Effect of quercetin on colon contractility and L-type Ca 2+ channels in colon smooth muscle of guinea-pig

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1 Acta Physiologica Sinica, December 25, 2009, 61 (6): Research Paper Effect of quercetin on colon contractility and L-type Ca 2+ channels in colon smooth muscle of guinea-pig HUANG Wei-Feng 1, OUYANG Shou 1,*, LI Shi-Ying 1, LIN Yan-Fei 1, OUYANG Hui 1, ZHANG Hui 1, LU Chun-Jing 2 1 Xiamen Medical Research Institute, Xiamen , China; 2 Department of Blood Transfusion, Maternal and Child Health Hospital of Xiamen, Xiamen , China Abstract: The aim of the present study was to investigate the effects of quercetin on colon contractility and voltage-dependent Ca 2+ channels in the single smooth muscle cell isolated from the proximal colon of guinea-pig and to clarify whether its effect on L-type Ca 2+ current (I Ca,L ) would be related to its myorelaxing properties. Colon smooth muscle strips were used to take contractile tension recordings. Smooth muscle cells were freshly isolated from the proximal colon of guinea-pig by means of papain treatment. I Ba,L (barium instead of calcium as current carrier) was measured by using whole-cell patch-clamp techniques. The results showed that quercetin relaxed colon muscle strips in a concentration-dependent manner and antagonized the contractile effect of acetylcholine and neostigmine. Preincubation with indomethcin [cyclooxygenase (COX) inhibitor] and methylene blue [guanylate cyclase (GC) inhibitor] significantly attenuated the relaxing effect of quercetin, respectively. Quercetin increased I Ba,L in a concentration- [EC 50 = (7.59±0.38) μmol/l] and voltage-dependent pattern, and shifted the maximum of the current-voltage curve by 10 mv in the depolarizing direction without modifying the threshold potential for Ca 2+ influx. Quercetin shifted the steady-state inactivation curve toward more positive potentials by approximately 3.75 mv without affecting the slope of activation and inactivation curve. H-89 (PKA inhibitor) abolished quercetininduced I Ba,L increase, while camp enhanced the quercetin-induced I Ba,L increase. The patch-clamp results proved that quercetin increased I Ba,L via PKA pathway. It is therefore suggested that the relaxing effect of quercetin attributes to the interaction of GC and COX stimulation, as well as the antagonism effect on acetylcholine, which hierarchically prevails over the increase in the Ca 2+ influx to be expected from I Ca,L stimulation. Key words: quercetin; colon; L-type Ca 2+ channels; guinea-pig 槲皮素对豚鼠结肠平滑肌收缩性和钙通道的影响 黄伟锋 1, 欧阳守 1,*, 李世英 1, 林燕飞 1, 欧阳晖 1, 张慧 1, 卢春敬 2 1 厦门市医药研究所, 厦门 ; 2 厦门市妇幼保健院输血科, 厦门 摘要 : 本文研究槲皮素对豚鼠结肠的收缩性及对结肠平滑肌细胞电压依赖性钙通道的影响, 以探讨槲皮素舒张结肠平滑肌的机制 实验中用张力换能器记录结肠肌条的收缩, 用木瓜蛋白酶分离近端结肠平滑肌细胞, 全细胞膜片钳技术记录 L 型钡电流 ( I Ba,L, 为了改善钙衰减, 细胞外液中用钡离子代替钙离子 ) 实验观察到, 槲皮素浓度依赖性地抑制结肠收缩, 且能拮抗乙酰胆碱和新斯的明 (neostigmine) 对结肠的收缩作用 吲哚美辛 [ 环氧合酶 (cyclooxygenase, COX) 抑制剂 ] 和亚甲蓝 [ 鸟苷酸环化酶 (guanylate cyclase, GC) 抑制剂 ] 可以拮抗槲皮素舒张结肠平滑肌的作用 槲皮素浓度 [EC 50 = (7.59±0.38) μmol/l] 和电压依赖性地增大结肠平滑肌细胞的 I Ba,L, 使电流 - 电压曲线的最大激活电压向去极化方向移动 10 mv, 但不改变 I Ba,L 的阈电位 槲皮素向去极化方向移动稳态失活曲线约 3.75 mv, 但不改变激活曲线和失活曲线的斜率 H-89 [ 蛋白激酶 A (protein kinase A, PKA) 抑制剂 ] 能够阻断槲皮素增大 I Ba,L 的作用, 而 camp 能够增强槲皮素增大 I Ba,L 的作用 这些结果说明槲皮素通过 PKA 途径增大 I Ba,L 槲皮素通过 GC 和 COX 途径以及拮抗乙酰胆碱的作用来抑制结肠的收缩, 而其增大钙离子内流的作用不足以抵 Received Accepted This work was supported by the Key Project of Department of Science and Technology of Fujian Province, China (No. 2009D026), the Science Foundation of Health Bureau of Xiamen Municipality (No. WZK21, 3502z ) and the Science Foundation of Health Department of Fujian Province, China (No ) * Corresponding author. Tel: ; Fax: ; xmmri@xmu.edu.cn

