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Inhibitory effects of indicaxanthin on mouse ileal contractility: Analysis of the mechanism of action
European Journal of Pharmacology 658 (2011) 200–205
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European Journal of Pharmacology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / e j p h a r
Pulmonary, Gastrointestinal and Urogenital Pharmacology
Inhibitory effects of indicaxanthin on mouse ileal contractility: Analysis of the mechanism of action Sara Baldassano a, Alessandra Rotondo a, Rosa Serio a, Maria Antonietta Livrea b, Luisa Tesoriere b, Flavia Mulè a,⁎ a b
Dipartimento di Biologia cellulare e dello Sviluppo, Università di Palermo, 90128 Palermo, Italy Dipartimento Farmacochimico Tossicologico Biologico, Università di Palermo, 90128 Palermo, Italy
a r t i c l e
i n f o
Article history: Received 9 September 2010 Received in revised form 21 January 2011 Accepted 15 February 2011 Available online 1 March 2011 Keywords: Indicaxanthin Cactus pear fruit Smooth muscle Contractility Phosphodiesterases
a b s t r a c t Recently, we have showed that indicaxanthin, the yellow betalain pigment abundant in the fruit of Opuntia ficus indica, has remarkable spasmolytic effects on the intestinal contractility in vitro. Thus, the purpose of the present study was to investigate the mechanism of action underlying the observed response. We used organ bath technique to record the mechanical activity of the mouse ileum longitudinal muscle and ELISA to measure the levels of cAMP. Indicaxanthin induced inhibitory effects on spontaneous mechanical activity, which were unaffected by indomethacin, a non-selective inhibitor of cycloxygenase; 1H-[1,2,4]oxadiazolo [4,3-a]quinoxalin-1-one, a selective inhibitor of nitric oxide-dependent guanylyl cyclase; 2′5′dideoxyadenosine, an adenylyl cyclase inhibitor; and zaprinast, a selective inhibitor of the cGMP phosphodiesterase isoenzyme. Indicaxanthin effects were reduced significantly in the presence of 3-isobutyl-1-methylxanthine (IBMX), a non selective inhibitor of phosphodiesterases (PDEs). Indicaxanthin and IBMX significantly reduced the carbachol-evoked contractions and the joint application of both drugs did not produce any additive effect. Indicaxanthin and IBMX increased the inhibitory effects of forskolin, an adenylyl cyclase activator, and the joint application of both drugs did not produce any additive effect. Indicaxanthin, contrarily to IBMX, did not affect the inhibitory action of sodium nitroprusside, a soluble guanylyl cyclase activator. Indicaxanthin increased both basal and forskolin-induced cAMP content of mouse ileal muscle. The present data show that indicaxanthin reduces the contractility of ileal longitudinal muscle by inhibition of PDEs and increase of cAMP concentration and raise the possibility of using indicaxanthin in the treatment of motility disorders, such as abdominal cramps. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Plant-derived natural products have recently attracted considerable attention of both health professionals and common population for improving overall well-being as well as for the prevention of diseases. It has been shown that polyphenol phytochemicals such as tannins and flavonoids are bioactive constituents of numerous medicinal plants commonly used to regulate gastrointestinal function (Palombo, 2006). In particular, a number of these compounds have been reported to reduce intestinal motility and secretion in vivo (Di Carlo et al., 1993; Palombo, 2006), as well as to inhibit in vitro the spontaneous and agonist-induced contractions in rodent gastrointestinal tract (Amos et al., 1998; Aviello et al., 2010; Capasso et al., 2008, 1991; Chen et al., 2009; Di Carlo et al., 1993; Mata et al., 1997; Rotondo et al., 2009). ⁎ Corresponding author at: Dipartimento di Biologia cellulare e dello Sviluppo, Laboratorio di Fisiologia generale, Università di Palermo, Viale delle Scienze, 90128 Palermo, Italy. Tel.: + 39 091 23897515; fax: + 39 091 6577501. E-mail address: [email protected] (F. Mulè). 0014-2999/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2011.02.034
Betalainic phytochemicals are nitrogen-containing pigments occurring in the Caryophyllales order of plants, including beetroot and cactus pear, and in some fungal genera (Stintzing and Carle, 2005; Strack et al., 2003). Indicaxanthin, the yellow betalain characterizing the edible fruit of the cactus Opuntia ficus indica, is highly bioavailable in humans reaching even plasmatic micromolar concentrations (Tesoriere et al., 2004), and it can bind to low density lipoproteins (LDL) and cells (Tesoriere et al., 2004, 2005). The redox potential and radical scavenging activity of this compound have been measured (Butera et al., 2002), and numerous data have been published about the antioxidant and protective effects of indicaxanthin in various biological environments (Gentile et al., 2004; Tesoriere et al., 2006, 2007). Recently, we have reported that indicaxanthin has remarkable spasmolytic effects on the intestinal contractility in vitro. The pigment reduces the smooth muscle contractility of mouse intestinal ileal segments through mechanisms which do not involve potassium channels or voltage-dependent calcium channels, rather indicaxanthin appears to interfere with pathways regulating intracellular Ca2+ release (Baldassano et al., 2010). Calcium levels in muscle cells are controlled by multiple signaling pathways, which allow Ca2+ cycling between sarcoplasm and
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sarcoplasmic reticulum, and, in turn muscle contraction or relaxation. Signal transduction pathways promoting smooth muscle relaxation involve increase of intracellular cAMP or cGMP concentrations, which leads to a decrease in intracellular Ca2+ concentration. Different mechanisms can be responsible for the Ca2+ reduction: enhanced Ca2+ uptake into the sarcoplasmic reticulum or extrusion through the cell membrane, inhibition of IP3 formation or inhibition of Ca2+ release from the sarcoplasmic reticulum (Abdel-Latif, 2001). In addition, prostaglandins (PGs), especially PGE2, produced throughout the gut, are considered important factors for the physiological functions of the gastrointestinal tract, including motility (Dey et al., 2006). Signaling through different receptors determines the various effects of PGE2 and stimulation of cAMP/protein kinase A associated with relaxation has been reported (Dey et al., 2006). In the present study, by using a number of activators or inhibitors of enzymes involved in the main signaling pathways regulating the intestinal contractility, we explored the mechanisms by which indicaxanthin induces inhibitory effects on mechanical activity of mouse intestinal longitudinal smooth muscle. 2. Materials and methods 2.1. Animals All animal procedures were in conformity with the Italian D.L. no. 116 of 27 January 1992 and associated guidelines in the European Communities Council Directive of 24 November 1986 (86/609/ECC). Adult male mice (C57BL/10SnJ) (Harlan Laboratories, San Pietro di Natisone Udine, Italy) (25 ± 2.1 g) were used for the study. Animals were maintained under controlled conditions of temperature (22 ± 2 °C) and humidity (55 ± 5%) and had free access to water and food. 2.2. Experimental procedure Mice were killed by cervical dislocation. The abdomen was immediately opened and the ileum was removed and placed in a modified Krebs solution of the following composition (mM): NaCl 119; KCl 4.5; MgSO4 2.5; NaHCO3 25; KH2PO4 1.2, CaCl2 2.5, glucose 11.1. Segments (20 mm in length) were suspended in a four channel organ bath containing 6 ml of oxygenated (95% O2 and 5% CO2) Krebs solution maintained to 37 °C. The distal end of each segment was tied to organ holder and the proximal end was secured with a silk thread to an isometric force transducer (FORT 25, Ugo Basile, Biological Research Apparatus, Comerio VA, Italy) to record contractions from the longitudinal axis. Mechanical activity was digitized on an A/D converter, visualized, recorded and analyzed on a personal computer using the PowerLab/400 system (Ugo Basile, Italy). The segments, longitudinally oriented, were subjected to an initial tension of 200 mg and were allowed to equilibrate for at least 30 min. Rhythmic spontaneous contractions developed in all preparations. Indicaxanthin (3–100 μM) was then tested cumulatively to obtain concentration–response curves. The contact time for each concentration was 5 min. The effect of indicaxanthin was also evaluated in the presence of: indomethacin (10 μM), an inhibitor of cycloxygenase, 2′5′ dideoxyadenosine (DDA) (10 μM), an inhibitor of adenylyl cyclase, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) (10 μM), a selective inhibitor of nitric oxide (NO)-stimulated guanylyl cyclase, 3isobutyl-1-methylxanthine (IBMX) (10 μM), a non-selective inhibitor of cyclic nucleotide phosphodiesterase, zaprinast (10 μM), a selective inhibitor of the cGMP phosphodiesterase isoenzyme (phosphodiesterase 5). The concentration of the inhibitors used in the present study was selected on the basis of literature data (Amira et al., 2008; Mulè et al., 1999; Rotondo et al., 2009; Sato et al., 2006; Zizzo et al., 2006). Each drug was added to perfusing solution at least 30 min before indicaxanthin was tested.
