Research Article Involvement of MAPKs and PLC Pathways in ...

Hindawi Publishing CorporationThe Scientific World JournalVolume 2013 Article ID 536350 10 pageshttpdxdoiorg1011552013536350

Research ArticleInvolvement of MAPKs and PLC Pathways in Modulation ofPacemaking Activity by So-Cheong-Ryong-Tang in InterstitialCells of Cajal from Murine Small Intestine

Min Woo Hwang1 Hee Jung Lee2 Ho Joon Song2 and Byung Joo Kim2

1 Department of Sasang Constitutional Medicine College of Korean Medicine Kyung Hee University Seoul 130-701 Republic of Korea2Division of Longevity and Biofunctional Medicine Pusan National University School of Korean Medicine Beomeori Mulgeum-eupYangsan Gyeongsangnam-do 626-870 Republic of Korea

Correspondence should be addressed to Byung Joo Kim visionpusanackr

Received 7 August 2013 Accepted 5 September 2013

Academic Editors A Beltsis M Kluppel and C Rizzetto

Copyright copy 2013 Min Woo Hwang et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Purpose Interstitial cells of Cajal (ICCs) are the pacemaker cells that generate slow waves in the gastrointestinal (GI) tract Wehave aimed to investigate the effects of Socheongryong-Tang (SCRT) in ICCs frommousersquos small intestineMethodsThe whole-cellpatch-clamp configuration was used to record membrane potentials from cultured ICCs Intracellular Ca2+ ([Ca2+]i) increase wasstudied in cultured ICCs using fura-2 AMResults ICCs generated pacemaker potentials inmousersquos small intestine SCRT producedmembrane depolarization in current clamp mode Y25130 (5-HT

3receptor antagonist) and RS39604 (5-HT

4receptor antagonist)

blocked SCRT-inducedmembrane depolarizationswhereas SB269970 (5-HT7receptor antagonist) did notWhenGDP-120573-S (1mM)

was in the pipette solution SCRT did not induce the membrane depolarizations [Ca2+]i analysis showed that SCRT increased[Ca2+]i In the presence of PD98059 (p4244 MAPK inhibitor) SCRT did not produce membrane depolarizations In additionSB203580 (p38 MAPK inhibitor) and JNK inhibitors blocked the depolarizations by SCRT in pacemaker potentials Furthermorethe membrane depolarizations by SCRT were not inhibited by U-73122 an active phospholipase C (PLC) inhibitor but by U-73343an inactive PLC inhibitor Conclusion These results suggest that SCRT might affect GI motility by the modulation of pacemakeractivity through MAPKs and PLC pathways in the ICCs

1 Introduction

Interstitial cells of Cajal (ICCs) are the pacemaking cells inthe gastrointestinal (GI) muscles that generate the rhyth-mic oscillations in the membrane potential known as slowwaves [1ndash3] Slow waves propagate within ICC networksare conducted into smooth muscle cells via gap junctionsand initiate phasic contractions by activating Ca2+ entrythrough L-type Ca2+ channels The pacemaker activity in themurine small intestine is due mainly to periodic activation ofnonselective cation channels (NSCC) [4 5] or Clminus channels[6 7] ICCs also mediate or transduce inputs from the entericnervous system Therefore in GI motility research ICCs are

the major tools to study In these days many new drugs aredeveloping in the area of GI motility

From ancient to modern history traditional plant-basedmedicines have played an important role in health care Inspite of the great advances of modem scientific medicinetraditional medicine is still the primary form of healingmethods readily available to the majority of the people inmany countries In fact many of todayrsquos popular drugs havetheir origins in traditional medicines [8]

So-Cheong-Ryong-Tang (SCRT) also called Xiao-Qing-LongTtang or Sho-Seiru-To contains eight species of medic-inal plants and has been a herbal medicine used to treatdiseases such as allergic rhinitis and asthma for hundreds

2 The Scientific World Journal

of years in Asian countries [9] However there were notmany attempts to investigate the efficacy of SCRT in digestivesystems

Of the pathways related to intestinal motility serotonin(5-hydroxytryptamine 5-HT a major neuromodulator) isknown to play a critical role in the GI tract Generally5-HT acts as a neurotransmitter in the central nervoussystem but most (95) 5-HT is found in the GI tract [10]Furthermore although 5-HT is known to interact with sevendifferent 5-HT receptor subtypes only three of these arefound in the ICCs in the murine small intestine [11] 5-HTcan modulate pacemaker activity through 5-HT3 4 and7 receptors In previous study we suggested that poncirustrifoliatemodulates pacemaker potentials through 5-HT

3and

5-HT4receptor-mediated pathways via external Na+ and

Ca2+ influx [6] However the effects of SCRT and the actionmechanism involved in the GI tract are not investigated

Therefore we undertook to investigate the effects of theSCRT on the pacemaker potentials of cultured ICCs derivedfrom murine small intestine and to identify the receptorsinvolved

2 Materials and Methods

21 Preparation of Cells and Cell Cultures Balbc mice (3ndash7 days old) of either sex were anesthetized with ether andkilled by cervical dislocation The small intestines from 1 cmbelow the pyloric ring to the cecum were removed andopened along the mesenteric border Luminal contents wereremoved bywashingwith Krebs-Ringer bicarbonate solutionThe tissues were pinned to the base of a Sylgard dish andthe mucosa removed by sharp dissection Small tissue stripsof the intestine muscle (consisting of both circular andlongitudinal muscles) were equilibrated in Ca2+-free Hankssolution (containing in mmolL KCl 536 NaCl 125 NaOH034 Na

2HCO3044 glucose 10 sucrose 29 and HEPES 11)

for 30min Then the cells were dispersed using an enzymesolution containing collagenase (Worthington BiochemicalCo Lakewood NJ USA) 13mgmL bovine serum albumin(Sigma Chemical Co St Louis MO USA) 2mgmL trypsininhibitor (Sigma) 2mgmL and ATP 027mgmL Cells wereplated onto sterile glass coverslips coated with murine col-lagen (25120583gmL FalconBD Franklin Lakes NJ USA) in a35-mm culture dish and then cultured at 37∘C in a 95 O

2

50mLL CO2incubator in a smooth muscle growth medium

(Clonetics Corp San Diego CA USA) supplemented with2 antibioticsantimycotics (Gibco Grand Island NY USA)and murine stem cell factor (SCF 5 ngmL Sigma) ICCswere identified immunologically with anti-c-kit antibody(phycoerythrin-conjugated rat anti-mouse c-kit monoclonalantibody eBioscience San Diego CA USA) at a dilution of1 50 for 20min [7] ICCs weremorphologically distinct fromother cell types in the culture and thus it was possible toidentify the cells by phase contrast microscopy once they hadbeen verified with anti c-kit antibody

