Induction of nitric oxide synthase in rat gastric smooth
muscle preparations
Xi-Long
Zheng1,
Keith A.
Sharkey2, and
Morley D.
Hollenberg1
1 Endocrine and
2 Neuroscience Research Groups,
Departments of 2 Physiology,
1 Pharmacology and
Therapeutics, and 1 Medicine,
Faculty of Medicine, University of Calgary, Calgary, Alberta,
Canada T2N 4N1
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ABSTRACT |
The induction in vitro of inducible nitric
oxide synthase (iNOS) in intact gastric circular (CM) and longitudinal
(LM) smooth muscle preparations was evaluated
1) pharmacologically, by the appearance of 1 mM L-arginine
(L-Arg)-induced relaxation in a precontracted tissue; 2)
biochemically, according to the appearance of iNOS mRNA using a reverse
transcriptase-polymerase chain reaction; and
3) immunohistochemically, using an
iNOS-specific antibody. Functionally, iNOS induction
affected the contractile properties of the CM but not the LM
preparation. The time course of iNOS induction monitored
pharmacologically paralleled exactly the appearance of iNOS mRNA. The
relaxant response to L-Arg in
iNOS-induced CM tissues was blocked by the iNOS inhibitor
aminoguanidine and by the guanylyl cyclase inhibitor LY-83583. The
addition of oxyhemoglobin to the organ bath also attenuated the
relaxant response, but tetrodotoxin had no effect. The transcriptional
inhibitor actinomycin D completely blocked iNOS induction as assessed
by both pharmacological and biochemical criteria. In iNOS-induced
preparations the iNOS immunoreactivity was not detected in the smooth
muscle elements but was localized in a layer of macrophage-related
cells that were in apposition to the CM smooth muscle elements. We
conclude that the spontaneous induction of iNOS in rat gastric tissue
can affect the pharmacomechanical reactivity of the CM elements and
that this regulation of the CM contractility is due to the induction of
iNOS in a set of macrophage-related cells that are closely apposed to
the CM elements so that they selectively affect only the contractility
of the CM preparation.
macrophage; circular muscle; inducible nitric oxide synthase; immunohistochemistry
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INTRODUCTION |
NITRIC OXIDE (NO) was found to be responsible for the
bactericidal and tumoricidal actions of macrophages (4-6, 20). The inducible form of the biosynthetic enzyme in macrophages responsible for the synthesis of NO from the precursor arginine [inducible nitric oxide synthase (iNOS)] has been cloned from a variety of tissues, including cultured rat smooth muscle cells (17). The iNOS
induced in macrophages and other cell types has been found to be
distinct in amino acid sequence and immunological cross-reactivity from
the constitutively produced calcium-dependent enzymes present in either
endothelial cells (eNOS3 or NOS3) or neural tissue (nNOS/bNOS or NOS1)
(8). The induction of iNOS in the smooth muscle elements of vascular
tissue in response to septicemia or lipopolysaccharide administration
in vivo is believed to underlie many of the untoward manifestations of
irreversible shock in humans and other animals. Much of the work
reported to date studying the induction of NOS in smooth muscle has
been done either with tissues harvested from animals pretreated with
endotoxin in vivo (9) or with cultured cell systems (1, 15). Although a
number of studies have examined the appearance of iNOS in
vascular-derived smooth muscle cells, there have not to date been
reports of the induction of iNOS in smooth muscle preparations derived
from the gastrointestinal tract. It was our working hypothesis that the
induction of NOS in gastric smooth muscle tissue may play a role in
gastric pathophysiology. In keeping with this working hypothesis, we
wished to study the possible induction of iNOS in an intact tissue
preparation derived from gastric smooth muscle. To this end, we
selected two rat gastric tissue preparations for our study:
1) a gastric longitudinal muscle (LM) preparation and 2) a gastric
circular muscle (CM) preparation, derived from the same tissue as the
LM preparation by cutting the LM bundles at right angles (7, 14). We
selected the LM and CM preparations for study both because of our
previous experience with the pharmacology of the two preparations and
because of the distinct signal transduction pathways that appear to be
present in the CM and LM elements (7, 14). Using the two tissue
preparations, we first evaluated our ability to monitor iNOS induction
pharmacologically by measuring the time-dependent appearance of an
arginine-mediated relaxant response during prolonged incubation in an
oxygenated, buffered organ bath. Second, we documented the
time-dependent appearance of iNOS mRNA in the incubated tissues
using a reverse transcriptase-polymerase chain reaction (RT-PCR)
approach. Finally, we used immunohistochemical methods, with antibodies
targeted specifically to iNOS and brain NOS (bNOS) to document the
sites of induction of iNOS within the gastric tissues.
