Department of Medicine, Division of Gastroenterology and Hepatology, Jefferson Medical College, Thomas Jefferson University, Philadelphia, Pennsylvania 19107
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ABSTRACT |
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Cholera toxin
(CTX), an activator of Gs protein,
is an important pharmacological tool in G protein research. The effect
and the mechanism of action of CTX in the gastrointestinal smooth muscle, including the internal anal sphincter (IAS), are not known. The
present investigation was carried out to examine the effects of CTX on
the signal transduction associated with the adenylate cyclase (AC)
pathway on the basal tone of the IAS smooth muscle. CTX caused a prompt
and dose-dependent fall in the basal tone of the IAS that was not
affected by the neurotoxins TTX and -conotoxin or the nitric oxide
synthase inhibitor
NG-nitro-L-arginine. The
cyclooxygenase inhibitor indomethacin, cAMP-dependent protein kinase
inhibitor Rp-8-bromoadenosine
3',5' cyclic monophosphorothioate inhibited CTX-induced IAS
smooth muscle relaxation. Furthermore, CTX caused a
concentration-dependent relaxation of the isolated smooth muscle cells
(SMC) of the IAS, which was blocked by
Gs
antibody
(Gs
-Ab). The IAS smooth muscle relaxation was accompanied with an increase in the GTPase activity that
was also specifically blocked by
Gs
-Ab. We conclude that a major
part of the inhibitory action of CTX in the IAS is via the direct
response of the SMC that is linked with
Gs protein to the AC pathway. A
part of the inhibitory action of CTX on the smooth muscle occurs via
the activation of cyclooxygenase pathway. The relative contribution of
such actions of CTX in the smooth muscle in the gastrointestinal
motility disturbances following cholera infection remains to be determined.
nonadrenergic noncholinergic; guanine nucleotide-binding protein; G protein; guanosine triphosphatase; adenylate cyclase; nitric oxide synthase
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INTRODUCTION |
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CHOLERA TOXIN (CTX) is well known to activate Gs protein and serves as an important pharmacological tool in G protein research. Additionally, CTX causes a number of gastrointestinal disturbances, including severe diarrhea. The effects of CTX on changes in secretion and absorption being responsible for the diarrhea are well established (4). The effects of CTX on the changes in the gastrointestinal motility, except for a few studies, have not been investigated. From in vivo studies, Cowles and Sarna (8-10) suggested that the primary effect of CTX on the canine intestine was an increase in the frequency of migrating motor complexes in the fasted state. The secondary effect of CTX may be due to an increase in the fluid volume in the small intestinal lumen and comprise an increase in the phase II duration and inhibition of migrating clustered contractions during this phase of migrating motor complexes. These actions of CTX take from 1 to 6 h to develop.
The anorectal region, especially the internal anal sphincter (IAS), plays an important role in the anorectal continence and incontinence and anorectal reflex-mediated relaxation of the IAS (2, 11, 31, 36). The effects and mechanism of action of CTX on any part of the large intestine including the anorectum have not been investigated before. Such studies may provide important insights into the pathogenesis of cholera-induced symptoms related to the changes in gastrointestinal motility.
In other systems it has been suggested that CTX may activate a membrane receptor that is linked to the G protein (12, 20, 23, 32, 38). A specific Gs protein has been shown to be involved in the signal transduction in response to CTX (20, 26). The activation of Gs protein in turn causes the activation of adenylate cyclase (AC) that is responsible for the action of CTX at the target site. An increase in cAMP may be responsible for the relaxation of a variety of smooth muscles (16, 34).
The secretory effects of CTX are well established and have been suggested to occur via both the enteric nerves (22) and the direct stimulation of the mucosa (5). There are no studies that examine the site of action of CTX on the gastrointestinal smooth muscle.
