Rr.s~i~io~
Phr~iolcq~~ ( 19 7X) 32. 79m90
@El sevier North-Holland
CHANGES
OF AIRWAY
Abstract. The present
animals
preparations
isolated of airway
from
control
substrate control
asthma
smooth
and asthmatic
asthmatic
by ED,,, (effective developed
smooth
to histamine.
asthma
abolished
Similarly.
muscle reactivity,
in grams.
removing
showed
a substrate
of
It was found that tracheal a significant significantly
stimulation.
response
by ED,,
stimulation.
and contractility
change
with preparations
and carbachol
contractile
as measured
(ITP) from control
50”,, response).
animals
ASTHMA’
and carbachol
as compared
asthmatic
after histamine
preparations
did not demonstrate
or carbachol
ITP from
tension
bath significantly
animals
airway
dose developing
with experimental
isometric
tracheal
to histamine
tension expressed
muscle to histamine
isolated decreased
The absence
of ITP isolated
from the experimental
to histamine.
of
from both solution
in both control
and
animals.
After tracheal
preparation
was contracted
was tested by isoproterenol(2.43 sensitization)
(Prrru.wi.s
x IO-’
smooth
of airway
muscle smooth
by carbachol.
lower (P < 0.001) ability
significant
isolated
the ability
of airway
M). It was found that ITP from animals
has a significantly
Itt sur~~trr~~. this study demonstrates es of airway reactivity
isometric
On the contrary.
maximal
in the experimental
did not change
IN EXPERIMENTAL
ofisolated
from animals
animals.
to develop
MUSCLE
the response
by maximal
in reactivity ability
SMOOTH
with two types of experimental
of ITP was measured
ITP uas measured
Press
study demonstrates
and animals
Reactivity
Biomedical
impairment
from guinea-pigs
muscle isolated
to relax following
of both contractility
with experimental
from asthmatic
smooth
animals
muscle to relax
with experimental and relaxation
asthma.
asthma
beta stimulation. process-
On the contrary.
was not found
to be different
the than
controls. Airway
smooth
Histamine
muscle
Carbachol Experimental
As frequently pointed out (Nadel, 1967). hyperreactivity of airways The nature of this hyperreactivity Acceptedfor
puhlicalion
8 August
’ This study was supported Grant
Substrate
dependency
asthma
~‘t al., 1965, 1975, 1976; Reed, 1974; Simonsson lung. is an important pattern of the asthmatic is not established, although several hypotheses
1977.
by the National
Institute
HL-17322. 79
of Health,
United
States
Public
Health
Service,
J. F. SOUHRADA
80
were suggested (Bouhuys, 1976; Gold ei trf., 1972a. b; Nadel, 1965, 1975, 1976; Reed, 1974; Richardson et al., 1973). Most often the irritant receptors and the activation of nerve pathways are implicated to explain this phenomenon (Gold rf al., 1972b; Mills and Widdicombe, 1970; Nadel, 1965, 1975; Simonsson et al., 1967). According to hypothesis introduced by Nadel (1965), the constriction of airway smooth muscle occurs as a result of reflex response mediated via vagus. This hypothesis does not imply direct involvement of airway smooth muscle assuming that the antigen-antibody reaction occurs on the surface of mast cells or irritant receptors. Airway smooth muscle isolated from lungs of asthmatic patients, however, demonstrates a significant response to agonists or antigen challenges (Schild ct ul., 1951). A significant degree of hyperplasia and hypertrophy of airway smooth muscle cells was also shown in the asthmatic lung (Dunnill rt al., 1969; Hossain, 1973) facts which could modify airway smooth muscle reactivity and its contractile response. The present study investigated the irr rim reactivity and contractile response of airway smooth muscle isolated from guinea-pigs with experimental asthma. Utilizing the in oitro preparation eliminates direct involvement of irritant receptors, epithelial, mucous and mast cells, and could demonstrate if the reactivity and/or contractility of asthmatic airway smooth muscle itself is changed. The role of substrate in the reactivity and/or contractility of airway smooth muscle isolated from animals with experimental asthma was also studied, since it was demonstrated (Altura and Altura, 1970) that the contractile response of vascular smooth muscle to some agonists is severely inhibited when no substrate is present in the experimental bath. In addition, the present study also investigated the activity of the beta adrenergic system of tracheal preparations, since it was previously suggested that loss ofactivity of beta adrenergic receptors might be an important factor in the pathogenesis of asthma (Szentivanyi, 1968).
