1 Department of Surgery, Helsinki University Central Hospital, 00029 Helsinki, Finland; and 2 Department of Gastroenterology, Juntendo University Central Hospital, School of Medicine, Tokyo 113, Japan
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ABSTRACT |
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Effects of nitric oxide (NO) on
gastric wound healing were investigated in primary rabbit gastric
epithelial cell cultures. We analyzed the speed of cell migration,
proliferation, and apoptosis after creating a round wound on
the cell cultures. The monolayers were incubated with or without the NO
donor sodium nitroprusside, oxatriazolimine 1,2,3,4-oxatriazolium,
5amino-3-(3,4-dichlorophenylchloride), or the peroxynitrite
generator 3-morpholinosydnomine-N-ethylcarbamide. The
possible role of cGMP as a second messenger of NO was investigated with
8-bromo-cGMP. The role of O
gastric epithelium; cell migration; cell proliferation; apoptosis; peroxynitrite; hydroxyl radicals; superoxide; guanosine 3',5'-cyclic monosphosphate
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INTRODUCTION |
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NITRIC OXIDE (NO), a uniquely diffusible messenger molecule, participates in many physiological and pathophysiological processes in the gastrointestinal tract. In the stomach, the constitutively expressed calcium-dependent NO synthase (cNOS) has been identified at least in the surface epithelial and chief cells, submucosal arteriolar plexus, and somatostatin-releasing D cells. cNOS activity has also been identified in enteric ganglia and in their processes (8). NO exerts a protective action against various mucosal injuries by regulating regional blood flow and increasing epithelial mucus production (13, 29, 36). In addition, high levels of NO produced by inducible NOS (iNOS) play a potent role as a cytotoxic agent during infection and inflammation, thus promoting lethal injury to the victim cells. NO is also involved in certain immune responses in acute and chronic infections, e.g., by increasing permeability of intestinal epithelium (38). NO may also have some role in the development of certain neoplastic diseases in the gastrointestinal tract (4, 5, 23, 42).
In apoptosis NO seems to have a dual regulatory function. In
certain cell types NO induces apoptosis, whereas in others it opposes this process (10, 17). It has been proposed that
apoptosis caused by NO or its metabolites results from DNA
damage and accumulation of tumor suppressor protein p53. Also, NO can
reversibly bind to cytochrome a3 leading to
inhibition of mitochondrial respiration and apoptosis
(2). It has become increasingly evident that other
reactive metabolites, e.g., peroxynitrite (PN), hydroxyl radicals, and
superoxide radicals (O
After superficial injury the gastric mucosa is rapidly restored. This is accomplished by migration of the surviving epithelial cells over the denuded mucosa (restitution) and, later, also by cellular proliferation. Some earlier reports suggested that NO somehow contributes to this healing process (1, 29). However, these studies were mostly performed with in vivo or ex vivo techniques, which provide multiple local targets and systemic interactions for the effects of NO, thus complicating the interpretation of the findings. The present study investigates the mechanisms by which NO affects wound repair in a primary monolayer cell culture with rabbit primary gastric oxyntic epithelial cells as the sole target for NO.
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MATERIALS AND METHODS |
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In this study isolated rabbit gastric mucosal monolayer cultures were used. All animals received humane care in compliance with the Principles of Laboratory Animal Care and the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH Pub. No. 86-23, revised 1985). The authorization to perform this study was given by the Provincial Government of Uusimaa in accordance with Finnish legislation.
