Helional Induces Ca2+ Decrease and Serotonin Secretion of QGP-1 Cells

57 3

b kalbe and others Ca2+ decrease and 5-HT 57: 3 201–210 Research release in QGP-1 cells

2+ Helional induces Ca decrease and serotonin secretion of QGP-1 cells via a PKG-mediated pathway

Benjamin Kalbe1, Marian Schlimm1, Julia Mohrhardt2, Paul Scholz1, Fabian Jansen1, Hanns Hatt1 and Sabrina Osterloh1 Correspondence should be addressed 1 Department of Cell Physiology, Ruhr-University Bochum, Bochum, Germany to B Kalbe 2 Department of Chemosensation, Institute for Biology II, RWTH Aachen University, Aachen, Germany Email [email protected]

Abstract

The secretion, motility and transport by intestinal tissues are regulated among others Key Words by specialized neuroendocrine cells, the so-called enterochromaffin (EC) cells. These ff olfactory receptor cells detect different luminal stimuli, such as mechanical stimuli, fatty acids, glucose and ff serotonin distinct chemosensory substances. The EC cells react to the changes in their environment ff enterochromaffin cells through the release of transmitter molecules, most importantly serotonin, to mediate the ff calcium signaling corresponding physiological response. However, little is known about the molecular targets of the chemical stimuli delivered from consumed food, spices and cosmetics within EC cells. In this study, we evaluated the expression of the olfactory receptor (OR) 2J3 in the human pancreatic EC cell line QGP-1 at the mRNA and protein levels. Using ratiofluorometric Ca2+ imaging experiments, we demonstrated that the OR2J3-specific agonist helional induces a transient dose-dependent decrease in the intracellular Ca2+ levels. This Ca2+ decrease Journal of Molecular Endocrinology is mediated by protein kinase G (PKG) on the basis that the specific pharmacological inhibition of PKG with Rp-8-pCPT-cGMPS abolished the helional-induced Ca2+ response. Furthermore, stimulation of QGP-1 cells with helional caused a dose-dependent release of serotonin that was comparable with the release induced by the application of a direct PKG activator (8-bromo-cGMP). Taken together, our results demonstrate that luminal odorants can be detected by specific ORs in QGP-1 cells and thus cause the directed release of Journal of Molecular Endocrinology serotonin and a PKG-dependent decrease in intracellular Ca2+. (2016) 57, 201–210

Introduction

Enterochromaffin (EC) cells, the most abundant endocrine peristalsis, inflammatory processes, osteogenesis and the cell type, regulate the important functions of the gut, transmission to afferent neurons, and peristalsis is also stomach, gall bladder, intestine and pancreas by secreting critically dependent on the activation of serotonergic biologically active signaling molecules (e.g. gastrin, neurons (Gershon 2004, 2013, Li et al. 2011, Heredia et al. chromogranins, secretin, somatostatin and cholecystokinin) 2013). In general, the release of serotonin is mediated by that are synthesized and stored within these cells (Moran an increase of the intracellular Ca2+ concentration via et al. 2008, Manocha & Khan 2012). One of the most influx of extracellular Ca2+ or liberation from intracellular prominent transmitters is serotonin (5-hydroxytryptamine, stores and exocytosis of vesicles (Racké et al. 1996). Cyclic 5-HT), which controls among others epithelial transport, adenosine monophosphate (cAMP) can also lead to the

http://jme.endocrinology-journals.org © 2016 Society for Endocrinology Published by Bioscientifica Ltd. DOI: 10.1530/JME-16-0063 Printed in Great Britain Downloaded from Bioscientifica.com at 10/01/2021 09:00:00PM via free access

