Department of Medicine, Division of Gastroenterology and Hepatology, Jefferson Medical College, Thomas Jefferson University, Philadelphia, Pennsylvania 19107
Submitted 27 December 2002 ; accepted in final form 30 April 2003
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ABSTRACT |
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internal anal sphincter smooth muscle; nitric oxide synthase; nitric oxide synthase inhibitors; nonadrenergic noncholinergic relaxation; Hirschsprung's disease
It is well known that stimulation of -ARs (
1,
2, or
3) causes release of
Gs
, which then activates adenylate cyclase to produce cAMP
(34,
41). Elevated levels of cAMP
produce relaxation of smooth muscle via activation of cAMP-dependent protein
kinase (PKA). There are significant differences in the intracellular signaling
pathways used by
1- and
2- vs.
3-AR subsequent to activation of PKA. Fully activated PKA may
cause phosphorylation of
1- and
2-ARs,
leading to their inactivation
(7,
26).
3-ARs
lack PKA phosphorylation sites; desensitization or internalization of
3-receptors is relatively less prevalent compared with
1- and
2-receptors
(15,
33). In addition, it has been
shown that endothelial nitric oxide synthase (eNOS) may partly modulate
relaxation of vascular smooth muscles in response to activation of the
3-AR (5,
40).
We determined the effect of selective 3- vs. nonselective
-AR agonists in the basal tone of the internal anal sphincter (IAS) and
its mechanism of action. Effects of such agents on the basal IAS tone and
understanding the basic mechanisms by which they induce relaxation of the
smooth muscle may provide a basis for development of potential therapeutics
for various conditions such as constipation with megarectum, anal fissures,
and Hirschsprung's disease. The purpose of the present investigation is
twofold: 1) to examine effect of
3-AR activation in
the basal IAS tone and 2) to examine the role of NOS in
3-AR-induced relaxation of the spontaneously tonic smooth
muscle of the IAS.
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MATERIALS AND METHODS |
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All protocols for use of animals were approved by Thomas Jefferson University's Animal Care and Use Committee.
Measurement of isometric tension. The smooth muscle strips were secured at both ends with silk sutures and transferred to muscle baths that contained 2 ml oxygenated Krebs solution (35°C). One end of each smooth 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 computerized PowerLab/800. The smooth muscle tension in the basal state and following different stimuli was amplified by ETH-400 Bridge Amplifiers (CB Sciences, Milford, MA). The signals were then processed through computer-based acquisition and data-analysis systems by use of a PowerLab/800 analog-to-digital converter (AD Instruments, Mountain View, CA) and recorded on a PC-based Hewlett-Packard computer (model HP Vectra VE, Hewlett-Packard, Palo Alta, CA) equipped with Microsoft Windows 98. The data were analyzed with PowerLab software. The smooth muscle strips were stretched at 10 mN tension initially and allowed to equilibrate for at least 1 h with intermittent washings. During this period, the muscle strips developed steady-state tone. Baseline and optimal length (Lo)of the smooth muscle strips were determined as described previously (32). Only smooth muscle strips that developed steady tone and relaxed in response to electrical field stimulation (EFS) were included in the study. The studies employed 32 adult opossums of either sex. Each animal usually yielded three to four usable smooth muscle strips, and only one antagonist was used in each smooth muscle strip. n Refers to different smooth muscle strips from different animals.
Nonadrenergic noncholinergic nerve stimulation with EFS. EFS was delivered with a Grass stimulator (model S88; Grass Instruments) connected in series to a Med-Lab Stimu-Splitter II (Med-Lab Instruments, Loveland, CO). We used optimal stimulus parameters for the neural stimulation (10-15 V, 0.5-ms pulse duration, 4-s train) at frequencies that varied from 0.5 to 20 Hz. The electrodes used for the EFS consisted of a pair of platinum wires fixed at both sides of the smooth muscle strip. Neurally mediated relaxation of the IAS smooth muscle strips was quantified in response to various frequencies.
Western blot studies. Western blot analysis of eNOS in the IAS
smooth muscle of opossums was determined according to the protocol of BD
Transduction Laboratories (San Diego, CA). Briefly, the circular smooth muscle
of the IAS (after the removal of the mucosa, muscular mucosa, serosal, and
longitudinal muscle layer) was carefully prepared. The IAS tissue thus
prepared was cut into small pieces (1- to 2-mm cubes) and rapidly homogenized
in boiling lysis buffer (1% SDS, 1.0 mM sodium orthovandate, 10 mM Tris, pH
7.4; 250 mg of tissue/3.5 ml of the buffer), then put into the microwave for
10 s. The homogenate was centrifuged (16,000 g at 15°C) for 5
min. The pellet was discarded, and the protein contents of the supernatants
were determined by the method of Lowry et al.
