Introduction
Flavonoids are naturally occurring polyphenolic compounds with a wide distribution in the plant kingdom. They possess
an-tioxidant, antitumor, antiangiogenic, anti-inflammatory, antial-lergic, and antiviral properties [1]. Luteolin (Fig.1), a flavone found in high concentrations in celery, green pepper, perilla leaf and seed, and chamomile, has been reported to inhibit
lipopoly-Mechanisms of Relaxant Action of Luteolin in
Isolated Guinea Pig Trachea
Wun-Chang Ko1 Chwen-Ming Shih2 I-Jung Leu1 Tzu-Ting Chen1 Jung-Pei Chang1 Affiliation
1Graduate Institute of Pharmacology, College of Medicine, Taipei Medical University, Taipei, Taiwan, R.O.C.
2Department of Biochemistry, College of Medicine, Taipei Medical University, Taipei, Taiwan, R.O.C.
Correspondence
Prof. Dr. Wun-Chang Ko ´ Graduate Institute of Pharmacology ´ College of Medicine ´ Taipei Medical University ´ 250 Wu-Hsing St. ´ Taipei 110 ´ Taiwan ´ R.O.C. ´ Fax: +886-2-2377-7639 ´ E-mail: [email protected] Received August 18, 2004 ´ Accepted November 15, 2004
Bibliography
Planta Med 2005; 71: 406±411 ´ Georg Thieme Verlag KG Stuttgart ´ New York DOI 10.1055/s-2005-864133
ISSN 0032-0943 Abstract
We have investigated the mechanisms of action of luteolin, a fla-vone found in Perilla frutescens, a Chinese herbal medicine for treating asthma. In fact, luteolin occurs mostly as a glycoside in many plant species. The tension changes of tracheal segments were isometrically recorded on a polygraph. Luteolin concentra-tion-dependently relaxed histamine (30 mM)-, carbachol (0.2 mM)- and KCl (30 mM)-induced precontractions, and inhibited cumulative histamine- and carbachol-induced contractions in a non-competitive manner. Luteolin also concentration-depen-dently and non-competitively inhibited cumulative Ca2+ -in-duced contractions in depolarized (K+, 60 mM) guinea-pig tra-chealis. The nifedipine (10mM)-remaining tension of histamine (30mM)-induced precontractions was further relaxed by luteo-lin, suggesting that no matter whether VDCCs were blocked or not, luteolin may have other mechanisms of relaxant action. The relaxant effect of luteolin was unaffected by the removal of epi-thelium or by the presence of propranolol (1mM), 2¢,5¢-dideoxy-adenosine (10mM), methylene blue (25 mM), glibenclamide (10 mM), Nw-nitro-l-arginine (20 mM), or a-chymotrypsin (1 U/mL). However, luteolin (10±20 mM) produced parallel and leftward shifts of the concentration-response curve of forskolin or nitro-prusside. Luteolin or IBMX at various concentrations (10±300
mM) concentration-dependently and significantly inhibited cAMP- and cGMP-PDE activities of the trachealis. The IC50values of luteolin were estimated to be 32.4 and 34.6mM, respectively. IBMX at various concentrations (10±300mM) selectively inhib-ited neither cAMP-, nor cGMP-PDE activity. In contrast to IBMX, luteolin at 100 and 300mM more potently (P < 0.05) inhibited cGMP-, than cAMP-PDE activity. The above results indicate that the mechanisms of relaxant action of luteolin may be due to its inhibitory effects on both PDE activities and its reduction on [Ca2+]
iof the trachealis. Key words
Luteolin ´ flavonoid ´ guinea-pig trachea ´ intracellular calcium ´ phosphodiesterase
Abbreviations
IBMX: 3-isobutyl-1-methylxanthine VDCCs: voltage dependent calcium channels cAMP: adenosine 3¢,5¢-cyclic monophosphate cGMP: guanosine 3¢,5¢-cyclic monophosphate PDE: phosphodiesterase
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saccharide (LPS)-induced tumor necrosis factor-a (TNF-a) and interleukin-6 production as well as inducible nitric oxide expres-sion [2]. Luteolin has been reported to interfere with LPS signal-ling by reducing the activation of several mitogen-activated pro-tein kinase family members, and to suppress TNF-a release by in-hibiting extracellular signal-regulated kinase, p38, casein kinase 2 activation [3]. In vivo, luteolin was found to be an active anti-inflammatory and anti-allergic constituent after orally adminis-tration of the extract of Perilla frutescens leaf, a component of the Chinese medicine ªTsai-Pu-Tangº for treating cough and bron-chial asthma, in mice [4]. Luteolin also attenuates TNF-a produc-tion and intracellular adhesion molecule-1 expression, and abol-ishes infiltration of leukocytes in the lung and liver of LPS-treat-ed mice [5]. Recently, luteolin has been reportLPS-treat-ed to alleviate bronchoconstriction and airway hyperreactivity in ovalbumin sensitized mice. Therefore, Das et al. [6] concluded that luteolin could be used either as a lead molecule to identify an effective anti-asthma therapy or as a means to identify novel anti-asthma targets. We have reported that luteolin has a high potency in re-laxing tracheal smooth muscle [7]. However, little is known about the influence of luteolin on tracheal smooth muscle. Therefore we were interested to investigate its mechanisms of relaxant action.
