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INFLAMMATION, IMMUNOPHARMACOLOGY, AND ASTHMA
Laboratory of Inflammation, Oswaldo Cruz Institute, Oswaldo Cruz Foundation, Rio de Janeiro, Rio de Janeiro, Brazil (L.P.C., M.F.S., A.L.d.A.P., R.S.B.C., P.M.R.e.S., M.A.M.); and Laboratory of Phytochemistry and Medicinal and Chemistry, Department of Pharmacy, Alfenas, Federal University of Alfenas, Minas Gerais, Brazil (M.H.d.S.)
Received for publication
February 13, 2008
Accepted
June 27, 2008.
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Abstract |
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-nitro-L-arginine methyl ester (L-NAME) (100 µM), or soluble guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) (10 µM). 7-Epiclusianone (1–100 µM) induced a dose-dependent increase in the intracellular cGMP levels of cultured tracheal rings. The relaxation effect of 7-epiclusianone was also inhibited by K+ channel blockers tetraethylammonium (10 µM), glibenclamide (1 µM), or apamin (1 µM), but not by 9-(tetrahydro-2'-furyl)adenine (SQ22,536) (100 µM), an adenylate cyclase inhibitor. In epithelium-intact tracheal rings, 7-epiclusianone also inhibited Ca2+-induced contractions in K+ (60 mM)-depolarized preparations, but it seemed ineffective in assays in which epithelium-denuded tracheal ring preparations were used. Oral administration of 7-epiclusinone (25–100 mg/kg) dose-dependently inhibited airway obstruction triggered by aerosolized methacholine (6–25 mg/ml), in a mechanism sensitive to L-NAME (20 mg/kg). In conclusion, the relaxation effect of 7-epiclusinone seems to be mediated by epithelium-, nitric oxide-, and cGMP-dependent mechanisms. Furthermore, oral administration of 7-epiclusianone reduces episodes of bronchial obstruction, warranting further research on this compound regarding a putative application in asthma therapy.
). The premise is that plants contain natural substances with marked health-promoting properties, although overall, limited scientific data supporting this premise are available (Phillipson, 2001).
Garcinia brasiliensis is a native plant of the Amazon region, typically tropical in distribution, and widespread in the Brazilian territory. The fruit of this species has been used in alternative medicine for the treatment of peptic ulcer, urinary, and tumor diseases (Corrêa, 1926). Garcinia species are a rich source of oxygenated and prenylated phenol derivatives (Bennett and Lee, 1988), and some of them have been demonstrated to exhibit antifungal, anti-inflammatory, antitumoral, antioxidant, and antilipidemic properties (Gopalakrishnan et al., 1997; Díaz-Carballo et al., 2003; Hay et al., 2004).
The pharmacological study of polyisoprenylated benzophenones has been shown to be of interest due to the wide spectrum of biological activities they provide and their putative applications in clinical conditions. Garcinol, isolated from the Garcinia indica fruit rind, is so far the best-studied compound of this class. It is a potent inhibitor of histone acetyltransferases and thereby gene expression (Balasubramanyam et al., 2004), being considered an interesting prototype in drug development for challenging diseases, including cancer, atherosclerosis, and human immunodeficiency virus.
Prior studies reveal that 7-epiclusianone, a tetraisoprenylated benzophenone, first isolated from Rheedia gardneriana Miers ex Planch and Triana (Santos et al., 1998), exhibits potent endothelium-dependent vasodilator effects on rat aortic rings (Cruz et al., 2006). We have further demonstrated that 7-epiclusianone, isolated from the fruit pericarp of G. brasiliensis, is also a potent inhibitor of allergen-induced tissue histamine release and ileum contraction triggered by either allergen, histamine, or acetylcholine (Neves et al., 2007), giving support to the interpretation that 7-epiclusianone should be further investigated as a putative lead compound in the context of allergic diseases and asthma therapy.
The present study was conducted to examine the putative mechanism(s) underlying the antispasmodic effect of 7-epiclusianone on isolated guinea pig trachea ring preparations. The potential effect of the oral treatment of 7-epiclusianone in the bronchospasm triggered by aerosolized methacholine in BALB/c mice was also investigated.