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METHODS |
Bioassay procedures.
The gastric LM and CM preparations were prepared essentially as
previously described (7, 14) with male Sprague-Dawley rats weighing
~250 g. Animals were cared for according to the recommendations of
the Canadian Council on Animal Care, in accordance with the
recommendations approved by the Council of the American Physiological
Society. After the rats were killed by rapid cervical dislocation, they
were exsanguinated from the common carotid arteries and the stomach
tissue was isolated. The stomach was opened along the lesser curvature,
and the smooth muscle component was carefully dissected free from all
overlying mucosa. It was not possible to assess the amount of residual
submucosal elements. The CM and LM strips (~3 × 10 mm) were
prepared by isolating the fundus tissue and by cutting either along or
at right angles to the visible CM bundles, respectively. This procedure
allows for a measurement of contractile responses of either the LM or
CM elements derived from the same tissue preparation (14). We have
found that anatomically and functionally the CM and LM preparations
obtained from either rats or guinea pigs are quite comparable. Each
preparation, secured at the ends with a silk suture, was mounted
vertically in a plastic cuvette organ bath containing 4 ml of
Krebs-Henseleit solution of the following composition (in mM): 118 NaCl, 4.7 KCl, 2.5 CaCl2, 1.2 MgCl2, 25 NaHCO3, 1.2 KH2PO4,
and 10 glucose in distilled deionized water. The bath medium was
maintained at 37°C and was gassed with a mixture of 95%
O2-5%
CO2 to maintain the pH at 7.4. A
resting tension of 1 g was applied initially, and the tissue was
allowed to equilibrate isometrically, at which time tension was in the
range of ~0.8 g. Changes in tissue tension were monitored isometrically using Grass or Statham force transducers. Routinely, tissue response was monitored by the addition of either 50 mM KCl or 1 µM carbachol (CCh) to the organ bath; after a contractile response
was monitored, tissues were washed and allowed to return to baseline
tension in fresh buffer. In experiments done to monitor the time of
induction of iNOS, tissues were incubated for prolonged time periods
(up to 10 h) in the organ bath, buffer was changed at ~45-min
intervals, and phenylephrine (PE) was used as a contractile agonist in
preference to CCh.
Preparation of tissue RNA and RT-PCR analysis.
The total RNA of the gastric CM tissue was extracted by using the TRI
Reagent protocol (Medical Research Center, Cincinnati, OH) before and
after prolonged incubation of the tissue in the organ
bath. The RT-PCR was used to amplify a 578-bp iNOS cDNA fragment from rat gastric RNA. The sequences of the forward
(5'-CCAGGGGCAAGCCATGTC-3') and reverse
(5'-CTCCAGGCCATCTTGGTGGC-3') primers were based on the
published rat aortic smooth muscle iNOS cDNA sequence (17). The PCR
product was cloned and sequenced by the dideoxy chain termination
reaction.
Pharmacological monitoring of iNOS induction using
L-arginine-mediated relaxation
response.
