Download Sympathomimetic Drugs Stimulate the Output of

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Clinical Science (1982) 63,23-28
Sympathomimetic drugs stimulate the output of secretory
glycoproteins from human bronchi in vitro
R. J. P H I P P S , I. P. WILLIAMS, P. S. R I C H A R D S O N , J. PELL, R. J. P A C K
A N D N. W R I G H T
Department of Physiology,St George’sHospital Medical School, London
(Received 10 June 1981f11 January 1982; accepted 27 January 1982)
Summary
Introduction
1. We describe a method for supporting pieces
of human bronchi in Ussing chambers, for
radiolabelling the contents of the secretory cells
with 35S, and for collecting radiolabelled macromolecules secreted on to the luminal aspect of
the tissue. This method has previously been used
to study airway secretions in animals [R. J.
Phipps, J. A. Nadel & B. Davis, American
Review of Respiratory Disease, (1980) 121,
359-3651. Evidence is given that the radiolabelled molecules are secretory glycoproteins,
probably mucus glycoproteins.
2. Phenylephrine, an a-adrenoceptor agonist,
increased the rate at which the bronchi secreted
radiolabelled glycoproteins. Thymoxamine, an
a-adrenoceptor antagonist, blocked this effect but
propranolol, a /I-adrenoceptor antagonist, did
not.
3. Dobutamine, a /I,-adrenoceptor agonist,
increased the rate of secretion of radiolabelled
glycoproteins. Propranolol blocked this but
thymoxamine did not.
4. Salbutamol, a /I,-adrenoceptor agonist, also
increased the rate of secretion of radiolabelled
glycoproteins. Propranolol blocked this effect.
5. We conclude that both (F and P
adrenoceptor agonists increase the rate of glycoprotein secretion in human bronchi in vitro and
that this almost certainly means that they
increase the rate of mucus secretion.
In animals both (lc- and Padrenoceptor agonists
stimulate the secretion of mucus into the airway .
[l-61. In man sympathomimetic drugs are used
widely in the treatment of lung disease but their
effects on mucus secretion are uncertain. Two
papers have claimed that such drugs have no
effect on the secretion rate of human bronchi [7,
81 but a recent study [91, using a method similar
to that of Boat & Kleinerman [81, found that
a-adrenoceptor agonists enhance secretion
whereas Padrenoceptor agonists do not and may
even inhibit secretion. In this paper we describe
the application of a different technique in vitro
[ 11 to the study of the effect of sympathomimetic
drugs on secretion by human bronchi. Preliminary accounts of some of these results have
been presented [ 10, 1 11.
Key words: a-adrenoceptor, Padrenoceptor,
airways, bronchi, glycoproteins, mucus.
Correspondence: Dr P. S. Richardson, Department
of Physiology, St George’s Hospital Medical School,
Cranmer Terrace, London SW17 ORE.
Methods
The methods, as applied to animal airways, have
been described previously [l, 21 but we describe
their application to human bronchi in some detail
here.
Tissue preparation
For this study we used a total of 30 pieces of
main-stem, lobar or segmental bronchus from
human lungs, surgically removed from 15
patients with bronchial carcinoma. After resection the lung tissue was placed in cold (OOC)
Krebs-Henseleit solution, gassed with 5% CO, in
air. With the lung tissue still immersed in ice-cold
Krebs-Henseleit solution pieces of bronchi well
away from the tumour were dissected free from
0143-5221/82/070023-06$01.50 0 1982 The Biochemical Society and the Medical Research Society
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R . J. Phipps et al.
Krebs 37'C
Time (h)
FIG. 1. Diagram showing a piece of bronchus mounted between the two halves of a modified Ussing
chamber. Oxygenated Krebs-Henseleit solution at
37°C was circulated through both half-chambers.
Secretions were collected by draining the luminal
half-chamber.
surrounding tissue, and opened. These pieces
were then mounted flat in modified Ussing
chambers. Both sides of the chamber were filled
with 15 ml of warm (37OC) Krebs-Henseleit
solution that was gassed with 0, + CO, (95 :5,
v/v) and continuously circulated. Two sizes of
chamber were used: the larger, of 12 mm internal
diameter, was used in 1 1 experiments, and the
smaller, of 6 mm internal diameter, in 19
experiments (Fig. 1).