22 Patch-Clamp Experiments The whole-cell patch-clampconfiguration was used to record membrane potentials

(current clamp) from cultured ICCs An axopatch ID(Axon Instruments Foster CA USA) was used to amplifymembrane currents and potentials The command pulsewas applied using an IBM-compatible personal computerand pClamp software (version 61 Axon Instruments)Data obtained were filtered at 5 kHz and displayed on anoscilloscope a computer monitor and using a pen recorder(Gould 2200 Gould Valley View OH USA) Results wereanalyzed using pClamp and Origin (version 60) softwareAll experiments were performed at 30ndash32∘C

23 Fura-2 Loading and Measurement of Intracellular FreeCalcium Ion Concentration [Ca2+]

119894 Cultured ICC clusters

were loaded with the acetoxymethyl ester form of fura-2(5 120583molL diluted from 1mmolL stock in dimethyl sulfoxide(DMSO)) in normal medium for 20 minutes at 37∘C Therecording of [Ca2+]i was performedwith amicrofluorometricsystem consisting of an inverted fluorescence microscope(Diaphot 300 Nikon Japan) with a dry-type fluorescenceobjective lens (40X numerical aperture 085) a photomul-tiplier tube (type R 1527 Hamamatsu Japan) and a PTI-Deltascan illuminator (Photon Technology InternationalInc) Cells were superfused at a flow rate of 15mLminLight was provided by a 75-W xenon lamp (Ushino Japan)To control excitation frequency a chopper wheel alternatedthe light path to monochromators (340 and 380 nm) with afrequency of 5 or 10Hz A short-pass dichroic mirror passedemission light of lt570 nm onto the photomultiplier tube andintensity was measured at 510 nm A mechanical image maskwas placed in the emission path to limit measurement to asingle cell Both data acquisition and control of light applica-tion were performed with computer software (Felix version11 Photon Technology International Inc Brunswick NJ)Because of uncertainties in calibrating the fura-2 signals inintact cells no attempt wasmade to calibrate [Ca2+]i insteadall results are reported as changes in the 340 nm380 nmsignal ratio

24 Solutions andDrugs Thephysiological salt solution usedto bathe cells (Na+-Tyrode) contained (mmolL) KCl 5 NaCl135 CaCl

22 glucose 10MgCl

212 andHEPES 10 adjusted to

pH 74 with NaOHThe pipette solution contained (mmolL)KCl 140 MgCl

25 K2ATP 27 NaGTP 01 creatine phosphate

disodium25HEPES 5 andEGTA01 adjusted to pH72withKOH

SCRT was purchased from I-WORLD Pharmaceuti-cals (South Korea) SCRT is composed of Rhizoma Pinel-liae Herba Ephedrae Radix Paeoniae Fructus SchisandraeHerba Asari Rhizoma Zingiberis Ramulus Cinnamomiand Radix Glycyrrhizae (Table 1) The dosage for adult is10ndash15 g (crude materials) per day More information aboutSCRT can be found in I-WORLDPharmaceuticalsHomepage(httpi-pharmkoreasmecom) The SCRT was dissolvedwith distilled water at the concentration of 05 g (crudedrug)mL and stored in refrigerator All other drugs wereobtained from Sigma (Sigma Chemical Co USA) Drugswere dissolved in distilledwater and added to bath solution tomake the desired concentrations just prior to use Addition

The Scientific World Journal 3

So-Cheong-Ryong-Tang 0mgmL (CTRL)

minus31mV

minus52mV

(a)

So-Cheong-Ryong-Tang 10mgmL

minus34mV

minus53mV

(b)

minus30mV

minus52mVSo-Cheong-Ryong-Tang 30mgmL

(c)

minus31mV


(d)

0

5

10

15

20

25

Deg

ree o

f dep

olar

izat

ion

(mV

)

CTRL 10 30 50

So-Cheong-Ryong-Tang (mgmL)

lowastlowast

lowastlowast

(e)

Figure 1 Effects of SCRT on pacemaker potentials in cultured ICCs from murine small intestine (a)ndash(d) show the pacemaker potentials ofICCs exposed to SCRT (0ndash50mgmL) in current clamping mode (119868 = 0) Responses to SCRT are summarized in (e) Bars represent meanvalues plusmn SEs lowastlowast119875 lt 001 Significantly different from untreated controls CTRL Control

Table 1 Amount and Composition of SCRT

Herb Scientific name Amount (g)Ban Ha Rhizoma Pinelliae 2 gMa Hwang Herba Ephedrae 1 gJak Yak Radix Paeoniae 1 gOmija Fructus Schisandrae 1 gSae Shin Herba Asari 1 gGun Kang Rhizoma Zingiberis 1 gKae Ji Ramulus Cinnamomi 1 gKam Cho Radix Glycyrrhizae 1 g

Total amount 9 g

of these chemicals to bath solution did not alter the pH of thesolution U-73122 U-73343 Y25130 RS39604 and SB269970were dissolved in DMSO for 50mmolL stock solution andadded to the bathing solution at the day of the experimentThe final concentration of DMSO in the bath solution wasalways lt01 and we confirmed that this concentration ofDMSO did not affect the results that were recorded

25 Statistics All data are expressed as mean plusmn SE Studentrsquos119905-test for unpaired data was used to compare control and

experimental groups The 119875 value of less than 005 was con-sidered statistically significant

3 Results

31 Effect of SCRT on Pacemaker Potentials in CulturedICCs To understand the relationship between SCRT andpacemaking activity in ICCs we examined the effects ofSCRTonpacemaker potentials Recording fromcultured ICCunder current clamp mode (119868 = 0) showed spontaneouspacemaker potentials The resting membrane potential wasminus521 plusmn 21mV and the amplitude was 20 plusmn 4mV In thepresence of SCRT (10ndash50mgmL) the membrane potentialswere depolarized to 13 plusmn 06mV at 10mgmL 116 plusmn 12mV at30mgmL and 203plusmn 15mV at 50mgmL (Figures 1(a)ndash1(e))The summarized values and bar graph of the SCRT effectson pacemaker potentials are indicated in Figure 1(e) (119899 = 4)Taken together these results show that SCRT havemembranedepolarization effects on ICC

32 Identification of SCRT Receptor Subtypes in CulturedICCs To investigate the relationship between SCRT and itsreceptors we studied about the 5-HT receptors because 5-HT receptors are known to mediate the motility of GI tract



minus31mV

minus53mV

(a)

So-Cheong-Ryong-Tang 50mgmLY25130 10120583M

minus32mV

minus53mV

(b)

So-Cheong-Ryong-Tang 50mgmLRS39604 10120583M

minus30mV

minus51mV

(c)

So-Cheong-Ryong-Tang 50mgmLSB269970 10120583M

minus15mV

minus53mV

(d)

0

10

20

30

40

Deg

ree o

f dep

olar

izat

ion

(mV

)

CTRL Y25130 RS39604 SB269970 So-Cheong-Ryong-Tang 50mgmL

lowastlowastlowastlowast

(e)

Figure 2 Effects of 5-HT receptor subtype antagonists on SCRT-induced pacemaker potential responses in cultured ICCs (a) SCRT(50mgmL) induced membrane depolarizations on ICCs (b) Pacemaker potentials of ICCs exposed to SCRT (50mgmL) in the presence of5-HT

