0022-3565/99/2881-0239$03.00/0
THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
Copyright © 1999 by The American Society for Pharmacology and Experimental Therapeutics
JPET 288:239 –246, 1999
Vol. 288, No. 1
Printed in U.S.A.
Mechanisms and Sites of Action of Endothelins 1 and 2 on the
Opossum Internal Anal Sphincter Smooth Muscle Tone In Vitro
SUSHANTA CHAKDER and SATISH RATTAN
Department of Medicine, Division of Gastroenterology and Hepatology, Jefferson Medical College, Thomas Jefferson University, Philadelphia,
Pennsylvania
Accepted for publication August 11, 1998
This paper is available online at http://www.jpet.org
Endothelins belong to a family of vasoconstrictor peptides
that were initially thought to be associated with the regulation of cardiovascular function. More recently, endothelins
have been suggested to play a broader role in diverse physiological actions (Rubanyi and Polokoff, 1994; Rae et al.,
1995). Endothelins have been shown to be localized in the
enteric nervous system (Takahashi et al., 1990; Inagaki et
al., 1991) and have been proposed to exert important modulatory actions in the gastrointestinal motility (Takahashi et
al., 1990; Allcock et al., 1995; Miasiro et al., 1995; Rae et al.,
1995). Most of the actions of endothelins in the gastrointestinal tract are contractile and occur via their direct actions at
the smooth muscle (Kitsukawa et al., 1994; Okabe et al.,
1995). Furthermore, endothelins have been proposed to play
Received for publication March 17, 1998.
1
This work was supported by National Institutes of Diabetes and Digestive
and Kidney Diseases Grant DK-35385 and an institutional grant from Thomas
Jefferson University.
hand, was not affected by any of the neurohumoral antagonists
but was significantly inhibited by the selective protein kinase C
inhibitor H-7 or the calmodulin inhibitor W-13. The combination
of H-7 and W-13 had no additive effect in attenuating the
contractile action of endothelin 1. There was clear evidence of
a cross-tachyphylaxis to the actions of endothelin 1 and endothelin 2. We conclude that the endothelins exert important
neuromodulatory effects on the basal tone of the IAS. The
contractile action occurs directly at the smooth muscle and the
relaxant action by the activation of neuronal nitric oxide synthase at the nerve terminals. The contraction and relaxation of
the smooth muscle caused by endothelins 1 and 2 may involve
distinct receptors that are similar for both endothelins. The
excitatory actions of endothelin 1 involve both the protein kinase C and the Ca11-calmodulin pathways that may lie in
series.
a role in the development of the enteric nervous system
(Baynash et al., 1994). It has been suggested that targeted
disruption of the endothelin B (ETB) receptor gene results in
an aganglionosis of the colon that resembles Hirschsprung’s
disease in humans (Puffenberger et al., 1994). In our recent
studies in an appropriate animal model, Ls/Ls mice, we have
shown that Hirschsprung’s disease is characterized by the
loss of nitrergic inhibitory neurotransmission in the internal
anal sphincter (IAS) (Chakder et al., 1997). In spite of their
widespread distribution, the actions of endothelins on the
gastrointestinal smooth muscle of the IAS are not known.
Two types of endothelin receptors, endothelin A (ETA) and
ETB, have been identified based on their actions on the
smooth muscle. In general, ETB receptor activation is known
to cause the smooth muscle relaxation; contraction, on the
other hand, may either be via both ETA and ETB receptors or
via ETA receptor only (Kobari et al., 1994; Irie et al., 1995;
Miasiro et al., 1995; Lucas et al., 1996; Higashi et al., 1997).
ABBREVIATIONS: IAS, internal anal sphincter; EFS, electrical field stimulation; ETA, endothelin receptor A; ETB, endothelin receptor B; NANC,
nonadrenergic noncholinergic; L-NNA, L-NG-nitro-arginine; TRIM, 1-(2-trifluoromethylphenyl) imidazole; NOS, nitric oxide synthase; TTX, tetrodotoxin; H-7, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride; W-13, N-(4-aminobutyl)-5-chloro-2-naphthalenesulfonamide hydrochloride; PKC, protein kinase C.
