Mechanisms of Relaxant Action of 3-O-Methylquercetin in Isolated Guinea Pig Trachea

• Abstract
• Abbreviations
• Abstract
• Abbreviations
• Introduction
• Materials and Methods
• Reagents and drugs
• Guinea-pig trachea
• Phosphodiesterase activity
• Statistical analysis
• Results
• Discussion
• Acknowledgements
• References

Abstract

We investigated the mechanisms of action of 3-O-methylquercetin (3-MQ), isolated from Rhamnus nakaharai (Hayata) Hayata (Rhamnaceae) which is used as a folk medicine for treating constipation, inflammation, tumors and asthma in Taiwan. The tension changes of tracheal segments were isometrically recorded on a polygraph. 3-MQ concentration-dependently relaxed histamine (30 μM)-, carbachol (0.2 μM)- and KCl (30 mM)-induced precontractions, and inhibited cumulative histamine-, and carbachol-induced contractions in a non-competitive manner. 3-MQ also concentration-dependently and non-competitively inhibited cumulative Ca²⁺-induced contractions in depolarized (K⁺, 60 mM) guinea-pig trachealis. The nifedipine (10 μM)-remaining tension of histamine (30 μM)-induced precontraction was further relaxed by 3-MQ, suggesting that no matter whether VDCCs were blocked or not, 3-MQ may have other mechanisms of relaxant action. The relaxant effect of 3-MQ was unaffected by the removal of epithelium or by the presence of propranolol (1 μM), 2′,5′-dideoxyadenosine (10 μM), methylene blue (25 μM), glibenclamide (10 μM), N ^ω-nitro-L-arginine (20 μM), or α-chymotrypsin (1 U/ml). However, 3-MQ (7.5 - 15 μM) and IBMX (3 - 6 μM), a positive control, produced parallel and leftward shifts of the concentration-response curve of forskoline (0.01 - 3 μM) or nitroprusside (0.01 - 30 μM). 3-MQ or IBMX at various concentrations (10 - 300 μM) concentration-dependently and significantly inhibited cAMP- and cGMP-PDE activities of the trachealis. The IC₅₀ values of 3-MQ were estimated to be 13.8 and 14.3 μM, respectively. The inhibitory effects of 3-MQ on both enzyme activities were not significantly different from those of IBMX, a non-selective PDE inhibitor. The above results reveal that the mechanisms of relaxant action of 3-MQ may be due to its inhibitory effects on both PDE activities and its subsequent reducing effect on [Ca²⁺]_i of the trachealis.

Abbreviations

3-MQ:3-O-methylquercetin

IBMX:3-isobutyl-1-methylxanthine

VDCCs:voltage dependent calcium channels

cAMP:adenosine 3′,5′-cyclic monophosphate

cGMP:guanosine 3′,5′-cyclic monophosphate

PDE:phosphodiesterase

Abstract

We investigated the mechanisms of action of 3-O-methylquercetin (3-MQ), isolated from Rhamnus nakaharai (Hayata) Hayata (Rhamnaceae) which is used as a folk medicine for treating constipation, inflammation, tumors and asthma in Taiwan. The tension changes of tracheal segments were isometrically recorded on a polygraph. 3-MQ concentration-dependently relaxed histamine (30 μM)-, carbachol (0.2 μM)- and KCl (30 mM)-induced precontractions, and inhibited cumulative histamine-, and carbachol-induced contractions in a non-competitive manner. 3-MQ also concentration-dependently and non-competitively inhibited cumulative Ca²⁺-induced contractions in depolarized (K⁺, 60 mM) guinea-pig trachealis. The nifedipine (10 μM)-remaining tension of histamine (30 μM)-induced precontraction was further relaxed by 3-MQ, suggesting that no matter whether VDCCs were blocked or not, 3-MQ may have other mechanisms of relaxant action. The relaxant effect of 3-MQ was unaffected by the removal of epithelium or by the presence of propranolol (1 μM), 2′,5′-dideoxyadenosine (10 μM), methylene blue (25 μM), glibenclamide (10 μM), N ^ω-nitro-L-arginine (20 μM), or α-chymotrypsin (1 U/ml). However, 3-MQ (7.5 - 15 μM) and IBMX (3 - 6 μM), a positive control, produced parallel and leftward shifts of the concentration-response curve of forskoline (0.01 - 3 μM) or nitroprusside (0.01 - 30 μM). 3-MQ or IBMX at various concentrations (10 - 300 μM) concentration-dependently and significantly inhibited cAMP- and cGMP-PDE activities of the trachealis. The IC₅₀ values of 3-MQ were estimated to be 13.8 and 14.3 μM, respectively. The inhibitory effects of 3-MQ on both enzyme activities were not significantly different from those of IBMX, a non-selective PDE inhibitor. The above results reveal that the mechanisms of relaxant action of 3-MQ may be due to its inhibitory effects on both PDE activities and its subsequent reducing effect on [Ca²⁺]_i of the trachealis.