3receptor antagonist (Y25130 10120583M) (c) Pacemaker potentials of ICCs exposed to SCRT in the presence of 5-HT

4receptor antagonist

(RS39604 10 120583M) (d) Pacemaker potentials of ICCs exposed to SCRT in the presence of 5-HT7receptor antagonist SB269970 (10 120583M)

Responses to SCRT in presence of different receptor antagonists are summarized in (e) Bars represent mean values plusmn SEs lowastlowast119875 lt 001Significantly different from untreated controls CTRL Control

and is of particular interest due to its strong associationwith potent prokinetic activity especially the 5-HT receptorsubtype 4 (5-HT

4R) [6 12] In the GI tract the stimulation

of 5-HT4R in the enteric nervous system results in the

release of acetylcholine which leads to the excitation ofsmoothmuscles in the myenteric plexus and thus 5-HT

4R is

regarded a prokinetic [12]Therefore we investigatedwhetherthe prokinetic action of SCRT involves 5-HT receptorsPrevious studies have shown that 5-HT interacts with sevendifferent 5-HT receptor subtypes but in another study onlythree (5-HT

3R 5-HT

4R and 5-HT

7R) were found in the

ICCs of the murine small intestine [6 11 13] To identify thereceptor subtypes of 5-HT involved in the effects of SCRTICCs were pretreated with various 5-HT receptor antagonistsand then treated with SCRT Y25130 (a 5-HT

3receptor

antagonist) RS39604 (a 5-HT4receptor antagonist) and

SB269970 (a 5-HT7receptor antagonist) were all pretreated

at a concentration of 10 120583M for 5min and SCRT was added

After pretreatment with Y25130 membrane depolarizationby SCRT was found to be blocked (Figure 2(b)) membranedepolarization produced in the presence of Y25130 by MPFwas 10 plusmn 02mV (119899 = 5 Figure 2(e)) RS39604 also blockedSCRT-inducedmembrane depolarization and themembranedepolarization produced in the presence of RS39604 by SCRTwas 12 plusmn 03mV (119899 = 5 Figures 2(c) and 2(e)) Howeverpretreatment with SB269970 did not block the effect of SCRT(119899 = 5 Figures 2(d) and 2(e)) These results show that SCRThas an effect on ICCs through 5-HT

3R and 5-HT

4R

33 The Involvement of G Protein on SCRT-Induced Depolar-izations in Pacemaker Potentials in Cultured ICCs Theeffectsof GDP-120573-S (a nonhydrolysable guanosine 51015840-diphosphateanalogue that permanently inactivates G-protein bindingproteins [14]) were examined to determine whether G-proteins are involved in the effects of SCRT on culturedICCs SCRT (30mgmL) induced membrane depolarizations



minus34mV

minus51mV

(a)


minus33mV

minus52mV

[GDP-120573-S 1mM]

(b)

Deg

ree o

f dep

olar

izat

ion

(mV

)

CTRL0

3

6

9

12

15


[GDP-120573-S 1mM]

lowastlowast

(c)

Figure 3 Effects of GDP-120573-S in the pipette on SCRT-induced pacemaker potentials in cultured ICCs of murine small intestine (a) SCRT(30mgmL) induced membrane depolarizations on ICCs (b) Pacemaker potentials of ICCs exposed to SCRT (30mgmL) in the presenceof GDP-120573-S (1mM) in the pipette Under these conditions SCRT (30mgmL) caused membrane depolarization (c) Responses to SCRT inthe presence of GDP-120573-S in the pipette are summarized in (b) Bars represent mean values plusmn SEs lowastlowast119875 lt 001 Significantly different fromuntreated controls CTRL Control

on ICCs (Figure 3(a)) However when GDP-120573-S (1mM) wasin the pipette solution SCRT (30mgmL) did not inducethe membrane depolarizations (Figure 3(b)) The membranedepolarizations induced by SCRT were significantly affectedby the presence of GDP-120573-S (1mM) in the pipette solution(119899 = 4 Figure 3(c)) These results show that G-protein is in-volved in SCRT-inducedmembrane depolarizations on ICCs

34 Response of the Intracellular Ca2+ ([Ca2+]119894) to SCRT To

investigate the effects of SCRT on [Ca2+]i oscillations wemeasured spontaneous [Ca2+]i oscillations in ICCs clustersbecause several authors have suggested that [Ca2+]i oscilla-tions in ICCs are primary responsible forGI pacemaker activ-ity Spontaneous [Ca2+]i oscillations were observed in ICCsclusters loaded with 5 120583M fluo-2 Figure 4 show the changesin the 340 nm380 nm signal ratio In normal conditionsspontaneous [Ca2+]i oscillations were induced (Figure 4(a))In the presence of SCRT (10 or 50mgmL) [Ca2+]i in ICCswas increased (Figures 4(b) and 4(c))These results show thatSCRT increase the [Ca2+]i in ICCs

35 Effects of Phospholipase C Inhibitor on SCRT-InducedDepolarizations in Pacemaker Potenials in Cultured ICCsSince the membrane depolarizations by SCRT was relatedto intracellular Ca2+ mobilization we examined whetherthe effects on pacemaker potentials require phospholipaseC (PLC) activation To test this possibility SCRT-induced

membrane depolarizations were measured in the absenceand presence of U-73122 an active PLC inhibitor [15] SCRT(30mgmL) induced membrane depolarizations on ICCs(Figure 5(a)) The pacemaker membrane depolarizationswere completely abolished by application of U-73122 (5120583M)and under these conditions SCRT-inducedmembrane depo-larizations were not produced (119899 = 4 Figure 5(b)) In thepresence of U-73122 the membrane depolarizations pro-duced by SCRT were 17 plusmn 06mV The value of membranedepolarizations by SCRT was significantly different whencompared with SCRT in the absence of U-73122 (119899 = 4Figure 5(d)) The treatment of U-73343 (5 120583M) an inac-tive analog of U-73122 had no influence on the pace-maker potentials and under these conditions SCRT-inducedmembrane depolarizations were not suppressed by U-73343(Figure 5(c)) These results show that PLC pathway isinvolved in SCRT-induced membrane depolarizations onICCs

36 Involvements of Mitogen-Activated Protein Kinases(MAPKs) in SCRT-Induced Depolarizations in PacemakerPotentials in Cultured ICCs Approximately 90 of end-ogenous 5-hydroxytryptamine (5-HT) in the body exists inthe digestive tract and 5-HT is believed to be involved inthe regulation of gastrointestinal motility Also it has beenreported that 5-HT activates MAPKs in many cell types andthus we examined whether or not MAPKs are involved in theeffects induced by SCRT by using PD98059 (a p4244MAPK


0 100 200 300 400

05

06

07

08F3

403

80

Control

Time (s)

(a)

0 50 100 150 200

05

06

07

08

F340

380

Time (s)