Mucus collection and estimation
We collected radiolabelled glycoproteins by
draining the luminal half-chamber, usually every
15 min. This chamber was then refilled with
Krebs-Henseleit solution. At the beginning of
each experiment we added 4 . 0 mCi of [3sSlsulphate (148 MBq as Na,35S0,, The Radiochemical Centre, Amersham, U.K.) to the submucosal half-chamber. Washings from the
luminal half-chamber subsequently contained a
mixture of macromolecularly bound 35Sand free
%O,. All samples were dialysed exhaustively
against distilled water to remove the latter, and
against urea solution (6 mol/l) to dissolve the
mucus. Such dialysis completely dispersed unbound [35Slsulphateeven in the presence of dilute
serum protein. The output of macromolecularly
bound 35S was measured by a liquid scintillation
counter (Intertechnique SL-30). A correction was
FIG.2. Rate of output of macromolecularly bound 35S
against time in one experiment. Phenylephrine (PHE),
dobutamine (DOB) and salbutamol (SAL) all increased the rate of output.
made for quenching by an external standard
channels ratio method, and the output expressed
as bound radioactive disintegrations per second
(becquerels) per minute of the collection period
(i.e. Bq/min).
Administration of drugs
The drugs used were phenylephrine hydrochloride, 4.9 x
mol/l (Boots Co. Ltd),
dobutamine hydrochloride, 4- 1 x
mol/l
(Dobutrex, Lilly), salbutamol sulphate, 4 . 2 x
mol/l (Ventolin, Allen and Hanburys Ltd),
mol/l
thymoxamine hydrochloride, 3.2 x
(Opilon, Warner) and propranolol hydrochloride, 3.4 x
mol/l (Inderal, I.C.I.).
To 12 tissues we gave agonists without
antagonists. After a control period of 2.5 h one of
the three agonists was added to both sides of the
chamber for one 15 min sampling period. Then
both sides of the chamber were drained and
refilled with Krebs-Henseleit solution; the [35Slsulphate on the submucosal side was also
replenished. This procedure was repeated for the
two other agonists (Fig. 2). Three 15 min control
periods were left between drug treatments. The
three agonists were given in different orders in
different preparations. In separate experiments
we examined the effects of either thymoxamine
(five tissues) or propranolol (1 3 tissues) on the
responses to the agonists. For these experiments
the antagonist was added to both sides of the
chamber and remained there throughout the
experiment. Here only two agonists were tested:
phenylephrine and one of the Padrenoceptor
stimulants.
Sympathomimetic drugs and bronchial glycoproteins
25
Analysis of results
The output of radioactivity during a 15 min
period, when an agonist was added to the
chamber, was calculated as a percentage change
of output from the immediately preceding control
period. Changes in mucus output are expressed
as median percentage changes (A). The percentage changes were not normally distributed so
non-parametric statistical tests have been used.
The significance of a particular change relative to
zero effect was tested with the ranked sign test:
the significance of the difference between effects
of an agonist with and that without a particular
antagonist was tested with the Mann-Whitney
U-test. A difference was considered statistically
significant if P < 0.05: N.S.means P > 0-05.
immersion in neutral-buffered 10% formol saline.
The tissues were then dehydrated, cleared and
embedded in wax. A series of six to ten sections
of 5 pm thickness were cut, mounted on
gelatinized slides and stained with periodic
acid/Schiff reagent. They were then prepared as
radioautographs by the stripping film technique
(Kodak AR10). After exposure in the dark at
4OC for 1-21 days, selected slides were
developed with Kodak D19 and examined for
silver grains. For seven tissues (from five
patients) it proved possible to calculate the Reid
index 1131. The mean value was 0.43 (range
0.29-0.60), consistent with a moderate hypertrophy of bronchial glands.
Biochemical analyses
Dialysed material collected from the luminal
chambers of the Ussing chamber was pooled and
concentrated on an Amicon XM 50 filter, which
retains molecules of more than 50 000 daltons. A
portion (4 ml) of the concentrated sample was
fractionated by gel exclusion chromatography on
a Sepharose CL2B column (dimensions 2.6 cm x
70 cm) and the specimen was eluted with sodium
phosphate buffer (10 mmol/l, pH 7.2), containing
urea (6 mol/l) and 0.01% (w/v) sodium azide.
The 3'S content of each fraction was measured.
The void volume of the column was measured
with Blue Dextran.
On four occasions we pooled all the washings
from an experiment, concentrated them as above
and measured the monosaccharide contents by
gas-liquid chromatography 121 and the amino
acid content on an LKB 4400 amino acid
analyser.