2 568 Acta Physiologica Sinica, December 25, 2009, 61 (6): 消上述的抑制作用, 从而表现为槲皮素对结肠的舒张作用 关键词 : 槲皮素 ; 结肠 ;L- 型钙通道 ; 豚鼠中图分类号 :R329 The flavonol quercetin is widely distributed in plants and, therefore, the most abundant flavonoid ingested by human and herbivorous animals. Interest in dietary phenolics has been increased greatly recently, owing to their antioxidant properties (free radical scavenging and metal chelating) and their possible beneficial implications in human health, such as in the treatment and prevention of cancer, cardiovascular diseases, and other diseases [1-4]. During the last two decades, it has been shown that quercetin is capable of inducing both endothelium-dependent [5-7] and endothelium-independent vasorelaxation in vitro [7-13]. However, most of these studies involve only the cardiovascular system. Since quercetin has protective effects on gastrointestinal system [14], we investigated the effects of quercetin on colon functions in vitro by comparing their mechanical and electrophysiological actions. Until now, the effects of this flavonoid on the electrophysiological properties of Ca 2+ channels in colon smooth muscle has not been investigated, although a decreased transmembrane Ca 2+ influx [15] was indicated as a possible mechanism for its relaxing and vasodilator effects. However, Summanen et al. have demonstrated that quercetin increases Ca 2+ current (I Ca ) in clonal rat pituitary GH 4 C 1 cells [16]. Moreover, quercetin induced stimulation of L-type Ca 2+ current (I Ca,L ) in GH3 [17] and in vascular smooth muscle cells [7,18,19]. Therefore, the aim of the present study was to investigate the effects of quercetin on voltage-dependent Ca 2+ channels in the single smooth muscle cell isolated from proximal colon of guinea-pig in an attempt to clarify whether quercetin might modulate I Ca of colon and whether this effect might contribute to its myorelaxing properties. 1 MATERIALS AND METHODS 1.1 Animal and chemicals Principles of laboratory animal care (NIH publication No , revised 1996) were followed. Male guinea-pigs were purchased from Shanghai Shengwang Animal Company (Certificate Number: SCXK: ), Grade II, g. Papain, CsCl, tetraethylammonium (TEA), CsOH, Na 2 ATP, GTP, Creatine phosphate, H-89 [protein kinase A (PKA) inhibitor], chelerythrine [protein kinase C (PKC) inhibitor], N-nitro-L-arginine methyl ester (L-NAME, NO synthase inhibitor), acetylcholine, indomethacin [cyclooxygenase (COX) inhibitor], methylene blue [guanylate cyclase (GC) inhibitor], camp, genistein (TPK inhibitor), and quercetin were purchased from Sigma. Quercetin was prepared as 0.1 mol/l stock solution in DMSO and stored at -20 ºC. DTT (BBI), HEPES, DMSO, bovine serum albumin were from Shanghai Sangon Biological Engineering Technology & Services Co., LTd. The other chemicals, if not stated, were all purchased from Chinese reagent companies. 