Clinical Science ( 1990) 79,3 15-323
315
Effect of sodium-transport inhibitors on airway smooth
muscle contractility in vitro
ALAN J. KNOX, PAUL AJAO, JOHN R. BRITTON AND ANNE E. TATTERSFIELD
Respiratory Medicine Unit, City Hospital,Nottingham, U.K.
(Received 29 June 1989/18 April 1990; accepted 22 May 1990)
SUMMARY
1. To determine whether alterations in membrane
sodium transport in airway smooth muscle can alter its
contractility, we studied the effect of ouabain (a N a + / K + adenosine triphosphatase inhibitor) and amiloride on
contractile responses in bovine trachea and human bronchial rings in a series of studies.
2. Ouabain ( 10-h-10-4 mol/l) caused concentrationrelated contraction of bovine trachea with a maximum
effect at 30 min; the mean increases in tension with lo-',
mol/l ouabain were 19, 27, and 32%,
and
respectively, of the maximum response seen with
mol/l histamine ( n = 6). In human bronchial rings,
mol/l) caused a mean contraction which
ouabain
was 40% of the maximum response to methacholine
( n = 8).
3. Calcium-free fluid (plus ethylenediaminetetraacetic acid) and nifedipine ( 10 - mol/l) inhibited ouabaininduced contractions, suggesting that contraction was
mediated in part by calcium entry via voltage-dependent
calcium channels. Phentolamine ( lo-' mol/l) was without
effect.
4. Ouabain
mol/l) did not alter histamine
responsiveness in bovine trachea or methacholine responsiveness in human bronchial rings.
5. Amiloride did not affect resting tone in bovine
trachea but caused a concentration-dependent relaxation
of bovine tracheal strips preconstricted with carbachol,
mol/l amiloride relaxing strips completely over 15
minutes ( n = 8). Pretreatment with amiloride significantly
inhibited contraction produced by both histamine and
carbachol in a dose-related manner, lo-',
and 10-3
mol/l amiloride shifting the concentration of histamine
producing 50% maximal contraction by 3-, 8- and 35fold ( n = 10)and that of carbachol by 1.4-, 6- and 86-fold
( n= 8),respectively.
'
Correspondence: Dr Alan J. b o x , Respiratory Medicine
Unit, City Hospital, Hucknall Road, Nottingham NG5 IPB,
U.K.
6. Amiloride also reduced the contraction produced
by lo-' mol/l ouabain from 32% (control) to 7% of the
maximum histamine response.
7. Our results suggest that alterations in cell membrane sodium transport modify the contractile properties
of airway smooth muscle.
Key words: airway smooth muscle, amiloride, ouabain,
sodium.
Abbreviations: DMSO, dimethylsulphoxide; EC,,, and
EClo, concentration producing 20% and 50% maximal
contraction, respectively; EDTA, ethylenediaminetetraacetic acid; KHS, Krebs-Henseleit solution; Na+/K ATPase, Na'/K+ -adenosine triphosphatase.
+
INTRODUCTION
Studies in vascular and cardiac smooth muscle have
suggested a role for different sodium-transport systems
in determining smooth muscle contractility. Na' /
K+-adenosine triphosphatase (Na+/K+ ATPase), Na+/
H C exchange and Na+/Ca'+ exchange have been shown
to be important in cardiac muscle [l-31, and Na+/K+
ATPase and possibly Na' /Ca2+ exchange in vascular
smooth muscle [4, 51. Recent studies have shown an
increase in bronchial reactivity with increasing dietary salt
intake [6, 71, raising the possibility that cell membrane
sodium-transport systems may be important in determining airway smooth muscle contractility and bronchial
reactivity in man. Little is known about the role of
sodium-transport systems in airway smooth muscle,
although ouabain, an Na+/K -ATPase inhibitor, has been
shown to contract animal airways in vitro [8, 91 and
increase histamine responsiveness in guinea pigs in vivo
[lo]. The only study of the effect of ouabain on human
airways in vitro [9] showed variable contractions in some
preparations, but as the specimens were obtained at post
mortem up to 12 h after death, the significance of these
findings is unclear. If Na+/K+-ATPase inhibitors were to
+
A. J. Knox et al.
316
contract human airways and increase bronchoconstrictor
responsiveness, it could provide an explanation for the
increase in bronchial reactivity induced by a high salt diet,
as plasma concentrations of endogenous Na /K -ATPase
inhibitors may be increased after oral sodium loading [ 1 1,
121.