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In a different set of experiments the influence of indicaxanthin (50 μM) or IBMX (10 μM), alone or in combination, was evaluated on the inhibitory responses induced by forskolin, an adenylyl cyclase activator, or by SNP, a soluble guanylyl cyclase activator. 2.3. Isolation of indicaxanthin Indicaxanthin was isolated from cactus pear (O. ficus-indica) fruits (yellow cultivar). The pigment was separated from a methanol extract of the pulp by liquid chromatography on Sephadex G-25 (Butera et al., 2002). Fractions containing indicaxanthin were submitted to cryodessiccation and purified according to Stintzing et al. (2002). Briefly, the desiccated material was re-suspended in 1% acetic acid in water and submitted to semi-preparative HPLC using a Varian Pursuit C18 column (250 × 10 mm i.d.; 5 mm; Varian, Palo Alto, CA), eluted with a 20-min linear gradient elution from solvent A (1% acetic acid in water) to 20% solvent B (1% acetic acid in acetonitrile) with a flow of 3 ml/ min. Spectrophotometric revelation was at 482 nm. The elution volumes relevant to indicaxanthin were collected. Samples after cryo-dessiccation were re-suspended in PBS at a suitable concentration and used immediately or stored at − 80 °C. Concentration of the samples was evaluated spectrophotometrically in a DU-800 Beckman spectrophotometer by using a molar coefficient at 482 nm of 42,800 (Piattelli et al., 1964). 2.4. Assay of cAMP The cAMP content of ileal muscle was measured by enzyme immunoassay. The muscular strips were weighted before starting the experiments. After incubation for 15 min with vehicle (Krebs solution), indicaxanthin (50–100 μM), IBMX (10 μM), forskolin (0.1 μM) alone or in combination with indicaxanthin (50 μM), the preparations were rapidly frozen in liquid nitrogen and homogenized in 5% trichloroacetic acid (TCA). The homogenate was centrifuged at 1500 ×g for 10 min. The supernatant was transferred to a clean tube and the TCA was extracted using water-satured ether. The residual of ether was removed by heating the sample to 70 °C for 5 min. The cAMP content was assayed with an enzyme immunoassay kit (Cyclic AMP EIA kit, Cayman). The detection limit of the assay was 0.1 pmol/ ml. The cAMP content was expressed as pmoles per gram of tissue wet weight. 2.5. Statistical analysis Mean amplitude of spontaneous contractions was measured prior to and following drug administration when a new steady state was reached. The results are expressed as the changes in mean amplitude of the phasic contractions and reported as percentages of the values obtained in the control (e.g. − 100% corresponds to the abolition of spontaneous activity). All data are expressed as mean values ± S.E.M. The letter n indicates the number of experimental animals. The concentration (IC50) with 95% confidence intervals (CIs) producing half maximum response was calculated using Prism 4.0, GraphPad (San Diego, CA, USA). Statistical analysis was performed by means of 2-way ANOVA, followed by Bonferroni's post hoc test. A probability value of less than 0.05 was regarded as significant. 2.6. Drugs Indomethacin, 3-isobutyl-1-methylxanthine (IBMX), forskolin, sodium nitroprusside (SNP), 2' - 5'-dideoxyadenosine (DDA) and 1H-(1,2,4) oxadiazolo-(4,3-a) quinoxalin-1-one (ODQ), zaprinast were purchased from Sigma (Milan, Italy). Indomethacin was dissolved in 2% Na2CO3, forskolin, IBMX, DDA and ODQ were dissolved in dimethyl sulfoxide (DMSO), SNP in distillated water. The working solutions were prepared freshly the day of the experiments by
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diluting the stock solutions in Krebs. Control experiments using DMSO alone showed that it did not significantly affect the contractility of the ileal segments. 3. Results 3.1. Indicaxanthin and spontaneous contractility As previously described (Baldassano et al., 2010), in mouse ileum indicaxanthin induced a concentration-dependent decrease in the amplitude of the spontaneous contractions, without affecting the frequency (control: 29 ± 2.1 cpm; 100 μM indicaxanthin: 30.2 ± 1.8 cpm; n = 6, P b 0.05) (Fig. 1). The effect was reversible after washing out. The threshold concentration of indicaxanthin was 3 μM and the maximum effect was observed at 75 μM (IC50 value: 24.9 μM; CIs 24.6– 25.2 μM) (Fig. 1). The effect of indicaxanthin was not affected by indomethacin (10 μM), DDA (10 μM), or ODQ (10 μM) (Fig. 1), indicating that indicaxanthin effects are not mediated by activation of cycloxygenase, adenylyl cyclase and NO-dependent guanylyl cyclase. Indeed, IBMX (10 μM), a non selective inhibitor of PDEs, which per se decreased the spontaneous phasic contractions (− 36.8± 5.6%; n = 5), significantly reduced the indicaxanthin inhibitory effect (Fig. 2), suggesting that the indicaxanthin relaxant action could be related to inhibition of PDEs leading to an increase of the cyclic nucleotide concentrations. To verify this hypothesis, we examined the influence of indicaxanthin on the effects induced by drugs which lead to increase the cAMP or cGMP intracellular concentrations. Forskolin (1 nM–1 μM), an adenylyl cyclase activator, reduced in a concentration-dependent
Fig. 1. (A) Typical recording showing the inhibitory effects induced by increasing concentrations of indicaxanthin on the spontaneous mechanical activity of mouse ileal longitudinal muscle. (B) Concentration–response curves for the inhibitory effects induced by indicaxanthin in the absence (control) or in the presence of ODQ (10 μM), DDA (10 μM), or indomethacin (10 μM). The inhibitory response is expressed as percent change of the resting activity (− 100% corresponds to the abolition of spontaneous activity). Each value is the mean ± S.E.M. of at least 4 separate experiments. S.E.M. is reported only if it exceeds the dimension of the symbol. The graphed values for the control curve are the means of the control data obtained before each treatment.
