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

    ABSTRACT
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Abstract
Introduction
Methods
Results
Discussion
References

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

    INTRODUCTION
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Abstract
Introduction
Methods
Results
Discussion
References

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.

    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

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.

    RESULTS
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Abstract
Introduction
Methods
Results
Discussion
References

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; black-down-triangle ) and then challenged with 1 mM L-Arg (square ), 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.

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 (open circle ), and a relaxation in response to addition of 1 mM L-Arg (square ) 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 (triangle ) to block relaxation caused by L-Arg but not by SNP is shown (A), as is ability of 20 µM LY (black-triangle) 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.

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; square ) to cause a relaxation in a PE (1 µM; open circle )-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 (open circle ) and assessed for a relaxation response caused by 1 mM L-Arg (square ), 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.

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.

    DISCUSSION
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Abstract
Introduction
Methods
Results
Discussion
References

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-1beta or tumor necrosis factor-alpha ) 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.

    ACKNOWLEDGEMENTS

We are grateful to Dr. C. R. Triggle for helpful discussions and to Winnie Ho for assistance with the immunohistochemical studies.

    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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Abstract
Introduction
Methods
Results
Discussion
References

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AJP Gastroint Liver Physiol 273(5):G1101-G1107
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