Respiration
Physiohgy,93 (1993)111-123
O 1993ElsevierSciencePublishemB.V. All rightsreserved.0034-5687/93/$06.00
111
RESP 02025
Effect of in vitro exposureto acroleinon carbachol
responses
in rat trachealismuscle
Abdellaziz Ben-Jebria, Roger Marthan, Max Rossetti, Jean-Pierre Savineau
and James S. Ultman
Laborutore de Physialogie,Facuhéde Médecineyictor Pachon,UniwÆitéde BordeauxII, Bodeaux. hance
(Accepted15 February1993)
Abstract,Isolâtedtrachealrings obtaited from male Wistar rats l0 !o 15 weeksold and weighing300 to
400 g were exposedto aqueoussolutionsof acroiein,and the resultingchangeof smoothmusclecontractility was evaluatedby measuringthe cumulâtivecarbacholconcentration-response
curve.Usingthe product of acroleincoûcertrationand time as a surrogatefor tàe acroleindosedeliveredto the smoothmuscle
cells,contractilitymeasuredaftera varietyofexposureconcenuâtions
from 0.01to 3.0/M and timesfrom
5 to 60 min could be correlatedin a dose-dependent
manner.In the rangeof dosesfrom 0.1 to 6 pM-min.
relativecontractilitycortinuouslyincreased
from 0 to 50% aboveunexposed
controlvalues.At dosesgreater
than 6 pM-min, the enhancement
in contractilitydeclined.This declinemay havebeendue to cell damage
or celldeathwhich was so severeat a doseof60 pM-min thât contractilityfell belorir'
controlvalues.Below
a lhresholddoseof0.I lM-min, acroleinhâdno effecton contÉclility.The rolearachidonicâcidmetabolism
in the enhancement
of smoothmusclereactivilyto cârbacholwas studiedusingindometacinto block the
pathwayard NDGA to block the lipoxygenâse
cyclo-oxygenase
pathway.At a concentratioD
of l0 lM of
eithe.indometacinor NDGA, the acrolein-induced
enharrcemenl
in airway reactivitywas completelyinhibited.At lower coûcentrations,
hhibition by thesetwo chemicalswâs panially additive,suggesting
that
pathwâysplay a role in lhe hyperreactive
both the lipoxygenase
and cyclo-oxygenase
response.
Airwây, smoothmuscle,acrolein;Mammals,rat; Pharmacological
âgeûts,acrolein,arachidonicacid, carbachol;Smoothmuscle,airway,acrolein
Acrolein, the most toxic respiratory irritant among several aliphatic aldehydes (Beauchamp et al., 1985; Kane and Alarie, 1977), is emitted into the environmenl by automobile exhaust, by cigarette smoke, a.nd by the burning of wood and fat-containing
foods (Altshuler and McPherson,1963;Beauchampetal., 1985).Due to its high
solubilityin aqueoussolution,the greatesteffectof acroleinis likely to take placein
the upper respiratoryairwaysalthoughinhalationof this aldehydeby laboratoryanimalscausesfunctionallesionsand hyperplasiaof epitheliumin both the proximaland
distal tracheobronchial
tree (Costaet q\.,1986; Egleand Richmond,1972).In guinea
pigsthat inhaled0.4-1.0 ppm acroleinfor 2 hours,pulmonaryresistance
increasedand
Correspondence
to:R.Marthan, MD, PhD, Laboratoirede Physiologie,
Faculréde MédecineVictorpâchon,
Univemitéde BordeauxII, 146rue Léo Saigûat,33076BordeauxCedex,France.Tel.: (33) 57 57 13 60;
Fax (33) 56 99 03 80.
1,12
respiratory frequency decreasedat the onset of exposure,but thereafter remained unchanged (Murphy et al.,1963). After exposure to 0.31-1.26 ppm acrolein, guinea pigs
increasedtheir bronchial reactivity to intravenously administeredâcetylcholine(Leikauf
et al., 1989a).
The enhancementof bronchial reactivity by acrolein and other air pollutants poses
a potenlially important dsk to human health, especially in those individuals whose
airways are already compromised by obstructive diseaseor sensitized by allergies.A
useful approach for constructing the local dose-responserelationships and for studying the underlying mechanisms by which a xenobiotic agent enha.ncesairway smooth
muscle contractility is to measure the response of an isolated bronchial airway to a
cholinergic agonist. For example, we recently demonstrated that in vitro nitrogen dioxide exposure causes a significant increasein the reactivity of human bronchial rings
to carbachol, and this sensitization occurs in a concentration-dependentmanner (BenJebria et a\.,1992). The current work applies a similar approach to acrolein exposure.
Our objectives wete 1o show that acrolein enhancesthe reactivity of isolated rat tracheal rings to carbachol, to determine the dose-responserelationship between acrolein
exposure ald reactivity to carbachol and to use modifiers of arachidonic acid metabolism as a means of understandins the mechanism of acrolein sensitization.
