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Role of airway smooth muscle in asthmaPossible relation to the neuroendocrine system.

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THE ANATOMICAL RECORD 236:152-163 (1993)
Role of Airway Smooth Muscle in Asthma: Possible Relation
to the Neuroendocrine System
NEWMAN L. STEPHENS, HE JIANG, AND ANDREW HALAYKO
Luzhou Uniuersity, Peoples’ Republic China (H.J.), Plant Science, University of Manitoba,
Winnipeg, Manitoba, Canada (A.H.);London, England (N.L.S.)
ABSTRACT
Though not yet firmly established, it appears likely that the
neuroendocrine system (NES)regulates airway smooth muscle function. As
it is the latter which is altered in asthma, the importance of the role of the
NES in this disease is clear. The fact that transmitters from the NE cells are
released from their basal aspect, and are in close proximity to the subjacent
airway smooth muscle, further indicates an interaction. The question then
arises as to what are the experimental desiderata for conducting studies of
the ASM. These should constitute what Sergei Sorokin has called the
“Koch’s postulates of airway smooth muscle research.”
As human tissues from asthmatics are difficult to obtain, animal models
have been developed. The requirements are that, in these animals, the allergy be IgE based, that a congenital or familial factor be operative, that a
noncholinergic nonadrenergic inhibitory system be a component of the
neural regulatory system, and that the antigen for immunization be of a
type commonly found in human asthmatics. Ideally, evidence of clinical
asthma and exercise-induced asthma and nocturnal attacks should also be
present. Unfortunately, no ideal animal models exist and one cannot talk
about asthmatic animals, but only of animals with allergic bronchospasm.
If in vitro research is to be conducted, there are additional requirements.
The tissue should be from a relevant location. The tracheal smooth muscle
which has been the favorite, purely because of its convenience, is not a
good model. For the early asthmatic attack, central bronchi (3-5 mm diameter) should be used. Muscle strips obtained from them should be parallelfibred and the cartilage plaques should be carefully dissected away, otherwise they contribute unwanted frictional forces when velocity is
measured. Care should be taken to ensure that the epithelial cell layer is
intact, as evidence indicates that it may regulate airway muscle function,
though this has not been established for all the animal species used in
asthma research.
The isolated muscle strip should be in a steady state, particularly with
respect to the functional variable under study, before definitive data are
collected. Most importantly, it is shortening capacity that must be studied,
as this is the in vitro analogue to in vivo narrowing of airways. Isometric
force development provides information about wall stiffness and is of very
little relevance to the elucidation of the mechanism of bronchospasm. Furthermore, as force is measured at the plateau of the record, it only yields
data relating to latch bridges which seem to play little role in narrowing the
airway. Unfortunately, isometric force is the most common measurement
made, purely because it is easy to carry out, while shortening is technically
more difficult.
Received October 23, 1991; accepted December 10, 1991.
Address reprint requests to N.L. Stephens, Prof. Dept of Physiology,
Faculty of Medicine, University of Manitoba, 425, Basic Medical Sciences Building, 770 Bannatyne Avenue, Winnipeg, Manitoba R3E
357 Canada.
Q 1993 WILEY-LISS, INC
AIRWAY SMOOTH MUSCLE AND THE NEUROENDOCRINE SYSTEM
153
The above notwithstanding, if force is the parameter being studied, its
correct normalization is a very important consideration. To make comparisons, force should be converted to stress. In tissues which are more than 5
mm in length and 5 mg in weight, the required cross-sectional area can be
obtained from the weight of the blotted tissue and its optimal length. For
tissues smaller than this measurement errors render the approach invalid
and direct measurements must be made using high performance optics. It
must be remembered too that the cross-sectional area of the tissue is only
appropriate when the entire tissue consists of muscle, as for example, in the
case of striated muscle. In smooth muscle, muscle content may only be 25%.
For this reason, tissue stress should be converted to muscle cell stress and
ultimately to myosin stress, as it is the myosin crossbridges that generate
the force. Estimation of muscle stress requires quantitative morphometry
while myosin stress requires morphometry of immunohistochemical micrographs.
Finally, in conducting studies of shortening and velocity, the nature of
the loading has to be kept in mind. Such studies are usually carried out
with isotonic loads as this holds that variable constant. However, in vivo
the load is more likely to be elastic or visco-elastic, hence, in vitro studies
should employ similar loading. In our own studies we have attempted to
pay attention to the desiderata mentioned above. In bronchial smooth muscle (central airways) from 4-month-old, ragweed pollen-sensitized dogs, we
have found that maximum shortening capacity and velocity are both increased while force production is normal. These changes are typical of
early disease. In addition, we have found that the compliance of the muscle’s so-called internal resistor is increased with sensitization. This could
account for the increased shortening capacity of the muscle. We have also
shown that the maximum velocity of the sensitized muscle is associated
with increased myofibrillar ATPase activity. This results, not from a
change in distribution in myosin heavy chain isozymes, but from increased
phosphorylation of the 20,000 dalton myosin light chain that is due to an
increased content of myosin light chain kinase. Our studies indicate that
this increase is due to increased gene translation rather than transcription,
as the content of messenger RNA for myosin light chain kinase is unchanged. o 1993 Wiley-Liss, Inc.
Key words: Airway smooth muscle, Sensitized airway smooth muscle,
Smooth muscle myosin light chain kinase
In a publication devoted to consideration of the structure and function of neuroendocrine (NE) cells in
health and disease, this chapter on the physiology and
pathophysiology of airway smooth muscle (ASM) is assuredly a n outlier. It has been included because the
importance of the NE system, whose function is still
not known, is “intuitively” felt to be in its putative
regulatory role in airway narrowing. If this is to be
substantiated, a clear understanding of airway smooth
muscle contractility is mandatory. Perhaps the most
important role for impaired interaction between the
NE system and ASM could be in the pathogenesis of
asthma or of the effects of hypoxia on pulmonary function.
When so much has been written about airway
smooth muscle in recent times, it is a curious anomaly
that we do not yet know the physiological function of
ASM, mainly because appropriate and reliable data are
not available. This has been clearly pointed out by Otis
(1983). Much of the physiology of ventilation and its
regional distribution can be accounted for by considering the airways as passive conducting tubes and the
respiratory skeletal muscles a s the driving pump for
ventilation, smooth muscle itself being not required.
Speculation has, however, provided a list of ASM
functions. Tracheo-bronchoconstriction,by reducing
anatomical dead space, would improve alveolar ventilation. This would only be of benefit if ventilation frequency did not increase too greatly, a s the increased
work of breathing would limit alveolar ventilation. Regional distribution of ventilation is also regulated by
local bronchoconstriction, as is the optimization of the
matching of ventilation to perfusion. Outpouring of
mucus from the glands into the airways has also been
ascribed to rhythmic ASM contraction.
All these speculations aside, there is little doubt
that, a t least, the early asthmatic response in patients
is the result of increased central airway smooth muscle
contraction. To what extent ASM contraction is involved in the late asthmatic response where bronchial
narrowing involves peripheral airways, is the source of
debate. The narrowing is felt to be due to geometrical
factors wherein local oedema and inflammation narrow the lumen.
154
N.L. STEPHENS ET AL
Study of Airway Smooth Muscle Function: The Nature
of Loading
Whether one wishes to study the role of ASM in
asthma or elucidate its relations to the function of neuroepithelial bodies, it is important to identify the correct parameter to study. This statement would be trivial were it not for the fact that the major portion of in
vitro research described in the literature is devoted to
measurement of isometric force development (Shioya et
al., 1989; Armour et al., 1985).The use of this approach
has been sanctioned by custom. It is difficult to justify
this, as isometric force development only provides insight into the stiffness of the tissue without providing
any information about shortening, which is the in vitro
counterpart to in vivo bronchoconstriction. In vivo
studies, which are mainly those of specific airway conductance, are free from this criticism.