10.1530/JME-16-0063 Journal of Molecular Endocrinology caused a significant suppression of the observed effect. caused asignificantsuppression oftheobserved levels. TheinhibitionofPKG throughaspecificblocker led todose-dependentdecreases inintracellularCa line QGP-1.Theapplication oftheodoranthelional levels ofspecificORsinthe humanpancreaticECcell et al. (MAPK), canbeinvolvedinectopicORsignaling( including Src kinaseandmitogen-activatedproteinkinases Additionally, diversedownstreamsignaling molecules, classical odorresponse( G has beenshownthatalternativeGproteins(othersthan alternative signalingcomponentshavebeendescribed. It pathway,addition tothecanonicalolfactory various cyclic nucleotide-gated(CNG)channels.However, in dependent signaltransductioncascadeandopeningof change ofthe OR is initialized, which triggersa cAMP- neurons(OSNs),aconformational sensory In theolfactory receptors (GPCRs)andareactivated by specificodorants. a supergenefamilywithintheclassofG protein-coupled processes, suchasproliferationormigration.ORsconstitute 2015 al. et cells ( as inprostatecancercells( ( expressed inmusclecells( 2006 tissues( types inadditiontotheolfactory that ORexpressioncanbefoundinvarioushumancell receptors, respectively. (ORs), transientreceptorpotential(TRP)channelsortaste (STC-1 cells)( irritants (QGP-1cells)( distinct odorants(BONcells)( EC cellscanactasspecializedchemosensorsbydetecting 2009 been establishedthatmimictheECfeatures( several celllines,includingKRJ-1,BONorQGP-1cells,have they constituteasmallsubpopulationwithingiventissue, Although humanECcellsaredifficulttoinvestigatebecause understand the physiology of the gastrointestinal tract. Therefore, thecharacterizationofECcellsisimportantto such asirritablebowelsyndrome( targets forthetreatmentofECcell-associateddiseases, 2012 stimulation withodorants( release ofserotoninaftermechanicalstimulationor Busse DOI: 10.1530/JME-16-0063 http://jme.endocrinology-journals.org olf Research ) can be activated, which lead to an inhibition of the ) canbeactivated,whichleadtoaninhibitionofthe In thisstudy, weevaluatedtheRNAand protein In recent years, emerging studies have demonstrated demonstrated years, emergingstudieshave In recent 2009 , ). Serotoninreceptorsareoftenusedastherapeutic ), in which they regulate important physiological ), inwhichtheyregulateimportantphysiological , 2007 Manteniotis Siddique Flegel et al. , 2014 ) and hepatocarcinoma cells ( Spehr et al. Wu Wu et al. ) and sperm cells ( et al. 2013 et al. t al. et 2009 2011 Doihara 2016 2002 ). These receptors are functionally ). Thesereceptorsarefunctionally ). A few studies have shown that ). Afewstudieshaveshownthat Spehr Neuhaus , Braun ), theECcelllineBON( Pavlath 2010 Maßberg ) through olfactory receptors receptors ) througholfactory Braun et al. t al. et b

Spehr kalbe t al. et © 2016SocietyforEndocrinology 2009 Bischoff et al. et al. 2002 et al. andothers 2007 et al. ) or bitter stimuli ) orbitterstimuli 2009 Feldmesser 2007 ), keratinocytes ), keratinocytes Printed inGreatBritain 2015 Maßberg , Pfragner 2003 t al. et Ache 2010 , ), leukemia ), leukemia Chin ), pungent ), pungent ). Neuhaus Neuhaus ) as well ) as well 2014 Braun Braun t al. et t al. et et al. et al. 2+ ). ). ). ).

