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1 Center for Swallowing and Motility Disorders, To identify the
enzymatic source of nitric oxide (NO) in the lower esophageal sphincter
(LES), studies were performed in wild-type and genetically engineered
endothelial nitric oxide synthase [eNOS(
nitric oxide; nonadrenergic noncholinergic neurotransmission; endothelial nitric oxide synthase lacking mutant mice
STIMULATION OF THE nonadrenergic noncholinergic (NANC)
inhibitory nerves causes relaxation of the lower esophageal sphincter (LES) in all animal species examined, including dog, cat, opossum, guinea pig, mice, frog, as well as in humans (1, 16-18,
24-26, 28). The NANC nerves have been shown to exert their effect
via the release of a product of the
L-arginine-nitric oxide synthase (NOS) pathway such as nitric oxide (NO) (3, 17). These conclusions are
supported by the fact that neurons in the myenteric plexus and the
motor nerve terminals express NOS (20). Moreover, NO has been shown to
be released with NANC nerve stimulation (16). There is also a
similarity of responses of the smooth muscle to NANC nerve stimulation
and exogenous NO donors. Both nitrergic transmitter and NO donor elicit
hyperpolarization of the smooth muscle cells (6, 25) and cause
intracellular accumulation of cGMP (5, 9). The most compelling argument
for NO as an inhibitory mediator is the block of nitrergic nerve
stimulation by the chemical inhibitors of NOS such as
N There are three isoforms of NOS that are products of distinct genes
(15). The inducible NOS(iNOS, also called type II) is not normally
present in tissues under physiological conditions but is induced during
tissue injury and inflammation. iNOS is the source of large quantities
of NO that is produced during inflammation. There are two types of
constitutive NOS that participate in normal physiological responses,
each with specific distribution in the gut. The neuronal NOS(nNOS, also
called ncNOS or type I) has been localized to intramural neurons and
nerve endings to the smooth muscle cells (4). In contrast, the
endothelial NOS(eNOS, also called ecNOS or type III) has been suggested
to be localized to smooth muscle cells (13). Targeted disruption of
nNOS and eNOS genes has led to production of mutant mice that lack nNOS
and eNOS, respectively (10, 11). These mutant mice allow distinction of
the roles of nNOS and eNOS in the nitrergic inhibitory
neurotransmission in the esophagus and the LES.
We report here our findings that nNOS( Mice and tissues.
Adult (C57BL/6J X 129/J)F1 mice were used as wild-type mice, and
nNOS(
ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
)] and neuronal NOS
[nNOS()] mice. Under nonadrenergic noncholinergic (NANC)
conditions, LES ring preparations developed spontaneous tone in all
animals. In the wild-type mice, electrical field stimulation produced
frequency-dependent intrastimulus relaxation and a poststimulus rebound
contraction. NOS inhibitor
N
-nitro-L-arginine methyl ester
(100 µM) abolished intrastimulus relaxation and rebound contraction.
In nNOS() mice, both the intrastimulus relaxation and rebound
contraction were absent. However, in eNOS() mice there was no
significant difference in either the relaxation or rebound contraction
from the wild-type animal. Both nNOS() and eNOS() tissues
showed concentration-dependent relaxation to NO donor
diethylenetriamine-NO and there was no difference in the sensitivity to
the NO donor in nNOS(), eNOS(), or wild-type animals.
These results indicate that in mouse LES, nNOS rather than eNOS is the
enzymatic source of the NO that mediates NANC relaxation and rebound contraction.
INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-nitro-L-arginine
(L-NNA) or
N-nitro-L-arginine methyl ester
(L-NAME) (16, 25). However, the
use of the nonselective inhibitors of NOS fails to identify the type of
NOS that is involved in the inhibitory neurotransmission. It also fails
to distinguish whether NO is a neurotransmitter released from nerve
endings or an intracellular mediator in the effector smooth muscle
cells (12).
) but not eNOS()
mice lack electrical field stimulation (EFS)-activated LES relaxation and rebound contraction. These studies show that
1) nNOS is the source of NO that is
responsible for EFS-induced LES relaxation and rebound contraction;
2) lifelong deficiency of nNOS is
not associated with any compensatory changes in the inhibitory
neurotransmission; and 3) because
nNOS is localized primarily to nerve ending and nNOS is the source of
NO involved in nitrergic neurotransmission and eNOS is present in
smooth muscle cells, these studies suggest that NO may serve as an
antegrade neurotransmitter in the nitrergic neurotransmission in the LES.
MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
) and eNOS() mice were generated as described
previously (10, 11). The nNOS() mice were prepared by deletion
of exon 2 that leads to loss of PDZ binding domain required for
membrane association of the enzyme. The PDZ domain also defines the
expression of nNOS-
but not the 5' splice variants nNOS-
and nNOS-
. The nNOS() mutant mice lack nNOS- but
still express nNOS- and nNOS- (2, 8). These two isoforms account
for 5-10% of the residual NOS activity in myenteric neurons in
nNOS() mutant mice (2). Therefore nNOS() mice represent
misexpression rather than lack of nNOS. The nNOS- and nNOS-
remain soluble cytosolic enzymes with activity that is ~80% and
~3% of the activity of full-length nNOS(2).
