The interaction of acetylcholine and ... on human bronchial smooth muscle ... D A. Knight*, M

Eur Reaplr J
1991' 4, 986-991
The interaction of acetylcholine and histamine
on human bronchial smooth muscle contraction
D.A. Knight*, M.J. Phillips*, G.A. Stewart**, P.J. Thompson***
The interaction of acetylcholine and histamine on human bronchial smooth
muscle contraction. D.A. Knight, M.J. Phi/lips, G.A. Stewart, PJ. Thompson.
ABSTRACT: The Interaction of histamine (Hist) and acetylcholine (ACh)
on human Isolated bronchial smooth muscle (HIBSM) contraction, and
the Influence of the epithelium, was assessed using HmSM obtained
from 15 patients undergoing thoracotomy. Cumulative concentration
effect curves for ACh and Hlst, together with combinations of equlpo·
tent concentrations of both agonlsts, were generated using both
epithelium-Intact and epithelium-denuded HmSM.
In epithelium-denuded HmSM both ACh (p<0.05) and Hist (p<O.OOS)
produced a significantly enhanced maximal response and a 2.1 fold in·
crease In the potency of ACh (p<0.02, n=l3). When ACh and Hist were
added simultaneously, in equipotent concentrations, to epithelium-Intact
HIBSM, a significantly less (p<O.OOOS, n=13) than additive response
occurred with only 60% of the predicted maximum response being
observed. However, following epithelium removal, an additive lnterac·
tlon between the two agonlsts (n=8) occurred.
Using IDBSM from five of the original 15 patients, similar experiments
were performed to determine the Influence of the muscarinic receptor
antagonist atropine (0.1 I!M> and the H 1 receptor antagonist mepyramine
(10 I!M). Both resulted In a signlflcantly less than additive Interaction
(40-50% of predicted tensions). Similar experiments were also
performed In the presence of the cyclo·oxygenase Inhibitor lndometh·
acln (5 JAM) and these failed to reverse the Inhibition observed In IDBSM
contraction (n=S).
The inhibitory Interaction between ACh and Hlst appears to be epi·
thelium dependent and is not mediated vio the release of prostanoids.
Thus, there appears to be a complex interaction between contractile
agonists and the epithelium, which Is not just a simple summation of the
activation of Individual receptors on HIBSM.
• University Dept of Medicine, Sir Charles Gairdner
Hospital, Perth, Western Australia.
•• The Western Australian Research Institute for
Child Health, Western Australia.
• • • Dept of Medicine, University of Western
Australia, Perth, Western Australia.
Correspondence: Dr P.J. Thompson, University Dept
of Medicine, Queen Elizabeth II Medical Centre,
Nedlands, Western Australia 6009.
Keywords: Acetylcholine; airway smooth muscle;
asthma; histamine; prostanoids.
Received: August 30, 1990; accepted after revision
April 30, 1991.
This project was supported by the National Health
and Medical Research Council of Australia. D.A.
Knight was the recipient of a Postgraduate
Scholarship from the University of Western
Australia.
Eur Respir J., 1991, 4, 985-991.
Airway smooth muscle contraction is recognized as
a central component of the asthmatic response. A variety of endogenous agents have been shown to cause
both bronchoconstriction in vivo and contraction of
airway smooth muscle in vitro and include histamine
(Hist), acetylcholine (ACh), prostaglandins, leukotrienes
and tachykinins (1-3]. These mediators are present
in a variety of cells resident in airways and are
liberated after allergen-immunoglobulin E (IgE)
interaction or neural stimulation (4]. The bronchoconstrictor response to inhaled agents such as histamine is
characteristically greater in asthmatic patients than in
non-asthmatic subjects (4]. The precise cause of this
bronchial hyperreactivity or enhanced smooth muscle
contraction is unknown. A variety of individual
mediators are known to be released simultaneously
following allergen challenge and it seems unlikely that
an increased sensitivity to any one bronchoconstricting agonist is the primary cause. It is more likely
that airway oedema, epithelial loss and/or the effect of
mediator interaction contribute to the overall hyperresponsiveness of the airway.
