THE EFFEGr OFCHEMICAL DENERVATION ON THE AGrION OF
ACETYLCHOLINEAND SUBSTANCE P
IN THE ISOlATED PERFUSEDMESENTERI C
ARTERIAL BED OFTHE RAT
by
C MargaretE.Miller,
ase,
Athesis submitted to theSchoolofGraduate Studies inpartialfulfilment of the
requirements forthedegree of Master ofScience
FacultyofMedicine Memorial UniversityofNewfoundland
September1989
St.John's Newfouudland
. ....
NalionalLibrary ofCanadaBibliOlheQue naticnete dU canada
canadian Thesessevce 5et'VlCe destheses cereoeooes 0 . - . . 0 -
kl"ONt
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a.
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ISBN 0-315-59 20 5- 2
ABSTRACf
Vascularendothe lialcellsrespond to certainvasoactiveagentsbyreleasing factorswhichact onmedialsmoothmuscleto causerelaxation orcontractionof blood vessels. One of thesubstancesresponsible forendothelium-dependent relaxation to acetylcholinehas recently been identified asnitricoxide. Wehavetestedthe hypothesisthatthe ability of vascularendotheliumtocause relaxation inresponse to stimulationbyvasoactiveagents isrelatedinsomewaytothe pattern of perivascular innervation.The actions ofacetylcholineand substancePweretested in the presenceof methoxamineinducedlone inthe isolat edperfusedmesenteric arterial bed of therat. Tissueswere testedfrom untreatednormalJ2 weekold Sprague -Dawleyratsand from ratswhichhadbeentreatedfrom birth withcapsaicin to prevent the development of peptidergic perivascular innervation or 6- hydroxydopamine to prevent developmentof catecbclamlnerglc innervation.
Concentrationdependen t endoth elium-dependentrelaxations wereobserved in response to acetylcholine. The concentrationresponse curveto acetylcholinewas shifted 1.2logunitsto the rightinthe capsaicin-treatedgroupbut nochange was observed with6-hydroxydopam inetreatment.SubstancePcauseda r'ose depende nt potentiationof themethoxamine inducedtone whichwas notdependent on the presence ofan intact endothe lium. Relaxations to substance P were notobserved atany dose.Sympathectomywithneonatal 6-0 HDA treatmentresultedin an increase
inthe substance Ppressor response, butno changeswereobservedwith capsaicin treatment. Thus, it appears thataltering thepeptidergie perivascular innervation resultsinadecreased sensitivityofthe mesentericarterialbed to acetylcholineand changesin the catechclaminergicfibre plexus result inanenhancementof the substanceP modulation of adrenergicvasoconstriction.
iii
ACKNOWLEDGEMENTS
Mysincerethanks gotomy supervisor, Dr.T,M.Scott, forhis supportand patience.Withouthis adviceandencouragement thisprojectwouldnothave been completed.Ithank also theother members ofmy supervisorycommittee,Dr.CR.
Triggle and Dr. T. Hoekmanfortheir guidancethroughout Ihis progra m.
1amindebted10Ms. JudyFoote, Mrs.MaryCrowleyand Mrs.JanetRobinson fortheirexpert technical assistanceaswell astheirfriendshipthroughout thisdegree program.ThanksalsogotothestaffoftheAnimalCareUnit,especiallyMr.Dennis Traversefor assistancewiththe rats.
Ithank M·, CliffordGeorge,AudiovisualServices,Faculty ofMedicine, Memorial Universityof Newfoundland,as wellasMrs.Vida MillerandMrs.
KimberleyRoss fortheir assistancein the preparationof Ihisthesis.Mythanks also go(0 Dr. D.W.McKayforthe use of thetransducer andchart recorder andto BurroughsWellcomeInc.fora supplyofmethoxamine.
Acknowledgement is alsodue10Dr.T.M.Scott,theFacultyofMedicine,and theSchool ofGraduate Studies,MemorialUniversityofNewfoundland,aswellas theDepartment of CareerDevelopment.Province ofNewfoundlandand Labrador forfinancial support throughout thisprogram.
Finally,myloveand appreciationgotomyhusbandDavid,forhis support andencouragement, and to myparents for believingin me always.
iv
TABLEOF CONTENTS
ABSTRACT'" •. • . ..••• • •. .• •••••... .. •.•.• • . • •...•. . .... ii
ACKNOWLEDGEMENTS. ..•..• •.•••.... ....• .... ... . ... iv
TABLE OF CONTENTS••,••...•.••.•. . ..•. • ••• • .. ... .• .•. •• v
LIST OF FIGURES ...•. • • ••..•••••. . . ... .•...••...viii
LIST OF TABLES ....•... . .• •...•.... ... • ... ...•. .•• . •• . x
LIST OF ABBREVIATIONS... . . .. ...• .••.... ... .. .. •....•.• xi
INTRODUCTION•. .• ....•... ....•••.. ...• .. . . ..•. 1.1. The Control of VascularTone... ... ... ... ... 1
1.1.1. VascularInnervation... ...•.. . ...•... . 1
(a) Adrenergic Innervationof the Vasculature.... .. 2
(b) Cholinergic Innervationof the Vasculature 3 (c) Peptidergic Innervat ionof the vasculature 3 SubstanceP•...•. ... .•. •• •. 4
CalcitoninGene-RelatedPeptide .•.• .. . •• ,. . 6
NeuropeptideY , 8 Vasoactive Intestinal Polypeptide.••. .. • • • • .. 9
1.1.2. Circulatingand Local Factors .••.• . .•••.... .•.• 11
(a) CirculatingFactorswhich RegulateBtood Flow. 12 (bJ LocalFactorsWhichRegulate Bloodflow .... 14
1.2. The Endothelium . . . . .. ... .. . ... .. ... 17
1.2.1. Receptors on theEndothelium. . .. . 17
1.2.2. Endothelium-DerivedRelaxingFactor. . . . ... . ... 20
(a) Characterizationand Identificationof EORF• •. 21 (b) Agents that Produce Endothelium-Dependent Relaxation 23 1.2.3. Endothelium-Derived Contracting Factor.. ... .. 32
(a) Characterization and Identification ofEOCF.. . 33
1.3. Objectives of the Studyand Review of Methods ....• • • •.. 36
1.3.1.The McGregorPrepa ration 36 1.3.2.Immunohistochemistry.... . ... . 38
1.3.3.ElectronMicroscopy. . ... ... . . ... . .. .. ... .. .. 39
1.3.4.Methods of Chemical Denervation 40 6-Hydroxydopamine 40 Capsaicin 43 MATERIALS AND METHODS... .. . . . .. ... ... .... ... 49
2.1. Tissue Preparation 49 2.2. Apparatus... ... •. ... .. .. .. ... .... . . ... .. 52
2.3, Drugs andChemicals•.••. ••••••. •.• •.•.••••.•. •.• 54 2.4. Animal Models 56 2.5. ExperimentProtocols... .... ... ... .•.... . ... . 58
2.5.1.Methoxamine ConcentrationResponse Experiment ... 58
25.2.AcetylcholineExperiments .... . .... .. . . .... .... 58
2.5.3.SubstancePExperiments... .... ... 59
(a) SubstancePin thePresence ofMethoxamine .. 61
(b) Substance P in Endothelium-Den uded Preparations ... . ... . ... ... ... 62
(c) SubstancePBeforeMethoxamine ... . .... . .. 63
(d) SubstancePin thePresence of AngiotensinII.. 64
(e) Substance P in the Presenceof Depolarizing Buffer 64 2.5.4.Immunohistochemical and ElectronMicroscopic Study. 65 (a) Immunohistochemistry... ... .. .. ... ... 66
(b) ElectronMicroscopy... ... . . ... . 67
25.5.Statlsncs ... ... ... . . . ...•. .... 68
RESULTS .... .• . ... .. ••.••••. • • •.••.. •• •.. . ••. ..•. .. ..• 70
3.1. Immunohistoche mistry Results ...•...••.• ... .. 70 3.2. MethoxamineConcentrationResponseExperiment 71 3.2.1.MethoxamineResponsein Capsaicin-treatedRats 71 3.2.2.MethoxamineResponse in6-0HDA·treated Rats 74
vi
3.3. AcetylcholineExperiments... ... . . .... . ... ... 74 3.3.1.Capsaicin-treatedAnimals .. ... 74 3.3.2.6-Hydroxydopamine TreatedAnimals. ... .. . . ... 79
3.4. SubstanceP Experiments 79
3.4.1.Substance P BeforeMethoxamine .. . ...• .. . . . .. . 84 3.4.2. SubstanceP in the Presenceof AngiotensinII... 89 3,..,3.SubstancePinthePresence ofDepolarizingBuffer. .. 89
DISCUSSION. .. ... ..•..•.... .... ...•.. .. •. ...•. •... 91 4.1. Considerationof DenervationTreatments .. •.... ....•... 91 4.2. FactorsContributingtothe Alterationof theACh Response . 93 4.2.1.ChangesinMuscarinicReceptorDensny•. .. ..••.•. 94 4.2.2.Changesin IntracellularEvents of theEndothelium... ,15 4.2.3.Changesinthe Diffusion of EDRF .. ... ... 97
4.2.4.Changesin SmoothMuscleCells 97
4.3. The Effect of6-0HDA Treatment en the ACh Response ... 103 4.4. The Responseto SPin the MesentericBed 104
4.4.1.The Mechanismof Action of SP in the Mesenteri c Bed...•....•••...••.• ...•... ... .•. .. 106 4.5. Conclusion.... . ... . . ... . .. . ... . ... ... 108
LISTOF REFERI;NCES ..•... .... ...•... ... .. ... .. • ...109
vii
INTRODUcnON
1.1. The Control of VascularTone
The controlof thecardiovascularsysteminvolvesavarietyof components which influencesmoothmuscle tone.The distribution of blood 10 tissuesin thebody
isdetermfnedbythe flowof blood from the heart and theregulation of blood vesseldiameter. Thecomplex systemresponsible for thisfunctionincludes the innervationof the vasculature,circulating factorsin theblood,and the endothelium.