1.2 Contractile tension recording Guinea-pigs were killed by cervical dislocation and distal colons were quickly excised. The colons were cut into several segments with the length of about 1.5 cm and serosa was dissected free of the muscle layer. Then the strips were rinsed by Krebs-Henseleit physiological salt solution [PSS (in mmol/l): NaCl 120.6, KCl 5.9, CaCl 2 2.5, MgCl 2 1.2, NaHCO , NaH 2 PO 4 1.2, glucose 11.5, ph 7.4 adjusted with NaOH] and were fixed on the perfused groove electrodes and energy transducers with the proximal ends upward and the distal ends downward, and stabilized in the oxygenated perfusates (PSS solution) containing 95% O 2 and 5% CO 2 at 37 ºC. Contractile tension was recorded using MP-150 (Biopac systems, USA) and processed with AcqKnowledge 3.7. The strips were allowed to equilibrate for 30 min at a resting tension of 0.5 g to test spontaneous contractions. During this equilibration period, PSS solution was changed every 15 min and the passive tension was re-adjusted to 0.5 g. Following the equilibration period, the strips were stimulated with 60 mmol/l K + (KPSS, replacing equimolar NaCl with KCl in PSS) until a sustained response was obtained (~15 min) in order to test their contractile capacity. Only the strips with regular and sustained response were used in the next session. The strips were then washed and equilibrated for another min before testing the various experimental settings. The control preparations were treated with the vehicle only. Cumulative concentration-response curves for quercetin were made. Amplitude and integral (area of the curve) were employed as indices. Each concentration of quercetin was left in contact with the strip for long enough to allow full development of the effect. Relaxation was then evaluated as a percentage of the initial tension.

3 HUANG Wei-Feng et al: Quercetin on Colon Smooth Muscle and L-type Ca 2+ Channels Cell dissociation Colon smooth muscle cells were enzymatically isolated using modified procedures described previously [20]. A segment of proximal colon of guinea-pigs (n=40), about 2 cm aboral to cecum, were rapidly excised. Under anatomical microscope, smooth muscle strips were dissected out, cut into small pieces and incubated for 30 min in low calcium solution containing (in mmol/l): HEPES 10, NaCl 135, KCl 6, CaCl , MgCl 2 1.2, ph 7.4 adjusted with NaOH. Pieces of muscles were then transferred to low calcium solution containing papain (3 g/l), DTT (2 g/l) and bovine serum albumin (2 g/l). Tissues were incubated at 36 ºC in enzyme solution for 15 min and then suspended in enzyme-free low calcium solution. Tissue pieces were gently agitated to create a cell suspension. Dispersed cells were stored at 4 ºC for later use. Experiments were performed at ºC within 10 h. 1.4 Whole-cell patch-clamp recording Relaxed single colon smooth muscle cells, with smooth appearance and spindle shape observed under an inverted microscope (IX70, Olympus), were used. These cells were superfused with bath solution containing (in mmol/l): HEPES 10, NaCl 135, TEA-Cl 6, CaCl , BaCl 2 10, MgCl 2 1.2, glucose 10, ph 7.4 adjusted with NaOH, in self-made small volume chamber in which solution can be exchanged completely in 30 s. Conventional whole-cell patch-clamp method was employed to voltage-clamp smooth muscle cells. Patch clamp micropipettes (borosilicate glass, WPI) were pulled by a programmable puller (P-97, Sutter Instruments) and their tips fire-polished (CPM-2, ALA Co.). The resistance of pipettes ranged 3-5 MΩ when filled with internal solution containing (in mmol/l): HEPES 10, CsCl 120, MgCl 2 1, TEA-Cl 10, EGTA 10, Na 2 ATP 5, Creatine phosphase 1.2, ph 7.2 adjusted with CsOH. Currents were amplified with a low-noise, high-performance Axopatch 200B (Axon Instruments), driven by a DELL computer in conjunction with an A/D, D/A board (Digidata 1320A interface, Axon Instruments, USA). Data were filtered at 1 khz, digitized at 5 khz and