Studies of the role of the epithelium and of mediator
interactions on airway smooth muscle responsiveness
in man have been limited. Assessments of mediator
interaction have primarily involved in vivo investigations using normal or asthmatic volunteers. These
studies have assessed various interactions of methacholine, histamine, leukotrienes and prostaglandins
[5-11]. Similar studies have been performed in animal
models both in vivo (12] and in vitro [13]. However,
in vitro studies of the interaction between putative
asthmatic mediators on human airway smooth
muscle responses are lacking. Thus, the effect of the
D.A. KNIGHT ET AL.
986
concomitant addition of Hist and ACh on isolated
human bronchi was investigated and the influence of
the epithelium on this response assessed.
Methods
Human bronchi were obtained from lungs resected
from 15 patients undergoing pneumonectomy or
lobectomy for the treatment of lung carcinoma.
Macroscopically normal bronchi (4-10 mm ID) remote
from the site of tumour were removed within 20 min
of resection and placed in ice-cold Krebs-Hen~eleit
solution previously aerated with 5% C02 in 0 2 and of
the following composition (mM) NaCl 118, KCl 5.4,
NaHC01 25, KHl04 1.1, MgSO, 0.57, D-glucose 11.1,
CaC1 2 2.5). The bronchi were dissected free of all
visible connective tissue and blood vessels and either
used on the day of resection or stored at 4°C in
Krebs-Henseleit solution for a maximum of 40 h.
Bronchi were cut longitudinally along the cartilage
plates and the resulting open segments (3-4 mm wide)
of human isolated bronchial smooth muscle (HIBSM)
were suspended in organ baths under 1 g tension in
Krebs-Henseleit solution.
Preparations were allowed to equilibrate (60-90 min)
under tension and the bath fluid exchanged every 15
min. Changes in isometric tension were measured
using a Grass FT03C force displacement transducer and
a Rikadenki L50 chart recorder. Any changes in
resting tone were readjusted to a tension to 1 g. Following equilibration, all HIBSM strips were exposed to
sub-maximal doses of ACh (10·5 M, final bath
concentration) to determine the magnitude and reproducibility of contraction. In order to ensure that
the mechanical properties of the muscle strips did not
alter during the course of the experiments, lengthtension relationships were also assessed at various
time intervals, by determining the response to a
maximal concentration of ACh using tensions of 0.5,
1.0 and 2.0 g.
Epithelium-intact experiments
HIBSM strips from 13 of the 15 patients were used.
Independent cumulative concentration-effect
curves (CCEC) were constructed for both Hist and
ACh by the cumulative addition of the specific agonist
(range 10·7 - 3x10·3 M) to the organ bath until a maximum affect (Emax) was obtained. Individual Emax
values were determined when responses to two
sequential doses of agonist were within 5% of each
other. Hist and ACh were added so as to produce
successive 1/2 log increases in agonist concentration.
The initial agonist used to generate the first CCEC
was randomly determined and the muscle strip was
then. allowed to return to baseline and re-equilibrate
before generating CCECs with the second agonist.
Using the data obtained from each of the single
agonist CCEC, the concentrations of each agonist
required to produce the 10, 20, 30, 40 and 50% of Emax
(EC10 - EC5J were calculated. From these data, the
individual equipotent concentrations (EC10 - EC5J of
ACh and Hist were tabulated and their responses
summated to produce a predicted additive CCEC.
After completing the single agonist CCECs, the same
muscle strip was washed and allowed to return to
baseline tension and then cumulatively stimulated
with combined equipotent concentrations of ACh and
Hist.
The above experiments were then repeated on
HIBSM from five of the 15 patients but with the
muscarinic antagonist atropine (0.1 ,....M), the H 1 receptor
antagonist mepyramine (10 ,....M) or the cyclooxygenase inhibitor indomethacin (5 ,_..M) being
added to the organ baths 30 min prior to the combined
addition of ACh and Hist.
Epithelium removed experiments
A similar set of experiments to those describe
above were performed in parallel, using adjacent
muscle strips from eight of the 15 subjects. In these
experiments the epithelium was removed prior to any
stimulation. This was achieved by gently rubbing
the mucosal surface with a moist cotton swab. Verification of successful epithelium removal was obtained
by light and scanning electron microscopy.