Total RNAwasextractedfromtheQGP-1cellsusingan Total RNAisolationandreverse transcriptase-PCR indicated. purchased fromThermoFisherScientificifnototherwise ( 5% CO and 100 in RPMI-1640mediumwith10%fetalbovineserum(FBS) Donadelli (UniversityofVerona, Italy).Theywerecultured The QGP-1cellswerethekindgiftofProf.DrMassimo Culture oftheQGP-1cells Materials andmethods dose-dependent releaseofserotoninfromtheQGP-1cells. Additionally, OR2J3 stimulation with helional induced a used: final extensionof10 by 40cyclesof45 β The followingtemperaturecycleprofilewasusedtodetect (Promega) inavolumeof20 RT-PCR wasperformedusingtheGoTaq qPCRMasterMix (−RT) toexcludegenomicDNA(gDNA)contamination. (RT)-PCR experiments,weincludedRNA-onlycontrols cDNA SynthesisKit(Bio-Rad).Forthereversetranscriptase DNA(cDNA)wassynthesizedusinganiScript complementary treatment withaTURBODNA-freeKit(ThermoScientific), I Spectrophotometer (Thermo Scientific). AfterDNase A280 ratio)wereanalyzedusingaNanoDropND-1000 instructions. TheRNAconcentrationandquality(A260/ RNeasy MiniKit(Qiagen)accordingtothemanufacturer’s the cells were fixed with ice-cold acetone for 5 they were80%confluent. After awashingstepusingPBS, The QGP-1cellswerecultured on30-mmcoverslipsuntil Immunocytochemical staining primer productswereconfirmedusingSangersequencing. control fortheprimers,humangDNAwasused.Theresulting 5 5 (forward: reverse: 5 5 (forward: reverse: 5 5 OR2J3 (forward: reverse: 5 release inQGP-1cells Ca Iguchi -actin, OR2J3,OR3A1andOR1A2:5 ′ Published byBioscientifica Ltd. -GGTGCAGAAGGCTTTGAATAGAC-3 2+ decreaseand5-HT β -actin (forward: 5 -actin (forward: 2 et al. humidifiedatmosphereasdescribedelsewhere U/mL penicillinandstreptomycinat37°Cina ′ ′ ′ -AGAGATGGTCTTTTCCGGGC-3 -TGAGCATCCTCCAGATGGCA-3 1990 -CTGGGTCTTGGGTGATTGGAA-3 s at95°C,45 ). Gibcosupplementsandmediawere min at 72°C. The following primers were min at72°C.Thefollowingprimerswere ′ -AGAAAGGGTGTAAAACGCAGC-3 ′ -TGCTAGCTCTGAGGGGTACTT-3 ′ ′ -TTCGCTCTGTAGAGGGCAGG-3 -GTACCCAGGCATTGCTGACA-3 Downloaded fromBioscientifica.com at10/01/202109:00:00PM μ L with10 s at60°C,45 min at 95°C followed min at95°Cfollowed pmols ofeachprimer. 57 ′ ). As a positive ). Asapositive : 3 s at 72°C and a s at72°Canda ′ ′ ), OR1A2 ), OR1A2 ), OR3A1 ), OR3A1 ′ , reverse: , reverse: min. To 202 via freeaccess ′ ), ), ′ ′ ′ , , , Journal of Molecular Endocrinology membranes were incubated with primary antibodydiluted membranes wereincubatedwithprimary TBS buffer and 50% casein in TBS, Thermo Scientific), the membranes. Afterablockingstepin50%casein(50% dodecyl sulfate(SDS)gelsandblottedtonitrocellulose al. et which was performed as described elsewhere ( prepared in Laemmli’s buffer for Western blot analysis, A sample of the whole protein fraction was collected and homogenized andcentrifuged(1000 of theQGP-1cells.Theextractwasthenmechanically appropriate volumeofRIPA bufferaftersedimentation A lysateofthetotalcellularproteinswasextractedinan Western blotting Corel DrawX5(Corel,Ottawa,ON,Canada). software (LAS,Leica).Theimageswereprocessedusing immersion objectiveandtheLeicaApplicationSuite LSM 510Meta,Oberkochen,Germany)witha40×oil signals weredetectedusingconfocalmicroscopy(Zeiss Antifade Gold(ThermoFisherScientific).Thefluorescent Scientific) wereused,andcellscoatedwithProLong 546 anti-rabbit) (#A-11034, Thermo FisherScientific) and antibodiesAlexaFluor488 coupled secondary fluorescent dyetolabelthenuclei.Thefluorophore- co-incubated with4 (SAB4501918, Sigma-Aldrich)wasused.Thecellswere antibody directedagainstOR2J3(rabbitpolyclonal) antibody.by incubationwiththeprimary Aprimary TBS with0.05%Triton X-100(Sigma-Aldrich),followed in 1%cold-waterfishgelatin(Sigma-Aldrich) dilutedin avoid nonspecificantibodybinding,thecellswereblocked with 3 approximately 80%confluence, thecellswereincubated dishes (Sarstedt,Nümbrecht, Germany).Afterreaching The QGP-1cellswerecultured in35-mmcell culture Calcium imaging used (ab8227,Abcam). control, from Sigma-Aldrich(SAB4501918).Asaproteinloading OR2J3 antibody (rabbit, polyclonal, 1:250) was purchased 3500-WL (Vilber Lourmat,Eberhardzell, Germany). The The chemiluminescencewasimagedusingFusion-SL antibody (goatanti-rabbit)(#170-6515,Bio-Rad)wasused. a horseradishperoxidase(HRP)-coupledsecondary in 75%TBSbufferand25%casein.Forimmunodetection, http://jme.endocrinology-journals.org DOI: 10.1530/JME-16-0063 Research nm (goatanti-rabbit)(#A-11010,ThermoFisher 2009 μ M fura-2-acetoxymethylester (MolecularProbes, β -actin antibody(rabbitpolyclonal,1:1000)was ). Thesampleswereloaded onto sodium ′ -6-diamidin-2-phenylindol (DAPI) b

kalbe © 2016SocietyforEndocrinology g for10 andothers Printed inGreatBritain min at4°C). nm (goat Neuhaus elsewhere ( The releaseofserotonin was measuredasdescribed Serotonin release measurement were initiallydissolvedinDMSO. Dr JPanten(SymriseAG,Holzminden,Germany)and 2012 3-hexen-1-ol (OR2J2,OR2J3andOR2W1( described previously( solution, andfluorometricimagingwasperformedas medium wasreplacedwithanextracellularRinger’s Thermo FisherScientific)for30 (OM4): ( OR2J2 ( OR2W1 ( ( eugenol methylether(OR51E1,OR10G7andOR5K1 ( odorant mixture3(OM3):eugenol(OR10G7andOR5K1 2005 al. et mixture 2(OM2):amylbutyrate(OR2AG1( al. et ( bourgeonal (OR1D2( Ca USA). As a negative control, DMSO (0.1%) was used in were obtainedfromEnzoLifeSciences(Farmingdale,NY, Sigma-Aldrich. 8-Bromo-cGMPandRp-8-pCPT-cGMPS pCPT-cGMPS (30 were preincubatedwiththePKG-specificinhibitorRp-8- 8-bromo-cGMP weredirectlyappliedfor60 well asallylisothiocyanate(AITC)andthePKGactivator concentration. Allodorantsandodorantmixtures,as dissolved intheextracellularsolutiontodesired in DMSO(maximalfinalconcentrationof0.1%)and application system. All the substances were prediluted experimental approachusingaspecializedmicrocapillary were exposedtotherelevantsubstances according tothe (OM1): according totheirchemicalstructures.Odorantmixture 1 known to be ligands for specific deorphanized ORs) (containing variousodorants(eachat300 The odorants were divided into four different mixtures Odorants release inQGP-1cells Ca Schmiedeberg Adipietro Adipietro Wetzel Published byBioscientifica Ltd. 2+ 2+ decreaseand5-HT imagingexperiments. )). All of the odorants were kindly provided by )) and PI-23472 (OR4D1 ( 2007 2006 γ Adipietro t al. et -decalacton (OR1G1( Saito β -ionone (OR51E2( ) andOR2J3( t al. et Doihara t al. et )), methyloctonoate(OR52D1( 1999 et al. t al. et 2012 2012 µM) for6 t al. et 2009 ), OR1A1andOR1A2( et al. Spehr 2007 ), OR4Q3( )), geraniol(OR2M7,OR1A1and Neuhaus 2012 )), menthol(OR8K3,OR51E1and Downloaded fromBioscientifica.com at10/01/202109:00:00PM 2009 Adipietro min. AITCwaspurchased from et al. ) n drn itr 4 )); andodorantmixture )) and ). QGP-1cellswereseeded Sanz Neuhaus min at37°C.Thegrowth 2003 Veitinger t al. et Mainland t al. et et al. S -citronellal (OR1A2 )), helional(OR3A1 57 2009 : 2012 3 2005 t al. et Schmiedeberg t al. et µM) thatare McRae t al. et ). Thecells s. Thecells Sanz )); odorant )) and 2009 Neuhaus 2011 2015 203 t al. et t al. et cis )); )), via freeaccess )) -