In different systems investigated so far it has been shown that CTX takes from several hours to days for the expression of its actions. This includes the tissues that otherwise are known to respond to different agonists within a matter of seconds. A long latency of action makes it rather difficult to examine in detail the mechanisms of actions of CTX and may be the reason for the lack of significant data on the mechanism of action of CTX in the gastrointestinal smooth muscle. The purpose of the present investigation was therefore to examine the effects and mechanism of action of CTX on the gastrointestinal smooth muscle. The IAS smooth muscle was used as a model since this smooth muscle is tonic and allows the investigation of both the inhibitory and excitatory actions (11, 29, 37). Our preliminary experiments suggested that the actions of CTX in the IAS smooth muscle were prompt. This allowed us to examine the mechanism of action of CTX in the IAS smooth muscle. Furthermore, the studies were designed to investigate the nature of G protein involved in the gastrointestinal smooth muscle relaxation by CTX.
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MATERIALS AND METHODS |
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Preparation of smooth muscle strips. Opossums (Didelphis virginiana) of either sex were used for the present studies. The animals were anesthetized with pentobarbital (40-50 mg/kg ip). Laparotomy was performed, and the entire anal canal was isolated and transferred to oxygenated (95% oxygen plus 5% carbon dioxide) Krebs physiological solution of the following composition (in mM): 118.07 NaCl, 4.69 KCl, 2.52 CaCl2, 1.16 MgSO4, 1.01 NaH2PO4, 25 NaHCO3, and 11.10 glucose. The anal canal was carefully freed of all the extraneous structures, including the striated muscle fibers, the adventitia, and the large blood vessels, and was opened and pinned flat with the mucosal side up on a dissecting tray containing oxygenated Krebs physiological solution. The mucosal and submucosal layers were removed by sharp dissection, and IAS circular smooth muscle strips (~1 × 10 mm) were prepared for the recording of isometric tension as described previously (21, 30).
Measurement of isometric tension.
The smooth muscle strips were secured at both ends with silk sutures
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, 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 10 mN of force 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 and the baseline of the smooth muscle strips were determined as
described previously (21). At the conclusion of the experiments, all
the tissues were cut below the suture material, blotted dry, and
accurately weighed. The force of all the smooth muscle tissues was
calculated in millinewtons as described before (19). The changes in the
force, whether the relaxation or contraction, were expressed as percent
of maximal changes in the basal tension at the end of the experiment
with 5 mM EGTA or 3 × 106 M phenylephrine, respectively.
Nonadrenergic noncholinergic nerve stimulation with 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 Stimu-splitter was used to amplify and measure the stimulus intensity using the optimal stimulus parameters for the nonadrenergic noncholinergic (NANC) neural stimulation (12 V, 0.5-ms pulse duration, 200-400 mA, 4-s train) at varying frequencies of 0.5 to 20 Hz. The electrodes used for the EFS consisted of a pair of platinum wires that were secured at both sides of the smooth muscle strip. Neurally mediated relaxation of the IAS smooth muscle was quantified in response to different frequencies.
Isolation of smooth muscle cells from IAS. The smooth muscle cells (SMC) from the IAS region were isolated following the method previously used in our laboratory (6). Briefly, the respective smooth muscle tissues were cleared from the adjoining blood vessels, serosa, and mucosa and cut into small pieces (1-mm cubes). The small pieces of tissues were incubated at 31°C in oxygenated Krebs solution containing 0.1% collagenase (CLS II, 140 U/mg), 0.01% soybean trypsin inhibitor, and mixtures of amino acids and vitamins for two successive 60-min periods and then filtered through 500-µm Nitex mesh. The tissues trapped on the mesh were rinsed with 50 ml of collagenase-free Krebs solution and incubated for another 30 min in oxygenated Krebs solution. The dispersed cells were harvested by filtration through 500-µm Nitex mesh, centrifuged at 350 g for 10 min, and resuspended as needed. For some experiments employing the permeabilized SMC, the enzymatically digested smooth muscle tissues were washed with an oxygenated cytosolic buffer of the following composition (in mM): 20 NaCl, 100 KCl, 5.0 MgSO4, 0.96 NaH2PO4, 25 NaHCO3, 1.0 EGTA, 0.48 CaCl2, and 1% BSA and were allowed to dissociate in this medium for 30 min.