Having indicated that shortening is the study parameter of choice, use of the correct loading regimen for
the muscle during shortening is a sine qua non. Classically, studies of shortening are carried out under isotonic loading conditions chiefly because information is
sought about intrinsic crossbridge activity; time-dependent load changes would constitute a confounding
variable. However, for studies that aim to relate to in
vivo function, the load that is imposed on the shortening muscle in vivo must be identified. A recent hypothesis about the pathogenesis of asthma is critically
based on the nature of this loading. As ASM contracts
and the airway narrows, the elastic properties of the
components of the airway wall (connective tissue and
cartilaginous plaques) and lung parenchyma provide
additional loads that limit the extent of shortening.
This is based on the idea that ASM is directly attached
to the cartilaginous plaques which in turn are the sites
of insertion of lung parenchyma. However, histologic
data published by von Hayek (1960) show that ASM is
not directly attached to the cartilage but via loose connective tissue. Furthermore, in the central airways the
lung parenchyma is not directly inserted into the bronchial wall but again via loose connective tissue. Thus,
the load on the ASM may not be elastic but isotonic.
This is a n important point to settle if meaningful
asthma research is to be undertaken. At any rate, the
hypothesis states that were parenchymal compliance to
increase, this would result in reduced bronchial loading and account for asthmatic bronchoconstriction.
The Concept of Contractility
What has been discussed above is the role of ASM
shortening and though this may help explain phenomena a t whole organ level, i t will not elucidate mechanisms which usually operate a t subcellular and molecular levels. To elucidate the former, auxotonic
shortening must be studied; for the latter, isotonic.
Measurement of shortening, however, does not provide complete information regarding muscle contractility, even though it is paramount with respect to
allergic bronchospasm. To obtain comprehensive information, all modalities of function should be measured
as shown by cardiac muscle investigators (Brutsaert e t
al., 1971).These consist of maximum shortening capacity (Almax),maximum shortening velocity (VJ, maximum force development (Po)and time. The importance
of time stems from the fact that maximum values of the
other factors can only be obtained if rates of energy
liberation are a t a steady maximum. The last named
varies quite considerably with time, maximally in the
case of cardiac muscle. To eliminate time-dependent
effects, tetanization has been used successfully. However, in the case of cardiac muscle, tetanization is not
possible, so steps must be taken to ensure that only
measurements taken at the moment of peak energy
liberation are used in defining contractility.
Special Considerations With Respect to Contractility
in Airway Smooth Muscle
Consideration of what mode of contraction to study
and how to assess contractility, though adequate for
the study of striated muscle, is not so for smooth muscle
which possesses another layer of complexity. In striated muscle the assumption can be made that average
crossbridge function is homogeneous throughout the
course of contraction. This is not so for smooth muscle
where the bridges are heterogeneous. Unlike striated
muscle, in which velocity of shortening, once maximum
is reached, remains unchanged, smooth muscle shows
progressive drop. In canine tracheal smooth muscle
(Stephens et al., 1985b) and in bronchial (Jiang and
Stephens, 1990) have shown that, as in other smooth
muscles (Dillon et al., 1981), velocity decreases considerably with time. This has been explained by the development of so-called latch bridges (LBR) which retard the velocity of normally cycling bridges (NBR)
that commenced operating during early contraction.
Alternatively it has been hypothesized that the mechanism is the uniform and progressive slowing of all the
bridges. This controversy has not yet been resolved.
That notwithstanding, we have reported (Stephens et
al., 1985) that peak velocity develops within 2 seconds
of onset of muscle shortening and is brought about by
the activity of NBR. Within this period the muscle
achieves 75% of its shortening; hence, in conditions
where shortening is altered, as for example asthma,
shortening must be studied within this 2 second period.
The major regulatory biochemical mechanism involved
is phosphorylation of myosin light chain by myosin
light chain kinase and it is that which needs study.
After 2 seconds, LBR replace NBR, at least in a functional sense. The former produce most of the force developed by the muscle and little shortening. If muscle
stiffness is to be studied, then studies a t about 8-10
seconds after stimulus onset should be undertaken.
The biochemical process underlying the development of
LBR (though controversial) is dephosphorylation of the
myosin light chain which entails study of myosin light
chain phosphatase (Dillon et al., 1981). From this, one
may conclude that the study to conduct and the parameter to choose depend critically on the question being
asked.
Smooth muscle presents yet another problem and
that relates to the existence of a so-called internal resistance to shortening (IRS). This is a hypothetical intracellular structure or process that resists shortening
as it becomes compressed. Its stiffness therefore dictates the magnitude of muscle shortening. Its presence
has been shown in cardiac muscle by Chiu et al. (1982).
We have reported its presence in airway smooth muscle
and have developed a method to delineate its compres-
AIRWAY SMOOTH MUSCLE AND THE NEUROENDOCRINE SYSTEM
155
sion-tension properties (Seow and Stephens, 1986). We
have also reported that its compliance is increased in
ASM from a dog model of asthma; this could account for
the increased bronchospasm seen in such preparations.
To sum up this section, to describe the change occurring in asthma one must measure the airway muscle’s
maximum shortening capacity (Al,,,), but to elucidate
the underlying mechanism, the internal resistance to
shortening must be studied.
Molecular Mechanisms of Airway Smooth Muscle
(ASM) Contraction
Both the physiology of normal ASM contraction and
the pathophysiology of asthma are based on biochemical changes involving processes which include excitation, excitation-contraction coupling, and contraction.
These involve the biochemistry of neuromuscular
transmitters and junctions (Burnstock, 1970) and peptides, the behavior of the resting membrane potential
(Small e t al., 1980; Kroeger and Stephens, 1975), the
activity of ion channels, the role of G-proteins, inositol
triphosphate, diacyl glycerol (Somlyo et al., 1987), the
release of calcium and its interaction with calmodulin,
and the activation of calcium-calmodulin-dependent
myosin light chain kinase (Kamm and Stull, 1988)
with resultant phosphorylation of the 20 kilodalton
myosin light chain (MLC). The process of relaxation
requires reversal of these processes with the addition of
dephosphorylation of MLC by a specific phosphatase.
Our own studies have revealed the following:
Airway smooth muscle model
Our original work was performed on canine tracheal
smooth muscle (Antonissen et al., 1979) even though
we knew the site of physiological regulation of ventiFig. 1 . Montage of a longitudinal section of canine tracheal smooth
lation was a t the level of 3 mm diameter airways. However, at the time, we felt that the presence of cartilage muscle that has been fixed at the peak of an isometric contraction
accounts for the dentrate cell outlines. The perifasicular space
plaques in those bronchi, by increasing visco-elastic which
above the muscle bundle contains college, a blood vessel, fibroblasts
loads, would render evaluation of force-velocity-and- and a mast cell typified by its granules. The calibration bar represents
shortening properties difficult. Figure 1shows tracheal 10 +m. (Taken from Stephens N.L. in Asthma: Basic Mechanisms and
smooth muscle in longitudinal section. The fibres are Clinical Managment. P.J. Barnes, I.W. Rodgers and N.C. Thomson,
parallel, which is a requirement for valid mechanical eds. Academic Press, New York, 1988, pp. 16.)
studies.
Figure 2a shows a section from a 5th generation ca- Length-tension properties of canine tracheal smooth
nine bronchus. The parallel arrangement of fibres is muscle (TSM). (Fig. 3)
evident a s is the fact that there is much less muscle
These were elicited by conventional techniques and
present (25% of tissue area) than in tracheal (75%). show that maximum stress development (1.1kg/cm2 or
Finally i t must be noted that the muscle is not directly = 1 x 105N/M2)was of the same value a s seen in other
attached to the cartilage but via loose connective tis- smooth and striated muscles. The curves enable idensue. Therefore, a s pointed out before, the role of carti- tification of optimal length (1, or l,,,)
which is the
lage as a source of loading of the shortening muscle is length a t which most studies should be conducted. The
debatable.
practice of applying a fixed, arbitrary load on the musFigure 2b shows a section similar to that shown cle to conduct pharmacological studies is incorrect, as
above but here the cartilage has been carefully dis- the length achieved is not correspondingly fixed but
sected away. The intactness of the subjacent muscle depends on the intrinsic compliance of the tissue. From
fibres is evident. Studies have shown that the maxi- the curves-see horizontal line labelled Alma,-the
mum isometric force (PJ, the maximum velocity of muscle’s maximum isotonic shortening capacity can be
shortening (V,) and the maximum shortening capacity deduced and is a surprising 90% of 1,. This, as i t is
(Al,,,)
are all increased after removal of the cartilage. derived from static measurements, is essentially a n
The reasons for the increase in V and Alma, are evi- overestimate.
dent; that for the increased P,/mm’ is not. We feel that
Maximum isotonic shortening measured dynamithe increase results from the normalization procedure, cally is about 65% which represents the true value.
as the tissue cross-section has been reduced by elimiFigure 4. One of our earliest findings (Antonissen et
nation of the cartilage.
al., 1979) was that in ASM from ragweed pollen- and/or
156
N.L. S T E P H E N S ET AL.