Results
Radioautography and histology
At the end of 11 experiments the tissues were
removed from the chambers and fixed by
Influence of drugs on glycoprotein secretion
A total of 30 tissues were studied: five were
pretreated with thymoxamine, 13 with propranolol and 12 with neither drug.
Phenylephrine, a relatively selective cradrenoceptor agonist, significantly increased the secretion of %-labelled glycoproteins (Table 1, Fig.
2). 8-Adrenoceptor blockade with propranolol
did not alter the response but the cradrenoceptor
antagonist, thymoxamine, abolished it (Table 1).
Dobutamine, a relatively selective &adrenoceptor agonist, significantly increased the secretion of radiolabelled glycoproteins (Table 1, Fig.
2). Thymoxamine did not significantly alter this
response but propranolol lessened it (Table 1).
Salbutamol, a relatively selective &adrenoceptor agonist, increased the secretion of radiolabelled glycoprotein (Fig. 2). Propranolol
abolished this response. The action of thymoxamine was not tested (Table 1).
With all the agonists the effects varied greatly
from one experiment to another. We consider
TABLE 1. Median changes in rate of secretion of 3sS-labelled glycoprotein produced by
adrenoceptor agonists
Ranges and number of trials (n) are given in parentheses. Significance of increase in secretory rate,
tested by ranked sign test: *P < 0.05; **P < 0.01. Significance of a decrease in effect with antagonist
compared with effect with agonist alone, tested by the Mann-Whitney U-test: tP < 0.05.
Median change (%)
Phenylephrine
Agonist alone
Agonist with thymoxamine
Agonist with propranolol
+41**
(-44 to +550, n = 12)
+2t
(-33 to +4, n = 5 )
+39'
(+5to+185,n=5)
+46**
( - 1 6 t o + 8 6 , n = 13)
-
+4t
(-25 to + 114. n = 6)
Dobutamine
+44**
Salbutamol
(-39 to +175, n = 11)
+33**
( + I 1 to +150. n = 9)
+8t
(-22 to + 107, n = 7)
R . J. Phipps et al.
26
TABLE2. Rates of secretion of amino acids
Rate of secretion (nmol h-l cm-' of tissue)
Specimen no. ...
.-
.-
0
0
5
a
0
0
100
200
300
400
Elution volume (mi)
FIG 3. Radiolabelled washings from an Ussing
chamber, fractionated in a column of Sepharose CL2B
in sodium phosphate buffer (10 mmol/l, pH 7.2)
containing urea (6 mol/l) and 0.01% (w/v) sodium
azide. V, shows void volume.
Aspartic acid
Threonine
Serine
Glutamic acid
Proline
Glycine
Alanine
Valine
Isoleucine
Tyrosine
Phenylalanine
Lysine
Histidine
Arginine
Leucine
1
2
3
4
3.8
7.9
6.1
4.9
4.2
5.7
4.9
2.6
1.1
0.5
1.4
1.0
1.1
1.8
3.5
1.1
0.6
2.1
2.1
0.5
2.2
1.3
0.8
0.3
3.5
2.0
2.3
4.6
1.9
2.2
3.3
2.8
0.7
9.6
7.3
7.7
15.2
6.0
7.6
11.3
8.5
2.3
0.8
4.5
-
I .6
2.6
0.8
1.3
2.2
0.3
0.5
0.3
0.3
0.6
8.8
2.4
4.0
9.3
TABLE
3. Rates of secretion of monosaccharides
The ratios with respect to sialic acid are given in parentheses.
Rate of secretion
(nmol h-l cm-] of tissue)
Specimen no. ...
Fucose
Mannose
Galactose
N-Acetylglucosamine
N-Acetylgalactosamine
Sialic acid
2
3
3.1 (0.15) 1.7(0.61)
4.0 (0.20) 1.2 (0.43)
3.8 (0.19) 7.2 (2.57)
0.8 (0.04) 1.4 (0.50)
4.25 (0.21) 1.5 (0.54)
20.3 (1.00) 2.8 (1.00)
4
4.2(0.19)
1.1 (0.05)
13.4 (0.60)
4.7 (0.21)
17.5 (0.78)
22.3 (1.00)
In each case there was a peak of radioactivity
which eluted in the void volume but no peak in
the included or partially included volumes (Fig.
3).
possible reasons for this in the Discussion
section.