Fig. 2. Concentration–response curves for the inhibitory effects induced by indicaxanthin on spontaneous contractions of mouse ileal longitudinal muscle in the absence (control) or in the presence of IBMX (10 μM). The inhibitory response is expressed as percent change of the resting activity (− 100% corresponds to the abolition of spontaneous activity). Each value is mean ± S.E.M. of 5 separate experiments. S.E.M. is reported only if it exceeds the dimension of the symbol. *P b 0.05 compared with control value.
manner the amplitude of the spontaneous contractions and this effect was significantly increased by pre-treatment with IBMX (10 μM) or indicaxanthin (50 μM) (Fig. 3). IBMX (10 μM) in combination with indicaxanthin (50 μM) did not produce further additive effect (Fig. 3). Sodium nitroprusside (SNP; 1–100 μM), which acts through generation of NO, leading in turn to activation of NO-dependent guanylyl cyclase and increase of cGMP, induced a concentrationdependent reduction of the amplitude of the spontaneous contractions. The SNP-induced inhibitory effects were potentiated by IBMX (10 μM) but not by indicaxanthin (50 μM) (Fig. 4), ruling out the possibility that the pigment inhibitory effects were related to an increase of the cGMP intracellular concentration. In addition, zaprinast (10 μM), a selective inhibitor of the cGMP phosphodiesterase isoenzyme (phosphodiesterase 5), which per se reduced the spontaneous mechanical activity (− 26.2 ± 3.5%; n = 3), did not affect the indicaxanthin-induced inhibitory effects (Fig. 5).
Fig. 3. Concentration–response curves for the inhibitory effects induced by forskolin on spontaneous contractions of mouse ileal longitudinal muscle in the absence or in the presence of IBMX (10 μM) or indicaxanthin (50 μM), added to the bath alone or in combination. The inhibitory response is expressed as percent change of the resting activity (− 100% corresponds to the abolition of spontaneous activity). Each value is mean ± S.E.M. of 6 separate experiments. S.E.M. is reported only if it exceeds the dimension of the symbol. The graphed values for the control curve are the means of the control data obtained before each treatment. *P b 0.05 compared with control value.
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Fig. 4. Concentration–response curves for the inhibitory effects induced by SNP on spontaneous contractions of mouse ileal longitudinal muscle in the absence or in the presence of IBMX (10 μM) or indicaxanthin (50 μM). The inhibitory response is expressed as percent change of the resting activity (− 100% corresponds to the abolition of spontaneous activity). Each value is mean ± S.E.M. of at least 4 separate experiments. S.E.M. is reported only if it exceeds the dimension of the symbol. The graphed values for the control curve are the means of the control data obtained before each treatment. *P b 0.05 compared with control value.
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Fig. 6. Histogram showing the effects of IBMX (10 μM), alone or in combination with indicaxanthin (100 μM), on carbachol (10 μM)-evoked phasic and tonic contractions of mouse ileal longitudinal muscle. Data are expressed as a percentage of the phasic responses obtained in control conditions. Each value is mean ± S.E.M. of 5 separate experiments. *P b 0.05 compared with control value.
3.3. Effects of indicaxanthin on cAMP content 3.2. Indicaxanthin and carbachol evoked contractions As previously described (Baldassano et al., 2010), carbachol (10 μM) induced in mouse ileum a contractile response, characterized by a fast initial increase of muscular tension (phasic component) followed by a decline to a tension sustained level (tonic component), being both phases reduced by indicaxanthin. IBMX (10 μM), as well, significantly reduced both components of the carbachol-induced contraction and IBMX (10 μM) in combination with indicaxanthin (100 μM) did not produce any additive reduction of the carbacholevoked contractions (Fig. 6), suggesting that the two treatments share a similar mechanism of action.
Fig. 5. Concentration–response curves for the inhibitory effects induced by indicaxanthin on spontaneous contractions of mouse ileal longitudinal muscle in the absence (control) or in the presence of zaprinast (10 μM). The inhibitory response is expressed as percent change of the resting activity (− 100% corresponds to the abolition of spontaneous activity). Each value is mean ± S.E.M. of 3 separate experiments. S.E.M. is reported only if it exceeds the dimension of the symbol.
To verify if the pigment increases cAMP intracellular level by inhibition of PDE we measured the cAMP amount in mouse ileal muscle after indicaxanthin treatment and compared it with IBMX treatment. Indicaxanthin (50–100 μM), induced a concentrationdependent increase in the cAMP content of ileal muscle compared to the control level, although it was lesser than cAMP increase induced by IBMX (10 μM) (Fig. 7). In addition, indicaxanthin (50 μM) enhanced the cAMP increase induced by forskolin (0.1 μM) (Fig. 7). 4. Discussion The results of the present study demonstrate that indicaxanthin, extracted from cactus pear, reduces the mouse ileal longitudinal
Fig. 7. Effects of indicaxanthin, IBMX, and forskolin alone or in combination with indicaxanthin on the cAMP content of mouse ileal muscle. Each value is mean ± S.E.M. of 4 separate experiments. * P b 0.05 when compared with control value. § P b 0.05 when compared to 50 μM indicaxanthin. # P b 0.05 when compared to 100 μM indicaxanthin. $ P b 0.05 when compared to 0.1 μM foskolin.