analyzed with pclamp software (version 8.2). Cell membrane capacitance (C m ) was obtained from the capacity compensation control of the amplifier after the whole-cell capacity current was eliminated. The series resistance was electronically compensated, up to 80%. Liquid junction potential was totally compensated. K + currents were blocked with internal solution containing TEA, Cs +, and external solution containing TEA, respectively. To better resolve the I Ca,L, Ba 2+ instead of Ca 2+ was added to the external solution as the current carrier. Therefore, we termed the actual recorded current as I Ba,L afterwards. I Ba,L was measured over a range of test potentials (400 ms) from -80 to 60 mv with 10 mv increments from a holding potential (V h ) of -80 mv, after each test pulse the clamp potential again to holding potential; and the interval between each pulse was 5 s. Data were collected once the current amplitude had been stabilized (usually 5-10 min after the whole-cell configuration had been obtained). I Ba,L did not run down during the following min under these conditions. Steady-state inactivation curves were obtained using the double-pulse protocol. Once various levels of the conditioning potential had been applied for 5 s, a test pulse (400 ms) up to 10 mv was delivered to evoke the current. Activation curves were derived from the current-voltage relationships. The points for I/I max were plotted against the membrane potential as relative amplitude. A two-pulse protocol was applied to measure the time course of recovery from inactivation: one-second clamp pulses to 10 mv from a V h of -80 mv were followed by a return to the V h of variable duration to allow some channels to recover from inactivation. A second pulse (400 ms) to 10 mv was delivered to determine how much recovery had occurred during the time interval. Current values were corrected for leakage using 300 μmol/l Cd 2+ which was assumed to completely block I Ba,L. 1.5 Statistical analysis Data were expressed as mean±sem. Differences in the data were evaluated by paired or independent t -tests when appropriate. P values less than 0.05 were taken as a statistically significant difference. Software Origin 7.0 and SigmaPlot 10.0 were used for statistical analysis and graph plotting. 2 RESULTS 2.1 Effect of quercetin on colon muscle contractility Colon smooth muscle of distal segment spontaneously contracted with frequency of (3.94±0.56) cycle/min. Quercetin relaxed colon in a concentration-dependent manner. Fitting with Hill equation, the IC 50 of amplitude and integral were (12.03±4.36) μmol/l (n=8, P=0.07), (5.06±1.52) μmol/l (n=7, P=0.04) respectively (Fig. 1). And the 95% confidence interval of amplitude and integral were μmol/l and μmol/l. In addition, quercetin antagonized the contraction induced by high K + (n=5,

4 570 P<0.01, Student s t test for paired samples, Table 1). Fig. 1. Effect of quercetin on colon muscle contractility. A: Original recordings of colon at contractile tension. Quercetin (50 μmol/l) relaxed colon contractility significantly. B: Quercetin relaxed colon in a concentration-dependent manner. The IC 50 of amplitude and integral was (12.03± 4.36) μmol/l (n=8), (5.06±1.52) μmol/l (n=7) respectively. Integral: area of the curve. Data points are mean±sem. Acta Physiologica Sinica, December 25, 2009, 61 (6): The antagonism effect of quercetin on acetylcholine and neostigmine To investigate the possible mechanisms of quercetin on colon smooth muscle, acetylcholine (1 