Fig. 2. a: Histological sections of 5th order canine from bronchi,
before removal of cartilage; smooth muscle is located right beneath
epithelium and does attach directly to cartilage external to it. EP1,
epithelium; SM, bronchial smooth muscle; CT, cartilage. Haematox-
ylin and eosin stain. x 200. (From Jiang and Stephens, J. Appl. Physiol., 69: 1990.) b Same as in a except that cartilage has been dissected
away.
AIRWAY SMOOTH MUSCLE AND THE NEUROENDOCRINE SYSTEM
157
the internal resistance to shortening (IRS). However,
to do that the force-velocity (FV) properties of the muscle should first be dealt with.
TISSUE WT. 0.015f0.002 G
M 1.04*
~ ~ 0.05CM.
L
Force-velocity (FV) relationships
LENGTH
PERCENT LMAX
Fig. 3. Length-tension curves from canine tracheal smooth muscle
(From Stephens e t al., J. Appl. Physiol., 26:685-692, 1969.)
ovalbumin-sensitized dogs, Al,,
was increased. The
increase, though only 10-15% of lo, accounted for a
50% increase in airway resistance. Studies to determine the causes of the increased Al,,
require consideration of the factors limiting muscle shortening, viz
7(
The dynamic properties of ASM, which are the really
physiological ones, are best assessed by delineating the
force velocity relationships of ASM (either tracheal or
bronchial) (Fig. 5 ) . However, as indicated before, independent FV curves for NBR and LBR are required.
These, elicited by standard quick-release techniques
during the course of a n isotonic contraction are shown
in Figure 6 . They reveal that the maximum velocity of
shortening for NBR is about 80% greater than that for
LBR; however, the pattern is reversed for Po;Al,,, for
NBR is, of course, greater than that for LBR.
In sensitized ASM (see test curve in Fig. 6 ) while P,
remains unchanged, V, and Al,,
are both increased.
For the latter refer back to Fig. 4.
An important point to be made here is that these are
changes seen in a model of early disease. At later
times, secondary changes obscure the picture. It is interesting to note also that had the hypothesis been
tested isometrically, the results would have been misleading, as the absence of difference in Po would have
led us to conclude the muscle was normal. This again
points up the superiority of studies of shortening.
In sensitized muscle, only the 2 second NBR show a n
increase in Al,,
and V,, the LBR remain normal. This
confirms that it is critically important to study the ap-
TSM SHORTENING(% I,,)
.(
5t
4c
%b
aa
10
10
0
%
Fig. 4.Plot of changes in length during isotonic shortening a t different loads for control and sensitized
canine trachealis. (From Antonissen et al., J. Appl. Physiol., 1974.)
158
-
N.L. STEPHENS ET AL.
0.1 0
500
0-
400
0.06
300
0.04
200
development only tell us about the development and
regression of wall stiffness and are limited in scope.
3. Alterations in shortening ability depend not only
on changes in crossbridge properties but also upon the
stiffness of the so-called internal resistor.
4. While velocity in skeletal muscle is not a limitation to extent of shortening, it is a limitation in smooth
muscle. I n the latter, most of the shortening is completed within 2 seconds by normally cycling crossbridge, so changes in V, could affect maximum shortening capacity.
"
Elucidation of the molecular mechanisms for
changes in ASM structure and function require biochemical studies. As our data showed that shortening
capacity (Alma*) and velocity (V,) were increased, the
following studies were undertaken.
Changes in AI,,,
This is a relatively new area and much needs to be
done. The internal resistor is situated either between
0
cells or between cell bundles and is likley to be made up
0
-aof collagen, elastin and other matrix substances. Alter4
8
12
16
20
0
natively, it could be the cytoskeleton of the smooth
LOAD IN GRAMS(P)
muscle cell. With respect to the latter, desmin, tubulin
(aand 6) and a-actinin are likely candidates. We have
currently set up methods to identify these by using
;ap!
specific antibodies and Western blotting techniques.
Figure 9 is a gel showing that vimentin and desmin
Fig. 5. Mean isotonic after-loaded force-velocity curves from canine
can be identified in canine tracheal smooth muscle.
tracheal smooth muscle, n = 32. P = load; V = maximum velocity of
shortening for a given load. Po = maximum load muscle can support Using densitometry, they can also be quantitated. Very
without shortening; a and b are constants with units of force and preliminary experiments suggest there is no difference
velocity, respectively. Note: the right hand ordinate applies to the between these proteins from control and sensitized
linear transform of Hill's equation (From Stephens et al., J. Appl. ASM. In hypertensive blood vessels, Berner et al.
Physiol., 26t685-692, 1969.)
(1981) have reported considerable increase in these
proteins; however, in these vessels, hypertrophy was
well advanced and the protein changes are likely late
propriate crossbridges in testing our hypotheses. TO changes. In our model there is no evidence of cellular
study the time course of crossbridge activity, maximum morphologic change and the changes we see are early
velocity of shortening by the application of so-called and therefore more likely to relate to early causes.
zero load clamps should be measured at second or half
second intervals throughout the course of a contracChanges in properties of normally cycling
tion.
The results of a n experiment are shown in Figures 7 crossbridge (NBR)
We have already alluded to the role of NBR in regand 8. The maximum slopes of the slow transient confirm that the velocity of the NBR is about 3-4 times ulating Alma,. The most likely cause of the increased V,
we have reported (Stephens et al., 1985) is increased
faster than that of the LBR (Stephens et al., 1985a).
The importance of V, in studies of asthma may well activity of actin-activated myosin M S +-ATPase activbe queried, as it is only Al,,
that relates to resistance ity.
in the intact airway, However, if one remembers that
Figure 9 shows a gradient polyacrylamide gel loaded
the bulk of shortening is achieved by NBR in the first with a crude extract of canine tracheal smooth muscle.
two seconds of the contraction, a reduced or perhaps The corresponding densitometrogram is also displayed.
The bands for the contractile proteins actin (A), myosin
even a normal V, may not permit any increase in Al,,
heavy chain (M), myosin light chain (MLC) and one of
to develop because of the time limitation.
To sum up what has been covered so far, the follow- the regulatory proteins, tropomyosin (T) are clearly
ing points are relevant.
seen. The bands for the cytoskeleton proteins filamin,
desmin and vimentin are also identifiable.
Figure 10 shows that myofibrillar ATPase activity is
1. If insight into human physiology and pathophysiology is sought, the choice of a n appropriate animal increased in sensitized TSM.
The next step was to determine the cause of the inmodel is critical.
2. If information is sought relating to normal abnor- creased ATPase activity. To this end we identified mymal airway function, studies of airway conductance are osin heavy chain isoforms (MW 204000 dalton and
all important. In vitro, this can best be assessed by 200,000) and compared their distributions in control
studies of muscle shortening. Studies of isometric force and sensitized muscles, a s this could account for al0.02
100
159
AIRWAY SMOOTH MUSCLE AND T H E NEUROENDOCRINE SYSTEM
\
.o
F-V CURVES
.4
.6
.8
1.0
1.a
1.4
LOAD( kg/trn')
Fig. 6. Mean isotonic after-loaded force-velocity curves from control and test (ovalbumen-sensitized)
canine tracheal smooth muscle strips. The control curve is similar to that shown in Figure 6. While
maximum velocity of shortening is increased in the test muscle, the maximum force developed (shown by
x axis) is not. (From Antonissen et al., J. Appl. Physiol., 46:681-687, 1974.)
tered ATPase activity. The fairly typical densitometrogram suggested no difference, and statistical analysis
of a series of experiments confirmed this.