After the gastric tissues (LM or CM preparation) were mounted in the
organ bath, tissues were precontracted with CCh (1 µM) or PE (1 µM). PE appeared more reliable than CCh to produce a sustained
contractile response that did not fade with time. Thus PE was used as a
contractile agonist for continued work. At the plateau of the
contractile response, L-arginine
(L-Arg, 1 mM) was added to the
organ bath; a relaxant response on adding
L-Arg served as a
pharmacological index of iNOS induction. Tissues were then washed three
times to remove agonist and excess
L-Arg from the organ bath. The
time course of the induction of
L-Arg-mediated relaxation was
determined by monitoring
L-Arg-induced relaxation every
hour during the incubation of tissue in the organ bath. Treatment with
the transcription inhibitor actinomycin D (1 µM) was started from the
beginning of the experiment. The guanylyl cyclase inhibitor LY-83583
(10 µM) and the iNOS inhibitor aminoguanidine (AG, 1 mM) were added
to the organ bath ~20 min before any contractile agonist.
Immunohistochemistry.
CM gastric tissues before and after 5 h of incubation were fixed by
overnight immersion in 4% paraformaldehyde in 0.1 M phosphate buffer
(pH 7.4) at 4°C. They were then washed with phosphate-buffered saline (PBS, pH 7.4; 3 × 10 min) and cryoprotected in PBS
containing 20% sucrose. The tissues were sectioned (12 µm) in a
cryostat and then processed for indirect immunofluorescence. Sections
were washed in PBS containing 0.1% Triton X-100 for 30 min at room temperature and incubated with the primary antibodies for 24-48 h
at 4°C in a moist chamber. The primary antibodies used were rabbit
anti-iNOS (Transduction Laboratories, Lexington, KY; 1:500), rabbit
anti-bNOS (Santa Cruz Biotechnology, Santa Cruz, CA; 1:1,000), and
mouse anti-rat macrophage (clone ED2, Serotec, Oxford, UK; 1:1,500)
(3). To double label iNOS and macrophages, the primary antibodies were
mixed before use as previously described (18). Sections were then
washed (3 × 10 min) in PBS and incubated with secondary
antibodies [donkey anti-rabbit immunoglobulin G (IgG) conjugated
to CY3 (1:100; Jackson ImmunoResearch Laboratories, West Grove, PA)
and/or goat anti-mouse IgG conjugated to fluorescein isothiocyanate (1:50; Incstar, Stillwater, MN)] for a further 1 h
at room temperature. Finally, they were washed in PBS containing 0.1%
Triton X-100 (3 × 10 min) and mounted in bicarbonate-buffered glycerol (pH 8.6). Sections were examined using a Zeiss Axioplan fluorescence microscope, and photographs were taken with Kodak TMax 400 ASA film.
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RESULTS |
Pharmacological analysis of iNOS induction: time course and
specificity for CM preparation.
For the pharmacological evaluation of iNOS induction, we hoped to take
advantage of the possibility that on prolonged incubation and washing
of the gastric smooth muscle preparation, free arginine stores might
not be sufficient to cause a prominent relaxant effect on the tissue
when iNOS was induced. Amino acid analysis of tissue extracts revealed
an ~10% reduction in L-Arg
content on prolonged incubation (3-6 h, data not shown). In
keeping with this supposition it was expected that the addition of 1 mM
L-Arg to a precontracted tissue
containing iNOS would cause a relaxation due to the conversion of
arginine to NO. This approach had been used previously in the study of
iNOS induction in rat aortic tissue (13). In fresh tissue
L-Arg caused no effect in either
the CM or LM preparations (Fig. 1).
Nonetheless, an L-Arg-induced
relaxation became apparent in the CM preparations after ~2 h of
incubation in the organ bath and appeared to be maximal at ~4-5
h of incubation (Figs. 1A and 2). In the CM tissue, a relaxation was
observed in tissues precontracted by either CCh or PE. In contrast,
prolonged incubation of the LM preparation did not result in a tissue
that was subject to L-Arg-induced relaxation,
despite the fact that the LM tissue did relax in response to the NO
donor sodium nitroprusside (SNP; Fig.
1B). It appeared therefore that
there was an induction of iNOS in the CM but not in the LM preparation;
for that reason, all further work focused on the properties of the CM
preparation.

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Fig. 1.
