PharmacologyoftheAutonomicNervousSystem:

UtilizingtheIsolatedIleumModelSystem
I.Introduction
Mammaliansmoothmuscletypicallyoccursassheetsofcontractilefiberssurroundingtheintestinal
tract,uterus,spleenandbloodvessels.Itisbothanatomicallyandphysiologicallydifferentfromskeletal
muscle.Thefibersareshort,narrow,spindleshapedcellswithlittlesarcoplasmicreticulum.Thehighly
orderedstructureofskeletalmuscleisabsent,andthecontractilepropertiesreflectthedifferencesin
structure.Smoothmusclecontractionisslow,sustained,andspontaneous.Ifsmoothmuscletissueis
stretched,itwillfirstrelaxtothisnewlength,andthenbegintoresumespontaneousactivity.Therefore,
themusclecanworkoverawiderangeoflengths,adefiniteadvantageforaholloworgan(e.g.,intestine,
uterusorbloodvessel)subjecttodistentionduetofilling.
Smoothmusclecontractionsoccurintheabsenceofanyinnervation(unlikeskeletalmuscle
contractions).However,smoothmuscleactivityismediatedbythetwodivisionsoftheautonomicnervous
system,sympatheticandparasympathetic.Thesympatheticinnervationisinhibitory,whilethe
parasympatheticisexcitatory.Intheintestinalsmoothmusclepreparationutilizedintheseexperiments,
theparasympatheticgangliaandthepostsynapticreceptorsarecontainedinthetissue.However,the
sympatheticgangliaareabsent.Whyisthis?
Reviewthestructureandpharmacologyoftheautonomicnervoussystemtofamiliarizeyourselfwith
thesedetails.
II.SummaryofExperimentalProcedures
Youwillisolateasegmentofratileumandsuspenditinanisotonicbufferedsalinesolution
(Tyrode's).Oneendofthemuscleisfixedwhiletheotherendisattachedtoaforcedisplacement
transducerforrecordingmusclecontractions.Youwillusethispreparationtodemonstrateandexamine:
1.TheprinciplesofabioassayandDoseResponsecurves;
2.Theeffectsofsympatheticandparasympatheticagonistsandantagonists;
3.Theuseofthispreparationinordertodeterminethesiteofactionofanunknownagent.
AllofthedatawillberecordedondiskusingtheWINDAQ/200programandthenanalyzedusing
theWINDAQplaybackfeature(seethesectionsinthismanualontheuseoftheseprograms).
III.METHODS
DataRecording:Whileonemembersofthegrouppreparesthetissue,anothermembercansetupthe
TransbridgewithaFORT10,forcedisplacementtransducerandreadytheWINDAQprogramfor
recordingononechannel.Sinceyouwillbeexaminingcontractionsthatinvokeanupwarddeflectionof
thecomputerscreentrace,setthebaseline(0tension)nearthebottomofthescreen.Theresultingchanges
inmuscletensionareslowtooccurbutlastupto4or5minutes.Selectaslowsamplingrate,e.g.,1050/
sec.
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Donottweak,flick,orbendthetransducer!!!Itisafragileinstrument!
DissectionTechniques:YourTAwillmercifullydispatchtheratwithCO2narcosis.Aftertheanimal
stopsbreathing,makeanabdominalmidlineincisiontoexposetheviscera.Takecaretoavoidcuttingany
visceralorgans.Exposethevisceraandcutthediaphragmatthetopoftheabdominalcavity.
Noteanyspontaneousmovementsoftheintestineinsitu.Carefullyliftouttheintestinewithout
stretchingitandlayitinaPetridishcontainingTyrode'ssolutionwarmedtoroomtemperature.Locatea
segmentofthesmallintestineasclosetotheileocecaljunctionaspossibleandcutawaythemesentery
beginningatthispointandproceedingfor1015cmtowardthestomach.Handletheintestinegentlyand
keepitmoist.(Feelfreetoperformadissectionoftherat.YourTAwillpointoutthemajororgans.)
Severtheileumasclosetothecolonaspossibleandagainabout1215cmtowardthemouth.
PlacethisisolatedsectionintoaseparatePetridishcontainingfreshTyrode'ssolution.Flushandrinseout
thelumenbygentlyforcingTyrode'sthroughitwitha5mlsyringe.Placethisrinsedsectionofintestine
intoanotherPetridishcontainingfreshTyrode'ssolution.Donotletittouchorsitinfluidcontaining
theintestinalcontents.
Attachsurgicalthreadtooneendoftheileum.Oneendofthisthreadshouldbeleftabout1012
incheslong(seeFigs1&2).Besuretotiesquareknots;nogrannyknots,please!Asecondthreadshould
betiedabout2.03.0cmdowntheileumfromthefirstthread.Theendsofthissecondthreadshouldbe
formedintoasmallloopwithanothersquareknot(Figs.1&2).Carefullyisolatethissegmentofileumby
cuttingitoutsideoftheattachedligatures.Repeatthisprocedureontheremaininglengthofileumuntil
onesegmenthasbeenpreparedforeachlabgroup.Figure1illustratesaresultingsectionofileum.