239
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ABSTRACT
Endothelins, localized in the enteric nervous system, may play
important roles in the morphogenesis of the gastrointestinal (GI)
tract and in the regulation of GI motility. However, the role of
endothelins in the GI sphincters, including the internal anal
sphincter (IAS) have not been examined. We examined the
actions of endothelins on the basal tone of the opossum IAS
circular smooth muscle strips before and after different neurohumoral antagonists or inhibitors. Endothelins 1 and 2 produced a concentration-dependent biphasic effect on the basal
tone of the IAS, an initial brief fall followed by a sustained rise.
The fall in the IAS smooth muscle tone was not modified by
atropine, guanethidine, or tetrodotoxin but was significantly
attenuated by the nitric oxide synthase inhibitor L-NNA, the
specific neuronal nitric oxide synthase inhibitor, 1-(2-trifluoromethylphenyl)imidazole, the N-type neuronal Ca11-channel
blocker v-conotoxin GVIA, and by the calmodulin antagonist
W-13. Endothelin-induced contraction of the IAS, on the other
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Chakder and Rattan
The purpose of the present investigation was to examine the
effects and mechanism of action of endothelins on the basal
tone of the IAS.
Materials and Methods
alcohol followed by the addition of Krebs’ solution to the final volume. Indomethacin solution thus prepared was added into the muscle bath so that the final concentration was 1 3 1025 M. The vials
and pipette tips were siliconized while the muscle baths were treated
with 2.5% bovine serum albumin to eliminate binding of the peptides
to the glass surface.
Drug Responses. The effects of different concentrations of endothelin 1 and endothelin 2 were examined using single concentrations
because of the tachyphylaxis problem when used cumulatively. The
concentrations of different antagonists were maximally effective in
blocking the actions of their respective agonists or enzymes and were
relatively selective against the intended effects. Once the concentration-response curve to an endothelin was determined, the smooth
muscle strips were washed 10 to 15 times over 2 h, and the resting
tension was allowed to recover to the preinjection levels. Different
antagonists or inhibitors except indomethacin were added 10 min
before the addition of the agonists to the muscle bath. The smooth
muscle strips were pretreated with indomethacin for 30 min before
testing the effects of endothelin 1. Endothelin 1 tachyphylaxis was
achieved by its frequent administration in the maximal effective
concentration.
Data Analysis. The results are expressed as means and S.E. of
different experiments. The fall of the resting IAS tension is expressed as the percentage of Emax (100%) in response to the supramaximal concentration (5 mM) of EDTA. The rise in tension is
expressed as the percentage of Emax (100%) obtained with phenylephrine (1 3 1025 M). Statistical significance between different
groups was determined by using paired or unpaired t test or analysis
of variance where applicable, and a p value smaller than 0.05 was
considered to be statistically significant.
Results
Effect of Endothelin 1 on the Basal Tone of the IAS
Smooth Muscle: Influence of Atropine and Guanethidine. In the initial experiments, we carried out concentration-response studies by the cumulative concentrations of
endothelin 1. In these experiments, endothelin 1 was found
to cause primarily concentration-dependent rise in the basal
tension of the IAS (Fig. 1). However, in some of these experiments, there was an indication of the fall in the basal IAS
tone, especially in the lower concentration range (1 3 1029–
1 3 1027 M). To further examine the divergent effects of
endothelins in detail, subsequent studies were performed
using single boluses. Under these experimental conditions,
we observed a clear dichotomy of the inhibitory and excitatory effects of endothelin 1. There was clear evidence of a
biphasic and concentration-dependent effect of endothelin 1
on the basal tone of the IAS. The biphasic effect consisted of
an initial brief relaxation followed by a sustained contraction
of the IAS smooth muscle. The initial fall was markedly more
prone to tachyphylaxis than the contraction. Therefore, for
the detailed pharmacological analyses of the concentrationresponse curves, extreme care was taken to wash the smooth
muscle repeatedly to ensure the reversal of the control responses before pursuing the studies. Furthermore, a given
smooth muscle was subjected to a limited experimental protocol. The actions of endothelin 2 were found to be similar to
those of endothelin 1.