Method
Tissuepreparation. Isolated tracheal rings were obtained from male Wistar rats l0 to
15 weeks old and weighing 300 to 400 g. In each experirnent,one rat was anesthetized
by intraperitoneal administration of 40 mg ethylcarbamate, its heart ând lungs were
removed en-bloc, and its trachea was immediately isolated and placed in KrebsHenseleit solution (composition in mM: 118.4 NaCl, 4.'7 KC|2.5 CaCl2 2Il2O, 1.2
MgSOo 7H.O, 1.2 KH2PO4, 25.0 NaHCO3, 11.1 ( + )-glucose)aeratedwith 957. 02
in CO2 at pH 7.410.05. As quickly as possible, the trachea was dissected into rings
of similar 2 mm diameter and 3-4 mm length. Employing 2 stirrup-shaped staineless
steel clips, each ffacheal ring was mounted horizontally in a separateorgan bath filled
with 20 ml of Krebs-Henseleit solution. The 4 baths were part ofan isolated organ bath
system {EMKA Technologies IOS1, Paris, France) that automated filling and emptying of solutions, and thermostated a[ the baths at 37 'C (Mahé et al., 1989).The organ
bath system also computerized the recording of isometric contrâction using built-in
force transducersto which the upper mounting clips ofthe tracheal rings were attached.
Each force transducer was mounted on a tridirectional positioning device that was used
to align the upper and lower mounting clips, and to adjust the resting tension of a
tracheal dng.
Protocol, The optimum resting tension was selecled on the basis of an exploratory
series ôf length-tension experiments using acetylchotine (ACh) as a convenient contraclile stimulus. After adjusting the resting tension ofa tracheal ring in Krebs-Henseleit
113
solutionto 0.5 g, a bolus of ACh was âddedto the bath (final concentrationl0 3 M
in the bath fluid), and the maximal contractile force was recorded.The ring was then
washedwith sufficientKrebs-Henseleitsolutionfor its restingtensionto return to
0.5 g. This processwas repeatedat alternativerestingtensionsof 1.0, 1.5,and 2.0 g
on setsof 3 rings lrom 5 diferent tracheae.The resultsindicatedthat a tensionof
1.5 g is the optimumrestingconditionfor rat trachealrings (Fig. 1).
Acroleindose-response
expedmentswerealsoreplicatedon setsof 4 dngs isolated
from 5 differenttracheae.At the beginningof eachexperiment,restingtensionswere
(ACh 10 3 M)
adjustedto 1.5g, and a supramaximalconcentrationof acetylcholine
was administeredto elicit the maximalacetylcholinecontractileforce for eachof the
4 dngs. After washingthe rings with fresh Krebs-Henseleitsolutionto eliminâtethe
acetylcholineresponse,2 of the rings were exposedto a solutionof acroleinat alternativeconcentrations
from 0.01to 3 pM and timesfrom 5 to 60 min. The 2 unexposed
rings servedas matchedcontrols. At the completionof exposure,all 4 rings were
solutionto removeunabsorbedacrolein,andsmooth
washedwith freshKrebs-Henseleit
muscle contractility was assessedby employingcarbachol.A seriesof aliquotsof
carbacholwere addedto eachorgan bath, allowingsufrcienttime to elapsebetween
- 4
4 -
ol
- o
a -
J l v
-E
'6
t =
gl
t>2o
c|l
LL
, o
l o
o
L-L
0 t
0.0
0.5
1.0
1.5
t
l
2.O 2.5
P os s i v e L o o d ( g )
Fig. 1. Optimâl restingload in rât trachealissmoothmusclerings. Raw valuesof force (left axis, open
column) as well as valuesof force normalized to dry tissueweight (ight axis,cross hatchedcolumn) induced
by iterativestimutationswith a suprânaxiûal concentrâtionof Acerycholine(10 I M bolus)are plotted
versuspassivepreload values achievedby stretchingthe tissueto increasinginitial lengths.Datâ âre
means1 SD for 15 rings originâtiûg from 5 difierent trâcheal specimens.
114
aliquotsso that the steadystatecontrâctileforce could be recorded.In this mânner,
the cumulativeincreasein musclecontractionwas recordedover a carbacholconcen-8
trationrangefrom l0 to 3 x 10 a M, and a cumulativeconcentration-response
curve
(CCRC) to carbacholwas then constructed.
In expedmentsdesignedto study the role of arachidonicacid metabolism,all 4
trachealringswereexposedto acroleinafterdeterminâtionofthe maximalacetylcholine
contractileforce in a ma:rnersimilarto that describedabovee.g.20 min exposureto
0.3 pM acrolein.Then, indometacinand/or NDGA (i.e., nordihydroguaiaratic
acid)
wereaddedto 2 rings,while the other2 rings servedas unmodulatedcontrols.Thirty
minuteslater,the smootJrmusclereactivityof all 4 ringswas assessed
usingcarbachol
as describedabove.Each of tlese experiments
was replicatedon 5 differenttracheas.