Differential responses of circular muscle (CM) and longitudinal muscle
(LM) preparations to L-arginine
(L-Arg). CM
(A) and LM
(B) preparations were precontracted
with 1 µM carbachol (CCh; ) and then challenged with 1 mM
L-Arg ( ), followed by washing
the tissue (W, arrows) and replacing the buffer. Response to
L-Arg was monitored both before
(left) and after
(right) a 5-h incubation period at
37°C. LM tissue, which unlike CM tissue did not relax on addition
of L-Arg, did relax in response
to the nitric oxide (NO) donor sodium nitroprusside (SNP, 0.01 µM,
*). Tracings are representative of 8-10 independently conducted
experiments done with tissues derived from 5 different animals. Scale
for time and tension is shown beside tracings in
A.
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Fig. 2.
Time course of L-Arg-induced
relaxation in CM preparation. CM preparations were incubated in the
organ bath at 37°C for a prolonged time period. At hourly
intervals, tissues were precontracted with 1 µM phenylephrine (PE; as
shown in Fig. 3), and a relaxation in response to addition of 1 mM
L-Arg was monitored. Relaxation
response was expressed as %relaxation relative to maximum tension
developed in response to PE in each tissue [% = 100 × (maximum tension, tension in presence of
L-Arg)/maximum tension].
Values at each time point are averages ± SE (bar) for results
obtained with 5 or more independently conducted experiments using
10-15 tissues derived from 5 or more different animals.
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In a CM preparation that had been incubated in the organ bath for
4-5 h, L-Arg caused a
reproducible relaxation response (Fig. 2) that was attenuated by the
iNOS inhibitor AG (Fig.
3A); the tissue was nonetheless still sensitive to the relaxant action of SNP in
the continued presence of AG (Fig. 3A,
right). The relaxation response to
L-Arg was unaltered in the
presence of 1 µM tetrodotoxin, which would be expected to inhibit a
nerve-mediated relaxation effect (data not shown). Because the NO
produced in response to the addition of
L-Arg would be expected to cause
its relaxant effect via the stimulation of soluble guanylyl cyclase, we
next examined the ability of the guanylate cyclase inhibitor LY-83583 to affect the L-Arg-induced
relaxation in the CM tissue. As shown in Fig.
3B, LY-83583 completely blocked
L-Arg-induced relaxation in CM
tissue that had been preincubated in the organ bath for 4-5 h;
similarly, LY-83583 also blocked the relaxant response to the NO donor
SNP (Fig. 3B,
right). Finally, if an NO-mediated relaxation were due to diffusible NO, as is the case for
endothelium-dependent relaxations caused by acetylcholine in aortic
tissue, the NO scavenger oxyhemoglobin would be expected to attenuate
the relaxant response. We found that the inclusion of oxyhemoglobin (5 µM) in the organ bath blocked the
L-Arg-induced relaxation in an
otherwise responsive preparation and that the addition of oxyhemoglobin
to the organ bath at the nadir of
L-Arg-induced relaxation in the
CM tissue reversed the relaxant response (data not shown).

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Fig. 3.
Effects of the inducible nitric oxide synthase (iNOS) inhibitor
aminoguanidine (AG; A) and the
guanylyl cyclase inhibitor LY-83583 (LY;
B) on
L-Arg-induced relaxation in CM
preparation. After a 5-h incubation at 37°C in the organ bath,
tissues were precontracted with 1 µM PE ( ), and a relaxation in
response to addition of 1 mM
L-Arg ( ) was monitored,
followed by washing tissues and replacing buffer (arrow). A relaxant
response in a PE-precontracted tissue due to addition of NO donor SNP
(0.01 µM, *) was also measured. Ability of 1 mM AG ( ) to block
relaxation caused by L-Arg but
not by SNP is shown (A), as is
ability of 20 µM LY ( ) to abolish relaxation caused by both
L-Arg and SNP
(B). Each set of tracings showing
effects of AG and LY illustrates response of a single tissue strip.
Data are representative of 8-10 independently conducted
experiments with tissue preparations derived from 4 or 5 different
animals. Scale for time and tension is shown at bottom right.
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Detection of iNOS induction using RT-PCR.