In some experiments using guanosine 5'-O-(2-thiodiphosphate) (GDPMeasurement of changes in SMC lengths (contraction or relaxation)
by scanning micrometry.
Individual SMC lengths were measured by scanning micrometry as
described before (28). An 0.1-ml aliquot of suspension containing 1 × 104 to 3 × 104 SMC/ml was treated with
bethanechol (1 × 107
or 1 × 10
6 M), and
the response was terminated after 30 s with 1% acrolein. The mean SMC
length of 30 cells was measured randomly by micrometry using phase
contrast microscopy, and the percent shortening of the SMC length in
the presence of bethanechol compared with the vehicle-treated SMC was
calculated. To examine the effect of CTX (50, 100, or 200 ng/ml),
GDP
S (1 mM), or Gs
antibody
(Gs
-Ab; 1:200 dilution) on the
SMC contraction caused by bethanechol, the SMC were first exposed to
these reagents for 1, 10, or 60 min, respectively, followed by the
bethanechol treatment in the manner similar to that previously described.
Preparation of SMC membranes.
SMC membranes were prepared according to the method of Pomerantz et al.
(25). Briefly, the dispersed SMC were washed in PBS containing 10 µM
phenylmethylsulfonyl fluoride and snap-frozen at 70°C. The
SMC were resuspended in 20 mM HEPES buffer (pH 7.2) containing 2 mM
MgCl2, 1 mM EDTA, 1 mM
benzamidine, and 10 µg each of leupeptin and pepstatin A, lysed by
passing through a 25-gauge needle, and then homogenized using a
teflonized glass homogenizer. Membrane fractions were isolated by
centrifugation at 75,000 g at 4°C
for 60 min (Beckman L8-70M Ultracentrifuge, Beckman Instruments,
Palo Alto, CA) through 40% sucrose. The pellets were rehomogenized in
the HEPES buffer and reisolated by centrifugation (110,000 g at 4°C for 60 min). The final
pellets were suspended in the HEPES buffer and stored at
70°C. The protein concentrations of the membrane suspensions
were determined by the method of Lowry et al. (18) using BSA as the standard.
Measurement of GTPase activity.
GTPase activity was determined by the method of Pomerantz et al. (25).
The principle for the assay is that
[-32P]GTP serves as
the substrate for GTPase, and GTPase activity is determined by the
simultaneous conversion of
[
-32P]GTP into GDP
and 32P. GDP and
32P are stoichiometrically
produced (1:1). Briefly, 10 µg of membrane proteins were added into
100-µl assay buffer containing (in mM) 100 NaCl, 5 MgCl2, 0.25 EGTA, 12.5 Tris
(pH 7.4), 2.0 dithiothreitol, 1.0 ATP, 0.5 5'-adenylylimidodiphosphate, and 10 phosphocreatine, 50 U/ml
creatine phosphokinase, and 1,000,000 dpm of
[
-32P]GTP (5,000 Ci/mmol). After 10 min of incubation at 37°C, 0.8 ml of ice-cold
acid charcoal solution (1 N HCl containing 10% Norit A) was added and
mixed well, and the samples were centrifuged at 14,000 g for 4 min. Radioactivity was counted
in 0.45 ml of supernatant plus 5 ml of scintillation liquid. To examine
the effects of CTX, GDP
S,
Gs
-Ab, or
Gi1-3-Ab, the mixtures of
membrane protein and assay buffer without
[
-32P]GTP were
pretreated with GDP
S or antibodies for 10 or 60 min, respectively,
and then incubated with CTX and
[
-32P]GTP. The
GTPase activity was calculated as femtomoles per minute per milligram
protein and expressed as percent of control. The control values were
determined in the absence of any treatment except
[
-32P]GTP.