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Materials and Methods |
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Drugs. Sodium chloride, potassium chloride, potassium dihydrogen phosphate, sodium hydrogen carbonate, magnesium sulfate heptahydrate, calcium chloride dehydrate, and dimethyl sulfoxide (DMSO) were purchased from Merck (Darmstadt, Germany). Glucose, EGTA, histamine, 5-hydroxytryptamine (5-HT), ovalbumin, carbachol, methacholine, theophylline, N-nitro-L-arginine methyl ester (L-NAME), tetraethylammonium (TEA), glibenclamide, apamin, iberitoxin (IbTX), and 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) were purchased from Sigma-Aldrich (St. Louis, MO). 7-Epiclusianone was isolated from the G. brasiliensis fruit pericarp, as reported previously (Neves et al., 2007) and provided by the Laboratory of Phytochemistry and Medicinal and Chemistry (Department of Pharmacy, Alfenas, Federal University of Alfenas, MG, Brazil). All solutions were freshly prepared in distilled water, DMSO (final concentration, 0.1%), or Tween 80 (final concentration, 0.2%), and they were protected from light.
Isolated Tracheal Preparation and Measurement of Tension. Guinea pigs used for the anaphylactic contraction assay were sensitized previously with a subcutaneous injection of a saline suspension containing 50 µg of ovalbumin and 5 mg of Al(OH)3, in a final volume of 0.2 ml. Animals were killed in a CO2 atmosphere 14 days after sensitization for tracheal removal, and trachea were quickly immersed in Krebs' nutritional solution (118 mM NaCl, 4.8 mM KCl, 2.5 mM CaCl2, 1.2 mM MgSO4, 1.2 mM KH2PO4, 24 mM NaHCO3, and 11 mM glucose). Tracheas were dissected free of adhering fat and connective tissue and then cut into rings. These tracheal rings were mounted in isolated organ baths filled with 10 ml of Krebs' solution, maintained at 37°C, and aerated with 95% O2 and 5% CO2. To achieve a steady spontaneous tone level, an initial tension of 1 g was applied. Contractions were measured isometrically with a force-displacement transducer (Ugo Basile, Comerio, Italy) and recorded by an Isolated Organs Data Acquisition program (Proto5; Letica Scientific Instruments, Barcelona, Spain).
Experimental Protocols. Tissues were allowed to stabilize for 60 min, whereas the bathing solution was exchanged at 10-min intervals. At the end of the equilibration period, the response to carbachol (2.5 µM) was recorded. After washout of carbachol and re-establishment of stable baseline tone, tissues were exposed to either carbachol (10-8–10-4 M), histamine (10-7–10-3 M), 5-HT (10-8–3 x 10-5 M), or antigen (ovalbumin; 0.001–100 µg/ml) in the presence or absence of 10 µM 7-epiclusianone. The preparations were preincubated with 7-epiclusianone 15 min before addition of the spasmodic agents. All responses were expressed as percentage of response to 2.5 µM carbachol.
In some experiments, the epithelial cells were removed mechanically by rubbing the internal tracheal surface with a fine silver wire (200 µm in diameter) as described previously (Wu et al., 2004). The removal of the epithelial layer was confirmed by histological examination. In brief, segments of trachea were fixed in Millong-formalin (Carson et al., 1973) and embedded with Paraplast-Plus paraffin. Sections (5 µm) were stained with hematoxylin and eosin and examined under light microscopy. During the experiment, the contractile response to 100 µg/ml ovalbumin or 2.5 µM carbachol was measured before and after a 15-min exposure to 10 µM 7-epiclusianone of intact or denuded epithelium tracheal rings.
To evaluate the putative interference of 7-epiclusianone with calcium influx, Ca2+ concentration-response curves were established according to Foster et al. (1984). In brief, the responses to 2.5 µM carbachol of tracheal ring segments from naive guinea pig were recorded. After washout of carbachol and re-establishment of stable baseline tone, tissues were exposed to successive cycles of 60 mM KCl stimulations/washouts in Ca2+-free Krebs' solution containing 2 mM EGTA until complete desensitization to the 60 mM KCl-evoked contractile response. Next, tracheal rings were immersed in Ca2+-free Krebs' solution containing 60 mM KCl, and the extracellular Ca2+ concentration was increased stepwise by the cumulative addition of CaCl2 (0.01–30 mM) in the presence or absence of 10 µM 7-epiclusianone or vehicle (0.1% DMSO). All responses were expressed as the percentage of response to 2.5 µM carbachol.