(b)

0 100 200 300 400

04

05

06

F340

380

Time (s)


(c)

Figure 4 Responses of intracellular Ca2+ to SCRT in cultured ICCs ICCswere suspended in 2mMCa2+-containing normal solution Increasein [Ca2+]i was plotted against SCRT concentrations (a) In normal conditions [Ca2+]i oscillations was induced (b) SCRT 10mgmL had noresponse but SCRT 50mgmL caused a sustained increase in [Ca2+]i (c)

inhibitor) SB203580 (a p38 MAPK inhibitor) or c-junNH2-terminal kinase (JNK) II inhibitor SCRT (30mgmL)induced membrane depolarizations on ICCs (Figure 6(a))In the presence of PD98059 (10 120583M) SCRT did not producemembrane depolarizations (119899 = 4 Figures 6(b) and 6(e))which indicated that p4244 plays a role in SCRT-inducedmembrane depolarization In addition SB203580 and JNK IIinhibitor blocked the depolarizations by SCRT in pacemakerpotentials (119899 = 4 Figures 6(c) 6(d) and 6(e)) These resultsshow that the regulation ofmitogen-activated protein kinasesis involved in SCRT induced membrane depolarizations onICCs

4 Discussion

In recent years there has beenmuch work done in the area oftraditional medicinal plants in Korea and many good results

or promising leads have been achieved [8] In GI motilityarea ICCs are good research tools to study GI motility andtherefore many labs use ICCs

In this study we used ICCs and are studying to developa new GI motility drug Until now SCRT was not used totreat GI motility diseases SCRT has been widely used totreat respiratory disease such as cough and asthma in orientalcountries [16] In this study we found the potentials of SCRTto treat in GI motility Because of the central role of ICCs inGImotility loss of these cellswould be extremely detrimentalResearch into the biology of ICCs provides exciting newopportunities to understand the etiology of diseases thathave long eluded comprehension Discovering the moleculesinvolved in the generation of pacemaker activity in ICCsmaylead to dramatic new therapies for chronic GI diseases thatresult in lifelong suffering Consequently ICCs are involvednot only in physiological GI motility but also in many bowel


minus33mV


(a)

minus33mV

minus52mV

So-Cheong-Ryong-Tang 30mgmLU-73122 5120583M

(b)


minus33mV

minus52mVU-73343 5120583M

(c)

Deg

ree o

f dep

olar

izat

ion

(mV

)

CTRL U-73122 U-733430

3

6

9

12

15


lowastlowast

(d)

Figure 5 Effects of SCRT on phospholipase C inhibitors in cultured ICCs (a) SCRT (30mgmL) induced membrane depolarizations onICCs (b) U-73122 (5120583M) a phospholipase C inhibitor abolished the generation of pacemaker potentials U-73122 blocked the SCRT-induced(30mgmL) membrane depolarizations (c) The application of U-73343 (5 120583M) did not show any influence on the generation of pacemakercurrents Also U-73343 did not block the SCRT-induced (30mgmL)membrane depolarizations (d)The responses to SCRT in phospholipaseC inhibitors are summarized in (d) Bars represent mean values plusmn SEs lowastlowast119875 lt 001 Significantly different from untreated controls CTRLControl

disorders including inflammatory bowel disease chronicidiopathic intestinal pseudo-obstruction intestinal obstruc-tion with hypertrophy achalasia Hirschsprung disease juve-nile pyloric stenosis juvenile intestinal obstruction andanorectal malformation [17]

5-HT plays a critical role in coordinating GI motility [18ndash21] In response to intestinal stretching stimulation 5-HTpromotes peristalsis via the activation of intrinsic primaryafferent neurons located in the submucous plexus [12 22 23]Of the 5-HT receptors believed to influence GI motility5-HT3and 5-HT

4receptors play an important role in the

regulation of GI motility [12 24ndash26] Also in guinea pigcolon 5-HT receptor was investigated [20] Furthermorethese receptors mediate peristaltic reflex and existed innervous systems in the guinea pig distal colon [27] Liu et al[13] suggested that 5-HT augments ICC pacemaker activityvia 5-HT3 receptors whereas Shahi et al [11] suggested that5-HT can modulate pacemaker activity via 5-HT3 4 and 7receptors On the other hand Wouters et al [28] suggestedthat 5-HT2B receptors regulate the proliferation of ICCs inmouse jejunum In Korea poncirus trifoliate (PT) is widelyused as a remedy for GI allergic and inflammatory diseases[29 30] In recent study Kim et al [6] suggested that PTexerts its prokinetic activity through a 5-HT

3and 5-HT

4

receptor-mediated pathway in ICCs inmouse small intestine

In the present study Y25130 (a 5-HT3receptor antagonist)

and RS39604 (a 5-HT4receptor antagonist) blocked SCRT-

induced membrane depolarizations whereas SB269970 (a 5-HT7receptor antagonist) did not Thus it appears that SCRT

modulates pacemaker potentials through 5-HT3and 5-HT

4

receptors-mediated pathways in the ICCs of mouse smallintestine (Figure 2)

In this study ICCs generated pacemaker potentials inmouse small intestine SCRT produced membrane depolar-ization in current clamp mode Y25130 (a 5-HT

3receptor

antagonist) and RS39604 (a 5-HT4receptor antagonist)

blocked MPF-induced membrane depolarizations whereasSB269970 (a 5-HT

7receptor antagonist) did not When

GDP-120573-S (1mM) was in the pipette solution SCRT did notinduce the membrane depolarizations Also SCRT increasedintracellular Ca2+ concentrations To examined whether ornot MAPKs are involved in the effects induced by SCRTwe used PD98059 (a p4244 MAPK inhibitor) SB203580 (ap38 MAPK inhibitor) or c-jun NH2-terminal kinase (JNK)II inhibitor In the presence of PD98059 (10 120583M) SCRTdid not produce membrane depolarizations In additionSB203580 and JNK II inhibitor blocked the depolariza-tions by SCRT in pacemaker potentials Furthermore themembrane depolarizations by SCRT were inhibited not byU-73122 an active phospholipase C (PLC) inhibitors but



minus34mV

minus53mV

(a)

minus34mV

minus53mV

So-Cheong-Ryong-Tang 30mgmLPD98059 10120583M

(b)


minus34mV

minus53mVSB203580 10120583M

(c)


minus34mV

minus53mVJNK II inhibitor 10120583M

(d)

Deg

ree o

f dep

olar

izat

ion

(mV

)

CTRL PD98059 SB203580 JNK II inhibitor 0

3

6

9

12

15


lowastlowastlowastlowast lowastlowast

(e)

