University of Groningen
Muscarinic receptor subtypes in airway smooth muscle
Roffel, Adriaan Frans
IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to
cite from it. Please check the document version below.
Document Version
Publisher's PDF, also known as Version of record
Publication date:
1990
Link to publication in University of Groningen/UMCG research database
Citation for published version (APA):
Roffel, A. F. (1990). Muscarinic receptor subtypes in airway smooth muscle: Binding, transduction, and
function s.n.
Copyright
Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the
author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).
Take-down policy
If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately
and investigate your claim.
Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons the
number of authors shown on this cover page is limited to 10 maximum.
Download date: 16-06-2017
SUMMARY
The airway diseases asthma, chronic bronchitis and emphysema all share
as a main characteristic shortness of breath, which is the result of airway
obstruction. The parasympathetic (cholinergic) nervous system, being the
major bronchoconstrictor neural pathway in (human) airways, has therefore
been implied in both the pathogenesis and the therapy of these disease
states. The effects of the cholinergic nervous system on target cells in the
airways (smooth muscle, exocrine glands, blood vessel walls) are elicited
via release of acetylcholine, which binds to muscarinic type receptors on
the target cells. The present thesis deals with investigations concerning
binding properties, transductional properties as well Írs functional
properties of these muscarinic receptors in airway smooth muscle (in
comparison with cardiac and brain tissue), in view of the notion emerged
during the past decade that muscarinic receptors can be distinguished into
discrete subtypes.
In the first part of the thesis (Chapters 2-5), the discovery of the
presence of a majority of M2 (cardiac type) as well as a minority of M3
(smooth muscle/glandular type) muscarinic binding sites in bovine tracheal
smooth muscle membranes is described. Initially it was found that the
binding properties of pirenzepine towards muscarinic receptors in bovine
cardiac and tracheal membranes agreed very well with affinities reported
in functional experiments using mainly guinea pig tissues (low affinity in
I 16. In cardiac
all cases), but that this was not the case for AF-DX
m e m b r a n e s ,t h e A F - D X l l 6 b i n d i n g a f f i n i t y w a s r e l a t i v e l y h i g h ( K a = 9 6
nM) in agreement with reported functional affinities (M1 receptor subtype).
However, in tracheal membranes trvo binding sites could be distinguished,
of which the majority (740/o)had high (Mr, heart-like) affinity and the
I16. Since these
minority low (N13, gland-like) affinity for AF-DX
membranes had been prepared from bovine tracheal smooth muscle with
intact epithelium and submucosa, it was suggested that the minority of low
affinity muscarinic binding sites represented glandular muscarinic receptors,
the majority of high affinity binding sites presumably representing smooth
muscle muscarinic receptors (Chapter 2). The binding properties of AF-DX
ll6 in bovine tracheal membranes were especially important because they
predicted discrepancies between binding and functional properties for
muscarinic receptors in smooth muscle, contraction being mediated by the
M3 subtype. This was further investigated in Chapter 3. It was established
that functional affinities of muscarinic antagonists(AF-DX l16 and six
others) in bovine tracheal smooth muscle correlated extremely well with
those reported for guinea pig smooth muscle preparations, suggesting
discrepancies between binding and functional properties of muscarinic
antagonists in bovine tracheal smooth muscle rather than species differences. These, occasionally marked, discrepancies between binding and
function indeed appeared to be present with all compounds tested.
However, in contrast to the non-selective (atropine, pirenzepine) and
smooth muscle selective antagonists (4-DAMP methobromide, hexahydro-
221
siladifenidol), u,hich gave monophasic displacement curi'es with M'-like
I 16, gallamine and
affinities, the cardioselective antagonists AF-DX
m e t h o c t r a m i n es t i l l d i s t i n g u i s h e d a m a j o r i t y o f M r - l i k e a n d a m i n o r i t y ' o f
M3-like binding sites, although the membranes had been prepared using
bovine trachealsmooth rnuscle fully denuded of epithelium and submucosa
( i . e . n o e x o c r i n e g l a n d s p r e s e n t ) . T h e s e r e s u l t s s u g g e s t e dt h a t i n s m o o t h
m u s c l e m e m b r a n e so n l y a s m a l l p r o p o r t i o n o f t h e m u s c a r i n i c b i n d i n g s i t e s
is involved in cont.raction, but the rather clear quantitative differences
between pA2 values for contraction and pI(1 values for binding to the
m i n o r i t - v o f M 3 - l i k e b i n d i n g s i t e s a s w e l l a s t h e p r e s e n c eo f t h e m a j o r i t y
of M' type binding sites precluded a definite conclusion.