The process for the isolation of gastric mucosal cells from rabbit stomach was previously described in detail (41). Briefly, fasted male rabbits (weight 2.0-3.0 kg) were euthanized with an overdose of pentobarbital sodium. The stomach was quickly removed, opened along the greater curvature, and rinsed with ice-cold isotonic saline. The oxyntic mucosa was separated with a razor blade and minced into small pieces (2-3 mm3). The pieces were incubated in a medium containing 0.07% collagenase (type I; Sigma, St. Louis, MO), 130 mM NaCl, 12 mM NaHCO3, 3 mM NaH2PO4, 2 mM MgSO4, 1 mM CaCl2, 0.1% bovine serum albumin, and 0.2% glucose for 15 min in a shaker bath at 370 C. After the incubation, the tissue was washed with Ca2+- and Mg2+-free Hanks' balanced salt solution (HBSS) with 1 mM EDTA. These procedures were repeated twice before the tissue was filtered through a metal mesh (mesh size 300 µm). The cells were then washed in Ca2+- and Mg2+-free HBSS containing 1 mM EDTA and 0.1% bovine serum albumin.
Cell Culture
Coon's modified Ham's F-12 medium supplemented with inactivated 10% fetal bovine serum, 10 × 104 U/l penicillin, 100 mg/l streptomycin, and 0.25 mg/l amphotericin was used. The isolated gastric mucosal cells were grown on collagen (type I)-coated plastic culture dishes (diameter 60 mm; Corning Glass Works, Corning, NY) at a concentration of 5 × 106 cells/dish. The cells were incubated in a humidified atmosphere containing 5% CO2-95% air at 37°C.Artificial Wounding and Restoration Assay of Monolayer
The cultured gastric epithelial cells formed a complete monolayer cell sheet within 48 h after cell inoculation. As reported earlier, >90% of the cells in the cell sheet were periodic acid-Schiff-positive mucous cells, 5% were succinic dehydrogenase-positive parietal cells, and 4% were Nile blue-positive chief cells (40). A round artificial wound with a cell-free area of constant size (3.0 mm2) was made in the center of the mucosal cell sheet using a modified pencil-type mixer with a rotating silicon tip without damaging the coated dish surface. After wounding, the total restoration assay including cell migration and cell proliferation was performed under fetal bovine serum-free conditions. The test agents, as described under Chemicals, were added immediately after the artificial wounding of the monolayer cell culture, and their effects were monitored by taking phase-contrast micrographs at 12-24 h intervals for 48 h. The cell-free area on the epithelial cell sheet was measured using an IBAS II image analyzer (Carl Zeiss, Oberkochen, Germany) and phase-contrast micrographs. The change in the cell-free area (round shaped) was converted to average migration speed (v) with the following formula
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Measurement of Nitrite/Nitrate Levels
NO production released by sodium nitroprusside (SNP) was assessed as combined accumulation of nitrite and nitrate into the culture medium. Total cumulative NO production over 24 and 48 h after artificial wounding of the monolayer was calculated from six different concentrations of SNP (10, 30, 100, 300, 500, and 1,000 µM) as described by Orpana et al. (26).Determination of Cellular Disruption by Trypan Blue Exclusion
Trypan blue dye [100 µl of 1% (wt/vol) solution; Sigma] was added directly to the monolayer culture. After 5 min, the number of stained and nonstained cells was estimated in a random manner by counting 100-200 cells from each culture under a microscope at ×40 magnification.Cell Proliferation Assay
DNA synthesis was determined in the control, 300 µM SNP, and 1 mM NG-nitro-L-arginine methyl ester (L-NAME) monolayer cultures at different time intervals. DNA-synthesizing cells were detected by indirect immunohistochemistry using monoclonal anti-5-bromodeoxyuridine (BrdU) antibody (Sigma, St. Louis, MO) (18). BrdU was added at the time of wounding (0 h) and 12, 24, 36, and 48 h later, and incubation was continued for 1 h. Samples were processed and stained for BrdU by standard techniques. Microscopic pictures were taken with a phase-contrast microscope (Nikon, Tokyo, Japan) using Fujichrome 100 ASA film. Photomicrographs were taken at the edge of the wounded monolayer, and the BrdU labeling index (BrdU-positive cell number/total cell number × 100) was calculated in a randomly selected unit area (1.47 mm2) from the edge of the wound.Apoptosis Assay