We have therefore performed a series of parallel
studies to determine whether drugs which inhibit sodium
transport can influence airway smooth muscle contractility in vitro and bronchial reactivity in vivo. The studies
in vitro are described in this paper and the studies in vivo
in the accompanying paper [ 131. We have looked at the
effect in vitro of ouabain, an inhibitor of Na+/K -ATPase
[l, 141, and amiloride, an inhibitor of Na+ entry, N a + / H +
exchange [15] and Na+/Ca2+ exchange [16, 171, on
smooth muscle tone and on the response to contractile
agents in bovine trachea and human bronchial rings. The
specific aims of this study were: (1) to determine the
mechanism of ouabain-induced contraction in bovine
trachea; (2) to study the effects of ouabain on human airway smooth muscle, obtained from thoracotomy specimens; (3) to see if pretreatment with ouabain increased
the response to histamine and methacholine, thus providing a possible explanation for the relationship between
dietary salt intake and bronchial reactivity; and (4) to
determine if amiloride altered the contractile properties
of airway smooth muscle.
+
+
+
MATERIALS AND METHODS
Sigma Chemicals, Poole, Dorset, U.K. Histamine acid
phosphate was obtained from BDH Chemicals, Poole,
Dorset, U.K., and phentolamine mesylate from
Ciba-Geigy, Horsham, West Sussex, U.K. All drugs were
dissolved in water, except nifedipine which was dissolved
in DMSO. Equal concentrations of the appropriate
diluent were used as controls ( 1 in 1000 bath concentration for DMSO).
Experiment 1: effect of ouabain on resting airway tone in
bovine trachea and effect of amiloride on the response
Tissue from six animals was studied. Four strips were
dissected from each trachea and studied in parallel. A
cumulative histamine concentration-response study was
performed on each strip, increasing amounts of histamine
being added to each bath to produce bath concentrations
over the range 10-7-10-3 mol/l. The tissue was then
washed repeatedly and left for approximately 1 h until the
tension had returned to baseline values. Amiloride was
then added to one bath (to a concentration of lo-' mol/l)
for 15 min before adding ouabain (bath concentration
mol/l). Ouabain was added to the other three strips
to achieve bath concentrations of
1 W 5 and lo-'
mol/l and the change in tension was observed over the
next hour. The contraction caused by ouabain was
expressed as a percentage of the maximum histamine
response in each strip. The effect of amiloride on the
maximum contraction produced by 10 - 4 mol/l ouabain
was assessed by using Student's paired t-test.
Methods
Fresh bovine tissue was obtained immediately after
death from the local abattoir. Tissue was transported in
ice to the City Hospital, where it was immediately dissected and bathed in Krebs-Henseleit solution (KHS) of
the following composition (mmol/l): Na+Cl-, 118;
K'CI-, 4.7; Mg2+ SO:-, 1.2; Na+H,PO,, 1.2; Ca2+C1-,
2.5; Na+HCO;, 25; glucose, 11.1; pH 7.4. Macroscopically normal human lung tissue was obtained from thoracotomy specimens of patients undergoing lung resection
for carcinoma and placed immediately in KHS. Bovine
tracheal strips (free of epithelium and connective tissue)
were suspended under 2 g tension, and human bronchial
rings (with epithelium intact) under 1.5 g tension, in organ
baths containing KHS at 37°C continuously gassed with
95% 0 , / 5 % CO,. These tensions had previously been
shown to produce optimal, repeatable responses in similar preparations. Changes in tension were recorded on 4
Grass F T 0 4 force displacement transducers (Grass
Instruments, Quincy, MA, U.S.A.) and displayed on two
CR600 two-channel flat-bed recorders (J. J. Instruments,
Southampton, U.K.). Tissue was allowed to equilibrate
under tension for 1 h before each experiment.
Experiment 2: effect of phentolamine, nifedipine and
calcium-free fluids (plus EDTA) on ouabain-induced
contraction of bovine trachea
Drugs
Experiment 3: effect of ouabain on histamine responsiveness in bovine trachea
Amiloride hydrochloride, ouabain octahydrate, carbachol, dimethylsulphoxide (DMSO), ethylenediaminetetraacetic acid (EDTA) and nifedipine were purchased from
Tissue from six animals was studied. Four strips were
dissected from each trachea and studied in parallel. A
cumulative histamine concentration-response study
(10-7-10-3mol/l) was performed on each strip. The
tissue was then washed repeatedly and left for approximately 1 h until the tension had returned to baseline
values. One of the following drugs was then added to each
bath (bath concentration in parentheses): phentolamine
(
mol/l), nifedipine (
mol/l), control (DMSO 1 in
1000) or calcium-free KHS (plus EDTA) ( 10-3 mol/l).
mol/l) was added to
Fifteen minutes later ouabain (
each bath and the change in tension was observed over
the next hour. The contraction caused by ouabain was
expressed as a percentage of the maximum histamine
response in each strip. The effects of phentolamine,
nifedipine and calcium-free fluids (plus EDTA) on the
maximal contractions produced by 10- mol/l ouabairi
were assessed by using Student's paired t-test.