Abbreviations

3-MQ:3-O-methylquercetin

IBMX:3-isobutyl-1-methylxanthine

VDCCs:voltage dependent calcium channels

cAMP:adenosine 3′,5′-cyclic monophosphate

cGMP:guanosine 3′,5′-cyclic monophosphate

PDE:phosphodiesterase

Introduction

3-O-Methylquercetin (3-MQ) was identified as one of the 3-methoxyflavones responsible for the pronounced antiviral activity of the extracts from Veronia amygdalina Del. (Compositae) and from different Euphorbia species, used as a traditional medicine in Central Africa [1]. The antiviral activity of 3-MQ on poliovirus RNA synthesis was reported by many authors [2], [3]. However, little is known about the influence of 3-MQ on smooth muscle. In 1986, Laekeman et al. reported the pharmacological effects of 3-MQ, including an anti-aggregatory effect on rabbit platelets, inhibitory effects on cyclooxygenase and lipooxygenase of the platelets, a vasodilating effect on rabbit central ear artery, an anti-arrhythmic effect on the left atrium and a positive chronotropic effect on the right atrium of guinea-pig. 3-MQ also potentiated the positive chronotropic effect of isoproterenol. However, they concluded that 3-MQ at a lower concentration (< 60 μM) did not significantly reveal any of the above effects [4]. Rhamnus nakaharai (Hayata) Hayata has been used as a folk medicine, similar to other Rhamnus species in Taiwan, for treating constipation, inflammation, tumors, and asthma [5]. Therefore, we are interested in investigating the mechanisms of the tracheal relaxant action of 3-MQ, a main constituent of the plant.

Materials and Methods

Reagents and drugs

3-MQ (Fig. [1]) was isolated from of Rhamnus nakahari (Hayata) Hayata [6], and identified by spectral methods, including UV, IR, MS, and NMR spectroscopic techniques (spectral data of 3-MQ are obtainable on request from the author of correspondence). The content and purity of 3-MQ were 0.01 % and 99 %, respectively. Fresh stem bark of the plant was collected at Ali, Wu-Tai Shian, Ping-Tung Hsien, Taiwan in July of 1990 and identified by Professor Chung-Nan Lin, School of Pharmacy, Kaoshiung Medical University, Kaoshiung, Taiwan. Voucher specimens (9001) are deposited in the herbarium of the School of Pharmacy, Kaoshiung Medical University. Aminophylline, carbachol, histamine, propranolol, 2′,5′-dideoxyadenosine, methylene blue, glibenclamide, N ^ω-nitro-L-arginine (L-NNA), α-chymotrypsin, nifedipine, indomethacin, ethylene glycol-bis(β-aminoethyl ether) N,N,N′,N′-tetraacetic acid (EGTA), Trizma base, DL-dithiothreitol, β-mercaptoethanol, cyclic AMP, cyclic GMP, calmodulin, Dowex resin, and Ophiophagus hannah snake venom, etc., were purchased from Sigma Chemical, St. Louis, MO, USA. [³ H]cAMP and [³ H]cGMP were purchased from DuPont, Boston, MA, USA. 3-Isobutyl-1-methylxanthine (IBMX) was purchased from Aldrich Chem., Milwaukee, WI, USA. All reagents, including KCl, were of analytical grade. Glibenclamide was dissolved in dimethyl sulfoxide (DMSO). 3-MQ, IBMX, cromakalim, forskolin, indomethacin, or nifedipine was dissolved in ethyl alcohol. Other drugs were dissolved 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

