Histamine

histamine (autocoids)bioassy

1. Br.J.Pharmac.(1976),56,39-41
BIOASSAYOFHISTAMINE
INTHEPRESENCEOFPROSTAGLANDINS
R.J.GRYGLEWSKI&R.KORBUT
DepartmentofPharmacology,CopernicusAcademyofMedicine,16Grzegorzecka,31-531Cracow,Poland
IAnAmberliteXAD-2columninaheated(370C)jacketwasincorporatedinbetweentwobanksof
bioassayorganssuperfusedwithKrebsbicarbonatesolutionincascade.Thecolumnremovedpro-
staglandinsEl,E2andF2aandalsorabbitaortacontractingsubstance(RCS)andpossiblyslow
reactingsubstanceofanaphylaxis(SRS-A).
2Thecolumngavefreepassagetohistamineinconcentrationsupto3000ng/ml.Themethod
describedimprovestheaccuracyofhistaminebioassayinthepresenceofprostaglandins.
Introduction
ThetechniqueofVane(1964)hasbeenusedfora
simultaneousbioassayofhistamine,slowreacting
substanceofanaphylaxis(SRS-A),prostaglandinsand
rabbitaortacontractingsubstance(RCS)inthe
effluentfromtheshockedperfusedlungsofsensitized
guinea-pigs(Piper&Vane,1969,1971;Palmer,Piper
&Vane,1973;Piper,1974).Thecatterminalileum
(Ferreira,Ng&Vane,1973)andthemepyramine-
treatedguinea-pigtracheawereusedtodifferentiate
betweenhistamineandSRS-A,andprostaglandins
andRCSwerebioassayedbystandardprocedures
(Ferreira&Vane,1967;Piper&Vane,1969).
Wehaveobservedthatthepeakconcentrationof
histaminereleasedduringanaphylacticshockfrom
perfusedguinea-piglungs,asindicatedbythe
differencebetweentheresponsesoftheantazoline-free
andantazoline-treatedguinea-pigileum(1.08jtg/ml,
s.e.+0.21,n=7)ismuchhigherthanthatdetermined
spectrofluorimetricallybythemethodofHakanson&
R6nnberg(1974)(0.23,g/ml,s.e.±0.04,n=7).One
ofthereasonsforthisdiscrepancymaybethe
presenceofprostaglandinsandRCSintheeffluent.
Herewedescribeatechnique,whichenables
histaminetobebioassayedbytheguinea-pigileumin
thepresenceofrelativelyhighconcentrationsof
prostaglandins.
Methods
gassedwith95%02and5%CO2.Krebssolution
(37°C)wasdrippedovertheorgansatarateof
2.5ml/min,orperfusedthroughtheisolatedlungsof
sensitizedguinea-pigsbeforereachingtheorgans
(Piper&Vane,1969).Allsubstanceswereinfused
directlyovertheorgansatarateof0.2ml/minexcept
forovalbumin(1Omg),whichwasinjectedintothe
pulmonaryartery.Thecombinedantagonists
(Gilmore,Vane&Wyllie,1968),minusmepyramine,
wereinfuseddirectlyoverthetissues.Themovements
oftheassayorgans(initialload2-3g)wererecorded
withPaton'sauxotonicleversconnectedtoHarvard
transducers(type386)andaWatanabemultirecorder.
AmberliteXAD-2(Keirse&Turnbull,1973)or
glasswool(incontrolexperiments)wereusedtofillthe
column(2x5cm).Thecolumnwasmaintainedat
37°Cbyawaterjacketandplacedbetweenthetwo
banksoftheassaytissues(Figure1).Beforebeing
usedtheAmberlitecolumnwaswashedtwicewith
100mlportionsofdistilledwaterandthenperfused
withKrebs(2.5ml/min)for15minutes.
Thefollowingsubstanceswereused:prostaglandins
E1,E2andF2a(UpjohnCo.),histamine
dihydrochloride(Polfa),antazolinemethane-
sulphonate(Polfa),mepyraminemaleate(Mayand
Baker)andAmberliteXAD-2(BDHChemicalsLtd).
Results
Twobanksoftheassayorgans,eachconsistingofa
rabbitaorticstrip,aratstomachstripandaguinea-pig
ileum,weresuperfusedwithKrebsbicarbonate
solutionofthefollowingcomposition(mM):NaCl
118.0,KCI4.7,KH2PO41.2,MgSO47H2O1.17,
CaCl26H202.5,NaHCO325.0andglucose8.4,
Inourexperimentsthethresholdconcentrationsof
prostaglandinsE1andE2whichcontractedtherat
stomachstripwere0.5-2ng/ml,andthoseofpros-
taglandinF2awere2-10ng/ml.Theguinea-pigileum
wasusuallycontractedbyhistamineinconcentrations
of5-10ng/ml,andalsobyprostaglandinsElandE2