(27) using BSA as the
standard. All of the samples were mixed with 2x sample buffer (125 mM
Tris, pH 6.8, 4% SDS, 10% glycerol, 0.006% bromophenol blue, 2%
-mercaptoethanol) and heated by placing samples in a boiling water bath
for 4 min. Total protein samples (0-40 µg) were loaded to commercially
available 7.5% polyacrylamide PAGEr Gold Gel (BioWhitaker Molecular
Applications, Rockland, ME) and applied to 7.5% SDS-PAGE by the method of
Laemmli (25). The separated
proteins were electrophoretically transferred to nitrocellulose membrane (NCM)
at 4°C. To block nonspecific antibody binding, the NCM were immersed
overnight at 4°C in Super Block Tris-buffered saline (TBS) blocking buffer
in TBS (Pierce Biotechnology, Rockford, IL). The NCM were incubated with
1:1,000 diluted primary eNOS antibody (Ab) (BD Transduction Laboratories)
for1hat room temperature. After being washed with the blocking buffer, the
membranes were incubated with horseradish peroxidase (HRP)-conjugated donkey
anti-rabbit IgG secondary Ab (1:1,500) (Amersham Biosciences, Piscataway, NJ)
for 1 h at room temperature. A separate NCM was incubated with primary Ab for
platelet endothelial marker [platelet endothelial cell adhesion molecule
(PECAM-1), Santa Cruz Biotechnology, Santa Cruz, CA] at a dilution of 1:1,000
and subsequently, after being washed, incubated at 1:1,500 dilution of
HRP-conjugated bovine anti-goat IgG secondary Ab (Santa Cruz). The membranes
were blotted semidry by placing them in between two filter papers, and bands
were identified by chemiluminescence using the enhanced chemiluminescence
(ECL) detection system and Hyperfilm ECL (Amersham Pharmacia Biotech, Little
Chalfont, UK). The bands were then scanned (Astra 1220S, UMAX, Dallas, TX),
and their relative densities were determined using Image-Pro Plus 4.0 software
(Media Cybernetics, Silver Spring, MD).
Determination of NO. Measurement of total NOx (NO + NO2- + NO3-) was used as a measure of release of NO by the tissues. Once released, NO is rapidly oxidized to NO2- > NO3-. Therefore, it is necessary to measure all forms of NO (17) to determine accurately the amount of NO released by tissues. Total NOx was determined with a nitrate/nitrite colorimetric assay kit (Cayman Chemical, Ann Arbor, MI) specifically designed for this purpose.
Perfusates were collected from the muscle baths before treatment of the smooth muscle strips with any agents (basal condition) and after treatment with either disodium (R,R)-5-[2-[2-3-chlorophenyl)-2-hydroxyethyl]-amino]propyl]-1,3-benzodioxole-2,2-dicarboxylate (CL 316243; 3 x 10-6 M) or isoproterenol (1 x 10-6 M). Such perfusates were collected before and after selective NOS inhibitors. After treatment with CL 316243 or isoproterenol, perfusates (100 µl) were collected at the onset of a fall in the basal IAS tone and transferred immediately to 1.5-ml vials at -20°C before analysis for content of NOx.
A commercially available (Cayman Chemical) nitrate/nitrite colorimetric assay kit was used to measure the total nitrate/nitrite concentration in a simple two-step process in which 1) nitrate in the sample is converted to nitrite by nitrate reductase and 2) nitrite is converted to a deep purple azo compound on the addition of Griess Reagent (17, 35). Eighty microliters of each sample were pipetted into a 96-well plate, and absorbance at 540 nm was determined using a spectrophotometer. Nitrite concentration in each sample was calculated by comparison with a standard curve generated immediately before analysis of the test samples. The standards and the test samples were analyzed in duplicate.
Drugs and chemicals. The following chemicals were used in the
study: N-nitro-L-arginine
(L-NNA);
L-N5-(1-iminoethyl)ornithine hydrochloride
(L-NIO; eNOS inhibitor)
(9);
1-[2-(trifluoromethylphenyl]imidazole (TRIM; nNOS inhibitor)
(19); CL 316243; isoproterenol
hydrochloride, atropine sulfate, guanethidine, pyrilamine, cimetidine,
indomethacin, sodium nitroprusside, sodium nitrate (Sigma, St. Louis, MO); TTX
(Calbiochem, San Diego, CA); VIP-(10-28) (Peninsula Laboratories, Belmont,
CA); N-(3-aminomethyl)benzyl acetamide 2HCl (1400DW; iNOS inhibitor;
Alexis Biochemicals, San Diego, CA)
(14); NO (Matheson Gas,
Bridgeport, NJ); and EGTA tetrasodium (Fisher Scientific, Pittsburgh, PA).