1.1.1.Vascular Innervation
Oneof themost powerful mechanismsfor the overallcontrol ofthe cardiovascular system is theinnervation providedbytheautonomicnervoussystem.
The nerve fibres whichsupply the blood vessels are calledperivascularnerves since theyform amesh-like terminalpiCKUSatthe adventitial-medialborderof theblood
vesselwall.
For a long timetheinnervationofthevasculature wasthought 10consistof sympatheticnervescontaining catechotamlnes.However,with the development of immunologicaltechniquescapable ofidentifyingand defining neurotransmitter substances, thesefibrenetworkshave beenshown tobe more complex(Kenigsberg andCuello,1987). Threedifferenttypes of perivascularnerves,adrenergic,
cholinergic, and peptldergtchavebeendistinguished.In addition,somenervefibres have beenshownto containmore than one ofthesetypesofneurotransmitter co- localizedat the nerveterminals(HckfeltetaL, 1987).
(a) AdrenergicInnervation of the Vasculature
The mostimportantcomponentofautonomicmotorcontrolofthe vasculature isIhe sympatheticnervous system, in which noradrenalineis the peripheral neurotransmitter.The adrenergicnervesupply10mostvessels inthe bodyoriginates inthe pre- or pare-vertebra!gangliaof the sympathetic nervous system.However, the innervationof some bloodvesselsin the brain may originatein central carechclaminergicneurons(Edvinssonet aL,1973).
There is a great variationinthepatternand densityof the sympathetic innervationofvascularsmoothmuscle (Burnstock, 1975). Itis likelythat such variationisrelated to the physiologicalrole of the blood vesselin the tissueit supplies.In general, large elasticarteriesshowa sparse patternof innervationand, as the sizeofthe artery decreases,the densityof innervationincreases. Capillaries, venulesandsmallveinsare considered tohave verylittle adrenergicnerve supply.
Veins usuallyhave a much lessdense innervationthan arteries.
(b) CholinergicInnervation ofthe Vasculature
Innervation of blood vessels by postganglionic cholinergicnerve fibres is generally thoughtto providean inhibitorycontrolof vascular tone.However,the nature of this vasodilatoryaction is understoodlessthanthe actionsofadre-e rgic nerves. Thesecholinergic nerveshave aless widespread distribution in the cardiovascularsystem than dothe sympathetic fibres.However,physiologicaland histochemicalstudieshavesuggested thepossibilityof cholinergic vasomotorcontrol in thebrain,heart, lung,kidney,skeletalmuscle and uterus (SchenkandEI Badawi, 1968; Bell, 1969; Iwayamaetal.,1970;Borodulyaand Pletcbkcva,1973).Thus, some vascular beds possessa dual adrenergicandcholinergic lnnervadon.
Cholinergic nerves intheblood vesselso~skeletal muscle originate from gangliaof the sympatheticchain,whilethose ofthe uterine arteryarise from the paracervicalganglion. The originof the cholinergicsupplytothe intracranial blood vesselsis not yetknown.Vessels ofthe gastrointestinal tractreceivetheircholinergic nerve supplyfrom the intramuralganglia(Owman,1983).
(c) Peptidergic Innervationof the vasculature
Inadditionto thesympatheticadrenergicandparasympatheticcholinergic divisions oftheautonomic nervoussystem,anetwork of peptidergicnerves which innervatethevasculatureofarange of mammals, including man,hasbeen shown
toexist (Furnesses aL, 1984).A variety cf smallpeptides,withup to about 40amino acidresidues,havebeenfound in peripheral nerves whichsupply autonomically innervatedorgans(Hokfeltelai,1980;Furnessd al,1982;Furnessand Costa,1982).
Within thecardiovascularsystem,thedistnbu tion ofthepeptidesappearstovary frombedtobedandfromone speciesto another.Whilemanyofthepeptidesheve beenfoundinthe card iovascular system,thosemostcommonlyinvolvedaresubstance P (SP). calcitoningene-relatedpeptide (CGRP),neuropeptide Y (NPY) and vasoactiveintestinalpolypeptide(VIP).
Substance P
Amongthe peptides listed.SPhas perhapsthelongesthistory. Itwasfirst descnbed as a biologicallyactivecompoundincrudegutextractsby von Euler and Gaddumin1931.Itwasnotuntil1971that Changandco-workersisolatedSPin itspureformfrom bovine hypothalamus andestab!:.:hed theeleven aminoacid sequenceofthepeptide. Shortly thereafterthe peptidewassynthesizedsuccessfully (Tregearel al,1971). Then, theavailabilityofapureandchemicallydefined SP madeit possibleto generate specificantibodiestothepeptideforradioimmunoassay andimmunohistochemistry.
SubstancePhasawidespreaddistributioninthe centraland peripheral nervoussystemsinavarietyof species. including man(Furnesset aJ.,1984).
Extensive studieshave establishedthe presenceof SPinmost parts ofthe central nervoussystemof allmammals.Detailedradioimmunoassay studies have indicated that thehighest concentrationsarepresentinthe mesencephalon,hypothalamus and preopticarea.whilethe cerebellumcontainsinsigni6cantamountsofSP(Brownstein etat.1976;Kanazz.wa andJessen,1976;GaleetaL,1978;EmscnaaL,1980;Cooper aaL,1981). Immunohistochemicalmappinghas so faridentified SPinover30 areas of the brain including the brainstem andspinalcord. Forathoroughaccountof studieswhichhavebeendonetolocalizeSP in thecentralnervoussystem the reader isreferredtoPcrnow(1983).
In the peripherysubstance Pismainly insensory nerveswith a widespread distributionto theheart andmostvascularbeds.Insomeanimalssuchasthe guinea- pig, virtuallyallvascularbedsaresupplied with SP-containing nervefibres.The presenceofSP fibres has been demonstratedin the guinea-pigheart(Whartonet aL,1981a),rat carotidsinus (Helkeet aL,1980),andin thearteries and veinsofthe tracheobronchialtissueofthe guinea-pig(SundleretaL,1977).lmmunohistochemical studieshave alsoidentifiedsubstance P-like immunoreactive(SPLI)nervesinthe guinea-pig aortaandpulmonaryartery(ReineckeetaL,1980) and inthevesselsof the urinary andreproductivesystems(AimetaL,1978;Whartonet01.,1981b).
SubstanceP fibreshave beenshownto innervatethe cerebral(Liu-Chenet aL,1986), coronary (Brumet01.,1986) and mesenteric vascularbeds(Scott€IaL, 1989).
Bothvasomotor andsensory functionshave been suggested 00accountfor the presence ofSP-fibresinbloodvessels.Incoronary.pulmonary,mesenteric,renal andcerebral bloodvessels SPhasbeenshowntocause vasodilation (Maxwen.1968;
Hallbergand Pemcw,1975; Ek.lundelat,19'n).VasoconstrictionbySPhasbeen reportedinthehepaticportal veinof therat (Hellstrandand Jarhult,1980;
Mastrangeloet aL,1980) and the anteriormesentericveinoftherabbit (Berubeet aL,1978).AsensoryfunctionCor SPin vascular nerve fibreshas also beensuggested, sincepretreatmentwith capsaicineliminatesSP-like immunoreactivity fromthese fibres(Furnesset01.,1982: Matthewsand Cuello,1982).
Calcitonin Gene-Related Peptide
Calcitonin gene-relatedpeptide(CGRP) is a:17·amino acidpeptideresulting from alternativeprocessingof theRNA duringexpressionofthecalcitonin gene (Amaraet al,1982;Rosenfeldetat,1983). The exi..tenceof CGRP wasflt'$t predictedbyanalysis ofa new RNAinarat cellline. A similarpeptidewas subsequentlyisolatedfromhumanmedullarycarcinoma ofthyroid tissue and sequenced in1984 (Morriset
a;
1984). CORPhas been showntohave strong vasodilatoryactions(BrainetaL,1985).Itexertsitsvasodilatoryeffect both through direct inlluence onvascularsmooth muscleandbyinhibitionof neurogenic vasoconstriction(Brainct al,193.5; FISCheret aL,1983;Hankoet al,1985).Immunohistochemicalstudieshaveshowna widespreaddistributionofCGRP in sensoryneuronesand nervefibresofthe cardiovascularsystem ofseveralspecies (Wharton and Gulbenkian,1987). Many studieshave been donein guinea-pig whereperivascularCGRPfibreshavebeenseeninallvascularbeds.ln an extensive studybyUddmanandco-workers (1986), the distributionof perivascularCGRPnerve fibresin theguinea-pigwas describedindetail.Theirfindings indicatea substantial CGRP nervesupplyto the heartand coronary bloodvessels. The carotidarteries and thoracicaorta receivenumerous CORP fibres,while the brachial and femoral arteries10the limbscontain fewfibres.In theguinea-pigbrain,the cerebralarteries wereshownto be surroundedbyadensenetwork of perivascularCGRP fibres, whereasthe smallpial arterieswereaccompaniedbyfew fibres. Vessels of the respiratory and genitourinarytracereceived a moderatesupplyof CORPfibres.