Tissue was obtained from eight animals. Two strips
from each trachea were dissected and studied in parallel.
Sodium transport and airway smooth muscle
A cumulative histamine concentration-response study
(10-7-10-3 mol/l) was performed on each strip. The
tissue was washed repeatedly and allowed to return to
baseline tension over 1 h. Ouabain was added, at a bath
concentration of
mol/l, to one of each pair of strips;
the other strip acted as a time-matched control. After 30
min, the cumulative histamine concentration-response
study was repeated. The concentration of histamine causing a 20% maximal contraction (EC,,,) before and after
ouabain was calculated by interpolation on a log concentration-response plot. The changes in the log EC,,, for
histamine between ouabain-treated and control strips
were compared by using Student's paired t-test. EC,,, was
measured on the assumption that if ouabain were to
potentiate histamine-induced contractions, the effect
would be more pronounced at lower histamine concentrations. The repeat histamine concentration-response
curve in the control strips provided evidence as to
whether or not the histamine response was maintained
over the 90 min of the study, with reference to experiments 1 and 2 .
317
Experiment 6: effect of amiloride on strips pre-contracted
with carbachol and on carbachol responsiveness in bovine
trachea
Tissue from eight animals was studied. Four strips were
dissected from each trachea and studied in parallel. A
cumulative carbachol concentration-response study
(10-7-10-3 mol/l) was performed on each strip. The
strips were washed twice after which they remained
80-90°/0 contracted. One strip then served as a timematched control and amiloride was added in different
doses to the other three strips to give bath concentrations
of 1W5,
and
mol/l. After 30 min the strips
were washed several times and allowed to relax back to
baseline over 1 h. Amiloride was then re-added to three
strips in the same doses as previously, with one strip again
acting as a time-matched control, and the cumulative
carbachol concentration-response study was repeated.
The effect of amiloride on precontracted strips, and the
change in the log EC,,, for carbachol between amiloride
and control was assessed by two-way analysis of variance.
RESULTS
Experiment 4: effect of ouabain on resting tension and
methacholine responsiveness in human bronchial rings
Fresh human tissue was obtained from eight thoracotomy specimens. Paired bronchial rings (2nd-5th order
bronchi) were dissected from each specimen and studied
in parallel. The experimental protocol was the same as in
experiment 3, except that methacholine was used as the
constrictor agent instead of histamine. Methacholine
was chosen, since this is the cholinergic agent most commonly used to challenge asthmatic subjects in viva
Contraction caused by ouabain was expressed as a percentage of the maximum methacholine response after
subtracting any change in tension that occurred in control
rings. The change in the log EC,,, for methacholine
between ouabain-treated and control rings was compared
by using Student's paired t-test.
Experiment 5: effect of amiloride on resting tension and
histamine responsiveness in bovine trachea
Tissue was obtained from 10 animals. Four strips were
dissected from each trachea. A cumulative histamine concentration-response study ( 10-'- lo-' mol/l) was performed on each strip. The tissue was washed repeatedly
and allowed to return to baseline tension over 1 h.
Amiloride at
and
mol/l was then added
to three of the strips, with the fourth strip acting as a timematched control; after 15 rnin the histamine concentration-response study was repeated as previously. The
ECs,,(concentration causing 50% maximum contraction)
values for histamine before and after drug were calculated
by interpolation on a log concentration-response plot.
The change in the log ECS0and in the maximum tension
generated were assessed by two-way analysis of variance.
Experiment 1: effect of ouabain on resting airway tone in
bovine trachea and effect of amiloride on the ouabain
response
Ouabain caused a concentration-related, slowly
developing contraction that was maximal at 25-35 min at
all concentrations tested. The mean (SEM) peak change in
tension after l W h ,
and 1 W 4 mol/l ouabain was 19
(6), 27 ( 5 ) and 32 (7)% of the maximal response to histamine, respectively (Figs. l a and l b). Contraction was
significantly less in the strips pretreated with
mol/l
arniloride, the mean (SEM) change in tension being 7
(1. ~ ) O / O of the maximal histamine response ( P = 0.015 )
(Fig. 1b ) .
