Guanidino Compounds in Biology and Medicine

185 Pages·2003·6.119 MB·English

by  Kazumasa Aoyagi, Yutaka Kuzure (auth.), Joseph F. Clark (eds.)

GUANIDINO COMPOUNDS IN BIOLOGY AND MEDICINE
Guanidino Compounds in Biology and
Medicine
Edited by
JOSEPH F. CLARK
Department of Neurology
University of Cincinnati Medical Center
45267-0536, Cincinnati, OH
USA
Reprinted from Molecular and Cellular Biochemistry, Volume 244 (2003)
W
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ISBN 978-1-4613-4985-3 ISBN 978-1-4615-0247-0 (eBook)
DOI 10.1007/978-1-4615-0247-0
Copyright © 2003 by Springer Science+Business Media New York
Originally published by Kluwer Academic Publishers in 2003
Softcover reprint of the hardcover 1st edition 2003
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Printed on acid-free paper
Molecular and Cellular Biochemistry:
An International Journal for Chemical Biology in Health and Disease
CONTENTS VOLUME 244, Nos. 1 & 2, February 2003
GUANTDINO COMPOUNDS INBIOLOGYAND MEDICINE
Dr.JosephF.Clark
Preface
K. Aoyagi, Y. Kuzure, S. Shahrzad,A. Hirayama, S. NagaseandA. Ueda: Inhibitionbyheparin ofproteinkinase Cactivationand
hydroxyl radical generationinpuromycinaminonuclcoside treatedisolatedrathcpatocytes 3-9
K. Aoyagi: Inhibitionofarginine synthesisbyurea: Amechanismfor arginine deficiencyinrenal failure which leads to increased
hydroxyl radical generation 11-15
S. NakayamaandJ.F. Clark: SmoothmuscleandNMRreview: Anoverviewofsmoothmusclemetabolism 17-30
B.D. Cohen: Methylgroup deficiency and guanidino productioninUremia 31-36
A.K. Bence,YR.Adams andP.A. Crooks: L-Canavanineas aradiosensitizationagent for humanpancreaticcancercells 37-43
TJ.deGrauw,K.M. Cecil,A.W.Byars,G.S.Salomons,W.S.BallandC.Jakobs: Theclinicalsyndromeofcreatinetransporterdeficiency 45-48
W. Derave,B.O. EijndeandP. Hespel: Creatine supplementationin healthanddisease: What isthe evidencefor long-termefficacy? 49-55
M. Hiramatsu: Arole for guanidinocompounds inthe brain 57-62
A. Hirayama, S. Nagase,A. Ueda, K. Yoh, T. Oteki, M. Obara, K. Takada, Y. Shimozawa, K. Aoyagi andA. Koyama: Electron
paramagneticresonance imagingofnitric oxideorgandistributionin lipopolysuccaridetreatedmice 63-67
J. Boero,W. Qin,J. Cheng,T.A. Woolsey,A.W. StraussandZ. Khuchua: Restrictedneuronalexpressionofubiquitousmitochondrial
creatinekinase: Changingpatternsindevelopment andwithincreasedactivity 69-76
T. Kitano,N.Nisimaru,E.Shibata,H. Iwasaka,T.NoguchiandI.Yokoi: Monocarboxylatesandglucoseutilizationasenergysubstrates
inratbrainslicesunder selectiveglialpoisoning- a31pNMRstudy 77-81
M. Greenwood, R.B. Kreider, C. Melton,C. Rasmussen, S. Lancaster,E. Cantler,P. MilnorandA.Almada: Creatinesupplementation
during collegefootball training does not increasethe incidenceofcrampingorinjury 83-88
R.B. Kreider: Effectsofcreatine supplementationonperformanceandtraining adaptations 89-94
R.B. Kreider, C. Melton,c.J.Rasmussen, M. Greenwood,S.Lancaster,E.c. Cantler,P. MilnorandA.L.Almada: Long-termcreatine
