Neuronal NOS provides nitrergic inhibitory neurotransmitter
in mouse lower esophageal sphincter
Chi Dae
Kim1,
Raj K.
Goyal1, and
Hiroshi
Mashimo1,2
1 Center for Swallowing and Motility Disorders,
Brockton/West Roxbury Veterans Affairs Medical Center, and
2 Gastrointestinal Unit, Massachusetts General
Hospital, and Harvard Medical School, Boston, Massachusetts 02132
 |
ABSTRACT |
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(
)] 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.
nitric oxide; nonadrenergic noncholinergic neurotransmission; endothelial nitric oxide synthase lacking mutant mice
 |
INTRODUCTION |
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
-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).
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(
) 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 |
Mice and tissues.
Adult (C57BL/6J X 129/J)F1 mice were used as wild-type mice, and
nNOS(
) 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).
Adult mice weighing between 25 and 30 g were killed by cervical
dislocation, followed by bleeding from the carotid arteries. The
stomach, including a portion of the esophagus, was quickly removed and
placed in a Sylgard-bottom petri dish filled with modified Krebs
solution (4°C) containing (in mmol/l) 118 NaCl, 4.7 KCl, 0.6 MgSO4, 25 NaHCO3, 1.0 NaH2PO4,
2.5 CaCl2, and 11 D-glucose, and bubbled with 95%
O2-5%
CO2. To isolate the LES, the
stomach was opened along the greater curvature to reveal the junction
between the esophagus and the stomach. The esophagus immediately
proximal to the gastroesophageal junction was dissected as a ring 1 mm
in width, and mounted in standard organ baths (Radnoti Glass). Among
these tissues, ring preparations showing spontaneous contraction were
considered as LES and used for the experiments.
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.
 |
RESULTS |
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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Fig. 1.
Effect of TTX (1 µM) on frequency-response relationship for
electrical field stimulation (EFS, 1-20 Hz, 60 V)-induced
intrastimulus relaxation (A) and
poststimulus rebound contraction (B)
in isolated mouse lower esophageal sphincter (LES) ring preparations.
Values represent means ± SE from 10 (Vehicle) or 3 (TTX)
experiments.
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Fig. 2.
A: representative recordings of EFS (5 Hz, 30 s)-induced intrastimulus relaxation and poststimulus rebound
contraction in U-46619 (1 µM)-contracted LES preparations of
wild-type mice in absence (Vehicle) and presence of 100 µM
N -nitro-L-arginine methyl ester
(L-NAME).
B and
C: EFS-induced responses of mouse LES
ring preparations in absence and presence of
L-NAME. %Change is expressed as
%change in grams of active tension before EFS-induced relaxation.
Values represent means ± SE of results obtained in 10 (Vehicle) or
6 (L-NAME) experiments.
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Table 1.
EC50 and Emax values for DNO-induced
relaxations in isolated LES ring preparations of wild-type mice in
presence and absence of 100 µM L-NAME
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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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Fig. 3.
A: representative recordings of EFS (5 Hz, 30 s)-induced intrastimulus relaxation and poststimulus rebound
contraction in LES preparations of wild-type (WT) and neuronal nitric
oxide synthase mice [nNOS( )] in absence
[nNOS( )] and presence [nNOS( ) + L-NAME] of 100 µM
L-NAME.
B and
C: EFS-induced responses of mouse LES
ring preparations in absence (Veh) and presence
(L-NAME) of
L-NAME. %Change is expressed as
%change in grams of active tension before EFS-induced relaxation.
Values represent means ± SE of results obtained from 5 experiments.
|
|
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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Fig. 4.
A: representative recordings of EFS (5 Hz, 30 s)-induced intrastimulus relaxation and poststimulus rebound
contraction in LES preparations of wild-type (WT) and endothelial NOS
mice [eNOS( )] in absence [eNOS( )] and
presence [eNOS( ) + L-NAME] of 100 µM
L-NAME.
B and
C: EFS-induced responses in LES ring
preparations of eNOS( ) mice in absence (Veh) and presence
(L-NAME) of
L-NAME. %Change is expressed as
%change in grams of active tension before EFS-induced relaxation.
Values represent means ± SE of results obtained in 6 experiments.
|
|
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 × 10
5 M),
and nNOS (
) (2.20 ± 1.47 × 10
5 M) mice were not significantly different among
these three kinds of mice.

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Fig. 5.
Relaxation induced by cumulative concentration of
diethylenetriamine-nitric oxide (DNO) in LES preparations of wild-type
(WT), eNOS( ), and nNOS( ) mice. Values represent means ± SE of results obtained in paired preparations from 4 mice.
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|
 |
DISCUSSION |
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.
 |
ACKNOWLEDGEMENTS |
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).
 |
FOOTNOTES |
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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