Another explanation was that increased phosphorylation of the regulatory 20,000 dalton myosin light
chain was responsible for the increased ATPase activity. A linear relation between the two has been reported. Figure 11shows 2-dimensional polyacrylamide
gels with 20,000 dalton myosin light chain bands; B is
the control and A the sensitized; no difference in densitometric pattern or area was detected. The two lower
gels show radioautograms taken after 32Ptreatment.
Increased phosphorylation of MLC2O is evident.
In smooth muscle, purified actin-activated myosin
ATPase has different properties from that of striated,
wherein the activity of the latter is not directly stimulated by calcium concentration and under resting conditions displays close to maximum activity. Smooth
muscles under these conditions displays very little activity. Thus, to evaluate any change in smooth muscle
myosin ATPase activity one has to also consider the
properties of the membrane and the excitation-contraction coupling mechanism, since they regulate calcium
concentration.
We have initiated studies of membrane electrophys-
iology including patch clamping. Preliminary results
in&@e that the resting sensitized membrane is depolarized by 5 mV and that three types of Ca2 activated
K + channels appear to be present with conductances of
200pS, 8OpS, and 20pS. Studies of Ca+ channels, especially the L type, are under way.
Returning to phosphorylation of the light chain, calcium-calmodulin and myosin light chain kinase
(MLCK) are the important regulators, with the latter
being the rate limiter. Using specific antibodies obtained from chicken gizzard (supplied by Dr. Mary
Pato) we have found that the content of MLCK is increased (Fig. 12). This could account for practically all
the changes we have described above.
To determine the cause of the increased content of
MLCK, measurements of the content of MLCK-mRNA
were made employing Western blot techniques. The
cDNA probe for the analysis was kindly sent to us by
Dr. Vince Guerrero of the University of Arizona a t Tucson, Arizona. Results revealed no increase in the content of the message. We thus concluded that increased
translation of the message, rather than transcription,
must be the underlying cause for increased MLCK content. Studies of stability of the message and of translation are to be conducted in the near future.
+
160
N.L. STEPHENS ET AL.
1
I
STIMULUS
.5.
0
5
TIME
10
IN
Fig. 7. Shortening in normalized units (1, = optimal muscle length)
is shown; it is plotted against time. The lowest sigmoidal curve represents lightly preloaded isotonic shortening. At different times during shortening, zero load clamps were applied. Each load clamp record
shows an initial fast transient stemming from the elastic recoil of the
ZERO
LOAD-CLAMP
DURING
SECs.
series elastic component and a shows one whole maximum slope provides the maximum velocity of shortening (V,) for that instant in
time. The progressive dimminution of V, with time is evident. (From
Stephens et al., Chest 88sr2235-2295, 1985.)
VELOCITIES
SHORTENING
3.:
A /
VELOCITY CURVE
2r
0
w
v)
\
E
E
v)
z
I.€
0.6
3
>
I0
0.7
0
w
>
>
z
I
-1
9
.8
0.8
0
f
Z
W
c
0.9 a
0
I
v)
0
1
4
TIME
1
6
IN
'
0
1.0
SEC
Fig. 8. Plot of V, vs. time. The original lightly preloaded isotonic shortening is also shown (right hand
ordinate). (From Stephens et al., 1985.)
161
AIRWAY SMOOTH MUSCLE AND THE NEUROENDOCRINE SYSTEM
Actin
I
I
Tropomyosin
20%
4%
Fig. 9. Gradient SDS-polyacrylamidegel (4-205) with densitometrogram shown contractile, regulatory
and cytoskeletal proteins.
to study of the latter by investigators primarily interested and possessing expertise in the former. In such a
context considerable care has to be taken in studies of
mechanical properties of smooth muscle. Some of the
requirements are listed below.
T
STSM
CTSM
Fig. 10. Myofibrillar ATPase activity of sensitized (STSM) and control (CTSM) canine tracheal smooth muscle.
The "Koch's Postulates" of Smooth Muscle Research
vis-a-vis the Neuroendocrine System
We stated a t the outset that the interrelation, a t
least topographically, between the neuroendocrine system and the subjacent airway smooth muscle must lead
1. As the main purpose is to obtain insight into human physiology and pathophysiology, the choice of a n
appropriate in vitro model is paramount. This is because, currently, it is very difficult to obtain human
specimens. The objectives must be to reproduce the human disease as closely a s possible. This dictates that
the disease in the animal model be IgE based. In the
guinea pig, for example, allergic bronchospasm is IgG
based which renders the model inappropriate. A genetic trait or familial influences in the animal are important, a s they very probably operate in humans.
Bronchospasm should develop on challenge with specific antigen as well as to exercise.
2. The correct segment of airway should be studied
with respect to the hypothesis being studied. If, for example, the early asthmatic response is to be studied
then the central airways (3rd to 5th generation) should
be chosen. For the late response the peripheral airways
(less than 1 mm in diameter) should be selected.
3. The epithelial layer should be intact. In some animals the epithelium regulates the contractile response
of the smooth muscle by liberating dilators such as
nitric oxide or constrictors such a s the endothelins. In
these, steps must be taken to insure epithelial function
is intact.
162
N.L. STEPHENS ET AL
Fig. 11. Two-dimensional gels electrophoresis of crude extracts from
control and sensitized canine tracheal smooth muscle (A and B in
upper panels). Radioautograms (32P)
of the same gels as shown above
are shown in the lower panels (A and B).
4. To compare muscle contractility between strips
from the same animal or in the same segmental strip
between animals or between animals a t different ages,
certain steps must be taken to assure that meaningful
comparisons can be made. These are:
i. Performance of all experiments a t optimal muscle
length (lo).This ensures that maximal contractile response is being studied. It is also known that excitability o r sensitivity of a muscle is length-dependent. If 1,
is not carefully monitored, horizontal shifts in doseresponse curves may not be the effect of treatment but
of length difference.
ii. The use of a supramaximal stimulus. This is because with time (this may amount to 4-6 hours) muscle excitability diminishes. A stimulus initially identified as maximal may prove inadequate by the end of
the experiment. Electrical field stimulation with mass
platinum electrodes is the best. The repeated washouts
required when chemical stimulation is used, result in
rapid muscle deterioration.
iii. The assurance that when data capture commences, the muscle is in satisfactory steady state. This
generally requires gentle handling of the tissue during
preparation, adequate oxygenation (and pH adjustment) and incubation a t the appropriate temperature
for one to three hours. Steady state is said to be
achieved when the maximum isometric force developed
(Po), the maximum rate of force development (dPidt)
and the contraction time do not vary by more than 10%.
iv. Utilizing animals, when comparison studies are
to be carried out, that are at the same level of maturity.
At different ages the excitability of the muscle membrane and the activity of enzymes (Na+-K+-ATPase,
myosin heavy chain ATPase) can vary quite considerably and influence the results of studies,
v. Measurement of the appropriate parameter with
respect to the exact functional modality to be studied. If
muscle stiffness is to be studied then isometric force
versus muscle length should be measured.
a) To quantitate muscle strength, maximal force
developed (Po)should be measured. However, for comparisons to be made between muscles,'normalization is
necessary. The most convenient is normalization to tissue stress, i.e., force developed per unit tissue crosssectional area. This is satisfactory for skeletal muscle
where more than 90% of the tissue cross-section is
made up of muscle. In smooth muscle, however, the
cross-sectional content of muscle cells, per unit area of
the whole tissue, varies from 75% (tracheal smooth
muscle) to 25% (bronchial smooth muscle). The normalization must therefore be made with respect to crosssectional area of muscle cell and not to that of the tissue, i.e., in lieu of tissue stress we need muscle cell
stress. Even this does not represent the final normalization, a s not all the tissue in a muscle cell is contractile; normalization should therefore be made with respect to amount of contractile protein (actomyosin)
present in the cross-section, i.e., the ultimate parameter is actomyosin stress.