In the gastrointestinal tract the mesenteric and gastroepiploicarterieshada dense supplyof CORP-containingnervefibres.Incontrast, smallbloodvesselsof skeletal musclereceiveda veryscarcesupplyof CORP fibres.Studiesin the rathave also indicateda widespreaddistributionof CGRP-fibresin the cardiovascularsystem (Mulderryet al.,1985).Thereare,however,some regionaland speciesvariations regarding the distributionofCGRP·immunoreactivenerve fibres (Whartontt01., 1986).
Thedistribution ofCG Rp·andSP-immunoreactivefibres isvery similarand severalstudieshavenowdemonstratedthattheyarefrequentlyco-localizedinsensory nerves (LundbergetaL,1985; Uddmann aL,1986;WanakaetaL, 1986 ).
Neuropeptlde Y
NeuropeptideY(NPY):~a36-amino acid peptideisolate dfromporcinebrain tissue(Tatemotoet aL, 1982). Thepeptidehas beensequenced(Tate moto,1982) andsynthesizedbya solidphasemethod[Balasubrarnaniamet aL,1987;Yamamoto et al.,1984). NPYhas been foundto be rich intyrosineandis structurallysimilar topancreatic polypeptide (PP )and peptide YY (PYY;Tatemot oetal.,1982).NPY iswidelydistributed in the centralandperipheral nervous systemsandhas been showntobeone ofthe most potentvasoconstrictor peptidesisolatedsofar (Emson and DeQuidt, 1984).
Inthe perip'.ery,NPYhasbeen localized in perivascular nerve fibresof severalspecies,includingman(Pemowetal.,1987).Itisoftencloselyassociated withorco-storedwith noradr e naline (Ekbladet al., 1984). Inthe rat,Allen and co-workers (1985)have identified high concentrations of NPYthrougho uttheheart and withinthemajor bloodvessels,in particular inthe renal andsuperiormesenteric arteries. This studyshoweda similarpatternof inne rvationbyNPY·and NA·
containingnervefibres,which wasdepleted aftertreatmentwith 6·hydroxydopamine
(6-0 H DA;Allenetal.,1985). Intheguinea-pig, NPY-immunoreactive nervefibres havebeenfoundtobewidely distribut ed in the vascularsyste m(Udd manet al., 1985) .Ingeneral,NPYfibres weremore numerousaroundarteries than in veins oftheguinea-pig.Theheart andcoronaryvessels,liketherat,wereshowntohave a richNPY-innervation.AwelldevelopedNPY plexuswasvisualized aroundlarg e elastic andmusculararteriessuc hasthe thoracic aortaandcommoncarotidarteries.
PerivascularNPYfibreswere numerousinarteriesoftherespiratory, gastrointestinal andgenitourinarytract s. Techniquesof surgicaland chemical sympathectomy,as wellas seque ntialimmunohistochemicalstaining,revea led thecoexistence ofNPY withNAhiperivascular nerve fibres oftheguinea-pig (UddmanelaL,1985).
VasoactiveIntestinalPolypeptide
Vasoactiveintestinalpolypeptide(VIP)is a 2S-aminoacidpeptidewhichwas originallyisola tedfromporcineintestine andnam edforitspotentvasodilatoryactions (SaidandMutt, 1970).VIPwas fitst synthesizedin197 3(BodanskyetaL)and has since becomecommerciallyava ilable.Thispep tide,whichisstructurallyrelated to secre tinandglucagon, wasoriginallytho ughttobelimited tothegastrointe stinaltract.
However,rad ioimmunoassay andimmunohistochemicalstudies haverevealedthat VIP has awidespreaddistributio n inthebody,localized inneurons(Fuxeet aI"1977;
LarssonetaL,1976).
10 Perivascular nervefibrescontainingVL.~-1!nmunoreacti\.ityjevebeenfound mainlyinregionalvascularbedsratherthaninthelargereonducrngbloodvessels (Whartonand Gulbenkian,1987).Inthe cat.VIP-contail"in~nervefibres werefound tobenume rousaro undart eriesof theuppe rrespiratory,gastrointe stinal,and genitourinarytracts(UddmanettlLt 1981).Veryfewfibreswere foundinblood vesse lsofske letal muscle andnonewere foundincoronaryarter ies. VIPfib res weresparsearoundlar gearteriesand veinsandappeare d tobeabsententiretyfro m blood vesselsof theliver,sple e nend kidney.Inthe guinea-pig, similar resultsha ve been reported(Dellael01.,1983).Thisstudydenc retrat edaspa rsesupplyof VIP·
immu noreact ivenervefibrestoIIIeheart andmajordistributingarte ries:ao rt a, subclavian,carotid,femoralandpulm onary.
or
thevascularbedsreceivingVIP fibre s,themostdenselysuppliedarteriawerethemesentericanduterinearteries.VIPinnervationofcerebralvesselsvaried fromthemostdenseintheanteriorand midd lecereb ralarteries 10verylillieintbebasilarart ery.Asinthecal,arteries runningtoskeletalmuscle and majororganswere lessdenselysup plied.Throughoul thebody'Veinsof theguinea-p igwere sparselysuppliedwith VIPnervefibres.
E\'idence forthecoexistenceofVIPwithACh hasbeensummarizedby Lundberg(1981). InadditionithasbeenshownthatVIP.containingfibres werenOI depleted when noradrenergicnervesweredegeneratedby6-0HDA or whenSP nervesweredisruptedbycapsa icin(Dena ttat,1983).Thissugge ststhat VIPnerves
11 innervatingthevasculature forma populat iondistinctfromsubstan ceP or sympatheticnerves.
Insummary.itcanbe seenthat theinnervatio nofthevasculatureismore complexthan wascommonlyaccepted untilrecently.It nowappea rstha t threetypes of perivascularnerveplexusexist:thecatecholamincrgicsystem whichhasNPYco- localizedwithNAin nervete rm inals,a system ofpeptidergicnerves containingSP and CORP,and thesystem ofcholine rgicand VIP<ontaining nervefibres.
1.1.2. CirculatingandLocal Factors
There are many fact ors which contrib ute to the ove rallco ntrol ofthe card iovascula rsystem,sometoa greate rdegree thanothers. In the circulation there areagentssuchaspe ptidesandhormoneswhichhave awide spreadinfluence on vasc ular tone . Localfactors suchasflow,pH,Po,and P~as well asthe concentratio nsofcertainionsinthe blood can determine the functional state ofa vescular beo.
12 (a) CirculatingFactorswhichRegulateBloodFlow
There arc certain naturallyoccurring vasoactiveamlnes,peptidesand lip ids which circulate in the bloodand canbegrouped underthetermlocal hormones or autacoids.Amongthese areacetylcholineandcarecholamineswhichmay befonned ex traneuronallyand releasedintothe blood.Acetylcholine has been showntocause relaxation of arteries,which is dependent upon the presenceofintactendothelium (FurchgotteraL,1981). Utile effecthasbeen observed in veins. Adrenaline, secreted from the adrenalmedulla,can causevasoconstrictionbyits actionona- adrenoceptocsorvasodilationbyanactionon,a-adrenoceptorsof vascularsmooth muscle.
Histamineis one ofthemost potentofthe endogenousbiogenicemines.Its actionsonbloodvesselsare complicated, since it actsontwo receptor subtypesand may alsointeractwiththe sympathetic nervous system(Brody,1980).In anumber ofspecieshistamine hasbeen showntoproducevasoconstrictionmediatedbyH, receptors,andvasodilation mediatedbyHzreceptors(Owen,1977).Serotonin is avasoactive amineidentified as S-hydroxytryptamine (S-HT).Inthe blood, S-HT is containedin platelets andisinvolved in theclottingprocess. Itis a potent vasoconstrictorexceptonbloodvesselsofskeletal muscle andcoronaryvesselsof theheart,whicharedilated(Cohenet01.,1981; VanNuetenet aL,1984).
13 Adenosineanditsphosphorylat ed derivatives,AMP, ADPandAlPhave vasodilatoractivity in the vascularbeds ofskeletalmuscle andthe coronaryvessels ofthe heart,andhavethus been shownto producea fallinblood pressureupon intravenousinjection.In many bloodvessels ATPand ADP have beenshownto cause endothelium-dependent relaxations while relaxations to adenosineand AMP wereindependentof the presenceof endothelialcells(vanhoutteandRlmele,1983;
reviewedbyFurchgott,1984).
Thepolypeptide familyofkininsincludes substanceP, kallidin and bradykinin, and angiotensin.SubstancePhas alreadybeenreviewed.The substanceskallidin and bradykininarecloselyrelatedpept idesof whichbradykinin has beenthemost studied.Bradykinin has beenshowntohave a potentvasodilatoryaction (Fox et aI.,1961;Haddyetal.,1970).This is an endothelium-dependent action whichwill bediscussedlater. Studieshave demonstratedthat thisaction is importantin the maintenanceof normalblood pressure(Gavras et al,1987).Kallidin isthought10 have thesame actions and similarpotency as bradykinin. Reninisan enzyme, secretedbythekidneys, which activates therenin-angiotensinsystemto generate angiotensin II.Whileangiotensin II is a directvasoconstr ictoragent,it also elicits the releaseofaldosteronefrom the adrenal co rtex. Aldosterone regulates sodium andpotassiumexcretionbythekidneysandthus contributes10the contra!of arterial bloodpressure(ReviewedinBowman andRa nd, 1980).