LES tension recording. Muscle tension recordings were performed using standard organ bath techniques. Ring preparations of the LES were suspended between two platinum electrodes in 3-ml organ baths containing modified Krebs solution pretreated with atropine (1 µM) and guanethidine (5 µM). One end of the ring preparation was anchored, and the other end was attached with stainless steel hooks to a force transducer (Grass FT03) and recorded by a MacLab acquisition (MacLab/8e AD Instruments) and computer system. The tissues were stretched to a resting tension of 0.2 g and allowed to equilibrate for 90 min. The muscle rings developed spontaneous force that declined after 1-2 h. Inclusion of prostaglandin agonist U-46619 (1 µM) in the bath resulted in increased tone that was steady over several hours. Selective neural stimulation of the tissue was achieved using a pulse generator (Grass Instruments S11) programmed to deliver 30-s duration trains of 60-V square-wave pulses, each of 2-ms pulse duration at 1-20 Hz. All these studies were performed in U-46619 (1 µM)-treated muscle preparations. For each parameter of stimulation, EFS was administered three times at 8-min intervals, and responses were averaged over the series. The relaxation responses by EFS were expressed as %change of active tension before EFS-induced relaxation. Active tension was defined as the force that was abolished by isoproterenol (100 µM). In a separate series of experiments, diethylenetriamine-NO (DNO) was added to assess the responses of LES rings to exogenous NO. At the end of the experiments, isoproterenol (100 µM) was added to attain the maximal relaxation.
Drugs. Drugs used in this study included U-46619 (1 µM), atropine (1 µM), guanethidine (5 µM), isoproterenol (100 µM), and L-NAME (100 µM) obtained from Sigma Chemical and the NO donor DNO from Research Biochemicals International. Each was prepared fresh on the day of the experiment. Tissues were treated with solution containing L-NAME (100 µM) for 45 min before study of its effect.
Statistics.
Data are expressed as means ± SE for wild-type, eNOS(), and
nNOS() tissues. Differences in the data were evaluated by
Student's t-test (nonparametric
analysis). P < 0.05 was considered
statistically significant, and n
represents the number of animals used for each protocol.
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RESULTS |
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DISCUSSION
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Responses in control wild-type mice. Ring preparations of LES were stimulated with EFS in the presence of atropine (1 µM) and guanethidine (5 µM) to reveal NANC responses. The muscle rings were contracted by inclusion of a prostaglandin agonist U-46619 (1 µM) to facilitate resolution of EFS-induced relaxation responses.
The amplitudes of both intrastimulus relaxation and poststimulus rebound contraction were frequency dependent over the range of 1-20 Hz in wild-type control tissues (Fig. 1). These responses were abolished by pretreatment with TTX (1 µM). Maximal intrastimulus relaxation and poststimulus rebound contraction were attained at 10 Hz (44.8 ± 5.0%) and 5 Hz (29.6 ± 3.3%), respectively, but higher frequencies of EFS showed diminished responses. The relaxation induced by frequency of 5 Hz applied every 8 min was reproducible. Therefore, experiments in the remainder of this study were carried out with this stimulation. L-NAME (100 µM) abolished the intrastimulus relaxation, converting this response to a contraction, and inhibited the poststimulus rebound contraction (Fig. 2). However, L-NAME did not affect the relaxations induced by direct application of DNO to the tissues (Table 1).
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Responses in nNOS() mice.
To clarify the enzymatic source of NO in the EFS-induced LES responses,
LES preparations from nNOS() mice were electrically stimulated
using the same conditions and parameters (60 V, 5 Hz). EFS-induced
responses of LES preparations of nNOS() mice were significantly
different from those of wild-type mice; these tissues failed to produce
EFS-induced relaxation or rebound contraction (Fig.
3) and resembled the responses of wild type
treated with L-NAME (100 µM).
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Responses in eNOS() mice.
To study the role of eNOS-derived NO in the EFS-induced LES responses,
LES preparations from eNOS() mice were electrically stimulated.
EFS(60 V, 5 Hz)-induced intrastimulus relaxation and rebound
contraction were not significantly altered in the preparations of
eNOS() mice (48.5 ± 9.6 and 27.1 ± 5.3%,
respectively) compared with those of wild-type control tissues (36.9 ± 3.9 and 29.6 ± 3.3%, respectively). In addition, the
EFS-induced relaxation and rebound contraction in the eNOS()
mice were completely abolished by pretreatment with
L-NAME (100 µM; Fig.
4).
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Comparison of tissue sensitivity to NO donor.