supplementationdoes notsignificantlyaffectclinical markers ofhealthinathletes 95-104
Y. Kurosawa, T. Hamaoka, T Katsumura, M. Kuwamori, N. Kimura, T. Sakoand B. Chance: Creatine supplementationenhances
anaerobicATPsynthesisduring a single 10sec maximal handgripexercise 105-112
S. Nagase, H. Suzuki,Y. Wang, S. Kikuchi, A. Hirayama, A. Ueda, K. Takada, T Oteki, M. Obara, K. Aoyagi andA. Koyama:
AssociationofecNOSgenepolymorphisms with endstagerenal diseases 113-118
A. Ueda, S. Nagase, H. Yokoyama, M. Tada, H. Noda, H. Ohya, H. Kamada,A. HirayamaandA. Koyama: Importance ofrenal
mitochondriainthe reductionofTEMPOL, anitroxideradical 119-124
J. Nikolic, G. Bjelakovic andI. Stojanovic: EffectofcaffeineonmetabolismofL-arginineinthebrain 125-128
M. Komurai,Y. Ishii,F. Matsuoka,K. Toyama,M. Ominato,T Sato,T Maeba,K. KimuraandS.Owada:Roleofnitricoxidesynthase
activity inexperimentalischemicacuterenal failure in rats 129-133
G. Ogimoto, T. Sakurada,K. Imamura, S.Kuboshima, T Maeba, K. Kimura and S. Owada:Alterationofenergyproductionbythe
heartinCRFpatientsundergoingperitonealdialysis 135-138
C.M. RemboldandC.R. Ayers: OralL-argininecanreverse digital necrosis inRaynaud'sphenomenon 139-141
A. Schulze: Creatine deficiencysyndromes 143-150
R.M. Murphy, R.J. Tunstall,K.A. Mehan, D. Cameron-Smith,M.J. McKenna,L.L. Spriet,M. Hargreaves andR.J. Snow: Human
skeletalmusclecreatinetransportermRNAandproteinexpressioninhealthy, youngmales andfemales 151-157
M.Tarnopolsky,G. Parise,M.-H.Fu,A. Brose,A.Parshad,O. SpeerandT Wallimann:Acuteandmoderate-termcreatinemonohydrate
supplementationdoes notaffectcreatinetransportermRNAorproteincontentin either young orelderlyhumans 159-166
L.Trezl,L. Hullan,Z.M. Jaszay,T. Szarvas,I.Petnehazy,B. Szende,1.Bocsi,Z.Takats,K.VekeyandL.Toke:Antagonisticreactions
ofarginine andlysineagainstformaldehyde andtheirrelationto cellproliferation, apoptosis, folate cyclcandphotosynthesis 167-176
D.N. Wheatley, R. PhilipandE. Campbell:Arginine deprivation andtumourcelldeath: Arginase anditsinhibition 177-185
Index to Volume 244 187-190
MolecularandCellularBiochemistry244:1,2003.
©2003KluwerAcademicPublishers.
Preface
Guanidinocompoundscomprisecreatine, arginine, and the clinical functions ofthese compounds. This translation of
guanidines.Inthepasttwoyearstherehavebeenover2000 science and clinical applications make this a valuable re
publishedarticleswiththenamesofthesecompoundsinthe source for learning the latestonthe subjectofGuanidino
title.Onecangotoanynutritionorhealthfoodstoreandbuy CompoundsinBiologyandMedicine.
these as supplements because itis believedthey improve The compilation ofthis importantwork has been made
healthandathleticperformance. easierbyassistancefromMuscleTechandAvicenaaswellas
InthisspecialissueofMolecularandCellularBiochem theJapaneseGuanidinoCompoundsinBiologyandMedicine
istrytheInternationalGuanidinoCompoundsinBiologyand Society andChildren'sHospitalMedicalCenterCincinnati.