Figure 6 Effects of various MAPK inhibitors on SCRT-induced pacemaker potentials responses in cultured ICCs (a) SCRT (30mgmL)induced membrane depolarizations on ICCs (b) Pacemaker potentials of cultured ICCs exposed to SCRT (30mgmL) in the presence of10120583M of PD98059 (a p4244 MAPK inhibitor) (c) Pacemaker potentials of cultured ICCs exposed to SCRT in the presence of 10120583M ofSB203580 (a p38 MAPK inhibitor) (d) Pacemaker potentials of an ICC exposed to SCRT in the presence of 10120583M of JNK II inhibitorResponses to SCRT in the presence of different MAPK inhibitors are summarized in (e) Bars represent mean values plusmn SEs lowastlowast119875 lt 001Significantly different from untreated controls CTRL Control

by U-73343 an inactive PLC inhibitors These results suggestthat SCRT might affect GI motility by the modulation ofpacemaker activity throughMAPKs and PLC pathways in theICCs

We think that SCRT activates PLCpathway (Figure 5) andincreases intracellular Ca2+ levels (Figure 4) IntracellularCa2+ increases induce the membrane depolarizations onICCs and then make ICCs contractions (Figure 7) Howeverthe exact mechanisms of membrane depolarizations on ICCsshould be investigated in future

The signaling pathways of MAPKs play important rolesin the mediation of cellular responses including visceralsmooth muscle contraction Three principal MAPKs areexpressed in various tissues (p4244 JNK and p38 MAPK)[31] Furthermore it has been demonstrated 5-HT

7receptors

activateMAP kinase in hippocampal neurons [32] and that 5-HT induces cyclooxygenase (COX)-2 by activatingMAPK in

vascular smoothmuscle cells [33] which show that 5-HT canregulate the MAPK system In the present study we do notknow whether this SCRT has a role of 5-HT or not Howeverin this study an inhibitor of p4244 and p38 and a JNK IIinhibitor inhibited the effect of SCRT which suggests thatp38 p4244 and JNKMAPK are involved in the modulationof pacemaker potentials by SCRT

Taken together our data suggest that the SCRT have anability to regulate the pacemaker potentials in ICCs and con-sidering the effects of this drug on the ICC further researchincluding finding active compound(s) and examining theiraction mechanisms is clearly needed

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper


G-proteinPLC

SR

ContractionMembrane depolarization

MAPKs

So-Cheong-Ryong-Tang (SCRT)

5-HT3R

[Ca2+]i

andor 5-HT4R

Figure 7 Hypothetical schematic signaling pathway of SCRT-induced membrane depolarizations on ICCs SCRT-induced membranedepolarization seems to be mediated by 5-HT

3andor 5-HT

4receptor coupled to G protein resulting in the activation of PLC pathway

which subsequently increases the intracellular Ca2+ level from SR The data also suggest that MAPKs are involved in the SCRT-inducedmembrane depolarizations

Acknowledgment

This study was supported by a Grant of the TraditionalKoreanMedicine RampDProjectMinistry ofHealthampWelfareRepublic of Korea (HI12C1886 (B120008))

References

[1] S M Ward A J Burns S Torihashi and K M SandersldquoMutation of the proto-oncogene c-kit blocks development ofinterstitial cells and electrical rhythmicity in murine intestinerdquoJournal of Physiology vol 480 no 1 pp 91ndash97 1994

[2] J D Huizinga L Thuneberg M Kluppel J Malysz H BMikkelsen and A Bernstein ldquoWkit gene required for intersti-tial cells of Cajal and for intestinal pacemaker activityrdquo Naturevol 373 no 6512 pp 347ndash349 1995

[3] KM Sanders ldquoA case for interstitial cells of Cajal as pacemakersand mediators of neurotransmission in the gastrointestinaltractrdquo Gastroenterology vol 111 no 2 pp 492ndash515 1996

[4] S D Koh J Y Jun T W Kim and K M Sanders ldquoACa2+-inhibited non-selective cation conductance contributes topacemaker currents in mouse interstitial cell of Cajalrdquo Journalof Physiology vol 540 part 3 pp 803ndash814 2002

[5] B J Kim H-H Lim D K Yang et al ldquoMelastatin-typetransient receptor potential channel 7 is required for intestinalpacemaking activityrdquoGastroenterology vol 129 no 5 pp 1504ndash1517 2005

[6] B J Kim H W Kim G S Lee et al ldquoPoncirus trifoliate fruitmodulates pacemaker activity in interstitial cells of Cajal fromthe murine small intestinerdquo Journal of Ethnopharmacology vol149 no 3 pp 668ndash675 2013

[7] K Goto S Matsuoka and A Noma ldquoTwo types of spontaneousdepolarizations in the interstitial cells freshly prepared from themurine small intestinerdquo Journal of Physiology vol 559 no 2 pp411ndash422 2004

[8] D Bai ldquoTraditional Chinese medicines and new drug develop-mentrdquo Pure and Applied Chemistry vol 65 no 6 pp 1103ndash11121993

[9] E Ko S Rho C Cho et al ldquoSo-Cheong-Ryong-Tang traditi-tional Korean medicine suppresses Th2 lineage developmentrdquoBiological and Pharmaceutical Bulletin vol 27 no 5 pp 739ndash743 2004

[10] D-Y Kim and M Camilleri ldquoSerotonin a mediator of thebrain-gut connectionrdquo American Journal of Gastroenterologyvol 95 no 10 pp 2704ndash2709 2000

[11] P K Shahi S Choi D C Zuo et al ldquo5-hydroxytryptaminegenerates tonic inward currents on pacemaker activity ofinterstitial cells of Cajal from mouse small intestinerdquo KoreanJournal of Physiology and Pharmacology vol 15 no 3 pp 129ndash135 2011

[12] M D Gershon and J Tack ldquoThe serotonin signaling systemfrom basic understanding to drug development for functionalGI disordersrdquoGastroenterology vol 132 no 1 pp 397ndash414 2007

[13] H-N Liu S Ohya Y Nishizawa et al ldquoSerotonin augments gutpacemaker activity via 5-HT

3receptorsrdquo PLoS ONE vol 6 no

9 Article ID e24928 2011[14] S M Ward T Ordog S D Koh et al ldquoPacemaking in

interstitial cells of Cajal depends upon calcium handling byendoplasmic reticulum and mitochondriardquo Journal of Physiol-ogy vol 525 no 2 pp 355ndash361 2000

[15] T Sakamoto T UnnoHMatsuyama et al ldquoCharacterization ofmuscarinic receptor-mediated cationic currents in longitudinalsmooth muscle cells of mouse small intestinerdquo Journal ofPharmacological Sciences vol 100 no 3 pp 215ndash226 2006

[16] S Jung S J Cho K I Moon H W Kim B Y Kim and SI Cho ldquoEffects of Socheongryong-tang on immunoglobulinproduction in asthmaticmicerdquoKorean Journal ofHerbology vol23 no 1 pp 23ndash28 2008

[17] B J Kim H-H Lim D K Yang et al ldquoMelastatin-typetransient receptor potential channel 7 is required for intestinal


pacemaking activityrdquoGastroenterology vol 129 no 5 pp 1504ndash1517 2005

[18] M B Hansen and E Skadhauge ldquoSignal transduction pathwaysfor serotonin as an intestinal secretagoguerdquo Comparative Bio-chemistry and Physiology vol 118 no 2 pp 283ndash290 1997

