Table Of ContentDYNAMICIMAGINGOFSECRETIONFROMPANCREATICBETA-CELLSBY
CONFOCALFLUORESCENCEMICROSCOPY
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WEIJUNQIAN
ADISSERTATIONPRESENTEDTOTHEGRADUATESCHOOL
OFTHEUNIVERSITYOFFLORIDAINPARTIALFULFILLMENT
OFTHEREQUIREMENTSFORTHEDEGREEOF
DOCTOROFPHILOSOPHY
UNIVERSITYOFFLORIDA
2002
Tomyparentsfortheirguidanceandsupportandmywifeforherlove,
supportandencouragement.
ACKNOWLEDGMENTS
Iwouldliketoexpressmysincerethankstomyadvisor,Dr.RobertT.Kennedy,
forhiscontinuousguidanceandsupportthroughoutmygraduatecareer. Iwouldalso
liketothankallthemembersoftheKennedygroup,pastandpresent,fortheirhelp
duringmytimeintheKennedylab. IwouldespeciallyliketothankDr.CraigA.
AspinwallandMr.MichaelG.Roperfortheirhelpandcollaborationinpartofmy
research,especiallytheamperometricdetectionofsecretionpresentedinChapter6. I
wouldalsoliketoexpressmygratitudetoDr.JenniferL.Petersforherhelpduringthe
writingofmydissertation.
IwouldliketoexpressmyappreciationtoDr.KyleR.GeeatMolecularProbes
forsupplyingsamplesofzincfluorophoresandforhiscollaboration. Iwouldliketo
thankDr.MerleA.Battisteforhisassistanceinthesynthesisofthezincfluorophore
—
Zinquin. Iwouldliketothankallofthemembersofmydoctoralcommittee Dr.Mark
—
A.Atkinson,Dr.JonD.Stewart,Dr.WeihongTan,andDr.RichardA.Yost fortheir
wisdom,assistance,andencouragement. 1wouldalsoliketothankEastmanChemical
Company,GEPlastics,theAmericanChemicalSocietyDivisionofAnalytical
Chemistry,andtheUFChemistryDepartmentDivisionofAnalyticalChemistryfor
sponsoringfellowshipstofundthiswork.
iii
TABLEOFCONTENTS
page
ACKNOWLEDGMENTS m
LISTOFABBREVIATIONS vii
ABSTRACT *
CHAPTERS
1 INTRODUCTION 1
SecretionofNeurotransmittersandHormones 1
InsulinSecretionandDiabetesMellitus 2
PancreaticIsletsandP-cells 5
Stimulus-secretionCouplinginp-cells 7
TechniquesforMonitoringInsulinSecretion 11
FluorescenceImagingTechniquesforMonitoringExocytosis 14
ConfocalFluorescenceMicroscopy 16
DissertationOverview 18
2 DETECTIONOFSECRETIONFROMSINGLEPANCREATICp-CELLS
USINGEXTRACELLULARFLUOROGENICREACTIONSAND
CONFOCALFLUORESCENCEMICROSCOPY 19
Introduction 19
Experimental 21
Results 28
Discussion 45
Conclusion 49
3 SPATIALORGANIZATIONOFCa2+ENTRYANDEXOCYTOSISIN
MOUSEPANCREATICp-CELLS 51
Introduction 51
Experimental 53
Results 55
Discussion 66
IV
,
4 DETECTIONANDIMAGINGZn2+RELEASEATTHELEVELOF
SINGLEEXOCYTOTICEVENTS 71
Introduction 71
Experimental 73
Results 77
Discussion 94
5 TEMPORALANDSPATIALPATTERNSOFOSCILLATORYZn2+
SECRETIONFROMCLUSTERSOFPANCREATICp-CELLSAND
ISLETSOFLANGERHANSREVEALEDBYFLUORESCENCEIMAGING 100
Introduction 100
Experimental 102
Results 105
Discussion 118
6 RPOHOLSEPSHOAFTEIDNYSULLIINNOSRIETCOELPT3-OKRINSAUSBES,TARNADTER-E1LEASEOF
INTRACELLULARCa2+STORESININSULIN-STIMULATEDINSULIN
SECRETIONINP-CELLS 124
Introduction 124
Experimental 127
Results 132
Discussion 144
7 SUMMARYANDFUTUREDIRECTIONS 149
Summary 149
FutureDirections 153
APPENDICES 157
A NIKONRCM8000CONFOCALLASER-SCANNINGSYSTEM 157