Materials and Methods Reagents and drugs
Luteolin (Fig.1), with a purity of 99%, was purchased from Indo-fine Chemical Co., Hillsborough, NJ, USA. Aminophylline, carba-chol, histamine, propranolol, 2¢,5¢-dideoxyadenosine, methylene blue, glibenclamide, Nw-nitro-L-arginine (L-NNA), a-chymotryp-sin, nifedipine, indomethacin, forskolin, sodium nitroprusside, ethylene glycol bis(b-aminoethyl ether) N,N,N¢,N¢-tetraacetic acid (EGTA), Trizma base, dl-dithiothreitol,b-mercaptoethanol, cyclic AMP, cyclic GMP, calmodulin, Dowex resin, and Crotalus atrox snake venom, etc., were purchased from Sigma Chemical, St. Louis, MO, USA. [3H]cAMP and [3H]cGMP were purchased from Amersham Pharmacia Biotech AB, Uppsala, Sweden. 3-Iso-butyl-1-methylxanthine (IBMX) was purchased from Aldrich Chem., Milwaukee, WI, USA. All reagents, including KCl, were of analytical grade. Glibenclamide was dissolved in dimethyl sulf-oxide (DMSO). Luteolin, IBMX, forskolin, indomethacin, or nife-dipine were dissolved in ethyl alcohol. Other drugs were dis-solved in distilled water. The final concentration of ethyl alcohol or DMSO was less than 0.1% and did not significantly affect the contraction of the trachea.
Guinea-pig trachea
Under a protocol approved by the Animal Care and Use Commit-tee of Taipei Medical University, male Hartley guinea pigs weigh-ing 250 to 450 g were killed by cervical dislocation and the
tra-cheas were removed. Each trachea was cut into six segments. Each segment consisted of three cartilage rings. All segments were cut open opposite the trachealis. After the segments were randomized to minimize regional variability, they were tied at one end to holders via silk suture, placed in 5 mLof normal or Ca2+-free Krebs solution containing indomethacin (3mM), gassed with a 95% O2±5% CO2mixture at 378C, and attached by the other end of each segment to force displacement transducers (Grass FT03) for the isometric recording of tension changes on a polygraph (Gould RS3200). The composition of the normal Krebs solution was (mM): NaCl 118, KCl 4.7, MgSO41.2, KH2PO41.2, CaCl22.5, NaHCO325, and dextrose 10.1. The isotonic high K+, Ca2+-free Krebs solution consisted of the above composition without CaCl2, but 60 mM NaCl was replaced by 60 mM KCl. The tissues were suspended in normal Krebs solution under an initial tension of 1.5 g and allowed to equilibrate for at least 1 h with washing at 15-min intervals. After the tissues were precontract-ed with histamine (30mM), carbachol (0.2 mM) or KCl (30 mM), Luteolin (1±300mM) was cumulatively added to the organ bath, and its tracheal relaxant effects were allowed to reach a steady state at each concentration. At the end of the experiment with-out washwith-out, 1 mM of aminophylline was added to standardize the tissue relaxing maximally. The relaxant potencies of luteolin were expressed as -log IC50values. To determine the antagonistic effects of luteolin against contractile agonists, either histamine or carbachol was then cumulatively added to the normal Krebs solution, and the procedure was repeated until the contraction reached constancy after washout. Then, cumulative concentra-tion-response curves were constructed. The maximal contrac-tions of the tracheas without incubation of drugs or their vehi-cles were set as 100%. After the tissues were preincubated with luteolin or its vehicle for 15 min, these two contractile agonists were also cumulatively added into the normal Krebs solution. The antagonistic potencies of luteolin were expressed as pD2¢ val-ues, when the antagonistic effect on these cumulative concentra-tion-response curves was in a non-competitive manner. In the case of isotonic high K+(60 mM)-depolarized tracheal prepara-tions, normal Krebs solution was replaced after equilibration by Ca2+-free Krebs solution without EGTA, and washed with the Ca2+-free solution with 2 mM EGTA after tracheal contraction reached constancy and then incubated for 5 min. After repeating the above procedure