Journal of Molecular Endocrinology i. 1 Fig. the intracellularCa ( −0.172 decrease intheQGP-1cells (OM1:−0.230 ( within theQGP-1cells. levels could indicate the specific activation of a certain OR inthe intracellularCa Therefore, anychangesobserved activators of ORs (see ‘Materials and methods’ section). of adifferentsetodorants,whichareallknown air pressure-drivensystem.Eachofthemixturesconsisted ‘Materials andmethods’section)throughacustom-made experiments. We appliedfourodormixtures(OM1-4, see cells, we conducted ratiofluorometric Ca To propertiesoftheQGP-1 evaluatethechemosensory QGP-1 cells Odorant mixtures elicita transientCa Results *** follows: n.s. figures, thesignificanceofdifferencesisrepresented as ( values represent mean tests were subjected to a Mann–Whitney test (Tukey). Thedatathatfailedtheaforementioned tailed unpaired variance andnormalitytestsweresubjectedtoatwo- quickcalcs/Grubbs1.cfm QuickCalcs (Grubb’s test)( variance. SignificantoutliersweredetectedviaGraphPad All theresultsweretestedfornormalityandequal Statistical analysis supernatant accordingtothemanufacturer’s protocol. performed tomeasuretheserotoninconcentrationin (ELISA) (EnzoLifeSciences,Farmingdale,NY, USA)was stored at−80°C.Enzyme-linkedimmunosorbentassay for 5 37°C. Theassaybufferwasthencollectedandcentrifuged 0.25 and 8-bromo-cGMP (prediluted in DMSO) were diluted in PBS containing 0.1% BSA and 2 the mediumwasremovedandcellswerewashedin in therange2 i. 1D Fig. 1C Fig. s DOI: 10.1530/JME-16-0063 http://jme.endocrinology-journals.org . Research e P . m

Application of OM1 ( < mL PBSandincubatedwithQGP-1cellsfor20 . min at1000 ) ofatleastthreeindependentexperiments.Inall , seesectionon 0.001. ± ) orRinger’s solutionwith0.1%DMSOaffected and

0.029), whereasneitherOM2 ( = × E not significant,* 10 ) initiatedatransientintracellularCa t -test or a one-way ANOVA with post hoc 5 g cells/wellina24-wellplate.After72 . Thesupernatantwasremovedand 2+ supplementary data supplementary concentration( ). Thedatathatpassedtheequal ±

standard error of the mean i. 1A Fig. b P μ

M fluoxetine. Helional kalbe

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compared with the effect of by helional (−0.118 decrease oftheintracellular Ca 2007 S and OR2J3)( odorants, only helional (an agonist for OR3A1, OR1A2 aforementioned effectivemixes.Ofallofthetested the Ca of OM1( concentration: anexampleisshownfortheapplication Ca end ofthisarticle).Thestrengththeodorant-induced unpaired two-sampleStudent’s representing themean experimental tracesindicatethedurationsofstimuli.Barsare applied attheendofeverymeasurement.Thebarsshowninall in theintracellularCa S (F) Thesingleodorantshelional(300 maximal decreaseintheCa application. (E)Bargraphofthe340 OM3 (C)producedstrongdecreasesintheintracellularCa odorant intheOMhadafinalconcentrationof300 (OM1 – 4). Theodorantmixtures(OM)wereappliedfor30 Ca intracellular Ca Odorant mixturesandsingleodorantseliciteddecreasesinthe Figure 1 release inQGP-1cells Ca -citronellal (300 -citronellal (OR1A1andOR1A2) ( Published byBioscientifica Ltd. 2+ 2+ 2+ imagingexperimentswithfourdifferent odorantmixtures decreaseand5-HT decrease was dependent on the cell´s basal Ca ) inducedanintracellularCa 2+ changesmediatedbysingleodorantsofthe upeetr i. 2 Fig. Supplementary 2+ Wetzel levelinQGP-1cells.(A–D)Sampletracesofratiometric µM, odorantofOM3)( 2+ ± level.Asaviabilitycontrol,ATP (100

s . et al. e . 2+ m ± whenOM1( . . Thesignificancewastestedusingan

0.015) was significantly higher t Downloaded fromBioscientifica.com at10/01/202109:00:00PM 1999 -test. n.s S µM, odorantofOM1)( nm/380 -citronellal (−0.054 , N Adipietro 2+ = N

