European Journal of Pharmacology 760 (2015) 20–26
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European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar
Pulmonary, gastrointestinal and urogenital pharmacology
Effects of flavone on the contractile activity of the circular smooth muscle of the rabbit middle colon in vitro Hassiba Benabdallah a,n, Kamel Gharzouli b a b
Department of Microbiology and Biochemistry, Faculty of Sciences, University Mohamed Boudiaf of M'sila, Algeria Department of Animal physiology, Faculty of Nature and Life Sciences, University Ferhat Abbes of Setif, Algeria
art ic l e i nf o
a b s t r a c t
Article history: Received 3 March 2015 Received in revised form 3 April 2015 Accepted 8 April 2015 Available online 17 April 2015
The circular smooth muscles of the middle colon of the rabbit generate giant contractions of high amplitude and low frequency. Flavone, at various concentrations, reduces the giant contractions and the tonic contraction induced by 10 mM carbachol and 80 mM KCl. The contractions induced by dequalinium and tetraethylammonium are reduced by flavone (30 mM). At 100 mM, flavone decreases the contraction induced by 100 mM methylene blue and 1 mM orthovanadate. These results suggest that flavone inhibit the giant contractions by (1) inhibition of voltage-dependent Ca2 þ channels, (2) activation of guanyl cyclase, (3) opening of K þ channels and (4) inhibition of tyrosines kinases. & 2015 Elsevier B.V. All rights reserved.
Chemical compounds studied in this article: Carbamylcholine chloride (PubChem CID: 5831) Tetraethylammonium chloride (PubChem CID: 5946) Dequalinium chloride (PubChem CID: 10649) Methylene blue trihydrate (PubChem CID: 104827) Sodium orthovanadate (PubChem CID: 61671) Keywords: Circular smooth muscle Colon Flavone Giant contractions Nitrergic transmission Potassium channels
1. Introduction The spontaneous mechanical activity of the proximal, middle and distal colon of the rabbit shows in vitro two types of contractions: phasic contractions with low amplitude and high frequency, giant contractions (GCs) with high amplitude and low frequency (Benabdallah et al., 2008). The circular smooth muscle in the colon of different species generate in vivo several types of spontaneous contractions which take part in mixture and propulsion: rhythmic phasic contractions, giant migrating contractions (GMCs), and tone (Sarna, 2006). The rhythmic phasic contractions are regulated by slow waves superimposed with spikes. Their maximum frequency is the same as that of slow waves (Gonzalez and Sarna, 2001b). In comparison with the phasic contractions,
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Corresponding author. Tel.: þ 213 773290384. E-mail address: [email protected] (H. Benabdallah).
http://dx.doi.org/10.1016/j.ejphar.2015.04.007 0014-2999/& 2015 Elsevier B.V. All rights reserved.
GMCs are ultrapropulsives contractions of high amplitude and long duration (Gonzalez and Sarna, 2001b; Sarna, 2006). They are not regulated by slow waves, as their duration is much longer than that of a single slow wave cycle (Sarna, 1987). The equivalent of these in vitro contractions are the giant contractions (GCs) which are distinguished from the phasic contractions by their high amplitude and low frequency (Gonzalez and Sarna, 2001a). Flavonoids are members of a class of natural compounds which recently constitute the subject of several studies with scientific and therapeutic interest. These polyphenolic compounds recognized by the presence of a C6–C3–C6 basic ring skeleton are widely distributed throughout the plant kingdom (vegetables, fruits, flowers, wine and tea) (Benavente-Garcia et al., 1997) and potentially interact with physiological processes. Flavonoids are known to exhibit various biological activities such as anti-inflammatory (Vongtau et al., 2000), anti-tumor (Ikemoto et al., 2000), antiproliferative (Zhang et al., 2002), antiviral, anticancer (Di Pietro et al., 2002), antioxidant (Calderone et al., 2004), antiulcer (Lewis and Shaw, 2001),
H. Benabdallah, K. Gharzouli / European Journal of Pharmacology 760 (2015) 20–26
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was adjusted to 1 g and the tissue was allowed to equilibrate for 45 min during which time the solution was renewed every 15 min. To verify the viability and the responsiveness of the smooth muscle, the strips were challenged with 1 mM carbachol for 3 min, a period sufficient for the development of maximal tension. After washing the carbachol, a second period of stabilization (45 min) with periodical washing was started. 2.2. Experimental protocols
Fig. 1. Structure of flavone.
After the second equilibration period, strips of colon were incubated with different treatment used in this study. antiallergic, and anti-hypertensive (Almeida et al., 2006) activities. The effect of different flavonoids on smooth muscle contraction of several tissues also have been described (Hammad and Abdalla, 1997; Ajay et al., 2003; Uydes-Dogan et al., 2005). Quercetin and kaempferol inhibit uterine smooth muscle contraction induced by KCl (60 mM) (Revuelta et al., 1997). Some flavonols, flavones, flavanones, isoflavones and flavanes dose-dependently inhibited the contractions induced by phenylephrine and high K þ in isolated rat thoracic aorta (Ajay et al., 2003). Several mechanisms, including protein kinases inhibition (Herrera et al., 1996), intracellular inhibition of cAMP and cGMP phosphodiesterase (Herrera et al., 1996; Revuelta et al., 1997) and inhibition of calcium ion influx (Ajay et al., 2003) could be involved in their inhibitory effect. A report by Benabdallah et al., (2008) indicates that circular muscle strips prepared from the rabbit colon generate regular GCs as well as phasic contractions. This in vitro finding and the fact that flavonoids inhibit smooth muscle contractions provide an opportunity to investigate the effects of these compounds on GCs in a muscle bath environment. The specific aim of the present paper was to study the effects of flavones as basic structure of flavonoids (Fig. 1) on the isolated circular muscle from rabbit colon and to examine his possible underlying mechanisms. For that, the interference of this flavonoid with: 1) intracellular Ca2 þ mobilization, 2) potassium channels, 3) nitrergic pathway, and 4) phosphorylation of proteins were studied.
2.2.1. Effects of flavone on the spontaneous mechanical activity Flavone in solution in the dimethyl sulphoxide (DMSO) or the vehicle (DMSO) were added to medium cumulatively 1–100 mM) at 15 min intervals. 2.2.2. Effects of flavone on the contraction induced by carbachol and KCl Strips from the middle colon were precontracted with either 10 mM carbachol or 80 mM KCl for 15 min and 30 min respectively and the relaxant responses to flavone (1–100 mM) were recorded by adding cumulative doses of flavonoid solutions to the tissue bath at 15 min intervals between sussessive concentrations.
2. Materials and methods
2.2.3. Effects of flavone after stimulation of the spontaneous mechanical activity with TEA, dequalinium, methylene blue, and sodium orthovanadate In order to investigate the mechanisms underlying the relaxant action of flavone, the strips from the middle colon were preincubated for 15 min with 5 mM tetraethylammonium (TEA), a non selective blocker of K þ channels (Dong et al., 2005), 10 mM dequalinium, an apamin-sensitive K þ channel blocker (Castle, 1999), 100 mM methylene blue, a guanylate cyclase inhibitor (Börjesson et al., 1999), or 1 mM sodium vanadate, a tyrosine phosphatase inhibitor (Alcon et al., 2000). After this period, the vehicle or the flavone was added to the organ bath separately (30 mM for 30 min) or cumulatively (1 mM for 5 min and 30 mM for 30 min or 30 and 100 mM for 15 min).