All the washings from each of four experiments
were pooled and their amino acids analysed. The
results, expressed as the mean output of amino
acid per cm2 of tissue per hour of secretion, are
shown in Table 2. Serine, threonine, proline
glutamic acid, aspartic acid, glycine and alanine
were the principal amino acids found. In three of
these specimens the monosaccharides were
analysed by gas-liquid chromatography. Six
sugars were consistently found. Their amounts
and their ratios with respect to sialic acid are
presented in Table 3. Uronic acids were never
detected.
Biochemical analyses of secreted macromolecules
Radioautography
On four occasions we pooled all the washings
from an Ussing chamber, concentrated them and
fractionated them on a Sepharose CL2B column.
Radioautography of the tissue after experiments showed that [3SSlsulphate was incorporated into all types of glycoprotein-producing
FIG. 4. The four main sites of bound '?3 incorporation. (A) A mucous (muc) and serous (ser) gland
acinus. Most of the silver grains are over the serous
(non-stained) acinus. The mucus in the duct is also
radiolabelled (+). (B) Epithelium of the bronchus
where both goblet cells (+) and the luminal surface
(*) are radiolabelled. (C) Chondrocytes also show
some radiolabelling
Bars = 20 ,urn.
(4).
Sympathomimetic drugs and bronchial glycoproteins
cells of the airway. Goblet cells and the surface
glycocalyx of the epithelium were both clearly
radiolabelled, together with a large proportion of
the gland acini (Fig. 4). The chondrocytes of the
cartilage and a few scattered cells in the lamina
propria also incorporated some 35S, but this was
much less than that taken up by secretory cells.
Discussion
The results show that adrenoceptor agonists
cause release of radiolabelled macromolecules
from human bronchi in uitro.
Origin and nature of the radiolabelled macromolecules
Radioautographs showed four principal sites
of 35S-radiolabellingin the airway wall: submucosal glands, luminal border of the airway
epithelium, epithelial goblet cells and chondrocytes. Submucosal glands and epithelial goblet
cells are known to secrete mucus glycoproteins
[141, and in this study radioautographs showed
radiolabelled material in the ducts of submucosal
glands. The microvillous border of the airway
contains a sulphated glycoprotein 115, 161, which
may be released into the lumen when the airway
is irritated [171. Chondrocytes synthesize
chondroitin sulphate, a proteoglycan 181, but
absence of uronic acids, major components of
chondroitin sulphate, in the washings confirms
that this proteoglycan was not a major source of
"S in the washings. The probable sources of the
radiolabelled macromolecules studied here were
submucosal glands and epithelial goblet cells.
These results do not allow us to say which of
these two the drugs stimulated, but in cat trachea
phenylephrine stimulates mucus glycoprotein
output from submucosal glands [51, and this may
also be true in human bronchi.
The 35Swas incorporated into macromolecules
which eluted in the void volume of a Sepharose
CL2B column even under dissociating conditions.
This implies that they were very large molecules
and is consistent with their identification as
mucus glycoproteins [2, 9, 19-221. Some of the
amino acids and sugars found in the washings are
more suggestive of serum glycoproteins than of
mucus glycoproteins (e.g. the relatively high
concentrations of glutamate, aspartate, mannose
and N-acetylgalactosamine). It is likely that the
early washings from the Ussing chambers contained some serum, from both exudate and
bleeding during surgery, but serum glycoproteins, which are not synthesized locally, would
not have become radiolabelled with 35S. Taken
27
together, the radioautographic and biochemical
evidence shows that the radiolabel was a marker
for secretory glycoproteins of high molecular
mass. These were probably mucus glycoproteins.
Adrenergic control of mucin secretion and its
clinical implications
Our results are consistent with the effects of aand &adrenoceptor agonists on airway mucus
output, which have been reported in cat and dog
[l-3, 5, 6, 231. Previous studies on human
bronchi in vitro have failed to show that
&adrenoceptor agonists alter the secretory activity of submucosal gland cells [71 or the
secretion of mucus glycoproteins [8, 91. One
possible explanation for the failure of other
workers to find the effects we report with
Padrenoceptor agonists is that they used sampling periods of 4 h and that this would make a
transient increase in secretion impossible to
detect. Our results do not establish whether
sympathomimetic drugs can have sustained
effects. Shelhamer et al. [9]recently reported that
a-adrenoceptor agonists stimulate mucus secretion and we confirm this study rather than
earlier ones [7,81, which denied such an effect.
One objection to these results is that peaks of
radioactivity might have occurred at random.
