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Seoul National University, Seoul, Korea 2 Department of Physiology, College of Medicine, Chungbuk National University, Cheongju, Korea Recently, we demonstrated that a large conductance Ca -activated

1 Lab. Anim. Res. 2010: 26(2), The Effect of Quercetin on the BK Ca in Umbilical Cord Vein-derived Mesenchymal Stem Cells Kyoung Sun Park 1 and Yangmi Kim 2 * 1 Department of Physiology, College of Medicine, Seoul National University, Seoul, Korea 2 Department of Physiology, College of Medicine, Chungbuk National University, Cheongju, Korea Recently, we demonstrated that a large conductance Ca -activated K + channel (BK Ca ) in human umbilical cord vein-derived mesenchymal cells (huc-mscs). In this study, we studied effects of quercetin, the flavonoid used for the modulator of cell cycle and the treatment of anti-tumor, on BK ca in huc-mscs using single channel and/or whole cell configuration. Single channel conductance was 277.8±36.2 ps in huc-mscs and the value is similar to that of typical BK Ca channel reported in other cells. The single channel current was activated by increasing of intracellular Ca. Quercetin (30~100 µm) increased BK Ca in whole cell patch configuration whereas EGCG was not affected on the channel. The activation effect of quercetin on BK Ca channel was also observed in inside-out configuration. The quercetin-induced BK Ca was increased a concentration-dependent manner with an IC 50 value of 19.9 µm. Taken together, BK Ca in huc- MSCs can be an important target for the action of quercetin and the channels are partly may contribute to maintaining homeostasis by regulation of K ion flux in huc-mscs. Key words: Human umbilical cord vein-derived mesenchymal cells, quercetin, BK Ca Received 27 April 2010; Revised version received 21 June 2010; Accepted 23 June 2010 f p(quercetin) v (flavonoid) s r w ƒ ù w w y. ƒ v 60-75%ƒ f p (Hertog and Hollman, 1996). f p y w x y z ùkü šx x j ù, w y w (Duarte et al., 2001; Sanchez et al., 2006). w f p w w w š s, s, š x (promyeloleukemic) s HL-60 s f p s sm (apoptosis) w š (Lee et al., 2002b; Wang et al., 2003). w s f p w s»ƒ š sm ƒ (Choi et al., 2001). f p w, w y ùküš» s w v y w š š» *Corresponding author: Yangmi Kim, Department of Physiology, College of Medicine, Chungbuk National University, 410 Seongbong-ro, Heungdeok-gu, Cheongju, Chungbuk Tel: Fax: yangmik@chungbuk.ac.kr wš. s f p q sx (osteoclastogenesis) w š (Rassi et al., 2005), v epigallocatechin gallate (EGCG) NF-kappaB w q s y wš(lin et al., 2009), hbm-mscs s y w š š (Shin et al., 2009). f p x sy m w. f p w NO/cGMP y BK Ca y yw wš w sü e (Ca ) š š(kuhlmann et al., 2005), (coronary arteries) f p y (H 2 ) w BK Ca ƒ k š š (Cogolludo et al., 2007).» s (human umbilical cord vein-derived mesenchymal stem cells, huc-mscs) BK Ca m (large conductance Ca - activated K channel), Na m š ƒ K m ƒ» x šw (Park et al., 2007). w huc-msc w BK Ca m m» w d y (amyotrophic lateral sclerosis) e riluzole w y y y 203