To further investigate the mechanisms of action of 7-epiclusianone, the tracheal rings were pretreated 10 min before its application with 10 µM ODQ, an inhibitor of guanylate cyclase; 100 µM L-NAME, an inhibitor of nitric-oxide synthase; 100 µM SQ22,536, an inhibitor of adenylate cyclase; 10 µM TEA, a nonselective K+ channel blocker; 1 µM glibenclamide, a KATP channel blocker; and 1 µM apamin or 0.1 µM IbTX, Ca2+-dependent K+ channel blockers of small and large conductance, respectively. All spasmolytic effects were expressed as a percentage of carbachol-induced maximal contractile responses.
Measurement of cGMP. Intracellular cGMP concentrations in guinea pig tracheal rings were assayed as described previously by Wu et al. (2004), with several modifications. Isolated tracheas were cut into rings cells, quickly immersed in Krebs' nutritional solution, and incubated with 7-epiclusianone (1–100 µM), L-NAME (100 µM), or ODQ (10 µM) in the presence of 100 µM 3-isobutyl-1-methylxanthine for 20 min. Some tracheal rings were pretreated with 100 µM L-NAME or 10 µM ODQ 10 min before addiction of 100 µM 7-epiclusianone. Tissues sections were rapidly frozen by immersion in liquid nitrogen. The frozen tracheal rings were homogenized in ice-cold 6% trichloroacetic acid (TCA). The homogenate was centrifuged at 2000g for 15 min at 4°C. To remove TCA, the supernatants were washed four times with 5 volumes of water-saturated diethyl ether. The top ether layer was discarded after each wash. Then, the supernatants were lyophilized, and the cGMP of each sample was determined by using commercially available enzyme immunoassay kits (GE Healthcare, Chalfont St. Giles, UK).
Assessment of the Effect 7-Epiclusianone on Airway Obstruction in Vivo. Using barometric whole body plethysmography (Buxco Research System, Wilmington, NC) and increases in enhanced pause (Penh) as an index of airway obstruction, we measured responses to inhaled methacholine (6, 12, and 25 mg/ml during 2.5 min, in 5-min intervals) in conscious, unrestrained naive BALB/c mice, as reported previously (Hamelmann et al., 1997). Penh measurements were performed within 1 and 4 h after treatment with 7-epiclusianone (25–100 mg/kg) or vehicle (1% polysorbate 80, diluted in 0.9% NaCl) administered via the oral route (gavage). The effect of oral treatment with theophylline (60 mg/kg) was also assessed for comparison.
Statistical Analysis. The results were expressed as mean ± S.E.M. Statistical differences were determined by using analysis of variance, followed by the Student-Newman-Keuls test. p values of 0.05 or less were considered significant.
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). Histological examination confirmed the epithelial layer removal as shown in Fig. 2B (epithelium denuded), compared with Fig. 2A (epithelium intact). The results showed that in the absence of tracheal epithelium, 10 µM 7-epiclusianone failed to inhibit contraction triggered by either ovalbumin (100 µg/ml) or carbachol (2.5 µM), as shown in Fig. 2, C and D, respectively.
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Effect of 7-Epiclusianone on cGMP Levels in Cultured Tracheal Rings. As illustrated in Fig. 4, 7-epiclusianone (1–100 µM) dose-dependently increased the tissue content of cGMP in cultured tracheal rings. The phenomenon was abolished by pretreatment with either the nitric-oxide synthase inhibitor L-NAME (100 µM) or the sGC inhibitor ODQ (10 µM).
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). As illustrated in Fig. 6B, mechanical removal of the tracheal epithelial barrier abrogated such protective effect.