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muscle contractility through inhibition of PDEs and increase of cAMP intracellular level. Recently, we have demonstrated that fruit extract from the yellow cultivar of cactus pear and its pigment indicaxanthin, exert a significant spasmolytic activity on the mouse intestinal smooth muscle (Baldassano et al., 2010). In fact, both cactus pear fruit extract and indicaxanthin were able to reduce reversibly and in a concentration-dependent manner the mouse ileum spontaneous phasic contractions or the carbachol-evoked contractions. Although we hypothesized that the extract inhibited the contractility of mouse ileum by interfering with pathways of intracellular Ca2+ release in the smooth muscle cells, the underlying mechanism remained to be elucidated. Cycloxygenase, and its product PGE2, may play an important role in gastrointestinal motility (Dey et al., 2006). In murine small intestine, suppression of pacemaker currents is mediated, at least in part, by PGE2 (Jun et al., 2005). Various phytochemicals (apigenin, quercetin, genistein) decrease the PGE2-induced contractions in rodent small intestine (Capasso et al., 1991; Grasa et al., 2006), and prostacyclins appear to participate in the vasorelaxant effects induced by flavonoids (Ajay et al., 2003). However, the observation that in our preparation, indomethacin, a non-selective inhibitor of cycloxygenase, did not reduce significantly the inhibitory effects of indicaxanthin, rules out a role of prostaglandins in the mechanism of action of the betalain. Cyclic nucleotides are important second messengers and have been associated with smooth muscle inhibitory effects (Diamond, 1978), including gastrointestinal smooth muscle relaxation (Makhlouf and Murthy, 1997). Intracellular concentrations of cyclic nucleotides are regulated by two families of enzymes, adenylyl- and guanylyl-cyclases which synthesize cAMP and cGMP from their corresponding triphospharilate nucleotides, and by the cyclic nucleotide phosphodiesterases which catalyze the hydrolysis of cAMP and cGMP. At first we test the hypothesis that the indicaxanthin effect was due to either activation of adenylyl cyclase or of guanylyl cyclase, leading in turn, to an increase in the intracellular cyclic nucleotide levels. Neither DDA, an adenylyl cyclase inhibitor, nor ODQ, a selective inhibitor of nitric oxide-sensitive guanylyl cyclase, did affect the indicaxanthin action, suggesting that the pigment does not stimulate cyclic nucleotide synthesis. On the contrary, a clear reduction of the indicaxanthin inhibitory effects was observed in the presence of IBMX, a non-selective inhibitor of PDEs, suggesting that PDE inhibition, and consequent increase in intracellular cyclic nucleotides, would play a role in the decrease of the ileal spontaneous contractions induced by indicaxanthin. Although the relaxation caused by different PDE inhibitors may be reduced by adenylyl cyclase inhibitors and/or guanylyl cyclase inhibitors in various smooth muscle preparations (Kaneda et al., 2004; Oger et al., 2010), other researchers have shown that adenylyl cyclase inhibitors does not affect smooth muscle relaxation induced by PDE inhibitors, similarly to our conditions (Koutsoviti-Papadopoulou et al., 2009; Lin et al., 2007). It might be argued that the reduction of the spontaneous contractions caused by IBMX pretreatment could account for the decreased inhibitory effects of indicaxanthin. However, this supposition can be ruled out because in the presence of IBMX, the reduction of the spontaneous contractions induced by forskolin, which acts stimulating adenylyl cyclase, was increased. Moreover, in our preparation the inhibitory effect of indicaxanthin on carbachol-evoked contractions was not observed in the presence of IBMX, further confirming that the two compounds share a similar mode of action. To support our hypothesis, we examined and compared the influence of indicaxanthin and IBMX on the effects of drugs able to increase cAMP or cGMP intracellular concentration, i.e. forskolin and SNP, respectively. Pretreatment with indicaxanthin enhanced the inhibitory effects induced by forskolin, similarly to IBMX pretreatment, and the application of IBMX in combination with indicaxanthin
did not produce any additive effects, providing further evidence that indicaxanthin possibly acts as an inhibitor of PDEs with consequent accumulation of cAMP. Moreover, contrarily to IBMX, indicaxanthin failed to enhance the inhibitory action of SNP, which acts through activation of guanylyl cyclase, and the inhibitory effects induced by indicaxanthin persisted in the presence of zaprinast, selective inhibitor of PDE5, suggesting that the increase of cGMP concentration is not involved in the pigment inhibitory action. This is not surprising considering that in longitudinal muscle of mouse ileum SNP inhibits the contractility through cGMP accummulation leading to activation of the large conductance Ca2+-dependent K+ channels (Zizzo et al., 2005), which are not involved in the effects induced by indicaxanthin (Baldassano et al., 2010). Finally, the observations that incubation with indicaxanthin significantly increased both basal and forskolininduced cAMP levels in mouse ileum are also consistent with the hypothesis that the indicaxanthin-induced inhibitory effects are due to an increase of the intracellular cAMP level, likely related to PDE inhibition. In particular, indicaxanthin increased the amount of cAMP in a concentration-dependent manner, although to a lesser extent than that obtained with IBMX. This finding could be explained taking into account that IBMX is a non selective PDE inhibitor, therefore it would inhibit more than one isozyme. Currently, it is known that PDEs comprise at least 11 distinct subfamilies which hydrolyze cAMP and/ or cGMP (Beavo, 1995; Keravis and Lugnier, 2010). PDE inhibitors have been reported to reduce contractility in diverse smooth muscle preparations (Kaneda et al., 2004; Polson and Strada, 1996; Qiu et al., 2001; Raeburn and Advenier, 1995) including ileal smooth muscle (Kaneda et al., 1997). Indeed, although our results provide evidence for involvement of PDEs hydrolyzing cAMP, they do not consent to conclude on the type of isozyme involved in the indicaxanthininduced response and further experiments are required to elucidate this point. The link between indicaxanthin and PDEs may be a matter of speculation. It could be hypothesized that the compound inhibit directly PDEs after traversing the membrane, as shown for betanin, a structurally related pigment (Sreekanth et al., 2007). Indeed, although the pigment is hydrophilic, it has been shown to bind to LDL either in vitro or in vivo (Tesoriere et al., 2003, 2004), as well as to microsomal membranes (Kanner et al., 2001). Moreover indicaxanthin locates to interface between hydrophobic core and hydrophilic head groups of dipalmitoyl-phosphatidylcholine vescicles (Turco Liveri et al., 2009), thus exhibiting amphiphilic properties that may allow this compound to cross the cell membranes and to each the cytosol. Lastly, incorporation of indicaxanthin in human red blood cells has been also demonstrated (Tesoriere et al., 2005). In conclusion, the present results provide evidence that the natural pigment indicaxanthin, a phytochemical isolated from the cactus pear yellow fruits, decreases the mouse ileal longitudinal muscle contractions through inhibition of PDEs and increase of cAMP levels. These findings raise the possibility of using indicaxanthin as regulator of intestinal motility in different disorders, such as abdominal cramps. Acknowledgment This work was supported by a grant from the Ministero dell'Istruzione, dell'Università e della Ricerca — Italy (ex 60%). References Abdel-Latif, A.A., 2001. Cross talk between cyclic nucleotides and polyphosphoinositide hydrolysis, protein kinases, and contraction in smooth muscle. Exp. Biol. Med. 226, 153–163. Ajay, M., Gilani, A.H., Mustafa, M.R., 2003. Effects of flavonoids on vascular smooth muscle of the isolated rat thoracic aorta. Life Sci. 74, 603–612. Amira, S., Rotondo, A., Mulè, F., 2008. Relaxant effects of flavonoids on the mouse isolated stomach: structure–activity relationships. Eur. J. Pharmacol. 599, 126–130. Amos, S., Okwuasaba, F.K., Gamaniel, K., Akah, P., Wambebe, C., 1998. Inhibitory effects of the aqueous extract of Pavetta crassipes leaves on gastrointestinal and uterine
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Contents lists available at ScienceDirect
European Journal of Pharmacology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / e j p h a r
Pulmonary, Gastrointestinal and Urogenital Pharmacology
Inhibitory effects of indicaxanthin on mouse ileal contractility: Analysis of the mechanism of action Sara Baldassano a, Alessandra Rotondo a, Rosa Serio a, Maria Antonietta Livrea b, Luisa Tesoriere b, Flavia Mulè a,⁎ a b