It must be stressed that to elucidate the muscular mechanisms that regulate distribution of ventilation or underlie the pathogenesis of allergic bronchoconstriction, it is shortening capacity that must be
studied. Study of force development can yield very little information about airway resistance.
b) To compare maximum shortening capacity and
velocity, allowance must be made for the different
lengths of muscle strips employed in different experiments. The best normalization is to relate length
changes to optimal length (lo). In elucidating shortening capacity one must bear in mind that in smooth and
cardiac muscle, this may be affected by maximal shortening velocity. This limitation does not hold for skeletal muscle. The smooth muscle limitation is due to the
fact that 75% of the muscle's shortening develops in the
first two seconds of the contraction. At this time, normally cycling bridges, with their relatively rapid velocities, are active. Changes in velocity could limit
maximum shortening capacity. In studying the velocity of shortening it is, therefore, the velocity at two
seconds that is important.
c) Muscle power (the product of force by velocity) is
normalized satisfactorily by referring the product to
the weight of the muscle.
ACKNOWLEDGMENTS
The work described in this communication was supported by a n operating grant from the Medical Research Council of Canada and the American Council
for Tobacco Research; J.H. was the recipient of a fellowship from the same council.
AIRWAY SMOOTH MUSCLE AND THE NEUKOENDOCRINE SYSTEM
Sensitized
Control
*
T
163
0
TSM
*
T
n=4
n=4
n=8
BSM
n=9
Fig. 12. Myosin light chain kinase content in control and sensitized tracheal (TSM) and bronchial
(BSM) smooth muscles.
LITERATURE CITED
Antonissen, L.A., R.W. Mitchell, E.A. Kroeger, W. Kepron, K.S. Tse,
and N.L. Stephens 1979 Mechanical alterations of airway smooth
muscle in a canine asthmatic model. J . Appl. Physiol., 46:681687.
Armour, C.L., J.L. Black, and P.R.A. Johnson 1985 A role for inflammatory mediators in airway hyperresponsiveness. In: Mechanisms in Asthma: Pharmacology, Physiology and Management.
Alan R. Liss, Inc., New York, pp. 99-108.
Berner, P.F., A.V. Somlyo, and A.P. Somlyo 1981 Hypertrophy-induced increase of intermediate filaments in vascular smooth muscle. 3. Cell Biol., 88:96-101.
Burnstock, G. 1970 Structure of smooth muscle and its innervation.
In: Smooth Muscle. A. Brading, A.F. Jones, and T. Tomita, eds.
Arnold, London, pp. 1-69.
Brutsaert, D.L., V.A. Claes, and M.A. Goethals 1971 Velocity ofshortening of unloaded heart muscle and the length-tension relation.
Circ. Res., 29t63-75.
Chiu, Y., E.W. Ballou, and L.E. Ford 1982 Velocity transients and
viscoelastic resistance to active shortening in cat oaoillarv muscle. Biophys. J., 40t121.
Dillon, P.F., M.O. Aksoy, S.P. Driska, and R.A. Murphy 1981 Myosin
phosphorylation and the crossbridge cycle in arterial smooth
muscle. Science, 211t495-497.
Jiang, H.. and N.L. Stephens 1990 Contractile Drouerties of bronchial
smooth muscle with and without cartilag; J.-Appl. Physiol., 69:
120-126.
Kamm, K.E., and J.T. Stull 1988 The function of myosin and myosin
light chain kinase phosphorylation in smooth muscle. Am. Rev.
Pharmacol. Toxicol., 25~593-670.
Kroeger, E.A., and N.L. Stephens 1975 Effects of tetraethylammonium on tonic airway smooth muscle: Initiation of phasic electrical activity. Am. J. Physiol., 228:633-636.
Otis, A.B. 1983 A perspective of respiratory mechanisms. J. Appl.
Physiol., 54t1183.
Seow, C.Y., and N.L. Stephens 1986 Force-velocity curves for smooth
muscle: Analysis of internal factors reducing
- velocity. Am. J .
Physiol., 25:C362-C366.
Shioya T., E.R. Pollack, N.M. Munoz, and A.R. Leff 1989 Distribution
of airway contractile responses in major resistance airways of the
dog. Am. J. Pathol., 129:102-117.
Small, R.C., J.P. Boyle, R.W. Foster, and D.M. Good 1990 Airway
Smooth Muscle: Electrophysiological properties and behaviour.
In: Airway Smooth Muscle: Modulation of Receptors and Response. D.K. Agrawal and R.G. Townley, eds. CRC Press, Boca
Raton, pp. 69-94.
Somlyo, A.V., M. Bond, A.P. Somylo, and A. Scarpa 1987 Inositol
triphosphate-induced calcium release and contraction in vascular
smooth muscle. Proc. Natl. Acad. Sci. USA, 82t523.
Stephens, N.L., C.S. Packer, and S.K. Kong 1985a Smooth muscle
contractility: Effects of hypoxia. Chest, 88~223s-229s.
Stephens, N.L., E.A. Kroeger, U. Kromer, and J.A. Mehta 1969 ForceI
I
.
I
velocity characteristics of respiratory airway smooth muscle. J.
Appl. Physiol., 26:685-692.
Stephens, N.L., G. Morgan, C.S. Packer, and S.K. Kong 1985b Smooth
muscle contractility: Effects of hypoxia. Chest, 88st2235-2295.
von Hayek, H. 1960 The Human Lung. Hafner Publishing Company,
New York. pp. 127-226.
DR. HOYT: Dr. Stephens, I wonder if I might lead
you out onto the ice and ask you to make a prediction?
From what we’ve seen in the last two days, I think most
people would agree that neuroepithelial bodies, no
matter whether they are autonomous or whether they
are innervated, are sited so as to release the bulk of
their granules across the basal cell membrane into the
peribronchial connective tissues. And of course we’re
interested in knowing what their targets are under
normal circumstances. There has been mention made
of adjusting ventilation and perfusion and of the possibility that there may be different functions for cells a t
different positions.
Can I ask you, with your knowledge of the bronchial
muscle and airflow patterns, to tell us where you
would site endocrine cells to adjust airflow significantly in the lungs to match perfusion? In other words,
are there critical places along the bronchial tree where
an effect on the muscle would be most striking and
produce the desired result?
DR. STEPHENS: Well, with respect to optimizing
ventilation, I would put them at the 3-5 millimeter
diameter airways in the human being.
The other issue that you raised was the matching of
ventilation and perfusion. That’s a little more difficult.
That’s going to occur at the alveolar level. And that’s
going to be either perhaps controlled by the neuroendocrine system, as I had mentioned earlier on in this
meeting, or by a group of cells described by Kapanci
(Kapanci et al., 1974), who called them interstitial
cells. They have contractile filaments in them, and he
figured that these made the final fine adjustments for
matching ventilation to perfusion.
DR. JOHNSON: This will sound extraordinarily naive, but can you make any generalizations about hy-
164
WORKSHOP ON PULMONARY NEUROENDOCRINE CELLS
pertrophied airway smooth muscle in terms of its mechanical properties?
DR. STEPHENS: Yes. Clinically, by the time the patient comes in there is almost certainly some hypertrophy and perhaps some hyperplasia, and what’s happening to the mechanical properties, of course, you can
assess by doing a time study. We don’t have the hard
information on airway smooth muscle, but we know
from high blood pressure that if you start early enough
the first change you see is increased shortening and
increased velocity, with no change in force development. Now, if you wait until later, when hypertrophy is
being developed, force development increases, so you
now have increased velocity, increased shortening and
increased force development. If you wait even longer,
the cytoskeleton starts to hypertrophy, and one of the
most striking things that occurs in high blood pressure
is that the whole cell gets simply stuck to the cytoskeleton such that the actomyosin gets reduced and, a t that
point, force development drops, velocity drops, and
shortening also drops. So there seems to be a whole
progression of these various modalities of function.