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Discussion |
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Garcinia is the most numerous genus of the Guttiferae (Clusiaceae)—a vegetal family typically tropical in distribution (Ampofo and Waterman, 1986). Garcinia species are rich in oxygenated and prenylated phenol derivatives (Bennett and Lee, 1988), and some of them exhibit interesting biological activities such as antifungal, anti-inflammatory, antitumoral, antioxidant, antilipidemic, and vasodilator activities (Gopalakrishnan et al., 1997; Díaz-Carballo et al., 2003; Hay et al., 2004; Cruz et al., 2006). In a recent study, we obtained a yellow crystalline solid from G. brasiliensis fruit pericarp, which was identified as the tetraprenylated benzophenone 7-epiclusianone (Neves et al., 2007), a substance previously isolated from Rheedia gardneriana Miers ex Planch and Triana (Santos et al., 1998). The current study was mainly motivated by the findings that 7-epiclusianone inhibited the anaphylactic contraction of isolated guinea pig ileum and prevented allergen-evoked histamine release with potency comparable with that of the antiallergic agent azelastine (Neves et al., 2007).
In this study, we found that 7-epiclusianone inhibited guinea pig tracheal contraction induced by allergen, histamine, 5-HT, or carbachol. That 7-epiclusianone impairs contraction by allergen as well as those triggered by histamine, 5-HT, and carbachol suggests that the manner by which 7-epiclusianone exerts its effect is by interfering with the mechanism of smooth muscle contraction. The next step was to investigate whether the effect of 7-epiclusianone could be dependent of the production of smooth muscle relaxation factors by epithelial cells. In fact, it was observed that the antispasmodic activity of 7-epiclusianone upon allergen- and carbachol-evoked contraction was unapparent after tracheal epithelium removal, clearly showing the crucial role of epithelial cells in this protective effect. We also studied the possibility that 7-epiclusianone would be acting via activation of nitric oxide production by epithelial cells, which is a mechanism implicated in the regulation of the airway tone, under physiological and pathological conditions in guinea pigs and humans (Folkerts and Nijkamp, 2006). In fact, our findings revealed that the 7-epiclusianone's protective effect upon carbachol-evoked contraction was abolished by prior exposure of tracheal rings to the nitric-oxide synthase inhibitor L-NAME.
It is well known that epithelium-derived nitric-oxide activates guanylate cyclase and increases cGMP intracellular concentration, leading to relaxation of airway smooth muscles (Katsuki and Murad, 1977). The effect of 7-epiclusianone under conditions of intact epithelium was then shown to be inhibited by pretreatment with the sGC inhibitor ODQ. It is noteworthy that the response to 7-epiclusianone was not modified by pretreatment with the adenylate cyclase inhibitor SQ22,536. In another set of experiments, we demonstrated that 7-epiclusinone was also able to augment the trachea ring content of cGMP, in a mechanism clearly sensitive to blockade of either nitric-oxide synthase or sGC, strongly supporting the interpretation that the activity of this compound depends on the activation of nitric oxide and sGC/cGMP, but not the cAMP pathway.
It has been reported that nitric oxide is capable of stimulating Ca2+-activated K+ channels in smooth muscles (Bolotina et al., 1994), including those in the airways (Abderrahmane et al., 1998), causing membrane hyperpolarization, decrease in calcium influx, and smooth muscle relaxation (Lincoln and Cornwell, 1991). The opening of several K+ channels, such small-conductance Ca2+-activated K+ and the KATP channels, produces relaxation of the smooth muscle (Kume et al., 1989; Kotlikoff, 1993; Archer et al., 1998). We then examined the effectiveness of distinct K+ channel blockers to interfere with the antispasmodic activity of 7-epiclusianone. We noted that tracheal relaxations were no more observed when 7-epiclusianone was preceded by the nonselective K+ channel blocker TEA, indicating the involvement of K+ channels in this process. The 7-epiclusianone relaxation response was also sensitive to the KATP channel blocker glibenclamide and to apamin, a specific inhibitor of the small-conductance Ca2+-activated K+ channel, but it remained unchanged after pretreatment with the specific inhibitor of high-conductance Ca2+-activated K+ channel iberiotoxin. These findings are consistent with the interpretation that the 7-epiclusianone tracheal effects do involve activation of the cGMP/protein kinase G signaling cascade, leading to the phosphorylation-dependent opening of both KATP and small-conductance Ca2+-activated K+ channels, blockade of Ca2+ entrance, and eventually tracheal relaxation. They also suggest, in line with a prior study (Wu et al., 2004), that the high-conductance Ca2+-activated K+ channel is probably not the predominant K+ channel expressed by the guinea pig tracheal tissue, despite that this channel is highly implicated in nitric oxide/sGC/cGMP pathway-dependent relaxation of several other smooth muscle cell systems (Kaczorowski et al., 1996).