Medicine Societyhas invitedthese authors to presenttheir Thecontributorsandindependentrefereeshaveprovidedan
findingswhichincludeanup-to-datesummaryofthescien invaluableserviceinproducingthiswork.
tific and clinical aspects ofessentially all thebiologically
active Guanidino compounds. The articles summarizethe JosephF. Clark
current scientificknowledge ofthese compounds with ref DepartmentofNeurology,UniversityofCincinnati
erencetorelevantclinicalconditions.Thereaderwillfinda Cincinnati,OH, USA
variety ofarticles discussing the chemical, biological and E-mail:[email protected]
MolecularandCellularBiochemistry244:3-9,2003.
©2003KluwerAcademicPublishers..
Inhibition by heparin of protein kinase C activation
and hydroxyl radical generation in puromycin
aminonucleoside treated isolated rat hepatocytes
3
Kazumasa Aoyagi,1Yutaka Kuzure,2 Siranoush Shahrzad,
Aki Hirayama,3 Sohji Nagase3 and Atsushi Ueda3
lDepartmentofTnternalMedicine, College Clinic, Tsukuba CollegeofTechnology, Tsukuba, Ibaraki; 2Bio-SystemResearch,
UniversityofTsukuba, Tsukuba; 3InstituteofClinicalMedicine, UniversityofTsukuba, Tsukuba, Japan
Abstract
Heparinhasbeenreportedtohavemanyactionssimilartocalcium-dependentproteinkinase(PKC)inhibitors.Wehavefound
thatpuromycinaminonucleoside(PAN)increaseshydroxylradicalgenerationandthiswaspreventedbyH-7,aPKCinhibitor
inisolatedrathepatocytes.Inthisstudy,weinvestigatetheeffectofheparinontheincreasedhydroxyl radical generation as
wellasPKCactivationbyPANinisolatedrathepatocytes.Toestimatetheamountofhydroxyl radicalgeneration,wemeas
uredmethylguanidine(MG)andcreatolwhicharetheproductsfromthereactionofcreatinineandhydroxylradical.Synthetic
rateofMGpluscreatolinisolatedrathepatocytesincubatedinKrebs-Henseleitbicarbonatebuffercontainingcreatinineand
testedreagentswererecorded.ThisratewithorwithoutPANwas231 ± 11or112±5.6nmoUgwetcells/4h(mean±S.E.,n
=5),respectively.Heparinconcentrationsof3.3,6.6and 10U/m]inhibitedMGpluscreatolsynthesisinthepresenceofPAN
by30,38and39%,andwithoutPANby8.4,27and34%,respectively.Statisticalsignificancewasobservedexceptfor3.3U/
ml withoutPAN.TheratioofPKCin membrane/cytoplasmicfraction, anindicatorofPKCactivation, increased2.8-and3
foldthatofthe0timeafter60and 120minincubationwithPANwhileheparinat 10Ulmlalmostcompletelysuppressedthis
increaseintheratio ofPKC.ThePKC ratio ofthe membrane/cytoplasmicfraction obtainedfrom hepatocytes with heparin
aloneorwithoutPANandheparinwasalmostunchangedduringthetestedperiod.VariationofPKClevelsinmembranefrac
tionissimilartothatofPKCratioofthemembrane/cytoplasmicfraction.Increasedcreato1synthesisbyPANanditsinhibition
by heparin wereobservedin thesamesamplesasthoseused forthePKCstudy.
