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Vol. 297, Issue 3, 1152-1159, June 2001
Department of Pharmacology, College of Medicine, University of California, Irvine, Irvine, California
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Abstract |
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 Top
 Abstract
 Introduction
Materials and Methods
Results
Discussion
References
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We have studied the role of M2 and M3
muscarinic receptors in acetylcholine-mediated desensitization of the
contractile response to histamine in the guinea pig ileum. Treatment of
the isolated ileum with acetylcholine (30 µM) for 20 min caused a
marked desensitization of the contractile response to histamine. When
measured 5 min after washout of acetylcholine, the EC50
value of histamine increased 5.8-fold compared with that estimated
before acetylcholine treatment, whereas the maximal response was
unaffected. This shift in the EC50 value of histamine was
maximal at the earliest time measured after acetylcholine treatment (5 min), and normal sensitivity recovered in approximately 20 min.
Acetylcholine-induced desensitization was prevented by uncoupling of
M2 receptors from Gi with pertussis toxin or by
selective inactivation of M3 receptors with
N-2-chloroethyl-4-piperidinyl diphenylacetate (4-DAMP
mustard). The shifts in the EC50 values of histamine
measured 5 min after acetylcholine treatment were only 2.0- and
1.8-fold in pertussis toxin- and 4-DAMP mustard-treated ilea,
respectively. Both pertussis toxin- and 4-DAMP mustard-treatment had
little or no effect on histamine-induced contractions in control ileum.
Measurement of histamine-stimulated inositol phosphate accumulation in
the longitudinal muscle of the ileum showed little or no inhibitory
effect of prior exposure to acetylcholine, indicating that the majority
of the heterologous desensitization occurs downstream from
phospholipase C
activation. Collectively, our results suggest that
activation of both M2 and M3 receptors is
required for heterologous desensitization of histamine-mediated
contractions in the guinea pig ileum.
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Introduction |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Muscarinic
M2 and M3 receptors are
abundantly expressed in gastrointestinal smooth muscle where their
distinct roles in mediating contraction are best appreciated under
different experimental conditions (for reviews, see Eglen et al., 1996
;
Ehlert et al., 1997). In the absence of other heterologous agents, the
contractile response to cholinergic agonists is pertussis
toxin-insensitive (Eglen et al., 1988; Thomas and Ehlert, 1994) and is
inhibited by muscarinic antagonists in a manner consistent with an
M3 receptor profile (Lambrecht et al., 1989;
Ehlert et al., 1997). Moreover, genetic studies have revealed that the
responsiveness of various smooth muscle types to muscarinic agonists is
greatly reduced in mice lacking the muscarinic M3
gene (Matsui et al., 2000), whereas a much smaller decrement in
contractile function was observed in mice lacking the
M2 gene (Stengel et al., 2000). The
M3 receptor's role in contraction can be
rationalized on the basis of its signaling mechanism. In
gastrointestinal smooth muscle (Candell et al., 1990; Zhang and Buxton,
1991) as well as in cell lines transfected with recombinant muscarinic
receptors (Peralta et al., 1988), M3 receptors
mediate a pertussis toxin-insensitive phosphoinositide response, which
presumably is involved in Ca2+ mobilization and
contraction, whereas M2 receptors interact with the Gi family of G proteins, which are not
directly linked to Ca2+ mobilization.
Nevertheless, activation of M2 receptors in
smooth muscle inhibits both the increase in cAMP and the relaxation
elicited by cAMP-stimulating agents, such as forskolin and
isoproterenol (Griffin and Ehlert, 1992; Reddy et al., 1995; Ostrom and
Ehlert, 1997). Unlike the contractile effects of the
M3 receptor, these latter responses of the
M2 receptor are pertussis toxin-sensitive (Thomas
and Ehlert, 1994).