2 204 Kyoung Sun Park and Yangmi Kim w (Park et al, 2008). huc-msc y» s w BK Ca v ƒ w w. huc-mscs w BK Ca w v z m whole cell patch configuration w y w. ¼ Ù e g z w. phosphate buffered saline (GIBCO-BRL, Invitrogen Corporation, Grand Island, NY, USA) x ü 0.5% collagenase (GIBCO-BRL, Invitrogen Corporation, Grand Island, NY, USA) š 37 C 5 ew o. x w ù s w. s 1,000 rpm 10 wš, Dulbecco s modified Eagle s medium-low glucose (GIBCO-BRL, Invitrogen Corporation, Grand Island, NY, USA), 100 U/mL penicillin, 100 µg/ml streptomycin, and 10% fetal bovine serum (GIBCO- BRL, Invitrogen Corporation, Grand Island, NY, USA) 75 cm 2 culture flasks 37 o C, 5% C» w. 2 ã š» w x w. j x w s 140 mm NaCl, 5,4 mm KCl, 0.5 mm MgCl 2, 1.8 mm CaCl 2, 10 mm glucose, 5 mm HEPES w š, ph 7.4 w w. m» (single channel recording) w inside-out patch cell-attached patch w (pipette) ü bath 150 mm KCl, 1 mm MgCl 2, 10 mm HEPES, 5 mm EGTA wš, ph 7.2 w. Whole cell patch š» w pipette 140 mm KCl, 1 mm MgCl 2, 5 mm Mg-ATP, 2 mm EGTA, 5 mm HEPES wš, ph 7.3 w w. s (whole-cell current) m (single channel current) gigaohm (GΩ)-seal x w» w. Whole-cell current» 1~3 MΩ w ƒ wš, m» 2~3 MΩ w w. qe ó sylgard (Dow Corning, Midland, MI, USA) coatingw z fire polishw w. m» m y qeš s»(patch clamp amplifier, axopatch 200B, Union city, CA, USA)» w.» y 8-pole bessel filter (-3 db, Frequency Device) w 2kHz filter w. Data computer ¼ 10 khz sampling rate digidata 1322A interface (Axon instrument, Union city, CA, USA) w computer w. data pclamp program 9.0 (Axon instrument, Union city, CA, USA) w w. x (23-25 C) o ww.» s (human umbilical cord vein-derived mesenchymal stem cells, huc-mscs) whole cell patch configuration w» x m ³ w (Park et al., 2007). largeconductance Ca -activated K + (BK Ca ) m ƒ v w w. Figure 1A huc-msc x. ü w ƒ ùkù w ƒ». üw š» w q e 30% Na y w (Park et al., 2007). qe 98% w w v z w. w nosy ql ƒ š BK Ca y (Park et al., 2007; Park et al., 2008). EGCG (50 µm) w üw w w x š ùkü (Figure 1A and 1A-1). f p w y y g y f p z š(figure 1B), š f p y z (Figure 1B-1). w x qe (n=16). 80 mv w 40 mv 50 mv¾» w. f p w y» w v 10 f p z» w (Figure 2A and 2A-1). 80 mv w š 120 mv 60 mv¾ v» w. f p w» ƒ ƒw ƒ f p w ƒw» w 2 ƒ ù w y w. Figure 2A-1 t š ùkü. j» +50 mv 6.7 ƒw (n=4), f p ü z ù z. f p w w ƒ ƒ» w ƒƒ

3 The effect of quercetin on BK Ca channels in huc-mscs 205 Figure 1. The effect EGCG (epigallocatechin 3-gallate) and quercetin on outward in huc-mscs. huc-mscs were voltageclamped under whole cell configuration at -80 mv. Voltage steps of 200 ms duration were then applied from -40 mv to 50 mv in 10- mv increments every 10 s, showing outward currents a rapidly activating current with noisy oscillation, similar to large conductance Ca activated K + current (BK Ca ). The pipette solution consisted of 140 mm KCl, 1 mm MgCl 2, 5 mm Mg-ATP and 2 mm EGTA; the bath solution contained 143 mm NaCl, 5.4 mm KCl, 0.5 mm NaH 2 PO 4, 0.5 mm MgCl 2, 1.8 mm CaCl 2, 5 mm HEPES and 10 mm glucose. (A) Membrane currents were recorded in the presence of EGCG. (A-1) The current-voltage relationship (I-V relationship) was plotted from A. Current-voltage relationship showed in the control (þ), 50 µm EGCG (ù) and wash ( ). (B) Quercetin increased outward current in huc-mscs. (B-1) The current-voltage relationship (I-V relationship) was plotted from B. I-V relationships showed in the control (þ), 100 µm quercetin (ù) and wash ( ).» w. f p w ƒw» w 50 µm š, IC µm (Figure 2B, n=3). f p w ƒ ƒ» w m» w» w. Figure 3A m large-conductance Ca -activated K + channel (BK Ca ) huc-mscs» w m 40 mv 277.8±36.2 ps m (11.6±1.5 pa), m s ü Ca y ƒw (Figure 2A). w (Park et al., 2008). 40 mv m y s³ (mean open time) 0.09 ms (n=4). 40 mv» s s ù ƒ w». m ƒ sü Ca w w» w sü Ca y j m y w. sü Ca k y m ƒ wù ƒ sü Ca ƒ 3 m ƒ, sü Ca ƒ ƒw