TheseresultsindicatethatheparininhibitstheincreaseinhydroxylradicalgenerationinducedbyPANthroughinhibitionof
PKCactivationinisolatedrathepatocytes. (MolCellBiochem244: 3-9,2003)
Key words: heparin puromycinaminonucleoside, proteinkinaseC,reactiveoxygen
Introduction
glomerularmessangialmatrixresemblingthefeaturesoffocal
glomerularsclerosis,anintractablenephrosis[7].Therefore,
Heparinhasbeenwidelyusedasan anticoagulantinhemo many investigatorshave usedthis reagent to createanimal
dialysisandcardiopulmonarybypassoperation.Manypapers modelsforthestudyofnephrosis.Besidesitsanticoagulant
reportfavorable effects ofheparinonkidney diseases such activity, therearemanyinhibitoryeffectsofheparinonthe
as glomerulonephritis [1], renal failure [2], diabetic vas following:smoothcellproliferation[8],tissuefactorexpres
cularcomplications[3,4] andchronicpuromycinamino sion[9]c-fosandc-micmRNAexpression[10], transcrip
nucleoside (PAN) nephrosis [5). PAN has beenknown to tion oftissue-type plasminogen activator [11] induction of
induce heavy proteinuria [6]as well as an increasein the matrixmetalloproteinase[12]PDGFstimulatedMAPkinase
Addressforoffprints:K.Aoyagi,CollegeClinic,CollegeofTsukubaCollegeofTechnology,Tsukuba,Ibaraki,305-0821.Japan
(E-mail:[email protected])
4
activation[13]andendothelin-1 synthesisinendothelialceUs IncubationofcellsforMGandcreatolsynthesis
[14lInmostofthesereports, activationofproteinkinaseC
(PKC)and/orinhibitionsofthesephenomenabyPKCinhibi Hepatocyteswereincubatedin6mlofKrebs-Henseleitbi
tors werereported. carbonatebuffercontaining 3% bovine serumalbumin, 10
Recently, increased reactive oxygen species (ROS) gen mMsodiumlactate,16.6mMcreatinine,indicatedsubstances
eration has been implicatedas the causeofthe toxicity in and withorwithout 1.9mM PAN.The incubation mixture
ducedbyPAN[15,16].We,also,reportedthatPANincreases wasshakenat60cycles/minina30-mlconicalflaskwitha
generationofhydroxylradical,themostreactiveROSiniso rubbercap under 95% oxygen and 5% carbon dioxide at
latedrathepatocytes[17-19]aswellasglomerulus[20].As 37°C. Equilibration ofthebuffer was repeatedevery hour.
for the molecular mechanism for the toxicity ofPAN, we Incubation was arrested bythe addition of0.6ml of100%
noted that 1-(5-isoquinoline sulfonyl)-2-methylpiperazine (w/v) trichloroaceticacid.
dihydrochloride(H-7),aPKCinhibitor,inhibitsthisincreases
ofhydroxylradicalgenerationinducedbyPAN[21].Wealso
foundthatPANactivatesPKCinisolatedrathepatocytes(in DeterminationofMGandcreatol
submission). Thisincreased hydroxyl radical generationis
indicatedbyanincreasedsynthesisofmethylguanidine(MG) Aftersonication, the supernatant ofcells and medium was
andcreatolinisolatedrathepatocytes withinanhourofthe obtainedbycentrifugationat1700xgfor15minatO°C,and
additionofPAN [19]. SinceweshowedthatMG, auremic 0.2 mloftheextractwasusedforMGandcreatolmeasure
toxin,isformed fromcreatininethroughahydroxylradical ments.MGwasdeterminedbyhigh-performanceliquidchro
adductofcreatinine [17-19]that was identified as creatol matographicanalysis using9,1O-phenanthrenequinonefor
later[22,23],ithasbecomeapparentthatthesyntheticrate thepost-labelingmethodasdescribedpreviously [24].Di
ofMGand/orcreatolfrom acertainlevel ofcreatininecan methylformarnideforfluorometricalusewaspurchasedfrom
beusedasamarkerfortherateofsynthesisofthehydroxyl WakoCo.,Japan.Creatolwasseparatedbyacationexchange
radical [24-26]. resincolumnlargerthan thatforMGanalysisandwascon
Inthepresentstudy,weinvestigatedtheeffectsofheparin verted to MG by heating at 125°C under strong alkaline
ontheactivationofPKCtogetherwiththehydroxylradical conditionusingthe HPLCsystemmodifiedfrom theMG
generationinPANtreated-isolatedrathepatocytes. determinationapparatus [30].