Short-term exposure of intestinal smooth muscle to muscarinic agonists
causes a desensitization to the contractile effects of both histamine
and muscarinic agonists (Cantoni and Eastman, 1946; Dale, 1958; Paton,
1961). A large part of this desensitization appears to be due to a
mechanism downstream from the receptor, which explains why continuous
activation of one receptor type leads to a subsensitivity of responses
to an agonist acting at a different receptor. With regard to muscarinic
agonist-induced desensitization of the contractile response to
muscarinic agonists, it has been shown that the desensitizing effect of
agonist exposure is prevented by coincubation with the
M3-selective antagonist p-fluorohexahydrosiladifenidol, but not with
M2- or M1-selective antagonists (Eglen et al., 1992). These results suggest that excessive activation of M3 receptors causes desensitization
in the guinea pig ileum.
In this report, we have investigated the role of M2 and M3 receptors in acetylcholine-mediated desensitization of contractions elicited to histamine in the guinea pig ileum. We found that treatment of the isolated ileum with acetylcholine (30 µM; 20 min) causes a large decrease in contractile sensitivity to histamine (6-fold increase in EC50 value), whereas it has little or no inhibitory effect on histamine-mediated phosphoinositide hydrolysis. These results show that the majority of the heterologous desensitization is the result of a change downstream from phosphoinositide hydrolysis. Acetylcholine-induced desensitization was prevented by pertussis toxin treatment or by selective inactivation of M3 receptors with N-2-chloroethyl-4-piperidinyl diphenylacetate (4-DAMP mustard). Since muscarinic M2 receptors are known to signal through pertussis toxin-sensitive G proteins in gastrointestinal smooth muscle (see above), our results suggest that activation of both M2 and M3 receptors is required for heterologous desensitization by muscarinic agonists.
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Materials and Methods |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Isolated Ileum.
Male guinea pigs (Hartley; 300-500 g) were
euthanized with CO2, and segments of the ileum
(approximately 2.5 cm) were removed starting at a point approximately
10 cm rostral from the caecum. Ileal segments were mounted
longitudinally in an organ bath containing Krebs-Ringer bicarbonate
buffer (KRB buffer; 124 mM NaCl, 5 mM KCl, 1.3 mM
MgSO4, 26 mM NaHCO3, 1.2 mM
KH2PO4, 1.8 mM
CaCl2, and 10 mM glucose) and indomethacin
(1.0 µM) gassed with
O2/CO2 (19:1). Tonic,
isometric contractions were measured with a force-displacement transducer and polygraph as described previously (Thomas et al., 1993).
Briefly, resting tension was adjusted to a load of 0.5 g, and ilea
were allowed to equilibrate for at least 60 min before contractions
were measured. Three test doses of the muscarinic agonist
oxotremorine-M (40 nM) were added to the bath in succession to ensure
the reproducibility and magnitude of contractile responses. The ileum
was allowed to rest 5 min between each test dose.
Concentration-response curves to histamine and acetylcholine were
measured using a cumulative technique as described previously (Thomas
et al., 1993). Approximately 5 to 7 min was required to measure a
complete concentration-response curve. All contractile responses are
expressed as mass equivalents (i.e., g) minus resting tension. Control
experiments showed that the EC50 value and
maximal response to histamine remained constant for at least 3 h
when measured every 30 min.
). The solution
was then placed on ice and used as soon as possible.
Phosphoinositide Hydrolysis.
Phosphoinositide hydrolysis was
measured in strips of the longitudinal muscle of the isolated ileum
using a procedure similar to that described previously by Thomas et al.
(1993). Our technique is based on the
[3H]inositol-labeling and ion exchange
separation method of Berridge et al. (1982), and it incorporates the
perchloric acid extraction method of Kendall and Hill (1990). Segments
of isolated ileum (approximately 10 cm) were removed from euthanized
guinea pigs (see above), washed with KRB buffer, and mounted on a glass
pipette. The outer longitudinal muscle layer was obtained by gentle
rubbing with a cotton swab as described by Paton and Vizi (1969). The tissue was cut into small strips (0.5 cm), and these were placed in an
Erlenmeyer flask (50 ml) containing
[3H]inositol (200 µCi; PerkinElmer Life
Science Products, Boston, MA) and KRB buffer (10 ml) gassed with
O2/CO2 (19:1) and sealed with a rubber stopper. The tissue was incubated at 37°C for 2 h
with gentle shaking. The atmosphere in the flask was flushed with
O2/CO2 every 30 min.