Neither the relaxant nor the contractile actions of endothelin 1 were modified by atropine (1 3 1026 M) or guanethidine
(3 3 1026 M). The percent fall and rise in the IAS tension in
response to endothelin 1 (1 3 1026 M) before atropine were
40.0 6 6.7 and 66.6 6 7.0, respectively. After treatment with
atropine, these values were 42.2 6 7.5 and 58.2 6 9.0%,
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Preparation of Smooth Muscle Strips. The IAS smooth muscle
strips from opossums (Didelphis virginiana) were prepared for the
recording of isometric tension as described previously (Rattan and
Chakder, 1992). Briefly, following anesthesia with pentobarbital (40
mg/kg, i.p.), the animals were sacrificed by exsanguination and the
anal canal along with a section of the rectum was isolated and
transferred to oxygenated (95% O2//5% CO2) Krebs’ solution of the
following composition: NaCl, 118.07 mM; KCl, 4.69 mM; CaCl2, 2.52
mM; MgSO4, 1.16 mM; NaH2PO4 , 1.01 mM; NaHCO3, 25 mM; and
glucose, 11.10 mM. The anal canal was carefully freed of all extraneous tissues, including the large blood vessels, opened, and pinned
flat with the mucosal side up on a dissecting tray containing oxygenated Krebs’ solution. The mucosal and submucosal layers were removed by sharp dissection and the IAS circular smooth muscle strips
(1 3 10 mm) were prepared.
Measurement of Isometric Tension. The smooth muscle strips
were tied at both ends with silk sutures (6 – 0; Ethicon Inc., Sommerville, NJ) and transferred to 2-ml muscle baths containing oxygenated Krebs’ solution (37°C). One end of the muscle strip was anchored at the bottom of the muscle bath and the other end was
attached to a force transducer (model FTO3; Grass Instruments Co.,
Quincy, MA) for the measurement of isometric tension on a Dynograph recorder (model R411; Beckman Instruments, Schiller Park,
IL). The muscle strips were stretched initially with 9.8 mN of tension
and then allowed to equilibrate for at least 1 h with regular washings
at 20-min intervals. Only the strips that developed spontaneous
steady tension and relaxed in response to electrical field stimulation
(EFS) were used. The optimal length (Lo) and the baseline of the
smooth muscle strips were determined as explained before (Rattan
and Chakder, 1992).
Nonadrenergic Noncholinergic (NANC) Nerve Stimulation
with Electrical Field Stimulation (EFS). EFS was delivered from
a Grass stimulator (model S88; Grass Instruments) connected in
series to a Med-Lab Stimu-Splitter II (Med-Lab Instruments, Loveland, CO). The Stimusplitter served an important purpose to amplify
and measure the actual stimulus intensity delivered to the tissues
under the existing experimental conditions, using the optimal stimulus parameters for the neural stimulation (12 V, 0.5-ms pulse
duration, 200 – 400 mA, 4-s train) at varying frequencies of 0.5 to 20
Hz. These parameters are known to cause the IAS smooth muscle
relaxation via selective activation of NANC myenteric neurons (Rattan and Chakder, 1992; Chakder and Rattan, 1993a; Rattan et al.,
1995). The electrodes used for the EFS consisted of a pair of platinum
wires fixed at both sides of the smooth muscle strip.
Drugs and Chemicals. The following chemicals were used in the
study: endothelin 1 and endothelin 2 (Bachem Bioscience Inc., King
of Prussia, PA); atropine sulfate (muscarinic antagonist), guanethidine (adrenergic blocker), spantide (Substance P antagonist), tetrodotoxin (TTX; sodium-mediated axonal conduction blocker), indomethacin, L-NG-nitro-arginine [nitric oxide synthase (NOS)
inhibitor] and N-type neuronal Ca11 channel blocker, v-conotoxin
GVIA (Sigma Chemical Co., St. Louis, MO); EDTA tetrasodium
(Ca11 chelator) (Fisher Scientific, Pittsburgh, PA); 1-(2-trifluoromethylphenyl) imidazole (TRIM; neuronal NOS inhibitor); 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H-7; protein
kinase C inhibitor), and N-(4-aminobutyl)-5-chloro-2-naphthalenesulfonamide hydrochloride (W-13; calmodulin inhibitor) (Research
Biochemicals International, Natick, MA).
All chemicals except indomethacin were dissolved and diluted in
Krebs’ solution and prepared fresh on the day of the experiment.