Analysk of data. Data were acquiredon-lineusingthe MOISE softwareand were
processedusingthe ANAMOISE software(Mahêet a1.,1989)that was providedwith
the automatedorgan bath system.Duplicatetrachealrings were studiedin eachexperimentalconditionand for eachexperiment.For eachring,the contractileresponses
to carbacholwere expressedas a percentageof the maximalcontractilerespons€to
ACh in that ring. From the individualCCRC constructedin eachring, a meanCCRC
was obtainedfor the 2 rings,eithercontrolor test,to be representative
of that trâcheal
specimenand r€peatedon 5 differenttrachealspecimens.
OverallmeanCCRC could
then be comparedpairewisebetweencontrol and testtissues,A geometricmeanECro
(the concentrationof drug producing50/" of the maximalresponse)and the 95%
conidenceintervallimits (95)2"CL) werecalculatedusinga least-square
linearregression method.The efficacyof carbacholin promotingsmoothmusclecontractionwas
definedasF-.,, the plateau(r'.e.,maximum)levelofthe contractileforceon the CCRC.
The potency of carbacholwas characterizedas ECro, the carbacholconcentration
producinga contractileforceof F-.*/2. The changein smoothmusclecontractilityby
acroleinwas definedas ôF-u,, the differencebetweenF-* in exposedand unexposed
control dngs expressedas a percentage
of F-u* in the control ring.
Statisticalcomparisonsof F-.,, ECro and responsesto eachconcentrationof agonists were carriedout usingboth ANOVA and paired Student'st-testsa.ndresults
wereconsideredsipificant when P< 0.05.
Drugsused. Carbamylcholinechloride(carbachol),acetylcholinechloride,indometacin and nordihydroguaiaratic
acid (NDGA) were obtainedfrom Sigma(St Louis,
MO). Acrolein, 97/" Etre and stabilizedwith 0.1 weightpercenthydroquinone,was
purchasedfrom Aldrich-Chimie(St Quentin Fallavier, France).All drugs, except
NDGA and indometâcin,were dissolvedin water to make 1 ml aliquotsof 10 I M
solutionwhich werekept frozenuntil dilutedin Krebs-Henseleitsolutionon the day
-2
ofuse.NDGA wasdissolvedin pureethanolto makea l0 M solution.Indomethacin
was dissolvedin 5% NaHCO3.Test measurements
indicatedthat the additionof the
requiredaliquotsof these solutionsincludinghydroquinoneto the Krebs-Henseleit
solution in ar organ bath had no effect on the resting force of the tracheal rings.
113
solutionto 0.5 g, a bolus of ACh was addedto the bath (final concentration10 3 M
in the bath fluid), and the maximalcontractileforce was recorded.The ring was then
washedwith sufficientKrebs-Henseleitsolution for its restingtensionto retum to
0.5 g. This processwâs repeâtedat alternativerestingtensionsof 1.0, 1.5,and 2.0 g
on sets of 3 rings from 5 differenttracheae.The resultsindicatedthat a tensionof
1.5 g is the optimumrestingconditionfor rat trachealrings (Fig. 1).
Acrolein dose-response
experimentswerealsoreplicatedon setsof 4 fings isolated
from 5 differenttracheae.At the beginningof eachexperiment,restingtensionswere
adjustedto 1.5g, and a supramaximalconcentrationof acetylcholine(ACh l0 3 M)
was administeredto elicit the maximal acetylcholinecontractile force for each of the
4 rings. After wâshingthe rings with fresh Krebs-Henseleitsolutionto eliminatethe
acetylcholineresponse,2 of the rings were exposedto a solutionof acroleinat alternativeconcentrations
from 0.01to 3 pM and timesfrom 5 to 60 min. The 2 unexposed
rings servedas matchedcontrols. At the completionof exposure,all 4 rings were
washedwith freshKrebs-Henseleit
solutionto removeunabsorbedacrolein.andsmooth
muscle contractility was assessedby employingcarbachol.A seriesof aliquotsof
carbacholwere addedto eachorgan bath, allowingsufrcienttime to elapsebetween
- 4
J \,/
-c
'ô
c',
e 2
, =
o
o
a
r q )
l
o
lr-
o.o
0.5
t
t
1.0
1.5
t
l
2.O 2.5
Pos s i v e L o o d ( g )
Fig. 1. Optimat restingload iû rât tracheâlissmoothmusclerings. Raw vâ.luesof force 0eft axis, open
column) as well as valuesolforce normalizedto dry tissueweight (right axis, cross hatchedcolumn) induced
by iterativestimulationsuath a suprama mal concentrationof Ac€tycholine(10 3 M bolus)are plotted
versuspassivepreload values achievedby stetching the tissue to increasinginitial lengths.Data are
means1 SD for 15 rings originating from 5 different trachea.lspecimens.
t14
aliquots so that the steady state contractile force could be recorded. In this manner,
the cumulative increasein muscle contraction was recorded over a carbachol concen-a
tration range from 10 Eto 3 x 10 M, and a cumulative concentration-responsecurve
(CCRC) to carbachol was then constructed.
