Properties of high-affinity L-Glutamate transport in glial- and neuronal-enriched fractions from rat brain

125 Pages·1997·4.6 MB·English

by  Daniels, Kellye K

PROPERTIESOFHIGH-AFFINITYL-GLUTAMATETRANSPORTINGLIAL-AND
NEURONAL-ENRICHEDFRACTIONSFROMRATBRAIN
By
KELLYEK.DANIELS
ADISSERTATIONPRESENTEDTOTHEGRADUATESCHOOL
OFTHEUNIVERSITYOFFLORIDAINPARTIALFULFILLMENT
OFTHEREQUIREMENTSFORTHEDEGREEOF
DOCTOROFPHILOSOPHY
UNIVERSITYOFFLORIDA
1997
ThisworkisdedicatedtoMauriceandAltaBennett,MaggieDaniels,andLouiseFerrell,
withgreatloveandfondremembrance.
ACKNOWLEDGMENTS
DuringmytimeasagraduatestudentattheUniversityofFlorida,Dr.WilliamG.
LuttgeandDr.MaryJoKorolygavemeexceptionalguidanceandsupport;such
generosityisrareandtrulyappreciated.
IextendmuchgratitudetotheCenterfortheNeurobiologyofAgingandtothe
BryanW.RobinsonNeurologicalFoundation,Inc.,forfinancialsupport.
iii
[
TABLEOFCONTENTS
page
ACKNOWLEDGMENTS
iii
ABSTRACT v
CHAPTERS
1 BACKGROUNDANDSIGNIFICANCE 1
2 ISOLATIONOFGLIALANDNEURONALFRACTIONS 22
Introduction 22
ExperimentalProcedures 24
Results 32
Discussion 47
3 EFFECTOFPHOSPHORLYATIONONL-[3H]GLUTAMATEUPTAKE 53
Introduction 53
ExperimentalProcedures 57
Results 62
Discussion 64
4 EFFECTOFAGINGONL-3H]GLUTAMATEUPTAKE 75
Introduction 75
ExperimentalProcedures 77
Results 82
Discussion 8?
5CONCLUSIONS 93
REFERENCES 98
BIOGRAPHICALSKETCH 116
iv
AbstractofDissertationPresentedtotheGraduateSchool
oftheUniversityofFloridainPartialFulfillmentofthe
RequirementsfortheDegreeofDoctorofPhilosophy
PROPERTIESOFHIGH-AFFINITYL-GLUTAMATETRANSPORTINGLIAL-AND
NEURONAL-ENRICHEDFRACTIONSFROMRATBRAIN
By
KellyeK.Daniels
December,1997
Chairman:ThomasW.Vickroy,Ph.D.
MajorDepartment:Neuroscience
PriorstudieshaveestablishedthatterminationofL-glutamate-mediated
neurotransmissionoccursthroughhigh-affinitytransporterslocatedonglialcellsand
neurons. ForinvitrostudiesofL-glutamatetransport,animprovedthree-stepdensity
gradientcentrifugationtechniquewasdevelopedtoisolateglial-(glialplasmalemmal
vesiclesorGPV)andneuronal-(synaptosomesorSYN)enrichedvesiclesfromratbrain
homogenates.Morphological,biochemical,andWesternblotanalysesconfirmedthehigh
degreeofseparationbetweenGPVandSYNtissuefractions.
GPVandSYNtissueswereusedtoevaluateL-glutamateuptake,toaddress
phosphorylation/dephosphorylationeventsaspossibleregulatorymechanismsofL-
glutamateuptake,andtodeterminetowhatextentagingmayalterthesemechanisms.L-
[3H]GlutamateuptakebyGPVandSYNrevealedremarkedlysimilarproperties. Kinetic
v
studiesindicatedamorerapidinitialrateforuptakeinGPV,aswellasaslightlyhigher
substrateaffinity. Conversely,SYNfractionsexhibiteda2-to3-foldgreatercapacityfor
L-[3H]glutamateuptake. InstudiesaddressingtheeffectsofphosphorylationonL-
[3H]glutamateuptake,GPVandSYNtissuefractionsrespondeddifferentiallyfollowing
incubationwithphorbol-12,13-dibutyrate(PDBu),anactivatorofproteinkinaseC(PKC),
anenzymewhichphosphorylatesserine/threonineresidues. GPVL-[3H]glutamateuptake
increasedsignificantlyabovecontrolvaluesafterincubationwithPDBu,whereasuptake
valuesinSYNfractionsdidnot. Likewise,L-[3H]glutamateuptakeinGPVsignificantly
increasedabovecontrolvalueswhenfractionswereincubatedwithokadaicacid,a
phosphoserine/phosphothreoninephosphataseinhibitor,whereasL-[3H]glutamateuptake
inSYNfractionsdidnotchangefromcontrollevels. InGPVandSYNfractionsobtained
fromanimalsaged5,25,31,and37months,onlyL-[3H]glutamateuptakeinSYN
fractionsfrom37montholdanimalssignificantlydecreasedfromuptakevaluesobtained
from5montholdanimals. WhenfractionswereincubatedwithPDBu,L-[3H]glutamate
uptakeincreaseddose-dependentlyacrossagegroupsinbothfractions. Thisresponsein
SYNfractions,however,decreasedwithincreasingage.