Analysis and statistics
At the end of the experiments, the strips were
weighed and their length measured for determination
of cross-sectional area and wet weight. Results were
expressed as the active tension developed in gig wet
weight of tissue.
The predicted CCEC data and the experimentally
generated CCEC were compared to determine whether
interactions between ACh and Hist were supra-additive,
additive or inhibitory. The mean (±SEM) tensions
were calculated for each datum point and the statistical
significance of differences between experimental
and predicted values determined using two-way
analysis of variance and Student's paired and unpaired
t-test. Data from the single agonist curves were also
used to determine Emax and the pD 2 (-log EC50)
values. These were compared using one way analysis of
variance and subsequent Student's paired t-test.
Drugs and solutions
The following drugs were used: histamine diphosphate
(British Drug Houses, Sydney, Australia); acetylcholine
chloride, atropine sulphate, mepyramine maleate and
indomethacin (Sigma Chemica~ Company Ltd, St
Louis, USA).
~tock solutions of ACh, Hist, atropine and
mepyramine were prepared by dissolving the reagents
in distilled water. Solutions were stored in 2 ml
aliquots at -20°C and used as required. For each
experiment a stock solution of indomethacin was
HUMAN BRONCHIAL SMOOTii MUSCLE CONTRACI10N
987
between predicted additive values and experimentally
generated data for the epithelium removed
preparations.
prepared in 5% (w/v) Na 2C03 and Krebs-Henseleit
solution was used for subsequent dilutions.
Results
The effect of selective antagonists
Single agonist CCEC
The muscarinic antagonist, atropine (0.1 ~-tM), had
little effect on the response to the combined addition of
ACh and Hist with there being a significantly reduced
contraction, equating to approximately 40% of the
predicted additive values (p<0.005 for Emax, n=5; table
2). Similarly, the addition of the H1 receptor antagonist
mepyramine (10 ~-tM) failed to enhance the responses
The CCEC for Hist was displaced to the left, relative
to ACh, resulting in the pD2 for Hist being significantly
greater than ACh (p<0.05, n=13; table 1). Although ACh
induced a greater maximal response (Emax) than Hist,
the difference between the two values was not statistically different (p<0.375; table 1).
Table 1. - The effect of epithelium removal on pD and Emax in
reponse to acetylcholine and histamine in human isofated bronchial
smooth muscle
Agonist
Histamine
Acetylcholine
Parameter
(+) Epi
(-) Epi
(+) Epi
(-) Epi
pDz
4.46::t0.1
4.88:tO.lt
5.16:tO.r
5.32:tO.l•
Emax
g·unir1 tissue
n
9.86:t:2.1
13
16.15:t:2.8*
8
7.95:t:l.O
13
13.3:t:2.7*
8
The mean:t:SEM values for both pD2 (-log EC,J and Emax following
cumulative addition of acetylcholine and histamine to isolated epitheliumintact and epithelium-denuded human bronchial smooth muscle. t: p<0.02
(epithelium intact compared to epithelium denuded preparations); •:
p<0.005 (epithelium intact compared to epithelium denuded preparations);
•: p<O.Ol (Hist pD2 compared to ACb pDJ.
Mechanical removal of the epithelium enhanced Emax
for both ACh and Hist when compared to responses
obtained from epithelium-intact preparations (p<0.005;
table 1, fig. lA and lB). Removal of the epithelium
also resulted in increases in pD 2 for both ACh and Hist
although this was significant only for ACh (p<0.02;
table 1).
Combined agonist data
The mean predicted additive CCEC and the subsequent
experimentally generated CCEC data for epithelium
intact preparations are shown in figure 2a. The combined
addition of ACh and Hist resulted in a significantly
inhibited response when compared to that predicted for
an additive interaction. The apparent degree of inhibition was between 40-50% of the predicted response at
each dosage increment (fig. 2A).
In contrast to these results, HBISM strips devoid of
epithelium demonstrated significantly greater responsiveness to the combined addition of ACh and Hist than did
the adjacent epithelium intact preparations (fig. 2B). As
a consequence there was no statistical difference
induced by combined addition of ACh and Hist with
the responsiveness (Emax) not exceeding 45% of the
predicted values (p<0.005; table 2).