2.1. Recording of spontaneous contractile activity
2.3. Drugs
The care and handling of the rabbits and the research protocol were in accordance with the institutional guidelines for the use of experimental animals. Adult rabbits of both sex and 1060 7339 g body weight were killed by bleeding from the carotid arteries. After a midline laparotomy the whole colon was removed and placed in a Petri dish containing HEPES buffered physiological solution (composition in mM: NaCl 126, KCl 6, MgCl2 1.2, CaCl2 2, EDTA 0.01, HEPES 10.5 and glucose 14, pH 7.4). The colon was divided into three different parts: proximal, middle and distal colon. Full thickness circular muscle strips of approximately 2 mm width and 10 mm length were prepared by cutting the tissue parallel to the circular axis. The strips were mounted vertically in organ bath chambers containing 25 ml of the physiological solution warmed at 37 1C and continuously oxygenated. The thread anchoring the upper end of the strips was connected to the lever of a force–displacement transducer (FSG-01/3, Experimetria, Budapest, Hungary) connected to an amplifier (EXP-D, Experimetria, Budapest, Hungary). Muscle activities were stored in a PC and simultaneously visualized (WinDaqLite software, DATAQ Instruments, OH, USA) using an external analog/digital conversion card with a sampling rate of 2 Hz (DI 700-PGL USB, DATAQ Instruments, OH, USA). Before the start of the experiments, the resting tension
The following drugs were used: carbamylcholine chloride (carbachol), tetraethylammonium chloride, dequalinium chloride, methylene blue trihydrate, sodium orthovanadate, and flavone: 2-phenyl-4- H-1-benzopyran-4-one (Sigma, St. Louis, USA). All drugs were prepared as stock solutions (carbachol 100 mM, TEA 500 mM, methylene blue 100 mM, sodium orthovanadate 1 M, and flavone 50 and 100 mM) in distilled water except for dequalinium, and flavone which were prepared in pure DMSO. All dilutions were made with the corresponding solvent of each drug and added in the bathing medium in volumes o0.5%. Preliminary experiments have shown that the vehicles (distilled water and DMSO) were without any observable effect on the mechanical activities of the preparation. 2.4. Statistical analyses Several parameters of the GCs were measured during the last three minutes of the incubation period: resting tone (the average minimal tension developed between GCs), maximal tension (g), amplitude (g), frequency (contractions.min 1, cpm) and duration (s). The results are expressed as mean 7S.E.M. with n indicating the number of animals. Differences between treatments were
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Fig. 2. Effects of cumulative addition of flavone on the giant contraction of the middle colon. (A) Typical recordings of the effect of flavone. (B) Effects of flavone on the amplitude and the frequency of giant contractions. Values are mean þS.E.M. (n¼ 6–8); nPr 0.05 with respect to basal control; ns: P 40.05.
compared using the test of student for paired data where appropriate, and by analysis of variance with Dunnett's test for the comparisons with the control or with Tukey's test for multiple comparisons. Means were considered significantly different when P values werer0.05.
3. Results 3.1. Effects of flavone on the spontaneous mechanical activity The circular muscle strips of the middle colon exhibited spontaneous contraction in the form of GCs (GC can be described by an initial phase of development of the maximum tension followed by a phase of relaxation) with high amplitude (0.7870.07 g) and low frequency (1.0870.08 GC/min). Flavone (1–100 mM) caused concentrationdependent reduction of the observed contractile activity (Fig. 2). Increasing the concentration of flavone to 30, 60 and 100 mM, reduced significantly the resting tone (0.5770.02 g, n¼7). The amplitude of the contractions was significantly reduced by 60 mM flavone (Fig. 2). The frequency of GCs is decreased significantly from 10 mM and reached very low values with 60 mM (0.1970.10 cpm, n¼7; Pr0.05; Fig. 2). This led to a significant prolongation of the phase of relaxation in 70% of the strips used. During the prolonged phase of relaxation a phasic activity was observed: 0.1870.01 g; frequency: 15.870.84 cpm (Fig. 2). At the highest concentration of flavone (100 mM), the rhythmic contractile activity was comppletely abolished. 3.2. Effects of flavone on carbachol and KCl-induced tonic contraction Carbachol (10 5 M) induced a phasic contraction followed by a tonic contraction (0.57 70.08 g; n¼ 10) of the strips. The Addition of flavone at the plateau of the tonic contraction induced the relaxation of the tissue in a concentration-dependent manner
(Fig. 3). A significant relaxing effect of flavone was observed with the concentration 10 mM (20.3 70.59%) and reached 62.72 73.93% with 100 mM flavone (Fig. 3). As carbachol, KCl (80 mM) produced a tonic contraction (0.55 70.04 g; n ¼8) which was reduced by the cumulative addition of flavones (Fig. 3); this effect was dependent of the concentration. Contrary to the relaxation of the tonic contraction induced by carbachol, 1 mM flavone was efficient in relaxing the tissue precontracted by KCl (8.80 70.86%). The KCl-induce tonic contraction was reduced by 81.32 72.2% with 100 mM flavone (Fig. 3). 3.3. Blockade of K þ channels by TEA and dequalinium Treatment of the strips from the middle colon with 5 mM TEA for 15 min maintained or converted the GCs into phasic contractions (Fig. 4). When the GCs were converted into phasic contractions, the addition of flavone (30 mM) to the organ bath had not significant effect on the resting tone and the frequency of the phasic contractions induced by the TEA. However, the amplitude of the phasic contractions was significantly reduced by 30 mM flavone (TEA: 0.17 7 0.04 g, flavone: 0.08 70.01, n¼ 8, P r0.05; Fig. 5). When the GCs were maintained in the presence of the TEA, flavone (30 mM) decreased significantly the tension and the frequency of GCs (Fig. 5). This last effect is due to a significant increase in the duration of GCs. Dequalinium 10 mM (apamin-sensitive K þ channel blocker) converted progressively the GCs into phasic contractions. The resting tone was increased whereas the amplitude of the contractions was significantly reduced (p r0.05; Fig. 6). Application of flavone (30 mM) induced a recovery of the basal activity at the end of the incubation period (incidence rate: 4/8; Fig. 6). The resting tone was significantly reduced by 30% in comparison with that noted in the presence of dequalinium alone (Fig. 6). The amplitude of the recovered GCs was significantly reduced with flavone (67.376.39%, n¼ 8, P r0.05; Fig. 6) compared to the basal period.
H. Benabdallah, K. Gharzouli / European Journal of Pharmacology 760 (2015) 20–26
Carbachol 10-5 M
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Flavone (µM) 10 30 100
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Flavone (µM) 10 30 100
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Fig. 3. Effects of the cumulative addition of flavone on the tonic contraction. (A) Typical recordings of the effect of flavones. Effects of the cumulative addition of flavone on the tonic contraction of the strips of the middle colon induced by 10 5 M carbachol (B) and 80 mM KCl (C). Values are mean 7S.E.M. (n ¼5–6); nP r0.05 with respect to tonic contraction induced by carbachol or KCl; ns: P4 0.05.
TEA 5 mM
Flavone (µM) 1 30
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Fig. 4. Typical recordings of the effect of the cumulative addition of flavone on the contraction of the strips of the middle colon in the presence of the TEA. (A) Conversion of GCs into phasic contractions. (B) Maintenance of GCs.