Method CHRONIC
EXPERIMENTAL
ASTHMA
Two different experimental models of asthma were used. In the first model a modified method of Stotland and Share (1974) was employed. Twenty male guinea-pigs (Hartley strain) were injected subcutaneously with 1.O mg of egg albumin (grade V, salt-free, crystallized and lyotilized, Sigma Chemical Company, St. Louis, MO.), and suspended in 1.O ml of saline. Then these animals received 1.O ml of ~UC~IIUS pertussis vaccine administered intraperitoneally (E. Lilly, Indianapolis, Ind.) containing approximately 16 billion killed bacilli. Twenty-one control animals were injected with saline. Two weeks were allowed for immunization, and then animals were exposed daily (5 times per week) to inhaled antigen, for a total period of 3 weeks. This procedure consisted of continuous exposure for 45 seconds to aerosolized 19., albumin administered through a nebulizer (USV Corporation) while animals were
ASTHMA
AND AIRWAY
SMOOTH
MUSCLE
81
placed in a special box (Souhrada and Dickey, 1976). The control group received corresponding treatment with aerosolized saline. In the second model the experimental protocol of Stein et al. (1961) was used. Ten male guinea-pigs (Hartley strain) were injected intraperitoneally three times on three alternating days with 0.25 ml of fresh, undiluted egg white. Two weeks were allowed for sensitization, the animals were exposed daily for a period of three weeks to inhaled antigen (Souhrada and Dickey, 1976). As in the previous group, this procedure consisted of a continuous exposure of aerosolized 1 ‘I0 albumin administered through the nebulizer (USV Corporation) for a period of 2 minutes. The control group (nine animals) received corresponding treatment with aerosolized saline. During exposure to aerosolized antigen, the animals were placed in a plastic box (30 x 18 x 25 cm) in the top portion of which a nebulizer was attached.
ISOLATED
TRACHEAL
PREPARATION
Technical details of this method were described in detail. (Souhrada and Dickey, 1976). Guinea-pigs were anesthetized (sodium pentobarbital i.p. 30 mg/kg BW), and tracheas were rapidly excised from larynx to carina. They were then immediately immersed in a warm (37 ‘C) physiological salt solution (PSS) aerated with a gas mixture of 20j:; oxygen, 5 “/, carbon dioxide, and 75 U; nitrogen (PO2 = 9OkO.5 mm Hg, PC@ = 24-11 mm Hg). The physiological salt solution had the following composition (in mmol/L): NaCl 117.5; KC1 5.37; CaCl, 2.52; MgSO, 7.0; H20 0.56; NaH,PO, 1.17; NaHCO, 15.51; glucose 5.50; and sucrose 13.65. In some experiments, those without substrate, a sucrose concentration of 19.15 mmol was used. The preparation was attached to an isometric force transducer (Grass FT 0.03) the output of which was displayed on a Beckman (411) recorder. The values of isometric tension are reported in grams. The initial length of the preparation (L,) was defined to be zero microns and the length at which no appreciable resting tension was recorded. Then each preparation was set at a tension of 0.5 grams and allowed to equilibrate for 75 minutes in oxygenated PSS. After this period, dose-response curves were performed.
DOSE-RESPONSE
CURVES
Essentially, the method of Van Rossum (1963) and Altura and Altura (1970) was used to generate cumulative log dose-response curves. Increasing doses of histamine (histamine dihydrochloride, J. T. Baker Co.) or carbachol (carbamylcholine chloride, Sigma) were administered into the experimental chamber in concentrations from below threshold to supramaximal levels, utilizing micropipettes (Beckman). Solutions of drugs in saline were prepared fresh every day, and concentrations are expressed on a molar basis.
J. F. SOUHRADA
82 RELAXATION
Tracheal
RESPONSE
preparations
beta adrenergic
with experimental (carbamylcholine mental chamber
isolated
receptors. asthma
(Prvtussis
chloride, Sigma), in two consecutive
preparations was expressed administration.
DETERMINATION
from guinea-pigs
When tracheal
OF
DNA
relax in response
preparations
sensitization)
to stimulation
from both controls were contracted
of
and animals
with a carbachol
isoproterenol was administered into the experidoses (2.43 x IOeh M). Relaxation of tracheal
in percent
of isometric
tension
achieved
after carbachol
CONTENT
The upper portion of trachea was rinsed in 0.6 N of perchloric acid, blotted and placed in liquid nitrogen. While frozen, tissue samples were weighed and placed in I ml of 0.6 N perchloric acid and analyzed. DNA was determined by a modified method of Zamenhof rt al. (1964) and Bevan rt uf. (1976), and DNA content was reported in Leg/g of wet weight. All assays were performed in triplicate with a 2”” coefficient of variation. All data are expressed as mean *SE, and the Student’s t-test for independent samples was used to determine the presence of any statistical differences. Analysis of variance was utilized to compare the doseeresponse curves. ED,, values (the dose that produces 50”. of the maximum response) represent geometric means of determination on individual were made on the logarithms
tracheal preparations. of the actual values.