Stock solution (4 ml) of indomethacin (10 mM) was prepared by
dissolving the appropriate amount of indomethacin in 200 ml of ethyl
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241
respectively (p . 0.05; n 5 5). In guanethidine experiments,
the percent fall and rise in the IAS tension caused by endothelin 1 (1 3 1026 M) in control were 36.0 6 10.4 and 61.8 6
11.4, respectively. In the presence of guanethidine, these
values were 30.8 6 5.2 and 51.7 6 6.4%, respectively (p .
0.05; n 5 5).
Influence of Substance P Antagonist Spantide on the
IAS Contraction Caused by Endothelin 1. Because the
major action of Substance P in the IAS was to cause contraction of the IAS smooth muscle, only the contractile action of
endothelin 1 was examined in the presence of spantide (3 3
1025 M). The data show that the rise in the IAS smooth
muscle tension by endothelin was not significantly modified
by spantide. The rise in the basal tension of the IAS in
response to endothelin 1 (1 3 1026 M) before and after
spantide were 54.8 6 11.6 and 49.6 6 4.4% respectively (p .
.05; n 5 4). The concentration of spantide used was found to
be maximally effective in antagonizing the actions of Substance P in the IAS smooth muscle.
Influence of TTX on the Actions of Endothelin 1. The
neurotoxin TTX, in the concentration (1 3 1026 M) that
abolishes the EFS-induced relaxation of the IAS, failed to
modify both the relaxant and the contractile actions of endothelin 1 on the basal tone of the IAS smooth muscle.
Influence of the Cycloxygenase Inhibitor Indomethacin on the Actions of Endothelin 1. Pretreatment with
indomethacin (1 3 1025 M) for 30 min had no significant
effect on either the fall or the rise in the basal IAS tension
caused by endothelin 1. In these experiments, the initial falls
in the basal tone of the IAS with 1 3 1027 M and 1 3 1026 M
endothelin 1 were 26.6. 6 6.9 and 38.9 6 4.0%, and the rises
were 33.4 6 8.9 and 47.4 6 6.0%, respectively. These values
for relaxation following indomethacin pretreatment were
25.9 6 6.4 and 38.9 6 4.0%, and for contraction were 50.0 6
7.0 and 60.6 6 7.6%, respectively (p . .05; n 5 5). Furthermore, indomethacin pretreatment failed to influence the
tachyphylaxis to the relaxation caused by endothelin 1.
Influence of the NOS inhibitor L-NG-nitro-arginine
(LNNA) and the Selective Neuronal Nitric Oxide Synthase (nNOS) Inhibitor 1-(2-Trifluoromethylphenyl)
Imidazole (TRIM) on the Actions of Endothelin 1. The
NOS inhibitor L-NNA (3 3 1025 M) that caused maximal
suppression of the NANC nerve-mediated IAS relaxation
(Rattan and Chakder, 1992) had no significant effect on the
contractile actions of endothelin 1, but it caused a significant
attenuation of the IAS smooth muscle relaxation by endothelin 1 (Fig. 2). It is well known that L-NNA, being a general
NOS inhibitor, may not be a good agent to discriminate the
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Fig. 1. Effect of endothelin 1 on the basal tone of the IAS smooth muscle. Endothelin 1 caused a biphasic effect in the IAS that consisted of an initial
fall followed by a sustained rise in the basal tone of the IAS smooth muscle. The data in this and other figures show mean and S.E. The data in this
figure represent mean and S.E. of five observations at each concentration level given in single boluses. The inset on the right is a typical example of
the action of endothelin 1 (1 3 1026 M) on the basal tone of the IAS smooth muscle.
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Chakder and Rattan
involvement of a specific type of NOS, i.e., neuronal or brain
(nNOS), endothelial NOS, and inducible NOS in a given
system (Moore and Handy, 1997). To specifically investigate
the role of nNOS in the mediation of inhibitory effects of
endothelin 1 in the IAS, we examined the influence of a
relatively selective nNOS inhibitor TRIM (Handy and Moore,
1997; Moore and Handy, 1997) on the effects of endothelin 1.