[19] J R Grider A E Foxx-Orenstein and J-G Jin ldquo5-hydroxy-tryptamine4 receptor agonists initiate the peristaltic reflex inhuman rat and guinea pig intestinerdquoGastroenterology vol 115no 2 pp 370ndash380 1998

[20] J-G Jin A E Foxx-Orenstein and J R Grider ldquoPropulsionin Guinea pig colon induced by 5-hydroxytryptamine (HT)via 5-HT

4and 5-HT

3receptorsrdquo Journal of Pharmacology and

Experimental Therapeutics vol 288 no 1 pp 93ndash97 1999[21] D E Baker ldquoRationale for using serotonergic agents to treat

irritable bowel syndromerdquo American Journal of Health-SystemPharmacy vol 62 no 7 pp 700ndash713 2005

[22] A L KirchgessnerH Tamir andMDGershon ldquoIdentificationand stimulation by serotonin of intrinsic sensory neurons ofthe submucosal plexus of the guinea pig gut activity-inducedexpression of Fos immunoreactivityrdquo Journal of Neurosciencevol 12 no 1 pp 235ndash248 1992

[23] A E Foxx-Orenstein J F Kuemmerle and J R Grider ldquoTheperistaltic reflex induced by mucosal stimuli in human andguinea pig intestine is mediated by distinct mucosal 5-HTreceptorsrdquo Gastroenterology vol 108 no 2 p A600 1995

[24] G J Sanger ldquo5-hydroxytryptamine and the gastrointestinaltract where nextrdquo Trends in Pharmacological Sciences vol 29no 9 pp 465ndash471 2008

[25] E J Dickson D J Heredia and T K Smith ldquoCritical roleof 5-HT

1119860 5-HT

3 and 5-HT

7receptor subtypes in the ini-

tiation generation and propagation of the murine colonicmigrating motor complexrdquo American Journal of PhysiologymdashGastrointestinal and Liver Physiology vol 299 no 1 pp G144ndashG157 2010

[26] K B Neal L J Parry and J C Bornstein ldquoStrain-specificgenetics anatomy and function of enteric neural serotonergicpathways in inbred micerdquo Journal of Physiology vol 587 no 3pp 567ndash586 2009

[27] M Kadowaki P R Wade andM D Gershon ldquoParticipation of5-HT

3 5-HT

4 and nicotinic receptors in the peristaltic reflex

of guinea pig distal colonrdquo American Journal of PhysiologymdashGastrointestinal and Liver Physiology vol 271 no 5 pp G849ndashG857 1996

[28] M M Wouters S J Gibbons J L Roeder et al ldquoExogenousserotonin regulates proliferation of interstitial cells of Cajal inmouse jejunum through 5-HT

2119861receptorsrdquo Gastroenterology

vol 133 no 3 pp 897ndash906 2007[29] C J Lee J H Lee J H Seok et al ldquoEffects of baicalein berber-

ine curcumin and hesperidin on mucin release from airwaygoblet cellsrdquo Planta Medica vol 69 no 6 pp 523ndash526 2003

[30] J-M Yi M-S Kim H-N Koo B-K Song Y-H Yoo and H-M Kim ldquoPoncirus trifoliata fruit induces apoptosis in humanpromyelocytic leukemia cellsrdquo Clinica Chimica Acta vol 340no 1-2 pp 179ndash185 2004

[31] G Pearson F Robinson T Beers Gibson et al ldquoMitogen-activated protein (MAP) kinase pathways regulation and phys-iological functionsrdquo Endocrine Reviews vol 22 no 2 pp 153ndash183 2001

[32] M Errico R A Crozier M R Plummer and D S Cowenldquo5-HT

7receptors activate the mitogen activated protein kinase

extracellular signal related kinase in cultured rat hippocampalneuronsrdquo Neuroscience vol 102 no 2 pp 361ndash367 2001

[33] T Machida M Ohta A Onoguchi et al ldquoHydroxytryptamineinduces cyclooxygenase-2 in rat vascular smooth muscle cellsmechanisms involving Src PKC and MAPK activationrdquo Euro-pean Journal of Pharmacology vol 656 no 1ndash3 pp 19ndash26 2011

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


of years in Asian countries [9] However there were notmany attempts to investigate the efficacy of SCRT in digestivesystems

Of the pathways related to intestinal motility serotonin(5-hydroxytryptamine 5-HT a major neuromodulator) isknown to play a critical role in the GI tract Generally5-HT acts as a neurotransmitter in the central nervoussystem but most (95) 5-HT is found in the GI tract [10]Furthermore although 5-HT is known to interact with sevendifferent 5-HT receptor subtypes only three of these arefound in the ICCs in the murine small intestine [11] 5-HTcan modulate pacemaker activity through 5-HT3 4 and7 receptors In previous study we suggested that poncirustrifoliatemodulates pacemaker potentials through 5-HT

3and

5-HT4receptor-mediated pathways via external Na+ and

Ca2+ influx [6] However the effects of SCRT and the actionmechanism involved in the GI tract are not investigated

Therefore we undertook to investigate the effects of theSCRT on the pacemaker potentials of cultured ICCs derivedfrom murine small intestine and to identify the receptorsinvolved

2 Materials and Methods

21 Preparation of Cells and Cell Cultures Balbc mice (3ndash7 days old) of either sex were anesthetized with ether andkilled by cervical dislocation The small intestines from 1 cmbelow the pyloric ring to the cecum were removed andopened along the mesenteric border Luminal contents wereremoved bywashingwith Krebs-Ringer bicarbonate solutionThe tissues were pinned to the base of a Sylgard dish andthe mucosa removed by sharp dissection Small tissue stripsof the intestine muscle (consisting of both circular andlongitudinal muscles) were equilibrated in Ca2+-free Hankssolution (containing in mmolL KCl 536 NaCl 125 NaOH034 Na

2HCO3044 glucose 10 sucrose 29 and HEPES 11)

for 30min Then the cells were dispersed using an enzymesolution containing collagenase (Worthington BiochemicalCo Lakewood NJ USA) 13mgmL bovine serum albumin(Sigma Chemical Co St Louis MO USA) 2mgmL trypsininhibitor (Sigma) 2mgmL and ATP 027mgmL Cells wereplated onto sterile glass coverslips coated with murine col-lagen (25120583gmL FalconBD Franklin Lakes NJ USA) in a35-mm culture dish and then cultured at 37∘C in a 95 O

2

50mLL CO2incubator in a smooth muscle growth medium

(Clonetics Corp San Diego CA USA) supplemented with2 antibioticsantimycotics (Gibco Grand Island NY USA)and murine stem cell factor (SCF 5 ngmL Sigma) ICCswere identified immunologically with anti-c-kit antibody(phycoerythrin-conjugated rat anti-mouse c-kit monoclonalantibody eBioscience San Diego CA USA) at a dilution of1 50 for 20min [7] ICCs weremorphologically distinct fromother cell types in the culture and thus it was possible toidentify the cells by phase contrast microscopy once they hadbeen verified with anti c-kit antibody