In experiments designed to study the role of arachidonic acid metabolism, all 4
tracheal rings were exposedto acrolein after deternination ofthe maximal acetylcholine
contractile force in a malner similar to that described above e.g.20 min exposure to
0.3 pM aoolein. Then, indometacin and/or NDGA (i.e., nordihydroguaiaratic acid)
were added to 2 rings, while the other 2 rings served as unmodulated controls. Thirty
minutes later, the smootl muscle reactivity of all 4 rings was assessedusing carbachol
as described above. Each of these experiments was replicated on 5 different tracheas.
Anelysis oJ data. Data were acquired onJine using the MOISE software and were
processedusing the ANAMOISE softw are (Mahê et al., 1989) that was provided with
the automated organ bath system. Duplicate tracheal rings were studied in each experimental condition and for each experiment. For each ring, the contractile responses
to carbachol were expressed as a percentage of the maximal contractile response to
ACh in that dng. From the individua.l CCRC constructed in each ring, a mean CCRC
was obtained for the 2 rings, either control or test, to be representativeof that trachea.l
specimen and repeated on 5 different tracheal specimens.Overall mean CCRC could
then be compared pairewise between control and test tissues. A geometric mean ECro
(the concentration of drug producing 50/" of the maximal response) and the 95 )2"
confidence interval limits (95l" CL) were calculated using a least-squarelinear regression melhod. The efrcacy of carbachol in promoting smooth muscle contraction was
defrnedas F-*, the plateau (i.e., maximum) level of the contractile force on the CCRC.
The potency of carbachol was characterized as ECro, the carbachol concentration
producing a contractile force of F^"*12. The change in smooth muscle contractility by
acrolein was defined as ôF-.", the differenc€between Fma*in exposed and unexposed
control rings expressedas a p€rcentageof F-., in the control ring.
Statistical comparisons of F-.*, EC56 and responsesto each concentration of agonists \ryerecârried out using both ANOVA and paired Student's f-tests and results
were considered significant when P< 0.05.
Drugs used. Carbamylcholine chloride (carbachol), acetylcholine chloride, indometacin and nordihydroguaiaratic acid (NDGA) were obtained from Sigma (St Louis,
MO). Acrolein, 9'l % ptûe and stabilized with 0.1 weight percent hydroquinone, was
purchased from Aldrich-Chimie (St Quentin Fallavier, France). All drugs, except
NDGA and indometacin, were dissolved in water to make I ml aliquots of 10 I M
solution which were kept frozen until diluted in Krebs-Henseleit solution on the day
-2
ofuse. NDGA was dissolved in pure ethanol to make a 10 M solulion. Indomethacin
was dissolved in 5/" NaHCOr. Test measurementsindicâted that the addition of the
required aliquots of these solutions including hydroquinone to the Krebs-Henseleit
solution in an organ bath had no effect on the resting force of the tracheal rings.
115
Moreover in control experiments,we checked that these diluents also had no eflect on
carbachol-induced tone.
R€sults
Effect of acrolein concentrqtionduring 20-min exposures. When added to the bath,
acrolein, whatever tlle concentration, did not alter baseline tone.
Judging from the CCRC (Fig. 2), exposuresof trâcheâl rings to acrolein for a fixed
interval of 20 min did affect the reactivity of airway smooth muscle to carbachol, but
a threshold concentration higher than 0.01 pM was required. Indeed, this latter concentration had no efect on the response to carbachol (Table 1, Fig.2A) whereas 0.1,
0.3 and 1 pM significantly increased the emcacy of cârbâchol compared to that in the
absence of pre-exposure to acrolein (Table i, Fig.2B-D). Examination of F-.* and
ECto values derived from the CCRC (Table 1) indicate that acrolein exposurechanged
carbachol efficacy without altering carbachol potency. Between exposure concentrations of 0.I and 0.3 pM, acrolein increasedthe reactivity ofthe tracheal rings (Fig. 3A).
However, this enhancementin reactivity declined during 1.0pM acrolein exposure,and
a decrement in reactivity was observed at the highestexposureconcentration of 3.0 pM.