Following this labeling phase, the tissue was washed three times with
warm KRB buffer and incubated at 37°C for 20 min in 10 ml of KRB
buffer containing 10 mM nonradioactive inositol. After this incubation,
the tissue was washed twice with warm KRB buffer.
).
After making this determination, all solvents were removed with a Speed
Vac concentrator connected to a waterjet aspirator (Savant Instruments,
Inc., Farmingdale, NY). The residual tissue was dissolved in 1 ml of
NaOH (0.5 M), and protein was estimated using the Bradford reagent
(Bio-Rad, San Diego, CA). Incorporation of
[3H]inositol into phosphoinositides is
expressed as counts per minute per milligram of protein. Estimates of
phosphoinositide hydrolysis are expressed as the percentage of
conversion of labeled phosphoinositides into
[3H]inositolphosphates.
Calculations.
The maximal response
(Emax), concentration of agonist
eliciting a half-maximal response (EC50 value),
and the Hill coefficient of histamine for eliciting contraction and
phosphoinositide hydrolysis were estimated by nonlinear regression
analysis of the concentration-response curves according to an
increasing logistic equation as described previously (Candell et al.,
1990). The paired Student's t test (two-tailed; minimum
level of significance, p = 0.05) was used to determine
whether the effect of acetylcholine treatment on the
EC50 value of histamine was significant. To
determine whether there was a significant difference between the
desensitizing effects of acetylcholine treatment on the
EC50 values of histamine in control, pertussis
toxin-treated, and 4-DAMP mustard-treated ilea, an unpaired
t test was used.
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Results |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Isolated Ileum.
Initial experiments were run using the
muscarinic agonist oxotremorine-M as the desensitizing agent; however,
it was difficult to establish resting tension quickly following washout
of concentrations of oxotremorine-M greater than 3 µM. Spontaneous
contractions persisted for several minutes following exposure of the
ileum to 3 to 10 µM oxotremorine-M for time periods ranging from 5 to 20 min. Similar observations have been reported for carbachol (Eglen et
al., 1992) and furtrethonium (Gosselin and Gosselin, 1973). However,
when we used acetylcholine as the desensitizing agent, resting tension
was restored quickly (within 2 min) following washout. Presumably, the
rapid hydrolysis of acetylcholine by acetylcholinesterase terminates
its action following washout.
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). The mechanism presumably
involves a selective ADP ribosylation of Gi.
Guinea pigs were injected in vivo with pertussis toxin (75 µg/kg
i.p.) 3 days prior to being euthanized for the subsequent in vitro
experiments. Figure 2 shows the effects
of pertussis toxin treatment on acetylcholine-induced heterologous
desensitization. Pertussis toxin treatment had no significant effect on
the control concentration-response curve to histamine (Fig. 2A). As
described above in connection with Fig. 1, acetylcholine treatment
caused a 5.8-fold increase in the EC50 value of
histamine (Fig. 2B) when measured 5 min after washout of acetylcholine.
This desensitization was largely prevented by pertussis toxin treatment
(Fig. 2C). Similar observations were made 10 min after washout of
acetylcholine, although the desensitizing effects of acetylcholine were
less at 10 min (Fig. 1). A summary of the effects of pertussis toxin
treatment on acetylcholine-induced desensitization of contractions to
histamine is given in Table 1.
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). We have also
shown in contractile assays that these effects of pertussis toxin are manifest by an increase in the relaxant action of isoproterenol on
contractions elicited to the muscarinic agonist oxotremorine-M (Thomas
and Ehlert, 1994; Ostrom and Ehlert, 1999; Sawyer and Ehlert, 1999a).
To verify the effectiveness of pertussis toxin treatment in the
experiments described in Fig. 2, we measured the effects of
isoproterenol on oxotremorine-M-mediated contractions in ilea taken
from the same guinea pigs used for the experiments shown in Fig. 2. In
control ilea, isoproterenol (1.0 µM) caused a 1.6-fold increase in
the EC50 value of oxotremorine-M and a nonsignificant 15% reduction in the maximal response. Following pertussis toxin treatment, the effects of isoproterenol were
significantly greater, corresponding to a 4.1-fold increase in the
EC50 value of oxotremorine-M with no change in
the maximal response. These effects of pertussis toxin treatment are
similar to those observed in a prior study in which the ADP
ribosylation of Gi was directly verified by
SDS-polyacrylamide gel electrophoresis (Thomas and Ehlert, 1994).