until no contraction was observed, cumula-tive Ca2+(0.01±10 mM) was added and contractions were elicit-ed in the depolarizelicit-ed trachealis. The maximal contractile re-sponse elicited by Ca2+(10 mM) was taken as 100%, and the cu-mulative concentration-response curve was constructed. The in-hibitory effects of luteolin on cumulative Ca2+-induced contrac-tions in isotonic high K+ (60 mM)-depolarized tracheas were expressed by -log IC50values. The tracheal relaxant effects of cu-mulative luteolin (10±100mM) on histamine (30 mM)-induced precontraction were allowed to reach a steady state at each con-centration. After the precontraction reached a steady state, all antagonists, including propranolol, glibenclamide, 2¢,5¢-dideoxa-denosine, methylene blue,L-NNA, anda-chymotrypsin or their
vehicles were incubated for 15 min prior to the first addition of luteolin. Similarly, nifedipine (10mM) was added at 15 min prior to the addition of luteolin (100mM) or its vehicle, after histamine (30mM)-induced precontraction reached a steady state. At the end of the experiment without washout, 1 mM of aminophylline was added to standardize the maximal tissue relaxation (100%). Fig. 1 Chemical structure of luteolin
(mol wt: 286.23).
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To observe the effect of luteolin on the relaxant response of for-skolin or nitroprusside to histamine (30mM)-induced precon-traction, luteolin (10±20mM) was incubated for 15 min prior to the addition of histamine. Forskolin or nitroprusside was cumu-latively added into the organ bath after the sustained contraction reached a constancy. At the end of the experiment, aminophyl-line (1 mM) was also added to maximally relax the tissue. To in-vestigate the effects of epithelium on the relaxant response of lu-teolin to histamine (30mM)-induced precontraction, some tra-cheal segments were denuded by rubbing with a moistened cot-ton-tipped applicator, while some were kept with the epithelium intact. At the end of the experiment, aminophylline (1 mM) was also added to maximally relax the tissue. The denuded and intact tissues were examined using light microscopy after staining with hematoxylin and eosin to determine the effectiveness of the epi-thelium removal procedure.
Phosphodiesterase activity
The isolated trachealis was homogenized with a glass/teflon homogenizer (Glas-Col, Terre Haute, IN, USA) in 20 volumes of cold medium (pH 7.4) containing 100 mM Tris-HCl, 2 mM MgCl2, and 1 mM dithiothreitol. cAMP- and cGMP-PDE activities in the homogenate were measured by a modification of the method of Cook et al. [8]. The homogenate was centrifuged at 9500 rpm for 15 min, and the upper layer was decanted. Twen-ty-five microliters of the upper layer were taken for determina-tion of enzyme activity in a final volume of 100mLcontaining 40 mM Tris-HCl (pH 8.0), 2.5 mM MgCl2, 3.75 mM mercaptoethanol, 0.1 unit calmodulin (PDE activator), 10mM CaCl2, and either 1mM cAMP with 0.2mCi [3H]-cAMP or 1mM cGMP with 0.2 mCi [3 H]-cGMP. In tests of enzyme inhibition, the reaction mixture con-tained various concentrations of luteolin (10±300mM) or IBMX (10±300mM), a positive control. The reagents and homogenate were mixed on ice, and the reaction was initiated by transferring the mixture to a water bath at 378C. Following a 30-min incuba-tion, the reaction was stopped by transferring the reaction vessel to a bath of boiling water for 3 min. After cooling on ice, 20mLof a 1 mg/mLsolution of Crotalus atrox venom were added to the re-action mixture, and the mixture was incubated at 378C for 10 min. Unreacted [3H]-cAMP or [3H]-cGMP was removed by the ad-dition of 500mLof a 1-in-1 Tris-HCl (40 mM) buffer suspension of Dowex resin (18±200) with incubation on ice for 30 min. Each tube was then centrifuged for 2 min at 6000 rpm, and 150 mLof the supernatant were removed for liquid scintillation counting. Less than 10 % of the tritiated cyclic nucleotide was hy-drolyzed in this assay.