ThisstudyhasdemonstratedthatwhilebothGPVandSYNfractionstransportL-
glutamatewithhigh-affinityandinasaturablemanner,theydifferinresponsetokinases
andphosphatases,ultimatelyindicatingthatphosphorylation/dephosphorylationevents
maymoretightlyregulateGPVratherthanSYNL-glutamatetransportandthatSYNL-
glutamatetransportismorevulnerabletotheagingprocess.
vi
CHAPTER
1
BACKGROUNDANDSIGNIFICANCE
PropertiesofEAATransporters
BiochemicalPropertiesandFunctions
TheaminoacidsL-aspartateandL-glutamaterepresentthemajorexcitatory
neurotransmittersinthemammalianbrain(Krnjevic,1970;Fonnum,1984). High-affinity
transportersfortheseexcitatoryaminoacids(EAA)areessentialforrecapturingthe
neurotransmitterL-glutamate,therebyterminatingitsextracellularactionsand
maintainingahighconcentrationgradientbetweentheextracellularspaceandthecytosol
ofnerveterminals(KannerandSchuldiner,1987;NichollsandAttwell,1990). Theability
ofL-glutamatetocauseneuronaldamageandcelldeath,however,posesaspecial
requirementforanefficientuptakesystem(Choi,1988,1994).
Forseveralyearsithasbeenknown,primarilythroughtheworkofKannerand
collaborators,thatL-glutamateuptakeiselectrogenicanddependentonexternalNa*and
internalK+ions(KannerandSchuldiner,1987;Danboltetal.,1990). Detailedstudieson
thestoichiometryofL-glutamateuptakehavebeencarriedoutonMullercells,a
specializedformofglialcells(Attwelletal,1991;Bouvieretal,1992). Sincethesecells
donotexpressL-glutamatereceptors,thecurrentelicitedbyL-glutamateprovidesa
convenientindexofelectrogenicuptake. Analysesbasedonwholecellpatchclamp
recordingsindicatethatL-glutamate(carryingonenetnegativechargeatphysiological
1
2
pH)istransportedintothecelltogetherwithtwosodiumionsandthatthereturnofthe
carriertotheoutsideofthecelliscoupledwithanoutwardtransportofonepotassiumion
andonehydroxylanion.
ThemaintenanceofalowlevelofexternalL-glutamateisoneobviousfunctionof
theL-glutamatetransporter. ThisfunctioniscrucialasL-glutamatebecomesneurotoxic
whenitsextracellularconcentrationexceedsacertainlevel(Choietai,1987;Beal,
1992a,b). TheconcentrationatwhichL-glutamateexertsexcitotoxicactionsishardto
determine,duetotheefficiencyofuptakeandthelackofnoncompetitive,irreversible
blockers. Estimatesrangefromseveralhundredmicromolar(NichollsandAttwell,1990)
toaslowas1micromolar(FrandsenandSchousboe,1990). Neuronaltoxicityisa
consequenceofexcessivemembranedepolarizationassociatedwithswellingandan
accumulationofcalciumrecruitedfromtheextracellularspaceorfromintracellularstores.