3.4. Inhibition of the guanylate cyclase by methylene blue The inhibition of guanylate cyclase by the cumulative addition of methylene blue (100 mM) converted the GCs into phasic contractions (Fig. 7). Flavone, added cumulatively (30 and 100 mM) to the organ bath, reduced progressively the resting tone (from 1.13 70.09 g to 0.42 70.03 g; n ¼6, Pr 0.05; Fig. 7). 3.5. Inhibition of tyrosine phosphatase by vanadate Incubation of the strips of the middle colon with vanadate (1 mM) converted the GCs into phasic contractions (Fig. 7). At 30 mM, flavone had not significant effect on the resting tone and the amplitude of contractions (Fig. 7). At 100 mM, the resting tone was significatively reduced (49.577.19%, n¼5, Pr0.05) and the amplitude of the contractions remains unchanged (P40.05; Fig. 7). 4. Discussion Flavone exerted an inhibitory effect on the GCs of the middle colon of rabbit in a concentration-dependent manner. This observation is
consistent with those reported for the effects of flavonoids on ileal and jejunum longitudinal smooth muscle of several animal species (Van Den Broucke and Lemli, 1983; Hammad and Abdalla, 1997; Rotondo et al., 2009). It was reported that (þ/ )-naringenin, a flavanone, induced concentration-dependent relaxation in endothelium-denuded rat aortic rings pre-contracted with either 20 mM KCl or noradrenaline. Naringin [(þ/ )-naringenin 7-β-neohesperidoside] caused a concentration dependent relaxation of rings pre-contracted with 20 mM KCl, although its potency and efficacy were significantly lower than those of (þ/)-naringenin (Saponara et al., 2006). In the other study, Rotondo et al. (2009) demonstrated that apigenin, a flavone, and quercetin, a flavonol, induced a concentration-dependent gastric relaxation. The relaxation of the rabbit colon induced by flavone is more significant than that induced by 100 mM quercetin (data not shown). This result is supported by the the study of which shows that apigenin being more potent than quercetin (Rotondo et al., 2009). In the present study, the relative importance of the effect of flavone can be due to its great permeability through the membrane (Dambros et al., 2005); once inside the cell, it could exert an effect on the contractile apparatus and/or influence the membrane mechanism responsible for the increase of intracellular Ca2 þ concentration. The relaxant effects of flavone have been attributed to the inhibition of
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Fig. 5. (A) Effects of the cumulative addition of flavone on the conversion of GCs into phasic contractions of the middle colon by the TEA. (B) Effects of the cumulative addition of flavone on the maintenance of GCs of the middle colon by the TEA. Values are mean 7 S.E.M. (n¼ (5–10)); nPr 0.05 with respect to TEA; ns: P4 0.05; #P r 0.05 with respect to the concentration of 30 mM.
Flavone (µM) Dequalinium 1 30
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Fig. 6. (A) Typical recordings of the effects of the cumulative addition of flavone on the contraction of the strips of the middle colon in the presence of dequalinium (10 mM). (B) Effects of flavone on the resting tone and the amplitude of the contractions. Values are mean7 S.E.M. (n¼ 9–11); nPr 0.05 with respect to dequalinium; ns: P 40.05; # P r 0.05 with respect to the concentration of 30 mM.
extracellular Ca2 þ influx as well as the inhibition of Ca2 þ release from intracellular store. Many studies agrees on the hypothesis of the mobilization of Ca2 þ in the inhibition of spontaneous and stimulated contraction of smooth muscle by flavonoids (Revuelta et al., 1997; Summanen et al., 2001; Dambros et al., 2005). Rotondo et al. (2009) demonstrated that the gastric relaxant activity of apigenin and quercetin is mainly due to their ability to block Ca2 þ influx through sarcolemma voltage-dependent Ca2 þ influx. The fact that the effect of galangin was partially eliminated when tested during treatment with verapamil, an L-type calcium channel blocker, suggests that the role of this flavonoid could involve those channels (Dambros et al., 2005). Flavones, naringenin and isoflavones (20 mg/ml) inhibit the 45Ca2 þ entry through the Ca2 þ voltage-dependent channels in rat pituitary GH4C1 cells (Summanen et al., 2001). The inhibitory effects of flavonoids on smooth muscle have been attributed to other mechanisms such as the inhibition of PKC (Herrera et al., 1996), myosin light chain kinase (Sohn et al., 2001), increase of level of cyclic nucleotides induced by phosphodiesterase inhibition (Herrera et al., 1996; Revuelta et al., 1997).
Flavone produced a concentration-dependent relaxation on carbachol-induced tonic contraction of rabbit colon. This effect could be due to interference with Ca2 þ influx. Several flavones inhibited carbachol and acetylcholin-induced ileal smooth muscle contraction (Van Den Broucke and Lemli, 1983). Flavone is also able to reduce the tonic contraction of the rabbit colon induced by neostigmine, an inhibitor of acetylcholinesterase. A myorelaxant effect of various flavonoids, like trimethoxyflavone and apigenin was also observed in the trachea (Leal et al., 2006) and thoarcic aorta (Ajay et al., 2003). The fact that the tonic contraction is due to the entry of extracellular Ca2 þ , flavone could interfere at this level. The blockade of the voltage-dependent Ca2 þ channels of ileum and rabbit colon by verapamil (results not presented) and nifedipine (Grasa et al., 2004) abolisched the phasic activity and reduced the tone of spontaneous contractions. The results of the present study showed that flavone concentration-dependently inhibited the contraction induced by 80 mM KCl. KCl-induced contraction of the smooth muscle result from increased Ca2 þ influx through voltage-dependent Ca2 þ channels (Buharalioglu and Akar, 2002; Grasa et al., 2004). The
H. Benabdallah, K. Gharzouli / European Journal of Pharmacology 760 (2015) 20–26
Methylene blue (µM) Flavone (µM) 10 30 100 30 100
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Vanadate (µM) Flavone (µM) 30 100 300 1000 30 100
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Fig. 7. Typical recordings of the effects of the cumulative addition of flavone on the contraction of the strips of the middle colon in the presence of the methylene blue (A) and the vanadate (B). Effects of the cumulative addition of flavone on the resting tone, the amplitude and the frequency of the phasic contractions in the presence of methylene blue (C) or vanadate (D). Values are mean 7 S.E.M. (n ¼5–6); nP r 0.05 with respect to methylene blue or vanadate; ns: P 40.05; #Pr 0.05 with respect to the flavone 30 mM.