Statistical analysis
The antagonistic effects of luteolin on these cumulative concen-tration-response curves were expressed as pD2¢ values, and the relaxing effects of forskolin and nitroprusside against histamine (30mM)-induced precontractions were expressed as pD2values, according to the method described by Arins and van Rossum [9]. The pD2values are the negative logarithm of the molar con-centrations of forskolin and nitroprusside at which half-relaxing effects on histamine (30mM)-induced precontractions were ob-served. pD2¢ = pDx¢ + log (x ± 1), where pDx¢ is the negative loga-rithm of the molar concentration of luteolin and x is the ratio be-tween the maximal effect of the agonist in the absence of luteolin and that in the presence of luteolin. The -logIC50value was
sidered to be equal to the negative logarithm of the molar con-centrations of luteolin at which a half-inhibitory effect on ago-nist-induced precontractions, Ca2+ (10 mM)-induced contrac-tion, or cyclic nucleotide PDE activity was observed. The IC50 val-ue was calculated by linear regression. All valval-ues are shown as means SEM. The differences among these values were statisti-cally calculated by one-way analysis of variance (ANOVA), then determined by least significant difference (LSD). The difference between two values, however, was determined by use of Stu-dent's unpaired t-test. The differences were considered statisti-cally significant if the P value was less than 0.05.
Results
Luteolin concentration-dependently relaxed the histamine (30 mM)-, carbachol (0.2 mM)-, and KCl (30 mM)-induced precontrac-tions (Fig. 2). The -log IC50values were 4.65 0.11 (n = 6), 4.64 0.06 (n = 7) and 4.58 0.13 (n = 7), respectively. The -logIC50 values did not significantly differ from each other. Luteolin (3± 100 mM) concentration-dependently inhibited the concentra-tion-response curves of cumulative histamine and carbachol in a non-competitive manner (Figs. 3A,B). The pD2¢ values were 4.41 0.13 (n = 6), and 4.03 0.08 (n = 6), respectively, which significantly differ from each other. This suggests that the anti-spasmodic effects of luteolin against histamine are more potent than those against carbachol. In isotonic Ca2+-free high K+ (60 mM)-depolarized tracheas, luteolin (10±100mM) concentration-dependently inhibited the concentration-response curves of cu-mulative Ca2+ (0.01±10 mM) in a non-competitive manner (Fig. 4). The -logIC50value was 4.56 0.13 (n = 6), which is not significantly different from that against KCl (30 mM)-induced pre-contractions. Nifedipine, a voltage-dependent calcium channels (VDCCs) blocker, at 10mM, however, only relaxed by 14.4 2.9% (n = 6) the histamine (30mM)-induced precontraction in the tra-cheas. The nifedipine (10mM)-remaining tension of the trachea was further relaxed by luteolin (100mM) to 98.8 3.8% (n = 6).
Fig. 2 The relaxant effects of luteolin on, histamine (*, 30mM)-, car-bachol (l, 0.2mM)-, and KCl (~, 30 mM)-induced precontractions in guinea pig trachealis. The relaxant effects do not include those of the vehicle. Each point represents the mean SEM of 6±7 experiments. AP: aminophylline.