To define further the time course of iNOS induction in the CM
preparation, CM tissue preparations were incubated as for the bioassay
measurements, and the appearance over time of an
L-Arg-induced relaxation was
monitored (Fig. 4). At the times indicated
the CM tissue preparations were withdrawn from the organ bath and rapidly processed for the isolation of tissue RNA. The RNA samples were
then reverse transcribed and subjected to PCR amplification using
primers targeted both to rat iNOS (578-bp product; Fig. 4A) and actin (247 bp). As shown in
Fig. 4 and as documented by the densitometry measurements recorded in
the figure legend, the time course of the appearance of the 578-bp PCR
product corresponding to iNOS was in step with the ability of
L-Arg to induce a relaxation in
the PE-precontracted CM preparations. The iNOS (PCR) signal was clearly
increased over the 4-h time period relative to the actin signal, which
was comparable in all samples tested. When the CM tissue was incubated
in the presence of the transcription inhibitor actinomycin D (1 µM),
there was no induction of iNOS PCR products and no appearance of
L-Arg-induced relaxation (Fig. 5). The nucleotide sequence of the 578-bp
iNOS PCR product was determined to be identical to the previously
published rat iNOS sequence derived from rat aortic smooth muscle (data
not shown).

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Fig. 4.
Correlation of the development of an
L-Arg-induced relaxation
(B) with appearance of iNOS mRNA
(A). Replicate CM tissue
preparations were incubated at 37°C in the organ bath for times
indicated (0-4 h). At hourly intervals, ability of
L-Arg (1 mM; ) to cause a
relaxation in a PE (1 µM; )-precontracted tissue was monitored,
followed by a tissue wash (W, arrows). At indicated times, replicate
tissues were also harvested for preparation of RNA, which was then
analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR)
using primer pairs targeted to either actin or iNOS. PCR products were
separated by agarose gel electrophoresis
(A). Arrows in
A indicate positions of iNOS and actin
PCR products; positions of standard markers [base pair
(bp)] are shown at right. Scale for time and tension is shown to
the right of contractile tracings
(B). By densitometry iNOS signals,
relative to the actin signals, were 0 at 0 h, 0.01 at 1 h, 0.06 at 2 h,
0.3 at 3 h, and 0.5 at 4 h.
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Fig. 5.
Effect of actinomycin D (ACTD) on development of
L-Arg-mediated relaxation
(B) and on iNOS message induction
(A) in CM tissue.
B: either freshly dissected CM tissue
(left) or tissues that had been
induced by incubation at 37°C in the organ bath for 5 h without
(middle) or with 1 µM ACTD
(right) were precontracted with 1 µM PE ( ) and assessed for a relaxation response caused by 1 mM
L-Arg ( ), followed by a
tissue wash (W, arrow). Tissue replicates were then processed for
preparation of RNA. A: presence of
iNOS message relative to that of actin was assessed by RT-PCR analysis
using appropriate primer pairs. Positions of iNOS (578 bp) and actin
(247 bp) PCR products separated by agarose gel electrophoresis are
shown. Positions of size markers (bp) are shown to right
(A). Scales for time and tension are
shown beside contractile tracings
(B). Results are representative of 2 independently conducted experiments.
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Immunohistochemical localization of iNOS and bNOS.
By pharmacological analysis, iNOS appeared to be induced in the CM but
not in the LM preparation. Unfortunately, the RT-PCR analysis, of
necessity, was done with RNA obtained from the intact tissue containing
both the CM and LM elements and could not therefore provide information
about the exact site of iNOS induction. Thus we next wished to localize
the site(s) of enzyme induction, using an immunohistochemical approach
employing iNOS-directed antibodies. Because bNOS is known to be present
in the neural plexus of the rat gastrointestinal tract (16), we also
analyzed the tissues for the presence of bNOS. As expected, bNOS was
detected in the samples, both in fresh tissue preparations (Fig.
6A) and
in "induced" tissue that had been incubated for 5 h in the organ
bath (labeling was comparable to Fig.
6A; data not shown). In contrast, no
staining for iNOS was observed for fresh tissue preparations (Fig.