4 206 Kyoung Sun Park and Yangmi Kim Figure 2. Effect of extracellular quercetin on the outward current in huc-mscs. (A) Ramp pulse was applied from -120 to 60 mv for 250 ms at a holding potential of -80 mv and the pulse was applied at every 10 s. The x-axis shows time and the y-axis shows current. The outward current was activated by quercetin. Currents were reached a stable level in 2 min after the application of quercetin (100 µm) and recovered after washing with quercetin-free solution. (A-1) Quercetin -induced typical outward-rectifying current vs. voltage. (B) Dose dependence of quercetin-induced outward current in huc-msc using the whole cell patch configuration. Currents were measured at 50 mv and IC 50 was 19.9 µm. (n=3 cells per concentration). s³ 0.09 ms 3.95 ms ƒw š, m y ƒ w. w sü Ca y y BK Ca m w ew (Park et al., 2008). m inside-out patch bath( sü) f p(30 µm) w w. f p w m y y y +60 mv» w. f p w» m y 0.035±0.001 ù f p z m y 0.056± ƒw. w m s³ 1.43±0.15 ms 2.63±0.42 ms ƒ w (n=5). w f p w w. f p w ƒ ƒ BK Ca y w» w f p z BK Ca p z w (Figure 4). 80 mv w whole cell patch 40 mv 50 mv¾ w w ƒ, w f p w y y š y y BK Ca p iberiotoxin w f p w ƒ BK Ca y w. y» s(huc-msc)» x m BK Ca ƒ f p w y y, f p w w m s» s y w s yù sü w y» w w. y» s(huc- MSCs) w w ƒ v f p w y y y w š, f p w y y ƒ BK Ca m y w. BK Ca m Ca m mw s Ca ù sü» s m mw e w sü Ca ƒw m

5 The effect of quercetin on BK Ca channels in huc-mscs 207 Figure 3. The effect of intracellular quercetin on large conductance Ca activated K + channels (BK Ca ) using single channel recording in huc-mscs. Data were obtained from estimates of relative channel activity. (A) The trace shown the effect of intracellular Ca. Single channel currents recorded at +40mV from an inside-out patch configuration. Pipette solution contained 150 mm KCl, 1 mm MgCl 2, 5 mm EGTA, and 10 mm HEPES. The bath solution consisted of 150 mm KCl 150 mm KCl, 1 mm MgCl 2, 5 mm EGTA, and 10 mm HEPES and 0 µm, 1 µm CaCl 2. The channel activity was increased by [Ca ] i dependently. The letter a and b show an expanded scale of the A. Current amplitude of 1 main peak is about 12 pa. (B) The traces were showed the effect of quercetin on BK Ca channels. The channel activity was increased by intracellular application of quercetin (30 µm). The letter a and b show an expanded scale of the A. The recording potential was +60mV. Current amplitude of 1 main peak is about 15 pa. The letter close represents the closed channel level and the letter open represents the open channel level. Abbreviations: min, minutes; ms, milliseconds; pa, picoampere. sü K s w sü y j w w. BK Ca m x sy x w w w š š x üv s ù s w (Wiecha et al., 1998; Roger et al., 2004). BK Ca sü w w BK Ca y s y j ù sü Ca w w w s w w ƒ. f p s sü y» w s»ù» w x sy x w. f p w BK Ca y x sy NO yù H 2 ƒ w ù š š (Kuhlmann et al., 2005; Cogolludo et al., 2007). huc-msc