= ). We furtheranalyzed not significant,* 2+ concentrationinduced

= 4) producedvisibledecreases nm ratioshowingthe decrease( 6) or3( Schmiedeberg 57 : µM. OM1(A)and 3 N et al. ) a applied. was =5) 2+ Fig. 1F s. Eachsingle duringthe N P 2012 µM) was

< ) and =5) 0.05. ± 0.018). ). The 204 t al. et ) and via freeaccess 2+

Journal of Molecular Endocrinology the Ca Because applicationofhelionaltriggeredareductionin QGP-1 cells Helional inducesadose-dependentCa staining ( antibodycontrolsampleshowednospecific A secondary against in thecytoplasm( protein levelandwaslocatedatthecellmembrane OR2J3. We showedthatOR2J3wasalsopresentatthe staining oftheQGP-1cellsusinganantibodyspecificfor we conductedWestern blottingandimmunocytochemical with humangDNA( OR1A2 and OR3A1 ( OR3A1. We detectedtranscriptsforOR2J3,butnot experiments usingspecificprimersforOR2J3,OR1A2and was present in the QGP-1 cells, we performed RT-PCR by helional.To examinewhethertheRNAfortheseORs Previous findingsidentifiedthreedifferentORsactivated OR2J3 isexpressed intheQGP-1cells effects onQGP-1cells. Therefore, we furthercharacterized the helional-induced secondary antibodycontrolshowed nospecificstaining(E).Thecellnucleiwerestainedwith4 was visible(C).(D,E)Immunocytochemical stainingoftheQGP-1cellsshowedthatOR2J3ismembraneand cytoplasmlocalized(D).Theanti-rabbit with anOR2J3-specificantibodyrevealed asinglebandat~40 qualitative PCR(qPCR)oftheQGP-1 cDNAusingtheOR2J3-specificprimers.(BandC) Western blottinganalysisoftheQGP-1proteinlysateincubated Expression ofolfactoryreceptors(ORs) atthetranscriptandproteinlevelsinQGP-1.(A)Abandspecificfor OR2J3(~250 Figure 2 http://jme.endocrinology-journals.org DOI: 10.1530/JME-16-0063 Research 2+ β levels,wemonitoredthiseffectinmoredetail. -actin wasusedfortheloadingcontrol( Fig. 2E ). i. 2B Fig. Fig. 2A upeetr i. 3 Fig. Supplementary and ). Primer specificity was tested D b

kalbe ). A primary antibody ). Aprimary © 2016SocietyforEndocrinology 2+ andothers decrease inthe Printed inGreatBritain ). Inaddition, kDa (B).Asaloadingcontrol, Fig. 2C ). cells, weappliedthedirectactivatorofprotein to thehelional-inducedCa To identifythepossiblemolecularmechanismsleading dependent The helional-inducedCa the intracellularCa from theineffectiveOM2,didnotelicitanychangesin Stimulation withacontrolodorant,500 Ca concentration inducedasignificantdecreaseinthe −0.063 −0.020 (100 µM: f dependent effect,asindicatedbydecreasesinthef application of100,300or800 induced transient Ca We analyzedthedosedependencyofhelional- The amplitudewascomparablewiththatevokedby areductioninCa cGMP toQGP-1cells,weobserved levels ( PKG caninduceadecreaseinthebasalCa imaging. Inpreviousstudies,ithasbeenshownthat and measuredtheintracellularCa G(PKG),8-bromo-cGMP (100 kinase release inQGP-1cells Ca 380 Published byBioscientifica Ltd. 2+ 2+ ratiowithincreasingconcentrationsoftheodorant decreaseand5-HT concentration compared with 100 ± Zhuang 2004 .2) ( 0.022) β ′ -actin-specific antibodywasused,and abandat~40 -6-diamidin-2-phenylindol (DAPI).Scale bars:50 ± i. 3A Fig. M −0.036 300 µM: 0.010; 2+ concentration( ). Afterapplying100 2+ 2+ decrease in QGP-1 cells. The Downloaded fromBioscientifica.com at10/01/202109:00:00PM and decrease inQGP-1cellsisPKG µM helionalcausedadose- bp) couldbedetectedafter 2+ B ). Helional at 800 decreaseinQGP-1 2+ Fig. 3A 57 levelsusingCa µM), tothecells µM amylbutyrate : µM and 500 ± 3 .1; 800 µM: 0.018; µM 8-bromo- ). 2+ calcium µM. 205 kDa µM. µM 340 2+ via freeaccess 2+ / .