Statistical
evaluations
of ED,,
Results Twenty-one days of repeated exposure of sensitized animals to aerosolized antigen did not significantly affect growth rate. Table 1 shows final body weights, wet, dry and relative weights of tracheal preparations, water content of trachea, and DNA content of trachea. In both experimental groups, larger tracheas as measured by wet and dry weights were seen ; however, this was due to the larger body size of experimental animals. In addition, the DNA content of tracheas from control and experimental groups was not different, which suggests that airway smooth muscle from asthmatic animals did not demonstrate any hypertrophy or hyperplasia. Figure 1 demonstrates histamine cumulative dose-response curves of tracheal preparations isolated from controls and both groups with experimental asthma. The gradual increase in tension of isolated tracheal preparations is proportional to the increasing dose of histamine. It can be seen that in the presence of substrate (5.50 mM of glucose) in PSS, the maximal values of isometric tension achieved in control groups were 7.86 kO.36 g. The absence of substrate in PSS significantly
ASTHMA
AND AIRWAY
TABLE Some characteristics
of tracheal
Final
preparations Tracheal
SMOOTH
83
MUSCLE
I
from controls
and animals
with experimental Relative
preparations ~
body weight Dry
(mg)
(mg)
DNA
wt.
of tracheal
‘I,, of Hz0
Wet
asthma
ng,‘g wet wt
preparations (mg of dry
(g) 360.00*
Controls
9.80
37.20+
1.40
wtikg of b. wt.)
10.20+0.37
70.90*
1.20
12.97iO.57
72.30+- 1.00
28.40&0.70
1.44*0.08
(n = 32) Chronic
asthma
462.3Ok24.10
47.6Oi2.20
28.50*
1.30
sensitization) Chronic
not determined
(egg albumin (n = IO)
asthma
455.20&
18.50
47.7Oi
1.70
13.10&0.62
P < 0.05 (controls
L‘Sasthmatic
72.30k0.84
29.OOi
I.19
1.36&0.08
( pcrtussis
sensitization) Data represent
(n = 20) mean
+SE.
animals).
decreased (P < 0.05) the maximal values of isometric tension of isolated tracheal preparations and was equal to 2.03 + 0.24 g only. Figure 1 also shows that isolated tracheal preparations from animals with both types of experimental asthma have a decreased response to increased doses of histamine, as measured by maximal isometric tension. These differences were statistically significant at P < 0.05. The reactivity of isolated tracheal preparations to histamine as measured by ED,,, (effective dose to achieve 5OY,, of maxima1 developed tension) is summarized in table 2. It can be seen that removing the substrate from the experimental medium did not affect the reactivity per se to the histamine. Secondly, no differences in ED,,,
TABLE EDsn in histamine
cumulative
doseeresponse
presence
curves
2 of isolated
(5.50 mM) and absence
tracheal
of glucose
preparations
in PSS
EDso.M Glucose
Controls
2.18f0.15~ (n = 24)
IO-’
2.81 iO.13 (n = 12)
x 10m5
Chronic
asthma
I
(egg white) Chronic
asthma
(Pertussis) Data represents
_
PSS
mean
+ SE.
II
(5.50 mM)
No glucose 1.47*0.29x
10m5
(n = 18)
2.51 kO.23 x 10m5 (n = 12)
3.80+0.97x
IO-’
(n = 8) 2.51+1.35x (n = IO)
lO-5
(guinea-pig)
in
-
control
----
/;
,’
!’ I’ !’ r’ I’ !’
,’
I’
,’
I’
,’
,I’
4
I’
log dose-response curve of tracheal preparations
6 5 -log M (Histamine)
A=20 n= 16
PSS:
(0)
= no substrate
asthma
7
0 Glucose 0 Sucrose
exp.
R =12 n = 10
-logM
6
n = 20 n = 16
control
lltistaminel
----
of tracbta
5
J,/J--I
present in PSS.