Drugs and chemicals.
The following chemicals were used in the study: CTX (Research
Biochemicals International, Natick, MA); bethanechol chloride, isoproterenol hydrochloride, phenylephrine, sodium nitroprusside, NG-nitro-L-arginine
(L-NNA), TTX, indomethacin,
GDPS (Sigma Chemical, St. Louis, MO);
-conotoxin GVIA, (Bachem
Bioscience, Torrance, CA); 8-bromoadenosine 3',5'-cyclic
monophosphate (8-BrcAMP),
Rp-8-bromoadenosine 3',5'-cyclic monophosphorothioate
(Rp-8-BrcAMPS), and
Rp-8-bromoguanosine 3',5'-cyclic monophosphorothioate
(Rp-8-BrcGMPS) (BioLog Life Sciences
Institute, La Jolla, CA); EDTA tetrasodium, and EGTA (Fisher
Scientific, Pittsburgh, PA).
Drug responses.
All experiments (except for bethanechol and phenylephrine effects) on
the IAS smooth muscle strips were carried out in the presence of
guanethidine (3 × 106
M) and atropine (1 × 10
6 M). All the agonists
were given in a cumulative fashion. Once the concentration-response
curve to an agent was determined, the smooth muscle strips were washed
at least six times and the resting tension was allowed to recover to
the preinjection levels.
Data analysis. The data were represented as means ± SE of different experiments. The basal tone and changes in the IAS smooth muscle tension in response to different agonists, stimuli, and CTX in control vs. different inhibitor and antagonist pretreatments as the case may be, were calculated as the force in millinewtons as previously described. The maximal fall (or the passive force) in the basal tension in each smooth muscle strip was determined at the end of each experiment by the addition of excess EGTA (usually 5 mM) until there was no further fall in the basal force. The data showing fall in the basal tension with the agonists and stimuli were expressed with reference to percent maximal (Emax) fall in the presence of EGTA. Percent fall in the basal tension was calculated by the ratio of decrease in force by the stimuli to the basal tone or active tone. The basal tone was calculated by subtracting the passive force from the total tension (15). The passive force was the residual force in the presence of EGTA.
The relaxation of the isolated SMC was determined by decrease in the percent maximal contraction caused by bethanechol as described before (28). Likewise, the contraction of the SMC was expressed as the percent decrease in the length of the SMC. Both of these responses were expressed as percent of maximal change as previously explained. Statistical significance of the differences between different groups was determined by Student's t-test. Two-way ANOVA was used for the comparison of the entire concentration or frequency response curve before and after the treatment. ![]() |
RESULTS |
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Effect of CTX on basal tone of IAS.
As shown in Fig. 1, CTX caused a prompt and
dose-dependent fall in the basal tone of the IAS. The threshold
concentration of 10 ng/ml caused 15.7 ± 2.3% fall in the basal
tension of the IAS. The maximal response of 88.6 ± 4.0% fall was
observed with 500 ng/ml of CTX in the muscle bath. The relaxation of
the IAS smooth muscle in response to 10 ng/ml CTX began within 15.8 ± 1.5 s and peaked within 91.3 ± 8.8 s. Likewise, the latency
of onset and peak of the fall in the basal tension of the IAS following 500 ng/ml were 14.3 ± 1.7 and 86.5 ± 9.1 s, respectively. Such an immediate response of CTX in the smooth muscle in comparison to its
actions in other systems was new and unexpected. The relaxation caused
by CTX in the IAS smooth muscle persisted for 1-2 h. The actions
of CTX were fully reversible on washing of the smooth muscles. An
example of the typical tracing showing the effect of different
concentrations of CTX on the basal tone of the IAS is shown as an inset
in Fig. 1.