Materials and methods Subcellularfractionation ofPKCfrom isolated
hepatocytes
Materials
Hepatocytes(1.5gwetcells)wereincubatedin35mlofthe
same bufferas used for MG and creatol synthesis with or
Puromycin aminonucleoside was purchased from Sigma
withoutPAN. Analiquot (5 ml)ofthecell suspensionwas
ChemicalCo.,St.Louis,USA.Heparinwaspurchasedfrom
takenat0,15,30,60and120minandthecellswerecollected
NovoCo.Ltd.ThecreatolstandardwaskindlydonatedbyDr.
bycentrifugation.Thecollectedcellswerehomogenizedwith
Ienagaofthe Nippon Zoki PharmaceuticalCo. Ltd. Mono
aDouncehomogenizer(100strokes)in2mlof50mMTris
clonalantibodiesspeci.ficagainstn, ~andyisozymeswere HClpH7.6containing0.25Msucrose,2mMEDTA,0.2mM
purchasedfromSeikagakuKogyoCo.(Tokyo,Japan).The
phenylmethanesulfonylfluorideand0.1 mMleupeptin.Af
cell lysate for the PKC standard was purchased from the
teran initialcentrifugation(600 xgfor 10min) toremove
TransductionLaboratory (Lexington, KY,USA).
nuclei, the supernatant was centrifuged at 12000 x g for
10 min to remove mitochondria and thesupernatants were
separated into cytoplasmic and insoluble fractions by cen
Preparationofisolatedrathepatocytes trifugation (l05,OOO x gfor 45 min). The insoluble (mem
brane) fractions werehomogenized in200mlofthebuffer
Male Wistarrats weighing 300-350g were used in allex usedforhomogenizationandwerekeptonicefor30minafter
periments. The rats were allowed free access to waterand theadditionof1%TritonX-100for solubilization.Thecy
laboratory chow containing 25% protein. Isolated hepato toplasmicandtheinsolublefractions wereboiledfor5min
cyteswerepreparedessentially accordingto the methodof inthepresenceof2%sodiumdodecylsulfate(SDS),andboth
BerryandFriend [27] asdescribedpreviously [28,29].We fractions werekept at-80°C. Proteininbothfractions was
calculatedthat9.8x107cellscorrespondto Igofwethepa determinedbytheBicinchoninicacidproteinassaymethod
tocytes. [31].
5
DeterminationofPKC gated. Thesyntheticrate ofMGinisolatedrathepatocytes
withandwithoutPANwere 177± 12and91.3 ±3.2nmollg
Bothfractions wereboiledfor 3min with 1% ~-mercapto­ ofwetcells/4h(mean±S.E.).Heparinattheconcentrations
ethanol,5%glyceroland0.006%bromophenolblue.Proteins of3.3,6.6and10U/mlsignificantlyinhibitedMGsynthesis
(110Ilgprotein)intheabovetwofractionswereseparatedby by38,39and41%inthepresenceofPANandby24,31and
electrophoresison8%SDS-polyacrylamidegels(14x14cm). 45%intheabsenceofPAN,respectivelyasshowninFig. lao
As apositivecontrolofPKC (1 Ilgprotein),ratbrainlysate TheMG plus creatol synthetic rate with and withoutPAN
obtainedfromTransductionLaboratorieswasused.Thepro were231±11and111.6±5.6nmollgofwetcells14h(mean
teinsweretransferredtonitrocellulosefilters.Immunoreactive ±S.E.),respectively.Heparinat3.3,6.6and 10U/mlinhib
proteinsweredetectedusingthemixtureofantibodiesspecific itedMGpluscreatolsynthesiswithPANby30,38and39%,
againsta,~andyisozymes.Thesewerediluted100timesand andwithoutPANby8.4,26.5and34%asshowninFig.1b.
mixed(1:1:1).Enhancedchemiluminescence(ECL)western MG plus creatol synthesis was significantly inhibited by
blottingdetectionreagents(RPN2106,AmershamPharmacia heparinatanytestedconcentrationexceptat3.3U/mlwith
Biotech,NJ,USA)wereusedfordetectingPKCs.Thebands outPAN.
ofPKCswerequantifiedbydensitometry. Theinhibitionrateof30%by heparinof3.3 Ulmlinthe
presence ofPAN is much higher than that of8.4% in the
absenceofPAN.Theinhibitionratedidnotincreasepropor
Statistics tional to the heparinconcentration. This indicates that the
inhibitionis not causedbya simplehydroxylradicalscav
Significanceofexperimentalresultsbetweentwogroupswas
engingactivityofheparin.
evaluatedusingtheunpairedStudent'st-test. Forcompari
sonsinvolvingmorethantwogroups,ANOVAwasapplied.