Journal of Molecular Endocrinology experiment, whichledtoanelevationofCa viability ofthecells,weappliedATP attheendofevery intracellular Ca AITC toconfirmTRPA1 arobust activationandobserved 2009 been describedpreviouslyinQGP-1cells( expression ofTRPA1 anditsstimulationwithAITChave helional 800 800 (Tukey). n.s. The significancewastestedusingaone-wayANOVA withposthoctest 800 induced asignificantCa every measurement.Thebarsindicatethestimulusduration.(B)Helional decrease. Asaviabilitycontrol,ATP (100 was testedasacontrolstimulusandinducednovisibleCa helional (100,500and800 of atracefromCa The helional-inducedCa Figure 3 similar tothehelional-evokedresponses.(A)SampletraceofCa 8-bromo-cGMP, aproteinkinase G(PKG)activator, ledto aCa Figure 4 (Tukey). n.s. significance wastestedusingaone-way ANOVA withposthoctest in theintracellularCa helional and8-bromo-cGMP. Incontrast,AITCledtoasignificant increase stimulus duration.(B)Bargraphshowing theCa was appliedattheendofeverymeasurement.Thebarsindicate the TRPA1-mediated Ca cells werethenstimulatedwithAITC(500 (800 imaging experimentshowingtheCa DOI: 10.1530/JME-16-0063 http://jme.endocrinology-journals.org Research µM comparedwiththatat100 µM; stimulus:60 µM helional (8-bromo-cGMP 100 ). We thereforestimulatedthecellswith500 = = not significant,* not significant,* µM: −0.051 2+ 2+ s) and8-bromo-cGMP(100 imagingexperimentwithvariousconcentrationsof 2+ increase(0.166 ( 2+ 2+ 2+ N increase.Asaviabilitycontrol,ATP (100 decreaseatconcentrationsof500 decreasewasdosedependent.(A)Anexample

= µM; stimulus:60 6). Barsrepresentmean P P

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significantly differentfrom the DMSO(0.1%)control. release ofserotonin(4.997 with thePKGactivator8-bromo-cGMP causedasimilar 12.658 1000 µM: 4.102 2.715 increase intheserotoninsecretion( The applicationofhelionalledtoadose-dependent 800 and1000 cells withvariousconcentrationsofhelional(10,300, serotonin concentrationafterthestimulationofQGP-1 activation affectedtheserotoninreleasebymeasuring serotonin-specific ELISA kit to determine whether OR al. et release serotoninuponTRPA1 activation( In previousstudies, it wasreported that QGP-1cells of thehelional-inducedCa Next, wewereinterestedinthephysiologicalrelevance from QGP-1cells Helional initiatesadose-dependentrelease ofserotonin in thehelional-inducedsignaling( −0.031 Ca led toasignificantsuppressionofthehelional-dependent 800 Rp-8-pCPT-cGMPS (Rp-8) (50 using anunpairedtwo-sampleStudent’s decrease ( caused asignificantinhibitionofthehelional(800 measurement. Thebarsindicatethestimulusduration.(B)Rp-8(30 again. Asaviabilitycontrol,ATP (100 with extracellularsolution,helional(800 co-application ofhelional(800 was appliedfor60 trace ofaratiometricCa a significantinhibitionofthehelional-inducedCa Co-application ofthePKG-specificblockerRp-8-pCPT-cGMPS (Rp-8)ledto Figure 5 release inQGP-1cells Ca Published byBioscientifica Ltd. 2+ 2+ µM helionalonQGP-1cells.TheinhibitionofPKG decreaseand5-HT Furthermore, wetestedthespecificPKGblocker, decrease (helional: −0.101 2009 ± ± ± M 10.028 800 µM: 0.229ng/mL; 2.901 10 µM: 0.12ng/mL; N

0.014), thusconfirmingtheinvolvementofPKG

= 6). Barsrepresentmean , Schulze s. Rp-8(30 µM) orwith8-bromo-cGMPfor15 ± ng/mL). Additionally,stimulation 1.039ng/mL). 2+ -imaging experiment.First,helional(800 t al. et µM) wasthenaddedfor6 µM; stimulus:60 Downloaded fromBioscientifica.com at10/01/202109:00:00PM 2013 2+ ± ± µM) wasappliedattheendofevery µM), in combinationwith

decreaseinQGP-1cells. s gm) wih was which 0.652ng/mL), . e µM; stimulus:60 t . -test. ** m ± ). We therefore used a . ± Thesignificancewastested .0; helional +Rp-8: 0.006; Fig. 5A gm; 300 µM: 0.188ng/mL; Fig. 6 s). After60 57 P 2+ µM)-evoked Ca