Since our results with pertussis toxin suggest that muscarinic
M2 receptor activation is required for
acetylcholine-mediated desensitization (under Discussion),
we wondered whether activation of M2 receptors by
themselves was sufficient to account for the desensitization. To test
this postulate, we treated ileal segments with 4-DAMP mustard to
inactivate all muscarinic receptor subtypes except the
M2 and measured the consequences on heterologous
desensitization. Ilea were incubated with the aziridinium ion of 4-DAMP
mustard (40 nM) in combination with AF-DX 116 (4 µM) for a total of
2 h. After 1-h incubation, the ileum was washed and exposed to a fresh solution containing 4-DAMP mustard and AF-DX 116 for the second
hour. The ileum was washed extensively and then used for contractile
studies. We first investigated the effects of 4-DAMP mustard treatment
on the contractile response to acetylcholine. Treatment with 4-DAMP
mustard caused a 182-fold increase in the EC50
value of acetylcholine without causing a significant effect on the
maximal response (Fig. 3). We have shown
that residual M3 receptors mediate the
contractile response to muscarinic agonists in the ileum after 4-DAMP
mustard treatment even though the mustard inactivates a great majority
of the M3 receptors (Thomas et al., 1993).
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Phosphoinositide Hydrolysis.
H1
histamine receptors are known to mediate the contractile effects of
histamine on the isolated guinea pig ileum (Black et al., 1972). This
receptor subtype signals through Gq to trigger phosphoinositide hydrolysis (Arrang et al., 1995). Thus, to investigate the extent of desensitization of H1 histamine
receptors in the ileum, we measured the effects of acetylcholine
treatment on histamine-stimulated phosphoinositide hydrolysis. Figure
5 shows the effects of acetylcholine treatment (30 µM; 20 min) on histamine-stimulated phosphoinositide hydrolysis in control and pertussis toxin-treated ilea, 5 min after
washout of acetylcholine. The phosphoinositide assay was run using a
5-min incubation with histamine. This incubation time was chosen
because it is equivalent to the time required to measure a
concentration-response curve for the contractile response. Thus, both
the contractile assay and the phosphoinositide assay were measured
during the same time interval after acetylcholine washout. In control
ilea, histamine stimulated phosphoinositide hydrolysis with an
EC50 value of 49 µM and an
Emax value corresponding to a 6.1%
conversion of labeled phosphoinositides into
[3H]inositolphosphates. Prior exposure to
acetylcholine had little influence on histamine
stimulated-phosphoinositide hydrolysis (Fig. 5B). The
EC50 value of histamine increased 2-fold;
however, this effect was not significant. Also, acetylcholine treatment had no significant effect on the maximal response to histamine, nor on
basal levels of [3H]inositolphosphates.
Similarly, acetylcholine treatment had no significant effect on the
EC50 and Emax
values of histamine for stimulating phosphoinositide hydrolysis in
pertussis toxin-treated ilea (Fig. 5C). Interestingly, pertussis toxin
treatment itself caused a 30% increase in the
Emax value of histamine for
stimulating phosphoinositide hydrolysis (Fig. 5A).
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Discussion |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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As described above, M2 and
M3 muscarinic receptors are abundantly expressed
in the smooth muscle of the guinea pig ileum where they have distinct
roles in eliciting contraction. However, their participation in the
desensitization process has not been carefully explored. Previous
studies from our laboratory have shown that 4-DAMP mustard is useful
for inactivating the M3 receptor selectively while having little influence on the M2 receptor.
Treatment of the isolated ileum with 4-DAMP mustard (40 nM) in
combination with AF-DX 116 (1 µM) caused a large inhibition of the
contractile response to the muscarinic agonist oxotremorine-M (Thomas
et al., 1992). It is known that, in the absence of other heterologous contractile or relaxant agents, muscarinic agonists elicit contraction through the M3 receptor (Lambrecht et al., 1989).
