Muscarinic Receptors in Ileum and Urinary Bladder Smooth Muscle

1
Muscarinic Receptors in Ileum and Urinary Bladder
Smooth Muscle
John A. Tran1, Minoru Matsui2, Michael T.
Griffin3, and Frederick J. Ehlert1 1Department of
Pharmacology, College of Medicine, University of
California, Irvine, Irvine, California
92697-4625 2Division of Neural Network,
University of Tokyo, Tokyo, Japan 113-003
3Department of Chemistry, Chapman University,
Orange, California 92866
Abstract We investigated the coupling of
muscarinic receptor subtypes to phosphoinositide
hydrolysis in ileum and urinary bladder using
mutant mice lacking M2 (M2 KO), M3 (M3 KO) and
both M2 and M3 (M2/M3 KO) receptors. In wild
type urinary bladder, the muscarinic agonist
oxotremorine-M (Oxo-M) elicited a robust
phosphoinositide response characterized by an
EC50 value of 0.22 mM and a maximal response
(Emax) of 32.7 conversion of 3Hinositol-labele
d phosphoinositides into 3Hinositol phosphates.
A similar response was observed in urinary
bladder from M2 KO mice, whereas no measurable
response was observed in M3 KO and M2/M3 KO mice.
In the ilea of wild type and M2 KO mice,
substantial Oxo-M-induced phosphoinositide
responses were measured, characterized by EC50
values of 0.37 and 0.52 mM and Emax values of
35.8 and 34.7 , respectively. In contrast to
our experiments on urinary bladder, the ilea from
M3 KO and M2/M3 KO mice exhibited significant
muscarinic phosphoinositide responses with EC50
values of 1.6 and 1.7 mM and Emax values of 31.2
and 20.6 , respectively. The phosphoinositide
response in the ileum from M3 KO mice was
substantially smaller than that of wild type, and
the response in the M2/M3 KO was significantly
smaller than that of the M3 KO. These data
suggest a major role for the M3 receptor and a
minor role for the M2. The competitive
antagonism of phosphoinositide hydrolysis in
ileum from M2/M3 KO mouse revealed an M1 profile.
Pertussis toxin-treatment had little effect in
any of the strains of mice implicating little or
no involvement of Gi/o in phosphoinositide
responses. Our results indicate that the M3
receptor plays a major role in muscarinic
phosphoinositide responses in mouse urinary
bladder and ileum while the M1 and M2 subtypes
play minor roles in the ileum. Supported by
Pfizer and NIH Grant 69829
Figure 1
Figure 2
M2/M3 KO Mouse Ileum
Dissociated cells
Intact ileal strip
Competitive antagonism of oxotremorine-M induced
phosphoinositide hydrolysis in M2/M3 KO mouse
ileum by AF-DX 116 and pirenzepine (a). The
antagonists were pre-incubated for thirty minutes
before the introduction of oxotremorine-M. Each
point represents the mean value SEM of three
separate experiments. Oxotremorine-M induced
phosphoinositide hydrolysis in dissociated ileal
smooth muscle cells (b). Data are expressed as
the conversion of inositol phosphates by 0.1 mM
oxotremorine-M over basal conversion. The mean
values SEM of three experiments are shown.
Figure 4
Introduction Contractions of the
gastrointestinal and urinary tract smooth muscle
are mediated by a variety of receptor types such
as muscarinic receptors. The major muscarinic
receptor subtypes that are thought to mediate the
contractile effects of acetylcholine in smooth
muscle are the M2 and M3 (Ehlert 2003a, 2003b).
Studies on cell lines have shown that the M3
subtype couples to Gq to mediate phosphoinositide
hydrolysis, whereas the M2 subtype couples to
Gi/o to mediate an inhibition of adenylyl cyclase
(Lai et al 1991, Peralta et al. 1988). A similar
pattern of muscarinic receptor coupling has been
observed in native smooth muscle of the ileum
(Candell et al. 1990), trachea (Roffell et al.