f p w BK Ca y NO y ù H 2 w. w NOù H 2 ƒ y inside-out patch configuration( m» wù ) f p m y ùkûš(figure 3), Kuhlman et al. (2005) Cogolludo et al. (2007) š f p w BK Ca y NO y ù H 2 w ý whole cell configuration (» w ) û ùk û» sü f p w H 2 y ù NO ƒ w ú ƒ w (Figure 3 and Figure 4). s f p w BK Ca y š ƒ. y» s y s y y, y (oxidative metabolism) y w yw w yƒ» s y v ƒ w ƒ. s» s(hbm-msc) v z ƒ w ƒ ew (Rassi et al., 2005; Lin et al., 2009; Shin et al., 2009). w sü» m ƒ» w w. w ¾» s m w w, ù ƒ v ƒ» s w m w š

6 208 Kyoung Sun Park and Yangmi Kim Figure 4. Effect of quercetin and Iberiotoxin (IBX, a blocker of large conductance Ca -activated K + current) on BK Ca in huc-mscs. (A) Current traces are shown for 200-ms depolarization pulses from -40 mv to +50 mv (in 10-mV increments) from a holding potential of -80mV. Quercetin activates noisy outward currents in huc-mscs. Currents were recorded before (control), after the exposure to quercetin (100 µm) reached a stable level (3 min) and after application with quercetin and IBX solution. (B) The currentvoltage relationship (I-V relationship) was plotted from A. I-V relationships of currents showed in the control (þ), 100 µm quercetin (ù) and 100 µm quercetin with 100 nm iberiotoxin ( ).. y» s (huc-mscs) v ƒ» s w m w v ƒ w w. y» s BK Ca ƒ v f p w ƒ y w m ƒ v w y» w ƒ š d wz ƒ. f p z BK Ca w y y K V Cl, Ca š. Kv1.5 m ƒ f p w y y š(yang et al., 2009), Cl j» w (Cermak et al., 1998). Õ sy (rat tail artery smooth muscle) L- type Ca m y y j (Saponara et al., 2002), f p Ca m y y w sü Ca w š š (Lee et al., 2002a). f p w sü Ca m mw sü Ca ƒw š huc-msc BK Ca y f p w sü Ca ƒw BK Ca m ƒ y y ƒ e w. w sü Ca-free k(10 7 M w) f p w y ùkû sü Ca ƒ w» f p ƒ w w (Figure 3). š huc-mscs w BK Ca m v w y y p, whole cell patch inside-out patch v w y y p, sy w BK Ca ƒ f p y ƒ. huc-msc v w m p ³ w v ƒ» s s y»» w ƒ w huc-msc w ù ù ƒ UC-MSC y w» œ ƒ. w» s y v ƒ m y ƒ w v w ƒ. q 2008 w w w. k Cermak, R., Follmer, U. and Wolffram, S. (1998) Dietary flavonol quercetin induces chloride secretion in rat colon. Am. J. Physiol. 275(5 Pt 1), G Choi, J. A., Kim, J. Y., Lee, J. Y., Kang, C. M., Kwon, H. J., Yoo, Y. D., Kim, T. W., Lee, Y. S. and Lee, S. J. (2001) Induction of cell cycle arrest and apoptosis in human breast cancer cells by quercetin. Int. J. Oncol. 19(4), Cogolludo, A., Frazziano, G., Briones, A. M., Cobeno, L.,

7 The effect of quercetin on BK Ca channels in huc-mscs 209 Moreno, L., Lodi, F., Salaices, M., Tamargo, J. and Perez- Vizcaino, F. (2007) The dietary flavonoid quercetin activates BKCa currents in coronary arteries via production of H 2. Role in vasodilatation. Cardiovasc. Res. 73(2), Duarte, J., Perez-Palencia, R., Vargas, F., Ocete, M. A., Perez- Vizcaino, F., Zarzuelo, A. and Tamargo, J. (2001) Antihypertensive effects of the flavonoid quercetin in spontaneously hypertensive rats. Br. J. Pharmacol. 