LOOP

LIGATURES
(tied with
square knots )

6-8
LIGATURE

Ileum

2 - 3 cm

FIGURE 1
Attachoneendofyoursegmenttothehookinthebottomofthetissuebath(seeFig.2&3)using
thesmallloopinthethread.Attachthelongthreadattachedtotheotherendofthemuscletotheleverof
thetransducer.TietheileumFIRST!!!!Then,movethetransducerupinordertoplaceaverysmall
amountoftensionontheileumsegment.Watchthescreeninordertodeterminewhenyoustarttoplace
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tensiononthetransducer.Becarefulnottodamagethetransducer!!! FillthebathwithaTyrode's
solutionbyclampingthetubingatthebaseofthetissuebathandaddingTyrodestothetop.

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Checkthesetuptomakecertainthatthesutureisasverticalaspossibleandisnottouchingtheside
ofthetissuebathandthemusclehasaslighttensiononit.Verygently,startaeratingthesolutionusingthe
attachedairpumpandrecordthecontractionsusingtheWINDAQdataacquisitionprogram.Thesampling
rateshouldbesetatalowfrequency.Aftersuspendingthemusclebutbeforeaddinganydrugs,
calibratethetransducerwitha10gweight.Youwillprobablyalsoneedtoturnthegaindownonthe
transbridgeamplifier,probablytox100orevenx10inordertoobservefullcontractionoftheileum
segment.

IV.EXPERIMENTS
A.BioassayforAcetylcholine(ACh)
AChinteractswithpostsynapticcholinergicreceptorsontheileumtoinitiateandpotentiate
contractions.Rememberthatthemostperipheralreceptorsareaffectedfirst.(So,arethesenicotinicor
muscarinic?)ThemagnitudeoftheresultingcontractionisproportionaltothedoseofAChappliedwithin
arestrictedrangeofAChconcentrationswhichvariesfrommuscletomuscle.Youwilltakeadvantageof
thistoconstructaDoseResponsecurveandthenusethisstandardcurve(seeBsci105andBsci230lab
manuals!)todeterminethe[ACh]ofan"unknown"solution.

Use Word 6.0c or later to

view Macintosh picture.

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StockAChsolutionwillbeavailableataconcentrationof1x101M(100mM).Makeaserial
dilutionofthissolutionbyplacing0.5mlofthestockAChin4.5mlofTyrode'ssolution;thisisequivalent
toa10folddilutionofthestockAChthatyieldsa102MAChsolution.Takea0.5mlofthis102M
solutionandadd4.5mlofTyrodeandyouwillnowhavea103MsolutionofACh.Continuethisuntil
youhaveAChsolutionsrangingfrom102Mto107M.Notethatthesedilutionsprovidearangeover

whichyoucantesttheileum'sresponse.YoucanmakeotherAChdilutionswithinamorenarrowrange
onceyouhavedeterminedhowyourparticularileumresponds.Youwillwanttouseatleast8different
[Ach]inordertogetacompletepictureofyourdoseresponsecurve.Whereshouldthese8[Ach]liealong
thexaxis?Thatiswhichconcentrationswillprovidethemostinformationabouttheshapeofyourdose
responsecurve?
Doyouunderstandlogdosesanddilutions??? Tobesure,completethefollowingcalculationsbefore
goinganyfurther.RememberthatyoucanusetheformulaC1xV1=C2xV2,(C=concentration,V=
volume).Thetrickisindetermininghowtoassignthevariables.YourTAwillelaborate.
1.

Ifyoudilute1.0mlofa105MAChsolutionwith1.0mlofTyrode'sbuffer,the[ACh]now=
__________________________________.

2.

Afteradding0.2mlof104MAChto20mlofTyrode'sbuffer,the[ACh]now=
_____________________________________.

3.

Afteraddinganother0.2mlof104MAChtothesame20mlofTyrode'sbufferin#2above,the
[ACh]now= _____________________________________.

4.

Ifyouadd0.4mlofanAChstocksolutionto20mlofTyrode'sandfoundthatyounowhaveafinal
[ACh]of2x106MinTyrode's,whatwasthe[ACh]intheoriginalstocksolution?
____________________________________________.

But,wehaven'tyetconsideredthefactthatthedoseofAchgets"diluted"whenitisaddedtothevolumeof
liquidinthetissuebath.Forexampleifyouaddthefollowingaliquots,whatisthefinal[ACh]inthebath
iftheinitialbathvolumeis20ml?
0.2ml of
0.2ml of
0.2ml of
0.2ml of

106MACh givinga[ACh]bath=___________M
105MACh givinga[ACh]bath=___________M
104MACh givinga[ACh]bath=___________M
103MACh givinga[ACh]bath=___________M

Afterconstructingyourprotocol(stockdoses,volumestobeadded,andresultingbathconcentrations):
1.