contraction of the smooth muscle of the colon, ileum and fundus induced by 60 mM KCl was inhibited by verapamil and nifedipine, antagonists of voltage-dependent Ca2 þ channels (Buharalioglu and Akar, 2002; Grasa et al., 2004). Flavone concentrationdependently reduced the tonic contraction induced by 80 mM KCl. A relaxant effect of quercetin and flavones was also reported on the contraction of the ileum, aorta, uterus and trachea induced by KCl or CaCl2 in depolarizing solution (Van Den Broucke and Lemli, 1983; Revuelta et al., 1997; Uydes-Dogan et al., 2005; Leal et al., 2006). When GCs are converted into phasic contractions by the TEA (5 mM), the application of 30 mM flavone does not modify the threshold reached by the phasic contractions, suggesting that the opening of some types of K þ channels insensitive to TEA may be involved in his mechanism of action (Vogalis, 2000). In addition, the application of dequalinium (10 mM) to inhibit specifically the K þ channels sensitive to the apamine revealed that the flavone converted the phasic contractions induced by dequalinium into GCs; although these last are reduced in amplitude and frequency. These data indicate that flavone is able to open channels other than the KCa channels - sensitive to the apamine. The channels candidates are the large-conductance calcium-activated potassium
channels (BK channels). The fact that GCs are affected compared to the GCs obtained in normal conditions, it is possible that this flavonoid continued to interfere with the mobilization of Ca2 þ . Both luteolin and 5-hydroxyflavone seem to possess a mechanism of vasorelaxaction, which involves the activation of at least two subtypes of potassium channels (calcium activated and ATPsensitive ones) (Calderone et al., 2004). Other studies showed that isokaempferide and apigenin are able to open the BK channels explaining their myorelaxant effect (Calderone et al., 2004; Leal et al., 2006). In rat tail artery myocytes, ( þ/ )-naringenin increased large conductance Ca2 þ -activated K þ (BKCa) currents in a concentration-dependent manner (Saponara et al., 2006). The inhibition of the guanylate cyclase by the methylene blue after pretreatment of the rabbit colon with flavone induced an increase in resting tone but on a level lower than that noted in control tissue, suggesting a possible activation of the guanylate cyclase by flavone. Apigenin (10 mM) increased the cGMP content of endothelium-intact tissues. Pretreatment with L-NAME (100 mM) or removal of endothelium significantly suppressed the effect of apigenin on cGMP production (Zhang et al., 2002). The inhibition of the soluble guanyl cyclase by 1H-[1,2,4]oxadiazolol [4,3-a] quinoxalin-1-one (ODQ) or methylene blue reduced the
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relaxation of the trachea and the aorta induced respectively by isokampferide (Leal et al., 2006). However, this pathway does not seem to be the single way of the relaxation induced by flavone in the rabbit colon since the flavone continues to relax the muscle pre-contracted with methylene blue. Vanadate converted GCs into phasic contractions and increased the resting tone. This effect was due primarily to the inhibition of the protein tyrosine phosphatises (Mori and Tsushima, 2004), which imply the role of tyrosins kinases in the contraction of the smooth muscle. Flavone (100 mM) relaxes the smooth muscle colon pre-contracted by vanadate, suggesting that the flavone would act like an inhibitor of tyrosine kinases. A similar effect was reported for the genistein, which inhibits tyrosine kinase activity by interacting with the ATP binding site (Alcon et al., 2000). The fact that vanadate induced the contraction of the smooth muscle by increasing the rate of intracellular Ca2 þ following the opening of the voltage-dependent Ca2 þ channels and/or the inhibition of Ca2 þ -ATPase of the reticulum sarcoendoplasmic (Kuriyama et al., 1998), it is possible that the myorelaxant effect of flavone is also due to the blockade of this type of channels. 5. Conclusions In conclusion, the present study provides evidence that flavone induced a concentration-dependent reduction of the spontaneous contractile activity of the middle colon of the rabbit. The influence of this flavonoid on the movements of Ca2 þ during contraction is explained by his capacity to inhibit the contraction induced by carbachol and KCl. The myorelaxant effect of flavone seems to interfere K þ channels insensitive to TEA and channels other than the KCa channels sensitive to the apamine owing to the fact that this flavonoid does not modify the threshold reached by phasic contractions in the presence of TEA and reduced the tonic contraction induced by dequalinium. Flavone continued to relax the muscle pre-contracted with methylene blue, and vanadate indicating that the nitrergic pathway and the blockade of the Ca2 þ channels do not seem to be the only ways of the relaxation induced by flavone. Acknowledgments These experiments were supported by the Agence Nationale pour le Développement de la Recherche en Santé. References Ajay, M., Gilani, A.U., Mustafa, M.R., 2003. Effects of flavonoids on vascular smooth muscle of the isolated rat thoracic aorta. Life Sci. 74, 603–612. Alcon, S., Camello, P.J., Garcia, L.J., Pozo, M.J., 2000. Activation of tyrosine kinase pathway by vanadate in gallbladder smooth muscle. Biochem. Pharmacol. 59, 1077–1089. Almeida, R.R., Raimundo, J.M., Oliveira, R.R., kaplan, M.A.C., Gattass, C.R., Sudo, R.T., Sudo, G.Z., 2006. Activity of Cecropia lyratiloba extract on contractility of cardiac and smooth muscles in wistar rats. Clin. Exp. Pharmacol. Physiol. 33, 109–113. Benabdallah, H., Messaoudi, D., Gharzouli, K., 2008. The spontaneous mechanical activity of the circular smooth muscle of the rabbit colon in vitro. Pharmacol. Res. 57, 132–141. Benavente-Garcia, O., Castillo, J., Marin, F.R., Ortuno, A., Del Rio, J.A., 1997. Uses and properties of citrus flavonoids. J. Agric. Food Chem. 45, 4505–4515. Börjesson, L., Nordgren, S., Delbro, D.S., 1999. K þ -induced neurogenic relaxation of rat distal colon. J. Pharmacol. Exp. Ther. 291, 717–724. Buharalioglu, C.K., Akar, F., 2002. The reactivity of serotonin, acetylcholine and KClinduced contractions to relaxant agents in the rat gastric fundus. Pharmacol. Res. 45, 325–331. Calderone, V., Chericoni, S., Martinelli, C., Testai, L., Nardi, A., Morelli, I., Breschi, M.C., Martinotti, E., 2004. Vasorelaxing effects of flavonoids: investigation on the