Male Hartley guinea-pigs weighing 250 to 450 g were killed by cervical dislocation and the tracheas 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 ml of normal or Ca²⁺-free Krebs solution containing indomethacin (3 μM), gassed with a 95 % O₂ - 5 % CO₂ mixture at 37 °C, 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, MgSO₄ 1.2, KH₂PO₄ 1.2, CaCl₂ 2.5, NaHCO₃ 25, 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 precontracted with histamine (30 μM), carbachol (0.2 μM) or KCl (30 mM), 3-MQ (1 - 30 μM) 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 without washout, 1 mM of aminophylline was added to standardize the tissue relaxing maximally. The relaxant potencies of 3-MQ were expressed as -logIC₅₀ values. To determine the antagonistic effects of 3-MQ 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 concentration-response curves were constructed. The maximal contractions of the tracheas without incubation of drugs or their vehicles were set as 100 %. After the tissues were preincubated with 3-MQ or its vehicle for 15 min, these two contractile agonists were also cumulatively added into the normal Krebs solution. The antagonistic potencies of 3-MQ were expressed as pD₂′ values, when the antagonistic effect on these cumulative concentration-response curves was in a non-competitive manner. In the case of isotonic high K⁺ (60 mM)-depolarized tracheal preparations, 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, cumulative Ca²⁺ (0.1 - 30 mM) was added and contractions were elicited in the depolarized trachealis. The maximal contractile response elicited by Ca²⁺ (30 mM) was taken as 100 %, and the cumulative concentration-response curve was constructed. The inhibitory effects of 3-MQ on cumulative Ca²⁺-induced contractions in isotonic high K⁺ (60 mM)-depolarized tracheas were expressed by -logIC₅₀ values. The tracheal relaxant effects of cumulative 3-MQ (1 - 100 μM) on histamine (30 μM)-induced precontraction were allowed to reach a steady state at each concentration. All antagonists, including propranolol, glibenclamide, 2′,5′-dideoxadenosine, methylene blue, L-NNA, and α-chymotrypsin or their vehicles were incubated after the precontraction reached a steady state for 15 min prior to the first addition of 3-MQ. In a similar manner, nifedipine (10 μM) was added after histamine (30 μM)-induced precontraction reached a steady state, at 15 min prior to the addition of 3-MQ (30 μM) or its vehicle. At the end of the experiment without washout, 1 mM of aminophylline was added to standardize the maximal tissue relaxation (100 %). To observe the effect of 3MQ on the relaxant responses of forskolin and nitroprusside to histamine (30 μM)-induced precontractions, 3MQ and IBMX, a positive control, were incubated for 15 min prior to the addition of histamine. Forskolin or nitroprusside was cumulatively added into the organ bath after the sustained contraction reached a constant value. At the end of the experiment, aminophylline (1 mM) was also added to maximally relax the tissue. To investigate the effects of epithelium on the relaxant response of 3-MQ to histamine (30 μM)-induced precontraction, some tracheal segments were denuded by rubbing with a moistened cotton-tipped applicator, while some were kept with the epithelium intact. At the end of 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 epithelium removal procedure [7].

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. Twenty-five microliters of the upper layer were taken for determination of enzyme activity in a final volume of 100 μl containing 40 mM Tris-HCl (pH 8.0), 2.5 mM MgCl₂, 3.75 mM mercaptoethanol, 0.1 unit calmodulin (PDE activator), 10 μM CaCl₂, and either 1 μM cAMP with 0.2 μCi [³ H]-cAMP or 1 μM cGMP with 0.2 μCi [³ H]-cGMP. In tests of enzyme inhibition, the reaction mixture contained various concentrations of 3-MQ (10 - 300 μM) or IBMX (10 - 300 μM), 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 37 ^oC. Following a 30-min incubation, the reaction was stopped by transferring the reaction vessel to a bath of boiling water for 3 min. After cooling on ice, 20 μl of a 1 mg/ml solution of Ophiophagus hannah venom were added to the reaction mixture, and the mixture was incubated at 37 ^oC for 10 min. Unreacted [³H]-cAMP or [³ H]-cGMP was removed by the addition of 500 μl of a 1-in-1 Tris-HCl (40 mM) buffer suspension of Dowex resin (1 × 8 - 200) with incubation on ice for 30 min. Each tube was then centrifuged for 2 min at 6000 rpm, and 150 μl of the supernatant were removed for liquid scintillation counting. Less than 10 % of the tritiated cyclic nucleotide was hydrolyzed in this assay.

Statistical analysis

The antagonistic effects of 3-MQ on these cumulative concentration-response curves were expressed as pD₂′ values, and the relaxing effects of forskolin and nitroprusside against histamine (30 μM)-induced precontractions were expressed as pD₂ values, according to the method described by Ariëns and van Rossum [9]. The pD₂ values are the negative logarithm of the molar concentrations of forskolin and nitroprusside at which half-relaxing effects on histamine (30 μM)-induced precontractions were observed. The pD₂′ = pD_x′ + log (x - 1), where pD_x′ is the negative logarithm of the molar concentration of 3-MQ and x is the ratio between the maximal effect of the agonist in the absence of 3-MQ and that in the presence of 3-MQ. The -logIC₅₀ value was considered to be equal to the negative logarithm of the molar concentrations of 3-MQ at which a half-inhibitory effect on Ca²⁺ (30 mM)-induced contraction was observed. The IC₅₀ value was calculated by linear regression. All values are shown as means ± SEM. The differences among these values were statistically 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 Student′s unpaired t-test. The differences were considered statistically significant if the P-value was less than 0.05.