First, we examined the influence of TRIM on the NANC
nerve-mediated relaxation of the IAS smooth muscle. Data
given in Fig. 3 show that TRIM causes a significant and
concentration-dependent attenuation of NANC nerve-mediated relaxation of the IAS. In control experiments, the fall in
the IAS tension with 0.5 and 1 Hz EFS was 46.4 6 5.4 and
62.5 6 4.5%, respectively; that was significantly attenuated
to 23.3 6 4.7 and 41.5 6 6.3%, respectively, following TRIM
(3 3 1024 M) (p , .05; n 5 8). The fall in the basal tension of
the IAS caused by NANC nerve stimulation by the higher
frequencies of EFS was likewise attenuated by TRIM.
Subsequently, we examined the effect of 3 3 1024 M TRIM
on the IAS smooth muscle relaxation caused by endothelin 1.
The data shown in Fig. 4 depict that TRIM (3 3 1024 M)
causes a significant attenuation of the IAS relaxation but not
the contraction caused by endothelin 1. The data shown in
Fig. 4 demonstrate that the nNOS inhibitor caused significant blockade of the relaxant action of endothelin 1 in the
IAS. The fall in the basal tension of the IAS with 1 3 1027
and 1 3 1026 M endothelin 1 was 29.2 6 4.6 and 52.4 6 6.0%,
respectively. Following the administration of TRIM, these
values were 1.0 6 0.5 and 9.5 6 5.5%, respectively (p , .05;
n 5 5).
Fig. 3. Data (mean and S.E.) demonstrate the attenuation of the IAS
smooth muscle relaxation by NANC nerve stimulation by different frequencies of EFS in the presence of different concentrations of the selective nNOS inhibitor TRIM (3 3 1024 M). The attenuation of the IAS
relaxation by TRIM was significant and concentration-dependent (p, p ,
.05; n 5 8).
Fig. 4. Differential influence of TRIM on the excitatory versus the inhibitory effects of endothelin 1 on the IAS. Data (mean and S.E.) shows a
significant blockade of the inhibitory (p, p , .05; n 5 5) but not the
excitatory effect (p . .05; n 5 5) of endothelin 1 on the basal tension of the
IAS smooth muscle in the presence of the nNOS inhibitor TRIM.
Influence of the N-Type Neuronal Ca11 Channel
Blocker v-Conotoxin GVIA on the Biphasic Effect of
Endothelin 1 on the IAS. First, we examined the effect of
v-conotoxin GVIA on the NANC nerve-mediated relaxation
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Fig. 2. Influence of the NOS inhibitor L-NNA on the biphasic effect of
endothelin 1 on the basal IAS tone. Data (mean and S.E.) show a significant blockade of the inhibitory (p, p , .05; n 5 11) but not the excitatory
(p . .05; n 5 11) effect of endothelin 1 on the basal tension of the IAS
smooth muscle in the presence of the NOS inhibitor L-NNA.
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Endothelin and Internal Anal Sphincter Smooth Muscle Tone
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of the IAS smooth muscle. It was determined that the concentration of 1 3 1025 M was optimal to inhibit EFS-induced
relaxation of the IAS. Under these experimental conditions,
the toxin was found to cause a significant blockade of the IAS
smooth muscle relaxation but not the contraction caused by
endothelin 1 (Fig. 5; p . .05; n 5 5). This concentration of the
toxin was found to have no adverse effect on the basal tone of
the IAS, 22.8 6 2.9 in control experiments versus 24.2 6 3.2
mN in the presence of the toxin (p . .05; n 5 5).
Influence of the Protein Kinase C (PKC) Inhibitor 1-(5Isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H-7) and the Calmodulin Inhibitor N-(4-Aminobutyl)-5-chloro-2-naphthalenesulfonamide hydrochloride
(W-13) on the IAS Contraction Caused by Endothelin 1.
Because the activation of PKC (Bitar et al., 1991; Hillemeier et
al., 1996; Sohn et al., 1997) and calmodulin (Hillemeier et al.,
1991; Yu et al., 1995) have been shown to be the two major
pathways involved in the basal tone and in the agonist-induced
contraction of the sphincteric smooth muscle, we examined the
influence of the commonly used PKC and calmodulin inhibitors
H-7 and W-13, respectively, on the increase in the basal IAS
tone by endothelin 1. H-7 and W-13 in the appropriate concentrations (3 3 1027 and 1 3 1026 M), when used alone caused a
significant attenuation of the rise in the basal tone of the IAS by
endothelin 1 (Fig. 6). To explore the involvement of PKC and
calmodulin pathways in the mediation of contractile response in
parallel, we examined the influence of these inhibitors in combination. Interestingly, the combination had no greater attenuation of the contractile response of endothelin 1 when compared to the individual inhibitors (p . .05; n 5 5; Fig. 6).