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Effects of flavonoids on rat aortic smooth muscle contractility: structure-activity relationships. Gen. Pharmacol. 27, 273–277. Ikemoto, S., Sugimura, K., Yoshida, N., Yasumoto, R., Wada, S., Yamamoto, K., Kishimoto, T., 2000. Antitumor effects of scutellariae radix and its components baicalein, baicalin, and wogonin on bladder cancer cell lines. Urology 55, 951–955. Kuriyama, H., Kitamura, K., Inoue, R., 1998. Physiological features of visceral smooth muscle cells, with special reference to receptors and ion channels. Physiol. Rev. 78, 812–920. Leal, L.K., Costa, M.F., Pitombeira, M., Barroso, V.M., Silveira, E.R., Canuto, K.M., Viana, G.S., 2006. Mechanisms underlying the relaxation induced by isokaempferide from Amburana cearensis in the guinea-pig isolated trachea. Life Sci. 79, 98–104. Lewis, D.A., Shaw, G.P., 2001. A natural flavonoid and synthetic analogues protect the gastric mucosa from aspirin-induced erosions. J. Nutr. Biochem. 12, 95–100. Mori, M., Tsushima, H., 2004. Vanadate activates Rho A translocation in association with contracting effects in ileal longitudinal smooth muscle of guinea pig. J. Pharmacol. Sci. 95, 443–451. Revuelta, M.P., Cantabrana, B., Hidalgo, A., 1997. Depolarization-dependent effect of flavonoids in rat uterine smooth muscle contraction elicited by CaCl2. Gen. Pharmacol. 29, 847–857. Rotondo, A., Serio, R., Mulè, F., 2009. Gastric relaxation induced by apigenin and quercetin: analysis of the mechanism of action. Life Sci. 85, 85–90. Saponara, S., Testai, L., Iozzi, D., Martinotti, E., Martelli, A., Chericoni, S., Sgaragli, G., Fusi, F., Calderone, V., 2006. ( þ/ )-Naringenin as large conductance Ca2 þ activated K þ (BKCa) channel opener in vascular smooth muscle cells. Br. J. Pharmacol. 149, 1013–1021. Sarna, S.K., 2006. Molecular, functional, and pharmacological targets for the development of gut promotility drugs. Am. J. Physiol. Gastrointest. Liver Physiol. 291, G545–G555. Sarna, S.K., 1987. Giant migrating contractions and their myoelectric correlates in the small intestine. Am. J. Physiol. Gastrointest. Liver Physiol. 253, G697–G705. Sohn, U.D., Cao, W., Tang, D.C., Stull, J.T., Haeberle, J.R., Wang, C.L.A., Harnett, K.M., Behar, J., Biancani, P., 2001. Myosin light chain kinase- and PKC-dependent contraction of LES and esophageal smooth muscle. Am. J. Physiol. Gastrointest. Liver Physiol. 281, G467–G478. Summanen, J., Vuorela, P., Rauha, J.P., Tammela, P., Marjamaki, K., Pasternack, M., Tornquist, K., Vuorela, H., 2001. Effects of simple aromatic compounds and flavonoids on Ca2 þ fluxes in rat pituitary GH4C1 cells. Eur. J. Pharmacol. 414, 125–133. Uydes-Dogan, B.S., Takir, S., Ozdemir, O., Kolak, U., Topcu, G., Ulubelen, A., 2005. The comparison of the relaxant effects of two methoxylated flavones in rat aortic rings. Vasc. Pharmacol. 43, 220–226. Van Den Broucke, C.O., Lemli, J.A., 1983. Spasmolytic activity of the flavonoids from Thymus vulgaris. Pharm. Weekbl. Sci. 5, 9–14. Vogalis, F., 2000. Potassium channels in gastrointestinal smooth muscle. J. Auton. Pharmacol. 20, 207–219. Vongtau, H.O., Amos, S., Binda, L., Kapu, S.D., Gamaniel, K.S., Kunle, O.F., Wambebe, C., 2000. Pharmacological effects of the aqueous extract of Neorautanenia mitis in rodents. J. Ethnopharmacol. 72, 207–214. Zhang, Y.H., Park, Y.S., Kim, T.J., Fang, L.H., Ahn, H.Y., Hong, J.T., Kim, Y., Lee, C.K., Yun, Y.P., 2002. Endothelium-dependent vasorelaxant and antiproliferative effects of apigenin. Gen. Pharmacol. 35, 341–347.
Contents lists available at ScienceDirect
European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar
Pulmonary, gastrointestinal and urogenital pharmacology
Effects of flavone on the contractile activity of the circular smooth muscle of the rabbit middle colon in vitro Hassiba Benabdallah a,n, Kamel Gharzouli b a b
Department of Microbiology and Biochemistry, Faculty of Sciences, University Mohamed Boudiaf of M'sila, Algeria Department of Animal physiology, Faculty of Nature and Life Sciences, University Ferhat Abbes of Setif, Algeria
art ic l e i nf o
a b s t r a c t
Article history: Received 3 March 2015 Received in revised form 3 April 2015 Accepted 8 April 2015 Available online 17 April 2015
The circular smooth muscles of the middle colon of the rabbit generate giant contractions of high amplitude and low frequency. Flavone, at various concentrations, reduces the giant contractions and the tonic contraction induced by 10 mM carbachol and 80 mM KCl. The contractions induced by dequalinium and tetraethylammonium are reduced by flavone (30 mM). At 100 mM, flavone decreases the contraction induced by 100 mM methylene blue and 1 mM orthovanadate. These results suggest that flavone inhibit the giant contractions by (1) inhibition of voltage-dependent Ca2 þ channels, (2) activation of guanyl cyclase, (3) opening of K þ channels and (4) inhibition of tyrosines kinases. & 2015 Elsevier B.V. All rights reserved.
Chemical compounds studied in this article: Carbamylcholine chloride (PubChem CID: 5831) Tetraethylammonium chloride (PubChem CID: 5946) Dequalinium chloride (PubChem CID: 10649) Methylene blue trihydrate (PubChem CID: 104827) Sodium orthovanadate (PubChem CID: 61671) Keywords: Circular smooth muscle Colon Flavone Giant contractions Nitrergic transmission Potassium channels
1. Introduction The spontaneous mechanical activity of the proximal, middle and distal colon of the rabbit shows in vitro two types of contractions: phasic contractions with low amplitude and high frequency, giant contractions (GCs) with high amplitude and low frequency (Benabdallah et al., 2008). The circular smooth muscle in the colon of different species generate in vivo several types of spontaneous contractions which take part in mixture and propulsion: rhythmic phasic contractions, giant migrating contractions (GMCs), and tone (Sarna, 2006). The rhythmic phasic contractions are regulated by slow waves superimposed with spikes. Their maximum frequency is the same as that of slow waves (Gonzalez and Sarna, 2001b). In comparison with the phasic contractions,
n
Corresponding author. Tel.: þ 213 773290384. E-mail address: [email protected] (H. Benabdallah).
http://dx.doi.org/10.1016/j.ejphar.2015.04.007 0014-2999/& 2015 Elsevier B.V. All rights reserved.
GMCs are ultrapropulsives contractions of high amplitude and long duration (Gonzalez and Sarna, 2001b; Sarna, 2006). They are not regulated by slow waves, as their duration is much longer than that of a single slow wave cycle (Sarna, 1987). The equivalent of these in vitro contractions are the giant contractions (GCs) which are distinguished from the phasic contractions by their high amplitude and low frequency (Gonzalez and Sarna, 2001a). Flavonoids are members of a class of natural compounds which recently constitute the subject of several studies with scientific and therapeutic interest. These polyphenolic compounds recognized by the presence of a C6–C3–C6 basic ring skeleton are widely distributed throughout the plant kingdom (vegetables, fruits, flowers, wine and tea) (Benavente-Garcia et al., 1997) and potentially interact with physiological processes. Flavonoids are known to exhibit various biological activities such as anti-inflammatory (Vongtau et al., 2000), anti-tumor (Ikemoto et al., 2000), antiproliferative (Zhang et al., 2002), antiviral, anticancer (Di Pietro et al., 2002), antioxidant (Calderone et al., 2004), antiulcer (Lewis and Shaw, 2001),
H. Benabdallah, K. Gharzouli / European Journal of Pharmacology 760 (2015) 20–26
3’ 1
8 7 6
O
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was adjusted to 1 g and the tissue was allowed to equilibrate for 45 min during which time the solution was renewed every 15 min. To verify the viability and the responsiveness of the smooth muscle, the strips were challenged with 1 mM carbachol for 3 min, a period sufficient for the development of maximal tension. After washing the carbachol, a second period of stabilization (45 min) with periodical washing was started. 2.2. Experimental protocols
Fig. 1. Structure of flavone.