6B). However, after preincubation of
the CM tissue in the organ bath for 4 h or more, we were able to
reproducibly observe a prominent appearance of immunoreactivity that
surprisingly was not in the smooth muscle elements. Rather,
immunohistochemistry revealed the presence of iNOS in a subset of cells
that was underneath the intestinal mucosa and closely apposed to the CM
elements (Fig. 6C,
top). We did not detect iNOS
immunoreactivity in either the circular or longitudinal smooth muscle
elements or in the myenteric plexus. The morphology of the iNOS
positive cells in the CM preparation suggested that they might be
macrophage related. As shown in Fig.7, double staining the iNOS-induced CM preparations both with iNOS and
with the macrophage-specific antibody ED2 (3) revealed a coincidence of
immunoreactivity in the same cell population.

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Fig. 6.
Immunohistochemical detection of iNOS and bNOS. Either freshly
dissected CM tissue (A and
B) or tissues that had been induced
by incubation in the organ bath at 37°C for 5 h
(C) were fixed and processed for
immunohistochemical detection of iNOS and bNOS.
A: bNOS is representative of either
fresh or induced tissue stained with anti-bNOS antibody.
B: iNOS-F shows fresh tissue stained
with anti-iNOS antibody. C: induced CM
tissue (iNOS-I) stained with same anti-iNOS antibody. Panels are
representative of 4 independently conducted experiments with tissues
from different animals. Scale bar, 50 µm; cm, circular muscle; sm,
submucosa.
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Fig. 7.
Colocalization of iNOS-positive immunoreactivity with
macrophage-related cells in induced CM tissue. CM tissue preparations
were induced by incubation for 5 h at 37°C in the organ bath, and
sections were then processed for double-labeling immunohistochemistry
using both anti-iNOS antibody (iNOS;
A) and macrophage-specific ED2
antibody (MAC; B). Pattern of
fluorescence labeling was virtually superimposable (compare staining
patterns in A and
B). Panels are representative of 3 independently conducted experiments. Scale bar, 50 µm.
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DISCUSSION |
The main finding of our study was that during prolonged incubation of a
gastric CM preparation in vitro there was a time-dependent spontaneous
induction of iNOS that was able to reduce tissue contractility in the
presence of sufficient L-Arg to
act as a substrate for the enzyme. In contrast, the LM preparation
derived from the same tissue was not affected functionally by the
induction of iNOS. The time-dependent induction of iNOS in the CM
preparation was substantiated using three sets of data:
1) pharmacological
(L-Arg-induced relaxation),
2) biochemical (RT-PCR analysis and
sequencing), and 3)
immunohistochemical. The relaxation response caused by L-Arg has been used previously
as an index of the appearance of iNOS in rat vascular tissue (13). The
ability of AG (a relatively selective inhibitor of iNOS, compared with
eNOS or bNOS) to block the
L-Arg-induced relaxation pointed
to the induction of iNOS, and the ability of the guanylate cyclase
inhibitor to block both the
L-Arg- and SNP-induced
relaxation in the CM tissue indicated that the metabolism of
L-Arg to NO was very likely the
cause of its relaxation effect via an elevation of tissue guanosine
3',5'-cyclic monophosphate. Because bNOS was detected in
the tissue both before and after the appearance of the relaxant
response caused by L-Arg, it was
unlikely that bNOS played any role in the functional response of the CM
preparation to L-Arg. This
conclusion was further substantiated by using tetrodotoxin to prevent
the possible action potential in bNOS-containing nerves; without
depolarization, in the presence of tetrodotoxin, there would be no
Ca2+ influx to stimulate NO
production by bNOS during exposure of the tissue to
L-Arg. The lack of effect of
tetrodotoxin on L-Arg-induced relaxation supported our conclusion that bNOS was not involved. The
RT-PCR data demonstrating an induction of a PCR product of the correct
iNOS nucleotide sequence, in keeping with the appearance of an
L-Arg relaxation, added further
evidence documenting the spontaneous appearance of iNOS in the CM
tissue. Because the addition of actinomycin D to the organ bath
prevented the appearance both of an
L-Arg-induced relaxation and of
an iNOS RT-PCR product, it can be concluded that the induction of iNOS
was at the transcriptional level. As is presumed to be the case for the
appearance of iNOS in aortic tissue during the course of organ bath
experiments (19), it is possible that endotoxin or a comparable
stimulus resulting from the tissue dissection procedure and from the
organ bath conditions may have been responsible for iNOS induction. In
the in vivo models of intestinal inflammation, iNOS induction has been
described in the myenteric plexus and the intestinal mucosa (12). These sites of induction would appear to differ from those we have observed in our studies. Whether a comparable induction of iNOS observed in the
CM tissue may occur in vivo in response to an inflammatory stimulus
remains to be determined.