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This suggests that no matter whether luteolin blocks the VDCCs or not, luteolin may have other relaxant action mechanism(s). However, the removal of epithelium, and the presence of antago-nist, such as propranolol (1mM), 2¢,5¢-dideoxyadenosine (10 mM), methylene blue (25mM), glibenclamide (10 mM), l-NNA (20 mM), ora-chymotrypsin (1 U/mL), did not affect the log concentration-relaxing response curves of cumulative luteolin to histamine (30 mM)-induced precontraction in normal Krebs solution (data not shown).
In contrast, luteolin (10 ±20mM) shifted in parallel leftwards the log concentration-response curves of forskolin (Fig. 5A) and ni-troprusside (Fig. 5B) to histamine (30mM)-induced precontrac-tions of the trachealis. Furthermore, at 20mM it significantly in-creased the pD2values of forskolin, and nitroprusside (Table 1). Luteolin at various concentrations (10 ±300mM), concentration-dependently and significantly inhibited cAMP- and cGMP-PDE activities. The IC50values of luteolin were 32.4 7.0 (n = 4) and 34.6 6.3 (n = 4)mM, respectively, which did not significantly
differ from each other, though the inhibitory effects of luteolin at 100 and 300mM on cGMP-PDE were more potent (P < 0.05) than on cAMP-PDE activity (Fig. 6). The IC50values of IBMX, a po-sitive control, were estimated to be 5.5 2.5 (n = 4) and 16.3 7.3 (n = 4)mM, respectively, which also did not significantly dif-fer from each other. In contrast to luteolin, IBMX at various con-centrations (10±300 mM) selectively inhibited neither cAMP-, nor cGMP-PDE activity (Fig. 6).
Discussion
The removal of epithelium did not affect the log concentration-relaxing response curve of cumulative luteolin to histamine (30 mM)-induced precontraction suggesting that the relaxant effect of luteolin is epithelium-independent. The log concentration-re-laxing response curve of cumulative luteolin to histamine (30 mM)-induced precontraction was not affected by propranolol (1 mM), a non-selective b-adrenoceptor blocker, suggesting that its relaxant effect is not via the activation ofb-adrenoceptor. 2¢,5¢-Dideoxyadenosine, an adenylate cyclase inhibitor [10], and me-thylene blue, a soluble guanylate cyclase inhibitor [11], also did not affect the log concentration-response curve of luteolin. This reveals that its relaxant effect is neither via the activation of ade-nylate cyclase nor via that of guaade-nylate cyclase. Glibenclamide, an ATP-sensitive potassium channel blocker [12], also did not af-fect the log concentration-response curve of luteolin, suggesting that its relaxant effect is not via the opening of ATP-sensitive po-tassium channels.L-NNA (20 mM), a nitric oxide (NO) synthase
inhibitor [13], did not affect the log concentration-response curve of luteolin, suggesting that its relaxant effect is unrelated to NO formation.a-Chymotrypsin (1 U/mL), a peptidase, also did not af-fect the log concentration-response curve of luteolin, suggesting that its relaxant effect is unrelated to the neuropeptides.
Luteolin (10±100mM) concentration-dependently and non-com-petitively inhibited cumulative Ca2+-induced contractions in the Fig. 3 The inhibitory effects of luteolin (*, vehicle; l, 3mM; !, 30
mM; n, 100 mM ) on cumulative histamine (A)-and carbachol (B)-in-duced contractions in guinea pig trachealis in normal Krebs solution. Each point represents the mean SEM of 6 experiments.
Fig. 4 The inhibitory effects of luteolin (*, vehicle;~, 10mM; !, 30 mM, n, 100 mM) on cumulative calcium-induced contractions in guinea pig trachealis depolarized by KCl 60 mM in Ca2+-free medium. Each point represents the mean SEM of 6 experiments.
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depolarized (K+, 60 mM) trachealis. Therefore, it may inhibit Ca2+ influx via VDCCs opened by 60 mM KCl. For example, nifedipine, a selective VDCCs blocker [14], at concentrations below 1mM, also inhibits those contractions in a non-competitive manner. Nifedipine at 1mM can completely inhibit those contractions. In the present study, nifedipine (10mM) relaxed the histamine-in-duced precontraction in normal Krebs solution by only 14.4%.