The effect of indomethacin
Exposure of muscle strips to indomethacin (5 ~otM)
failed to modify the inhibitory interaction between
ACh and Hist. The magnitude of the responses obtained
in the presence of this antagonist was approximately
50% of the predicted values for an additive interaction
(p<O.Ol for Emax, n=5; table 2).
Control data
In all of the experiments length-tension relation·
ships were preserved. Similarly, responses to a maximal dose of ACh at the commencement and completion
of each experiment were not statistically significantly
different, suggesting that the results obtained were not
influenced by alterations in muscle responsiveness
during the course of the experiment. Histological
D.A. KNIGHT ET AL.
988
-
20
A
10
A
18
.r:.
0)
·~
J
16
14
8
12
6
~ 10
§
8
'iii
6
4
2
0
c
t!!
<l
•••
4
2
0
10"5
EC1o
ACh cone M
20
:E
18
·~
16
14
0)
i
<l
EC3o
EC4o
ECso
Effective concentration
8
20
.E
8
18
C)
'i
16
14
12
~ 10
c
8
.Q
(/)
6
c
4
~
<l
2
0
Qj
~
12
~ 10
§ 8
'iii
6
c
t!!
EC2o
4
2
0
10'5
10"'
EC1o
Hist cone M
EC2o
EC3o
EC4o
ECso
Effective concentration
Fig. 1. - A) The mean tension (~sEM) generated following
cumulative addition of acetylcholine (ACh) to epithelium-intact
(n=13)
and epithelium-denuded (n=8) (.&.) human isolated
bronchial smooth muscle. B) The mean tension (~SEM) generated
following cumulative addition of histamine (Hist) to epitheliumintact (n=13)
and epithelium-denude d (n•8) (.&.) human
isolated bronchial smooth muscle.
<•>
<•)
Fig. 2. - A) The mean tension (:tsEM) generated following cumulative addition of equipotent concentrations (EC1o.-~J, of acetylcholine
and histamine in epithelium-intact, isolated human bronchial
smooth muscle strips (.&.) compared to predicted curve
(n=13).
•: p<O.OS; .. : p<O.Ol; ... : p<O.OOS. B) The mean tension (~SEM)
generated following cumulative addition of equipotent concentrations (EC 1~, of acetylcholine and histamine in epithelium-denuded
isolated human bronchial smooth muscle strips (.&.) compared to
predicted curve
(n=8).
<•>
c-)
Table 2. - The observed mean (±seM) response in epithelium intact preparations, obtained for the cumulative addition of equipotent concentrations
(EC 10-60 ) of acetylcholine and histamine in the presence of selective
antagonists
EC value
30
10
20
40
50
Control
n=13
72±17•
74%11•
66:t7•
61:t6••
57:t4•••
Atropine
n=5
28:t15•
35:tlS••
36:t15•••
35:t15•••
36±12···
Mepyramine
n:::5
3l:t18•
27:t10..
27:t9•••
41:t8···
44:t9•••
Indomethacin
n:::5
45:t16•
57:t19•
58:t14•
55:tlO•
57:t9 ..
Data are expressed as a percentage of the predicted response for an additive
interaction. Atropine (0.1 ~A-M); mepyramine (10 J.lM); indomethacin (5 J.lM).
•: p<0.05; .. : p<0.01; •••: p<0.005 compared to predicted value unpaired
t-test.
HUMAN BRONCHIAL SMOOTH MUSCLE CONTRACTION
assessment of bronchial strips following experimentation revealed complete removal of epithelial cell layer,
without obvious damage to underlying muscle.
Discussion
The combined addition of ACh and Hist to HIBSM
bronchial smooth muscle strips resulted in a 40%
reduction of expected smooth muscle contraction.
In contrast, when the epithelium was removed, the
combined effect of ACh and Hist on muscle contraction
was essentially additive. These results suggest an interaction between Hist, ACh and the epithelium which
results in the inhibition of smooth muscle contraction,
although the underlying mechanism(s) is unclear.