Dipartimento di Biologia cellulare e dello Sviluppo, Università di Palermo, 90128 Palermo, Italy Dipartimento Farmacochimico Tossicologico Biologico, Università di Palermo, 90128 Palermo, Italy
a r t i c l e
i n f o
Article history: Received 9 September 2010 Received in revised form 21 January 2011 Accepted 15 February 2011 Available online 1 March 2011 Keywords: Indicaxanthin Cactus pear fruit Smooth muscle Contractility Phosphodiesterases
a b s t r a c t Recently, we have showed that indicaxanthin, the yellow betalain pigment abundant in the fruit of Opuntia ficus indica, has remarkable spasmolytic effects on the intestinal contractility in vitro. Thus, the purpose of the present study was to investigate the mechanism of action underlying the observed response. We used organ bath technique to record the mechanical activity of the mouse ileum longitudinal muscle and ELISA to measure the levels of cAMP. Indicaxanthin induced inhibitory effects on spontaneous mechanical activity, which were unaffected by indomethacin, a non-selective inhibitor of cycloxygenase; 1H-[1,2,4]oxadiazolo [4,3-a]quinoxalin-1-one, a selective inhibitor of nitric oxide-dependent guanylyl cyclase; 2′5′dideoxyadenosine, an adenylyl cyclase inhibitor; and zaprinast, a selective inhibitor of the cGMP phosphodiesterase isoenzyme. Indicaxanthin effects were reduced significantly in the presence of 3-isobutyl-1-methylxanthine (IBMX), a non selective inhibitor of phosphodiesterases (PDEs). Indicaxanthin and IBMX significantly reduced the carbachol-evoked contractions and the joint application of both drugs did not produce any additive effect. Indicaxanthin and IBMX increased the inhibitory effects of forskolin, an adenylyl cyclase activator, and the joint application of both drugs did not produce any additive effect. Indicaxanthin, contrarily to IBMX, did not affect the inhibitory action of sodium nitroprusside, a soluble guanylyl cyclase activator. Indicaxanthin increased both basal and forskolin-induced cAMP content of mouse ileal muscle. The present data show that indicaxanthin reduces the contractility of ileal longitudinal muscle by inhibition of PDEs and increase of cAMP concentration and raise the possibility of using indicaxanthin in the treatment of motility disorders, such as abdominal cramps. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Plant-derived natural products have recently attracted considerable attention of both health professionals and common population for improving overall well-being as well as for the prevention of diseases. It has been shown that polyphenol phytochemicals such as tannins and flavonoids are bioactive constituents of numerous medicinal plants commonly used to regulate gastrointestinal function (Palombo, 2006). In particular, a number of these compounds have been reported to reduce intestinal motility and secretion in vivo (Di Carlo et al., 1993; Palombo, 2006), as well as to inhibit in vitro the spontaneous and agonist-induced contractions in rodent gastrointestinal tract (Amos et al., 1998; Aviello et al., 2010; Capasso et al., 2008, 1991; Chen et al., 2009; Di Carlo et al., 1993; Mata et al., 1997; Rotondo et al., 2009). ⁎ Corresponding author at: Dipartimento di Biologia cellulare e dello Sviluppo, Laboratorio di Fisiologia generale, Università di Palermo, Viale delle Scienze, 90128 Palermo, Italy. Tel.: + 39 091 23897515; fax: + 39 091 6577501. E-mail address: [email protected] (F. Mulè). 0014-2999/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2011.02.034
Betalainic phytochemicals are nitrogen-containing pigments occurring in the Caryophyllales order of plants, including beetroot and cactus pear, and in some fungal genera (Stintzing and Carle, 2005; Strack et al., 2003). Indicaxanthin, the yellow betalain characterizing the edible fruit of the cactus Opuntia ficus indica, is highly bioavailable in humans reaching even plasmatic micromolar concentrations (Tesoriere et al., 2004), and it can bind to low density lipoproteins (LDL) and cells (Tesoriere et al., 2004, 2005). The redox potential and radical scavenging activity of this compound have been measured (Butera et al., 2002), and numerous data have been published about the antioxidant and protective effects of indicaxanthin in various biological environments (Gentile et al., 2004; Tesoriere et al., 2006, 2007). Recently, we have reported that indicaxanthin has remarkable spasmolytic effects on the intestinal contractility in vitro. The pigment reduces the smooth muscle contractility of mouse intestinal ileal segments through mechanisms which do not involve potassium channels or voltage-dependent calcium channels, rather indicaxanthin appears to interfere with pathways regulating intracellular Ca2+ release (Baldassano et al., 2010). Calcium levels in muscle cells are controlled by multiple signaling pathways, which allow Ca2+ cycling between sarcoplasm and
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sarcoplasmic reticulum, and, in turn muscle contraction or relaxation. Signal transduction pathways promoting smooth muscle relaxation involve increase of intracellular cAMP or cGMP concentrations, which leads to a decrease in intracellular Ca2+ concentration. Different mechanisms can be responsible for the Ca2+ reduction: enhanced Ca2+ uptake into the sarcoplasmic reticulum or extrusion through the cell membrane, inhibition of IP3 formation or inhibition of Ca2+ release from the sarcoplasmic reticulum (Abdel-Latif, 2001). In addition, prostaglandins (PGs), especially PGE2, produced throughout the gut, are considered important factors for the physiological functions of the gastrointestinal tract, including motility (Dey et al., 2006). Signaling through different receptors determines the various effects of PGE2 and stimulation of cAMP/protein kinase A associated with relaxation has been reported (Dey et al., 2006). In the present study, by using a number of activators or inhibitors of enzymes involved in the main signaling pathways regulating the intestinal contractility, we explored the mechanisms by which indicaxanthin induces inhibitory effects on mechanical activity of mouse intestinal longitudinal smooth muscle. 2. Materials and methods 2.1. Animals All animal procedures were in conformity with the Italian D.L. no. 116 of 27 January 1992 and associated guidelines in the European Communities Council Directive of 24 November 1986 (86/609/ECC). Adult male mice (C57BL/10SnJ) (Harlan Laboratories, San Pietro di Natisone Udine, Italy) (25 ± 2.1 g) were used for the study. Animals were maintained under controlled conditions of temperature (22 ± 2 °C) and humidity (55 ± 5%) and had free access to water and food. 2.2. Experimental procedure Mice were killed by cervical dislocation. The abdomen was immediately opened and the ileum was removed and placed in a modified Krebs solution of the following composition (mM): NaCl 119; KCl 4.5; MgSO4 2.5; NaHCO3 25; KH2PO4 1.2, CaCl2 2.5, glucose 11.1. Segments (20 mm in length) were suspended in a four channel organ bath containing 6 ml of oxygenated (95% O2 and 5% CO2) Krebs solution maintained to 37 °C. The distal end of each segment was tied to organ holder and the proximal end was secured with a silk thread to an isometric force transducer (FORT 25, Ugo Basile, Biological Research Apparatus, Comerio VA, Italy) to record contractions from the longitudinal axis. Mechanical activity was digitized on an A/D converter, visualized, recorded and analyzed on a personal computer using the PowerLab/400 system (Ugo Basile, Italy). The segments, longitudinally oriented, were subjected to an initial tension of 200 mg and were allowed to equilibrate for at least 30 min. Rhythmic spontaneous contractions developed in all preparations. Indicaxanthin (3–100 μM) was then tested cumulatively to obtain concentration–response curves. The contact time for each concentration was 5 min. The effect of indicaxanthin was also evaluated in the presence of: indomethacin (10 μM), an inhibitor of cycloxygenase, 2′5′ dideoxyadenosine (DDA) (10 μM), an inhibitor of adenylyl cyclase, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) (10 μM), a selective inhibitor of nitric oxide (NO)-stimulated guanylyl cyclase, 3isobutyl-1-methylxanthine (IBMX) (10 μM), a non-selective inhibitor of cyclic nucleotide phosphodiesterase, zaprinast (10 μM), a selective inhibitor of the cGMP phosphodiesterase isoenzyme (phosphodiesterase 5). The concentration of the inhibitors used in the present study was selected on the basis of literature data (Amira et al., 2008; Mulè et al., 1999; Rotondo et al., 2009; Sato et al., 2006; Zizzo et al., 2006). Each drug was added to perfusing solution at least 30 min before indicaxanthin was tested.