14 VasopressinIs a peptidehormonereleased from the neurohypophysis into thecirculation in response to nerve impulses. It isalso known as antidiuretic hormone (ADH). ADH isapotentvasoconstrictorof all types of bloodvessel, especiallycapillaries and venules. A major role played by ADH is in the response to haemorrhage. Atrial natriureticfactor (ANF) is anotherpeptide hormone belongingto a recentlydiscoveredgroupof peptides (de Boldet al.,1981).ANF is released Crom granulesintheatria ofmammalsand has beenshownto be a potent vasodilator in pre-contractedblood vessels (Currie et aL, 1983).
Prostaglandinsare anothergroupof local hormonesthat exert an effectat ordistal 10the siteof.their synthesis(Va ne andMcGiff,1975). Prostaglandins are released froma varietyof tissuesincludingbloodvesselsthemselves (Tuvemo and Wide,1973).Prostaglandinshave a wide varietyof cardiovascular actionsincluding reductionof blood pressure,vasodilation of many vascularbedsand constrictionof cerebralbloodvessels.
(b) Local Factors Which RegulateBlood Flow
In additionto circulatingaminesand peptides in the blood,there are certain local factors which may influence thecirculation(Haddy and Scott, 1968). The balance between dissolvedoxygen(Ol)and carbon dioxide(COl)can haveboth a directand indirect effect on vascularto ne. When levelsare elevated,COlacts
15 directly10dilatebloodvessels and depresscardiacfunction (Price.1960).Through reflexactions,CO2causesthe releaseofadre naline from the adrenal medulla to resul t in peripheralvasoconstrictionand an increase inblood pressure. Similarly, stud ieshaveshownthatareduction in oxygen tension oftheperfusingblood lowers theresistance toflowin thecoronary.hindlimb,forelimb.lntestinel,gracilisandrenal vascularbeds(Berneet aL,1957;Daughertyet aL,1967; Allingeretal.,1967; Skinner and Powell,1967).
ThepHof blood is another factor involvedinlocal regulation offlow.
Althoughthe bloodis a bufferedsolution,metabolicchangesduringexercisecan cause slightvar iations in hydrogenionconcentration.Studieshaveshownthatlocal administrationofacidproduceschangessimilar to thosewhichoccurduring increased COztension. Infactsome studies indicatethattheactionof COz ismediated via thehydrogenion(Hiltonand Eichholtz,1925; McElroyeta/.,1958).Ithas thusbeen shown thatthehydrogenion is a vasoactiveagent, with highconcentrationscausing dilationand low concentrations causing constriction.
Theconcentrationof potassiumion intheblood canalso influencevascular lone. IIhaslong been known that a small increase in potassium inthe blood or in aperfusing solution causesvasodilation(Dawes, 1941).Itseems,however,that the effectofpotassiumonthetone ofvascularsmoothmuscledependsonits concentration.Konoldand co-workers(1968) founda vasodlletory responseup to a potassiumconcentrationof about 35mMbutat higherconcentrations a contractile
16 response was obtained. Vasoconstrictionwasalsoobserved upon decreasing potassiumconcentrationbelownormal (2.68mM).
The dilator response of conduitarteries10 anincrease inblood flowwasfir..t observedin 1933 (Schretzenmayr,A:quotedbyPohletaL,1986). Morerecently the vasodilatorresponse to increased flowhas been shownto be mediatedbythe endothelium(Pohletal.,1986; Rubanyietal,1986 ). BevanandJoyce(1988a) have shown in rabbit eararte ry thatincreasedflow can causecontractionof anintact segmentwithor withoutthe presenceofendothelium.Inthesamepreparation,they haveshown flowinduced vasodilationafteractivetonehad been inducedby noradrenaline(Bevanand Joyce,1988b).
Manyofthe factorsinvolvedin the controlofvasculartonedepend onor are influencedby the endothelium. Whilethis celllayerdoesact asabarrier between thebloodandvascular smoothmuscle,itis also anactive tissueinvolved inthesyn thesis and releaseof compoundswhich actto contractor dilatethe blood vessel.
17 1.2. The Endothelium
End otheli al cellsfonn a continuous layerasinglecellthick thatlinestheentire circulatorysystem. These cellsactnot onlyas abarrierbetweenthe blood andtile vascular wall,butas an activetissuereceiving a varietyof signals,both blood-borne and tissue-derived.Vasoactivecompoundsinteract withendothelialcells to cause the release ofproslacyclin(Crutchleyet aL,1983), platelet activatingfactor(McIntyre et aL,1985),endothelium-d erived vasodila tors(r e viewedbyFurchgott,1984) and endothelium-derivedvasoconstrictingfactors (reviewedbyRubanyi, 1988). Thus, the endothelium playsan important functional roleinthe controlofcardiovascular tone.
1.2.1.Receptors on the Endothelium
Avariety ofreceptorshave beencharacte rized onthe intimalenduthe lial surface of bloodvessels bythe use of pharmacological techniques and autoradiography.Itisthroughthe mediation ofthesereceptorsthat manyvasoactive agentsact.
Severalstudieshaveshownthatthepresence ofa functionalendothelium limits the vasoconstrictor responsetoadrenergic agonistssuchasnoradrenaline(Cocksand Angus,1983;Millerand Vanhoutte, 1985;Anguselal.,1986). These authors proposedthata-adrenergicagonistsstimulatetherelease ofan endothelium-de rived
18 relaxing(actor(EDRF)through an Clfreceptor locatedonthe endothelial cell surface.The vascularendotheliumhas also been showntohave p-adrenoceptors, stimulationof which leads to elevationof cyclic AMP(SchaferetaL,1980). These p-receptorsaTCofthe132subtype anddonotmediatethereleaseofEDRF when stimulated(MacDonaldaal,1987).
The releaseofEDRF from endothelialcells in response to ACh hasbeen shownto be mediatedby a muscarinic receptor on the endothelium (Furchgottand Zawadzki,1980a). Furchgottand Cherry(1984)couldfindno evidence to indicate that this muscarinicreceptor was of a novel subtypein their rabbit aortic ring preparations. On the basis of affinity {or pirenzepine,a selectivemuscarinic antagonistfor theM,subtype,the muscarinicreceptorof the endothelium which mediatesvasodilationhasbeen classified as Mz(Hyneset al , 1986,Chooel 01.,1986).
However,more recently it has been suggestedthattwosubtypesof muscarinic receptor may beresponsible for the releaseofdifferent typesof EDRF(Rubanyi etat.1987). Horst and colleagues have also suggestedthis possibility and have indicatedthat furtherstudieswith new and novel M2antagonistsmay clarifythe situation(Horste' 01.,1987).
Receptorsfor substanceP havealsobeen localizedonthe intimalsurfaceof bloodvessels. SubstanceP relaxedpreparationsof preconstrictedporcinecoronary arteryonlywhenanintact endotheliumwaspresent,thus suggestingthepresence of SPreceptors on the endothelialcells(Gulatielat.,1986).In dogcarotidartery,
19 autoradiographic techn iqueshavebeen used todemonstratethe presence of SP receptors on the endothelium (StephensonetaL, 1986).
Theaction of bradykinin in canine and human arteries hasbeenshownto dependon the presenceofintact endothelium(Altura and Chand,1981;Cherryet
01"1982). These studies suggestthat the vasodilatory effectof bradykinininthese
vesselbeds istheresult ofaninteractionwithsome receptivesubstance ofthe endotheliumto elicit thereleaseofEDRF.
Histaminerecep torshavebeen studied ina varietyof blood vessel preparations. In ratthoracicaorta, H(receptorswerefoundtobe responsible for mediatingthe releaseofEDRF in responsetohistamine(VandeVoorde and Leusen,1983).RadioJigandbindingstudiesindicateadifferentialreceptor population betweenarteriesandveins.Inguinea-pigaorta,HI-receptorswerefound to bemore concentratedonthe endotheliumthan on thesmooth muscle (Hideet al; 1988).
ThepopulationofH2:-receptorswas found to be ofhigherdensityon theendothelium invenules ofmouse diaphragmthan inarteriolesorcapillaries(Antoheetat,1986).
StudieswithATP haveindicatedthaisubtypesof receptorsforpurines exist on the vascularendothelium. TheP2"receptorwhich respondsto the purine nucleotides, ATPand ADP,bas been differentiatedinto2 subtypesonthe basis of affinity forthe antagonists,reactive blue2and a,,B-methylene-ATP (Houstoneta1., 1987).Theseexperiments indicatethat it isthe P2Ysubtypewhichislocated on the endothelium and is responsible forthe releaseof EDRF elicitedbyATPand ADP.
20 Similarly,HopwoodandBurnstock(1987)have foundcoronaryvasodilationto be mediatedbytheP2y·purinoceptorsubclass.
Althoughsome agents act directlyon thevascularsmoothmuscle to elicittheir response, it is evidentthat receptors on the intimalsurfaceof the endotheliumplay an activeroleinmediatingvascularreactivity to a wide variety of vasoactive substances.
1.22. Endothelium-DerivedRelaxingFactor
Itwas first discoveredin 1980 thatan intactendotheliumwasrequired (or therelaxation of isolated arteriesto occur inresponse 10 ACh (Furchgott and Zawadzki, 19808,1980b; Furchgott etal,1981). This finding explainedwhy ACh, a potentvasodilator of arteriesinvivo.often produced norelaxation or even contractionofisolated preparationsof arteriesin vitro(Furchgcn,1981;Furchgott, 1982).In thosetissues whichfailed to respondto ACh, theendotheliumwas often damagedor strippedfromthe vesselwallduring preparation. Intheiroriginalreport of thesefindings,Furchgott and Zawadzki(198Oa) proposedthai ACh was acting on a muscarinicreceptoron the endothelium10 stimulateIhe release of one ormore factorswhich caused relaxation of vascularsmooth muscle.Theterm endothelium- derived relaxing faclor(s) (EDRF)was coinedfor this substance(s)(CherryetaL, 1982).