2. 40 R.J.GRYGLEWSKI&R.KORBUT
PerfusedLungs LungsII
lungs 10min
b 750OmV
RbALJ
RSS -J\*
GPI
Off On
XAD-2
RbAL_J
RSS
b **4 U* 4*
E2H E2H E2H
mgmlr1minr13020050400 40400
OVA OVA
10mg 10mg
Figure1Isolatedlungsoftwosensitizedguinea-
pigs(lungsIandlungs11)wereperfusedwithKrebs
bicarbonatesolution(2.5ml/minute).Theeffluent
wasusedtosuperfusetwobanksoftheassaytissues.
Eachconsistedofarabbitaorticstrip(RbA),arat
stomachstrip(RSS),andapieceofguinea-pigileum
(GPI).Alltissueswereblockedwithcombined
antagonistsminusmepyramine(Gilmoreetal.,
1968).Calibrationamounts(U)ofprostaglandinE2
(E2)andhistamine(H)wereinfuseddirectlyintothe
effluentatthetopofthecascade.Antigen(ovalbumin
10mg)wasinjectedintothepulmonaryartery(OVA).
Duringtheperfusionoflungs11theXAD-2column
wasplacedbetweenthetwobanksoftissues.The
differenceintheresponsesoftheguinea-pigilea
aboveandbelowthecolumnshowstheinfluenceof
theinterferingsubstancesonhistaminebioassayby
theupperguinea-pigileum.
inconcentrationsof10-20ng/ml.Aspartially
exemplifiedinFigure1,prostaglandinsE1,E2andF2a
at10-150ng/mlandalsoRCSfromshockedguinea-
piglungsarereducedtoundetectablelevelsduringone
passagethroughXAD-2.Amberliteabsorbsneither
histamine(10-3000ng/ml)norcatecholamines
(10-100ng/ml).Theguinea-pigileumpretreatedwith
antazoline(0.5jig/ml)orwithmepyramine
(1.0jg/ml)andsituatedbeneaththeXAD-2column
wascontractedneitherbyhistaminenorbythe
effluentfromshockedlungs(5experiments).Itseems
likelythereforethatSRS-Aisalsoabsorbedby
XAD-2.
Usingtheabovemethodwehavefoundthatthe
effluentfromshockedlungsofsensitizedguinea-pigs
containshistamineintherange225-700ng/ml
(470±54ng/ml,mean±s.e.,n=10)togetherwitha
prostaglandin-likesubstance(43.1+3.7ng/mlpros-
taglandinE2equivalents).
Discussion
AnAmberliteXAD-2columnremovespros-
taglandins,RCSandpossiblySRS-A,butnot
histaminefromtheeffluentfromtheshockedlungsof
sensitizedguinea-pigs,andthereforetheguinea-pig
ileumsituateddistaltothecolumnregisterstheactual
amountofhistamineintheeffluent.Asimilar
absorbentcolumn,butfilledwithaluminiumoxide,
hasbeenproposedbyusforthebioassayofpro-
staglandinsinthepresenceofhighconcentrationsof
catecholamines(Korbut,1975;Grodzifiska,
Panczenko&Gryglewski,1975).
Differentialassayofbiologicallyactivesubstances
inamixtureispossiblewithorgancascadesbecause
oftheselectivesensitivityoftheassayorganstowards
individualhormones(Vane,1964).Thismaybe
furtherimprovedbyadministrationofcombined
antagonists(Gilmoreetal.,1968).Theprocedure
describedhereprovidesanotherwayoffacilitatingthe
specificbioassayofhormonesandautacoidsin
mixtures.
WethanktheTrusteesofTheWelicomeTrust(London)for
thegenerousgrantofequipment,andDrJ.Pike,Upjohn
Co.,Kalamazoo,U.SA.forkindlysupplyinguswithpros-
taglandins.
References
FERREIRA,S.H.,NG,K.K.&VANE,J.R.(1973).The
continuousbioassayofthereleaseanddisappearanceof
histamineinthecirculation.Br.J.Pharmac.,49,
543-553.
FERREIRA,S.H.&VANE,J.R.(1967).Prostaglandins:their
disappearancefromandreleaseintothecirculation.
Nature,Lond.,216,868-873.
GILMORE,N.,VANE,J.R.&WYLLIE,J.H.(1968).
Prostaglandinsreleasedbythespleen.Nature,Lond.,
218,1135-1137.
GRODZINSKA,L.,PANCZENKO,B.&GRYGLEWSKI,RJ.
(1975).GenerationofprostaglandinE-likematerialby
theguineapigtracheacontractedbyhistamine.J.
Pharm.Pharmac.,27,88-91.

3. HISTAMINEASSAYINTHEPRESENCEOFPGs 41
HAKANSON,R.&RONNBERG,A.L.(1974).Improved
fluorimetricassayofhistamine:condensationwitho-
phthalaldehydeat-20°C.Anal.Biochem.,60,560-567.
KEIRSE,M.J.N.C.&TURNBULL,A.C.(1973).Extractionof
prostaglandinsfromhumanblood.Prostaglandins,4,
607-617.
KORBUT,R.(1975).Bioassayofprostaglandinsinthe
presenceofhighconcentrationsofcatecholamines.Pol.
J.Pharmac.Pharm.(inpress).
PALMER,M.A.,PIPER,PJ.&VANE,J.R.(1973).Releaseof
rabbitaortacontractingsubstance(RCS)andpros-
taglandinsinducedbychemicalormechanical
stimulationofguineapiglungs.Br.J.Pharmac.,49,
226-242.
PIPER,P.J.&VANE,J.R.(1969).Releaseofadditional
factorsinanaphylaxisanditsantagonismbyanti-
inflammatorydrugs.Nature,Lond.,223,29-35.
PIPER,P.J.&VANE,J.R.(1971).Thereleaseof
prostaglandinsfromlungandothertissues.Ann.N.Y.
Acad.Sci.,180,363-385.
PIPER,P.J.(1974).Releaseandmetabolismof
prostaglandinsinlungtissue.Pol.J.Pharmac.Pharm.,
26,61-72.
VANE,J.R.(1964).Theuseofisolatedorgansdetecting
activesubstancesinthecirculatingblood.Br.J.
Pharmac.,23,360-373.
(ReceivedApril25,1975.
RevisedJuly1,1975)