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In a different set of experiments the influence of indicaxanthin (50 μM) or IBMX (10 μM), alone or in combination, was evaluated on the inhibitory responses induced by forskolin, an adenylyl cyclase activator, or by SNP, a soluble guanylyl cyclase activator. 2.3. Isolation of indicaxanthin Indicaxanthin was isolated from cactus pear (O. ficus-indica) fruits (yellow cultivar). The pigment was separated from a methanol extract of the pulp by liquid chromatography on Sephadex G-25 (Butera et al., 2002). Fractions containing indicaxanthin were submitted to cryodessiccation and purified according to Stintzing et al. (2002). Briefly, the desiccated material was re-suspended in 1% acetic acid in water and submitted to semi-preparative HPLC using a Varian Pursuit C18 column (250 × 10 mm i.d.; 5 mm; Varian, Palo Alto, CA), eluted with a 20-min linear gradient elution from solvent A (1% acetic acid in water) to 20% solvent B (1% acetic acid in acetonitrile) with a flow of 3 ml/ min. Spectrophotometric revelation was at 482 nm. The elution volumes relevant to indicaxanthin were collected. Samples after cryo-dessiccation were re-suspended in PBS at a suitable concentration and used immediately or stored at − 80 °C. Concentration of the samples was evaluated spectrophotometrically in a DU-800 Beckman spectrophotometer by using a molar coefficient at 482 nm of 42,800 (Piattelli et al., 1964). 2.4. Assay of cAMP The cAMP content of ileal muscle was measured by enzyme immunoassay. The muscular strips were weighted before starting the experiments. After incubation for 15 min with vehicle (Krebs solution), indicaxanthin (50–100 μM), IBMX (10 μM), forskolin (0.1 μM) alone or in combination with indicaxanthin (50 μM), the preparations were rapidly frozen in liquid nitrogen and homogenized in 5% trichloroacetic acid (TCA). The homogenate was centrifuged at 1500 ×g for 10 min. The supernatant was transferred to a clean tube and the TCA was extracted using water-satured ether. The residual of ether was removed by heating the sample to 70 °C for 5 min. The cAMP content was assayed with an enzyme immunoassay kit (Cyclic AMP EIA kit, Cayman). The detection limit of the assay was 0.1 pmol/ ml. The cAMP content was expressed as pmoles per gram of tissue wet weight. 2.5. Statistical analysis Mean amplitude of spontaneous contractions was measured prior to and following drug administration when a new steady state was reached. The results are expressed as the changes in mean amplitude of the phasic contractions and reported as percentages of the values obtained in the control (e.g. − 100% corresponds to the abolition of spontaneous activity). All data are expressed as mean values ± S.E.M. The letter n indicates the number of experimental animals. The concentration (IC50) with 95% confidence intervals (CIs) producing half maximum response was calculated using Prism 4.0, GraphPad (San Diego, CA, USA). Statistical analysis was performed by means of 2-way ANOVA, followed by Bonferroni's post hoc test. A probability value of less than 0.05 was regarded as significant. 2.6. Drugs Indomethacin, 3-isobutyl-1-methylxanthine (IBMX), forskolin, sodium nitroprusside (SNP), 2' - 5'-dideoxyadenosine (DDA) and 1H-(1,2,4) oxadiazolo-(4,3-a) quinoxalin-1-one (ODQ), zaprinast were purchased from Sigma (Milan, Italy). Indomethacin was dissolved in 2% Na2CO3, forskolin, IBMX, DDA and ODQ were dissolved in dimethyl sulfoxide (DMSO), SNP in distillated water. The working solutions were prepared freshly the day of the experiments by
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diluting the stock solutions in Krebs. Control experiments using DMSO alone showed that it did not significantly affect the contractility of the ileal segments. 3. Results 3.1. Indicaxanthin and spontaneous contractility As previously described (Baldassano et al., 2010), in mouse ileum indicaxanthin induced a concentration-dependent decrease in the amplitude of the spontaneous contractions, without affecting the frequency (control: 29 ± 2.1 cpm; 100 μM indicaxanthin: 30.2 ± 1.8 cpm; n = 6, P b 0.05) (Fig. 1). The effect was reversible after washing out. The threshold concentration of indicaxanthin was 3 μM and the maximum effect was observed at 75 μM (IC50 value: 24.9 μM; CIs 24.6– 25.2 μM) (Fig. 1). The effect of indicaxanthin was not affected by indomethacin (10 μM), DDA (10 μM), or ODQ (10 μM) (Fig. 1), indicating that indicaxanthin effects are not mediated by activation of cycloxygenase, adenylyl cyclase and NO-dependent guanylyl cyclase. Indeed, IBMX (10 μM), a non selective inhibitor of PDEs, which per se decreased the spontaneous phasic contractions (− 36.8± 5.6%; n = 5), significantly reduced the indicaxanthin inhibitory effect (Fig. 2), suggesting that the indicaxanthin relaxant action could be related to inhibition of PDEs leading to an increase of the cyclic nucleotide concentrations. To verify this hypothesis, we examined the influence of indicaxanthin on the effects induced by drugs which lead to increase the cAMP or cGMP intracellular concentrations. Forskolin (1 nM–1 μM), an adenylyl cyclase activator, reduced in a concentration-dependent
Fig. 1. (A) Typical recording showing the inhibitory effects induced by increasing concentrations of indicaxanthin on the spontaneous mechanical activity of mouse ileal longitudinal muscle. (B) Concentration–response curves for the inhibitory effects induced by indicaxanthin in the absence (control) or in the presence of ODQ (10 μM), DDA (10 μM), or indomethacin (10 μM). The inhibitory response is expressed as percent change of the resting activity (− 100% corresponds to the abolition of spontaneous activity). Each value is the mean ± S.E.M. of at least 4 separate experiments. S.E.M. is reported only if it exceeds the dimension of the symbol. The graphed values for the control curve are the means of the control data obtained before each treatment.