21 (a) Characterizationand IdentificationofEDRF
These original findingsbyFurchgott and Zawadzkiprompted many investigationsintothe identity and mode of actionof EDRF. Earlystudies demonstratedthelack ofeffect of cyclooxygenase inhibitors on therelaxingaction ofAChindicatingthat EDRFwas nota prostaglandin(FurchgottandZawadzki, 198Oa). EDRF isalabile{actor with measuredhalflivesof 6 to 49seconds (Griffith et aL, 1984;Ferstermann et 01.,1984;Rubanyiet 01.,1985; Angusand Cocks, 1987).
Like thenitrovasodilators,suchas nitroglycerin andsodiumnitroprusside (SNP), EDRFhasbeenshownto causevasodilationbystimulation of the solubleguanylate cyclase ofvascularsmoothmuscle, resultinginelevatedcyclicGMP levels (Fcrstermannetal.,1986; Holtzmann,1982;Ignarroet 01.,1984;Rapoportand Murad,1983a). CyclicGMPthen mediatesrelaxation, probablybymultipleactions onthe controlofintracellularfree calcium(Collinselal., 1%6).EDRFand the nitrovasodilatorsalso inhibit plateletaggregation (Ayumael aL,1986),presumably via a similarcyclicGMP-mt:diatedaction.
In the searchfor theidentity of EDRF,severalagents and conditionswhich inhibitendothelium-dependentrelaxations werefound. It was discoveredthat the actionsof EDRFon vascularsmoothmuscle arepotentiatedbysuperoxidedismutase and cytochromeC(Gryglewskiet01.•19800;Rubanyiand Vanhoutte,1986;Moncada etaL,1986) andare inhibitedbyFeZ" (Gryglewskiet al, 1986b),hydroquinone
22 (Griffithet aL,1984; Moncadattat..J986) and by pyrogallol (Moncadatlat, 1986). This ledtothescgges-ionthat these agentsinactivateEDRF via the generation of superoxidcanions (MoncadaetaL,1986).Haemoglobin also inhibits theactionsof EDRF(GriffithdoL,1984;MartintlQL, 1985)probablythrougha differentmechanismof action involving the bindingofthe molecule (Cocks and Angus.,1985; Martinet aL,1986).
Finally,as theactionsof EDRF were found tobe quite similar tothose of the nitrovasodilators,itwas suggestedbyFurchgott(1988)thatEDRFmight f>imply benitricoxide(NO). Nitric oxideisthoughtto bethe active metabolite thro uge which nitrovasodilatcrs stimulatesolubleguanylatecyclase.Evidence hasbeengiven byPalmer,Ferr ige, and Moncada(1987)to show that NOreleased fromendothelial cellsisindistinguishablefromEDRFinterms of biologicalaClivityandstability, as well assusceptibility to inhibitionbyhaemoglobin and enhancementbysuperoxide de mutase.Since thisfirstreport.furthercontirmationthat EDRFisNO hasbeen providedbystudies of the comparativepharmacologyofthetwoagents{Hutchinson etal,1987;Radomskiaat.,1987}. Mechanisms other thantherelease of NOmay also play arole inendothelium-dependent relaxation. Mechanismshavebeen suggestedincludingmediators suchas ammonia{~~H3:Vanhoutte,1987aj,or the involvement ofion channels which mayregulateEDRF release orpropagate endothelialsignalsvia gapjunctions (Olesenelal,1988;Lansman etal,1987).
Anendothelium-dependenttransienthyperpolarization of vascularsmooth mwcle
23 cells,whichmaybeindependentof NO,basalso beensuggested (Vanhoutte, 1987a). These observationsmust be fullyinvestigatedbeforeitisknownifnon-prostanoid endothelium-dependent mediators otherthan NO exist.
(b) AgentsthaiProd uce Endothelium-Dependent Relaxation
Although acetylcholinewasthefirstagent shownto elicit endothelium.
dependentvasodilation,a variety of agents,bethhumoralandsynthetic,have also been shownto cause therelease of EDRF.Someofthese agents also causethe releaseof prostaglandins{romendothelialcells.However,prostaglandinsdo not appear to have a significantroleas mediatorsofthese relaxingeffects. In addition, withsomeofthese agents,endothelium-dependentrelaxationof isolatedarterieshas beenlimited to certainspecies. Inscmecases itislimited to specific arteriesina particularspecies.
Acetylcholinewas first shownto causerelaxationswhich weredependenton the presence of intactendotheliumin isolatedringsofrabbit thoracic aorta using isometrictension measurements(Furcbgott, 1981;Furcbgouand Zawadzki,198Oa, 1980b;Furchgoltet01.,1981). In addition tothe actionsofACh on rabbitaorta, it wasfoundthat AChcausedendothelium-dependent relaxationsinisolated arteries from allmammalianspecies.These tissuesincluded:rabbit superior mesenteric.
pulmonary,and eararteries;rat thoracicaorta;guinea-pigthoracic aorta;cat thoracic
24 and abdominalaortae, superior mesenteric,pulmonary,and ex ternal iliacarteries;
and dogcircumflexandleft anteriordescending coronal)'arteries(Furchgott and Zawadzki,1980a).Otherinvestigatorshavefound relaxations toAChin caninerenal (Chandand Altura,1981) andfemoral arteries (DeMey andVanho ulte,1981), rat (DaviesandWilliams,1984)andpig aorta(Gordonand Martin.1983), as well as cat cerebralarteries(Lee, 1981) andbovinecoronaryarteries(Holtzmann,1982).
Studieson human blood vessels have shown that ACh causesrelaxations in the presenceof intactendotheliumin isolatedrenaland peripheralarterialrings(Luscher etal.,1987a) andinisolatedrenal, splenic;gastric,pulmonary,brachial,transverse cervical and coronaryarterialrings(ThemetaL,1985).Theendothe lium-de pe nde nt vasodilatory effectof AChis much lesspronouncedin isolatedvenous preparations (De Meyand Vanhoutte,1982).
Asynthetic compound,the calciumionophore A23187 is even more potent than AChinits endothelium-dependentvasodilatoryactions(Zawadzkia at,1980).
Againsthighlevelsof tone,the maximalrelaxation by A23187isalways greaterthan thatbyACh (Furchgottet al, 1983). An187 has been shownto cause endothelium- dependent relaxationsin rabbitthoracicaorta (Furchgou,1981;ZawadzkietaL,1980;
SingerandPeach,1982), ratthoracicaorta (Rapoport andMurad,1983b), pigaorta (Gordon and Martin,1983) and in a varietyof humanarteries(Thornetat,1985;
Thornet al.,1987a). It isinterestingto note that inthe presence of a maximally effectiveconcentrationofA23IS7,additionsofACh andother endothelium-
2S dependentvasodilators produceno relaxationof contractedarteries (Furchgontt at,1983).Thisbesbeenattributed to A23181fullyactivating the mechanism(or productionand release of EDRFinendothelial cells.
NoradrenalineandselectiveQl-8c"renoceptoragonists can cause relaxationof canine and porcinecoronaryand systemicarteriesandcaninepulmonaryarteries and veinsifthe endotheliumispresent(CocksanaAngus,1983;Miller and vanhoutre,1984). In the caninecoronary artery the relaxing effects of thep.
adrenergicagonists,noradrenaline andisoproterenol arereduced following removal ofthe endothelium(Rubanyiandvanhcune,1985a).Itisnot certainwhetherthis greatereffectisdueto anactionons-receptorsontheendotheliumorifrelaxation iscausedbythebasalreleaseofEDRF.The presence of Q2-inhibitoryreceptors on theendotheliumdoeshelp10explainwhythecontractile responseto nonselective c-adrenoceproragonistsisenhanceduponremoval of theendothelium.
ThepurinesATPandADP,whicharereleasedbyplatelets duringaggregation, also induceendothelium-dependentrelaxationsin arteries.Removal ofendolhelial cellswasshowntomarkedlyreduce thegradedconcentr:".tion-dependent relaxations to ATP orADPinisolated preparationsof rabbit aorta(Furchgcu, 1981; Furchgott etal.,1983;FurchgoltandZawadzki,198Oc),dogfemoral artery(DeMey and Vanhoutte,1981) and pig aorta (Gordonand Martin,1983). Vasodilationby adenosine and AMPin rabbitaorta anddogfemoral arterywereshown 10be independent of the presenceofendothelialcells (DeMeyandvanbouue,1981;
26 Furchgottel af.,1983;Furchgottand Zawadzki,198Oc). However,in pig aorta a significantpariofthe relaxationbyadenosineand AMPhas beenreported tobe endothelium-dependent(Gordon andMartin,1983). Relaxations to adenosine in caninecoronaryarteryarc alsomediatedbythe endothelium(Rubanyiand Vanhoutte,1985a).