GENERAL DISCUSSION: FUTURE DIRECTIONS
DR. SOROKIN: We’ve saved a little time for discussion of what we think are important areas for study,
given our current knowledge about pulmonary neuroendocrine cells. The National Heart, Lung, and Blood
Institute is also interested to know what kinds of
things we recommend for future research in this field.
Dr. Polak has a number of suggestions which she will
offer presently. Meanwhile, I’ll just say that for me the
outstanding characteristic of this Workshop has been
that people trained in very different research disciplines were able to come together and present findings
that stimulated us all and enriched our understanding
of the subject. Hence I believe that for future studies on
the pulmonary neuroendocrine system all sorts of approaches should be encouraged, because no single discipline or approach has so far had a monopoly on significant new findings.
DR. HOYT: In other words, you think that the door
should not be slammed on any particular technique
just because it’s two years old. Is that the idea?
DR. SOROKIN: Exactly.
DR. CUTZ: Well, I think what has transpired, a t
least from my perspective, is that there is a consensus
now that the function of these cells perhaps varies with
development. In other words, they may function during
intrauterine life and postnatal life, so that studies
should still focus on both those areas.
DR. SOROKIN: On a biphasic manner of function in
these two periods. Yes.
DR. AGUAYO: I think one of the important things is
that we try to reach a consensus about nomenclature. I
don’t believe anybody here is confused about what is a
neuroendocrine cell, but in terms of making this literature accessible to other people interested in getting
into the field, perhaps we could devise some kind of
common nomenclature so that we all mean roughly the
same thing when we’re talking about neuroepithelial
bodies, single neuroendocrine cells or whatever.
The same is true with respect to the roster of neuropeptides or markers that are going to be used to define
what is a neuroendocrine cell. I think everybody agrees
on what is the morphology and the location and the
specific characteristics of these cells. But when we report and discuss our findings, we have to be very clear
about what type of marker we’re using and about recognizing the limitations of such markers versus others
that you may or may not want to use.
It’s also very important that we start recognizing, as
Dr. Battey was saying, the heterogeneous families of
peptides and mediators and receptors. When we start
reporting our data we should try to be a little bit more
specific about what is bombesin-like immunoreactivity, for example, and not say, “This cell makes bombesin.” If you don’t have mRNA present in the cell you
have to be careful about saying these cells produce the
peptide, a s opposed to having peptide immunoreactivity.
Attention to such small details in nomenclature and
definitions should facilitate our ability to exchange information and reach consensus.
DR. SCHEUERMANN: I think it is important that
we continue receptor research on these cells and also
make in situ hybridization a high priority.
DR. HOYT: I’d like to go back to what Sergei has said
about approaches. We now see that it’s possible to isolate these cells and deal with them under controlled
conditions. Dr. Cutz and others clearly show us that
now, at last, we’ll be able to get at some of their receptor biology and electrophysiology.
That’s fine. But I wouldn’t like the powers that be to
get the impression that this should be the only emphasis. As always, we have the whole animal before us,
and so we still need a broad approach. These cells are
not all that easy to find. They’re not all that easy to pin
down. But by their particular siting, they may perform
different functions, and so we have to push both aspects
a t once, in vivo and in vitro.
I also think we should talk together more often.
DR. POLAK: Well, following what you’ve said, I’d
like to say this Workshop has been really a n excellent
idea on Dorothy Gail’s part. As Sergei pointed out,
there hasn’t been a meeting of this kind for more than
10 years, and in fact most of the faces are new. Not new
in terms of age, but new to me. I haven’t met them. In
fact, some of the people said that it was very nice to
come and meet the oldest-I think they thought I
might be 70 and in a wheelchair.
It’s absolutely necessary that we try, if possible, to
meet at regular intervals. Not too frequently, of course,
because there are far too many meetings. But something like every 2 years. Since the initiative of 10 years
ago, we haven’t met, and lots of developments have
occurred. One of the biggest diseases of this century is
lack of communication. I am sure in the next two years
we will see, following this meeting, a n enormous
amount of data being generated. From talking to various people, I have the impression they’re all going
back enthusiastically to continue their work and to answer a number of the questions raised here. I see two
points that need special attention. One relates to technology and one to the questions we all want to answer.
In terms of technology, over and above classical histological methods and immunocytochemistry we need
to consider new means of quantifying these cells in a
reliable way. The idea we had in the early days, that
biochemistry can give us quantitative answers, is not
WORKSHOP ON PULMONARY NEUROENDOCRINE CELLS
enough. We need to know precisely where cells and
molecules are in the tissues. But of course there are
problems in that as well. We have very gently skimmed
over molecular biological approaches to the question of
hormone receptors, and as Dr. Scheuermann said,
these have been little used in the lung. And so in terms
of technology, we’ve got it. It’s with us, but we need to
apply it.
In terms of questions and answers, it is clear, as has
been pointed out all along, that looking a t these cells in
isolation is extremely useful because i t simplifies a
number of things, but we cannot extrapolate the data
completely to what happens in vivo where there are
lots of nerves and where we now have regulatory factors in the endothelium. We need to relate cellular
mechanisms to the structure of the tissue. So I would
expect we will have meetings where the innervation as
well as endothelial and vascular aspects will be thoroughly discussed.
DR. HOYT: I think the innervation is absolutely critical.
DR. POLAK: Yes. I think so. We can’t discuss these
cells in isolation. Many times in this meeting we have
asked what will happen to the innervation as well as
the endocrine cells. We can already recommend to the
Institute more frequent meetings and use of novel technology to study the pulmonary neuroendocrine system
in its integral relations with nerves and the vasculature.
The use of genetic models has been mentioned in a
number of instances. These horrify me because, of
course, they are complex models. But I think they will
play a n important role in answering certain questions.
Again, we cannot simply extrapolate from them to
what happens in a genetically normal animal or human being, but these techniques can be helpful. There
aren’t very many models as yet, and very little has
been investigated, practically nothing.
DR. HOYT: Something that has emerged over the
last two days is a desire to know more about the mechanisms that induce formation of the endocrine cells and
that maintain their population. This is a difficult question that will require not only work in vitro but also
studies in situ, in the tissues. It is extremely important: if you can understand that, then I think you’re on
the verge of understanding what happens in the lungs
in many situations where there has been a pathologic
challenge.
DR. POLAK: I think you are saying in a very kind
way that you don’t think we’ve been intellectual
enough in this meeting.
DR. HOYT: No. It’s just that many people are working towards this problem from different approaches,
and yet we still know virtually nothing about it. I think
this is the central event that links the normal development and normal functions of pulmonary neuroendocrine cells with clinical and pathological disorders.
DR. POLAK: Phenomenology is useful, but it’s sort
of, “Gee whiz, how exciting!” We need to put it to work,
and not just in trying to understand development. I
think Elizabeth-is she there?
DR. McDOWELL: I’m here.
DR. POLAK: She brought up something that we
must not forget. She asked, “Can you look a t the airway epithelium in its integrity?” and I’m making a
165
plea that we look at the whole lung in its integrity. It’s
really necessary if we want to interpret, let’s say, pulmonary hypertension and hyperplasia of endocrine
cells, to know what happens with the oncogene expression in the early phases, and-again I’ll come back to
novel technology-what is the receptor, is it up or down
regulated, et cetera. We need, a s Dr. Hoyt has said, to
try to interpret the mechanisms, which is the most difficult thing to do. But I think it goes two ways. If you
can understand what’s happening in pathology you
then begin to unravel what could happen in normal
circumstances.
DR. HOYT Yes. I think they’re absolutely intertwined.
DR. POLAK: The way this Workshop has been organized is very good in that we have a mixture of people
with different knowledge. I mean, who would know
what Frank Cuttitta knows? He’s made a fantastic contribution; physicians and pathologists are defining relevant clinical entities, etcetera, etcetera. So I think we
need this multidisciplinary approach, but let’s not wait
10 years for another meeting. I, myself, I’m in a hurry.
(Laughter.)