After the second equilibration period, strips of colon were incubated with different treatment used in this study. antiallergic, and anti-hypertensive (Almeida et al., 2006) activities. The effect of different flavonoids on smooth muscle contraction of several tissues also have been described (Hammad and Abdalla, 1997; Ajay et al., 2003; Uydes-Dogan et al., 2005). Quercetin and kaempferol inhibit uterine smooth muscle contraction induced by KCl (60 mM) (Revuelta et al., 1997). Some flavonols, flavones, flavanones, isoflavones and flavanes dose-dependently inhibited the contractions induced by phenylephrine and high K þ in isolated rat thoracic aorta (Ajay et al., 2003). Several mechanisms, including protein kinases inhibition (Herrera et al., 1996), intracellular inhibition of cAMP and cGMP phosphodiesterase (Herrera et al., 1996; Revuelta et al., 1997) and inhibition of calcium ion influx (Ajay et al., 2003) could be involved in their inhibitory effect. A report by Benabdallah et al., (2008) indicates that circular muscle strips prepared from the rabbit colon generate regular GCs as well as phasic contractions. This in vitro finding and the fact that flavonoids inhibit smooth muscle contractions provide an opportunity to investigate the effects of these compounds on GCs in a muscle bath environment. The specific aim of the present paper was to study the effects of flavones as basic structure of flavonoids (Fig. 1) on the isolated circular muscle from rabbit colon and to examine his possible underlying mechanisms. For that, the interference of this flavonoid with: 1) intracellular Ca2 þ mobilization, 2) potassium channels, 3) nitrergic pathway, and 4) phosphorylation of proteins were studied.
2.2.1. Effects of flavone on the spontaneous mechanical activity Flavone in solution in the dimethyl sulphoxide (DMSO) or the vehicle (DMSO) were added to medium cumulatively 1–100 mM) at 15 min intervals. 2.2.2. Effects of flavone on the contraction induced by carbachol and KCl Strips from the middle colon were precontracted with either 10 mM carbachol or 80 mM KCl for 15 min and 30 min respectively and the relaxant responses to flavone (1–100 mM) were recorded by adding cumulative doses of flavonoid solutions to the tissue bath at 15 min intervals between sussessive concentrations.
2. Materials and methods
2.2.3. Effects of flavone after stimulation of the spontaneous mechanical activity with TEA, dequalinium, methylene blue, and sodium orthovanadate In order to investigate the mechanisms underlying the relaxant action of flavone, the strips from the middle colon were preincubated for 15 min with 5 mM tetraethylammonium (TEA), a non selective blocker of K þ channels (Dong et al., 2005), 10 mM dequalinium, an apamin-sensitive K þ channel blocker (Castle, 1999), 100 mM methylene blue, a guanylate cyclase inhibitor (Börjesson et al., 1999), or 1 mM sodium vanadate, a tyrosine phosphatase inhibitor (Alcon et al., 2000). After this period, the vehicle or the flavone was added to the organ bath separately (30 mM for 30 min) or cumulatively (1 mM for 5 min and 30 mM for 30 min or 30 and 100 mM for 15 min).
2.1. Recording of spontaneous contractile activity
2.3. Drugs
The care and handling of the rabbits and the research protocol were in accordance with the institutional guidelines for the use of experimental animals. Adult rabbits of both sex and 1060 7339 g body weight were killed by bleeding from the carotid arteries. After a midline laparotomy the whole colon was removed and placed in a Petri dish containing HEPES buffered physiological solution (composition in mM: NaCl 126, KCl 6, MgCl2 1.2, CaCl2 2, EDTA 0.01, HEPES 10.5 and glucose 14, pH 7.4). The colon was divided into three different parts: proximal, middle and distal colon. Full thickness circular muscle strips of approximately 2 mm width and 10 mm length were prepared by cutting the tissue parallel to the circular axis. The strips were mounted vertically in organ bath chambers containing 25 ml of the physiological solution warmed at 37 1C and continuously oxygenated. The thread anchoring the upper end of the strips was connected to the lever of a force–displacement transducer (FSG-01/3, Experimetria, Budapest, Hungary) connected to an amplifier (EXP-D, Experimetria, Budapest, Hungary). Muscle activities were stored in a PC and simultaneously visualized (WinDaqLite software, DATAQ Instruments, OH, USA) using an external analog/digital conversion card with a sampling rate of 2 Hz (DI 700-PGL USB, DATAQ Instruments, OH, USA). Before the start of the experiments, the resting tension
The following drugs were used: carbamylcholine chloride (carbachol), tetraethylammonium chloride, dequalinium chloride, methylene blue trihydrate, sodium orthovanadate, and flavone: 2-phenyl-4- H-1-benzopyran-4-one (Sigma, St. Louis, USA). All drugs were prepared as stock solutions (carbachol 100 mM, TEA 500 mM, methylene blue 100 mM, sodium orthovanadate 1 M, and flavone 50 and 100 mM) in distilled water except for dequalinium, and flavone which were prepared in pure DMSO. All dilutions were made with the corresponding solvent of each drug and added in the bathing medium in volumes o0.5%. Preliminary experiments have shown that the vehicles (distilled water and DMSO) were without any observable effect on the mechanical activities of the preparation. 2.4. Statistical analyses Several parameters of the GCs were measured during the last three minutes of the incubation period: resting tone (the average minimal tension developed between GCs), maximal tension (g), amplitude (g), frequency (contractions.min 1, cpm) and duration (s). The results are expressed as mean 7S.E.M. with n indicating the number of animals. Differences between treatments were
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Fig. 2. Effects of cumulative addition of flavone on the giant contraction of the middle colon. (A) Typical recordings of the effect of flavone. (B) Effects of flavone on the amplitude and the frequency of giant contractions. Values are mean þS.E.M. (n¼ 6–8); nPr 0.05 with respect to basal control; ns: P 40.05.
compared using the test of student for paired data where appropriate, and by analysis of variance with Dunnett's test for the comparisons with the control or with Tukey's test for multiple comparisons. Means were considered significantly different when P values werer0.05.
3. Results 3.1. Effects of flavone on the spontaneous mechanical activity The circular muscle strips of the middle colon exhibited spontaneous contraction in the form of GCs (GC can be described by an initial phase of development of the maximum tension followed by a phase of relaxation) with high amplitude (0.7870.07 g) and low frequency (1.0870.08 GC/min). Flavone (1–100 mM) caused concentrationdependent reduction of the observed contractile activity (Fig. 2). Increasing the concentration of flavone to 30, 60 and 100 mM, reduced significantly the resting tone (0.5770.02 g, n¼7). The amplitude of the contractions was significantly reduced by 60 mM flavone (Fig. 2). The frequency of GCs is decreased significantly from 10 mM and reached very low values with 60 mM (0.1970.10 cpm, n¼7; Pr0.05; Fig. 2). This led to a significant prolongation of the phase of relaxation in 70% of the strips used. During the prolonged phase of relaxation a phasic activity was observed: 0.1870.01 g; frequency: 15.870.84 cpm (Fig. 2). At the highest concentration of flavone (100 mM), the rhythmic contractile activity was comppletely abolished. 3.2. Effects of flavone on carbachol and KCl-induced tonic contraction Carbachol (10 5 M) induced a phasic contraction followed by a tonic contraction (0.57 70.08 g; n¼ 10) of the strips. The Addition of flavone at the plateau of the tonic contraction induced the relaxation of the tissue in a concentration-dependent manner
(Fig. 3). A significant relaxing effect of flavone was observed with the concentration 10 mM (20.3 70.59%) and reached 62.72 73.93% with 100 mM flavone (Fig. 3). As carbachol, KCl (80 mM) produced a tonic contraction (0.55 70.04 g; n ¼8) which was reduced by the cumulative addition of flavones (Fig. 3); this effect was dependent of the concentration. Contrary to the relaxation of the tonic contraction induced by carbachol, 1 mM flavone was efficient in relaxing the tissue precontracted by KCl (8.80 70.86%). The KCl-induce tonic contraction was reduced by 81.32 72.2% with 100 mM flavone (Fig. 3). 3.3. Blockade of K þ channels by TEA and dequalinium Treatment of the strips from the middle colon with 5 mM TEA for 15 min maintained or converted the GCs into phasic contractions (Fig. 4). When the GCs were converted into phasic contractions, the addition of flavone (30 mM) to the organ bath had not significant effect on the resting tone and the frequency of the phasic contractions induced by the TEA. However, the amplitude of the phasic contractions was significantly reduced by 30 mM flavone (TEA: 0.17 7 0.04 g, flavone: 0.08 70.01, n¼ 8, P r0.05; Fig. 5). When the GCs were maintained in the presence of the TEA, flavone (30 mM) decreased significantly the tension and the frequency of GCs (Fig. 5). This last effect is due to a significant increase in the duration of GCs. Dequalinium 10 mM (apamin-sensitive K þ channel blocker) converted progressively the GCs into phasic contractions. The resting tone was increased whereas the amplitude of the contractions was significantly reduced (p r0.05; Fig. 6). Application of flavone (30 mM) induced a recovery of the basal activity at the end of the incubation period (incidence rate: 4/8; Fig. 6). The resting tone was significantly reduced by 30% in comparison with that noted in the presence of dequalinium alone (Fig. 6). The amplitude of the recovered GCs was significantly reduced with flavone (67.376.39%, n¼ 8, P r0.05; Fig. 6) compared to the basal period.