SubstanceP is one ofthe most potent endothelium-dependentvasodilators studied(Zawadzki eraL,1981). SPhas been shown,usingan electromagnetic flowmeter technique,to increasebloodflowin the aorta,carotid,hepatic,superior mesenteric, and femoral arteries of the dog (Hallbergand Pemow, 1975). SubsequentlySPhas beenshownto giveendothelium-dependentrelaxation inisolated ringsofrabbit aorta anddog superior mesenteric arteries whichhad been preconstricted with noradrenaline (Furchgott et al. 1983). The threshold concentrationfor relaxation by SP was 10 pMin rabbit aorta and IpM in dog superior mesentericartery.In these experimentsadesensitization to SP at InM occurredwithin10minutes. This was overcomebywashoutof the preparationand did n01have any effect onrelaxation10 ACh or A23IS7.Endothelium-dependent vasodilationbySPhas alsobeenstudied in isolateddog (D'Orleans-Justeet al, 1985) and pig (Gelatie! aL,1987) coronary arteries. Inthe dog, SP relaxed isolated coronary arterial rings whichhad beenpreconstrictedwith noradrenaline(20nM) at a thresholdconcentrationof 65 pM.Inisolated ringsof pig coronaryartery;
which,like human coronaryartery,contractsto acetylcholine(50JIM), thethreshold
27 forendothelium-dependent relaxationto SPwas30 pM. Substance P has alsobeen shownto causevasodilation upon intravenousadministration in thepreconstricted pulmonaryvasculature of thedog (ArcheretaL,1986).Inhumans, SPhasbeen shownto Increase blood
n ow
intheskinandskeletalmuscleof thefore annupon close arterial infusion of 1·2ng/mimne (Eklundet al,1977).Experiments on isolated branches ofhumanmesenteric arteries haveshownthat relaxationsbySPare strictly dependentonthepresence ofendoth elial cells (Furchgoltet aI., 1983).Bradykininis alsoknownas apotent vasodilator. Bradykininhasbeenshown toproduceconcentration-dependent relaxation ofcanine intrapulmonaryandrenal arterieswhich is dependentonthe presenceof anintactendothelium (Alturaand Chand, 1981).Removal oftheendotheliumresulted in either a contractileresponse tobradykinin orno responseatall. In isolatedrings ofdog carotid arteries preconstrictedwithnoradrenaline (20nM),bradykininelicitsconcentration-dependent relaxations ata thresholdconcentrationof2n M(D'Orjeans-Juste eral., 1985).These relaxations occurredonlyinthe presence ofan intactendothelium. Thelackof inhibition ofthis vasodilatoryresponsetobradykininbycyclooxygenase and llpoxygenase inhibitorsindicated thatitwasnot mediatedbyprostaglandins.An extensivestudybyCherryand co-workers(1982)evaluated the actionsof bradykinin inisolated arterial ringsfrom cats, dogsand rabbits.In the superiormesentericand celiacarteriesfromrabbit and cat removal of endothelial cellsdidnotconsistently result in loss of therelaxations to bradykinin. Thisrelaxationwasblockedby
28 cyclooxygenaseinhibitorsandthusinthesevesselsthe actionof bradykininBppean;
tobemediatedbyprostaglandins. Incontrast,allarteries testedfromthedog:
splenic,gastric, celiac,femoral,renal,coronary.pulmonary,and superior mesenteric, showed a requirementfor endoth elialcells in the relaxation bybradykinin.
Cyclooxygenaseinhibitorsdidnotinterferewith the relaxationrespo nseand50
bradykininis thoughtto act via EDRFin bloodvessels of the dog.Bradykinin was alsostudiedon rings of mesentericartery fromhumans andfound10elicitrelaxations withsimilar mecha nismto thoseindog arteries.Inadd ition,Gordo nand Martin (1983) have reported that bradykinin inducesrelaxations ofpig aorta which are also endothelium-dependentandprostaglandin-independent.
Vasoactive intestinalpolypeptideisa potentvasodilatorwhichacts bydifferent mechanisms in a varietyofspecies. Inrat aorta VIPcausedrelaxationswhichwere endothelium-dependent(Daviesand Williams,1984). Ina studyofhuman blood vessels,VIPproducedendothelium-dependentrelaxationincoeliacbrancharteries, butillisolated pulmonaryarteriesits relaxing effectwasindependent of thepresence ofendothelial cells(ThornelaJ.,1987a). Anothergroup has alsoreported an endothelium-independentmechanismfor relaxation ofhuman pulmonaryarteriesby VIP (BarnesetaJ.,1986).VIP has also been showntorelaxhuman gastricand transversecervicalarteriesinanendothelium-dependent matter (ThometaJ.,1987a).
The vasodilationof bovinepulmonaryartery andcaninecoronaryarteryhasalsobeen reported to beindependent of the presenceof endothelialcells(GriffitheraJ.,1985).
29 Calcitonin gene-relatedpeptideisanotherpeptidewhichhas vasodilator activityin man andothermammals. In astrip preparation of ratthoracicaorta CORP gavedose-dependentrelaxationsof noradrenaline-inducedconewhichwere depend ent onthepresence of endothelialcells(Brain~taL,1985). Thisrelaxation was partiallysuppressedbycyclooxygenaseinhib!10nandso prostaglandinsmaybe responsiblefor someof theresponseto CGRP. In this study CGRPalsocaused vasodilationofthe cutaneousmicrovascula ture of rabbitand man invitro.CGRP was1000timesmorepotent than ACh,ATP,ADP,adenosine,S-HT andSP inthis action.Anotherstudyhas confirmedendothelium-dependentrelaxations to CGRP inringsof ratthoracic aorta(KubotattaL.1985).RecentlyCGRP has been shown to relaxpreconstrictedsegmentsofhuman radial,coronary.gastric and cerebral arteriesinanendothelium-dependent manner(ThornttaL,1987b).Cyclooxygenase inhibitors didnotblock the responses to CGRPandthus EDRFisthought10 be the mediatorof theserelaxations.
Histamineisavasodilatorwhichhasbeenshownto actviaEDRFin certain isolated bloodvesselpreparations. Itwas fint demonstratedto cause a dose- dependentrelaxation inringsof ratthoracicaorta, precontractedwithnoradren- aline(Vande VoordeandLeusen, 1983).Thisrelaxationtohistamine (10-1000IJM) wasdependent on thepresence of anintactendothelium andwas notmediatedby prostaglandins.The use ofspecific histamine receptor antagonistsIndicatedthat histamine wasactingon anH,receptor of the endothelium.Rapoport andMurad
30 (1983) havereported similar results for histamine in rataorta. Histamine also producesendothclium-dependentrelaxationof guinea-pigpulmonaryartery (Salah andInui, 1984). This responseisthoughtto involve theH, receptoronthe endotheliumin thistissue as well.Itis alsoof interest tonotethat histamine causes the releaseof EDRFfromhuman umbilical bloodvesselsvia an H,receptor subtype (Vande VoordeetaL,1987).This is atissue whichdoes notrelease EDRF in response toACb.
Thrombin, whichis generatedduringthecascade reactions of coagulation, causes endothelium-dependent relaxation in basilar,coronary, femoral, saphenous, splenic,andpulmonaryarteriesof the dog (De Mey et aL,1982; De Mey and Vanh outte, 1982;Katuslcel aL,1984) and inaortaof therat (Rapoportet 01.,1984).
Relaxation was not inhibitedby inhibitorsof cycJooxygenase and prostacyclin synthetase,butwas inhibited by heparin. Similarfindings have been madeon thrombin-Induced endct hellum-dependentrelaxation of isolated canine coronal')' arteries (Ku, 1982), In these arteries,the relar.ation to each additionofthrombin was transient,andwas counteracted by a directcontract ileactiononthe smooth muscle.
Platelet activatingfactor(PAF) is releasedduring platelet aggregation and has been shown to cause vasodilation ina numberof animalmodels (Vargaftigand Benveniste,1983). Atveryhighconcentrations,PAPinducesendot helium-dependent
31 relaxations in isolatedcaninecoronaryand femoral arteriesandrat aortae (Kamitani etal.,1984;KasuyaetaL, 1984).
Serotoninis also released duringplateletaggregation.Precontractedcanine coronary arteries have been foundtorelaxto serotoninonlyif endothelialcellsare present (Cohenet al.,1983).In theabsenceofendothelium, serotonin produces onlya contractionin the caninecoronaryarterial rings. Endothelium-dependent relaxationbyserotoninhas norbeenreported for anyotherarteries.
Hydralazineis an antihypertensivedrug whichproducesits relaxant effecton isolated ringsofrabbitaorta mainly throughanendothelial cell-dependentmechanism (Spokaset01., 1983). This effectoccursat relativelylow concentrationsof O.1-1I£M.
Thisis ofinterest because most other antihypertensivedrugs; nitroglycerin, nitroprusside,minoxidilanddtazoxtde, are thought to actdirectly onthe vascular smooth muscle,independent of thepresenceof endothelialcells.
Endothelium-derivedrelaxing factor is alsoreleasedfrom arteriesin response to incrementsinflow (Holtzetat.,1984a). Effluentfrom perfused rabbitaorta (Griffithetat.,1984), or caninefemoralarterypreparations withendothelium (Rubanyietal.,1986), inresponse to increasedflow,caused relaxationsof bioassay coronaryarterieswithoutendothelium.Incrementsin flow througharteriesin\'ivo (Gerovaetal., 1983;Hintz andVatner,1983;HoltzetaI.,1984a; Kaiseretal ,1985;
Rubanyiet al,1986)orin vitro (Holtzeral, 1984b)inducevasodilation, whichcannot be preventedbycyclooxygenaseinhibitors (Holtzetal, 1984a),but can be abolished
32 byremovalofthe endothelium(HoltzetaL,1984b)ordepressedbymethylene blue (KaisereraL,1985). Thus, theEDRFreleasedbylarge arteries inresponseto flow has characteristicsvery similarto that released by ACh.