Effect of exposuretime at fxed auolern concentatipn. The consequenceof varying the
duralion of exposure from 5 to 60 min was exarlined at fixed acrolein concentrations
of 0.01 pM and 0.3 pM. The relationship between enhancementin airwây reactivity
and time of exposure is different at these two concentrations (Fig. 3B). An acrolein
concentration of0.0l pM was below the threshold necessaryfor enhancementairway
reactivity when exposure times were 20 min or less. At longer exposure times, however, the value of ôF-"* increased monitonically. On the other hand, an acrolein
TABLE 1
Values of mean maximal force, F-,, (% of acetylcholine ma.\.) and of geometric mean EC5o in response
to carbachol in non- and Dre-treatedrat tracheal rines with acrolein.
Control
0.01tM acrolein
Con!rol
0.1!M acrolein
Control
0.3pM acrolein
Control
1.0pM acroleir
F*., (1sD)
% mâ,\.ACh
EC5o(95% conûdencelimits)
M
146.6(3.5)
145.4(4.0)
t44.4(1.0)
182.8(9.1F
133.8(8.6)
20t .2 (t4.6)*
134.4(4.s)
16r.6(6.9)*
3 . 5x l 0 - 7 ( 2 . 3x 1 0 ? , 5 . 3x 1 0 ? )
3 . 6x l 0 - 7 ( 1 . 6x 1 0 - ? , 8 . x4 l 0 7 )
5.5x l0 7 (3.2x 10 1,9.4\ to-')
5.2x 10 7 (4.3x 10 7,6.5x 10 ')
2 . 3x l 0 7 ( 9 . 5 x 1 0 3 , 5 . 5 xl 0 ? )
2 . 1 x 1 0 - 7( ? . 4x 1 0 3 , 5 . 9 x 1 0 ? )
3 . 8x 1 0 ? ( 1 . 6 xl 0 - ? , 9 . 3 xt 0 - 7 )
5.0x l0 ? (3.5x 10 1, 7.2x t0-1)
* Pairs of values were considered significantly diferent from each other (acrolein and control) when P < 0.05.
116
220
o 200
E
= 180
-c 1 6 0
o 140
;
120
100
80
x
A
*
OU
*
À
E
40
às 2 0
l
220 ZUU -
o
.: 'l 80 o
-c I O U
;
Q)
o
x
E
1 4 0lzv
c
'
t
'
l
r ' t ' t , t ' l
, *.*
x--f
D
*
*5'
*5'
-
1 o o80OU -
40-
àq
o r ' t , l , t ' l
-a -7 -6 -5 -4
l o g C o r b o c h o (l M )
|
-8
'
l
-7
'
t
-6
,
l
-5
'
l
-4
l o g C o r b o c h o(l M )
Fig.2. Effectof a 20 min exposureto acroleinon the subsequent
CCRC for carbacholin rat trachealis
smoothmusclerings.Altemativeacroleinconcentrations
were:A: 0.01,B: 0.1, C: 0.3 â.ndD: I pM. Each
opencircle representsthe meanvalueof contractilelorce (expressed
as a percentage
of the acetylcholine
maximum) for 5 experimentsin acrolein-exposedtissues.Each closed circle representsthe mean value for
5 pairedexperiments
in non-exposed
tissues.VerticalbarsindicateSD. Absenceof bârs indicâtesâ deviâtion lessthân the diâmeterol the svmbol.*(P<0.05).
concentrationof 0.3 pM eliciteda maximumin the enhancement
of airwayreactivity
at 20 min of exposure.Consideringthe combinedresultsin figures3A ând 38, it is
appaxentthat immersionof rat trachealrings in 0.3 pM acroleinfor 20 min is the
optimum conditionfor enhancement
of smoothmusclereactivity.
rt1
ou I
B
*
o
tr
40-
o
àR
i
E
Z U -
0 -
L!
0.01 0.1
1
10 0 1020 30 40 50 60
A c r o l e i n ( , u r . M ) T i m e e x p o s u r e( m i n )
Fig.3. Effectol concentration
ol andtimeof exposureto acroleinon theefrcacyof cdbâcholin rat trâcheâlis
smootàmusclerings.A: The effectof acroleinconcentrationon the efficacyof carbacholafter a 20-min
exposure.Eachpoint represents
tàe meanvalue( I SD, r = 5) of the changein carbacholefficacy(ôF-..)
calculatedâs the rnâximalresponseto carbacholin acrolein-exposed
tissueminus that in pairedtissues
unexposedto acroleinto the lâtter.B: The effectof time on the efrcacyofcarbachol.Mean values( I SD,
n = 5) of the changein emcacyof carbachol(ôF",.,) are plottedvenus durationof exposureto 0.3 (closed
squares)or 0.01 (closedcircles)lM acrolein.*P<0.05 between€xposedand non-expos€d
tissùes.
Efect
of modulators of arachidonic acid metabolism.
The role of arachidonic
acid
metabolit€s in the enhancementof smooth muscle reactivity to carbachol îollowing a
20-min exposure to 0.3 /lM acrolein was studied using indometacin to block the
cyclo-oxygenasepathway and NDGA to block the lipoxygenasepathway.
