response is significant (p < 0.001).
Nitric oxide is an important factor in the regulation of several physiological and pathological processes including airway smooth muscle tone. Nitroglycerin and SNP have been used as pharmacological tools to study the NO effects. In the respiratory system, NO-donors are known for many years to induce bronchial relaxation. Further more, the administration of exogenous nitric oxide is known to attenuate the response to bronchoconstricting agents in both laboratory animals and humans (Janssen et al., 2000; Elmedal et al., 2005; Toque et al., 2010). In this study, the effect of the new NO-donor TERPY is demonstrated to decreases intracellular Ca2+ concentration and dilates the tracheal smooth muscle. The most interesting findings in this study are that this ruthenium complex shown higher efficacy than SNP. Moreover, the mechanism by which TERPY induces airway relaxation is different from SNP, and seems to act on Ca2+ and K+ currents, leading to decreasing in cytosolic Ca2+ concentration.
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Fig. 8. Decrease of cytosolic Ca2+ concentration ([Ca2+]c) induced by TERPY and SNP (100 lM) in rat tracheal smooth muscle. A serial Ca2+ images of Fluo 3-AM fluorescence in slices of rat trachea were recorded at t = 0 (before addition of NO-donor), 90 and 180 s after addition of TERPY (A, B, C, respectively) or SNP (D, E, F, respectively). (G) % DFI indicates the average of % [Ca2+]c reduction after 180 s. Statistic ⁄p < 0.05.
Since the discovery that NO containing substances, such as SNP induce relaxation of the isolated airways, the ability of exogenous NO to provoke a bronchodilator effect in animals and man has been studied. In the present study, SNP relaxed contracted rat trachea in a concentration-dependent way, however, in a lesser proportion than the new NO-donor TERPY, which induced full relaxation. Our results with SNP corroborates with previous study in which this drug only induced a half-relaxation in airways (Corompt et al., 1998; Hjoberg et al., 1999; Vaali et al., 2000; Toque et al., 2010). On the other hand, our experiments have shown that TERPY induce 100% relaxation in pre-contracted rat trachea, and this can be a major advantage in relation to the other NO-correlated drugs. Although their mechanism of action is controversial, it is believed that NO (which in the airway is produced largely by epithelial cells) activates sGC and increases intracellular cGMP concentration, leading to relaxation of airway smooth muscles (Belik et al., 2007; Laursen et al., 2006; Toque et al., 2010). In fact, our results obtained with SNP corroborates with this authors, since that relaxation induced by SNP in rat trachea was decreased after inhibition of sCG by the selective inhibitor ODQ. However, in this tissue, the role of cGMP in mediating this relaxation is not clear and not all studies support the role of sGC/cGMP pathway in mediating the relaxant effects stimulated by NO, suggesting participation of other cellular pathways. In canine airway, both
relaxation and the increase in intracellular cGMP induced by NOdonors are inhibited by the putative sGC inhibitor methylene blue (Zhou and Torphy, 1991; Jones et al., 1994). In contrast, relaxation of human bronchi is unaffected by methylene blue, although increases in intracellular cGMP are attenuated (Gaston et al., 1994). Stuart-Smith et al. (1994), have shown that the relaxation induced by three different NO-donors in tracheal preparations may occur without a concomitant increase in cGMP, and whereas relaxation induced by some NO-donors may increase intracellular cGMP, the relaxation is not completely dependent upon it, since that sGC inhibition did not alter the relaxation. Our data suggest that the relaxation produced by TERPY do not involves sCG/cGMP pathway, since that ODQ did not modify the response, unlike the standard drug SNP. Several types of K+ channels have been identified in plasma membrane of airway smooth muscle. Opening of K+ channels will cause plasma membrane hyperpolarization by increasing K+ conductance (Standen and Quayle, 1998). Membrane hyperpolarization would reduce cell excitability, thereby promoting smooth muscle relaxation (Quast, 1993; Malerba et al., 2010). To investigate whether K+ channels are also involved in the trachea relaxation stimulated by SNP and TERPY, the preparation was pretreated with TEA, a non-selective K+ channel blocker. TEA was able to reduce significantly the relaxant effect of TERPY. Thus, K+