Results

3-MQ concentration-dependently relaxed the histamine (30 μM)-, carbachol (0.2 μM)-, and KCl (30 mM)-induced precontractions (Figs. [2] A, B, C). Their -log IC₅₀ values were 4.92 ± 0.09 (n = 7), 4.67 ± 0.04 (n = 6) and 4.68 ± 0.08 (n = 9), respectively. The -log IC₅₀ value against histamine was significantly different from that against carbachol. 3-MQ (1 - 30 μM) concentration-dependently inhibited concentration-response curves of cumulative histamine and carbachol in a non-competitive manner (Figs. [3] A, B). The pD₂′ values were 5.07 ± 0.12 (n = 12), and 4.55 ± 0.21 (n = 12), respectively, which are significantly different from each other. It suggests that the antispasmodic effects of 3-MQ against histamine are more potent than those against carbachol. In isotonic Ca²⁺-free high K⁺ (60 mM)-depolarized tracheas, 3-MQ (3 - 30 μM) concentration-dependently inhibited concentration-response curves of cumulative Ca²⁺ (0.1 - 30 mM) in a non-competitive manner (Fig. [4]). The -log IC₅₀ value was 5.02 ± 0.14 (n = 18), which is not significantly different from that against KCl (30 mM)-induced precontraction. Nifedipine (1 μM), a voltage-dependent calcium channel blocker, can completely inhibit the calcium-induced contractions in the deporalized trachealis [10]. In this present experiment, nifedipine (10 μM), however, only relaxed 30.2 ± 5.6 % (n = 6) of the histamine (30 μM)-induced precontraction in the tracheas. The nifedipine (10 μM)-remaining tension of the trachea was further relaxed by 3-MQ (30 μM) to 92.3 ± 3.8 % (n = 6). This suggests that no matter whether 3-MQ blocks the voltage-dependent calcium channels (VDCCs) or not, 3-MQ may have other relaxant action mechanism(s).

However, the removal of epithelium, and the presence of antagonist, such as propranolol (1 μM), 2′,5′-dideoxyadenosine (10 μM), methylene blue (25 μM), glibenclamide (10 μM), L-NNA (20 μM), or α-chymotrypsin (1 U/ml), did not affect the log concentration-relaxing response curves of cumulative 3-MQ to histamine (30 μM)-induced precontraction in normal Krebs solution (data not shown).

In contrast, 3-MQ (7.5 - 15 μM) and IBMX (3 - 6 μM) parallelly shifted left-ward the log concentration-response curves of forskolin (Fig. [5] A, B) and nitroprusside (Fig. [5] C, D) to histamine (30 μM)-induced precontractions of the trachealis, and significantly increased the pD₂ values of forskolin, and nitroprusside (Tab. [1]). This reveals the relaxant effect of 3-MQ may be via the inhibition of cAMP- and cGMP-PDE, and the subsequent increase of these two cyclic nucleotides. Indeed, in this present study, 3-MQ or IBMX, a positive control, at various concentrations (10 - 300 μM), concentration-dependently and significantly inhibited cAMP- and cGMP-PDE activities. The inhibitory effects of 3-MQ were not significantly different from those of IBMX. The IC₅₀ values of 3-MQ or IBMX were estimated to be 13.8 or 8.3 and 14.3 or 9.9 μM, respectively. IBMX at various concentrations (10 - 300 μM) selectively inhibited neither cAMP-, nor cGMP-PDE activity. In contrast to IBMX, 3-MQ at 300 μM more selectively (P < 0.01) inhibited cGMP-, than cAMP-dependent PDE activity (Fig. [6]).

Discussion

The removal of epithelium did not affect the log concentration-relaxing response curve of cumulative 3-MQ to histamine (30 μM)-induced precontraction suggesting that the relaxant effect of 3-MQ is epithelium-independent. The log concentration-relaxing response curve of cumulative 3-MQ to histamine (30 μM)-induced precontraction was not affected by propranolol (1 μM), a non-selective β-adrenoceptor blocker, suggesting that its relaxant effect is not via the activation of β-adrenoceptor. 2′,5′-Dideoxyadenosine, an adenylate cyclase inhibitor [11], and methylene blue, a soluble guanylate cyclase inhibitor [12], also did not affect the log concentration-response curve of 3-MQ. This reveals that its relaxant effect is neither via the activation of adenylate cyclase nor via that of guanylate cyclase. Glibenclamide, an ATP-sensitive potassium channel blocker [13], also did not affect the log concentration-response curve of 3-MQ, suggesting that its relaxant effect is not via the opening of ATP-sensitive potassium channels. L-NNA (20 μM), a nitric oxide (NO) synthase inhibitor [14], did not affect the log concentration-response curve of 3-MQ, suggesting that its relaxant effect is unrelated to NO formation. α-Chymotrypsin (1 U/ml), a peptidase, also did not affect the log concentration-response curve of 3-MQ, suggesting that its relaxant effect is unrelated to the neuropeptides.