In control experimentswe verfied tlat indometacin or NDGA, at the concentration
of 10 pM, altered neither the emcacy nor the potency of carbachol in unexposed tracheal rings (z = 5 in each case, data not shown).
Indomethacin reduced the ability of acrolein to enhance airway reactivity, ard this
inhibition was diectly dependent on indometacin concentrations from 0.1 to 10 pM
(Fig. 4A). At the highest indometacin concentration, a comparison of Fig. 4A (triangles up) to Fig. 2 (closed circles) sugg€ststhat the enhancementof airway reactivity by
acrolein exposure is completely blocked. However, a paired comparison of
indometacin-modulated exposedrings to unexposedrings was not possibl€ since measurem€nts on unexposed controls from the same tracheae were not made. There was
also a reduction of enhanced airway reactivity when tracheal rings were treated with
NDGA at concentrations of I and 10 pM (Fig.4B). As in the case of indometacin,
the inhibition was directly dependent on NDGA concentration with virtual eliminatiôn
ofthe effect ofacrolein exposureat the highest NDGA concentration. Treating tracheal
rings with a combination of 0.1 pM indometacin and 1 pM NDGA (i. e. with th€ lowest
118
zzu
-
A
1 8 0()
=
-c
o
;
q.)
x
E
èR
I O U -
1 4 0:
lzv
-
8oOU -
40-
-
20 1
'l
o r
r ' t
-8 -7
' t
-6
, t ' l r ' r
-5 -4 -8 -7
l o g C o r b o c h o (l M )
' t , t ' l
-6 -5 -4
l o 9 C o r b o c h o (l M )
Fig. 4. Efect of enzymemodùlatorson the CCRC for carbacholin rât trachealissmoothmuscledngs.A:
Effectofthe cyclo-oxygenase
inhibitor,hdometacin,in ringsexposedfor 20 min to 0.3 pM acrolein.Each
symbolrepresentsthe meanvalue( t SD, ,?= 5) in the absence(closedcircles)and in the presenceof 0.1
(closedsquares),1 (closedtrianglesdown) a[d l0 ÊM (closedtrianglesup) indometacin.B: Effectolthe
lipoxygenase
inhibitor,NDGA, in ringsexposedfor 20 min to 0.3lM acrolein.Eachs],rnbolrepresetrts
the
meÂûvalue( t SD, /, = 5) in the absence(closedcircles)md in the presenceol I (closedsquares),l0 pM
(closedtrianglesdown) NDGA. *P<0.05 betweentissuesin the p.esenceof enzymeinhibitors(wathever
the concentration)and tissuesin the absenceof enz)meinhibitors.
concentrâtion
of each of these drugs used alone) demonstrated that inhibition
by the
two drugsis partially additive(Fig. 5). At theseconcentrations,
the reactivitywas reducedapproximatelyby 50/. for indometacinalone,by 40/. for NDGA alone,and
by 10/. in the presenceof both inhibitors.
Discussion
To our knowledge,this is the fust studythat has exarninedthe responsiveness
of airway smoothmuscleafter in vitro exposureof trachealtissueto acrolein.Our results
119
zzu -
2oo:
() 1 8 0 .: 1 6 0 -c 1 4 0 ;
C)
lzu -
x
80-
E
OU -
x
40:
200 t
-8
'
I
-7
'
|
-6
'
|
-
-5
t
l
l
-4
l o g C o r b o c h o l( M )
Fig. 5. Effectof the combinatonof indometacinand NDGA on the CCRC for carbacholin rat trachealis
smoothmuscleringsexposedfor 20 min to 0.3pM acrolein.Eachsymbolrepresents
the meanvalue( I SD,
,?= 5) in the absence(closedcircles)and in the presence
of 0.1 ÉM indometacinand 1 pM NDGA (closed
*P< 0.05betwe€ntissuesin the presence
squares).
olindometacincombinedwith NDGA and tissuesin the
absenceof enz)'rneinhibitors,
showsthat subsequent
to acroleinexposure,tlrereis an enhancement
in the contractile responseofexcisedrat tracheato the cholinergicagonistcarbachol.This effect,that
dependson both the acroleinconcentrationand the durationofthe exposure,appears
to involvearachidonicacidmetabolismvia bolh the cyclo-oxygenase
ard lipoxygenase
pathways.