Calmodulin has been suggested to be an important cofactor
for the activation of nNOS, and W-13, in addition to being a
calmodulin inhibitor in the smooth muscle cells may inhibit
the activation of nNOS in the non-neuronal cells. We examined this possibility by examining influence of W-13 on the
Fig. 6. Influence of the calmodulin inhibitor W-13 (upper panel) and the
protein kinase C inhibitor H-7 (middle panel) used alone, and in combination (lower panel), on the rise in the basal IAS tension by endothelin 1.
The data (mean and SE) show a significant blockade of the control
excitatory effect (p, p , .05; n 5 5) of endothelin 1 on the basal tension of
the IAS smooth muscle by either H-7 or W-13. The combination of H-7
and W-13 caused no further attenuation of endothelin 1-induced rise in
IAS tension.
Fig. 5. Effect of v-conotoxin GVIA on the biphasic effect of endothelin 1
on the IAS. The toxin caused a significant blockade of the fall (p, p , .05;
n 5 5) but not of the rise (p . .05; n 5 5) in the basal tension of the IAS.
IAS smooth muscle relaxation by endothelin 1. Interestingly,
the fall in basal IAS smooth muscle tension by endothelin 1
was significantly attenuated by the calmodulin inhibitor
(Fig. 7). The data suggest the involvement of the constitutive
nNOS in the endothelin-induced relaxation of the IAS
smooth muscle. The fall in the basal IAS tone in response to
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Chakder and Rattan
1 3 1027 M endothelin 1 before and after W-13 were 34.5 6
4.8 and 0.5 6 0.5%, respectively (p , .05; n 5 5). Interestingly, this concentration of W-13 had no significant effect on
the basal IAS tone. In these experiments, the basal IAS tone
before and after 1 3 1027 M W-13 were 17.9 6 2.2 and 18.4 6
2.2 mN, respectively (p . .05; n 5 5).
Influence of Endothelin 1 Tachyphylaxis on the Actions of Endothelin 2 in the IAS. Endothelin 1 tachyphylaxis caused a significant diminution of both the relaxant and
contractile actions of endothelin 2 on the IAS smooth muscle
(p , .05; n 5 5; Fig. 8). In control experiments, the initial fall
in the basal tension of the IAS with 1 3 1027 and 1 3 1026
M endothelin 2 were 18.3 6 6.3 and 57.3 6 2.6%, respectively. The later rise in the IAS tension with the same concentrations of endothelin 2 were 42.9 6 7.7 and 65.5 6 7.1%,
respectively. The fall in the basal tension of the IAS caused
by 1 3 1027 and 1 3 1026 M endothelin 2 in the presence of
endothelin 1 tachyphylaxis were 4.1 6 2.5 and 3.7 6 2.5%,
respectively. The rise in the IAS tension caused by the same
concentrations of endothelin 2 in the presence of endothelin 1
tachyphylaxis were 6.8 6 4.3 and 2.0 6 2.0%, respectively
(p , .05; n 5 5; Fig. 8).
Discussion
The present studies on the basal tone of the IAS smooth
muscle show a biphasic effect of endothelins on the spontaneously tonic smooth muscle of the gastrointestinal tract.
The biphasic action of endothelin 1 consisted of an initial
brief relaxation followed by a sustained contraction.