Treatment with 4-DAMP mustard also greatly inhibits the
phosphoinositide response to oxotremorine-M while having little or no
effect on its ability to inhibit cAMP accumulation (Thomas and Ehlert,
1994). The latter two responses are mediated by
M3 and M2 muscarinic
receptors, respectively (Candell et al., 1990; Griffin and Ehlert,
1992). Thus, the heterologous desensitization of histamine-induced
contraction by acetylcholine most certainly requires activation of
M3 receptors because the desensitization is
blocked by 4-DAMP mustard treatment.
Pertussis toxin is a useful tool for discriminating between
M2 and M3 muscarinic
responses in the guinea pig ileum as well as in cell lines transfected
with recombinant muscarinic receptors. The M2
receptor is known to signal through the pertussis toxin-sensitive Gi family to inhibit adenylyl cyclase, whereas
the M3 receptor signals through the pertussis
toxin-insensitive Gq to stimulate phosphoinositide hydrolysis (Peralta et al., 1988; Lai et al., 1991;
Dell'Acqua et al., 1993). Accordingly, pertussis toxin has no
inhibitory effect on muscarinic agonist-mediated contraction and
stimulation of phosphoinositide hydrolysis in the guinea pig ileum,
both of which are mediated via the M3 receptor
(Thomas and Ehlert, 1994). In contrast, pertussis toxin treatment
inhibits M2 receptor-mediated inhibition of the
cAMP accumulation elicited by forskolin and isoproterenol in the ileum
(Thomas and Ehlert, 1994; Ostrom and Ehlert, 1999). Moreover, pertussis
toxin treatment inhibits M2 receptor
mediated-inhibition of the relaxant effects of isoproterenol and
forskolin on histamine-induced contractions (Thomas and Ehlert, 1994;
Ostrom and Ehlert, 1999). The ability of pertussis toxin to block the
desensitizing effect of acetylcholine on histamine-mediated contraction
strongly suggests that activation of M2 receptors
is required for acetylcholine-mediated heterologous desensitization.
Collectively, our results indicate that activation of both
M2 and M3 receptors is
required for heterologous desensitization and that activation of either
receptor by itself is insufficient to cause desensitization. This
situation is reminiscent of a contractile phenomenon that we have
previously observed in gastrointestinal smooth muscle. After
inactivation of a majority of the M3 receptors in
the guinea pig colon with 4-DAMP mustard, it is still possible to
elicit a contractile response to a muscarinic agonist, albeit with
greatly reduced potency (Sawyer and Ehlert, 1998, 1999b). Under this
condition, the contractile response is pertussis toxin-sensitive, suggesting a role for the M2 receptor.
Nevertheless, the contractile response is relatively insensitive to the
M2-selective antagonist AF-DX 116. Thus, the
contractile response under this condition is enigmatic; it is
M2-like in its sensitivity to pertussis toxin yet
is M3-like in its profile for pharmacological
antagonism. We have previously shown that this behavior can be
rationalized by a model based on an interaction between
M2 and M3 receptors (Ehlert
et al., 1999; Sawyer and Ehlert, 1999b). According to the model,
activation of the M2 receptor by itself does not
cause contraction; nevertheless, M2 receptor
activation does potentiate the contractile response elicited through
the M3 receptor. It is possible that a similar
phenomenon is operative with regard to the heterologous desensitization
observed in this study. If both M2 and
M3 receptors interact with each other to elicit
contraction in the presence of high concentrations of acetylcholine,
then it is possible that the downstream signaling mechanisms interact with each other in a manner that reflects this interaction. The resulting subsensitivity of the contractile machinery and its dependence on an M2-M3
receptor interaction could account for acetylcholine-mediated
desensitization of histamine-induced contraction.
The heterologous desensitization observed in this study appears to be
due to a subsensitivity in the signaling pathway downstream from the
receptor and phosphoinositide hydrolysis. Several different mechanisms
may be involved in excitation-contraction coupling in the guinea pig
ileum, including activation of a nonselective cation conductance
(Bolton, 1979), activation of voltage-dependent Ca2+ channels, activation of phospholipase
A2 (Wang et al., 1993), and activation of
phospholipase C and the consequent hydrolysis of phosphoinositides
(Berridge, 1984; Abdel-Latif, 1986). Although we think it is likely
that phosphoinositide hydrolysis is an early step in the signaling
pathway for the contractile response to histamine and muscarinic
agonists, it is possible that another mechanism is more important.