< decrease.(A)Sample : 3 0.01. and ) (0.1%DMSO: ± min beforethe 0.775ng/mL; s) wasapplied s ofwashing B ). Doihara 2+ 206 µM) µM)

min. via freeaccess Journal of Molecular Endocrinology presence ofOR2J3attheprotein levelviaWestern blotting neuroendocrine tumor cell line QGP-1. We confirmed the expressed atthemRNAlevel inthehumanpancreatic et al. 2009 carried outatpresent( however, only afewphysiologicalstudieshavebeen the nose( celltypesand tissuesoutside various nonchemosensory In thelastdecade,humanORshavebeenidentified in Discussion ** tested usinganunpairedtwo-sampleStudent’s with theDMSOcontrol.Barsrepresentmean produced asignificantincreaseintheserotoninconcentrationcompared control ( significant ataconcentrationof300 serotonin inducedbyhelionalwasconcentrationdependentand commercially availableELISAkit.Theobviousincreaseinthesecretionof and theextracellularserotoninconcentrationwasmeasuredusinga serotonin concentrationinng/mL.Thecellswerestimulatedfor15 ( µM 1000 and (N =6) 800 µM Stimulation ofQGP-1cellswithhelional10 Figure 6 http://jme.endocrinology-journals.org DOI: 10.1530/JME-16-0063 P Research

< We present the first data thatshow thatOR2J3 is .1 *** 0.01, 2016 , Busse N

= 3). ThePKGactivator8-bromo-cGMPS(300µM)( ). P Feldmesser

et al. < 0.001. 2014 N ,

= t al. et Spehr Maßberg 6) ledtoanincreaseintheextracellular µM comparedwiththeDMSO(0.1%) 2006 t al. et b µM ( et al.

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expressed ORs( investigated indetailbyotherstudiesthatusedectopically Previous studieshavedemonstrated that theactivation muscle cellsthroughareductionoftheintracellularCa regulates thevascularreactivityandtoneinsmooth monophosphate (cGMP)-dependentproteinkinase(PKG) Importantly, ithasbeenshownthatthecyclic guanosine We thereforedidnotanalyzethoseinthe QGP-1 cells. induced Ca increases intheCa molecules, suchasphospholipaseCorSrc kinases,causes signaling that the activation of other alternative olfactory Manteniotis Ca Bakalyar &Reed1990 proteins, suchastheadenylylcyclaseIII( signaling did notfurtherconcentrateonclassicalolfactory that distinct ORs initiate not onlythecanonical Ca et al. ( decreases havebeendemonstratedpreviouslyforfeline in QGP-1cells.Odorant-dependentintracellularCa to atransientconcentration-dependentCa and OR3A1 ( and the single odorant helional, which activates OR2J3 that the applicationof four different odorant mixtures al. et Ca Both ORsandTRPchannelstriggeranintracellular has beenidentifiedinQGP-1cells( EC BONcellline( and hOR17-210) has alreadybeenreported for thehuman expression of other ORs (hOR73, hOR17-7/11, hOR1G1 in the membrane and the cytoplasm of the cells. The OR2J3-specific antibodyandsawaspecificORstaining analysis andimmunocytochemicalexperimentswithan et al. neuroblastoma cells,cardiomyocytes andatrialcells( Ca pathways can inhibit the opening of the TRP and N-type levels ( onlydecreasesintheintracellularCa As weobserved possible signalingcomponentsinvolvedinthisprocess. remains unresolved.We thereforeaimedtoelucidatethe the underlyingsignal transduction pathway currently Torres-Padilla 1999 lead todecreasesinthebasalCa are abletomodulatetheCa Furthermore, itisalreadyknownthat system. increases butalsodecreaseswithintheolfactory release inQGP-1cells Ca Gomez Published byBioscientifica Ltd. 2+ 2+ 2+ 2+ decreaseand5-HT channel( increasefollowedbyareleaseofserotonin( channels and modulate the L-type Ca 2000 2000 2007 Taylor et al. , ) andmudpuppy( , D’Ascenzo 2+ Doihara Wetzel t al. et 2005 increases( Nakamura &Gold1987 t al. et Busse 2016 Braun ), human( ). However, the molecular identity of 2004 et al. 2+ t al. et et al. levelsorinhibitstheodorant- t al. et ), orthecyclicnucleotide-gated ). Previousstudieshaveshown Downloaded fromBioscientifica.com at10/01/202109:00:00PM Spehr et al. 1999 ). Furthermore, PKG-mediated 2009 2002 2014 Delay 2002 2+ Rawson 2007 , levelsinfibroblaststhat t al. et ). Remarkably, we found Jacquier , 2+ , Koitabashi level( Maßberg ). Inaddition,TRPA1 Doihara 2002 57 ), whichhavebeen et al. α : ) OSNs,showing 1D 3 Lowe et al. García-Sáinz & -adrenoceptors 1997 2+ , channels in Ache 2010 et al. t al. et et al. 2+ 2006 et al. decrease , 2009 Gomez 2010 2015 1989 Braun Wang 207 2+ ), led , we via freeaccess 2+ 2+ 2+ ). ). ). , ,