T h e s i m u l t a n e o u so c c u r r e n c e o f M 2 a n d M 3 t y p e m u s c a r i n i c b i n d i n g
sitcs in bovine tracheal smooth muscle could also be demonstrated using a
k i n e t i c a p p r o a c h , i . e . r a d i o l i g a n d d i s s o c i a t i o n m e a s u r e m e n t s( C h a p t e r 4 ) . I t
a p p e a r e dt h a t [ r H ] d e x e t i m i d e d i s s o c i a t i o n f r o m m u s c a r i n i c r e c e p t o r si n t h i s
tissuewas biphasic, with a fast phase (half*life < I min) followed after 45 min by a slor,,,phase (half-life - 40 min). The fast phase was shor.r'nto
represent the cardiac t],pe receptors in bovine tracheal smooth muscle
b e c a u s e( i ) a s i m i l a r d i s s o c i a t i o n h a l f - l i f e w a s f o u n d f o r [ 3 H ] d e x e t i m i d ei n
bovine cardiac lefl r'cntricular membranes, (ii) the relative proportion of
muscarinic binding sites in bovine tracheal smooth muscle exhibiting this
fast dissociation behaviour agreed with the proportion of cardiac (Mr)
muscarinic sites as detected in equilibrium binding studies, and (iii) a
virtuallf identical allosteric (negative cooperative) modulation of radioligand dissociation from M1 muscarinic receptors in bovine cardiac and
[ r a c h e a l s m o o t h m u s c l e m e m b r a n e sb y A F - D X 1 1 6 , g a l l a m i n ea n d m e t h o c tramine was observed. The slow phase was concluded to represent the
s m o o t h m u s c l e / g l a n d u l a rt y p e r e c e p t o r s i n t h i s t i s s u e b a s e d o n i t s r e l a t i v e
p r o p o r t i o n ( 3 0 V 0 )a n d c o m p a r i s o n t o [ r H ] N - m e t h y l s c o p o i a m i n e d i s s o c i a t i o n
behaviour in cardiac and glandular membranes (as reported bv others).lt
was concluded that the M2 type muscarinic binding sites in bovine tracheal
smooth muscle membranes resemble M2 musc?rinic receptors in cardiac
membranes noI only regarding equilibrium binding affinities but also
regarding radioligand dissociation and allosteric modulation. Moreover, M-,
type binding sites in bovine tracheal smooth muscle appeared much iess
sensitive to allosteric modulation than the cardiac receptor subtype. Thc
studies concerning the M2 and M3 muscarinic binding sites in (bovine
tracheal) smooth muscle were completed with Chapter 5. Using cornpuÍ.er
fitting and simulation procedures, it was shown that the presumed non*
s e l c c t i v em u s c a r i n i c r a d i o l i g a n d a p p l i e d i n o u r e x p e r i m e n t s ,[ r H ] d e x e t i m i d c ,
a c t u a l l y p o s s e s s e sa s m a l l ( a b o u t 6 - f o l d ) s e l e c t i v i t y f o r M 3 o v e r M 2
'Ihe
receptors.
assumption of non-selecÍ.ivity of this radioligand (as in
Chapters 2 and 3) was shown to result in erroneous estimates of binding
parameters for cardioselective muscarinic antagonists, and the small
radioligand selectivity actuaily present was shown to lead to an inability to
detect both M, and M3 receptor populations using smooth rnuscle selective
muscarinic antagonists with selectivities less than 30-fold. Using fore-
222
knowledge acquired
m o n o p h a s i cd i s p l a c e
antagonists could br
populations. As a cc
obtaincdin Chapters
muscle membranesc<
(smooth muscleT/gla
r e c e p t o r si n v o l v e d i n
The secondpart o
t h e r c c o g n i it t t n b y
ipratropium, N-mcth
discrete binding sites
was found that this
occurs in horinc slr
brain mcmbranes, in
b i n d i n g p o p u l a t i o n sd
It was also í'ound tha
t h c p i r e n z e p i n ch i n d i
tissue distribution of
that quaternarv musc
binding sitcs in bov
hor inc cardiac (atria
Chaptcr 6 were exten
binding heterogcncit
of this binding beha
(Chapter 8). Using
r c c c p t o rb i n d i n g{ N a ' ;
no indications werc tl
a r e m o l e c u l a r l l ,d i f í ' e
differcntialll' modula
agcnts (sodium lau
c h o l c s t c r y lh c n r i s u c c
affinity (Q2) binding
difÍ'erent membrane r
compounds, since th
a p p e a r a n c co Í t h e h e t
correlation could bc
c o c Í ' f i c i c n t s( a s a m c a
binding hetcrogenei
thereforc the lorv aff
c a r i n i c r e c c p t o r sl o c a t
I n b o v i n e t r a c h c a lr
nary muscarinic anta
antagonizing metha
mcthylscopolamineo
,x
Schild plot.s r,"'itli slc
i n d i c a li n g u n c o m p c t ir
knowledge acquired with the cardioselective compounds, however, the
monophasic displacement curves obtained with the smooth muscle selective
antagonists could be resolved into high (Ms) and low (Mz) affinity
populations. As a consequence of the findings in Chapter 5, all results
obtained in Chapters 2-4 could be accomodated into the model that smooth
muscle membranes contain a majority of M2 (cardiac) and a minority of M3
(smooth muscle/glandular) type binding sites, the latter representing the
receptors involved in smooth muscle contraction.