Experiment 2: effect of phentolamine, nifedipine and
calcium-free fluid (plus EDTA) on ouabain-induced
contraction of bovine trachea
As in the previous experiment, ouabain caused contraction, the mean (SEM) change in tension reaching a peak
of 47 (6)% of the maximal response to histamine. Phentolamine did not alter the response, the mean (SEM) change in
tension with phentolamine being 39 (24)% ( P = 0.5). Both
nifedipine and calcium-free fluid (plus EDTA) reduced
the response to ouabain, to 16 ( 13)"/o ( P= 0.000 1) and 13
(11)% ( P= 0.0008), respectively (Fig. 1c).
Experiment 3: effect of ouabain on histamine responsiveness in bovine trachea
mol/l) contracted the tracheal tissue as
Ouabain (
previously, the mean (SEM) tension at 30 min being
30 (7)% of the maximum response to histamine. There
was no contraction in the time-matched control strips.
Values of the EC2,,for histamine [geometric mean (SEM in
log mol/l)] before and after ouabain were 5.2 X
(0.2)
A. J. Knox et al.
T
40
501
T
C
0
.-c
E
+
3
0
10
30
20
40
log([Histamine](mol/l))
Time (min)
"1
T
(b'
40
20
I
10-6
10-5
10-~
Ouabain concn. (mol/l)
mol/l
Arniloride +
mol/l
ouabain
50
T
40
30
20
10
0
Control
Nifedipine Phentolamine Ca2+free
ouabain
contraction
Fig. 1. ( a )Time course of contraction produced by lo-'
mol/l ouabain in bovine trachea. Values are means fSEM
( n= 6 experiments). ( b ) Contraction produced by different concentrations of ouabain and the effect of amiloride
pretreatment. Values are means fSEM ( n= 6). (c) Effect
of nifedipine (
mol/l), phentolamine (
mol/l) and
calcium-free fluid (plus EDTA) ( l o - ' mol/l) on contracmol/l). Values are
tion produced by ouabain
means fSEM ( n = 6).
-9
-8
-7
-6
-5
-4
-3
log([Histamine](mol/l)}
Fig.2. Histamine concentration-response studies before
and after ouabain and subtracted curve ( b ) compared
with repeat histamine concentration-response studies in
control strips ( a ) in bovine trachea. ( a ) 0 , Control 1; 0 ,
control 2. ( b )0 , Before ouabain; 0 , after ouabain; 0 , subtracted curve. Values are means f SEM ( n= 8).
mol/l and 1.1 x
(0.38) mol/l compared with
6.4~
(0.18) mol/l and 6.9 x
(0.28) mol/l in the
control strips. The histamine response was thus well
maintained over the time course of this experiment which
was similar in time course to experiments 1 and 2 (Fig.
2a). The change in the EC,,, for histamine in ouabaintreated and control rings did not differ significantly
( P = 0.38). Fig. 2( b ) shows the histamine dose-response
study before and after ouabain. As ouabain caused
contraction, the histamine dose-response study after
ouabain was performed from a pre-constricted baseline.
The effect of subtracting the ouabain-induced change in
tension from the histamine response is shown as the subtracted curve.
Experiment 4: effect of ouabain on resting tension and
methacholine responsiveness in human bronchial rings
Ouabain caused a slowly developing contraction of
human airways over 30 min, the mean (SEM) peak change
Sodium transport and airway smooth muscle
in tension being 40 (7)0/0of the maximum response to
methacholine at 30 min. The geometric mean values of
the EC,, for methacholine (SEM in log mol/l) before and
after ouabain were 4.5 x lo-' (0.18) mol/l and 4.4 X
(0.2) mol/l compared with 5.3 x lo-' (0.14) mol/l and
2.3 x
(0.29) mol/l in the control rings. There was no
significant difference in the change in EC2, for methacholine between ouabain-treated and control rings ( P = 0.2).
Fig. 3(b ) shows the methacholine dose-response studies
before and after ouabain, with the subtracted curve.
319
1201 ( a )
h
am
100
E
LI
'g
2
40
3
20
Experiment 5: effect of amiloride on resting tension and
histamine responsiveness in bovine trachea
Amiloride had no effect on resting tension, but caused
a significant increase in the EC,, for histamine and a
reduction in the maximum tension generated with histamine. The mean change in the EC,, for histamine was
1.5-fold in the control strip and 2.6-, 7.7- and 34.7-fold
after
and
mol/l amiloride, respectively
(Figs. 4a-4d) (P<O.OOl). The mean change in the maximum tension generated was + 13% in the control strip,
and -670, -28%and -41%after lo-', 10-4and
mol/l amiloride, respectively ( P < 0.001).