When possible, all chemicals were dissolved and diluted in Krebs solution on the day of the experiment. Vials and pipette tips were siliconized, and the muscle baths were treated with 2.5% bovine serum albumin to reduce binding of peptides to the surface of the glass or plastic.
Drug responses. Pretreatment with different concentrations of NOS inhibitors (1 x 10-6 to 1 x 10-4 M) was used to examine their effects on decrease in the basal IAS tone by CL 316243, isoproterenol, or an NO donor [sodium nitroprusside (SNP)]. All agonists were given in a cumulative fashion. Once the concentration response curve (CRC) for an agent was determined, the smooth muscle strips were washed at least six times and resting tone was allowed to recover to the original level. The effects of the various agonists, administered at their ECmax, on NO release were determined in the presence or absence of selective inhibitors (1 x 10-6 to 1 x 10-4 M) of NOS.
Data analysis. The fall in the basal IAS tone is expressed as the percent maximal fall (100%) induced by a supramaximal concentration (5 mM) of EGTA (4). Statistical significance between different groups was determined by using paired or unpaired t-tests. ANOVA was performed to compare the entire frequency-response curve or CRC before and after administration of NOS inhibitors. The results are expressed as means ± SE of different experiments. A P value <0.05 was considered statistically significant.
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RESULTS |
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The IAS smooth muscle relaxation caused by CL 316243 but not that by
isoproterenol was selectively antagonized by L 748337 in a
concentration-dependent manner (*P < 0.05; n =
5-8; Fig. 1). The basal IAS
tone in CL 316243 and isoproterenol experiments was 1.48 ± 0.18 and
1.52 ± 0.12 g, respectively. The smooth muscle tone was not
significantly modified by the 3-antagonist. The corresponding
values of basal IAS tone in these experiments in the presence of L 748337 was
1.35 ± 0.23 and 1.38 ± 0.18 g, respectively (P >
0.05; n = 5-8). Conversely, the IAS smooth muscle relaxation by
isoproterenol but not by CL 316243 was significantly and concentration
dependently antagonized by propranolol (*P < 0.05;
n = 5-8; Fig. 2). The
lower concentration of propranolol (that causes a significant rightward shift
in the CRC of isoproterenol; Fig.
2B) had no significant effect on CL 316243-induced fall
in the basal IAS tone (P > 0.05; n = 5-8;
Fig. 2A). Only a
higher concentration of propranolol caused a moderate antagonism of CL 316243
effect (*P < 0.05; n = 5-8).
Relaxation of the IAS smooth muscle induced by CL 316243 was not modified
by neither neurohumoral inhibitors nor the neurotoxin TTX (1 x
10-6 M; P > 0.05; n = 5-8).
Different neurohumoral inhibitors examined were atropine (muscarinic
antagonist; 1 x 10-6 M), guanethidine (adrenergic
blocking agent; 3 x 10-6 M), pyrilamine and
cimetidine (H1 and H2 histamine receptor antagonists,
respectively; 1 x 10-5 M), indomethacin
(prostaglandin synthetase inhibitor; 1 x 10-5 M),
and VIP-(10-28) (VIP antagonist; 1 x 10-6 M).
These data suggest that CL 316243 directly induces relaxation of the IAS
smooth muscle via selective activation of 3-ARs. Further
support for the notion that CL 316243 acts directly on smooth muscle cells
(SMC) of the IAS is the observation that it induced relaxation of the isolated
SMCs (data not shown).
Time course for the effects of CL 316243 and isoproterenol on the basal
tone of the IAS smooth muscle. Relaxation of the IAS smooth muscle
induced by CL 316243 was significantly longer in duration than that induced by
isoproterenol (Fig. 3). The IAS
relaxation by isoproterenol recovered to near original baseline within 20 min.
By contrast, the total duration of IAS relaxation with CL 316243 was 240
± 20 min. In these series of experiments with CL 316243, the basal tone
recovered to 70% of the original level at the end of 3 h.