Indeed some washings contained unexpectedly
large quantities of radiolabel even in the absence
of an applied stimulus. The sudden freeing of a
bleb of mucus which had stuck to the bronchial
wall probably accounts for such 'noise' in the
method. Even so, the consistency of effects of the
agonists and their abolition only by the appropriate antagonist provides powerful evidence that
the effects described here were real. Another
reason for the variability in results may be that
the tissues had various degrees of submucosal
gland hypertrophy. Sturgess & Reid [71 showed
that cholinergic drugs stimulated the cells of
hypertrophied submucosal glands more than
those from normal glands and suggested that the
number of acetylcholine receptors increased with
gland hypertrophy. The same may apply to
adrenoceptors but we have insufficient results to
test this hypothesis.
&Adrenoceptor agonists are widely used in the
treatment of lung disease so effects on airway
mucus secretion may be important. A typical
dose (two puffs) of salbutamol from a metered
dose aerosol contains 200 pg of the drug, of
which about 20 pg is deposited in the lower
airways [241. It is dimcult to assess the volume of
lung tissue that dilutes the drug, but the concentration in submucosal gland is likely to reach 10
28
R . J. Phipps et al.
pg/ml (that used in this study) at least transiently
in patches of heavy aerosol deposition. PAdrenoceptor agonists may increase the rate of
mucociliary transport [25, 261, although this has
not been found consistently [271. Enhancement of
mucus secretion by the agonist might explain the
change.
Patients with asthma have slowed mucociliary
transport 1281 and mucus and cell debris accumulates in their airways [291. On the basis of the
results presented here it is likely that treatment of
asthma with adrenoceptor agonists, such as
isoprenaline and salbutamol, would increase the
rate of secretion and exacerbate the plugging of
the bronchi.
In other conditions the effects of adrenoceptor
agonists on glycoprotein secretion might be used
beneficially. If adrenoceptor agonists stimulate
glycoprotein secretion, they may change the
physical properties of airway mucus in such a
way as to hasten its clearance from the lungs
1301. This property seems worth testing because
adrenoceptor agonists, unlike cholinergic drugs,
do not narrow the airways.
References
Ill PHIPPS,R.J., NADEL,J.A. & DAVIS,B. (1980) Effect of
alpha-adrenergic stimulation on mucus secretion and on ion
transport in cat trachea in vitro. American Review of
Respiratory Disease, 121,359-365.
(21 GALLAGHER,
J.T., KENT,P.W.,
PASSATORE,
M., PHIPPS,R.J.
& RICHARDSON,
P.S. (1975) The composition of tracheal
mucus and the nervous control of its secretion in the cat.
Proceedings of The Royal Society of London B, 192,49-76.
131 DAVIS,B., CHIN, R., GRAP, P., POPOVAC,D. & NADEL,J.
(1979) Effects of phenylephrine and superior laryngeal nerve
stimulation on submucosal gland secretion in canine trachea in
vivo. Clinical Research, 27,55a.
[41 UEKI,I., GERMAN,V.F. & NADEL,J.A. (1980) Micropipette
measurement of airway submucosal gland secretion.American
Review of Respiratory Disease, 121,35 1-357.
[51 QUINTON,P. (1979) Composition and control of secretions
from tracheal bronchial submucosal glands. Nature (London),
279.55 1-552.
P.S. (1980) Receptors
l6l PEATFIELD,A.C. & RICHARDSON,
involved in sympathomimeticstimulation of mucus secretion in
the cat trachea. Journal of Physiology (London),3 0 3 , 4 7 ~ .
J. & REID,L. (1972) An organ culture study of the
[71 STURGESS,
effect of drugs on the secretory activity of the human bronchial
submucosal glands. Clinical Science, 43,533-543.
I81 BOAT,T.F. & KLEMERMAN,
J.L. (1975) Human respiratory
tract secretions. 2. Effect of cholinergic and adrenergic agents
on in vitro release of protein and mucus glycoprotein. Chest,
67,32S-34S.
[91 SHELHAMER,
J.H., MAROM,Z. & KALMER,M. (1980)
Immunologic and neuropharmacologic stimulation of mucous
glycoprotein release from human airways in vitro. Journal of
Clinical Investigation, 66, 1400-1408.
I101 PHIPPS,
R.J. (1979) Adrenergic stimulation of mucus secretion
in the human bronchus. Journal of Physiology (London),296,
44P.
1111 WILLIAMS,
I.P., PHIPPS,R.J.,WRIQHT,N.L., PACK, R.1. &
RICHARDSON,
P.S. (1981) Sympathomimetic agonists stimulate mucus secretion into human bronchi. Thorax, 36.23 1.