μmol/l) and neostigmine (1 μmol/l) were pretreated respectively. Quercetin antagonized contractile effect of acetylcholine (Integral: 12.91%±1.48% of acetylcholine, n=8, P<0.01; Amplitude: 13.65%±1.57% of acetylcholine, n=8, P<0.01, Student s t test for paired samples, Table 1) and neostigmine (Integral: 16.06%±1.85% of neostigmine, n=6, P<0.01; Amplitude: 21.40%±2.01% of neostigmine, n=6, P<0.01, Student s t test for paired samples, Table 1). Moreover, quercetin enhanced 10 μmol/l atropine s relaxing effect (n=5, P<0.05, Student s t test for paired samples, Table 1). 2.3 Pharmacological interaction between indomethacin, methylene blue and quercetin In order to test pharmacological interactions between indomethacin, methylene blue and quercetin, the effects of indomethacin and methylene blue were studied on colon muscle. Indomethacin (10 μmol/l) alone did not affect colon contractility (n=6, P>0.05, Student s t test for unpaired samples), however, preincubation with indomethacin attenuated the relaxing effect of quercetin (n=6, P<0.01, Student s t test for unpaired samples, Table 2). All in the same, pretreatment with methylene blue (10 μmol/l) attenuated the relaxant effect of quercetin significantly (n=8, P<0.01, Student s t test for unpaired samples, Table 2). 2.4 The effect of L-NAME and chelerythrine on the relaxant effect of quercetin L-NAME (100 μmol/l) alone enhanced colon contractile capacity (n=9, P<0.05, Student s t test for unpaired samples, Table 2), while preincubation with L-NAME did not influence the relaxant effects of quercetin (n=9, P>0.05, Student s t test for unpaired samples). PKC inhibitor (chelerythrine, 10 μmol/l) did not enhance the relaxant effects of querce- Table 1. Effect of pretreatment with acetylcholine, neostigmine or atropine on the myorelaxing action of quercetin, and effect of quercetin on extracellular high K + Group n Integral (%, of control) Amplitude (%, of control) Quercetin ± ±1.89 Acetylcholine ± ±15.14 Acetylcholine+Quercetin ± 4.23 ** 19.13±3.85 ** Neostigmine ± ±20.47 Neostigmine+Quercetin ±1.86 ## 27.94±3.26 ## Atropine ± ±3.83 Atropine+Quercetin ± ± High K ± ±9.87 High K + +Quercetin ±4.01 ΔΔ 31.24±4.42 ΔΔ The control was treated with the vehicle only. Quercetin: 50 μmol/l; Acetylcholine: 1 μmol/l; Neostigmine: 1 μmol/l; Atropine: 10 μmol/l; High K + : 30 mmol/l. Data are mean±sem. ** P<0.01 vs Acetylcholine; ## P<0.01 vs Neostigmine; + P<0.05 vs Atropine; ΔΔ P<0.01 vs High K +. Student s t test for paired samples.

5 HUANG Wei-Feng et al: Quercetin on Colon Smooth Muscle and L-type Ca 2+ Channels 571 Table 2. Effect of pretreatment with L-NAME, methylene blue, chelerythrine or indomethacin on the myorelaxing action of quercetin Group n Integral (%, of control) Amplitude (%, of control) Quercetin ± ±1.89 Indomethacin ± ±8.75 Indomethacin+Quercetin ± ±9.30 Methylene blue ± ±8.15 Methylene blue+quercetin ± ±5.44 Chelerythrine ± ±8.34 Chelerythrine+Quercetin ±1.96 ΔΔ 16.15±1.80 ΔΔ L-NAME ±8.75 * ±9.17 * L-NAME+Quercetin ±3.32 ## 20.51±2.43 ## The control was treated with the vehicle only. Quercetin: 50 μmol/l; L-NAME: 100 μmol/l; Methylene blue: 10 μmol/l; Chelerythrine: 10 μmol/l; Indomethacin: 10 μmol/l. Data are mean±sem. * P<0.05 vs control; ## P<0.01 vs L-NAME; ++ P<0.01 vs Quercetin; P<0.01 vs Methylene blue; ΔΔ P<0.01 vs Chelerythrine; P<0.01 vs Quercetin. Student s t test for unpaired samples or paired samples. tin at contractile tension recording (n=5, P>0.05, Student s t test for unpaired samples, Table 2). 