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Carbachol 10-5 M
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Fig. 3. Effects of the cumulative addition of flavone on the tonic contraction. (A) Typical recordings of the effect of flavones. Effects of the cumulative addition of flavone on the tonic contraction of the strips of the middle colon induced by 10 5 M carbachol (B) and 80 mM KCl (C). Values are mean 7S.E.M. (n ¼5–6); nP r0.05 with respect to tonic contraction induced by carbachol or KCl; ns: P4 0.05.
TEA 5 mM
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Fig. 4. Typical recordings of the effect of the cumulative addition of flavone on the contraction of the strips of the middle colon in the presence of the TEA. (A) Conversion of GCs into phasic contractions. (B) Maintenance of GCs.
3.4. Inhibition of the guanylate cyclase by methylene blue The inhibition of guanylate cyclase by the cumulative addition of methylene blue (100 mM) converted the GCs into phasic contractions (Fig. 7). Flavone, added cumulatively (30 and 100 mM) to the organ bath, reduced progressively the resting tone (from 1.13 70.09 g to 0.42 70.03 g; n ¼6, Pr 0.05; Fig. 7). 3.5. Inhibition of tyrosine phosphatase by vanadate Incubation of the strips of the middle colon with vanadate (1 mM) converted the GCs into phasic contractions (Fig. 7). At 30 mM, flavone had not significant effect on the resting tone and the amplitude of contractions (Fig. 7). At 100 mM, the resting tone was significatively reduced (49.577.19%, n¼5, Pr0.05) and the amplitude of the contractions remains unchanged (P40.05; Fig. 7). 4. Discussion Flavone exerted an inhibitory effect on the GCs of the middle colon of rabbit in a concentration-dependent manner. This observation is
consistent with those reported for the effects of flavonoids on ileal and jejunum longitudinal smooth muscle of several animal species (Van Den Broucke and Lemli, 1983; Hammad and Abdalla, 1997; Rotondo et al., 2009). It was reported that (þ/ )-naringenin, a flavanone, induced concentration-dependent relaxation in endothelium-denuded rat aortic rings pre-contracted with either 20 mM KCl or noradrenaline. Naringin [(þ/ )-naringenin 7-β-neohesperidoside] caused a concentration dependent relaxation of rings pre-contracted with 20 mM KCl, although its potency and efficacy were significantly lower than those of (þ/)-naringenin (Saponara et al., 2006). In the other study, Rotondo et al. (2009) demonstrated that apigenin, a flavone, and quercetin, a flavonol, induced a concentration-dependent gastric relaxation. The relaxation of the rabbit colon induced by flavone is more significant than that induced by 100 mM quercetin (data not shown). This result is supported by the the study of which shows that apigenin being more potent than quercetin (Rotondo et al., 2009). In the present study, the relative importance of the effect of flavone can be due to its great permeability through the membrane (Dambros et al., 2005); once inside the cell, it could exert an effect on the contractile apparatus and/or influence the membrane mechanism responsible for the increase of intracellular Ca2 þ concentration. The relaxant effects of flavone have been attributed to the inhibition of
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Fig. 5. (A) Effects of the cumulative addition of flavone on the conversion of GCs into phasic contractions of the middle colon by the TEA. (B) Effects of the cumulative addition of flavone on the maintenance of GCs of the middle colon by the TEA. Values are mean 7 S.E.M. (n¼ (5–10)); nPr 0.05 with respect to TEA; ns: P4 0.05; #P r 0.05 with respect to the concentration of 30 mM.
Flavone (µM) Dequalinium 1 30
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Fig. 6. (A) Typical recordings of the effects of the cumulative addition of flavone on the contraction of the strips of the middle colon in the presence of dequalinium (10 mM). (B) Effects of flavone on the resting tone and the amplitude of the contractions. Values are mean7 S.E.M. (n¼ 9–11); nPr 0.05 with respect to dequalinium; ns: P 40.05; # P r 0.05 with respect to the concentration of 30 mM.
extracellular Ca2 þ influx as well as the inhibition of Ca2 þ release from intracellular store. Many studies agrees on the hypothesis of the mobilization of Ca2 þ in the inhibition of spontaneous and stimulated contraction of smooth muscle by flavonoids (Revuelta et al., 1997; Summanen et al., 2001; Dambros et al., 2005). Rotondo et al. (2009) demonstrated that the gastric relaxant activity of apigenin and quercetin is mainly due to their ability to block Ca2 þ influx through sarcolemma voltage-dependent Ca2 þ influx. The fact that the effect of galangin was partially eliminated when tested during treatment with verapamil, an L-type calcium channel blocker, suggests that the role of this flavonoid could involve those channels (Dambros et al., 2005). Flavones, naringenin and isoflavones (20 mg/ml) inhibit the 45Ca2 þ entry through the Ca2 þ voltage-dependent channels in rat pituitary GH4C1 cells (Summanen et al., 2001). The inhibitory effects of flavonoids on smooth muscle have been attributed to other mechanisms such as the inhibition of PKC (Herrera et al., 1996), myosin light chain kinase (Sohn et al., 2001), increase of level of cyclic nucleotides induced by phosphodiesterase inhibition (Herrera et al., 1996; Revuelta et al., 1997).