IntroductionoftheSystem 157
ImagingOperation 160
DataAnalysisandImagePresentation 164
InstrumentalMaintenance 165
v
B CONFOCALIMAGINGOFINTRACELLULARFREECa2+
CONCENTRATIONS 167
FluorescentCa2+Probes 167
ConfocalImagingofCa2+inPancreaticP-cells 167
LISTOFREFERENCES 171
BIOGRAPHICALSKETCH 183
vi
LISTOFABBREVIATIONS
5-HT 5-hydroxytryptamine
5-HTP 5-hydroxytryptophan
ADP Adenosinediphosphate
AM Acetoxymethylester
ATP Adenosinetriphosphate
pTC3 Insulinsecretingtumorline
[Ca2+]i IntracellularfreeCa2+concentration
cAMP Cyclicadenosinemonophosphate
CLSM Confocallaser-scanningmicroscope
DMEM Dulbecco’smodifiedEagle’smedium
DMSO Dimethylsulfoxide
EDTA Ethylenediaminetetraaceticacid
EGTA Ethyleneglycol-bis(2-aminoethyl)-N,N,N',N'-tetraaceticacid
ELISA Enzyme-linkedimmunosorbentassay
ER Endoplasmicreticulum
FBS Fetalbovineserum
GABA y-aminobutyrate
GLUT-2 Type2glucosetransporter
GSIS Glucose-stimulatedinsulinsecretion
Vll
HBSS Hanks’BalanceSaltSolution
HEPES N-(2-hydroxyethyl)piperazine-N’-(2-ethanesulfonicacid)
IDDM Insulin-dependentdiabetesmellitus
IP3 Inositol1,4,5-triphosphate
IRS Insulinreceptorsubstrate
KRB Krebs-Ringerbuffer
NA Numericalaperture
NEDDM Non-insulin-dependentdiabetesmellitus
NMDA N-methyl-Daspartate
OMDR OpticalMonochromeDiscRecorder
PI-3K Phosphoinositol3-kinase
PIP3 Phosphatidylinositol3,4,5-triphosphate
PKB ProteinkinaseB
PKC ProteinkinaseC
PLC PhospholipaseC
PMT Photomultipliertube
RIA Radioimmunoassay
ROI Regionofinterest
RRP Readilyreleasablepool
S.D. Standarddeviation
SEM Standarderrorofthemean
SERCA SarcoplasmicandendoplasmicreticulumCa -ATPases
S/N Signal-to-Noiseratio
Vlll
SNAP-25 Synaptosomalproteinof25kDa
SNAREs SolubleA-ethylmaleimide-sensitivefactorattachmentproteinreceptors
SSCE Sodium-saturatedcalomelreferenceelectrode
TPEN Tetrakis-(2-pyridylmethyl)ethylenediamine
UV Ultraviolet
IX
AbstractofDissertationPresentedtotheGraduateSchool
oftheUniversityofFloridainPartialFulfillmentofthe
RequirementsfortheDegreeofDoctorofPhilosophy
DYNAMICIMAGINGOFSECRETIONFROMPANCREATICBETA-CELLSBY
CONFOCALFLUORESCENCEMICROSCOPY
By
WeijunQian
May2002
Chair: RobertT.Kennedy
MajorDepartment: Chemistry
InsulinisstoredinsecretoryvesiclesofpancreaticP-cellsandsecretedby
exocytosisinresponsetoexternalstimuli,mostnotablyglucose. Abetterunderstanding
ofstimulus-secretioncouplingisofgreatinterestduetothepossibleroleofdefective
insulinsecretionintype2diabetes. Studiesofstimulus-secretioncouplingrequire
analyticaltechniquesthatallowthemonitoringofsecretionwithhightemporaland
spatialresolution.
Inthiswork,afluorescenceimagingapproachhasbeendevelopedforspatially
andtemporallyresolvedmeasurementsofsecretionfromP-cellsusingextracellular
fluorogenicreactionsandconfocallaser-scanningfluorescencemicroscopy. Inthis
method,Zn2+effluxfromP-cellsismonitoredasamarkerofinsulinsecretionbecauseof
theco-releaseofZn2+andinsulinfromP-cellsbyexocytosis. TodetectZn2+release,
cellsareincubatedinbuffercontainingthefluorogenicZn2+indicatorZinquin,while
x