Ilnc);
(0
1=
substrate Vertical barb indlcatc +SE.
asthma (B) (solld
(5.50 mM of glucose) present m
laolated 1‘1~111 control animal, (broken line); t’rom ammals with chrome (egg white
and from animals with chronic (PWIILLG.\ sensltlzatlon)
histamine
I = 6
Glrcost I =11
0 Sucrose
l
exp. asthma
Dose rtsponst curvt to Histamine :
of track
‘Dost rtsptnst cum to Histamine :
asthma (A);
I. Cumulative
sensitization)
FIN.
6
I,,
8.
6. Pertussis
A. Albumin
85
ASTHMA AND AIRWAY SMOOTH MUSCLE
8
-logM
Fig. 2. Cumulattve animals (broken performed
carbachol
line) and animals in presence
log dose
response
with chronic
of substrate
5
6
7
I
Xarbacholl
curve
of tracheal
(Pcrr~r.\.~.\ sensitization)
(5.50 mM of glucose)
preparation asthma
isolated
from
control
(solid line). All experiments
in PSS. Vertical
bars indicate
+SE.
were observed in tracheal preparations from controls and both groups with experimental asthma. This finding suggests that airway smooth muscle isolated from animals with experimental asthma did not demonstrate different reactivity to histamine administration as compared with controls. Figure 2 shows a carbachol cumulative dose-response curve of isolated tracheas in the presence of substrate (5.50 mM glucose) in PSS. In the control experiments, the maximal isometric tension was 7.5 k 0.50 g, as compared with the experimental group
(Pertussis
sensitization)
where
this value
was 5.8f0.60.
This difference
is
statistically significant at P < 0.05. No significant differences between control and asthmatic groups were found in ED,,; in controls this value was equal to 1.69 +0.33 x IO- ’ M, and in the case of tracheal preparations isolated from asthmatic animals, this value was 2.63kO.31 x lo-’ M, To determine relaxation abilities of tracheal preparations, carbachol was administered first into the experimental chamber. This was followed by a significant increase in the isometric tension. As seen in fig. 3, when isoproterenol was administered tracheal preparations from animals with experimental asthma (Pertunis sensitization) exhibited a significantly lower ability (P < 0.001) to relax. Even when repeated doses of isoproterenol were administered 10 minutes later, tracheal preparations isolated from asthmatic animak demonstrated a significantly lower (P < 0.05) ability to relax as compared with controls.
J. F. SOUHRADA
86
90
p-y01
~--P-q.001
80
I= .O 3 g
70
Z g
60 50 40
m
1
i
m
I
2.43~10.~
4.86 x lO-6 Ilsoproterenol)
Fig. 3. A comparison guinea-pigs contract isometric
of the relaxant
with experimental with carbachol, tension
steady-state
followed
achieved tension
asthma
after
of tracheal
beta response
of tracheal
preparations
isolated
(Pwrussis
sensitization).
Tracheal
preparations
by administrations carbachol
of isoproterenol
administration
preparations
observed
was designed
from controls
in two consecutive
doses.
IOO”,,. Bars represent
after isoproterenol
and
were made
administration
to The
a new
?SE.
Discussion A guinea-pig sensitized with egg albumin represents the classical model of experimental asthma. Several weeks after sensitization, a minute amount of inhaled antigen caused severe airway constriction as seen by dyspnea, coughing and general poor being of animals. Functionally this response resembles severe bronchoconstriction seen in patients with bronchial asthma, but it differs from human asthma in several points. First, the antibody is part of another immunoglobulin fraction (IgG); second, a reaction can be evoked by soluble antigen-antibody complexes; and finally, chemical mediators may differ, histamine being quantitatively more important in the guinea-pig than in man (Bouhuys, 1974). On the other hand, it would appear that human bronchi and guinea-pig tracheas demonstrate pharmacologically similar responses (Fleisch and Calkins, 1976). Airway hyperreactivity is one of the most important aspects concerning pathogenesis of bronchial asthma. Despite numerous in oivo studies concerning hyperreactivity of asthmatic lungs (Gold et al., 1972b; Nadel, 1965, 1975; Simonsson et al., 1967), airway smooth muscle reactivity per se has received limited attention. It has been suggested that in addition to neurogenic and humoral factors (Bouhuys, 1974; Nadel, 1965, 1973), hyperreactivity of asthmatic airways may be due to alteration in the Birway smooth muscle itself (Nadel, 1976). If this assumption is correct, the dose-response curve to histamine or carbachol of a tracheal preparation
ASTHMA
AND
AIRWAY
SMOOTH
MUSCLE
87