Resultswereexpressedasmean±S.E.Thedifferenceswere
InhibitionbyheparinofPKCactivationinisolatedrat
consideredstatisticallysignificantwhenthecalculatedPvalue
hepatocytes
waslessthan5%.
The translocationofPKC from the cytoplasmto the mem
Results branefractionhasbeenreportedasthemostreliableindica
toroftheactivationoftheconventionaltypeofPKCincells
InhibitionbyheparinofMGandcreatolsynthesisin [32-34].BandsofPKCinthemembranefractionswerede
isolatedrathepatocytes tected by the Western blotting method and their densities
obtainedby densitometry from isolatedrathepatocytes in
Effectofheparinon the synthesis ofcreatol andlorMGin cubatedincontrolsolution(withoutPANandheparin),with
isolated rat hepatocytes with or without PAN was investi- PAN, withPANandheparin, orwithheparinalonerespec-
180 (a)
160
~140
01 *
~120
C)
~
100
~80
o
],60
~40
20
o , , ! I I I o , I ! ! , !
012345678910 012345678910
Heparin(unit/m1) Heparin(unit/m1)
Fig.1. InhibitionofMGorMGpluscreatolsynthesisbyheparininisolatedrathepatocytes.Isolatedrathepatocytes(0.1gwetcells)wereincubatedwith
variousconcentrationsofheparinwith(0)orwithout1.9ruMPAN(e)asdescribedin'Materialsandmethods'.Valuesareexpressedasthemean±S.E.
(n=5).*Indicatesapvaluelessthan0.05vs.thecontrolvalue(withoutheparin).DataweresubjectedtoANOVAwithpairwisecomparisonbytheBonferroni
method.
6
tively. PKC in the membranefractions increased at60and Ontheotherhand,thePKCratioofthehepatocytesincubated
120 min incubations in PAN-treated hepatocytes. As for withheparinandPAN,withheparinaloneandwithoutPAN
thePKCbandsfromhepatocytesincubatedwithPANplus orheparindidnotchangeorchangedlittleduringthetested
heparin,inhibitionoftheincreaseofPKCinthe membrane incubationperiod(Fig.2a).PKClevelsinthemembranefrac
fractionsat60and120minwasnoted.Sincethetotalamount tionafteradjustingforthedensitiesbasedonassumptionthat
ofPKCismorethan 10timesofthatofthemembranefrac otimedensityisaconstantisshown(Fig.2b).Thereareno
tion,activationofPKCwillnotaffectthePKCamountwithin majordifferencesbetweentheresultsusingcytoplasmicPKC
ashortperiod.Thereafter,cytoplasmicPKCcan beusedto or0 time membrane PKC for the correction. Creatol was
correctthevariationindensitycausedbythedifferentexpo determinedinthesamesamplesasthoseusedfortestingPKC
surestoluminescencebytheWesternblotmethod.ThePKC activation. Creatollevels inthe presence ofPANat 30, 60
ratioofthemembrane/cytoplasmicfractionoftheisolated and 120min incubationincreasedto 3.2, 3.0and2.3 times
rathepatocytesdeterminedafter0, 15,30,60and 120min ofthat seen without PAN (Fig. 2c). Heparin almost com
incubationswithorwithout 1.9 mM PAN wereas follows. pletelyinhibitedthisincreasedsynthesisofcreatol.
InthepresenceofPAN,thePKCratiointhemembrane/cy TheseresultsindicatethatheparininhibitsbothPKCacti
toplasmicfraction after60and 120min incubationwas 2.8 vationinducedby PAN andMGgeneration inisolated rat
and3.0timeshigherthanthatofthe0timecontrol(Fig.2a). hepatocytes.