The initial relaxant action of endothelin 1 was found to be
via the activation of nNOS at the myenteric nerve terminals
of the IAS. Among all of the neurohumoral antagonists investigated, the relaxant action of endothelin 1 was only
Fig. 8. Influence of endothelin 1 tachyphylaxis on the biphasic effect of
endothelins 2 on the basal IAS tension. Note that endothelin 1 tachyphylaxis caused almost complete obliteration of the fall as well as the rise in
the IAS tension caused by not only endothelin 1 but also by endothelin 2
(p, p , .05; n 5 5).
blocked by the NOS inhibitors, N-type neuronal Ca11 channel blocker v-conotoxin GVIA and the calmodulin inhibitor
W-13. The neurotoxin TTX and cycloxygenase inhibitor indomethacin were found to have no effect on the endothelin
response. We (Chakder and Rattan, 1996) and others
(Mashimo et al., 1996) have shown previously that a part of
the relaxant action of vasoactive intestinal polypeptide that
was not affected by the neurotoxin TTX, occurs via the activation of specific receptors on the nerve terminals of the
myenteric neurons. The data suggest that the relaxant action
of endothelin occurred via the activation of endothelin receptor below the axonal level. The studies by Kitsukawa et al.
(1994) on the isolated smooth muscle cells further corroborate the thesis that the relaxant action of endothelin 1 is
indirect. To further verify the involvement of nNOS in the
inhibitory action of endothelin, we examined the influence of
a selective nNOS inhibitor TRIM. Interestingly, TRIM, like
L-NNA, caused near obliteration of the IAS smooth muscle
relaxation caused by endothelin 1.
It is well known that calmodulin plays an important role in
the activation of constitutive nNOS (Matsuoka et al., 1994;
Stevens-Truss et al., 1997). We found that the calmodulin
inhibitor W-13, in the concentrations known to inhibit calmodulin, caused near obliteration of the fall in the basal tone
caused by endothelin 1. These data further corroborate the
involvement of nNOS in the mediation of the IAS smooth
muscle relaxation by endothelin 1.
The exact intracellular pathway of the IAS smooth muscle
relaxation by endothelin was not investigated in the present
study. However, in general, it is well known that the smooth
muscle relaxation involving nNOS occurs primarily via the
activation of guanylate cyclase and an increase in the
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Fig. 7. Influence of W-13 (1 3 1026 M) on the fall in the basal tension of
the IAS caused by endothelin 1 (1 3 1027 M). The data (mean and S.E.)
show a significant suppression of the IAS smooth muscle relaxation in the
presence of W-13 (p, p , .05).
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1999
Endothelin and Internal Anal Sphincter Smooth Muscle Tone
shown in different systems that the activation of ETB may be
associated with the activation of constitutive NOS (endothelial NOS or nNOS) and release of NO (Kobari et al., 1994;
Lucas et al., 1996; Higuchi and Satoh, 1997). The earlier
studies have shown that the brain contains exclusively the
ETB receptors (Lysko et al., 1995) and further suggest the
possibility of endothelins-induced IAS smooth muscle relaxation via the activation of ETB receptors on nNOS-containing
nerve terminals.
The characterization of the endothelin receptor subtype/s
in the smooth muscle contraction or relaxation was not
within the scope of the present project. However, cross-tachyphylaxis between the relaxant and contractile actions of endothelin 1 and endothelin 2 suggest the involvement of a
common receptor subtype/s in the responses of the two endothelins. Furthermore, our preliminary data with the selective
antagonists of ETA and ETB BQ-123 and IRL-1038, respectively, show that these antagonists were almost equally effective in blocking the responses to endothelins 1 and 2. The
influence of these antagonists on the relaxant responses of
the endothelins were not investigated. The relative involvement of ETA and ETB receptors in the endothelin-mediated
relaxation and contractile responses remains to be determined.
Although, there are numerous studies in different smooth
muscle systems, including the gastrointestinal tract, to report the contractile actions of endothelins, there is limited
data that report the smooth muscle relaxation by endothelin.
In general, the studies reporting the smooth muscles relaxation by endothelins were done following their contraction by
another agonist or on the spontaneously phasic smooth muscles. To our knowledge, the relaxant response of endothelin
on the spontaneously tonic smooth muscle has not been reported before.
In summary, these studies show that endothelin causes the
relaxation and contraction of the spontaneously tonic sphincteric gastrointestinal smooth muscle. The relaxation by endothelins was observed as an initial and transient response
that was followed by the sustained contraction. The data
suggest that the IAS smooth muscle relaxation by endothelin
is primarily NOS-mediated by the activation of receptor at
the myenteric nerve terminals. The IAS smooth muscle contraction in response to endothelin, on the other hand, may be
caused by the direct activation of the smooth muscle cells.