Gastric epithelial cell apoptosis was detected by terminal deoxytransferase-mediated dUTP-biotin nick end labeling (TUNEL) with an in situ apoptosis detection kit (Apo-BrdU, no. 6576kk. PharMingen). The apoptosis assays were performed 24 and 48 h after wounding of the monolayer. The colored cells were analyzed using Becton-Dickinson FACScan flow cytometry and Compu Cyten laser scanning cytometry (LSC). Altogether 5,000-10,000 events/culture dish were collected by flow cytometry and analyzed using the Cell Quest program. Cell clusters and subcellular particles were removed by gating (i.e., only the single cells were analyzed). The measurements of LSC were performed by using CytoSpin preparation; the data were analyzed by using Win Cyte analysis. The labeling index for TUNEL-positive cells was calculated by the formula labeling index (%) = (positive cell number/total cell number) × 100.Chemicals
Two kinds of NO donors in three different concentrations were used, SNP (100, 300, and 1,000 µM; Sigma) and 1,2,3,4-oxatriazolium, 5-amino-3-(3,4-dichlorophenylchloride) (GEA-3162, 1, 10, and 30 µM; gift of the Dept. of Pharmacology, University of Helsinki). In other experiments 3-morpholinosydnomine-N-ethylcarbamide (SIN-1, 100, 500, and 1,000 µM; GEA-Pharmaceuticals, Copenhagen, Denmark) was used as a donor of NO and OSNP and SIN-1 were directly dissolved in culture medium. GEA-3162 was first dissolved in a DMSO solution. The final concentration of DMSO in the culture medium was 0.01%, which was also added to the appropriate controls. No significant differences were observed in wound healing between normal and 0.01% DMSO-containing controls.
To investigate the role of endogenous production of NO · in the cell monolayers NO synthesis was inhibited with a nonselective NOS inhibitor, L-NAME (ICN, Costa Mesa, CA), in three different concentrations (100, 300, and 1,000 µM).
To investigate the role of cGMP as the second messenger of NO,
8-bromo-cGMP (8-BrcGMP) (Sigma) was used in two concentrations (100 and
1,000 µM). To investigate whether the cytotoxic effects of NO are, at
least in part, mediated by PN (a metabolite of NO and
O
Statistics
The results are expressed as means ± SD. Student's unpaired t-test for equal and unequal variances was used for statistical analysis of the raw data. General linear modeling was used in migration data to adjust for the possible differences in migration speed between different cultures. The SAS program GLM (SAS Institute, Cary, NC) was used to fit a model in which the dependent variable was the speed of migration and the independent variables were the group variable and a categorical variable for different cultures. A P value <0.05 was considered a statistically significant difference. ![]() |
RESULTS |
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Effects of NO and PN Donors
GEA-3162.
The highest concentration of the NO donor GEA-3162 (30 µM) caused a
statistically significant (P < 0.01) retardation in
the speed of cell migration and wound healing (21.4 ± 4.9 vs.
12.4 ± 8.9 µm/h), whereas the two lower concentrations of
GEA-3162 had no significant effect on this process (Fig.
1).
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SIN-1. The effects of SIN-1 were less pronounced than those of the NO donors. However, the two highest concentrations of SIN-1 (500 µM and 1 mM) had a significant inhibitory effect on the speed of cell migration and wound healing (Fig. 1).
Neither GEA-3162 nor SIN-1 caused any cellular damage in the monolayer cultures as judged from the trypan blue exclusion experiments.Production of Nitrite/Nitrate by SNP
SNP produced nitrite and nitrate in a time- and concentration-dependent manner (Fig. 2). The most frequently used concentration of SNP (300 µM) produced 73.3 ± 7.7 µM nitrite/nitrate in 24 h. This concentration did not cause any destruction in the cell cultures as judged from the trypan blue exclusion experiments. In control monolayers (without SNP) there was no detectable nitrate/nitrite production.