3-MQ (3 - 30 μM) concentration-dependently and non-competitively inhibited cumulative Ca²⁺-induced contractions in the depolarized (K⁺, 60 mM) trachealis. Therefore, it may inhibit Ca²⁺ influx via VDCCs opened by 60 mM KCl. For example, nifedipine, a selective VDCC blocker (15), at concentrations below 1 μM, also inhibits those contractions in a non-competitive manner. Nifedipine at 1 μM can further completely inhibit those contractions (10). In the present study, nifedipine (10 μM) only (25 - 39 %) relaxed the histamine-induced precontraction in normal Krebs solution. The nifedipine-remaining tension was further (87 - 96 %) relaxed by 3-MQ at 30 μM suggesting that no matter whether it blocked the VDCCs or not, it may have other mechanisms of relaxant action.

3-MQ concentration-dependently relaxed the histamine (30 μM)-, carbachol (0.2 μM)-, and KCl (30 mM)-induced precontractions. The -log IC₅₀ value against histamine was significantly greater than that against carbachol. The pD₂′ value of 3-MQ against cumulative histamine-induced contractions was also significantly greater than that against carbachol. This suggests that the antispasmodic effects of 3-MQ 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 M₂ receptors, a major (80 %) receptor population, via a pertussis-toxin-sensitive G protein, G_i, to inhibit adenylate cyclase activity [16] and cause an indirect contraction which attenuates the relaxant effects of 3-MQ. 3-MQ (7.5 - 15 μM) and IBMX (3 - 6 μM) parallelly shifted left-ward both the log concentration-response curves of forskolin, an activator of adenylate cyclase [17], and those of nitroprusside, an activator of guanylate cyclase (18), to histamine (30 μM)-induced precontractions of the trachealis, and significantly increased the pD₂ values of forskolin, and nitroprusside (Table [1]). This reveals that the relaxant effect of 3-MQ 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-messenger pathway is not known, but it may result from decreased intracellular Ca²⁺ ([Ca²⁺]_i). The decrease of [Ca²⁺]_i may be due to reduced influx of Ca²⁺, enhanced Ca²⁺ uptake into the sarcoplasmic reticula, or enhanced Ca²⁺ extrusion through the cell membrane (19). In this present study, indeed, 3-MQ or IBMX, a positive control, at various concentrations (10 - 300 μM), significantly inhibited cAMP- and cGMP-PDE activities. The inhibitory effects of 3-MQ were not significantly different from those of IBMX. The IC₅₀ values of 3-MQ were 13.8 and 14.3 μM, therefore, the -log IC₅₀ values of 3-MQ were 4.86 and 4.84, respectively. These -log IC₅₀ values were similar to those of 3-MQ on relaxant effects in the trachealis, precontracted by histamine, carbachol or KCl (see Results). It has been reported that there is a strong positive correlation between the IC₅₀ values of IBMX either on cAMP- [20] or on cGMP-PDE activity [21] and its EC₅₀ values for the tracheal muscle relaxation. Therefore, we cannot exclude the possibility that the relaxant effects of 3-MQ may be due to its inhibitory effect on both enzyme activities and its subsequent reducing effect on [Ca²⁺]_i of the trachealis.

Acknowledgements

This work was supported by a grant (NSC 89 - 2320-B038 - 045) from the National Council of Science, ROC.

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Prof. Wun-Chang Ko

Graduate Institute of Medical Sciences

Taipei Medical University

250 Wu-Hsing St.

Taipei 110

Taiwan

R.O.C.

Email: wc_ko@tmu.edu.tw

Fax: +886-2-2377-7639

References

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  PubMedSearch in Google Scholar
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Prof. Wun-Chang Ko

Graduate Institute of Medical Sciences

Taipei Medical University

250 Wu-Hsing St.

Taipei 110

Taiwan

R.O.C.

Email: wc_ko@tmu.edu.tw

Fax: +886-2-2377-7639