22 Patch-Clamp Experiments The whole-cell patch-clampconfiguration was used to record membrane potentials

(current clamp) from cultured ICCs An axopatch ID(Axon Instruments Foster CA USA) was used to amplifymembrane currents and potentials The command pulsewas applied using an IBM-compatible personal computerand pClamp software (version 61 Axon Instruments)Data obtained were filtered at 5 kHz and displayed on anoscilloscope a computer monitor and using a pen recorder(Gould 2200 Gould Valley View OH USA) Results wereanalyzed using pClamp and Origin (version 60) softwareAll experiments were performed at 30ndash32∘C

23 Fura-2 Loading and Measurement of Intracellular FreeCalcium Ion Concentration [Ca2+]

119894 Cultured ICC clusters

were loaded with the acetoxymethyl ester form of fura-2(5 120583molL diluted from 1mmolL stock in dimethyl sulfoxide(DMSO)) in normal medium for 20 minutes at 37∘C Therecording of [Ca2+]i was performedwith amicrofluorometricsystem consisting of an inverted fluorescence microscope(Diaphot 300 Nikon Japan) with a dry-type fluorescenceobjective lens (40X numerical aperture 085) a photomul-tiplier tube (type R 1527 Hamamatsu Japan) and a PTI-Deltascan illuminator (Photon Technology InternationalInc) Cells were superfused at a flow rate of 15mLminLight was provided by a 75-W xenon lamp (Ushino Japan)To control excitation frequency a chopper wheel alternatedthe light path to monochromators (340 and 380 nm) with afrequency of 5 or 10Hz A short-pass dichroic mirror passedemission light of lt570 nm onto the photomultiplier tube andintensity was measured at 510 nm A mechanical image maskwas placed in the emission path to limit measurement to asingle cell Both data acquisition and control of light applica-tion were performed with computer software (Felix version11 Photon Technology International Inc Brunswick NJ)Because of uncertainties in calibrating the fura-2 signals inintact cells no attempt wasmade to calibrate [Ca2+]i insteadall results are reported as changes in the 340 nm380 nmsignal ratio

24 Solutions andDrugs Thephysiological salt solution usedto bathe cells (Na+-Tyrode) contained (mmolL) KCl 5 NaCl135 CaCl

22 glucose 10MgCl

212 andHEPES 10 adjusted to

pH 74 with NaOHThe pipette solution contained (mmolL)KCl 140 MgCl

25 K2ATP 27 NaGTP 01 creatine phosphate

disodium25HEPES 5 andEGTA01 adjusted to pH72withKOH

SCRT was purchased from I-WORLD Pharmaceuti-cals (South Korea) SCRT is composed of Rhizoma Pinel-liae Herba Ephedrae Radix Paeoniae Fructus SchisandraeHerba Asari Rhizoma Zingiberis Ramulus Cinnamomiand Radix Glycyrrhizae (Table 1) The dosage for adult is10ndash15 g (crude materials) per day More information aboutSCRT can be found in I-WORLDPharmaceuticalsHomepage(httpi-pharmkoreasmecom) The SCRT was dissolvedwith distilled water at the concentration of 05 g (crudedrug)mL and stored in refrigerator All other drugs wereobtained from Sigma (Sigma Chemical Co USA) Drugswere dissolved in distilledwater and added to bath solution tomake the desired concentrations just prior to use Addition



minus31mV

minus52mV

(a)


minus34mV

minus53mV

(b)

minus30mV


(c)

minus31mV


(d)

0

5

10

15

20

25

Deg

ree o

f dep

olar

izat

ion

(mV

)

CTRL 10 30 50


lowastlowast

lowastlowast

(e)




Total amount 9 g




3 Results





minus31mV

minus53mV

(a)


minus32mV

minus53mV

(b)


minus30mV

minus51mV

(c)


minus15mV

minus53mV

(d)

0

10

20

30

40

Deg

ree o

f dep

olar

izat

ion

(mV

)



(e)










4R is


3R 5-HT

4R and 5-HT



3receptor





3R and 5-HT

4R




minus34mV

minus51mV

(a)


minus33mV

minus52mV

[GDP-120573-S 1mM]

(b)

Deg

ree o

f dep

olar

izat

ion

(mV

)

CTRL0

3

6

9

12

15


[GDP-120573-S 1mM]

lowastlowast

(c)









0 100 200 300 400

05

06

07

08F3

403

80

Control

Time (s)

(a)

0 50 100 150 200

05

06

07

08

F340

380

Time (s)


(b)

0 100 200 300 400

04

05

06

F340

380

Time (s)


(c)



4 Discussion





minus33mV


(a)

minus33mV

minus52mV


(b)


minus33mV

minus52mVU-73343 5120583M

(c)

Deg

ree o

f dep

olar

izat

ion

(mV

)

CTRL U-73122 U-733430

3

6

9

12

15


lowastlowast

(d)






3and 5-HT

4






4



3receptor







minus34mV

minus53mV

(a)

minus34mV

minus53mV


(b)


minus34mV

minus53mVSB203580 10120583M

(c)


minus34mV


(d)

Deg

ree o

f dep

olar

izat

ion

(mV

)


3

6

9

12

15



(e)





7receptors







G-proteinPLC

SR


MAPKs


5-HT3R

[Ca2+]i

andor 5-HT4R


3andor 5-HT



Acknowledgment


References

























4and 5-HT








1119860 5-HT

3 and 5-HT





3 5-HT


















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















minus31mV

minus52mV

(a)


minus34mV

minus53mV

(b)

minus30mV


(c)

minus31mV


(d)

0

5

10

15

20

25

Deg

ree o

f dep

olar

izat

ion

(mV

)

CTRL 10 30 50


lowastlowast

lowastlowast

(e)




Total amount 9 g




3 Results





minus31mV

minus53mV

(a)


minus32mV

minus53mV

(b)


minus30mV

minus51mV

(c)


minus15mV

minus53mV

(d)

0

10

20

30

40

Deg

ree o

f dep

olar

izat

ion

(mV

)



(e)










4R is


3R 5-HT

4R and 5-HT



3receptor





3R and 5-HT

4R




minus34mV

minus51mV

(a)


minus33mV

minus52mV

[GDP-120573-S 1mM]

(b)

Deg

ree o

f dep

olar

izat

ion

(mV

)

CTRL0

3

6

9

12

15


[GDP-120573-S 1mM]

lowastlowast

(c)









0 100 200 300 400

05

06

07

08F3

403

80

Control

Time (s)

(a)

0 50 100 150 200

05

06

07

08

F340

380

Time (s)


(b)

0 100 200 300 400

04

05

06

F340

380

Time (s)


(c)



4 Discussion





minus33mV


(a)

minus33mV

minus52mV


(b)


minus33mV

minus52mVU-73343 5120583M

(c)