TheimportanceofglialL-glutamatetransportersforreducingL-glutamateneurotoxicityis
suggestedbyexperimentsshowingthatneuronsaremuchmoresensitivetoL-glutamate-
inducedtoxicitywhengrownalonethanwhengrowninco-culturewithglia(Rosenberget
ai,1992). Inconditionssuchashypoxiaandischemia,thedrivingforceforL-glutamate
uptakecollapsesduetothebreakdownoftheelectrochemicalsodiumandpotassium
gradients,andL-glutamatetransportiscompromisedorreversed(Szatkowskiand
Attwell,1994). Infactthereisincreasingevidencetosuggestthattheextracellular
overflowofL-glutamateinischemicandanoxicconditionsprimarilyreflectsinadequateor
reversedL-glutamatetransportandthatexocytoticreleaseplaysaminorrole(Nicholls
andAttwell,1990).
1
3
L-Glutamatedysfunctionhasbeenimplicatedinotherneurologicdisorderssuchas
amyotrophiclateralsclerosis(ALS)(Plaitakis,1990;Rothsteinetai,1992;Shawetai,
1994;Rothsteinetal,1995;BristolandRothstein,1996;LeighandMeldrum,1996).
High-affinity,sodium-dependenttransportofL-glutamatewasfoundtobemarkedly
impairedinALSpatientsinthespinalcord,motorcortex,andsomatosensorycortex
(Rothsteinetai,1992). ThesearethebrainregionsmostaffectedinALS. Thechanges
werespecificforALS,astheywerenotobservedinHuntington'sdisease,Alzheimer's
disease,ornonneurologicdiseasecontrols. ThisfindingledRothsteinandcolleagues
(1992)toconcludethatdefectsintheclearanceofextracellularL-glutamate,secondaryto
faultytransporterfunction,couldleadtoneurotoxiclevelsofextracellularL-glutamate
andthusbepathogenicinALS.
Regional/CellularLocalization
Threesodium-dependenttransportersubtypeshavebeenidentifiedinratforebrain:
L-glutamate/L-aspartatetransporter(GLAST)(Storcketai,1992),glialtransporter-
(GLT-1)(Pinesetai,1992),andexcitatoryaminoacidcarrier-1(EAAC1)(Velaz-
Fairclothetai,1996). Threesimilartransportershavebeenclonedfromhumanbrain,and
theprovisionalhumanhomologshavebeentermedexcitatoryaminoacidtransporters
(EAAT)-1,-2,and-3,correspondingwithgreaterthan90%sequencehomologyto
GLAST,GLT-1,andEAAC1,respectively(Arrizaetai,1995). Afourthsubtype,
EAAT4,hasbeenidentifiedinhumancerebellum(Fairmanetai,1995),andafifth
subtype,EAAT5,hasbeenidentifiedinhumanretina(Arrizaetai,1997).
4
WithregardtothecellularandregionallocationsofL-glutamatetransporters,
resultsfromimmunocytochemistry(Lehreetal,1995)andinsituhybridization(Torpet
al,1994)indicatethattherearepronouncedregionaldifferencesintheexpressionofthe
transporters. ThesestudieshavemappedGLASTmRNAtothePurkinjecelllayerofthe
cerebellum,apparentlyassociatedwithBergmannglia(Storcketal,1992),aswellasto
thecortexandhippocampus(Rothsteinetal,1994). Otherstudies,usinginsitu
hybridizationforthecarboxyl-terminaldomainsofeachtransporter,initiallyreporteda
lowlevelofGLASTimmunoreactivityinbothneuronsandastroglia(Rothsteinetal,
1994). Amino-terminaloligopeptideantibodieslocalizedGLASTtoastrocytes(Rothstein
etal,1995;Lehreetal,1995;Schmittetai,1997). Inaddition,GLT-1ispresentin
astrocytesthroughoutthebrain,withpredominantexpressionintelencephalicstructures,
includingthehippocampus,neocortex,andstriatum(Danboltetal,1992;Rothsteinetal,
1994). However,bothradioactiveaswellasnon-radioactiveinsituhybridization
techniqueshaveshownthatselectedneuronalpopulations(i.e.,thalamus,hypothalamus,
andpyramidalcellsofthehippocampus)expressGLT-1mRNA(Torpetal,1994;
Schmittetal,1996). TheobservationsthatGLT-1mRNAisexpressedinorbyneurons
seemstobeatvariancewithotherstudies(Danboltetal,1992;Rothsteinetal,1994;
Lehreetal,1995)whichfailedtodetectGLT-1proteininimmunoelectronmicroscopic
preparations. Schmittandcolleagues(1996)maintainthatafailuretodetectGLT-1
proteininneuronalmembranesmaybearesultoftheproteinconcentrationbeingbelow
themethod'sdetectionlimitsandthatattheelectronmicroscopicleveltheextremely