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Effects of L-NNA, a general inhibitor of NOS, on IAS smooth muscle relaxation by either CL 316243 or isoproterenol. IAS smooth muscle was treated with L-NNA, a general inhibitor of NOS, followed by either CL 316243 or isoproterenol to determine whether NOS had a role in agonist-induced relaxation of this muscle. L-NNA caused significant attenuation of the CL 316243-induced fall in basal IAS tone (*P < 0.05; n = 5-8) (Fig. 4A) but had no significant effect on the isoproterenol-induced fall in IAS tone (Fig. 4B). In these experiments, CL 316243 (3 x 10-6 M) caused a 75.03 ± 1.7% fall in the basal IAS tone that was attenuated to 35.6 ± 2% following the pretreatment with L-NNA.
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Effects of different NOS inhibitors on IAS smooth muscle relaxation induced by either CL 316243 or isoproterenol. The decrease in IAS smooth muscle tone induced by CL 316243 was not significantly affected by a selective inhibitor of either nNOS (TRIM) or of iNOS (1400DW; 1 x 10-6, 1 x 10-5, and 1 x 10-4 M; Figs. 5, A and B; P > 0.05; n = 5-8).
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In contrast, L-NIO (1 x 10-6 and 1 x 10-5 M) significantly attenuated the CL 316243-induced fall in IAS tone in a concentration-dependent fashion (*P < 0.05; n = 5-8; Fig. 6). L-NIO caused no significant effect on IAS smooth muscle relaxation by SNP (P > 0.05; n = 5-8; Fig. 7). The IAS smooth muscle relaxation caused by EFS was, however, partially but significantly attenuated by L-NIO (*P < 0.05; n = 5-8).
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In these series of experiments, the overall CRC for the fall in the basal IAS tone by different concentrations of isoproterenol was not affected significantly by any of the inhibitors of NOS (P > 0.05; n = 5-8; Fig. 8).
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Presence of eNOS protein in the IAS smooth muscle. Western blot studies revealed the presence of eNOS-specific protein in the circular smooth muscle of the IAS (Fig. 9). The expression of eNOS in the IAS smooth muscle was found to be significant and specific, because PECAM-1 (an endothelial cell marker) was poorly expressed under the identical experimental conditions.
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Effect of CL 316243 on NO release in the IAS smooth muscle before and after different inhibitors: comparison with isoproterenol and EFS. To ascertain the direct release of NO and the nature of NOS involved, NO release was determined by measuring the amount of total NOx as explained in MATERIALS AND METHODS. The standard curves for NOx generated by the addition of sodium nitrate (from 1 to 100 nmol) were linear with a coefficient of correlation (r) of 0.993. CL 316243 (3 x 10-6 M) caused a significant increase in the basal levels of NO in the IAS smooth muscle. The eNOS inhibitor L-NIO selectively and significantly blocked this increase in NO release (Fig. 10). The basal release from the IAS smooth muscle tissues was 19 ± 4 nmol, and CL 316243 increased these levels to 38 ± 9 nmol. L-NIO (1 x 10-5 M) attenuated CL 316243-induced increased NO release to 20 ± 3 nmol (*P < 0.05; n = 4; Fig. 10). Conversely, neuronal and inducible NOS inhibitors TRIM and 1400DW, respectively, had no significant effect on CL 316243-induced increase in the levels of NO release (Fig. 10; P < 0.05; n = 4).
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We also monitored the release of NO following isoproterenol (1 x 10-6 M) and EFS (10-15 V, 0.5-ms pulse duration, 4-s train, and 5 Hz). EFS caused a significant increase in the NO release; this was partly attenuated by TRIM but not by 1400DW and L-NIO. Isoproterenol did not produce a significant increase in NO release in the IAS smooth muscle.
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DISCUSSION |
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Data suggest that the IAS relaxation by CL 316243 is caused by the
activation of the 3-AR in the smooth muscle. Relaxation of
the IAS smooth muscle induced by CL 316243 was not antagonized by propranolol
in a concentration-dependent manner known to primarily block the effects of
1- and
2-AR activation. This finding
supports the suggestion that CL 316243 is an agonist for
3-AR
and induces relaxation of the IAS smooth muscle via those specific receptors.
This suggestion is supported further by our finding that
3-AR
antagonist L 748337 (6)
inhibits CL 316243-induced relaxation of the IAS smooth muscle in a
concentration-dependent manner. Conversely, the smooth muscle relaxation by
isoproterenol is antagonized by propranolol but not by L 748337. Different
neurohumoral inhibitors and neurotoxin TTX do not modify the smooth muscle
relaxation caused by CL 316243. The direct action of
3-agonist at the smooth muscle is further demonstrated by its
inhibitory action at the SMC.