It isevident thata widerange of agentscause the releaseof a relaxing substancefrom the endothelium.Some ofthese agents arc found in perivascular nerve fibresand some are endogenousto the circulationunder variousconditions, while some compounds which elicitthe release of EDRF are synthetic. Itseems that even theflowrate of blood throughthe vasculatureisinvolvedin the control of smooth muscle tone through the mediation of the endothelium. Whether all
these agents releasenitricoxide or othertypes of a non-prostanoidEDRFisnot known.However,basedon common susceptibilities to inhibitoryagentsitislikely that manyof theseagents actto releasethe samerelaxing factorthatAChproduces (Furchgotlelal.,1983; Furchgou,1984).
1.2.3.Endothelium-DerivedCcntraetingFactor
In additionto the vasodilator substances whichare released bythe endothelium,evidencehas beengiven for the release of one or more vasacontractile factors fromvascular endothelialcells,Itwasfirstreponedin1982 that an intact endotheliumwas necessaryfarcontractionof canine systemic and pulmonaryveins inresponsetoarachidonicacid (De Meyand Vanhoutte,1982). Vasoconstriction,
33 dependenton or enhancedby anintactendothelium.has beenobserved in response
to variouschemicaland physical stimuli (reviewedby Vanhoutte,1987b;Vanhoutte and Katusic,1988).
(a) Characterization and Identificationof EDCF
Partoftheevidencesupportingthe release otvasoconstrictcr substances from the endothelium comesfromexperiments in which anoxia produced endothelium- dependentcontractionsof peripheral,coronary,and cerebralarteries(Rubanyi and Vanhoutte,1985b;De Mey and Vanhoutte,1983;Katusic and veuhoutte ,1986).
These responseswere notsensitive toinhibition of cyclooxygenase and thuswere attributedtoan unidentifiedvasoconstrictorsubstancetermed EDCF1(Vanhoutte 1987b).Ithasalsobeen demonstratedthatculturedendothelialcellsproduce a vasoconstrictorpeptide(HickeyttaL,1985; Gillespieet al; 1986), which hasrecently beencharacterizedand namedendothelin (Yanagisawaet aL,1988). Endothelinhas, uponfurther study,been showntoconsist of a group of three structurally distinct isopeptideswhich have different pharmacologicalprofiles(Inoueet al.,1989).Since theproductionof endothelinand itscontractile effect donot depend on cycle- oxygenasc activity, it hasbeen proposedthat EDCF, maybeendothelin.Thereis, however,evidenceto indicate that endothelin isunlikely tobethe mediatorof the so-canedhypoxicresponseof vascular smooth muscle(Vanhoutteand Katusic,1988).
34 Anotherseries of experiments has led to thediscovery of endothel ium- dependent contractions thai are sensitivetoinhibitorsof cyclooxygenasc. These studiesbegan withthefind ingsofDeMeyandve nhouue(1982) that arac hidonic addcaus edanendothelium-dependentaugmentation of noradrenaline-induced contractioninsystem and pulmonary veinsofthedog. Furtherstudiesrevealed endothelium-dependentcontractionsin canine veins andcerebralarteriesandinthe aortaofspontaneouslyhypertensive rats to avariety of stimuli,includingarachidonic acid,acetylcholine,A23187,serotoninandsudden stretch (MillerandVanhoutte, 19853;Luscher and Vanbouue,1986a;LuscherandVanhounc,1986b;Katusicet 01.,1987;KatusicitaL,1988). The mediatorof theseendothelium-dependent contractions wassuggested10bean unidentified productof thecyclooxygenase pathway, possiblethromboxane
Az.
andwastentativelyreferred10 asEDCF2 (Vanhouuc, l987b).Vanhoutte and Katusichavenotedtha tsupercddeanicncauses contractionsin canine basilarartery andthat superoxide dismutase plus catalase abolishenJothelium-dependent contractions toA23181inthesame preparation (Vanhoutte andKatusic,1988). On the basisor this and otherstudieswith cyclooxygenase(KontosetaL,1985;Rosenblum,1982)it has beensuggestedthat EDCFzis thesuperoxideanion (vanhouue and Katuslc,1988).Thus,itcanbeseenthatthe endotheliumcanberesponsiblefor mediating both vasodilationIhroughtherelease ornitricoxide andpossibleotherdilator substances. and vasoeonstrictcnthrough therelease or endothelinpeptides,
lS thromboxane
Az.
superoxideanion andpossibleadditionalvasoconstrictorsubstances.Thephysiologicalsignificanceof theseprocessesisbeginningtobeunderstoodand theendotheliumisnowrecognized as animponantccmr ibutortothecontrolof vascularton e.
Thisreviewhas attemptedtoencompassthewide number ofcomponents involvedin thecontrolof thecardiovascularsystem. From thedirectcontrolof vascular and cardiacmuscleprovidedby theautonomic nervoussystem,10the influenceoffactors in thecirculation,therate of flowofblooddepe ndson the cnd result ofa balancebetweenvascular contractionand relaxation. Although much progresshasbeenmadetoward anunderstandingof themechanisms involved inthis control,muchremainstobestudied with respecttocardiovascular controlindisease stalessuchas atherosclerosis.diabetes andhypcn ension.
36 1.3. Objectivesof the StudyandReviewof Methods
The objectiveofthisstudy was to tes t thehypothesisthatthe abilityof vascularendo theliumtoreleaseEDRF inrespo nse10stimulationbyvasoactive agents isrelatedinsomewayto thepatternof perivascularinnervatio n.From thiswor k it is hoped to determ inewha tinfluence,ifany, thepe rivascular innervationofa vascularresistancebed hasuponthe abilityof endothelialcellsto cause relaxation of medial smooth muscle. Indiseasestatessuchashypertension and diabetes, changesoccurinthe structureand fun ctionof thevasculature(ScottandPang,1983;
Bro dyelal.,1980; Gabbay, 1911; McMillan, 1975). Information aboutthe mecha nisms involved in the control ofthe vasculatu remay be utilizedinthe developmentof pharmacologi calagt.-ttsusedto treatcardiovasculardisease.
1.3.1.TheMcGregor-Prepara tion
Thetissuechosenforthisstudy wastheMcGregorprepar-ati onof theisolat ed perfusedmesentericarterialbedoftherat(McGregor,1965). There areseve r-al reasons for this choice.First,an isolatedtissue was chosensothat card iovascular reflex mechanismsdid not con tributeto theme asuredresult.Somewhole animal preparationshavebee nusedtoinvestigate the effectsof vasoactive agentsonblood flowofvascularbeds, buttheseresultscanbecomplicatedbyregulatorymechanisms
31 insitu(Hallbergand Pemow,1975;SmieskoetQL,1985 ;Anguset aL,1983;Archer el al.,1986). Second,an in tactarte rialresistancebedwasstudiedinorderto simu latethein vivosituationinthe determinationof the effectofth e vasoactive agents used.Whilethere are problemsinherentinthesettingupand maintaining of anisolatedvascularbed,thistissu eisacomparati velysimplepreparationof perfused mesenteric blood ve ssels. The majority'Ifthe stud iesof endothelium- dependent rel axationhave beencarriedout onisolate dring orstrippreparati ons oflar ge cond uitarte ries(Fur chgouandZawadzki,19808; reviewedbyFurchgott, 1984). Allhougnthesepreparationsareeasily setupandrespondwell to vasoactive agents,the entiretissueisbat hedintheadministereddrugsolutions. In theisolated perf used mesenteric arterialbed, onlytheintimalsurface of the bloodvesselis exposedto administe redagen ts.tnaddition,the roleofthe Conduitarteryin the contribution toblood pressure is thoughttobelessimportanttha nth£ltofthesmaller resist ance vessels ofthevascularbeds(Prewittet aL,1987;Longhurstelal.,1986).
Ring andhelicalstripprepar a tions ofresistancebloodvessels.includi ngsupe rior mese nteric artery, havealso been studied(Toda,1974; FurcbgouandZawadzki.
198Oa; reviewedbyFurchgott,1984).However, thesmallerdiame terof thesevessels makesildifficultto avoidendo thelialdamageduringpre paration. These tissuesare also completelybathedintheagent beingtested.
Thechoice of the mes enteric bed oftheratwas basedonthebodyof anatomicaland histologicalin forma tio nalrea dyavailablein this labora tory(SCali,
38 RobinsonandFoote,1986, 1989). Thereisalsoextensive information in the titcrn.tureaboutnutrition,endocrinology, meta bolis m,andphysiologyoftheratwhich, alon gwithits size,makeit a usefulanimalforc:ardiovascularre search(reviewedby GilletaL,1989~
TheMcGregorprepara tionof the isola tedperfusedmesentericancrialbed is,the refore,asuitable tissuein which to stud- 'beaction of vasoactive agentson theendotheliu m inavascularresistance bed. However, inanisolatedvascularbed suchas this,therear e many compone nts whichreactto vasoa ctivesu bs tances 10 cont ributetothe overallmeasuredchangesinperfusionpress ure. Itispossible thatinpharmarologicalexperi ments withIhistissue thevariatio ninmeasurements maybehigh.