The second part of the thesis (Chapters 6-8) presents investigations on
the recognition by classical quaternary muscarinic antagonists (like
ipratropium, N-methylscopolamine and oxyphenonium bromide) of two
discrete binding sites, as initially observed in calf and rat brain tissue. It
was found that this binding heterogeneity, which we have called Q1/Q2,
occurs in bovine striatal membranes in a manner similar to bovine total
brain membranes, indicating that the high (Qr) and low (Q2) affinity
binding populations do not originate from different brain areas (Chapter 6).
It was also found that the Qr/Qz binding heterogeneity is independent of
the pirenzepine binding heterogeneity (M1/M2). In search of the peripheral
tissue distribution of the Qt/Qz binding heterogeneity it was established
that quaternary muscarinic antagonists also distinguish the two classesof
binding sites in bovine tracheal smooth muscle membranes but not in
bovine cardiac (atrial and ventricular) membranes. The findings from
Chapter 6 were extended by investigating the molecular basis of the Q1/Q2
binding heterogeneity (Chapter 7) and the possible functional consequences
of this binding behaviour in bovine tracheal smooth muscle contraction
(Chapter 8). Using agents that are known to modulate (muscarinic)
r e c e p t o rb i n d i n g ( N a + a n d M g 2 * , G p p N H p , d i t h i o t h r e i t o l , N - e t h y l m a l e i m i d e ) ,
no indications were obtained that the Q1 and Q2 binding site populations
are molecularly different muscarinic receptor subtypes since they were not
differentially modulated by these agents. Using membrane modulating
agents (sodium laurylsulfate, digitonine,
polyethylene glycol 6000,
cholesteryl hemisuccinate), no evidence was obtained either that the low
affinity (Qz) binding sites represent muscarinic receptors located in a
different membrane environment less accessible to (charged) quaternary
compounds, since these agents did not bring about changes in the
appearance of the heterogenous binding behaviour. However, a qualitative
correlation could be observed between the octanol:buffer partition
coefficients (as a measure of lipid solubility) and the appearanceof Q1/Q2
binding heterogeneity for six quaternary muscarinic antagonists and
therefore the low affinity binding sites may after all be (normal) muscarinic receptors located in a hydrophobic membrane domain (Chapter 7).
In bovine tracheal smooth muscle contraction experiments, some quaternary muscarinic antagonists showed anomalous behaviour as well. When
Nipratropium,
antagonizing
methacholine-induced
contraction,
methylscopolamine, oxyphenonium, and N-methyldeptropine bromide gave
Schild plots with slopes significantly greater than unity (up to 2.O),
indicating uncompetitive interaction. By contrast, the tertiary analogues
223
a t r o p i n e a n d s c o p o l a m i n ea s u ' e l l a s t w o o t h e r q u a t e r n a r y c o m p o u n d s , 4 D A M P m e t h o b r o m i d e a n d t h i a z i n a m i u m , b e h a v e d a s c l a s s i c a la n l a g o n i s t s
u ' i t h S c h i l d s l o p e s o f u n i t y , a s d i d i p r a t r o p i u m a n d N - m e t h , v l d e p t r o p i n ei n
guinea pig trachea. The high Schild plot slopes in bovine tracheal smooth
muscle were found not to be due to inadequate equilibration of the
a n t a g o n i s t s o r t o t h e p r e s e n c e o f a n a t r o p i n e s t e r a s ei n t h e t i s s u e . b u t
p o s i t i v e c o o p e r a t i v i t y o f t h e q u a t e r n a r y m u s c a r i n i c a n t a g o n i s t sw a s s h o w n
to be the underlying mechanism instead (Chapter 8). It should be ment i o n e d t h a t a p p a r e n t l y 'n o r e l a t i o n s h i p e x i s t s b e t w e e n h e t e r o g e n e o u sb i n d i n g
behaviour of classical quaternary muscarinic antagonists and positivc
cooperativity in contraction studies, since N-methyldeptropine gave the
highest Schild plot slopcs (Chapter 8) but did not exhibit Qr/Qz binding
h e t e r o g e n e i t y( C h a p t e r 7 ) .