We further investigated the intracellular mechanism of IAS smooth muscle
relaxation by 3-AR activation. In our preliminary studies,
the smooth muscle relaxation by CL 316243 was inhibited by the general NOS
inhibitor L-NNA, suggesting the involvement of NOS. To investigate
the nature of NOS involved, we examined the influence of selective NOS
inhibitors. Detailed experimentation with selective inhibitors of different
NOS isozymes revealed that only L-NIO causes a substantial and
concentration-dependent attenuation of the smooth muscle relaxation by CL
316243. Inhibitors of inducible and neuronal NOS have no significant effect.
Our Western blot studies using specific eNOS antibody further show the
presence of eNOS in the circular smooth muscle of the IAS. The conclusion is
supported by recent findings in a different system, where it was shown that
activation of
3-AR causes vascular smooth muscle relaxation
by activation of eNOS (5,
40).
It is quite clear that the enzymatic activity of eNOS in IAS smooth muscle
is increased on stimulation of the 3-adrenoreceptor and is
associated with relaxation of that smooth muscle. That is, inhibitor of eNOS
attenuates the production of NO and the relaxation of the IAS smooth muscle in
response to CL 316243. Furthermore, increased production of NO is unique to
the
3-AR, because treatment of IAS smooth muscle with
isoproterenol (primarily
1/
2-AR) does not
stimulate production of NO. eNOS responsible for
3-AR-mediated relaxation of IAS smooth muscle may be present
in the SMC. The role of eNOS from endothelial cells in our smooth muscle
preparations is expected to be minimal because of the limited presence of
endothelial cells. In addition, the inhibitory effect of CL 316243 is
preserved in the presence of different neurohumoral inhibitors and is
demonstrated directly in the isolated SMC. Therefore, we speculate that the
3-AR are present in the SMC and that relaxation of the IAS
smooth muscle is partly mediated by direct interaction of agonists with those
receptors. Western blot studies further delineate the presence of eNOS in the
IAS smooth muscle. PECAM-1, on the other hand, was not found in significant
amounts. Other investigators
(13) have also found eNOS to
be present in SMCs of the GI tract and to be involved in smooth muscle
relaxation by a number of neurohumoral agonists. However, the role for NOS
pathway in
-AR agonist-induced relaxation of the GI smooth muscle has
not been examined before.
Recent studies by König et al.
(24) in the GI tract are in
general agreement with the concept that eNOS is not restricted to endothelial
cells and that it also occurs in other cells such as interstitial cells of
Cajal (ICC) (42) and SMC
(39). The proposed role of SMC
eNOS in the present studies is different from previous studies in the vascular
smooth muscle where 3-agonist-induced relaxation is mediated
primarily by eNOS in the endothelium
(5,
40). Interestingly, in heart,
eNOS present in cardiac myocytes has been shown to be involved in the negative
inotropic effect of
3-AR activation
(16). Additional studies are
needed to ascertain the definitive and relative roles of eNOS in the SMC vs.
the endothelial cells and ICC in the IAS smooth muscle relaxation by
3-AR activation.
3-agonist-induced prolonged relaxation of the IAS smooth
muscle may have important therapeutic implications in conditions such as anal
fissures, Hirschsprung's disease, irritable bowel syndrome, severe
constipation due to spastic gut, and achalasia. Presently, such conditions are
being treated with topical glyceryl trinitrate (GTN), cardizem ointment,
botulinum toxin injection, and surgical intervention
(21,
22,
29,
37,
38). One of the deleterious
effects of botulinum toxin injection includes fecal incontinence, whereas
topical nitrates cause headaches in 20-100% of cases. Surgical intervention
may lead to long-term complications (notably incontinence)
(30). It is of interest that
both GTN and
3-agonist act via the release of NO, but
3-agonist has fewer cardiovascular side effects
(10).
-ARs, especially
3-AR activation-mediated smooth
muscle, involve a number of intracellular mechanisms. In the present
investigation, we did not examine the relative role of other intracellular
mechanisms such as cAMP/PKA
(34,
41), decrease in free
intracellular Ca2+ levels
(18,
20,
23), and lack of PKA
phosphorylation sites in
3- vs.
1- and
2-receptors
(7,
26). We speculate that a
combination of the NOS activation and other intracellular events may be
responsible for the prolonged IAS smooth muscle relaxation by
3-agonists.
In summary, 3-AR agonists possess unique characteristics
such as prolonged IAS smooth muscle relaxation and a novel mechanism of action
via the activation of NOS. This combined with other intracellular pathways may
be responsible for the prolonged IAS smooth muscle relaxation. These
attributes and minimal cardiovascular side effects suggest that selective
3-agonists may have clinical relevance for treating anorectal
motility disorders.
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DISCLOSURES |
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ACKNOWLEDGMENTS |
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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. Section 1734 solely to indicate this fact.
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REFERENCES |
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