[ 121 HALL,R.L. (1978) Gas-liquid chromatographic determination
of the carbohydrate content of tracheal mucus samples.
Journal of Physiology (London), 2 7 5 , 1 1 ~ - 1 2 ~ .
[I31 REID,L. (1960) Measurement of the bronchial mucous gland
layer: a diagnostic yardstick in chronic bronchitis. Thorax, IS,
132-141.
H.M.& WELLS,A.Q. (1932) Mucus
[141 FLOREY,
H., CARLETON,
secretion in the trachea. British Journal of Experimental
Pathology, 13,269-284.
[I51 SPICER, S.S., CHAKRIN, L.W., WARDELL,J.R. & KENDRICK,
W. (1971) Histochemistry of mucosubstances in the canine
and human respiratory tract. Laboratory Investigation, 25,
483-490.
S.S.,MOCHIZUKI,
I., SETSER,M.E. & MARTINEZ,
J.R.
I161 SPICER,
(1980) Complex carbohydrates of rat tracheobronchial surface
epithelium visualised ultrastructurally. American Journal of
Anatomy, 158,93-109.
J.T., HALL, R.J., JEPPERY,
P.K., PHIPPS, R.J. &
1171 GALLAGHER,
RICHARDSON,
P.S. (1978) The nature and origin of tracheal
secretions released in response to pilocarpine and ammonia.
Journal of Physiologv (London),2 7 5 , 3 6 ~ - 3 7 ~ .
[IS] GALLAGHER,
J.T. & KENT, P.W. (1975) Structure and
metabolism of glycoproteins and glycosaminoglycanssecreted
by organ cultures of rabbit trachea. Biochemical Journal, 148,
187-196.
1191 FELDHOPP,P.A., VEERASINOHAM,
P.B. & DAVIDSON,E.A.
(1979) Puriflcation, properties and analysis of human asthmatic bronchial much. Biochemistry, 18,243&2436.
I201 LIAO, T., BLUMENPELD,
0.0.
& PARK, S.S. (1979) Isolation
and characterisation of glycoproteins from canine tracheal
pouch secretions. Biochimica et Biophysica Acta, 577,
442453.
I211 ROUSSEL,P., DEGAND, P., LAMBLM,G., LAINE, A. &
LAPI=, J.J. (1978) Biochemical definition of human tracheobronchial mucus. Lung, 154,241-260.
I221 CREETH,M., BHASKAR,K.R. & HORTON,J.R. (1977) The
separation and characterization of bronchial glycoproteins by
density-gradientmethods. Biochemical Journal, 167,557-569.
[231 PEATFIELD,
A.C. & RICHARDSON,
P.S. (1980) Sympathetic
nerve activity stimulates cat tracheal much output via
padrenoceptors. Journal of Physiologv (London), 305, 7 8 ~
79P.
E.W., BRIANT,R.H., CONOLLY,
M.E., DAVIES,
1241 BLACKWELL,
C.T. (1974) Metabolism of isoprenaline
D.S. & DOLLERY,
aRer aerosol and direct intrabronchial administration in man
and dog. British Journal of Pharmacology, 50,587-59 1.
I251 CAMNER,P., STRANDBERQ,K. & PHILIPSON,K. (1976)
Increased mucociliary transport by adrenergic stimulation.
Archives of Environmental Health, 31,79-82.
B., STRANDBERG,
K., PHILIPSON,
K. & CAMNER,
[26l MOSSBERG,
P. (1976) Tracheobronchial clearance and beta-adrenoceptor
stimulation in patients with chronic bronchitis. Scandinavian
Journal of Respiratory Disease, 57,281-289.
1271 PAVIA,D., BATEMAN,J A M . & CLARKE,S.W. (1980)
Deposition and clearance of inhaled particles. Bulletin
Europien de Physiopathologie Respiratoire, 16,335-366.
R.J., JANUSZKIEWICZ,
D8l MEZEY,R.J.,COHN,M.A., FERNANDEZ,
A.J. & WANNER,
A. (1978) Mucociliary transport in allergic
patients with antigen induced bronchospasm. American
Review of Respiratory Disease, 118,677-684.
M.S. (1960) The pathology of asthma with specific
l291 DUNNILL,
reference to changes in the bronchid mucosa. Journal of
ClinicalPathology, 13,27-33.
[301 KING, M.,GILBOA,A., MEYER,F.A. & SILBERBERG,
A.
(1974) On the transport of mucus and its rheologic simulants
in ciliated systems. American Review of Respiratory Disease,
110,740-745.