Journal of Molecular Endocrinology to beinvestigatedinfurther studies. components involvedinthe releaseofserotonin.Thishas there might be currently uncharacterized signaling with helionalseemstobe conceivable.Furthermore, 2006 dependent onacGMP/PKGsignalingpathway( al. et secretion ofserotonininECcells( mitogen-activated proteinkinase(MAPK)ERKleadstothe of in QGP-1cells.TheGPCR-mediatedphosphorylation release canalsobemediatedbytheactivationofPKG Ca is mediatedbycGMPandthereforeindependentofthe of of serotonin.IntheECcelllineBON,vesicularrelease activator of PKG,ledtoa comparable increase in therelease flux. Furthermore, we showed that 8-bromo-cGMP, the processes candirectlyleadtoexocytosiswithoution B cells ( Ca that insulinexocytosisisindependentoftheintracellular based onasofarunknownmechanism.Onestudyshowed pathway mightleadtoanincreaseoftheserotoninrelease in serotoninrelease.We suggestthataCa that besidetheCa et al. to anincreaseinintracellularCa that serotonin release from EC cells by odorants is coupled identified signalingcascade.Previousstudiesdemonstrated demonstrating thephysiologicalrelevanceofthisnewly release ofserotonininadose-dependentmanner, thus open ( TRPV5 andTRPV6),whichareknowntobeconstitutively targets mightbetheTRPchannels(forexampleTRPA1, then leadtoadecreaseinthebasalCa inhibited through the activation of PKG, which would It ispossiblethatconstitutivelyopenchannelsmightbe pathway. However, thedetailedpathwayremainselusive. PKG wasinvolvedinthehelional-inducedsignaling odorant-dependent Ca blocker Rp-8-pCPT-cGMPS significantlyinhibitedthe response. Inaddition,treatmentwiththePKG-specific a Ca activator 8-bromo-cGMPtotheQGP-1cellsandobserved Koitabashi thereby decreasestheinfluxofCa TRPC3andTRPC6 of PKGdirectlyphosphorylates DOI: 10.1530/JME-16-0063 http://jme.endocrinology-journals.org Research 2+ 2+ γ We foundthattheapplicationofhelionalledtoa level( increaseormembranedepolarizationinpancreatic aiouyi cd(AA n hoornn A -aminobutyric acid(GABA)andchromogranin 2+ 2007 ). AsimilarpathwayinQGP-1 cellsuponactivation 2012 decreasecomparable with thehelional-dependent Nilius 2007 Lang , ). In platelets, the phosphorylation of ERK is ). Inplatelets,thephosphorylation et al. John Doihara t al. et 2010 t al. et ). 2+ reduction,helionalledtoanincrease 1998 et al. ). Thus,weappliedtheknownPKG 1998 2+ decrease, thus proving that 2009 ). Therefore, GPCR-mediated ). We suggestthatserotonin ). Incontrast,weshowed 2+ b

the releaseofserotoninandledtoadecreaseinCa signaling pathwayinhumanQGP-1cellsthattriggered Ache BW 2010Odorant-specificmodesofsignalinginmammalian Ache BW References technical support. regarding molecular biological techniques and Simon Pyschny for the of Verona, Italy).TheauthorsthankDrGünterGisselmannforhissupport QGP-1 cellswerekindlyprovidedbyProfDrMassimoDonadelli(University Acknowledgements the manuscriptforimportantintellectualcontent. S O carried out the analysis and interpretation; B K, H H and S O drafted B K,HandSOhelpedinconceptiondesign;MS,JM,PF Author contributionstatement Ruhr-University Research School. Forschungsgemeinschaft grantnumbersSFB874and642the This researchprojectwasfinanciallysupportedbytheDeutsche Funding perceived asprejudicingtheimpartialityofresearchreported. The authorsdeclarethatthereisnoconflictofinterestcouldbe Declaration ofinterest JME-16-0063. This islinkedtotheonlineversionofpaperat Supplementary data OR-related signalingtoanodorantstimulusinECcells. ofan Our studythereforedescribesthenoveldiscovery intracellular Ca time, weshowedthattheORsareabletoreduce the ORsasnovelputativeclinicaltargets.Forfirst irritable bowelsyndrome.Theseresultsalsoemphasize of severaldiseasesassociatedwiththesecells,including the activatingmechanismsforECcellsandsymptoms information that willleadtoabetterunderstanding of induced byPKG.Thus,thisstudyprovidesimportant Bischoff SC, Barbara G, Buurman W, Ockhuizen T, Schulzke J-D, Serino M, Buurman W, Serino M, Ockhuizen T, Barbara G, Schulzke J-D, Bischoff SC, 1990 Identificationofaspecializedadenylyl &Reed RR Bakalyar HA 2012Functional evolution &Matsunami H Mainland JD Adipietro KA, release inQGP-1cells Ca Published byBioscientifica Ltd. 2+ Tilg H, Watson A &Wells JM Tilg H, 2014Intestinalpermeability–a new (doi:10.1126/science.2255909) cyclase thatmaymediateodorantdetection. (doi:10.1371/journal.pgen.1002821) of mammalianodorantreceptors. olfaction. decreaseand5-HT In summary, wedescribedanodorant-activated Chemical Senses 2+ concentration in nonolfactory cells. concentrationinnonolfactory

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