0
-9
-8
-7
-6
-5
-4
log([Methacholine](mmol/l)}
-3
l2O1 ( b )
u
o
80
60
40
Experiment 6: effect of amiloride on strips preconstricted with carbachol and on carbachol responsiveness in bovine trachea
Amiloride caused a concentration-related relaxation,
maximal at 20 min, of strips pre-contracted with carbachol ( P < O . O O l at all time points) (Fig. 5). The EC,, for
carbachol was increased after amiloride, the mean change
being 0.6-fold in the control strip and 1.4-, 5.6- and 86fold after
and
mol/l amiloride, respectively (Fig. 6a-6d) (P<O.OOl). The mean change in the
maximum tension generated was + l o % in the control
strip and +1%, -2% and -31% after lo-,,
and
10- mol/l amiloride, respectively ( P= 0.1 ).
DISCUSSION
The aim of this study was to determine whether drugs
which alter membrane sodium transport affect airway
smooth muscle contractility. We used ouabain, which inhibits Na+/K+-ATPase [l, 141, and amiloride, which
inhibits Na+ entry, Na+/H+exchange [15] and Na+/Ca2+
exchange [16, 171. Alterations in the activity of these
sodium-transport systems would be expected to alter
smooth muscle contractility by one of three mechanisms.
First, any alteration of intracellular sodium concentration
would cause an alteration in intracellular calcium via
Na+/Ca2+exchange [18].Secondly, the Na+/H+antiport
regulates pH and alterations in intracellular pH have been
shown to alter the activity of enzymes involved in contractile protein phosphorylation, such as calmodulin [19].
Thirdly, alterations in membrane potential would alter
contractility [20].
20
0
-9
-8
-7
-6
-5
-4
log([Methacholine](mol/l))
-3
Fig. 3. Methacholine concentration-response studies
before and after ouabain and subtracted curve ( b )compared with repeat methacholine concentration-response
studies in control rings ( a )in human bronchi. ( a )0 , Control 1; 0, control 2. ( b )
Before ouabain; 0 , after
ouabain; 0 , subtracted curve. Values are means f SEM
(n=8).
.,
There has been relatively little work done to determine
which sodium-transport systems are important in controlling the function of airway smooth muscle and most of
this has concentrated on the Na+/K+ pump. Souhrada et
al. [21] first demonstrated the presence of Na+/K+ATPase in guinea-pig and bovine airways, and Bullock et
al. [8] showed that ouabain caused contraction of bovine
airways. Bateman et al. [22], however, were unable to
show any effect of ouabain in the rat lung, suggesting a difference in Na+/K+ pump activity between species. More
recently, Chideckel et al. [9] looked at the effect of
ouabain in airway tissue from several species and showed
that it produced contraction in guinea-pig and canine
trachea and in some human post-mortem tracheal specimens and that this was not inhibited by atropine or antihistamines. The present study confirms that ouabain
causes a dose-related contraction of bovine airways.
Ouabain also contracted human airways in our studies,
A. J. Knox et al.
320
(b)
-9
-8
-7
-6
-5
-4
-3
-9
-8
log{[Histamine](mol/l)]
-7
-6
-5
-4
-3
log{[Histamine](mol/l)}
120,
-9
-8
-7 - 6 -5 -4 - 3
log{[Histamine](mol/l)}
.,.,
-9
-8
- 7 - 6 -5 -4 - 3
log{[Histamine](mol/l)}
.,
Fig. 4. Effect of different concentrations of amiloride on the histamine concentration-response
mol/l
curve. Values are mean sf^^^ ( n = 10).( a ) Control 1; 0 , control 2 . ( b ) Before
amiloride; 0 , after 1WSmol/l amiloride. ( c ) Before
mol/l amiloride; 0 , after lW4mol/l
mol/l amiloride; 0 , after 10 - 3 mol/l amiloride.
amiloride. ( d ) Before
.,
3
-80- 100,
0
I
I
5
10
Time (min)
15
20
Fig. 5. Effect of different concentrations of amiloride on
tissue preconstricted with carbachol. Values are
lo-' mol/l amiloride;
means k SEM ( n= 8). 0 , Control;
mol/l amiloride; 0 ,
mol/l amiloride.
.,
+,
suggesting that modification of Na+/K+-ATPase activity
can alter airway smooth muscle tension in man. In addition, we studied the possible mechanism of contraction.