The nifedipine-remaining tension was further (98.8%) relaxed by luteolin at 100 mM suggesting that no matter whether it blocked the VDCCs or not, it may have other mechanisms of re-laxant action. Luteolin concentration-dependently relaxed the histamine (30mM)-, carbachol (0.2 mM)-, and KCl (30 mM)-in-duced precontractions. The -logIC50values against these three contractile agents did not significantly differ from each other, suggesting that the ability of luteolin to inhibit calcium influx from the extracellular space may be similar. It has been reported that tonic, but not phasic, contraction is maintained by calcium influx [15]. However, The pD2¢ value of luteolin against cumula-tive histamine-induced contractions was significantly greater than that against carbachol. This suggests that the antispasmo-dic effects of luteolin against histamine are more potent than those against carbachol. Although the exact reason is not clear, it has been established that carbachol may activate muscarinic M2receptors, a major (80%) receptor population, via a pertus-sis-toxin-sensitive G protein, Gi, to inhibit adenylate cyclase ac-tivity [16] and cause an indirect contraction which attenuates the relaxant effects of luteolin. Luteolin (10 ±20mM) shifted in parallel leftward both the log concentration-response curves of forskolin, an activator of adenylate cyclase [17], and those of ni-troprusside, an activator of guanylate cyclase [18], to histamine (30mM)-induced precontractions of the trachealis, and signifi-cantly increased the pD2values of forskolin and nitroprusside (Table 1). This reveals that the relaxant effect of luteolin may be via the inhibitions of cAMP- and cGMP-PDE, and the subsequent increase of these two cyclic nucleotides. The increased cAMP or cGMP level subsequently activates cAMP- or cGMP-dependent protein kinase which may phosphorylate and inhibit myosin light-chain kinase, thus inhibiting contraction [19]. The precise mechanism by which relaxation is produced by this second-mes-senger pathway is not known, but it may result from decreased intracellular Ca2+([Ca2+]
i). The decrease of [Ca2+]imay be due to reduced influx of Ca2+, enhanced Ca2+uptake into the sarcoplas-mic reticula, or enhanced Ca2+extrusion through the cell mem-brane [19]. In this present study, indeed, luteolin or IBMX, a posi-Fig. 5 The potentiating effects of luteolin (*, vehicle; l, 10mM;~,
20mM) on the relaxant responses of cumulative forskolin (A) and nitro-prusside (B) to the histamine (30mM)-induced precontractions in the guinea pig trachealis. Each point represents the mean SEM of 6 ex-periments. AP: aminophylline.
Fig. 6 The inhibitory effects of luteolin (&, n) and IBMX (*, l), a po-sitive control, on cAMP-(*, &) and cGMP-PDE (l , n) activities. The inhibitory effects do not include those of their vehicle. Each point re-presents the mean SEM of at least 4 experiments. * P < 0.05, ** P < 0.01 when compared with corresponding value on cAMP-PDE activity. Table 1 The pD2values of forskolin and nitroprusside against
hista-mine (30mM)-induced precontractions in the absence and presence of luteolin Forskolin Nitroprusside Luteolin Vehicle 7.33 0.08 (6) 5.85 0.18 (6) 10mM 7.57 0.19 (6) 6.20 0.18 (6) 20mM 7.81 0.23 (6)* 6.39 0.07 (6)*
Values are presented as means SEM (n); n is the number of experiments. * P < 0.05 when compared with their corresponding values of vehicle.
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tive control, at various concentrations (10±300mM), significant-ly inhibited cAMP- and cGMP-PDE activities. The -logIC50values of luteolin were 4.49 and 4.46, respectively. These -logIC50values were similar to those of luteolin on relaxant effects in the tra-chealis, precontracted by histamine, carbachol or KCl (see Re-sults). It has been reported that there is a strong positive correla-tion between the IC50values of IBMX either on cAMP- [20] or on cGMP-PDE activity [21] and its EC50values for the tracheal mus-cle relaxation. Therefore, we cannot exclude the possibility that the relaxant effects of luteolin may be due to its inhibitory effect on both enzyme activities and its subsequent reducing effect on [Ca2+]
iof the trachealis.
Acknowledgements
This work was supported by a grant (NSC 87-2314-B038-039) from the National Science Council, Taiwan, ROC.
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