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Compared with the controls, SNP caused a significant inhibition of cell
migration during the initial 24 h of wounding (Fig. 3). The highest concentration (1,000 µM) caused even diffuse cellular damage with cell death and
detachment from the collagen matrix. The inhibition of the wound
healing was, at least in part, reversible: when SNP was removed from
the culture medium 24 h after its addition, the wound healing
process was gradually normalized (Fig.
4).
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BrdU staining displayed that SNP almost totally inhibited cellular
proliferation at 24-36 h (Table 1).
SNP also caused a statistically significant increase in
apoptosis index. After 24 h the apoptosis index
was 9.3 ± 2.9% in the control cultures and 24.4 ± 4.8% in
SNP cultures (n = 12, P < 0.001; Table
2).
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Effects of 8-BrcGMP
As compared with the controls, 8-BrcGMP had no significant effects on the speed of wound healing or cell migration (21.7 ± 6.3 vs. 22.7 ± 6.5 and 21.1 ± 5.6 vs. 21. ± 5.9 µm/h for controls and 100 and 1,000 µM 8-BrcGMP, respectively).Effects of O
DDC.
DDC is an agent capable of inhibiting endogenous SOD activity, which in
turn leads to increased amounts of O
PG.
PG (which undergoes spontaneous autooxidation under physiological
conditions, resulting in generation of O
Effects of O
SOD + CAT.
To further explore whether O
AP. AP acts as an inhibitor of XO and inhibits production of intracellular reactive oxidation metabolites. Incubation of monolayers with AP partially abolished the effects of SNP on the speed of cell migration and the apoptosis index (Table 2).
Effect of Inhibition of NO Synthesis: L-NAME
To examine the effects of total elimination of NO in the wound healing, three concentrations of L-NAME (n = 7 each) were used in the monolayer cultures. Compared with the controls, the speed of cell migration during the first 24 h of wound healing was essentially the same in all L-NAME groups (Fig. 5). Even with the highest concentration of L-NAME (1 mM) the number of BrdU-positive cells at different time intervals was essentially the same as in the controls. At 24 h BrdU-positive cells were identified only rarely, and at 36 h the number of BrdU-positive cells was at its maximum. The number of these cells decreased after complete healing of the wound (Table 1).
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DISCUSSION |
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NO seems to have a protective action against gastric mucosal injury provoked by hypertonic salt solution (33), hydrochloric acid (31), ethanol (43), taurocholate (32, 34), ischemia-reperfusion (28), water immersion (11), or cold restraint-induced stress ulcer (6). Furthermore, NO seems to contribute to gastric adaptive cytoprotection induced by mild irritants (18). Inhibition of NOS with the nonselective NOS inhibitor L-NAME impairs the recovery of transmucosal electric resistance in L-arginine-treated isolated gastric mucosa (43). Constitutively produced NO has an important role in maintaining mucosal integrity under normal and pathophysiological conditions (38), and inhibition of NO production increases intestinal permeability (16, 19). The starting phase of migration is also NO dependent in renal tubular epithelium (24). It has been suggested that in in vivo conditions the most important protective mechanism of NO is improvement of mucosal blood flow (12, 14, 19, 21, 30).
The effects of NO seem to be "dose dependent." Small amounts of NO act as classic neurotransmitters in regulating gastrointestinal motility, regional blood flow, and neuroendocrine function, whereas excessive amounts of NO can act as cytotoxic agents in several pathophysiological conditions. According to some other experiments NO can also damage the gastrointestinal mucosa. For instance, NO seems to play a role in the development of gastric mucosal lesions in association with portal hypertension in the rat (25). Furthermore, administration of L-arginine (the substrate for NOS) dose dependently enhanced gastric mucosal damage caused by 20% ethanol in the rat (9). Also, high levels of NO formed by iNOS seem to contribute to the damage associated with colon and small intestinal mucosal inflammation (35). However, there are also findings divergent from these results. Exogenous NO generated by NO donors [3-(cis-2,6-dimethyl piperidino)-sydnonimine (CAS 754), SIN-1] did not cause cellular, vascular, or mucosal dysfunction in normal or infectious small intestine in the cat (20).