1990, Yang 1991) and urinary bladder
(Noronha-Blob et al. 1989). The signaling
pathways of M2 and M3 muscarinic receptors in
smooth muscle are consistent with their role in
contraction. The M3 receptor is known to mediate
a direct contractile response in
gastrointestinal, airway and urinary bladder
smooth muscle from a variety of species (Candell
et al. 1990, Noronha-Blob et al. 1989, Yang
1991). In contrast, two indirect roles for the
M2 receptor in contraction have been
demonstrated 1) a high potency inhibition of
forskolin- and isoproterenol-mediated relaxation
(Thomas et al. 1993) and 2) a low potency
potentiation of M3 receptor-mediated contractions
(Sawyer and Ehlert, 1998). Recent studies on M2
muscarinic receptor knockout (M2 KO) mice have
confirmed that the M2 receptor mediates an
inhibition of isoproterenol- and forskolin-
induced relaxation of smooth muscle (Matsui et
al. 2003) and a conditional potentiation of M3
receptor mediated contraction (Griffin et al.
2004). Nevertheless, evidence for a direct,
albeit weak, contractile response to the M2
receptor has been obtained in the ileum, but not
in urinary bladder, in studies on M3 KO (Matsui
2000) and M2/M3 double KO mice (Matsui 2002).
These results suggest that in mice ileum, the M2
receptor may be able to elicit Ca2 mobilization
directly through stimulation of phosphoinositide
hydrolysis. To address this hypothesis, we
investigated the coupling of muscarinic receptors
to phosphoinositide hydrolysis in ileum and
urinary bladder from wild-type, M2 KO, M3 KO and
M2/M3 double KO mice. Our results suggest that
M1, M2 and M3 receptors may couple to
phosphoinositide hydrolysis in mice ileum.
Oxotremorine-M induced phosphoinositide
hydrolysis in wild type, M2 KO, M3 KO, and M2/M3
KO mouse urinary bladder strips (a) and
longitudinal muscle strips of the ileum (b).
Each point represents the mean values SEM from
six to ten separate experiments.
Figure 3
The effect of pertussis toxin-treatment on the
competitive inhibition of the binding of 3HNMS
by oxotremorine M in the longitudinal muscle of
wild type mouse ileum (a). Specific binding was
measured in the absence and presence of GTP.
Pertussis toxin (70-100 mg/kg body weight) was
injected three days prior. Data are expressed as
a percentage of the specific binding measured in
the absence of oxotremorine-M. Each data point
represents the mean value SEM of three
experiments. Pertussis toxin-treatment caused a
reduction in both the affinity of oxotremorine-M
and the negatively cooperative effects of GTP.
The specific binding of 3HNMS in control and
pertussis toxin-treated wild type mouse urinary
bladder (b). Pertussis toxin (70-100 mg/kg body
weight) was injected intraperitoneally three days
prior. The specific binding of 3HNMS was
measured at a single concentration of 0.25 nM in
control and pertussis-toxin treated urinary
bladder. The data represent the percent increase
in the specific binding of 3HNMS caused of GTP
(1 mM). Mean values SEM of three separate
experiments are shown. Pertussis toxin-treatment
significantly reduced the increase in 3HNMS
binding caused by GTP.

• Conclusions
• Only the M3 receptor mediates muscarinic
  phosphoinositide hydrolysis in the mouse urinary
  bladder, because the response is completely lost
  in the M3 KO mouse.
• At least three different muscarinic receptor
  subtypes contribute to phosphoinositide
  hydrolysis in the longitudinal muscle of the
  mouse ileum. Using a method akin to Furchgott
  analysis, it is possible to attribute 8 of the
  response to the M2 receptor, 80 of the response
  to the M3 receptor, and 12 to the residual
  receptor mediating the response in the M2/M3 KO
  mouse.