possessing hyperreactivity should lie significantly to the left of curves obtained from controls. The present study, however, failed to demonstrate increased reactivity of airway smooth muscle isolated from asthmatic guinea-pigs to histamine or carbachol. Quantitatively, the ability of two tested agonists to combine with its membrane receptor is not different in airway smooth muscle of asthmatic animals. As previously shown (Hansen et a/.. 1974), ED,, is a suitable measure of smooth muscle reactivity. and readily reflects changes in the position of doseeresponse curves. On the other hand, this study demonstrated that the contractility of airway smooth muscle isolated from asthmatic animals is severely impaired. The term ‘contractility’ is used to refer to the force generating ability of the muscle to contract when it is fully activated (Altura and Altura. 1970; Hansen et al., 1974). A decrease of intrinsic ability to develop isometric tension after agonist administration (both histamine and carbachol) was seen in both models of experimental asthma. In this aspect, the experimental asthma as caused by Perrussis sensitization and which probably induced a partial beta blockade in target tissues (Reed, 1967; Szentivanyi, 1968), does not seem to be qualitatively different from the experimental group sensitized with egg white only. During histamine or carbachol dose-response curves, it can be assumed that activation of membrane receptors is proportional to developed tension. Thus one possible explanation for a significant decrease in maximal developed tension after agonist administration might be that repeated antigenantibody reaction interferes in some way with the action of agonist at the receptor side. This, however, does not seem to be the case. considering normal reactivity of airway smooth muscle isolated from asthmatic animals as measured by ED,,. Secondly, a consequent change in cyclic nucleotides observed in tissue of sensitized animals (Krzanowski et al., 1976; Polson et al., 1974) could modify tissue metabolism and probably interferes with excitation-contraction processes of airway smooth muscle. Removing substrate from the experimental medium significantly abolished contractility of tracheal preparations isolated from both control and experimental animals. In histamine dose-response curves without substrate, EDSo was not significantly changed as compared with those with substrate present in PSS, which suggests that the absence of substrate does not affect the airway smooth muscle reactivity to histamine. These findings correlate well with the author’s recent data (unpublished observation) which suggested that glucose metabolism of airway smooth muscle is an important factor maintaining the normal ability of smooth muscle to develop isometric tension. Finally, these studies show that tracheal preparations isolated from asthmatic animals have decreased ability to relax, following beta stimulation with isoproterenol. This finding seems to correlate well with the assumption that asthmatic airway smooth muscle indeed has impaired contractile machinery. In addition, it seems probable that this can also reflect partial beta blockade of adrenergic receptors, hypothesis introduced by Szentivanyi (1968).
88
J. I.. SOUHRADA
Available data concerning reactivity and contractility of airway smooth muscle in abnormal lungs is lacking. Simonsson et al. (1970) isolated airway smooth muscle from patients with chronic bronchitis. A five-fold increase in sensitivity (reactivity) to carbachol. response
and a lOO-fold increase
seen in a normal
to bradykinin
lung. The credibility
was found
as compared
of this data may be argued,
to the
however,
due to the fact that the mucosa of bronchi was scraped away from the muscular layer, a fact which could modify the performance of smooth muscle cells. There is an apparent analogy between presented data and data received with vascular smooth muscle isolated from animals with systemic hypertension. Both reactivity and contractility of isolated vessels from hypertensive animals after agonist stimulation was decreased (Hansen et al., 1974; Spector et al., 1969). The relevance of this data to human bronchial asthma could be questioned, since no airway smooth muscle hypertrophy was detected in the present experimental model. This is probably due to relatively short exposure of animals to repeated antigen administration, time probably being insufficient to stimulate development of airway hypertrophy. On the other hand, it is a clinical experience that the airway hyperreactivity can develop and disappear in a relatively short time (Empey et c/I., 1976). These findings do not support the assumption that airway smooth muscle per se is responsible or is a significant component of airway hyperreactivity in the asthmatic lung. However, they are compatible with the ‘reflex’ theory of airway hyperreactivity in bronchial asthma (Mills and Widdicombe, 1970; Nadel, 1965, 1975; Simonsson rt al., 1967) implying that the hyperreactive response of irritant receptors and/or nerve pathways together with consequent mediator release are factors responsible for clinically observed airway hyperreactivity in asthmatic lungs.
Acknowledgements
The technical assistance of Ms. J. Loader and Ms. G. Kaub clerical assistance of Ms. Georgia Sear is greatly appreciated.
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