2.5 Effect of quercetin on I Ba,L 10 μmol/l quercetin, when applied to single smooth muscle cells, caused a significant and sustained increase of I Ba,L from (-9.55±0.75) pa/pf (control) to (-12.58±0.72) pa/ pf (n=42, P<0.01, Student s t test for unpaired samples). The stimulating effect of quercetin on I Ba,L was reversible upon wash-out (Fig. 2A). Quercetin increased I Ba,L in a concentration-dependent manner with a EC 50 of (7.59±0.38) μmol/l (Fig. 2B). We recorded no T-type Ca 2+ current in guinea-pig colon smooth muscle cells (data not shown). 2.6 Effect of quercetin on I-V curves of I Ba,L There was a typical recording of I Ba,L under control conditions and following the addition of 10 μmol/l quercetin (Fig. 3A, B). The current-voltage curve (Fig. 3C) showed that quercetin significantly increased the peak inward current in the range of -20 to 60 mv (n=15, P<0.05, Student s t test for unpaired samples), and shifted the maximum by 10 mv in the depolarizing direction without, however, varying the threshold potential at about -30 mv. 2.7 Effect of quercetin on steady-state inactivation and activation curves of I Ba,L The voltage-dependence of quercetin stimulation was analyzed by determining the steady-state inactivation and activation curves for I Ba,L. The activation curves were fitted to the Boltzmann equation (Fig. 4A). The 50% activation potential [(-2.06±0.88) mv, control, n=10] was not changed by quercetin [(-1.28± 0.38) mv, n=8, P>0.05, Student s t test for unpaired samples]. Moreover, the slope factor (5.71±0.71, control) was not affected by quercetin (5.34±0.31, P>0.05, Fig. 2. The effect of quercetin on I Ba,L in single colon smooth muscle cells. A: Original recordings of conventional whole-cell I Ba,L in colon smooth muscle cells elicited with 400-ms clamp pulses to 10 mv from a V h of -80 mv (see schematic diagram), measured in the absence (control) or presence of quercetin (10 μmol/l). I Ba,L suppression by 5 μmol/l nifedipine is also shown. B: Concentration-dependent effect of quercetin on I Ba,L. On the ordinate scale, response is reported as relative to control. The curve shows the best fit of the points [EC 50 =(7.59±0.38) μmol/l]. Data points are mean±sem.

6 572 Acta Physiologica Sinica, December 25, 2009, 61 (6): Fig. 3. The effect of quercetin on current-voltage curves of I Ba,L in colon smooth muscle cells. A, B: Original recordings of conventional whole-cell I Ba,L in colon smooth muscle cells elicited with 400-ms clamp pulses from a V h of -80 mv to test potentials of -80 to 60 mv (see schematic diagram), measured in the absence (control, A) or presence of quercetin (10 μmol/l, B). C: Current-voltage curves of I Ba,L influenced by quercetin. Data points are mean±sem. (n=15, * P< 0.05 vs control). Student s t test for unpaired samples. Fig. 4. The effects of quercetin on both the activation and inactivation curves of I Ba,L in colon smooth muscle cells. Activation curves (A) were obtained from the current-voltage relationships of Fig. 3 and fitted to the Boltzmann equation. The steady-state inactivation curve (B) was obtained using the double-pulse protocol (condition potential, from -80 to 40 mv, 5 s; test potential, 10 mv, 400 ms, C) in the absence (control) and presence of 10 μmol/l quercetin. Peak current values were used. The current measured during the test pulse was plotted against membrane potential and expressed as relative amplitude, which were fitted to the Boltzmann equation. Each point represents the mean±sem.