Given the functional consequence of the induction of iNOS in terms of
the ability of L-Arg to modulate
CM (but not LM) contractility, we were surprised to find that the iNOS
immunoreactivity could not be detected in the CM smooth muscle
elements. Rather there was an unequivocal induction of iNOS
immunoreactivity that was localized in cells that also were visualized
by a macrophage-specific antibody. Such macrophagerelated cells
have been detected previously in the muscularis externa of mouse small
intestine (10, 11) but have yet to be documented in rat gastric tissue.
Although not detected by immunohistochemistry in the CM smooth muscle
elements, iNOS in that location might possibly have played a role in
the relaxation caused by L-Arg.
However, we would not have expected that oxyhemoglobin would have
attenuated the relaxation response if smooth muscle iNOS were the only
source of NO. We therefore suspect a major contribution by the
macrophage-related cells to the
L-Arg-induced relaxation
response we observed in the CM tissue. Given the localization of the
macrophage-related cells in which iNOS was induced, in close proximity
to the CM layer but physically quite remote from the LM layer, it is
possible to rationalize the selective functional effect of iNOS
induction on the mechanical properties of the CM preparation but not
the LM preparation. Given the physical distance and tissue barrier
between the macrophage-related cells and the LM elements, it is likely
that the NO produced by the macrophage-related cells on the addition of
L-Arg was not able to diffuse
far enough to affect LM function. This rationale was supported by the
ability of oxyhemoglobin to block the
L-Arg-mediated relaxation in the
"induced" CM tissue, presumably by scavenging NO and preventing
the diffusion of NO from one part of the tissue to another. Whether the
induction of iNOS in such macrophage-related cells in the submucosal
layer of the intestinal tract in vivo might play a pathophysiological
role in intestinal motility remains an intriguing question that merits
further study.
It can be noted that iNOS was spontaneously induced in the gastric
tissue in the absence of added cytokines (interleukin-1
or tumor
necrosis factor-
) or other inducers (e.g., lipopolysaccharide). The
induction may have resulted from the spontaneous production of
cytokines in the tissue, triggered by the "injury" of the
dissection process, or from the presence of endotoxin in the reusable
bioassay cuvettes. Alternatively, the preparation of the tissue for
bioassay may have removed an NO-mediated inhibitory signal present in
vivo (e.g., nerve-released NO) (2) that may have suppressed iNOS induction until the tissue was transferred to the bioassay environment. Further work will be required to identify the factors responsible for
the induction of iNOS in the CM tissue in vitro.
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ACKNOWLEDGEMENTS |
We are grateful to Dr. C. R. Triggle for helpful discussions and to
Winnie Ho for assistance with the immunohistochemical studies.
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FOOTNOTES |
These studies were supported by funds from the Medical Research Council
of Canada (M. D. Hollenberg and K. A. Sharkey). X.-L. Zheng was
supported in part by a William H. Davies Research Scholarship. K. A. Sharkey is an Alberta Heritage Foundation for Medical Research Senior
Scholar.
Address for reprint requests: M. D. Hollenberg, Dept. of Pharmacology
and Therapeutics, Univ. of Calgary, Faculty of Medicine, 3330 Hospital
Drive NW, Calgary, Alberta, Canada T2N 4N1.
Received 15 April 1997; accepted in final form 5 August 1997.
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