DR. CUTZ: Well, I’d like to come back to normal
functions. It’s important to decide whether there’s
enough evidence to say, for example, that the neuroepithelial body is a chemoreceptor. Or to say that i t has
some function regulating growth and differentiation.
Or that it functions only during development and then
gets switched off. Or that i t can be switched off and
switched on again later.
If you can decide this is a chemoreceptor in the same
class as the carotid body, then you can study i t along
those same lines. Now, how do you prove something is
chemoreceptor? You can use classical physiology or you
can bypass it looking a t the membrane properties, and
this is why you need isolated cells. It helps you get
around the difficulty that this is a widely dispersed
system. You can’t simply remove i t for study as you can
the pituitary or the carotid body, but if you are able to
characterize the membrane properties of the cells in
isolation, you can compare them with those of better
known systems.
DR. SCHEUERMANN: The functional histological
approach is very important also. Perhaps we are thinking about a n electrophysiological approach to the nerve
endings near these cells as one of our aims?
DR. SOROKIN: I think there’s a clear case for studying these cells in isolation to get the sort of information
Dr. Cutz has mentioned. Since we are dealing with
neuroepithelial bodies, however, it’s equally clear that
to get the full picture we have to understand how
they’re related to the nerves. It seems that patterns
may differ from one species to another, perhaps along
Cartesian coordinates of afferent and efferent neurites
interfaced with neuroendocrine cells. There are still
huge gaps in our detailed knowledge that must be repaired if we are fully to understand the role of these
cells in chemoreception.
DR. POLAK: Yes.
DR. LINNOILA: Well, I certainly agree with all of
this. But in addition to methods I think it would be
useful to note currently the functions that are postulated and half-proven, those for which there is some
kind of intellectual basis, or those actively being
166
WORKSHOP ON PULMONARY NEUROENDOCKINE CELLS
worked on. We also mentioned that it would be helpful
for other researchers if we tabulate all the peptides,
amines, and the like, and tabulate the functions as
well. It might be useful conceptually to put the fetal
and newborn functions in one list, adult functions in
another, and maybe then the pathologies. Just create
lists of functions. One would be the chemoreceptor
function, but the question is whether we would put
chemoreception in all the lists.
DR. POLAK: I think you are very optimistic. Ideally
one should define them broadly as chemoreception and
then growth and development. Otherwise you will list
control of smooth muscle, control of endothelium, vascular and airway smooth muscle, epithelium, blood gas
levels. The whole lot. Then, as you were saying, you
have to think which one is real and point a n accusing
finger a t the others. The main divisions which appear
to be the most solid ones are growth and development
and tissue repair and chemoreceptor function. The nitpicking of whether they are vasodilators or vasoconstrictors and whether endothelin is-it’s impossible
right now!
DR. LINNOILA: Yes. It may be impossible. But if we
also want t o be of service to the outside community,
then listing some of these functions is useful. And if we
can attach a reference, or abstract, or something of that
kind, it would be even better. I agree, you know, that it
may be a n impossible task to list all these functions.
DR. SCHEUERMANN: But all the functions are not
known momently.
DR. CUTZ: Perhaps those are hypotheses?
DR. HOYT: Some we’ve begun to test, some have not
been tested, and some have been tested a little bit and
found to be either too difficult to deal with right now or
perhaps-DR. POLAK: Well today a t lunch time some of us
were discussing the paper published on the effect of
bombesin on human bronchial epithelium. There
haven’t been many papers on the subject, and when
people have tried to reproduce these results they’ve
encountered difficulties. But it was such a predictable
answer. It fit so nicely with the theory that people do
quote it. I mean, i t may be true but i t needs [confirmation]. As you said, at the moment the functions need a
little more work, whereas we can start building up a
table on peptides in the different species.
DR. HOYT: I think Ernest is right. We should perhaps, a t this stage, not become hung up because we
don’t find a given peptide in all species, or because we
have evidence for a function in one species but not yet
in another. The first priority ought to be to figure out
what these cells do. To me it’s obvious they’re going to
do different things a t different times of life. It’s not a n
accident that they appear soon after the airway is laid
down, a very long time before birth, and they’re well
recognized in adult life where their morphological and
physiological circumstances are quite different.
DR. SUNDAY: I was going to say that it’s just by the
nature of the organ that we’re probably several decades
behind fields like hematology or even pituitary cell biology, where you can get normal and abnormal tissue
a t any time you wish. As Dr. Polak was saying, a lot of
this is sort of nonintellectual; I think she means nonmolecular, in that a lot of this(Laughter.)
DR. POLAK: What I meant was, we could go on and
on in a complacent way, saying, “Oh, gee whiz, this is
fascinating. They twitch on the left and they twitch on
the right.” And remember electron microscopy-well
you don’t, you weren’t born-but electron microscopy,
just describing and describing and describing.
DR. SUNDAY: But the descriptive work is extremely
important and will lay the foundation. It’s not completed. It would be very helpful if we could make a
chart with the different peptides in different species,
potential roles, and, as Dr. Cutz says, antibody
sources-which are good antibodies, which are bad antibodies. It would be like the codification of human leukocytic antigens. Common reagents can be very important in trying to understand what’s going on.
DR. POLAK: But I’m sure if we tried to determine
the possible roles of pulmonary endocrine cells we’ll
conclude that we need to do a basic, systematic, histological analysis of a species.
DR. SUNDAY: Well, you know, now a lot of the molecular work is going back to morphology.
DR. POLAK: Exactly.
DR. SUNDAY: But I say, do the descriptive work and
then perhaps in two years we’ll have more ideas, not
just about molecules that are involved in pathways, but
cell to cell interactions, like what goes on between the
neuroendocrine cells and the nerve endings and the
epithelial cells.
DR. POLAK: Exactly.
DR. SUNDAY: And across muscle to fibroblasts.
DR. HOYT: What about gap junctions?
DR. SCHEUERMANN: The membrane biology of
these cells? But I think also our attention must be,
should be, focused on the relationship of these neuroendocrine cells to the autonomic nervous system. It
should be included in this large field of research.
DR. HOYT: Well, the source of the innervation is
still a n open question. Allusions have been made to
possible sensory feedback loops between the endocrine
cells and the intrinsic nervous system in the lung.
Their innervation shouldn’t simply be viewed as vagal
afferents with local axon reflexes.
Take the heart. I remember seeing a poster some
years ago a t a meeting in Montreal. There was a picture of a muscle spindle in the myocardium, and the
fiber from that muscle spindle very definitely synapsed
on a cardiac ganglion cell. That’s a good example of
local sensory feedback, not upstairs but right a t the
local level. It isn’t just a matter of what the endings are
like and how they relate to the endocrine cells; you
have to have the rest of the wiring diagram.
DR. SCHEUERMANN: But perhaps we must also
focus our attention on the relationship between these
neuroendocrine cells and the chromaffin cells in the
ganglia located in the connective tissue. I think it is
necessary.
DR. CUTZ: Obviously when you talk about a receptor you have to know its innervation. That’s fundamental. Since you know the innervation of the carotid body
you can manipulate it. I would agree 100 percent that
the innervation of the endocrine cells has to be worked
out; the species differences have to be worked out.
There are some wonderful neuroanatomical methods
which Dr. Polak uses, retrograde tracing and these
kinds of things, which could be easily-
WUKKSHUI‘ VN Y U L M O N A K I
DR. HOYT: Hallmark preparations.
DR. CUTZ: Right. And I don’t think this has been
done in the lung, and it would help you to understand
the connections. By no means do you get all the answers looking a t the cells. It’s the whole receptor organ.
DR. POLAK: If you do as you suggest-retrograde
tracing, which is so important and very little investigated-and use confocal microscopy, how do we get
Dorothy to fund all that?
DR. GAIL: I’ll try. I need your ideas.
DR. POLAK: No problem with ideas. The cash is
missing.
DR. SUNDAY: Well i t seems that animal models
would also be very welcome. Dr. Polak described the
emphysema model, but there may be many models that
nobody has analyzed. There are a lot of inbred strains
of mice, and maybe some of these have abnormalities in
the neuroendocrine cells, or there are a lot of transgenic mice with various homozygous deletions.