Deg

ree o

f dep

olar

izat

ion

(mV

)

CTRL U-73122 U-733430

3

6

9

12

15


lowastlowast

(d)






3and 5-HT

4






4



3receptor







minus34mV

minus53mV

(a)

minus34mV

minus53mV


(b)


minus34mV

minus53mVSB203580 10120583M

(c)


minus34mV


(d)

Deg

ree o

f dep

olar

izat

ion

(mV

)


3

6

9

12

15



(e)





7receptors







G-proteinPLC

SR


MAPKs


5-HT3R

[Ca2+]i

andor 5-HT4R


3andor 5-HT



Acknowledgment


References

























4and 5-HT








1119860 5-HT

3 and 5-HT





3 5-HT


















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















minus31mV

minus53mV

(a)


minus32mV

minus53mV

(b)


minus30mV

minus51mV

(c)


minus15mV

minus53mV

(d)

0

10

20

30

40

Deg

ree o

f dep

olar

izat

ion

(mV

)



(e)










4R is


3R 5-HT

4R and 5-HT



3receptor





3R and 5-HT

4R




minus34mV

minus51mV

(a)


minus33mV

minus52mV

[GDP-120573-S 1mM]

(b)

Deg

ree o

f dep

olar

izat

ion

(mV

)

CTRL0

3

6

9

12

15


[GDP-120573-S 1mM]

lowastlowast

(c)









0 100 200 300 400

05

06

07

08F3

403

80

Control

Time (s)

(a)

0 50 100 150 200

05

06

07

08

F340

380

Time (s)


(b)

0 100 200 300 400

04

05

06

F340

380

Time (s)


(c)



4 Discussion





minus33mV


(a)

minus33mV

minus52mV


(b)


minus33mV

minus52mVU-73343 5120583M

(c)

Deg

ree o

f dep

olar

izat

ion

(mV

)

CTRL U-73122 U-733430

3

6

9

12

15


lowastlowast

(d)






3and 5-HT

4






4



3receptor







minus34mV

minus53mV

(a)

minus34mV

minus53mV


(b)


minus34mV

minus53mVSB203580 10120583M

(c)


minus34mV


(d)

Deg

ree o

f dep

olar

izat

ion

(mV

)


3

6

9

12

15



(e)





7receptors







G-proteinPLC

SR


MAPKs


5-HT3R

[Ca2+]i

andor 5-HT4R


3andor 5-HT



Acknowledgment


References

























4and 5-HT








1119860 5-HT

3 and 5-HT





3 5-HT


















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















minus34mV

minus51mV

(a)


minus33mV

minus52mV

[GDP-120573-S 1mM]

(b)

Deg

ree o

f dep

olar

izat

ion

(mV

)

CTRL0

3

6

9

12

15


[GDP-120573-S 1mM]

lowastlowast

(c)









0 100 200 300 400

05

06

07

08F3

403

80

Control

Time (s)

(a)

0 50 100 150 200

05

06

07

08

F340

380

Time (s)


(b)

0 100 200 300 400

04

05

06

F340

380

Time (s)


(c)



4 Discussion





minus33mV


(a)

minus33mV

minus52mV


(b)


minus33mV

minus52mVU-73343 5120583M

(c)

Deg

ree o

f dep

olar

izat

ion

(mV

)

CTRL U-73122 U-733430

3

6

9

12

15


lowastlowast

(d)






3and 5-HT

4






4



3receptor







minus34mV

minus53mV

(a)

minus34mV

minus53mV


(b)


minus34mV

minus53mVSB203580 10120583M

(c)


minus34mV


(d)

Deg

ree o

f dep

olar

izat

ion

(mV

)


3

6

9

12

15



(e)





7receptors







G-proteinPLC

SR


MAPKs


5-HT3R

[Ca2+]i

andor 5-HT4R


3andor 5-HT



Acknowledgment


References

























4and 5-HT








1119860 5-HT

3 and 5-HT





3 5-HT


















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0 100 200 300 400

05

06

07

08F3

403

80

Control

Time (s)

(a)

0 50 100 150 200

05

06

07

08

F340

380

Time (s)


(b)

0 100 200 300 400

04

05

06

F340

380

Time (s)


(c)



4 Discussion





minus33mV


(a)

minus33mV

minus52mV


(b)


minus33mV

minus52mVU-73343 5120583M

(c)

Deg

ree o

f dep

olar

izat

ion

(mV

)

CTRL U-73122 U-733430

3

6

9

12

15


lowastlowast

(d)






3and 5-HT

4






4



3receptor







minus34mV

minus53mV

(a)

minus34mV

minus53mV


(b)


minus34mV

minus53mVSB203580 10120583M

(c)


minus34mV


(d)

Deg

ree o

f dep

olar

izat

ion

(mV

)


3

6

9

12

15



(e)





7receptors







G-proteinPLC

SR


MAPKs


5-HT3R

[Ca2+]i

andor 5-HT4R


3andor 5-HT



Acknowledgment


References

























4and 5-HT








1119860 5-HT

3 and 5-HT





3 5-HT


















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















minus33mV


(a)

minus33mV

minus52mV


(b)


minus33mV

minus52mVU-73343 5120583M

(c)

Deg

ree o

f dep

olar

izat

ion

(mV

)

CTRL U-73122 U-733430

3

6

9

12

15


lowastlowast

(d)






3and 5-HT

4






4



3receptor







minus34mV

minus53mV

(a)

minus34mV

minus53mV


(b)


minus34mV

minus53mVSB203580 10120583M

(c)


minus34mV


(d)

Deg

ree o

f dep

olar

izat

ion

(mV

)


3

6

9

12

15



(e)





7receptors







G-proteinPLC

SR


MAPKs


5-HT3R

[Ca2+]i

andor 5-HT4R


3andor 5-HT



Acknowledgment


References

























4and 5-HT








1119860 5-HT

3 and 5-HT





3 5-HT


















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















minus34mV

minus53mV

(a)

minus34mV

minus53mV


(b)


minus34mV

minus53mVSB203580 10120583M

(c)


minus34mV


(d)

Deg

ree o

f dep

olar

izat

ion

(mV

)


3

6

9

12

15



(e)





7receptors







G-proteinPLC

SR


MAPKs


5-HT3R

[Ca2+]i

andor 5-HT4R


3andor 5-HT



Acknowledgment


References

























4and 5-HT








1119860 5-HT

3 and 5-HT





3 5-HT


















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















G-proteinPLC

SR


MAPKs


5-HT3R

[Ca2+]i

andor 5-HT4R


3andor 5-HT



Acknowledgment


References

























4and 5-HT








1119860 5-HT

3 and 5-HT





3 5-HT


















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















4and 5-HT








1119860 5-HT

3 and 5-HT





3 5-HT


















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Research Article Involvement of MAPKs and PLC Pathways in ...

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Transcript of Research Article Involvement of MAPKs and PLC Pathways in ...