133(1), Hertog, M. G. and Hollman, P. C. (1996) Potential health effects of the dietary flavonol quercetin. Eur. J. Clin. Nutr. 50(2), Kuhlmann, C. R., Schaefer, C. A., Kosok, C., Abdallah, Y., Walther, S., Ludders, D. W., Neumann, T., Tillmanns, H., Schafer, C., Piper, H. M. and Erdogan, A. (2005) Quercetininduced induction of the NO/cGMP pathway depends on Ca -activated K + channel-induced hyperpolarizationmediated Ca -entry into cultured human endothelial cells. Planta Med. 71(6), Lee, E. H., Meissner, G. and Kim, D. H. (2002a) Effects of quercetin on single Ca release channel behavior of skeletal muscle. Biophys. J. 82(3), Lee, L. T., Huang, Y. T., Hwang, J. J., Lee, P. P., Ke, F. C., Nair, M. P., Kanadaswam, C. and Lee, M. T. (2002b) Blockade of the epidermal growth factor receptor tyrosine kinase activity by quercetin and luteolin leads to growth inhibition and apoptosis of pancreatic tumor cells. Anticancer Res. 22(3), Lin, R. W., Chen, C. H., Wang, Y. H., Ho, M. L., Hung, S. H., Chen, I. S. and Wang, G. J. (2009) (-)-Epigallocatechin gallate inhibition of osteoclastic differentiation via NF-kappaB. Biochem. Biophys. Res. Commun. 379(4), Park, K. S., Jung, K. H., Kim, S. H., Kim, K. S., Choi, M. R., Kim, Y. and Chai, Y. G. (2007) Functional expression of ion channels in mesenchymal stem cells derived from umbilical cord vein. Stem Cells 25(8), Park, K. S., Jung, K. H., Chai, Y. G., Kim, C. H., Shin, E. Y. and Kim. Y. (2008) Riluzole-induced activation of BKCa channels and inhibition of voltage-gated Na + channel in human umbilical cord vein-derived mesenchymal cells. Lab. Anim. Res. 24(3), Rassi, C. M., Lieberherr, M., Chaumaz, G., Pointillart, A. and Cournot, G. (2005) Modulation of osteoclastogenesis in porcine bone marrow cultures by quercetin and rutin. Cell Tissue Res. 319(3), Roger, S., Potier, M., Vandier, C., Le Guennec, J. Y. and Besson, P. (2004) Description and role in proliferation of iberiotoxinsensitive currents in different human mammary epithelial normal and cancerous cells. Biochim. Biophys. Acta 1667(2), Sanchez, M., Galisteo, M., Vera, R., Villar, I. C., Zarzuelo, A., Tamargo, J., Perez-Vizcaino, F. and Duarte, J. (2006) Quercetin downregulates NADPH oxidase, increases enos activity and prevents endothelial dysfunction in spontaneously hypertensive rats. J. Hypertens. 24(1), Saponara, S., Sgaragli, G. and Fusi, F. (2002) Quercetin as a novel activator of L-type Ca channels in rat tail artery smooth muscle cells. Br. J. Pharmacol. 135(7), Shin, D. W., Kim, S. N., Lee, S. M., Lee, W., Song, M. J., Park, S. M., Lee, T. R., Baik, J. H., Kim, H. K., Hong, J. H. and Noh, M. (2009) (-)-Catechin promotes adipocyte differentiation in human bone marrow mesenchymal stem cells through PPAR gamma transactivation. Biochem. Pharmacol. 77(1), Wang, S., Degroff, V. L. and Clinton, S. K. (2003) Tomato and soy polyphenols reduce insulin-like growth factor-i-stimulated rat prostate cancer cell proliferation and apoptotic resistance in vitro via inhibition of intracellular signaling pathways involving tyrosine kinase. J. Nutr. 133(7), Wiecha, J., Munz, B., Wu, Y., Noll, T., Tillmanns, H. and Waldecker, B. (1998) Blockade of Ca -activated K + channels inhibits proliferation of human endothelial cells induced by basic fibroblast growth factor. J. Vasc. Res. 35(5), Yang, L., Ma, J. H., Zhang, P. H., Zou, A. R. and Tu, D. N. (2009) Quercetin activates human Kv1.5 channels by a residue I502 in the S6 segment. Clin. Exp. Pharmacol. Physiol. 36(2),

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