1.3.2. lmmunohinochemistry
TheperivasculariMc:rva lionof themesentericarterieswasvisua lizedusing immunohistochemicalstaining10verify thepresence orabsence of nervefib res containingSP ornt The methodofimmunohistochem icalstaini ngwasathreestep peroxidase ann-peroxidase(P AP) pro cedurewhichutilizesdia minoben zidine to producea coloredreac tionpro ductwhich canbe viewe datalaterdate with light micro scopy.Thismethod hasbeen rout inelyusedinthis laboratory(Scott, Robinson, and Foote,1987) and is quitespecificfortheneuropeptidesbeingvisualized.
39 Although primary and linking antibodies arereadilyavailable forthismethod of staining. these materials can be expensive. II was alsopossible to do immunoh istochemical staining using primary antibodies which arelinked to a fluorescent marker. Thismethod is alsospecflcfor the neuropeptid esbutismore expensive and the tissues must be viewedimmediately asthefluor escence may fade.
1.3.3.ElectronMicroscopy
Transmission electronmicroscopywas utilizedto checkthe integrityofthe endothe lial lining of the blood vessels of the tissue preparations. This typeof electronmicroscopy allowedus to visualizea cross sectionof eventhe smallestof the jejuna larteries.Whileeach ]pm section is only a very smallsample ofthe vessel bed,a large number of samples were taken randomly. Inthis wayit was possibleto determineifendothelial cellswerebeing removed fromany...essels in the preparation. There are other methodsfor viewingtheendothelialsurfaceof bloodvessels. Scanning electronmicroscopy can alsobe usedto check the endothelium.Aswell,a methodofell facesil...er stainingallowsvisualizationof the endotheliumby lightmicroscopy (Pooleet aJ.,1958). Neitherofthe se methodsis suitablefor the smalljejunalvesselsof the mesentericbedas thevessels mustbe cutlengthwiscto visualize theintimal surface.
40 1.3 .4.Methodsof ChemicalDenerva uoa
6-Hy<hmydopanilile
Developmentof thesympatheticnervo ussystem wasprevented inagroup of ratsforthis studybyneona talad ministrat ionof&-hydroxydopamine(6-0HDA~
an isomerofnorad renaline . This treatmen twasuse d topro vi deanexperimental modelofblood vesselswithanalte r e dsympa thetic nervefibre plexus.
In 1959,while stu dying the enzymaticconversions ofdopamine to nor a drenaline inra t tissuehomogenates,Senoh andco-workers dis covered and isola tedthemetabo lite,6-0 HDA The initialbiologicaleffec tsof6-0 HDA were reported shortlythereafte r(Porte ret01.,1963;Porteretat.,19 6 5;Ston eelaL,1963;
Stoneet0/.,1964). These esperimeoudemonstratedthat 6-0HD A produ ced dep letionof noradre naline in theheartsofmice and dogs and that thiseffectla sted longe rthanthat causedbyotherage nts.Itwas alsofound that certainother agents couldantago nizetheeffect of6-0HD A,indicatingthat uptakeintothencradre ncr gic neu ronwasnecessary forthisactionof(}.OHDA. Nextcame thedisco verythat6- OH DAcaus edanactualdestruction oftheterminal endingsofthesympath etic neurons(Tra nzeran dThcen en ,1967, quotedbyKostrzewa an d Jacobowitz.1974).
Thisresult was later definedas"chemicalsympathect omy" (TranaerandThoe nen, 1968) . Sincethese origina lfindings,avastamou ntofinformation has been
41 accumulated on 6-0HDA and its pharmacology. For detailed reviews thereader is referred to Thoenenand Tranzer (1973)35wellas Kostrzewa andJacobowitz (1974).
The sequenceofeventswhichlead 10 the destructionof noradrenergicfibres after administration of 6-0HDA hasbeenwell described, beginning withthe active uptakeinto sympathetic neurons.6-0HDA is actively transportedto inteaneuronal siteswhere it acts as a falsetransmitter,displacing noradrenaline(Thoenenand Tranter,1968). When criticalinteaneuronalconcentrations of &.OHDA or metabolitesare attained,destructiveprocesses begin and cellularenzymes and elementsof therespiratoryelectron transport chainare destroyed.It is atthis point tha t thesympathetic nerve terminals lose their abilityto conductaction potentials but maystillhavea relativelyintactmonoamineuptakemechanism (Haeusler,1971). Because of the internal destruction,noradrenalineis releasedinto the synapse, resulting in a host ofsympathomimetic effects(StoneetaL,1%4). After a period oftime,thenerveterminalsmaybecompletelydestroyedand there willbea marked reduction of noradrenaline,tyrosinehydroxylase activity, and monoamineuptake capacityin these tissues(IversenandUretsky,1970).
The action of 6-0HDA is selectivefor terminalfibresof noradrenergic sympatheticnerves.Blecucn microscopic studies haveindicatedthat cholinergicand noradrenergic neurons,myelinatedaxons, smooth musclecells,Schwann cellsand endothelialcellsappear normal while noradrenergicneurons are in theprocess of
42 degeneration(BloomttaL,1969;Tranzerand Thoeeen,1967,1968).However,it wasdiscoveredthatafteraperiodofweekstheadrenergicterminalplexusappeared to rege nerate intissues inwhich theNAcontent hadbeenmarkedly reducedby6- OHDA treatment (Thoenenand Tranze r, 1968). Thus, while destruct ionof noradrenergicterminalshad beenachieved. the perikaryaappearedintactto bring
abouttheregenerationoffibres. Thisre versibility was foundtoberelated 10 the age ofthe animalsat the timeof6-0HDAtreatment. When6-0HDA is administeredtonewborn animals its neuro toxiceffects arc accentuated,Angeletti andLevi-Montalcini(1970) found thatwhe n~HDAwasgivento newborn mice or ratsitcausedextensive lesions, resultinginthedestruction ofthesympathetic perikaryainthe superiorcervical, stellate,celiac,mesenteric gangliaand allofthe thoracic paravertebralchain.Thus thedistinctionwasmadebetweenthe"reversible chemical sympathectomy"inadult animals and the "irreversible chemical sympathectomy"in newbornanimalsaftertreatmentwith6-OHDA(Thoenen, 1971).
Wlu1e6-0HDA hasproven to be avaluablepharmacologicaltool,therehas been somecontroversyastoitsusefulnessincardiovascularresearch.Somestudies indicate that6-0HDA doesnotproduceacomplete sympathectomyof the vasculature(Berkowitzn al.,1972;Finchet al.,1973a,1973b;Kurnjeket al., 1984).
However,the degreeofchemicalsympathectomyattained dependsupon a variety offactors. While theage at whichtreatmentisinitiatedisclearly important,the doseof 6-OHDA,its routeand frequencyofadministration,aswellasthe tissue
43
being exam inedare allimportant factorswhich contribu te to theeffectiveness of6- OHDAtreatme nt.
Capsaicin
Capsaicintreatmentwas used inthis studyto destroy peptidergic peripheral innervationcontainingthe peptidesSPand CGRP.Thistreatmentproduceda group ofratswithan alteredpeptidergicnerve fibreplexus.
Capsaicinis thetrivialname for8-methyl·N-vanillyl~nonenamide.themajor pungent componentof hot peppersof thegenusCapsicum.Hot peppershave been used asfoodeddltives and preservatives,as medicines and in social ritua ls for centuries. In the late 1940's an extensive characterization of the pharmacologica l effectsof capsaicin and its congeners on sensory processes in mammalswas undertakenbyJancso andcolleaguesin Hungary.Thesestudiesrevealedthat most of the biologicaleffectsof capsaicinresult froman initialintenseexcitationof certain sensoryneurons, followed byaprolongedperiod of insensitivity to physicochemical stimuli. Forreviews of this earlyresearch on capsaicinthe readerisreferr ed to Jancso (1968),VirusandGebhart (1979)and Szolcsanyi(1982).
The observation that capsaicindep letes fluoride-resistantacidphosphatase activity, known tobeassociated withce ntral terminalsof some primary afferent neurons from the dorsalhorn of the spinalcord, suggestedthatcapsaicinacts on
44 the biochemi calprocessesof thesesensoryneurons(Jancsc and Knyihar,1975).This had beensuggested earlierbyGasparovicandco-workers(1964)whodiscoveredthat capsaicin treatmentreducedthe amountofbioassayableSPinthespinalcord but not inthebrain.Furtherconfirmation of thespecificityofthe effects ofcapsa icin for SP-containingprimaryafferent neuronscamefrom the results of Jessel andco- workers(1978). They observed thatsystemic treatmentof adult ratswitha cumulativedoseof capsaicindepletedSPfro mthe dorsalhorn of thespinalcord without a ge neraleffectonsplnalneuronsand withoutdestructionofprimaryaffe rent neuronterm inals in the cord. Nume rous studies have confirmed by immunohist ochemical or radioimmunoassaytechniques,the SP depleting actionsof capsaicinin laboratory animals.Inparticular , astudybyFurnessand co-work ers (1982)found thatcapsaicinmarkedly deplete dSPfrom nearlyevery vascularbed ofthe guinea -pig. The exceptionto thiswas a groupofSP-conlainingfibreson arter iestothedistalcolonandrectum.These investigatorsalsoconfirmed the depletion of SP in the ureter ,atrium andsupe rior mesenteric artery ofcapsaicin- treated anima ls(Murphyt taL,1982).Theseresultsindicatethattheneurochemical effectsof capsaicin arespecific forprimal}'afferentneurons.
Since theoriginalresearchonthe denervatlon of SP-containingsensorynerves withcapsaicin,furtherstudies using anatomical, histochemical andimmunological methodshaverevea led thepresence ofa variety of peptidesin afferentneurons (reviewedby Holzer.1988). Functionalevide nce indicatesa possible mediatoror