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Influence of nitric oxide synthase inhibitor
L-NNA and neurotoxins TTX and
-conotoxin on CTX-induced fall in basal tone of IAS.
To determine the site of action of CTX in causing the relaxation of the
IAS smooth muscle, first we investigated the influence of the
neurotoxin TTX on the IAS smooth muscle relaxation by CTX. TTX had no
significant effect on the fall in the IAS tension caused by CTX (Fig.
2). In these experiments, in
control studies, 250 ng/ml of CTX produced 81.9 ± 1.8% fall in the
basal tone of the IAS. In the presence of TTX, the fall in the IAS
tension by CTX was 80.0 ± 2.0% (P > 0.05, n = 4). It has been shown
previously that the neurally mediated relaxation of the IAS smooth
muscle is mediated primarily by the nitric oxide synthase (NOS) pathway (27). There are examples where the IAS smooth muscle relaxation in
response to an agonist may be neurally mediated at the nerve terminal
site via the activation of NOS and is resistant to TTX (29). Such
smooth muscle relaxation is sensitive to
-conotoxin and the NOS
inhibitor. To examine those possibilities for the CTX-induced IAS
smooth muscle relaxation, we tested the effects of
-conotoxin and
the NOS inhibitor L-NNA on the
relaxant effect of CTX in the IAS smooth muscle. Neither
-conotoxin
nor L-NNA were found to have any
significant effect on the IAS smooth muscle relaxation caused by CTX
(P > 0.05, n = 4; Fig. 2). In some experiments, we also examined the effect of
-conotoxin plus
L-NNA on CTX-induced fall in the
basal tension of the IAS smooth muscle. The combination had no
significant effect on the inhibitory effect of CTX on the basal IAS
tone. In these experiments, 250 ng/ml of CTX caused 85.2 ± 3.0 and
81.3 ± 6.0% fall in the IAS tension before and after the
combination of
-conotoxin plus
L-NNA, respectively (P > 0.05, n = 4). The comparison of entire
dose-response curve in control vs. in the presence of
-conotoxin
plus L-NNA experiments also
revealed no significant effect of the combination (data not shown). The
findings suggest that the major site of the relaxant action of CTX in
the IAS smooth muscle is directly at the SMC.
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Influence of cyclooxygenase inhibitor indomethacin on CTX-induced
fall in basal tone of IAS.
Indomethacin was used as a cyclooxygenase inhibitor in concentrations
that have been shown to be effective before (17). Indomethacin caused a
significant and concentration-dependent suppression of the IAS smooth
muscle relaxation by CTX (P < 0.05, n = 6; Fig.
3A). In
control experiments, 100 ng/ml CTX caused 68.2 ± 6.2% fall in the
basal tone of the IAS. The fall in the IAS tone by the same
concentration of CTX in the presence of 3 × 106 and 1 × 10
5 M indomethacin were
48.9 ± 5.1 and 39.4 ± 5.1%, respectively. The
comparison of the latencies of the onsets of the relaxant actions of
CTX before and after indomethacin also provided important information.
The cyclooxygenase inhibitor caused a significant delay in the
inhibitory action of CTX (500 ng/ml) in the IAS, from 18.4 ± 2.0 s
(observed in control experiments) to 36.0 ± 3.7 s (in the presence
of indomethacin; P < 0.05, n = 6). Indomethacin in the
concentrations used does not appear to have neural actions because it
had no significant effect on the IAS smooth muscle relaxation by neural
stimulation with EFS as shown in Fig.
3B (P > 0.05, n = 4). The data suggest
that the fall in the basal tone of the IAS by CTX is via its action
directly at the smooth muscle cell. However, in the IAS SMC, the
inhibitory action of CTX is partly mediated via the cyclooxygenase
pathway.
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Effect of permeant cyclic nucleotides 8-BrcAMP on basal tone of IAS:
influence of cAMP-dependent protein kinase inhibitor
Rp-8-BrcAMPS on fall in basal IAS tension
by CTX.