Sincepreconditioningof airwaypreparationsis an importart factor in determining
the lorce generatedby agoniststimulation,we conductedexploratoryexperimentsto
determinethe passiveload requiredto stretcha rat trachealring to its optimallength
(Fig. 1).The resultingvalueof 1.5g wassubsequently
usedasthe standardrestingload,
as wâs âlso suggested
by Mitchell et al. (1991).Another important issuerelativeto
standardizingour resultsis the vadation of contentand orientationof smoothmuscle amongdiferent trachealrings. It has recentlybeensuggested
that a rigorousdefinition of contractilityis the ratio of the raw forceto the fractionalunits of myosinin
the cross-section(Jrangetal., 1991).To normalizeour force measurements
in this
mannerwould requiremorphometricand electrophoreticdeterminationsof myosin
-
120
units,techniqueswhich are currentlyunavailablein our laboratory.A simplerway to
standardizeforce measurements
is to normâlizethem by the weight of the tissue
specimen.Unlike observationsfrom humar airwây tissueresearch(Marthan etal,
1988),however,our datafrom a singlebreedofrats of similarweightsandagesindicate
that it makeslittle differencewhetheror not this typeof normalizationis made(Fig. 1).
Ultimately, we employed â functional method of standârdizingforce measur€ments.
That is, the lorce was expressedas a percentageof the maximalcontractionelicited
by a standard agonist. We selectedacetylcholineas a convenientstandaxdbecauseit
is rapidely metabolizedby âirwây tissue, and its effect on smootl muscle contraction
was reversible.Cholinergicresponses
werethen assessed
usingthe cholinergicmalog
carbachol which, unlike acetylcholine,is not metabolizedby acetylcholinesteraseand
thus enableto constructsteadystat€cumulativeconcentrationresponsecurves.
Two types of acrolein exposurewere performed in this study: fixed-time exposures
in which trachealrings wereexposedlor 20 min at 5 concentrations
from 0.01to 3.0
pM; a"ndconstart-concentration
in which concentrationwas fixed at either
exposures
0.01 pM or 0.3 pM during 4 âltemativeexposuretimesbetween5 and 60 min. The
changein airwayreactvity relativeto unexposedcontrols,ôFûax,was a function of
both acroleinconcentration(Fig.3A) ard exposuretime (Fig.3B). Moreover, tlle
dependenceon exposuretine was drastically diflerent at the two acrolein concentrations thât w€retested.This apparentdisparitycan be understoodby viewingthe data
within a dose-response
framework.In other words, we hypothesizethat ôF-", (1.e.,
the response)is uniquely related to tlte uptake of acrolein by airway tissue (i.e., the
dose).In our experimants,
acroleinuptakecouldnot be measuredand,in the absence
of a realistc dosimetry tleory, uptake carnot be accuratelyestimated.Nevertheless,
it is likely that the transport of acrolein from the Krebs-Henseleitsoluton to the interior of the airway tssue is limited by linear difrrsion processes.In that case, it is
reâsonâbleto âssumethat acrolein uptake will be proportional to the product of exposure concentration multiplied by exposuretime, C x T. Using this vaxiable as a
sllrrogatefor dose,we constructeda dose-response
cwve (Fig.6) which doesa reasonablejob of corelating the two setsof constânt-concentrationdata with eachother
(squaresand triangles) as well as with the fixed-time exposuredatâ (circles). The important featuresof this dose-responsecuxvea.rea threshold va.lueof C x T of approximately 0.1 pM-nin, a peak ôF-* value of 50l. at a CxT of 6 pM-min, and the
appeârânce
of negativeôF-* valueswhen CxT is greâterthen 30 pM-min. We believe that this behavior results primarily from two opposingprocesses.First, acrolein
causesa hglersensitizationof airwaysmoothmuscle,ând this accouûtsfor the rising
portion ofthe curve.Second,acroleinmay causedamageor cell death,ard this could
explain the decline of the curve ât higher doses; extremecell damageor death would
occur when contractionof the exposedmuscleis less than that of the unexposed
controls(i.e.,when ôF-"* is negative).It is alsopossiblethat the decliningportion of
the curve between6 and 30 pM-nin is due to ân adaptationprocesswhich reduces
eitherthe sensitivityof the musclecells to acroleinor reducesthe actuâl dosethat
reachesthe a.ffectedcells. Alternatvely, acrolein may increase the concentration of
t2r
60-
I
o
I
40-
c
o
àR
20-
x
E
LL
ro
0 -
-20 I
0.01
|
"
l
"
|
0.1
1
10
A cr ol ei n I C x T ] ( p M x m i n )
I
'100
as the percentincreasein manimalcontractionrelativeto the conFig.6. Dose-response
curveexpressed
trol vâlùe(ôF--) versusthe productof acroleinexposueconcentrationand exposuretime. Closedcircles
reprcsenta fixed 2o-minuteexposuretme at 5 concenfatronsfrom 0.01-3 gM acrolein;closedsquares
representa constantexposureconcentrationof0.01 pM acroleindudngaltemativeexposuetimesof5, 20,
40 or 60 min; and closedtdanglesrepresenta cotstant exposureconcentrâtionof0.3 pM acroleinduring
altemative erposure times of 5, 20, 40 or 60 min.
endogenous
inhibitorymediâtorsor decrease
that of endogenous
excitatorymediators
to accountfor this decliningportion of the curve.