Several reports suggest that an excessive amount of NO, whether endogenous or exogenous, deteriorates cellular viability and can be cytotoxic to gastrointestinal mucosa (4, 9). Brown and coworkers (4) showed that surface cells are more susceptible to damage by NO than other cells of the gastric epithelium. Furthermore, it has been shown that high levels of NO generated by bacterial endotoxin challenge in rats cause gastrointestinal cell injury both in vivo and in vitro in gastric mucosal cell suspensions. This response seems to be causally related to an increase in intracellular Ca2+ concentration and protein kinase C activation (3, 37).
In the present model of rabbit primary gastric epithelial cell monolayer cultures, exogenous NO retarded wound healing in a time- and concentration-dependent manner by inhibiting cell migration and proliferation and by inducing cell apoptosis. The inhibition of wound healing was reversible after the NO donor SNP was removed from the culture medium. The highest concentration of SNP completely and irreversibly inhibited wound healing, provoking cellular ruffling and detachment from the collagen matrix. It is possible that this concentration might have a direct toxic action on the cell cultures. For this reason, a smaller concentration of SNP was used as the standard concentration in subsequent experiments.
In the present study the gastric epithelial cell monolayer cultures did not produce detectable amounts of endogenous NO, and the nonselective NOS inhibitor L-NAME did not have any influence on the speed of wound healing. This finding is in accordance with earlier reports suggesting that, for some reason, the cultured cells lose their ability to produce NO (26).
It has been assumed that cGMP acts as a second messenger of NO, and an increase of intracellular cGMP concentration has even been used as an indirect indicator of the amount of NO produced (45). However, this concept has been questioned, because in vitro even very high concentrations of NO (10- to 100-fold increase) have only a minor influence on the level of a cGMP (7). In the present study a cGMP analog, 8'-BrcGMP (which readily penetrates the cell membrane and is rather resistant against phosphodiesterases), was used to further elucidate these effects. The data demonstrate that excess of cGMP is not the cause of the inhibitory effects of NO on wound healing.
The present findings suggest that the toxicity of NO donors is
mediated, at least in part, by its reaction with
O). This view is supported by the findings that the
XO inhibitor AP, which blocks the formation of O
NO also promotes apoptosis, which obviously contributes to the
delayed wound healing provoked by NO. It seems that this action of NO
requires simultaneous intracellular generation of
O
It was reported earlier that NO alone is almost nontoxic against cultured rat liver endothelial cells under low O2 partial pressures but highly cytotoxic in the presence of nonphysiological high O2 partial pressure (15). In the present experiments the O2 partial pressure was kept under 20% as recommended by those authors. Thus our results are not explicable in terms of high O2 partial pressure.
As yet, there are only a few reports on the effects of NO on cell
proliferation in general (15) and, to our knowledge, none as regards gastric epithelial primary cultured nontransformed cells.
The present finding of an inhibitory effect of NO on cellular proliferation is in accordance with that of Yang and coworkers (44), who demonstrated that exogenous NO inhibits the
proliferation of cultured vascular endothelial cells. Furthermore,
SIN-1, a donor of NO and O
In conclusion, NO donors retard gastric wound healing by inhibiting
cell migration and proliferation and inducing cell apoptosis. These effects do not seem to be mediated via cGMP, but
O
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ACKNOWLEDGEMENTS |
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We thank Paula Kokko for valuable technical assistance.
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FOOTNOTES |
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This work was supported in part by grants from the Research Institute, Helsinki University Central Hospital, Sigrid Jusélius Foundation, and the Academy of Finland.
Address for reprint requests and other correspondence: T. Kiviluoto, Helsinki Univ. Central Hospital, Dept. of Surgery, PL 340, 00029 Helsinki, Finland (E-mail: tuula.kiviluoto{at}hus.fi).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 14 March 2001; accepted in final form 3 July 2001.
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