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channels appear to be a molecular target for the relaxant activity of TERPY. Moreover, the relaxant effect of TERPY was significantly inhibited in the muscle pre-contracted by KCl 75 mM, which indirectly shows the dependence of K+ channels in the relaxant response of TERPY. Differently from this, pretreatment with TEA augmented the relaxant effect of SNP; and the pre-contraction with KCl 75 mM did not alter the relaxant response to SNP, which indirectly shows the independence of K+ channels to the effects of SNP. Take together, these results suggest that the relaxant effect of the TERPY is in part mediated via K+ channels. Contrary to this, these channels do not seem to be important for SNP-induced relaxation. Is difficult explain (and is not the objective of this study) the unexpected effect of TEA on SNP-induced relaxation. TEA is also a know activator of gap junctions communications in smooth muscle cells, including trachea (Kannan and Daniel, 1978; Watts et al., 1994). Direct cell-to-cell communication along the muscular tissue is governed by gap junctions, enabling the production of coordinated multicellular responses (Christ, 1995; Rocha et al., 2008). Some studies also have shown that NO, through the cGMP/PKG pathway, regulates and further increases gap junctions communications (Lampe and Lau, 2004; Gönczi et al., 2009). Since gap junctions mediates the spread of ions and second messengers, such as cGMP, from one cell to another (Kumar and Gilula, 1996; Kanaporis et al., 2008), the increase in gap junction coupling should enhance the diffusion potential, leading to direct activation of a greater fraction of smooth muscle cells in the preparation (Kenakin, 1980; Christ, 1995). Christ et al. (1994) have proposed that NO-induced dilation in peripheral tissues is dependent on the cell-to-cell diffusion of cGMP through the gap junction. The effect of TEA potentiating NO-induced relaxation did not was seen to TERPY, once for TERPY, the cGMP/PKG pathway is not being recruited, unlike to SNP, which appears to activate the cGMP pathway. Although we think these hypotheses are reasonable, we cannot state here the real mechanism by which TEA increases the relaxant effect of SNP. Previously, it has been reported that calcium uptake into intracellular stores by SERCA is normally involved in the smooth muscle relaxation induced by NO. In the current study, we have shown that thapsigargin did not modify the relaxant response to both the SNP and for TERPY, suggesting that the relaxation to both NO-donors does not rely on SERCA. These findings are in agreement to previous results indicating that, although Ca2+ pumping back by SERCA seems to be active, it is not involved in intracellular Ca2+ decrease in airway myocytes (Roux and Marhl, 2004). In biological systems, the main mechanism for NO release from SNP is enzyme-dependent (Harrison and Bates, 1993). A membrane-bound enzyme may be involved in the generation of NO from SNP in biological tissues, and either NADH or NADPH appears to be required as a cofactor (Bates et al., 1991; Kowaluk et al., 1992). The same do not occur for NO-related ruthenium compounds. For those complexes, the NO release has been achieved by chemical or electrochemical reduction (Toledo et al., 2002; Sauaia et al., 2005). In this context, Bonaventura et al. (2008) verified in rat aortas that the relaxation induced by SNP is potentiated by the presence of vascular endothelium. The modulation of the endothelium on the vasorelaxation evoked by SNP is due to the activation of constitutive enzyme systems present in endothelial cells, particularly NO-synthase, which in turn could culminate in the generation of endogenous NO (Bonaventura et al., 2008). Moreover, human airway epithelium expresses endothelial NO-synthase (Shaul et al., 1993) and inducible NO-synthase, which continuously synthesizes NO in vivo (Guo et al., 1995). Therefore, in this study we have analyzed the epithelial modulation on the relaxant responses to NO-donors SNP and TERPY in isolated rat trachea. The results confirmed that epithelial cells modulate SNP-induced relaxation, since the relaxation induced by SNP after epithelium