7 HUANG Wei-Feng et al: Quercetin on Colon Smooth Muscle and L-type Ca 2+ Channels 573 Student s t test for unpaired samples). 10 μmol/l quercetin shifted the steady-state inactivation curve to more positive potentials by approximately 3.75 mv (Fig. 4B). The 50% inactivation potentials, evaluated by means of Boltzmann fitting, were (-41.77±0.95) mv (control, n=7) and (-37.60±1.07) mv (quercetin, n=6; P= , Student s t test for unpaired samples), respectively. On the contrast, the slope factor (-15.23±0.79, control, n=7) was not affected by quercetin (-13.03±0.75, n=6, P=0.0672, Student s t test for unpaired samples). 2.8 Effects of quercetin on recovery from inactivation Fitting with exponent equation I/I max =1- exp(-t/τ), quercetin (10 μmol/l) did not influence the rate of recovery from inactivation (P=0.1396, Student s t test for unpaired samples, Fig. 5). The value of τ was (196.08±23.07) ms (n=6) in control session in comparison of (263.16±34.63) ms (n=5) with quercetin. (n=5, P= , Student s t test for paired samples, Fig. 6A). Moreover, 20 μmol/l camp induced an additional increase of I Ba,L [from (-12.15±0.75) pa/pf to (-15.57± 0.81) pa/pf, n=5, P=0.0147, Student s t test for paired samples, Fig. 6B]. Chelerythrine (1 μmol/l) did not affect quercetin-induced activation of I Ba,L [from (-11.85±0.89) pa/pf to (-11.43±0.93) pa/pf, n=5, P=0.7526, Student s 2.9 The pathway of quercetin's stimulating I Ba,L In order to investigate via which pathway quercetin induced the activation of I Ba,L, genistein, H-89 and chelerythrine were used. Quercetin (10 μmol/l) caused a significant increase of I Ba,L, however, the subsequent addition of H-89 (5 μmol/l) abolished the effect of quercetin Fig. 5. The effects of quercetin on recovery from inactivation of L- type Ca 2+ channel. Recovery at V h of -80 mv was obtained using a double-pulse protocol (see schematic inset). One-second clamp pulse to 10 mv from a V h of -80 mv (pulse 1; P1) was followed by a second pulse (400 ms) to 10 mv from a V h of -80 mv (pulse 2; P2). Delay (t) between P1 and P2 was variable in duration. Relative peak I Ba,L, measured in the absence (control) or presence of quercetin (10 μmol/l), was plotted against recovery interval. Each point represents the mean±sem. (control, n=6; quercetin, n=5, P>0.05). Student s t test for unpaired samples. Fig. 6. The effect of PKA, PKC, tyrosine protein kinase (TPK) on quercetin s increasing I Ba,L in colon smooth muscle cells (original recordings). A: PKA inhibitor (H-89, 5 μmol/l) could abolish the quercetin s effect (n=5, P=0.0072). B: camp (20 μmol/l) could enhance the quercetin s effect (n=5, P=0.0147). C: Chelerythrine (1 μmol/l) couldn t influence the quercetin s effect on I Ba,L in colon smooth muscle cells (n=5, P=0.7526). D: Genistein (25 μmol/l) inhibited I Ba,L (n=6, P=0.0153), but pretreatment with it did not abolish the stimulating effect of quercetin on I Ba,L (n=5, P<0.05 vs control). Student s t test for paired samples or unpaired samples.

8 574 t test for paired samples, Fig. 6C]. Genistein (25 μmol/l) inhibited the activation of I Ba,L [(-11.45±0.78) pa/pf vs (-8.56±0.61) pa/pf, n=6, P=0.0153, Student s t test for paired samples], but it did not abolish the stimulating effect of quercetin on I Ba,L (n=5, P<0.05 vs control, Student s t test for unpaired samples, Fig. 6D). 3 DISCUSSION This study provides the direct electrophysiological evidence that quercetin, a naturally-occurring flavonoid widely represented in the human diet, is an activator of L-type Ca 2+ channels in colon smooth muscle. In this study, quercetin induced a peak current enhancement which was smaller at weak depolarization and became progressively greater with increasing depolarization, and shifted the maximum of the I Ba,L -V curve towards more positive potentials, without, however, affecting the threshold for I Ba,L. Moreover, quercetin shifted the inactivation curve to more positive potentials. These results suggest that quercetin may alter the voltage sensitivity of L-type Ca 2+ channel. In a recent paper, Summanen et al. demonstrated that quercetin increased I Ca in clonal rat pituitary GH 4 C 1 cells, possibly via a camp-induced activation of PKA [16], which is in agreement with our results. In vascular smooth muscle, however, both camp and its related kinase have been shown to inhibit L-type Ca 2+ channels [21]. The discrepancy may be from using different cells. Flavonoids belong to a large group of plant polyphenols, and many polyphenols compounds regulate ion channels activity via tyrosine protein kinase (TPK) pathway. We investigated whether genistein could abolish the stimulating effect of quercetin on I Ca,L. The result provided us an evidence that quercetin