Previous investigations of the interactive effects of
ACh and Hist on bronchial smooth muscle contraction
have been of varying design and results. In animals,
in vivo studies in the guinea-pig and the dog [12, 14]
have demonstrated synergism between Hist and
methacholine. However, in the guinea-pig isolated
trachea, graded doses of Hist did not enhance the
CCEC to carbachol and indeed, as with the results
reported here, at higher Hist concentrations there was a
significant reduction in the potency of carbachol
[15). In man, in vivo studies have been limited and
conflicting. MITCHELL and BouHUYs [10] reported an
additive effect on forced expiratory volume in one
second (FEV ) when non-asthmatic patients were
challenged with single concentrations of Hist and ACh,
and STERK et al. [11] reached a similar conclusion
by demonstrating that Hist produced a small but
additional contractile response following a maximum
response to inhaled methacholine.
In vivo studies are difficult to interpret since a
variety of tissue responses enhance airway obstruction
and, thus, Hist and ACh as well as causing smooth
muscle contraction can stimulate mucus production
and airway wall oedema [16). More importantly, the
geometry of the airway is such that small changes
in airway radius will have substantial effects on resistance to flow [17). Therefore, a small dose of one
agonist may appear to sensitize the airway to the effect
of another, suggesting apparent pharmacological
synergism. Equally, if the concentrations of agonist
are chosen because of similar weight or molar concentration, it is difficult to interpret the results as each
agonist may be acting at a different position on their
respective CCEC. Ideally, similarly effective concentrations of each agonist should be compared to
determine if true synergy exists.
In this study, it seems unlikely that the responses
obtained were a reflection of de novo alteration in
muscle responsiveness or fatigue. All strips could be
maximally contracted by ACh at the conclusion of the
experiment and this response was not statistically
significantly different from the original responses to
ACh. Additionally, although the experiments involved
the same muscle strips being studied over 36 h,
previous studies from our laboratory (18] and from
989
others (19] have shown storage for this period of
time does not alter tissue responsiveness. Similarly,
removal of the epithelium did not appear to alter
length-tension relationships.
Hist is currently thought to act via three receptor
subtypes, H 1, H2 and H 3 [16). H~ receptors cause
bronchoconstriction in man and altnough H 2 and H 3
receptors have been demonstrated in lung parenchyma
and airways, their role is unclear [20] . ACh may act
on autonomic ganglia as well as directly via muscle
receptors. Nine different muscarinic receptor
subtypes have been identified but only three (M1-M;>
have been described in human airways. M3 receptors
are found on human airway smooth muscle and
submucosal glands, whilst the presence of muscarinic
receptors on human airway epithelium has yet to be
confirmed (21, 22].
Despite the knowledge of different receptor
subtypes, the relative effect of eo-stimulating these
receptors and the role of the epithelium is unknown.
To determine the relative contributions of each
agonist, the selective receptor antagonists atropine
and mepyramine were employed to block ACh and
Hist responses, respectively. The apparent contribution
made by ACh in the presence of mepyramine, was
approximately 35% of predicted response. For Hist,
in the presence of atropine, a similar result was obtained with the average response also being
approximately 35% of the predicted additive response.
This would suggest that both ACh and Hist are
being equally inhibited when HBISM is eo-exposed
to both agonists and that this is abolished when the
epithelium is removed. Recently, several investigators
have postulated the presence of epithelially-derived
inhibitory factor(s) (EpDif) which may be protective
against hyperstimulation of airway smooth muscle
[23-28). The regulation of EpDif release, its nature
and its mode of action have yet to be defined.
However, an enhanced generation of EpDif occurring
only in response to multiple agonist challenge
could provide a possible explanation for the data
discussed above.
In some animal species, cyclo-oxygenase metabolites
are thought to be at least partially responsible for
regulating muscle tone. Hist stimulation generates the
release of a variety of prostanoids from the airways
of a number of species, including man [28, 30). In
human airways, inhibitory prostaglandins such as
PgE 2 and Pgi2 appear to be preferentially released
both under basal and agonist-induced conditions [29].
In this study, the cycle-oxygenase inhibitor indomethacin did not significantly reverse the inhibited response
observed following the simultaneous addition of ACh
and Hist, suggesting that under these conditions
prostaglandins were not significantly involved in
modulating airway smooth muscle responses. These data
are compatible with the data from HAYE-LEGRAND et al.