removal was lesser to that observed in the presence of epithelial cells, similarly as observed in vascular bed (Bonaventura et al., 2008). However, the relaxation induced by TERPY was not affected by the epithelial layer removal, maybe because it does not need be metabolized to release NO (Toledo et al., 2002; Sauaia et al., 2005). In airway smooth muscle, extracellular Ca2+ influx is a very important determinant of intracellular Ca2+ elevation during the contraction phase and can to be stimulated by muscarinic agonist (Du et al., 2005; Tazzeo et al., 2008). Several different mechanisms for NO-induced inhibition of Ca2+ influx through of the plasma membrane have been proposed, including their inhibition by cGMP-dependent mechanisms (Blatter and Wier, 1994) or by membrane hyperpolarization due to direct (Bolotina et al., 1994) or indirect cGMP-dependent activation of K+ channels (Robertson et al., 1993; Ay et al., 2006). Our experiments also revealed the importance of extracellular Ca2+ influx inhibition induced by NO. We found that the both SNP and TERPY are able to inhibit the contraction evoked by extracellular Ca2+ influx stimulated by agonist, which can contribute to relaxant effect of these drugs. Moreover, TERPY was more effective in promoting this inhibition than SNP. Based on these results, we can suggest that TERPY mediates the relaxation by mechanisms that alter the mobilization or sensitivity to extracellular Ca2+. In confocal microscope, membrane permeable Fluo 3-AM readily enters the cells and is subsequently hydrolyzed by cytosolic esterases releasing free Fluo 3, which does not leak into the medium. At physiological pH, Fluo 3 is relatively non-fluorescent; however, in the presence of Ca2+, it forms a fluorescent product. This approach allows the direct visualization and semi-quantitative analysis of the basal Ca2+ availability at the cell level. The decrease in cytosolic Ca2+ concentrations in response to TERPY is sharper than SNP, these results support the tracheal reactivity study shows in Fig. 2. In summary, the new NO-donor TERPY is more effective in reducing cytosolic Ca2+ concentration and inducing relaxation in rat trachea than the SNP. Different of SNP, NO released from the ruthenium complex, induces airway smooth muscle relaxation by cGMP-independent mechanisms, appears to involve Ca2+ and K+ fluxes across the membrane, leading to decreased cytosolic Ca2+ concentrations. Conflict of interest None declare. Acknowledgments This study was supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES). References Arnold, W.P., Longnecker, D.E., Epstein, R.M., 1984. Photodegradation of sodium nitroprusside: biologic activity and cyanide release. Anesthesiology 61, 254– 260. Arnold, W.P., Mittal, C.K., Katsuki, S., Murad, F., 1977. Nitric oxide activates guanylate cyclase and increases guanosine 30 :50 -cyclic monophosphate levels in various tissue preparations. Proc. Natl. Acad. Sci. USA 74 (8), 3203–3207. Ay, B., Iyanoye, A., Sieck, G.C., Prakash, Y.S., Pabelick, C.M., 2006. Cyclic nucleotide regulation of store-operated Ca2+ influx in airway smooth muscle. Am. J. Physiol.: Lung Cell. Mol. Physiol. 290, L278–L283. Barnes, P.J., Belvisi, M.G., 1993. Nitric oxide and lung disease. Thorax 48, 1034– 1043. Bates, J.N., Baker, M.T., Guerra, R.J.R., Harrison, D.G., 1991. Nitric oxide generation from nitroprusside by vascular tissue: evidence that reduction of the nitroprusside anion and cyanide loss are required. Biochem. Pharmacol. 42, S157–S165. Belik, J., Hehne, N., Pan, J., Behrends, S., 2007. Soluble guanylate cyclase-dependent relaxation is reduced in the adult rat bronchial smooth muscle. Am. J. Physiol. 292 (3), L699–L703.
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