Startrecordingthedatainthecomputerandquicklyaddthefirstsample(lowestdose)ofACh,
waitingapproximately1min.foraresponse.Waitapproximately1fullminbeforeassumingno
response.Onceyouwitnessacontraction,youhaveaveryroughestimateofthelogunitthreshold
doseofAChforthissectionofthisparticularileum.PlanyourprotocolofAChadditionsto
includeseveral(>8)dosesofAChthatarebetweenthresholdandmaximum.
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2.

Addthenextvolumeincrement,withoutflushingthebath.

3.

Onceacontractionisevoked,thetimingofyournextadditionbecomescritical.Assoonasthis
initialcontractionreachesamaximumandthetracebeginstoplateau,addthenextdose.Aseries
ofdosescanthenbeaddedtothemuscle,withoutflushingouttheorganbathbetweeneach
addition.Thismethodisbasedontherelativelyslowresponseandrecoverytimeofthesmooth
muscle.Bythetimethemusclehasreachedmaximalcontraction,theaddedAChhasessentially
equilibratedamongtheaccessibletissuecompartments.Atthispoint,themuscleisinastateof
contractionproportionaltotheconcentrationofexogenousACh.Therefore,ifthenextdoseof
AChisthenadded(attheplateaupointoftheproceedingdose),themusclewillcontracttoa
lengthproportionaltothetotaldosenowinthebath.HowcanyoudeterminetheTOTAL[ACh]
inthebathateachstep?(HINT:mustconsidertotalAMOUNTofAChaddedandthencalculate
thetotalvolumeofliquidinthebathateachstep.)
Inordertodeterminethefinal[ACh]inthetissuebath,youwillhavetoaddthedosesofACh(in
moles),beginningwiththemostdilutesolution,aswellasconsidertheincrementalincreasein
bathvolumewitheachadded0.2mldose.

4.

Nowgraphyourresultsonsemiloggraphpaper.SimplyplaceyourdosesofAChontheXaxis
(thelogarithmicaxis)andthecorrespondingmuscleresponses(ingramsoftension)ontheYaxis
(linearaxis).Whyisitoftennecessarytoplotthedoseonalogarithmicscale?

B.

*AChAntagonist*
1.

2.
antagonist.

C.

ObtainarepresentativeAChdoseresponse(D/R)curve.
Duplicatethecurveinthepresenceof107M(bathdose!)atropine,amuscarinic
PlotthecurvesobtainedinA&Bonthesameaxes.

3.

Predictwhatwillhappenifthedoseoftheantagonistwasincreasedordecreased.

4.

PredictwhatwillhappeniftheAChD/Rcurveweregeneratedinthepresenceof
hexamethonium.(AfterC,tryit!!!!)

InhibitionofAcetylcholinesterase(AChase)

AChesteraseisanenzymelocatedoncholinergicpostsynapticmembranes.AChesterase
hydrolyzesAChintocholineandacetatewhichdonotinteractwiththeAChreceptors.Demonstratethe
effectofanAChaseinhibitor,eserine(106107M),ontheileum'sresponsetoACh.Reportyourresults
asdoseresponsecurvesforA,B&Cplottedonthesameaxes.
D.

(WEEK2)DeterminingtheSiteofActionofanUnknownAgent

Youwillbegivenasolutioncontaininganunknownagent.Youmustusetheileumpreparationto
determineitssiteofaction.Theagentmaybeanyoneofthefollowing:
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muscarinicagonistorantagonist
nicotinicagonistorantagonist
alphaadrenergicagonistorantagonist
betaadrenergicagonistorantagonist
nonspecificmusclecontractionorrelaxingagent

Beforeyouarriveinlabforweek2,constructaflowchartofallpossibleresultsinducedbyanunknown
agent.Thus,thedrugmayelicitcontraction,relaxation,ornoresponse.Ifitinducescontractionofthe
ileum,whatarethepossibilities?Itcouldbeamuscarinicagonistoranicotinicagonist.Whatdrugsor
sequenceofdrugswouldyouapplytodistinguishamongthesepossibilities?Considerthisscenario:ifthe
unknownwasblockedbyatropinebutnotbyhexamethonium,whatwouldyourconclusionbe?Whatifit
weren'tblockedbyeither?
Nowconstructaflowchartfordeterminingtheidentityofanunknownagentthatproducesileum
relaxation.noresponse
SuggestedStrategy:
1.

TestmusclewithAChtodeterminetissueviability.Washthoroughly.

2.

Applyunknown.

3.

Employyourflowchartofpossibleresultsandtheisolatedileumpreparationtoactuallydetermine
thesiteofactionofanunknowndrug.NotethatyoumayneedtoapplyasmalldoseofAchin
ordertogeneratesometensionbeforeyouareactuallyabletoseeanyrelaxationelicitedbyan
adrenergicagonist.

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