Phentolamine, an a-adrenergic blocker, had no effect on
ouabain-induced contractions, suggesting that release of
noradrenaline from sympathetic nerve endings was not
responsible for the contraction, unlike some vascular
smooth muscle preparations [23]. Calcium-free KHS and
nifedipine, an inhibitor of the voltage-dependent calcium
channel, inhibited oubain-induced contraction. This suggests that ouabain-induced contraction is partly due to
membrane depolarization with calcium influx via voltagedependent calcium channels, as is the case in some vascular smooth muscle preparations [4].
We found that ouabain-induced contraction was
markedly inhibited by amiloride. These findings have
several possible explanations. A similar sequence of
events may take place in airway smooth muscle as has
been described in cardiac muscle, whereby inhibition of
Na+/K+-ATPase leads to accumulation of intracellular
sodium via the N a + / H + antiport, and the increase in
intracellular sodium causes a subsequent rise in intracellular calcium via Na+/Ca2+exchange [2]. As amiloride
inhibits both N a + / H +exchange and Na+/Ca*+ exchange,
the inhibitory effect of amiloride on ouabain-induced
contraction could be due to inhibition of either or both of
these channels. Inhibition of the voltage-dependent calcium channel has also been reported with amiloride in
cardiac tissue [24] and this is an alternative explanation
for its effect. However, the fact that amiloride was more
effective than nifedipine in our studies suggests that its
effects are not solely on the voltage-dependent calcium
32 1
Sodium transport and airway smooth muscle
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5
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20-
7
-9
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-8 -7 - 6
-5 -4
log([Carbachol](mol/l)}
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.,.,
04-9
-9
-8 - 7 - 6 - 5 - 4 -3
log([Histamine] (mol/l)}
-8
-7
- 6 - 5 - 4 -3
log([Carbachol](mol/l)}
Fig. 6 . Effect of different concentrations of amiloride on the carbachol concentration-response
mol/l
curve. Values are mean sf^^^ ( n = 8 ) . ( a ) Control 1; 0 , control 2. ( b )U, Before
mol/l amiloride; 0 , after
mol/l
amiloride; 0 , after lo-' mol/l amiloride. (c) Before
mol/l amiloride; 0 , after lO-'mol/l amiloride.
miloride. ( d )a, Before
channel. Further contractile studies with more selective
drugs and more complex electrophysiological studies are
needed to resolve the issue.
We also studied the effect of ouabain on the contractile
response to histamine. One possible explanation for the
association between dietary salt and bronchial reactivity
[6, 71 is that increased dietary salt intake leads to
increased levels of one or more circulating N a + / K + ATPase inhibitors from the hypothalamus [ l l , 121. These
would cause an increase in smooth muscle sodium and
calcium concentrations and thus increase airway smooth
muscle contractility and bronchial reactivity. Ouabain did
not alter histamine o r methacholine responsiveness in airway smooth muscle in our study in contrast to its effect in
vascular smooth muscle, where several studies have
shown that ouabain and digoxin increase the sensitivity to
contractile agents in animals and man [4,25,26].
In addition to our studies with ouabain, we performed
several studies looking at the effect of amiloride on
contractile responses in bovine trachea. Amiloride, at
concentrations of 10 pmol/l and above, caused marked
inhibition of both histamine and carbachol responses in
our studies and a dose-dependent relaxation of strips precontracted with carbachol. The effect of amiloride on
histamine and carbachol responses has not been studied
previously, although in a study by Souhrada & Souhrada
[27] amiloride reduced the contractile response to antigen
in sensitized guinea-pig airways. Several mechanisms
need to be considered to explain the effect of amiloride.
In concentrations of approximately 1 pmol/l amiloride
inhibits a sodium-entry channel [15]. An effect on this
channel is unlikely to explain our results as concentrations
of amiloride of 10 ,umol/l or above were required to produce an effect. Amiloride, at these concentrations, has
been shown to inhibit the Na+/H antiport [ 151 and Na+/
Ca2+exchange [16, 171. It has also been reported to have
other effects such as inhibition of protein kinase C [28],
inhibition of the voltage-dependent calcium channel [24]
and weak a-adrenergic blocking activity [29]. In view of
the multiplicity of the reported actions of amiloride it is
impossible to determine precisely which of these mechanisms was responsible for the effects seen in our study.
Inhibition of either N a + / H + exchange or protein kinase C
would appear to be the two most likely. Protein kinase C
is thought to be responsible for the maintenance phase of
airway smooth muscle contraction [30] and protein kinase
C-induced activation of N a t / H + exchange is thought to
be an integral part of the signal transduction pathway in
platelets [311. Inhibition of the voltage-dependent calcium
channel would seem less likely as the effects of this group
of drugs on receptor-operated contraction in airway
smooth muscle have been small in most species that have
been studied [32-351. Electrophysiological studies looking at the effects of amiloride on calcium influx through
+
322
A. J. Knox et al.
this channel are, however, needed to resolve this issue, as
are studies using analogues of amiloride with a greater
specificity for the Na+/Ca2+and N a + / H + channels [36,
371. However, irrespective of the mechanism of action of
amiloride, our studies raise the question of whether
amiloride may have a role in preventing the airway
response to bronchoconstrictor stimuli in uivo in man.