Leikaufand colleagues
demonstratedthat eicosanoidsmay be involvedin the broninducedby acroleinin live guineapig. In their fust study
chial hyperresponsiveness
(Leikauf et al., 1989a),they found that the levelsof prostaglandinF2a and thrompathwayof arachidonicacid metabolism,
boxane82, formedby the cyclo-oxygenase
rapidly increasedin the bronchioalveolar
lavage(BAL) fluid ofguineapigswhich have
in the partsper million range.In a subsequent
inhaledacroleinvapor at concentrations
study (Leikauf et a/., 1989b),theseinvestigatorsdiscoveredthat levelsof leukotriene
pathway,increasedin BAL fluid and,also,
C4, a metaboliteformedby the lipoxygenase
bronchialhypeûesponthât an LTC4 receptorantagonistdiminishedacrolein-induced
siveness.Althoughwe employeddifferentânimalsand differentexposureconditions,
the presentresultsfrom an in vitro preparationconfirm that eicosanoidsplay a role
We found that indometacinand
in acrolein-inducedairway hyperresponsiveness.
122
and lipoxygenase,
respectively(Shore
NDGA, potent inhibitors of cyclo-oxygenase
of carbacholefrcacyby a degreethat was diet al., 1985),reducedthe enhancement
rectlydependenton the inhibitor concentration.We alsofound that whenboth inhibitheir effectswerepartially additive(Fig.5), suggesting
tors wereus€dsimultaneously,
and lipoxygenase
act in combinationto
thât eicosanoidsproducedby cyclo-oxygenase
As an evidencefor the specificityof
mediateacrolein-induced
airwayresponsiveness.
the modulationof arachidonicacid metabolitesto the actionof acrolein,it shouldbe
usedin
remindedthat neitherindometacinnor NDGA, at the highestconcentrations
in unexposedtrachealrings.
this study(1.e.10 pM), alteredcarbacholresponsiveness
The differences
betweenanimalspeciesand betweenexposureconditionsemployed
by us and by Leikauf et al. (1989a,b)deservefurther discussion.First, the detailsof
arachidonicacidmetabolismdifer betweenthe rat andguineapig. For example,Chang
and Voelkel(1991)found that eicosanoidprotles following stimulationwith the ionophoreA23187werediferent in thesetwo animals.Sincewe usedenzymeinhibitors
and not eicosanoidreceptorantagonists,the speciûcprostaglandinsand leukotrienes
that mediatedthe acroleineffectin isolatedtracheaewere not identified,unlike the
resultsin live guineapigs whereprostaglandinF2d, thromboxaneB2 and leukotriene
C4 were implicated.Further studiesin which both the concentrationof variousleukotrienesis measuredin the efluent of the organbath and specificleukotrienereceptor antagonistsare usedare requiredto preciselydetermineeicosanoidprofilesin this
condition. Second,we exposedrat tracheaeto aqueoussolutionscontaining0.01 to
3.0 pM acroleinwhereasthe live guineapigs inhaledpaft per million concentrations
of acroleinvapor mixed with air. Sincepure acroleinliquid has a vapor pressureof
214 mmHg at 20'C (Commiteeon Aldehydes,1981),we estimatethat the gasphase
concentrationin equilibriumwith our exposuresolutionswould be on the order of
one-thousanthof a part per million. Thus, the effec1of acroleinâppears10 be much
strongerwhenit is deliveredby an aqueoussolutionthan whenit is inhaledas a vapor.
reactioninduced
Thereare plausibleexplanationsfor this. Althoughthe infla.rnmatory
by acrolein inhaled rr?vivois limited i, u?roand hencethe amount of inflammatory cells
within isolatedairwaysis somewhatsmaller,irnmersionofthe rat tracheaein liquid may
lift off the protectivemucuslayer,resultingin an increasedacroleindoseto underlying tissue.Also, a large portion of inhaled acroleinis normally absorbedby upper
airwayswhereas,whenisolatedtracheaearedirectlyexposedto aqueoussolution,this
form of protectionis absenl.
Acknowledgements.
This studywas supporledby grantsfrom "Intitut National de la Sanléet de la Recherche
Etudeset Techniques"(DRET
Médicale",(INSERM, CRB N" 91.04.11)and "Dfecton desRecherches,
N" 91-1205).Dr JamesS. Ultman was the recipientof a visititrg ProfessorScholarshipfrom the "Unité
de Formaton et de Rechercheen SântéPublique,Universitéde BordeauxII". His permaûentaddressis
StateUniversity,Pennsylvania-UsA.
the Departmentol ChemicalEngineeringat the Pennsylvania
The authors are gratefirl to "EMKA Technologies" (Paris, France) lor the supply of complementary
equipmeûtsto the computerizedorgan bath system.
r23
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