In the third and last part of the thesis (Chapters 9-ll), experiments
are described concerning the transduction mechanism(s) coupled to
muscarinic receptors in bovine tracheal smooth rnuscle as r,lell as the
c h a r a c t e r i z a t i o no f t h e m u s c a r i n i c r e c e p l o r s u b t y p e m e d i a t i n g c o n t r a c t i o n
of hwrnn airway' smooth muscle. In Chapter 9, the relationship between
b o v i n e t r a c h e a l s m o o t h m u s c l e c o n t r a c t i o n a n d p h o s p h o i n o s i t i d em e t a b o l i s m
was studied using muscarinic agonists. It was establishedthat with all
three agonists used, methacholine (non-selective), McN-A -343 (M rs e l e c t i v e ) , o x o t r e m o r i n e ( M 7 - s e l e c t i v e ) , 5 0 0 / oc o n t r a c t i o n w a s e l i c i t c d b y
c o n c e n t r a t . i o n su ' h i c h o n l y g a v e 3 . S o / cotf t h e m a x i m a l a t t a i n a b l e i n o s i t o l
p h o s p h a t e sa c c u m u l a t i o n , i n d i c a t i n g ( i ) a c o n s i d e r a b l e r e s e r v e o f i n o s i t o l
phosphatesproduction for the full contractile agonists methacholinc and
oxotremorinc, and (ii) a direct relationship between phosphoinositidc
mctabolism and smooth muscle contraction. such a direct relationship
could also be concluded from Chapter 10, u,here the muscarinic receptor
s u b t v p e m c d i a t i n g p h o s p h o i n o s i t i d em e t a b o l i s m i n b o v i n e t r a c h e a l s m o o t h
muscle was pharmacologicallf identified using the competitive antagonists
pirenzepine (Mr-selcctive), AF-DX
116 (Mr-selective), and 4-DAMP
m e t h o b r o m i d e ( M 3 - s e l e c t i v e ) .T h e M 3 c h a r a c t e r f o u n d i n t h e s e e x p e r i m e n t s
coincided with the M3 muscarinic receptor subtl'pe rnediating smoolh
muscle contraction and thus provided additional evidence for the involvem e n t o Í ' p h o s p h o i n o s i t i d em e t , a b o l i s mi n t h e p h a r m a c o m e c h a n i c a cl o u p l i n g
between muscarinic receptor stimulation and contraction of (bovine
tracheal) smooLh muscle. Hence, the function of the major population of
c a r d i a c t y p e ( M 2 ) n r u s c a r i n i c r e c e p t o r s a n d t h e t . r a n s d u c t i o nm e c h a n i s m
involved remain to be elucidated.
A s a " G r a n d c F i n a l e " , t h e t h e s i s c l o s e s w i t h t h e c h a r a c t e r i z a í i o no f t h e
muscarinic receptor subtype involved in contraction of human airw.ay
s m o o t h m u s c l e ( C h a p t e r I I ) " I t w a s f o u n d , u s i n g a S c h i l d a n a l y s i sw i t h f i v e
select.ive muscarinic antagonists, that contraction of human peripheral
(small bronchi) and central (trachea) airway smooth muscle is mediated tr_v
the M, muscarinic receptor subtype, just as is the case with bovine anci
guinea pig tracheal smooth rnuscle. These animal tissues therefore provide
e x c e l l e n t m o d e l s f o r t h e d e v e l o p m e n t o f M , s u b t l ' p e - s e l e c t i v em u s c a r i n i c
224
antagonists lo be ust
receptor) blocking prc
In conclusion, thr
significantly to the ir
over
the past yearl
lmportant questionsr,
smooth muscle and thr
sm0oth muscle. cspec
relationship
betueen
SubscqucntlV, studics
tional) muscarinic rec
hand.
antagonists to be used as bronchodilators free of M2-(cardiac or autoreceptor) blocking properties.
In conclusion, the studies described in this thesis have contributed
significantly to the increasing knowledge on muscarinic receptor subtypes
over the past years, especially concerning (airway) smooth muscle'
Important questions remaining include the function of the M2 receptors in
smooth muscle and the extrapolation of selected findings to human (airway)
smooth muscle, especially regarding the presence of M2 receptors and the
and contraction.
relationship
metabolism
betu'een phosphoinositide
Subsequently, studies on the possible disturbance of (pre- and postjunctional) muscarinic receptor mechanisms in obstructive airway disease are at
hand.
225