DR. GOSNEY: The thing that strikes me sitting here
these past two days is that there is no real justification
for a revolution. I think that we’re all working away a t
different aspects of the problem, most of us making
some progress and each contributing in a different way.
I think it would be a shame if anybody felt that what
they were contributing was somehow redundant in the
major task of sorting all this out.
DR. POLAK: Speak for yourself.
(Laughter.)
DR. GOSNEY: No. I mean I feel, personally, speaking for the other people with a pathological bent, the
amateur scientists amongst us, it’s a question of more
of the same but with greater care, perhaps using some
of the new technology, more in situ hybridization of
receptors and so on and so forth. My own feeling is that
I should be going back, for instance in these pulmonary
hypertension cases, looking a t them more carefully, reexamining them, doing things that have been suggested, teasing them out more and more. But I don’t
think there is any justification for a huge revolution.
Let’s not put all our eggs in one basket. I think we all
need to carry on.
DR. POLAK: I think the important thing is, as you
said, to keep your identity. It’ll be foolish if you become
a cell biologist and, you knowDR. GOSNEY: I couldn’t do it.
(Laughter.)
DR. POLAK: I think we all play a very key role.
DR. GOSNEY: I agree. I think one of the interesting
things has been how many differentDR. P0LAK:-Aspects.
DR. GOSNEY: Yes.
DR. SUNDAY: The multidisciplinary approach is
very important.
DR. POLAK: The problem is how do we cure the jet
lag? That’s the big problem.
(Laughter.)
DR. GAIL: Sunshine, isn’t it?
DR. SOROKIN: In a way we have solved the problem. If Dr. Polaks suggestion to meet more frequently
is taken up, then the multidisciplinary approach
makes a lot of sense. When we sit down and talk together it’s much clearer what each has to contribute.
DR. POLAK: As I said before, I think the lack of
communication is a big disease.
DR. GAIL: Thank you all very, very much.
DR. POLAK: Thank you.
DR. GAIL: Thank you, Sergei.
(Applause.1
(Whereupon, a t 3:lO p.m., on September 6, 1991, the
workshop concluded.)
WORKSHOP CONSENSUS:
CURRENT STATE OF KNOWLEDGE
A consensus of the Workshop was that pulmonary
neuroendocrine cells are biphasic in function, having:
(1) a “mitogenic” role i n controlling the growth o f the
airway epithelium, particularly during the morphogenesis of the bronchial tree i n prenatal life, and (2) a
chemoreceptiveleffector role that predominates in postnatal life and appears directed to several target cells,
especially airway smooth muscle. New evidence tends to
confirm their role as chemoreceptors for oxygen; their
mitogenic role may be reasserted postnatally as the population undergoes hypertrophy in regions o f the lung
severely damaged by certain lung diseases (e.g., bronchopulmonary dysplasia, cystic fibrosis, and among a
damage-susceptible subgroup o f cigarette smokers), accompanied by developing fibrosis, epithelial metaplasia,
and airway and vascular smooth muscle hypertrophy.
These functions are associated with secretion of regulatory peptides that the cells synthesize (bombesinlGRP,
calcitonin, CGRP, etc.) and store i n cytoplasmic granules, although the spectrum o f peptides synthesized is
not the same in all species. Many pulmonary neuroendocrine cells are elaborately innervated by visceral sensory and autonomic fibers which play a role i n integrating chemosensory responses; work on endocrine cellenriched cultures, however, indicates that i n the absence
o f innervation, cells will degranulate in response to hypoxia, and during lung formation noninnervated cells
appear responsible for growth regulation. Apart from
some 10 regulatory peptides known to be associated with
these cells in different species, pulmonary endocrine
cells likely contain, or can synthesize, a number of additional, hitherto unrecognized ones, O n the one hand,
electron microscopic immunocytochemistry indicates
that no regulatory peptides have yet been found for certain morphological subtypes of these cells existing in the
lungs; o n the other, neuroendocrine cells are provided
with specific enzymes of the peptidyl glycine alpha-amidating monooxygenase ( P A M ) group, which are capable
o f making biologically active peptides from larger prohormones or molecules like chromogranins, present in
the secretory granules. Once released from the cells,
these substances gain ready access to lung tissues and
act mainly at specific local sites, although a select subunit of pulmonary endocrine cells may reach more distant targets because of especially favorable access to the
circulation. Receptors for some of the peptides secreted
by these cells have been found i n the lungs, but much of
the critical work i n this area has been done o n cultures
ofpulmonary small-cell carcinoma cells, and the picture
in the intact lung is yet far from clear. From evidence of
bombesiniGRP receptors presented at the Workshop,
however, it appears likely that pulmonary cells possess
both high- and low -affinity receptors for a given peptide,
and that control over the effects of the neuroendocrine
cell peptides also occurs at the receptor level.
168
WORKSHOP ON PULMONARY NEUROENDOCRINE CELLS
RECOMMENDATIONS
1. More work is needed to characterize the “mitogenic” and “chemoreceptive” roles of pulmonary neuroendocrine cells. What are the normal times of onset
and duration of these functions, do they overlap, and
are they carried out by the same cells or by distinct
subpopulations?
2. Investigation should be encouraged into factors or
mechanisms regulating pulmonary neuroendocrine
cell population size in normal and diseased lungs.
What causes their hyperplasia after certain types of
lung injury, and does this cause renewed growth of the
lungs, or is it only a consequence of the repair process?
3. New openings in research should be followed up.
As presented a t the Workshop, these included: (1) a
role for pulmonary neuroendocrine cells in regulation
of lung development, ( 2 ) improved ways to isolate the
cells for experimental study, and (3) experimental
studies on the response of cells to hypoxia (and other)
stimuli, the mechanism of secretion, and stimulus-secretion coupling, now made more feasible by improvements in technology. Some of these studies can be performed on neuroendocrine cell-enriched cultures or
lung organ cultures, but related matters can also be
investigated in intact lungs through application of
newer microscopic techniques and analytical methods
(immunocytochemistry, mRNA probes, receptor localization, and cell blot assays for secretion), enhanced by
confocal microscopy.
4.Further studies are needed to identify new bioactive peptides in pulmonary neuroendocrine cells and
the mechanism of their synthesis, since the full range
of peptides that these cells are capable of producing is
not fully appreciated. Furthermore, it is not known to
what extent the cells normally are capable of shifting
production from a prevalence of one type of peptide
(e.g., for regulating growth) to another (e.g., for influencing smooth muscle contraction), or whether autocrine mechanisms may be involved in effecting the
change.
5. Comprehensive studies are needed on the relationship among pulmonary endocrine cells, the central and
peripheral nervous system, and likely local target cells
like bronchial epithelium or smooth muscle, in order to
understand how all of these elements interact to promote regulation of the lungs. These should include investigations into the “wiring” pattern of neuroepithelial bodies and the nature of synaptic connections
present and neurotransmitters within them, as well as
the location of receptor proteins for transmitters and/or
endocrine cell peptides on target cells and on the endocrine cells themselves. Current work has not yet
opened up this area, but this can be anticipated for the
near future.
6. In relation to the preceding, specific information is
needed about the molecular biology of peptide receptors, together with their binding affinities, in the intact
lungs of human beings and laboratory animals.
7. Because of its diffuse distribution and activity predominantly a t small focal sites along airway branches,
the pulmonary neuroendocrine system has been very
difficult to study. In consequence, much of what is
known about its function necessarily has been obtained
by indirect, idiosyncratic, time-consuming, and occasionally ingenious procedures rather than by more conventional physiological research methods. The presentations and discussions at the Workshop made i t clear
to the participants that our current understanding
about these cells is based on contributions of investigators using very diverse approaches. Continued use of
a variety of quantitative methods, microscopic techniques, and molecular biologic methods to relate structural features of pulmonary neuroendocrine cells to
their biological functions should be encouraged.
8. In view of the currently increased interest and
pace of work in this field, Workshop participants recommend that future meetings of a similar kind be held
a t approximately two year intervals.
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