Fig. 2. Concentration–response curves for the inhibitory effects induced by indicaxanthin on spontaneous contractions of mouse ileal longitudinal muscle in the absence (control) or in the presence of IBMX (10 μM). The inhibitory response is expressed as percent change of the resting activity (− 100% corresponds to the abolition of spontaneous activity). Each value is mean ± S.E.M. of 5 separate experiments. S.E.M. is reported only if it exceeds the dimension of the symbol. *P b 0.05 compared with control value.
manner the amplitude of the spontaneous contractions and this effect was significantly increased by pre-treatment with IBMX (10 μM) or indicaxanthin (50 μM) (Fig. 3). IBMX (10 μM) in combination with indicaxanthin (50 μM) did not produce further additive effect (Fig. 3). Sodium nitroprusside (SNP; 1–100 μM), which acts through generation of NO, leading in turn to activation of NO-dependent guanylyl cyclase and increase of cGMP, induced a concentrationdependent reduction of the amplitude of the spontaneous contractions. The SNP-induced inhibitory effects were potentiated by IBMX (10 μM) but not by indicaxanthin (50 μM) (Fig. 4), ruling out the possibility that the pigment inhibitory effects were related to an increase of the cGMP intracellular concentration. In addition, zaprinast (10 μM), a selective inhibitor of the cGMP phosphodiesterase isoenzyme (phosphodiesterase 5), which per se reduced the spontaneous mechanical activity (− 26.2 ± 3.5%; n = 3), did not affect the indicaxanthin-induced inhibitory effects (Fig. 5).
Fig. 3. Concentration–response curves for the inhibitory effects induced by forskolin on spontaneous contractions of mouse ileal longitudinal muscle in the absence or in the presence of IBMX (10 μM) or indicaxanthin (50 μM), added to the bath alone or in combination. The inhibitory response is expressed as percent change of the resting activity (− 100% corresponds to the abolition of spontaneous activity). Each value is mean ± S.E.M. of 6 separate experiments. S.E.M. is reported only if it exceeds the dimension of the symbol. The graphed values for the control curve are the means of the control data obtained before each treatment. *P b 0.05 compared with control value.
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Fig. 4. Concentration–response curves for the inhibitory effects induced by SNP on spontaneous contractions of mouse ileal longitudinal muscle in the absence or in the presence of IBMX (10 μM) or indicaxanthin (50 μM). The inhibitory response is expressed as percent change of the resting activity (− 100% corresponds to the abolition of spontaneous activity). Each value is mean ± S.E.M. of at least 4 separate experiments. S.E.M. is reported only if it exceeds the dimension of the symbol. The graphed values for the control curve are the means of the control data obtained before each treatment. *P b 0.05 compared with control value.
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Fig. 6. Histogram showing the effects of IBMX (10 μM), alone or in combination with indicaxanthin (100 μM), on carbachol (10 μM)-evoked phasic and tonic contractions of mouse ileal longitudinal muscle. Data are expressed as a percentage of the phasic responses obtained in control conditions. Each value is mean ± S.E.M. of 5 separate experiments. *P b 0.05 compared with control value.
3.3. Effects of indicaxanthin on cAMP content 3.2. Indicaxanthin and carbachol evoked contractions As previously described (Baldassano et al., 2010), carbachol (10 μM) induced in mouse ileum a contractile response, characterized by a fast initial increase of muscular tension (phasic component) followed by a decline to a tension sustained level (tonic component), being both phases reduced by indicaxanthin. IBMX (10 μM), as well, significantly reduced both components of the carbachol-induced contraction and IBMX (10 μM) in combination with indicaxanthin (100 μM) did not produce any additive reduction of the carbacholevoked contractions (Fig. 6), suggesting that the two treatments share a similar mechanism of action.
Fig. 5. Concentration–response curves for the inhibitory effects induced by indicaxanthin on spontaneous contractions of mouse ileal longitudinal muscle in the absence (control) or in the presence of zaprinast (10 μM). The inhibitory response is expressed as percent change of the resting activity (− 100% corresponds to the abolition of spontaneous activity). Each value is mean ± S.E.M. of 3 separate experiments. S.E.M. is reported only if it exceeds the dimension of the symbol.
To verify if the pigment increases cAMP intracellular level by inhibition of PDE we measured the cAMP amount in mouse ileal muscle after indicaxanthin treatment and compared it with IBMX treatment. Indicaxanthin (50–100 μM), induced a concentrationdependent increase in the cAMP content of ileal muscle compared to the control level, although it was lesser than cAMP increase induced by IBMX (10 μM) (Fig. 7). In addition, indicaxanthin (50 μM) enhanced the cAMP increase induced by forskolin (0.1 μM) (Fig. 7). 4. Discussion The results of the present study demonstrate that indicaxanthin, extracted from cactus pear, reduces the mouse ileal longitudinal
Fig. 7. Effects of indicaxanthin, IBMX, and forskolin alone or in combination with indicaxanthin on the cAMP content of mouse ileal muscle. Each value is mean ± S.E.M. of 4 separate experiments. * P b 0.05 when compared with control value. § P b 0.05 when compared to 50 μM indicaxanthin. # P b 0.05 when compared to 100 μM indicaxanthin. $ P b 0.05 when compared to 0.1 μM foskolin.