[30), who suggested that although human airways
generate significant quantities of prostaglandins
following Hist challenge, they are not involved in
modifying subsequent responses to Hist.
990
D.A. KNIGHT ET AL.
In this study, the interaction of Hist, ACh and the
epithelium produced an inhibitory effect with all
three components being required. Whether this reflects
the possibility that both ACh and Hist synergistically
enhance the release of a non-prostanoid epitheliallyderived inhibitory factor is uncertain. However, recent
in vivo studies indicate that tachyphylaxis to repeated
airway stimulation with Hist in mild asthmatic
patients can occur [31) and, thus, it is also possible
Hist may be causing a down-regulation of its own
receptor, which is modulated via the epithelium and
thereby contributes to an apparent reduction in additive
response.
Since asthmatic patients demonstrate increased
sensitivity to inhaled stimuli when compared to normal
subjects, and since epithelial damage and desquamation
are histological features frequently seen in the
asthmatic airway [32), the results from this study may
be clinically significant. The difference between
asthmatic patients and normal individuals may relate to
the enhanced interactive responsiveness as seen with
ACh and Hist when the epithelium is damaged, and it
remains to be determined whether this is related to the
postulated epithelially-derived inhibitory factor(s).
Nevertheless, it would appear that the regulation of
smooth muscle contraction by epithelial factors and
the interaction of contractile agonists is more complex
than is currently accepted. Further investigation of the
pathways and the mediators involved is required.
Acknowledgements: The authors thank the
surgeons and pathologists at Royal Perth, Sir
Charles Oaird.ner and Mount Hospitals for their
contributions to this study.
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L 'interaction de l'acetylcholine et de /'histamine sur la contraction du muscle lisse bronchique humain. D.A. Knight,
M.J. Phi/lips, G.A. Stewart, P.J. Thompson.
RESUME: L'interaction de }'histamine (Hist) et de
l'acetylcholine (ACh) sur la contraction du muscle lisse
bronchique humain isole (HIBSM) et !'influence de
l'epitMlium ont ete appreciees en utilisant le muscle Jisse
bronchique isole obtenu chez 15 patients ayant subi une
tboracotomie. L'on a elabore les courbes d'effets cumulatifs
des concentrations pour ACh et Hist, en meme temps que
pour des combinaisons de concentrations equivalentes des
991
deux agonistes, en utilisant ~ la fois du muscle lisse avec
epithelium intact ou epithelium denude.
Dans le muscle lisse avec epithelium denude, tant l'ACh
(p<0.05) que l'Hist (p<O.OOS) ont produit une reponse
maximale significativement accrue et une augmentation de
2.1 fois dans la puissance de l'ACh (p<0.02, n=13). Quand
l'ACh et l'Hist soot additionnees simultanement ~ des concentrations equivalentes au muscle lisse avec epithelium intact, une reponse significativement inferieure ~ la reponse
additive (p<0.0005, n=13) est survenue: 60% seulement de la
reponse maximale predite etant observee. Toutefois, apr~s
ablation de !'epithelium une interaction additive des deux
agonistes (n=8) survient.
En utilisant le HIBSM provenant de 5 des 15 patients
originaux, des experiences similaires ont ete conduites pour
determiner !'influence de l'antagoniste des recepteurs
muscariniques (!'atropine: 0.1 J!M) et de l'antagoniste des
recepteurs HI (la mepyramine: 10 J.LM). Les deux produits
ont entraine une interaction significativement plus faible que
!'interaction additive (40 ~ SO% seulement des tensions
prevues). Des experiences similaires ont ete egalement ete
conduites en presence de l'indomethacine (5 J.LM), inbibiteur
de la cyclooxygenase, et celles-ci n'ont pas reussi ~ inverser
!'inhibition observee dans la contraction de HIBSM (n=5).
L'interaction inhibitrice entre ACb et Hist s'av~re done
dependante de l'epith61ium et n'est pas mediee par la liberation
de prostanoldes. Done, il semble qu'il existe une interaction
complexe entre les agonistes constricteurs et l'epithelium, qui
n'est pas une simple sommation de !'activation des recepteurs
individuels de HIBSM.
Eur Respir J., 1991, 4, 985-991.