Regardless, the phosphoinositide response should be useful as an
endogenous reporter mechanism for assessing desensitization of
histamine receptors. Accordingly, we observed that acetylcholine
treatment caused a small, 2-fold increase in the
EC50 value of histamine for triggering
phosphoinositide hydrolysis. The variance of the data was such that
there was insufficient statistical power to resolve this potential
difference. Nevertheless, there was a large, highly significant, 6-fold
increase in the EC50 value of histamine for
eliciting contraction after acetylcholine treatment. Collectively,
these results show that the main effect of acetylcholine treatment was
to cause desensitization at a level downstream from the receptor and
phosphoinositide hydrolysis. Such a locus is consistent with
heterologous desensitization.
We noted that pertussis toxin treatment caused an increase in the
Emax value of histamine for
stimulating phosphoinositide hydrolysis in the guinea pig ileum. We
have previously noted a similar phenomenon in the guinea pig colon
where pertussis toxin treatment caused a small increase in the maximal
response of the muscarinic agonist oxotremorine-M for eliciting
phosphoinositide hydrolysis (Sawyer and Ehlert, 1999b). In that study,
the effect on phosphoinositide hydrolysis was associated with a similar
pertussis toxin-mediated enhancement of the contractile response to
oxotremorine-M. However, in this study, pertussis toxin was without
effect on the contractile response to histamine even though it enhanced the phosphoinositide response. We have no adequate explanation for this
discrepancy. In studies on cell lines, pertussis toxin treatment has
been shown to enhance receptor-mediated phosphoinositide hydrolysis
(Hermans et al., 2000).
It has been noted previously that muscarinic agonist-mediated
heterologous desensitization of histamine-induced contractions in the
guinea pig ileum is greater than the converse histamine-mediated desensitization of muscarinic agonist-induced contractions (compare Leurs et al., 1991; Eglen and Whiting, 1987; and Ishii and Kato, 1987).
In other words, muscarinic agonists are more effective at causing
heterologous desensitization than histamine. The explanation for this
phenomenon might be related to the types of G proteins activated by the
different agonists. In ileal smooth muscle, muscarinic agonists
interact with both M2 and
M3 muscarinic receptors, resulting in an
activation of Gi and Gq,
respectively. In contrast, histamine signals mainly through the
H1 receptor to activate only
Gq. In this study, activation of the
M3-Gq pathway alone by
acetylcholine was insufficient to cause heterologous desensitization as
shown by the prevention of desensitization by pertussis toxin
treatment, which selectively inactivates
M2-Gi signaling. By
analogy, histamine would not be expected to cause much heterologous
desensitization because it only activates Gq and
not Gq in combination with
Gi. Thus, the inability of histamine to activate
Gi in the ileum may explain its reported weak
desensitizing action.
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Footnotes |
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Accepted for publication February 17, 2001.
Received for publication November 13, 2000.
This work was supported by National Institutes of Health Grant NS30882.
Send reprint requests to: Frederick J. Ehlert, Department of Pharmacology, College of Medicine, University of California, Irvine, Irvine, California 92697-4625. E-mail: fjehlert{at}uci.edu
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Abbreviations |
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4-DAMP mustard, N-2-chloroethyl-4-piperidinyl diphenylacetate; KRB, Krebs-Ringer bicarbonate; AF-DX 116, [[2-[(diethylamino)methyl]-1-piperidinyl]acetyl]-5,11-dihydro-6H-pyrido[2,3b][1,4]-benzodiazepine-6-one.
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References |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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) and pertussis toxin-treated (
) guinea pigs.
B, contractile response to histamine was measured before (
) exposure to acetylcholine (30 µM, 20 min) in ilea
from untreated guinea pigs. C, contractile response to histamine was
measured before (
) exposure to acetylcholine
(30 µM, 20 min) in ilea from pertussis toxin-treated guinea pigs. The
data represent the mean values ± S.E.M. from four control ilea
and three pertussis toxin-treated ilea, each from a different guinea
pig.