The data suggest the involvement of both Ca11-calmodulin
and PKC pathways for the smooth muscle contraction by
endothelin. These pathways appear to lie in series rather
than in parallel. The studies suggest important neuromodulatory influences of endothelins on the basal tone of the IAS
smooth muscle. Future studies should focus on the localization of endothelins in the neuronal and non-neuronal structures of the IAS. Additional studies on the role of endothelins
in the inhibitory neurotransmission and morphogenesis of
the gastrointestinal tract may provide important information
on the pathophysiology of the gastrointestinal motility disorders.
Acknowledgments
We thank Dr. Ya-Ping Fan for his valuable suggestions.
Downloaded from jpet.aspetjournals.org at ASPET Journals on June 17, 2017
cytosolic cyclic GMP (Chakder and Rattan, 1993b; Luo et al.,
1995; Franck et al., 1997).
The contractile action of endothelin 1 unlike its relaxant
action on the IAS was caued by the direct activation of
endothelin receptor at the smooth muscle. This conclusion
was inferred because none of the neurohumoral blocking
agents investigated, including L-NNA and TRIM, had any
significant effect on the IAS smooth muscle contraction
caused by endothelin 1. The direct contractile action of endothelin 1 in the IAS smooth muscle was similar to that of the
rat colon (Moummi et al., 1992) and guinea pig gastric
smooth muscles (Kitsukawa et al., 1994), and other regions of
the gastrointestinal tract (Okabe et al., 1995; Bitar et al.,
1992).
For in-depth examination of the intracellular mechanism
of action of endothelin in producing IAS smooth muscle contraction, we investigated the influence of specific inhibitors of
two major pathways responsible for the smooth muscle contraction in the basal state as well as in response to certain
agonists. For this, the influences of the calmodulin and PKC
inhibitors W-13 and H-7, respectively (Biancani et al., 1994),
on the IAS smooth muscle contraction by endothelin 1 were
investigated. The data revealed the involvement of both
pathways for smooth muscle contraction by endothelin 1
because both W-13 and H-7 caused substantial suppression
of the smooth muscle contraction. We raised the issues
whether both pathways are involved in the endothelin 1-mediated IAS smooth muscle contraction in parallel and
whether these pathways together may account fully for the
smooth muscle contraction by endothelin 1. Interestingly, the
combination of W-13 and H-7 caused no further attenuation
of the IAS smooth muscle contraction observed by the individual use of these agents. The observations suggest that the
calmodulin and PKC pathways, for the endothelin-mediated
IAS smooth muscle contraction, may lie in series rather than
parallel. It is possible that PKC-activated smooth muscle
contraction is mediated via the Ca11-calmodulin pathway
(Singer, 1990; Miura et al., 1997). The studies further raise
the possibility of intracellular mechanism other than Ca11calmodulin and PKC for the remaining smooth muscle contraction by endothelin 1 in the presence of the combination of
W-13 and H-7. The participation of exact intracellular mechanisms independent of PKC and Ca11-calmodulin pathways
responsible for the endothelin-induced contraction of the
smooth muscle is not known. In this regard, the relative roles
of phospholipase D activation independent of PKC activation,
Ca11 influx, release of intracellular Ca11, Na1/H1 exchange, increased sensitivity to Ca11, adenylate and guanylate cyclase inhibition, in the mediation of smooth muscle
contraction remain to be determined.
To date, in general, two types of endothelin receptors (ETA
and ETB) have been recognized to explain the actions of
endothelins in different systems (Kitsukawa et al., 1994;
Masaki et al., 1994; Rubanyi and Polokoff, 1994; Allcock et
al., 1995; Gray et al., 1995; Okabe et al., 1995). There is some
data to suggest that different receptor subtypes may mediate
the inhibitory and excitatory smooth muscle responses to
endothelin. ETB receptor activation may mediate the smooth
muscle relaxation, whereas both ETA and ETB may be involved in the smooth muscle contraction by endothelins (Kobari et al., 1994; Irie et al., 1995; Miasiro et al., 1995; Lucas
et al., 1996; Higashi et al., 1997). Furthermore, it has been
245
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Chakder and Rattan
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