increased I Ca,L not via TPK pathway. In addition, we also found out that quercetin increased I Ca,L not via PKC pathway. Therefore, we conclude that the quercetin-induced activation of I Ba,L observed here in colon smooth muscle cells should be attributed to changes in the intracellular levels of camp (Fig. 6). Quercetin was shown in our study to antagonize K + - induced contraction (Table 1). K + -induced contraction is the result of an increased Ca 2+ influx through voltagedependent Ca 2+ channels and is specifically inhibited by Ca 2+ -antagonists. Therefore, the inhibition of K + -induced contraction by quercetin might be interpreted as a consequence of the blockade of voltage-dependent Ca 2+ channels, as previously hypothesized by others [9]. Surprisingly enough, however, the electrophysiological data presented here showed a clear, potent activation of I Ca,L by quercetin. Acta Physiologica Sinica, December 25, 2009, 61 (6): Activation of I Ca,L by quercetin contradicts its myorelaxing properties, as it would be expected to cause the contraction of the colon smooth muscle. As such, we suggest that the myorelaxing effect of quercetin in tissue preparations originates from its reaction with a second target, beyond the Ca 2+ channel, which hierarchically prevails over the increase in the Ca 2+ influx to be expected from I Ca,L stimulation. Since it has been well shown that quercetin is capable of inducing both endothelium-dependent [5-7] and endothelium-independent [7-13] vasorelaxation in vitro, we investigated the role of NO in quercetin s myorelaxing effect. In our study, the preincubation with 100 μmol/l L-NAME did not influence the relaxant effects of quercetin, while the pretreatment with methylene blue attenuated the relaxant effect of quercetin significantly (Table 2). NO is also well known to relax smooth muscles via NO-GC-PKG pathway. Quercetin may activate GC directly to relax colon muscle rather than activate NOS [22]. Since prostaglandin (PG) is an important endogenous material in humoral regulation of gastrointestinal tact, we used COX inhibitor (indomethacin) to investigate whether quercetin could be via activating COX to relax colon. The results showed indomethacin attenuated the myorelaxing effect of quercetin. Therefore, activation of COX is one of the pathways, through which quercetin relaxes colon [9]. What is more, the results of this study showed that quercetin could also inhibit the effect of acetylcholine, which is in agreement with Fanning s report [23]. Atropine could inhibit colon smooth muscle contraction significantly, which gives us an elucidation that acetylcholine plays a role in basic tonicity. Since PKC plays a key role in maintenance of tonic contraction of vascular smooth muscle [24], and Duarte et al. have demonstrated that quercetin produced a vasodilator effect in isolated rat aorta that had been attributed to the inhibition of PKC [9,10]. PKC inhibition by quercetin is furthermore independent of Ca 2+ [25]. We also investigated whether PKC could enhance the effect of quercetin in contractile tension. But we did not observe the expecting result. In addition, the differences between the effects of methylene blue and methylene blue+quercetin, indomethacin and indomethacin+quercetin suggest quercetin may relax colon via not only GC pathway or COX pathway, but also other pathways. Cogolludo et al. and Pérez-Vizcaíno et al. have demonstrated that quercetin induced vasorelaxation via the activation of BK Ca channels and inhibition of myosin light chain kinase (MLCK), respectively [8,13]. In summary, owning to its diverse biological activities, quercetin may relax colon smooth muscle due to several pathways interaction, which is more complicated than its I Ca,L

9 HUANG Wei-Feng et al: Quercetin on Colon Smooth Muscle and L-type Ca 2+ Channels 575 stimulation. Although quercetin and dietary flavonoids have come into the focus of medicinal interest, data on their bioavailability after oral intake are scarce and contradictory [26]. Quercetin aglycon and its metabolites have been detected in plasma of human volunteers in the range of μmol/l [27-32]. Furthermore, the rate of elimination of quercetin is relatively low and, therefore, repeated consumption of quercetin-containing foods might cause accumulation of quercetin in blood [33] at levels extremely similar to those found to be active in the present study. In conclusion, the present electrophysiological data proved quercetin is a Ca 2+ channel activator in colon smooth muscle cells. The relaxant effect of quercetin attributes to the interaction of GC and COX stimulation, as well as the antagonism effect on acetylcholine. 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