There is a significant body of evidence for the existence of voltage-dependent Ca2+ channels in airway smooth muscle (Marthan et al., 1989; Hisada et al., 1990; Worley and Kotlikoff, 1990). In guinea pig airway smooth muscle, contraction evoked by KCl is due to membrane depolarization with influx of Ca2+ through voltage-dependent Ca2+ channels (Knox and Tattersfield, 1995). It is noteworthy that in epithelium-intact trachea rings, 7-epiclusianone dose-dependently inhibited Ca2+-induced contractions in K+ (60 mM)-depolarized preparations, suggesting that 7-epiclusianone could inhibit Ca2+ influx via blockade of voltage-dependent Ca2+ channels (Neves et al., 2007). Nevertheless, this interpretation does not find support in similar experiments carried out in epithelium-denuded tracheal rings. Under this condition, 7-epiclusianone failed to alter contractile response induced by changes in extracellular Ca2+ concentration during high K+ depolarization, discarding the involvement of voltage-dependent Ca2+ channels while reinforcing the crucial role of the epithelial layer in the antispasmodic effect of 7-epiclusianone.
Finally, while trying to gain insight in the putative therapeutic application of this benzophenone derivative, we have used noninvasive barometric plethysmography to assess the effectiveness of 7-epiclusianone in animals. Essentially, we measured methacholine-evoked airway obstruction in BALB/c mice subjected to oral treatment with 7-epiclusianone, theophylline, or vehicle. Both compounds significantly attenuated methacholine-induced increases in Penh at 1 h compared with the vehicle-treated group, but only 7-epiclusianone remained active 4 h after treatment, showing for the first time that this natural product was active orally and displayed a longer lasting effect in comparison with theophylline. Moreover, it was interesting to find that the blockade of nitric-oxide synthesis in the animal model also reversed the antispasmodic effect of 7-epiclusianone, adding support to the interpretation that nitric oxide is indeed mediating the phenomenon. In contrast, the perspective of having some systemic cardiovascular repercussions for the oral 7-epiclusianone treatment should not be ruled out. This possibility is also supported by in vitro studies in which 7-epiclusianone exhibited an endothelium-dependent vasodilator effect in rat aortic rings (Cruz et al., 2006). A better characterization of the clinical value of 7-epiclusianone for the treatment of allergic and inflammatory diseases requires additional investigations.
In conclusion, this study suggests that the airways relaxation effect of 7-epiclusinone is accounted for by epithelium-, nitric oxide-, and sGC/cGMP-dependent mechanisms. The fact that oral administration of 7-epiclusianone can inhibit airways spasm in animal model emphasizes the possibility that this compound may be beneficial for the treatment of airflow limitation triggered by allergen challenge in humans.
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Acknowledgements |
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Footnotes |
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: DMSO, dimethyl sulfoxide; 5-HT, 5-hydroxytryptamine; L-NAME, N-nitro-L-arginine methyl ester; TEA, tetraethylammonium; KAPT, ATP-sensitive K+; IbTX, iberiotoxin; ODQ, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one; SQ22,536, 9-(tetrahydro-2'-furyl)adenine; TCA, trichloroacetic acid; Penh, enhanced pause; sGC, soluble guanylate cyclase.
Address correspondence to: Dr. Marco Aurélio Martins, Laboratory of Inflammation, Oswaldo Cruz Institute, Oswaldo Cruz Foundation, Av. Brasil, 4365, Manguinhos, CEP 21045-900, Rio de Janeiro, RJ, Brazil. E-mail: mmartins{at}ioc.fiocruz.br
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), 10 µM (
), or 100 µM (
) 7-epiclusianone on the guinea pig tracheal contraction induced by ovalbumin (10-9–10-4 g/ml) (A), histamine (10-7–10-2 M) (B), carbachol (10-8–10-4 M) (C), or 5-HT (10-9–3 x 10-5 M) (D). Each point represents the mean ± S.E.M. of eight experiments. All results were expressed as a percentage of contractile responses induced by 2.5 µM carbachol. *, p < 0.05 compared with tracheal responses from vehicle-treated preparations (
).