We also showed that ouabain, an N a + / K + pump inhibitor, causes airway smooth muscle contraction, suggesting that it might be possible to alter airway smooth
muscle contractility and airway reactivity in man by
administering drugs which modulate sodium transport.
We have investigated this in a series of studies in normal
and asthmatic subjects [ 131.
ACKNOW LEDGMENTS
We thank Mr P. Kemp for assistance with the technical
aspects of initiating these studies, and the Asthma
Research Council for supporting A.J.K. We are also
grateful to Mason Brothers, Nottingham, U.K., for donating bovine tissue, and Mr W.E. Morgan, Mr ED. Salama
and Professor S.P.S. Jones at Nottingham City Hospital
for providing us with human tissue.
13. Knox, A.J., Britton, J.R. & Tattersfield, A.E. Effect of
sodium-transport inhibitors on bronchial reactivity in vivo.
Clin. Sci. 1990; 79, 325-30.
14. Smith, T.W., Antman, E.M., Friedman, P.L., Blatt, C.M. &
Marsh, J.D. Digitalis glycosides: mechanisms and manifestations of toxicity. Prog. Cardiovasc. Dis. 1984; 26,495-523.
15. Benos, D.J. Amiloride: a molecular probe of sodium transport in tissues and cells. Am. J. Physiol. 1981; 242,
c 13 1-45.
16. Floreani, M. & Luciani, S. Amiloride: relationship between
cardiac effects and inhibition of Na+/Ca2+exchange. Eur. J.
Pharmacol. 1984; 105,317-22.
17. Debetto, P., Floriani, M., Carpenedo, F. & Luciani, S. Inhibition of the Na+/Ca'+ exchange in cardiac sarcolemmal
vesicles by amiloride. Life Sci. 1987; 40, 1523-30.
1 8. Reuter, H., Blaustein, M.P. & Haeusler, G. Na+-Ca2+
exchange and tension development in arterial smooth
muscle. Philos. Trans. R. Soc. London Ser. B 1973; 265,
87-94.
19. Busa, W.B. & Nuccitelli, R. Metabolic regulation via intracellular pH. Am. J. Physiol. 1984; 246, R409-38.
20. Bolton, T.B. Mechanisms of action of transmitters and other
substances on smooth muscle. Physiol. Rev. 1979; 59,
606-7 18.
21. Souhrada, M., Souhrada, J.F. & Cherniak, R.M. Evidence
for a sodium electrogenic pump in airway smooth muscle. J.
Appl. Physiol.: Respir. Environ. Exercise Physiol. 1981; 51,
346-52.
22. Bateman, C.J., Twort, C.H.C. & Cameron, I.R. The effect of
23.
REFERENCES
1. Smith, T.W. Digitalis. Mechanisms of action and use. N.
Engl. J. Med. 1988; 318, 358-65.
2. Frelin, C., Vigne, P. & Lazdunski, M. The role of the Na'/
H' exchange system in cardiac cells in relation to the control of the internal Na+ concentration. J. Biol. Chem. 19x4;
259,8880-5.
3. Reeves, J.P. & Sutko, J.L. Sodium-calcium exchange in
cardiac membrane vesicles. Proc. Nat. Acad. Sci. U.S.A.
1970; 76, 590-4.
4. Mikkelsen, E., Anderson, K.E. & Pederson, O.L. Effects of
digoxin on isolated human mesenteric vessels. Acta
Pharmacol. Toxicol. 1070; 45,25-3 1 ,
5. Brading, A.F. & Lategan, T.W. Na+-Ca?+ exchange in
vascular smooth muscle. J. Hypertens. 1985; 3, 109- 16.
6. Burney, P.G.J., Neild, J., Twort, C. et al. The effect of changing dietary sodium on the bronchial response to histamine.
Thorax 1989; 44,36-41.
7. Javaid, A., Cushley, M.J. & Bone, M.F. Effect of dietary salt
on bronchial reactivity to histamine in asthma. Br. Med. J.
1988; 297,454.
8. Bullock, C.G., Fettes, J.J.F. & Kirkpatrick, C.T. Tracheal
smooth muscle:- second thoughts on sodium-calcium
exchange.J. Physiol. (London) 1981; 318,461'.