In a separate series of experiments, we wished to assess possible
differences in LES tissue sensitivity of wild-type, eNOS(), and
nNOS() mice to the NO-releasing agent DNO. DNO was used because it is a donor of the NO · redox form of NO that has
been shown to be an inhibitory neurotransmitter (9). As shown in Fig. 5, DNO induced relaxation in the LES
preparations of these three kinds of mice in a dose-dependent manner.
The ED50 for DNO in wild-type
(8.05 ± 1.67 × 10
5 M), eNOS() (8.75 ± 3.44 × 105 M),
and nNOS () (2.20 ± 1.47 × 105 M) mice were not significantly different among
these three kinds of mice.
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DISCUSSION |
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DISCUSSION
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The mouse LES is similar to that of other animal species. It is composed of smooth muscle fibers and develops spontaneous tone that is not abolished by TTX. Moreover, EFS produces frequency-dependent relaxation that is followed by rebound contraction under NANC conditions. Both the relaxation and rebound contractions are suppressed by NOS inhibitor L-NAME, suggesting that both relaxation and rebound contractions involve NO. The main finding of this study is that both relaxation and aftercontraction in response to EFS under NANC conditions are missing in nNOS-deficient mice. These observations suggest that nNOS is the enzymatic source of NO that is involved in inhibitory nitrergic neurotransmission.
The various structures involved in neuromuscular inhibitory transmission include the motor nerve endings, intramuscular interstitial cells of Cajal (ICC) that may be interposed between the nerve endings and the smooth muscle cells, and the smooth muscle cells themselves (7). In mice, nNOS immunoreactivity has been localized to the myenteric neurons and nerve endings but was not reported in ICC or smooth muscle cells (20). However, Ward and colleagues (25) found NADPH reactivity and NOS staining in ICC but not in smooth muscle cells. In the smooth muscle cells in other species, gene expression for nNOS has been reported in some (4) but not in other studies (19, 23). NOS in the ICC was proposed to provide a major mechanism for amplification of neural signals to the smooth muscle cells. However, it is now thought that the ICC may act to transduce nitrergic chemical signals into electrical hyperpolarization to smooth muscle cells (25). Lack of ICC, as occurs in WWv mutant mice, leads to loss of nitrergic relaxation and aftercontraction in the LES even though nitrergic neurons and nerve endings are well preserved in these mutant mice (25).
eNOS has been shown to be localized in some neurons in the central nervous system where NO derived from eNOS may be the physiological mediator (21). eNOS has also been reported to be present in smooth muscle cells (13, 23). Smooth muscle eNOS has been proposed as a major source of NO that is released by the action of putative inhibitory neurotransmitter on the smooth muscle cells (13). The present study shows that nitrergic responses of the LES are not affected in eNOS deficiency.
The localization of nNOS in the nerve endings suggests that the product of L-arginine-nNOS pathway serves as an antegrade neurotransmitter. Recent studies have shown that NO · redox form of NO is the nitrergic neurotransmitter (9). The gastric smooth muscle studies have shown that nNOS-deficient animals have selective loss of nitrergic inhibitory junction potential (IJP), but purinergic IJP is present (14). It was suggested that electrically independent pharmaco-mechanical inhibition may occur due to alternative mechanisms (14). Moreover, opossum LES relaxation is not fully blocked by L-NNA in vitro and L-NAME in vivo (27). In guinea pig LES L-NNA-resistant IJP was blocked by apamin (28) and in the rat stomach L-NNA-resistant delayed relaxation has been reported to be due to vasoactive intestinal peptide (22).
Mice lacking nNOS revealed complete block of EFS-evoked relaxation and
rebound contraction of the LES. The lack of LES relaxatory responses to
EFS in the nNOS-deficient mice was qualitatively and quantitatively
similar to that produced by acute suppression of NOS by chemical
blocker of NOS. The findings suggest that the responses in
nNOS() mice represent simple lack of NO. These observations are
somewhat surprising because we expected lifelong deficiency of nNOS
would lead to some alternative or redundant inhibitory pathways.
However, no such compensation was observed. We did not perform
morphological studies to show any structural changes in the connections
of the inhibitory nerves with ICCs. Further morphological and
functional studies are needed to identify the presence of possible
parallel transmitters and additional role of NOS as an intracellular
mediator in the inhibitory neurotransmission in the mouse LES.
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ACKNOWLEDGEMENTS |
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These studies were supported by the National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-31902 (R. K. Goyal) and DK-02462 (H. Mashimo), and Department of Veterans Affairs Medical Research Services Merit Review (R. K. Goyal and H. Mashimo).
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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: H. Mashimo, Research and Development (151), VA Medical Center, 1400 VFW Parkway, West Roxbury, MA 02132 (E-mail: mashimo{at}helix.mgh.harvard.edu).
Received 16 February 1999; accepted in final form 16 April 1999.
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DISCUSSION
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