It is well known that CTX in a number of systems causes an activation
of Gs protein, leading to an
activation of AC and an increase in intracellular cAMP (20, 26). In the
IAS SMC, AC pathway has been shown to play a significant role in the
relaxation of the smooth muscle (7, 14). To investigate the signal
transduction involved in the CTX-induced relaxation of the IAS smooth
muscle, we examined the role of AC pathway in the relaxation of the IAS smooth muscle. In this process, we examined the actions of 8-BrcAMP before and after Rp-8-BrcAMPS
(cAMP-dependent protein kinase inhibitor) (1) on the basal IAS tone.
Then we examined the influence of the specific G protein antibody
(Gs-Ab) on the smooth muscle relaxation by CTX.
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Effect of CTX on isolated SMC of IAS: influence of
Gs-Ab.
CTX caused a concentration-dependent relaxation of the isolated SMC of
the IAS in a manner similar to that in the IAS smooth muscle strips
(Fig. 5). Figure
5A shows that 1 × 10
6 M bethanechol caused
maximal contraction
(Emax or 100%)
in the intact SMC of the IAS. CTX produced a concentration-dependent relaxation of the SMC (Fig. 5B). CTX
(250 ng/ml) produced 76.6 ± 1.6% relaxation of the SMC maximally
contracted by bethanechol.
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Effect of CTX on GTPase activity: influence of
Gs-Ab.
To examine and compare the role of specific
Gs protein on the CTX-induced
changes in the GTPase responsible for the relaxation of the IAS SMC, we
first determined the presence of GTPase activity in the IAS smooth
muscle in the basal state and then the effect of different
concentrations of CTX on the GTPase activity. This was followed by the
determination of GTPase activity in the presence of CTX (100 ng/ml)
with or without Gs
-Ab. The data
show the presence of GTPase activity in the IAS and that CTX causes a
concentration-dependent increase in the GTPase activity (Fig.
7A).
Furthermore, the GTPase activity in the basal state and the increased
GTPase activity in the presence of CTX were dose dependently inhibited
by the universal G protein inhibitor GDP
S (data not shown). We then examined the influence of CTX (100 ng/ml) on the GTPase activity before
and after Gs
-Ab. To determine
the specificity of Gs
-Ab in
inhibiting the action of CTX, we also examined the effect of Gi1-3-Ab. The data show that
the increase in GTPase activity by CTX was blocked specifically by
Gs
-Ab, since
Gi1-3-Ab had no significant
effect (Fig. 7B).
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DISCUSSION |
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These studies for the first time show a prompt and direct relaxation of gastrointestinal smooth muscle of the IAS in response to CTX. Furthermore, the studies show that the relaxant response of CTX on the IAS smooth muscle occurs via two pathways: 1) the activation of Gs protein that is linked to the AC and 2) the activation of cyclooxygenase pathway in the SMC.
The relaxation of the IAS smooth muscle in response to CTX was found to occur within a matter of seconds. This rapid response of CTX, especially on the smooth muscle, has not been shown before. Most of the actions of CTX in different systems have been shown to take from two to several hours, and this includes changes in the gastrointestinal motility (8, 9, 13). The fastest action of CTX so far reported has been on the rabbit vascular smooth muscles of the ear artery, thoracic aorta, and saphenous vein (35). Such actions of CTX on the vascular smooth muscles occurred on the order of 10 min and were seen only in the smooth muscles that were precontracted with an agonist. These blood vessels are otherwise well known to relax immediately with agonists other than CTX. Because of the long latencies of the action, the exact mechanism of the smooth muscle relaxation by CTX might have been difficult to ascertain. It is possible that the vascular smooth muscle relaxation by CTX is the result of the interference of the toxin with the contractile agonist at the receptor level or at the intracellular level. The immediate relaxation of the IAS smooth muscle is a novel finding, and the responses were highly reproducible and concentration dependent. Moreover, the smooth muscle relaxation by CTX was also observed in the isolated SMC via the mechanisms similar to those proposed earlier for the other secretory and nonsecretory cells (20, 26).