9. Chideckel, E.W., Frost, J.L., Mike, P. & Fedan, J.S. The
effect of ouabain on tension in isolated respiratory tract
smooth muscle of humans and other species. Br. J. PharmaCOI.1987; 92,609- 14.
10. Agrawal, K.P. & Hyatt, R.E. Airway response to inhaled
ouabain and histamine in conscious guinea pigs. J. Appl.
Physiol. 1986; 60, 2089-93.
11 De Wardener, H.E., MacGregor, G.A., Clarkson, E.M.,
Alaghband-Zadeh, J., Bitensky, L. & Chayen, J. Effect of
sodium intake on ability of human plasma to inhibit renal
Na+-K+ adenosine triphosphatase in v i m . Lancet 198 I ; i,
411-12.
12 Swann, A.C. Na+-K+-ATPase regulation and Na+Cl+
intake: effects on circulating inhibitor and sensitivity to noradrenaline. Clin. Sci. 1985; 69,441-7.
24.
25.
26.
alteration of the transmembrane sodium gradient on airways
smooth muscle contraction. Clin. Sci. 1986; 70 (Suppl. 13),
65P.
Karaki, H. & Urakawa, N. Possible role of endogenous catecholamines in the contractions induced in rabbit aorta by
ouabain, sodium depletion and potassium depletion. Eur. J.
Pharmacol. 1977; 43,65-72.
Kleyman, T.R. & Cragoe, E.J. Amiloride and its analogues
as tools in the study of ion transport. J. Membr. Biol. 1988;
105, 1-21.
Mir, M.A., Morgan, K., Chappell, S. et al. Calcium retention
and increased vascular reactivity caused by a hypothalamic
sodium transport inhibitor. Clin. Sci. 1988; 75, 197-202.
Broekaert, A. & Codfraind, T. The actions of ouabain on
isolated arteries. Arch. Int. Pharmacodyn. Ther. 1973; 203,
393-5.
27. Souhrada, M. & Souhrada, J.F. Sensitisation induced
sodium flux in airway smooth muscle cells. Am. Rev. Respir.
Dis. 1985; 131. A356.
28 Besterman, J.M., May, W.S., Levine, H. & Cragoe, E.J. &
Cuatrecasas, P. Amiloride inhibits phorbol ester-stimulated
N a + / H + exchange and protein kinase C. J. Biol. Chem.
1985; 260, 1155-9.
29 Bhalla, R.C. & Sharma, R.V. Competitive interaction of
amiloride and verapamil with alpha- 1 adrenoceptors in
vascular smooth muscle. J. Cardiovasc. Pharmacol. 1986; 8,
927-32.
30. Siffert, W. & Akkerman, J.W.N. Activation of sodium-
proton exchange is a prerequisite for Ca mobilisation in
human platelets. Nature (London) 1987; 325,456-8.
31 Rasmussen, H., Takuwa, Y.& Park, S. Protein kinase C in
the regulation of smooth muscle contraction. FASEB J.
1987; 1,177-85.
32. Fanta, C.H., Venugopalan, C.S., Lacouture, P.G. & Drazen,
J.M. Inhibition of bronchoconstriction in the guinea-pig by a
calcium channel blocker, nifedipine. Am. Rev. Respir. Dis.
1982; 125,61-6.
33 Cheng, J.B. & Townley, R.G. Pharmacological characterisation of the effects of nifedipine on isolated guinea-pig
and rat tracheal smooth muscle. Arch. Int. Pharmacodyn.
Ther. 1983: 236.228-42.
34. Farley, J.M. & Miles, P.R. The sources of calcium for acetylcholine induced contractions of dog tracheal smooth
muscle. J. Pharmacol. Exp. Ther. 1978; 207,34 1-6.
I
,
Sodium transport and airway smooth muscle
35. Drazen, J.M., Fanta, C.H. & Lacouture, P.G. Effect of
nifedipine on constriction of human tracheal strips in v i m .
Br. J. Pharmacol. 1983; 78,687-91.
36. Siegl, P.K.S., Cragoe, E.J., Trumble, M.J. & Kaczorowski,
G.J. Inhibition of Na+/Caz+exchange in membrane vesicles
323
and papillary muscle preparations by analogues of
amiloride. Proc. Natl. Acad. Sci. U.S.A. 1984; 81,3238-42.
37. Vigne, P., Frelin, C., Cragoe, E.J. & Ladzunski, M.
Structure-activity relationships of amiloride and certain of
its analogues in relation to the blockade of the Na+/H+
exchange system. Mol. Pharmacol. 1983; 25, 131-6.