Flavone produced a concentration-dependent relaxation on carbachol-induced tonic contraction of rabbit colon. This effect could be due to interference with Ca2 þ influx. Several flavones inhibited carbachol and acetylcholin-induced ileal smooth muscle contraction (Van Den Broucke and Lemli, 1983). Flavone is also able to reduce the tonic contraction of the rabbit colon induced by neostigmine, an inhibitor of acetylcholinesterase. A myorelaxant effect of various flavonoids, like trimethoxyflavone and apigenin was also observed in the trachea (Leal et al., 2006) and thoarcic aorta (Ajay et al., 2003). The fact that the tonic contraction is due to the entry of extracellular Ca2 þ , flavone could interfere at this level. The blockade of the voltage-dependent Ca2 þ channels of ileum and rabbit colon by verapamil (results not presented) and nifedipine (Grasa et al., 2004) abolisched the phasic activity and reduced the tone of spontaneous contractions. The results of the present study showed that flavone concentration-dependently inhibited the contraction induced by 80 mM KCl. KCl-induced contraction of the smooth muscle result from increased Ca2 þ influx through voltage-dependent Ca2 þ channels (Buharalioglu and Akar, 2002; Grasa et al., 2004). The
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Fig. 7. Typical recordings of the effects of the cumulative addition of flavone on the contraction of the strips of the middle colon in the presence of the methylene blue (A) and the vanadate (B). Effects of the cumulative addition of flavone on the resting tone, the amplitude and the frequency of the phasic contractions in the presence of methylene blue (C) or vanadate (D). Values are mean 7 S.E.M. (n ¼5–6); nP r 0.05 with respect to methylene blue or vanadate; ns: P 40.05; #Pr 0.05 with respect to the flavone 30 mM.
contraction of the smooth muscle of the colon, ileum and fundus induced by 60 mM KCl was inhibited by verapamil and nifedipine, antagonists of voltage-dependent Ca2 þ channels (Buharalioglu and Akar, 2002; Grasa et al., 2004). Flavone concentrationdependently reduced the tonic contraction induced by 80 mM KCl. A relaxant effect of quercetin and flavones was also reported on the contraction of the ileum, aorta, uterus and trachea induced by KCl or CaCl2 in depolarizing solution (Van Den Broucke and Lemli, 1983; Revuelta et al., 1997; Uydes-Dogan et al., 2005; Leal et al., 2006). When GCs are converted into phasic contractions by the TEA (5 mM), the application of 30 mM flavone does not modify the threshold reached by the phasic contractions, suggesting that the opening of some types of K þ channels insensitive to TEA may be involved in his mechanism of action (Vogalis, 2000). In addition, the application of dequalinium (10 mM) to inhibit specifically the K þ channels sensitive to the apamine revealed that the flavone converted the phasic contractions induced by dequalinium into GCs; although these last are reduced in amplitude and frequency. These data indicate that flavone is able to open channels other than the KCa channels - sensitive to the apamine. The channels candidates are the large-conductance calcium-activated potassium
channels (BK channels). The fact that GCs are affected compared to the GCs obtained in normal conditions, it is possible that this flavonoid continued to interfere with the mobilization of Ca2 þ . Both luteolin and 5-hydroxyflavone seem to possess a mechanism of vasorelaxaction, which involves the activation of at least two subtypes of potassium channels (calcium activated and ATPsensitive ones) (Calderone et al., 2004). Other studies showed that isokaempferide and apigenin are able to open the BK channels explaining their myorelaxant effect (Calderone et al., 2004; Leal et al., 2006). In rat tail artery myocytes, ( þ/ )-naringenin increased large conductance Ca2 þ -activated K þ (BKCa) currents in a concentration-dependent manner (Saponara et al., 2006). The inhibition of the guanylate cyclase by the methylene blue after pretreatment of the rabbit colon with flavone induced an increase in resting tone but on a level lower than that noted in control tissue, suggesting a possible activation of the guanylate cyclase by flavone. Apigenin (10 mM) increased the cGMP content of endothelium-intact tissues. Pretreatment with L-NAME (100 mM) or removal of endothelium significantly suppressed the effect of apigenin on cGMP production (Zhang et al., 2002). The inhibition of the soluble guanyl cyclase by 1H-[1,2,4]oxadiazolol [4,3-a] quinoxalin-1-one (ODQ) or methylene blue reduced the
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relaxation of the trachea and the aorta induced respectively by isokampferide (Leal et al., 2006). However, this pathway does not seem to be the single way of the relaxation induced by flavone in the rabbit colon since the flavone continues to relax the muscle pre-contracted with methylene blue. Vanadate converted GCs into phasic contractions and increased the resting tone. This effect was due primarily to the inhibition of the protein tyrosine phosphatises (Mori and Tsushima, 2004), which imply the role of tyrosins kinases in the contraction of the smooth muscle. Flavone (100 mM) relaxes the smooth muscle colon pre-contracted by vanadate, suggesting that the flavone would act like an inhibitor of tyrosine kinases. A similar effect was reported for the genistein, which inhibits tyrosine kinase activity by interacting with the ATP binding site (Alcon et al., 2000). The fact that vanadate induced the contraction of the smooth muscle by increasing the rate of intracellular Ca2 þ following the opening of the voltage-dependent Ca2 þ channels and/or the inhibition of Ca2 þ -ATPase of the reticulum sarcoendoplasmic (Kuriyama et al., 1998), it is possible that the myorelaxant effect of flavone is also due to the blockade of this type of channels. 5. Conclusions In conclusion, the present study provides evidence that flavone induced a concentration-dependent reduction of the spontaneous contractile activity of the middle colon of the rabbit. The influence of this flavonoid on the movements of Ca2 þ during contraction is explained by his capacity to inhibit the contraction induced by carbachol and KCl. The myorelaxant effect of flavone seems to interfere K þ channels insensitive to TEA and channels other than the KCa channels sensitive to the apamine owing to the fact that this flavonoid does not modify the threshold reached by phasic contractions in the presence of TEA and reduced the tonic contraction induced by dequalinium. Flavone continued to relax the muscle pre-contracted with methylene blue, and vanadate indicating that the nitrergic pathway and the blockade of the Ca2 þ channels do not seem to be the only ways of the relaxation induced by flavone. Acknowledgments These experiments were supported by the Agence Nationale pour le Développement de la Recherche en Santé. References Ajay, M., Gilani, A.U., Mustafa, M.R., 2003. Effects of flavonoids on vascular smooth muscle of the isolated rat thoracic aorta. Life Sci. 74, 603–612. Alcon, S., Camello, P.J., Garcia, L.J., Pozo, M.J., 2000. Activation of tyrosine kinase pathway by vanadate in gallbladder smooth muscle. Biochem. Pharmacol. 59, 1077–1089. Almeida, R.R., Raimundo, J.M., Oliveira, R.R., kaplan, M.A.C., Gattass, C.R., Sudo, R.T., Sudo, G.Z., 2006. Activity of Cecropia lyratiloba extract on contractility of cardiac and smooth muscles in wistar rats. Clin. Exp. Pharmacol. Physiol. 33, 109–113. Benabdallah, H., Messaoudi, D., Gharzouli, K., 2008. The spontaneous mechanical activity of the circular smooth muscle of the rabbit colon in vitro. Pharmacol. Res. 57, 132–141. Benavente-Garcia, O., Castillo, J., Marin, F.R., Ortuno, A., Del Rio, J.A., 1997. Uses and properties of citrus flavonoids. J. Agric. Food Chem. 45, 4505–4515. Börjesson, L., Nordgren, S., Delbro, D.S., 1999. K þ -induced neurogenic relaxation of rat distal colon. J. Pharmacol. Exp. Ther. 291, 717–724. Buharalioglu, C.K., Akar, F., 2002. The reactivity of serotonin, acetylcholine and KClinduced contractions to relaxant agents in the rat gastric fundus. Pharmacol. Res. 45, 325–331. Calderone, V., Chericoni, S., Martinelli, C., Testai, L., Nardi, A., Morelli, I., Breschi, M.C., Martinotti, E., 2004. Vasorelaxing effects of flavonoids: investigation on the
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