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muscle contractility through inhibition of PDEs and increase of cAMP intracellular level. Recently, we have demonstrated that fruit extract from the yellow cultivar of cactus pear and its pigment indicaxanthin, exert a significant spasmolytic activity on the mouse intestinal smooth muscle (Baldassano et al., 2010). In fact, both cactus pear fruit extract and indicaxanthin were able to reduce reversibly and in a concentration-dependent manner the mouse ileum spontaneous phasic contractions or the carbachol-evoked contractions. Although we hypothesized that the extract inhibited the contractility of mouse ileum by interfering with pathways of intracellular Ca2+ release in the smooth muscle cells, the underlying mechanism remained to be elucidated. Cycloxygenase, and its product PGE2, may play an important role in gastrointestinal motility (Dey et al., 2006). In murine small intestine, suppression of pacemaker currents is mediated, at least in part, by PGE2 (Jun et al., 2005). Various phytochemicals (apigenin, quercetin, genistein) decrease the PGE2-induced contractions in rodent small intestine (Capasso et al., 1991; Grasa et al., 2006), and prostacyclins appear to participate in the vasorelaxant effects induced by flavonoids (Ajay et al., 2003). However, the observation that in our preparation, indomethacin, a non-selective inhibitor of cycloxygenase, did not reduce significantly the inhibitory effects of indicaxanthin, rules out a role of prostaglandins in the mechanism of action of the betalain. Cyclic nucleotides are important second messengers and have been associated with smooth muscle inhibitory effects (Diamond, 1978), including gastrointestinal smooth muscle relaxation (Makhlouf and Murthy, 1997). Intracellular concentrations of cyclic nucleotides are regulated by two families of enzymes, adenylyl- and guanylyl-cyclases which synthesize cAMP and cGMP from their corresponding triphospharilate nucleotides, and by the cyclic nucleotide phosphodiesterases which catalyze the hydrolysis of cAMP and cGMP. At first we test the hypothesis that the indicaxanthin effect was due to either activation of adenylyl cyclase or of guanylyl cyclase, leading in turn, to an increase in the intracellular cyclic nucleotide levels. Neither DDA, an adenylyl cyclase inhibitor, nor ODQ, a selective inhibitor of nitric oxide-sensitive guanylyl cyclase, did affect the indicaxanthin action, suggesting that the pigment does not stimulate cyclic nucleotide synthesis. On the contrary, a clear reduction of the indicaxanthin inhibitory effects was observed in the presence of IBMX, a non-selective inhibitor of PDEs, suggesting that PDE inhibition, and consequent increase in intracellular cyclic nucleotides, would play a role in the decrease of the ileal spontaneous contractions induced by indicaxanthin. Although the relaxation caused by different PDE inhibitors may be reduced by adenylyl cyclase inhibitors and/or guanylyl cyclase inhibitors in various smooth muscle preparations (Kaneda et al., 2004; Oger et al., 2010), other researchers have shown that adenylyl cyclase inhibitors does not affect smooth muscle relaxation induced by PDE inhibitors, similarly to our conditions (Koutsoviti-Papadopoulou et al., 2009; Lin et al., 2007). It might be argued that the reduction of the spontaneous contractions caused by IBMX pretreatment could account for the decreased inhibitory effects of indicaxanthin. However, this supposition can be ruled out because in the presence of IBMX, the reduction of the spontaneous contractions induced by forskolin, which acts stimulating adenylyl cyclase, was increased. Moreover, in our preparation the inhibitory effect of indicaxanthin on carbachol-evoked contractions was not observed in the presence of IBMX, further confirming that the two compounds share a similar mode of action. To support our hypothesis, we examined and compared the influence of indicaxanthin and IBMX on the effects of drugs able to increase cAMP or cGMP intracellular concentration, i.e. forskolin and SNP, respectively. Pretreatment with indicaxanthin enhanced the inhibitory effects induced by forskolin, similarly to IBMX pretreatment, and the application of IBMX in combination with indicaxanthin
did not produce any additive effects, providing further evidence that indicaxanthin possibly acts as an inhibitor of PDEs with consequent accumulation of cAMP. Moreover, contrarily to IBMX, indicaxanthin failed to enhance the inhibitory action of SNP, which acts through activation of guanylyl cyclase, and the inhibitory effects induced by indicaxanthin persisted in the presence of zaprinast, selective inhibitor of PDE5, suggesting that the increase of cGMP concentration is not involved in the pigment inhibitory action. This is not surprising considering that in longitudinal muscle of mouse ileum SNP inhibits the contractility through cGMP accummulation leading to activation of the large conductance Ca2+-dependent K+ channels (Zizzo et al., 2005), which are not involved in the effects induced by indicaxanthin (Baldassano et al., 2010). Finally, the observations that incubation with indicaxanthin significantly increased both basal and forskolininduced cAMP levels in mouse ileum are also consistent with the hypothesis that the indicaxanthin-induced inhibitory effects are due to an increase of the intracellular cAMP level, likely related to PDE inhibition. In particular, indicaxanthin increased the amount of cAMP in a concentration-dependent manner, although to a lesser extent than that obtained with IBMX. This finding could be explained taking into account that IBMX is a non selective PDE inhibitor, therefore it would inhibit more than one isozyme. Currently, it is known that PDEs comprise at least 11 distinct subfamilies which hydrolyze cAMP and/ or cGMP (Beavo, 1995; Keravis and Lugnier, 2010). PDE inhibitors have been reported to reduce contractility in diverse smooth muscle preparations (Kaneda et al., 2004; Polson and Strada, 1996; Qiu et al., 2001; Raeburn and Advenier, 1995) including ileal smooth muscle (Kaneda et al., 1997). Indeed, although our results provide evidence for involvement of PDEs hydrolyzing cAMP, they do not consent to conclude on the type of isozyme involved in the indicaxanthininduced response and further experiments are required to elucidate this point. The link between indicaxanthin and PDEs may be a matter of speculation. It could be hypothesized that the compound inhibit directly PDEs after traversing the membrane, as shown for betanin, a structurally related pigment (Sreekanth et al., 2007). Indeed, although the pigment is hydrophilic, it has been shown to bind to LDL either in vitro or in vivo (Tesoriere et al., 2003, 2004), as well as to microsomal membranes (Kanner et al., 2001). Moreover indicaxanthin locates to interface between hydrophobic core and hydrophilic head groups of dipalmitoyl-phosphatidylcholine vescicles (Turco Liveri et al., 2009), thus exhibiting amphiphilic properties that may allow this compound to cross the cell membranes and to each the cytosol. Lastly, incorporation of indicaxanthin in human red blood cells has been also demonstrated (Tesoriere et al., 2005). In conclusion, the present results provide evidence that the natural pigment indicaxanthin, a phytochemical isolated from the cactus pear yellow fruits, decreases the mouse ileal longitudinal muscle contractions through inhibition of PDEs and increase of cAMP levels. These findings raise the possibility of using indicaxanthin as regulator of intestinal motility in different disorders, such as abdominal cramps. Acknowledgment This work was supported by a grant from the Ministero dell'Istruzione, dell'Università e della Ricerca — Italy (ex 60%). References Abdel-Latif, A.A., 2001. Cross talk between cyclic nucleotides and polyphosphoinositide hydrolysis, protein kinases, and contraction in smooth muscle. Exp. Biol. Med. 226, 153–163. Ajay, M., Gilani, A.H., Mustafa, M.R., 2003. Effects of flavonoids on vascular smooth muscle of the isolated rat thoracic aorta. Life Sci. 74, 603–612. Amira, S., Rotondo, A., Mulè, F., 2008. Relaxant effects of flavonoids on the mouse isolated stomach: structure–activity relationships. Eur. J. Pharmacol. 599, 126–130. Amos, S., Okwuasaba, F.K., Gamaniel, K., Akah, P., Wambebe, C., 1998. Inhibitory effects of the aqueous extract of Pavetta crassipes leaves on gastrointestinal and uterine
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