The relaxation of the IAS smooth muscle caused by CTX was found to be
by its action directly at the SMC since it was modified by neither of
the neurotoxins TTX and -conotoxin. Furthermore, the CTX-induced
relaxation of the IAS smooth muscle was independent of the NOS pathway
and was not modified by the NOS inhibitor
L-NNA. These inhibitors used in
the same concentrations have been shown before to block the IAS smooth
muscle relaxation caused by the NANC nerve stimulation (27, 29).
Additionally, CTX also caused a relaxation of the SMC isolated from the IAS.
A major component of the CTX-induced relaxation of the IAS SMC seems to
involve the activation of G protein linked to AC. There were several
lines of evidence to that effect. The relaxation of the IAS SMC by CTX
was blocked specifically by the introduction of
Gs-Ab and was not affected by
similar pretreatment of the cells with antibodies against other G
proteins. The activation of G protein in causing the smooth muscle
relaxation was also reflected by an increase in the GTPase activity
following CTX pretreatment in the time frame that causes the smooth
muscle relaxation. The relaxation of the IAS smooth muscle was
significantly antagonized by the specific inhibitor of cAMP-dependent
protein kinase.
The studies show that the major part of the IAS smooth muscle relaxation in response to CTX occurs via the AC pathway. However, the studies suggest an additional signal transduction pathway involving cyclooxygenase in partial response to the toxin. The relaxation of the IAS smooth muscle by CTX was significantly attenuated by the cyclooxygenase inhibitor indomethacin. Similar concentrations of indomethacin were found to have no significant effect on the NANC nerve-mediated relaxation of the IAS smooth muscle. The dual pathway in the relaxation of the smooth muscle has not been shown before. However, this effect is similar to that observed in the murine smooth muscle-like cells (BC3HI) (24). The role of prostaglandins in the mediation of pathogenesis of secretory actions leading to diarrhea is well known (3). Whether there is an association or an overlap between these two pathways in the IAS smooth muscle remains to be determined. The present studies also did not examine the role of guanylate cyclase pathway in the CTX-mediated relaxation of the IAS smooth muscle.
The immediate and direct relaxation of the IAS smooth muscle provides significant new information and will facilitate the understanding of basic mechanisms underlying CTX. The studies also provide important insights into the mechanism of the inhibitory action of CTX in the gastrointestinal smooth muscle. The actions of CTX on the gastrointestinal smooth muscles that are not spontaneously active are not known. The relaxant actions of CTX on the IAS smooth muscle were immediate and reversible. Because of these characteristic responses, the IAS smooth muscle may serve as an important model to investigate the mechanism of actions of CTX. A considerable delay in the order of hours for the action of CTX has been reported in other cell types. In-depth studies in the IAS smooth muscle may help in defining the intracellular mechanisms for the differences in the signal transduction of CTX in different cell types. Moss and Vaughan (20) have reviewed a number of possible mechanisms for the delay in the action of CTX in different systems.
In summary, CTX exerts potent, prompt, and direct inhibitory actions on the IAS smooth muscle via the activation of Gs protein-linked AC and cyclooxygenase pathways. The actions of CTX on the IAS smooth muscle may provide further insights into the basic mechanisms of the pathophysiology of CTX-associated gastrointestinal motility disorders.
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ACKNOWLEDGEMENTS |
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This work was supported by the National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-35385 and an institutional grant from Thomas Jefferson University.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: S. Rattan, 901 College, Dept. of Medicine, Div. of Gastroenterology and Hepatology, 1025 Walnut St., Philadelphia, PA 19107.
Received 28 October 1998; accepted in final form 1 April 1999.
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