• The modest phosphoinositide response noted in the
  M2/M3 double KO mouse exhibited an M1 profile in
  competitive antagonism experiments suggesting
  that it is primarily the M1 receptor that
  mediates phosphoinositide hydrolysis in the M2/M3
  KO ileum. This response occurs in the muscle
  itself, because it persists in a preparation of
  dissociated smooth muscle cells from the M2/M3 KO
  mouse ileum that presumably lacks neurons.
• Pertussis toxin-treatment had little influence on
  muscarinic receptor-mediated phosphoinositide
  hydrolysis in ileum and urinary bladder.
• Since the M2 receptor is expected to signal
  through pertussis toxin-sensitive Gi/o, our
  inability to detect a pertussis toxin-sensitive
  component of the muscarinic phosphoinositide
  response in the ileum may be attributed to 1) the
  small M2 component of the response, 2) a
  secondary stimulating effect of pertussis
  toxin-treatment on phosphoinositide hydrolysis in
  the ileum (see Ehlert 2001), 3) the lack of a
  role of Gi/o in the response.
• Pertussis toxin-treatment was effective in
  ADP-ribosylating Gi/o as evidenced by its
  inhibitory effect on GTP modulation of muscarinic
  receptor binding properties in ileum and urinary
  bladder.

References Candell, L.M., Yun, S.H., Tran, L.L.,
Ehlert, F.J. 1990. Mol. Pharmacol. 38
689-697. Ehlert F.J., Ansari K.Z., Shehnaz D.,
Sawyer G.W. and Griffin M.T. 2001. J. Pharmacol.
Exp. Ther. 2991126-1132. Ehlert, F.J. 2003a.
Life Sci. 74 355-366. Ehlert, F.J. 2003b.
Receptors and Channels 9 261-277. Griffin, M.T.,
Matsui, M., Shehnaz, D., Ansari, K.Z., Taketo,
M.M. Manabe, T., Ehlert, F.J. 2004. J.
Pharmacol. Exp. Ther. 308 339-349. Lai, J.,
Waite, S.L., Bloom, J.W., Yamamura, H.I., Roeske,
W.K. 1991. J. Pharmacol. Exp. Ther. 258
938-944. Matsui, M., Motomura, D., Karasawa, H.,
Fujikawa, T., Jiang, J., Komiya, Y., Takahashi,
S., Taketo, M.M. 2000. Proc. Natl. Acad. Sci.
USA. 97 9579-9584. Matsui, M., Motomura, D.,
Fujikawa, T., Jiang, J., Takahashi, S., Manabe,
T., Taketo,M.M. 2002. J. Neurosci. 22
10627-10632. Matsui, M., Griffin, M.T., Shehnaz,
D., Taketo, M.M., Ehlert, F.J. 2003. J.
Pharmacol. Exp. Ther. 305 106-113. Noronha-Blo
b, L., Lowe, V., Patton, A., Canning, B.,
Costello, D., Kinnier, W.J. 1989. J. Pharmacol.
Exp. Ther. 249 843-851. Peralta, E.G., Ashenazi,
A., Winslow, J.W., Ramachandran, J., Capon, D.J.
1988. Nature 334 434- 437. Roffel, A.F., Meurs,
H., Elzinga, C.R., Zaagsma, J., 1990. Brit. J.
Pharmacol. 99 293-296. Sawyer, G.W., Ehlert,
F.J. 1998. J. Pharmacol. Exp. Ther. 284
269-277. Thomas, E.A., Baker, S.A., Ehlert, F.J.
1993. Mol. Pharmacol. 44 102-110. Yang, C.M.
1991. J. Auton. Phamacol. 11 51-61.
Effects of pertussis toxin in wild type (a), M2
KO (b), M3 KO (c) and M2/M3 KO (d) mouse ileum.
Pertussis toxin (70-100 mg/kg body weight) was
injected intraperitoneally three days prior to
experiments. Mean values SEM from three to ten
experiments are shown.