3.5
I (a) 300 (b)
-a. 3 ~250
..
~2.5 '"
.~..200
!
2
1.&>1.5 !150
u
~
100
'j I .i
a: 5
50
UO. ~
~ E
ll. ° 0
° 20 40 60 III 100 120 0 20 40 60 80 100 120
IncubationTime(min) IncubationTime(min)
250 (c)
oO°-::~E:fOI.""""'_..L------l._-'-----'
20 40 60 III 100 120
IncubationTime(min)
Fig.2. InhibitionofPKCactivationinPAN-treatedhepatocytesbyheparin.TheratioofPKCinthemembrane/cytoplasmicfractionisshownin(a).The
PKClevelinthemembranefractionafteradjustingthelevelbasedontheideathat0timedensityisaconstantisshown(b).Creatolsynthesisinisolated
hepatocytesinthesamesampleasthatforPKCstudyundervariousconditionsareshownin(c).Thesymbols0 representsPANandheparin.0 represents
PAN alone•• represents no additives and. represents heparin alone.
7
Discussion acidmetabolismwhichisknowntogeneratehydroxylradi
calsingallbladdermusclecells [49].
Inthis study,wedemonstrate thatheparininhibitsthe PKC As a mechanism ofinhibitionofPKC activation, it was
activationas wellas MGsynthesisinPAN-treatedisolated reported thatheparin acts as an inositol 1,4,5-triphosphate
rathepatocytes.Theheparinconcentrationof3.3Vlml that receptorantagonistinreceptorcoupledGprotein-mediated
significantlyinhibitedMGsynthesisinPAN-treatedhepato contractionofisolatedintestinalmuscle cells [50]and de
cytesislowerthanthatinthebloodofthepatientsundergo creasesintracellularCa2+concentration[51].
inghemodialysistherapyorcardiopulmonarybypasssurgery Thus, heparin, whichboth scavenges ROS and inhibits
[35].TheinhibitionrateofMGsynthesisdidnotincreaseat PKCactivationorPKCactivity,representsapotentialtreat
concentrationsofheparinhigherthan6.6and10Vlml.These mentformanydiseasesinwhichPKCactivationisamajor
datasuggestthattheinhibitiondoseisnotdependentonthe element.
ROSscavengingactivityofheparin,sincetheROSscaveng
ingactivityofheparinisrelativelylow [36].Withregardto
theheparineffectonROS,ithasbeenreportedthatheparin Acknowledgements
inhibitROSgenerationinleukocytes[37]andinrheumatoid
synovial cellA (macrophage-like) and B (fibroblast-like)
ThisstudywassupportedinpartbytheScientificResearch
stimulatedbysubstanceP[38].Itisreportedthatheparinre FundsoftheMinistryofEducation,ScienceandCultureof
duces hepatic ischemia-reperfusion injury with decreased
Japan(C-05670942,C-0667I126),HeparinConference,and
lipidperoxidein vivo [39]. However, inthisreport, antico
GrantforScientificResearchExpensesforHealthandWel
agulantrole ofheparin was notexcludedas acauseofthis
fare Programs, Ministry ofHealth andWelfare, andJapan
favorable effect. This reportmay be the secondinstancein
Societyfor the Promotion ofScience (P99168). We thank
whichheparininhibitsROSgenerationinnon-inflammatory
ProfessorBurtonD.Cohen(DepartmentofMedicine,Albert
cellsfollowingsynovialBcells.
EinsteinCollegeofMedicine)forhiscriticalcomments.We
Favorableeffectsofheparinonglomerulonephritis[40]as
thankDr.KenjiNomoto,TsukubaInstituteoftheEisaiPhar
well as chronic PAN-nephrosis[5] has been reported at
maceuticalCo.Ltd.forthistechnicaladviceinthePKCstudy.
concentrationsinsufficienttoinhibitcoagulation.Similar
favorable effects ofheparin or non-coagulant heparin like
substancesonthevascularcomplicationsindiabetesmellitus
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