RGM1 Cells Research Articles

Suppression of Growth Arrest and DNA Damage-Inducible 45α Expression Confers Resistance to Sulindac and Indomethacin-Induced Gastric Mucosal Injury

Nonsteroidal anti-inflammatory drugs (NSAIDs) such as sulindac and indomethacin are a major cause of gastric erosions and ulcers. Induction of apoptosis by NSAIDs is an important mechanism involved. Understanding how NSAIDs affect genes that regulate apoptosis is useful for designing therapeutic or preventive strategies and for evaluating the efficacy of safer drugs being developed. We investigated whether growth arrest and DNA damage-inducible 45alpha (GADD45alpha), a stress signal response gene involved in regulation of DNA repair and induction of apoptosis, plays a part in NSAID-induced gastric mucosal injury and apoptosis in vivo in mice and in vitro in cultured human AGS and rat RGM-1 gastric epithelial cells. Intraperitoneal administration of sulindac and indomethacin both resulted in up-regulation of GADD45alpha expression and induction of significant injury and apoptosis in gastric mucosa of wild-type mice. GADD45alpha(-/-) mice were markedly more resistant to both sulindac- and indomethacin-induced gastric mucosal injury and apoptosis than wild-type mice. Sulindac sulfide and indomethacin treatments also concentration-dependently increased GADD45alpha expression and apoptosis in AGS and RGM-1 cells. Antisense suppression of GADD45alpha expression significantly reduced sulindac and indomethacin-induced activation of caspase-9 and apoptosis in AGS cells. Pretreatments with exogenous prostaglandins and small interfering RNA suppression of cyclooxygenase (COX)-1 and -2 did not affect up-regulation of GADD45alpha by sulindac sulfide and indomethacin in AGS cells. These findings indicate that GADD45alpha up-regulation is a COX-independent mechanism that is required for induction of severe gastric mucosal apoptosis and injury by NSAIDs, probably via a capase-9-dependent pathway of programmed cell death.

A putative role for AHNAK1 in maintaining gastric epithelial permeability barrier properties

The organization and integrity of the plasma membrane of gastric epithelial cells is crucial for providing a protective barrier for the stomach. AHNAK1 is a scaffolding protein that has been described as a protein marker of cells with barrier properties and is implicated in the regulation and organization of the cytoskeleton and the plasma membrane of epithelial cells. Previously, we reported AHNAK1 protein is expressed in different regions of the stomach of healthy human organ donors, and in cultured non‐transformed rat gastric mucosal (RGM1) polarized epithelial cells. We showed AHNAK1 protein is highly expressed in the cytoplasm and at the plasma membrane of RGM1 cells. Here, we investigated a possible function for AHNAK1 in mediating the selective paracellular permeability properties of gastric epithelial cells. An siRNA mediated knockdown of AHNAK1 protein in RGM1 cells by approximately 70% was determined by Western blot and densitometric analysis. Immunofluorescence studies revealed AHNAK1 protein colocalizes with the tight junction associated protein ZO2. In vitro cell permeability assays showed an increase in permeation of fluorescein isothiocyanate (FITC)‐dextran across RGM1 monolayers after transfecting cells with a smartpool of siRNAs targeting AHNAK1 compared to transfecting cells with nontargeting siRNAs or treating cells with EDTA. Taken together, our results suggest a role for AHNAK1 in maintaining the integrity of the permeability barrier properties of gastric mucosal cells. This research was supported by an American Heart Association Predoctoral Fellowship and a Veterans Affairs Merit grant.

Opposite effects of two thiazolidinediones, ciglitazone and troglitazone, on proteinase-activated receptor-1-triggered prostaglandin E 2 release

Thiazolidinediones, known as peroxisome proliferator-activated receptor-γ (PPARγ) agonists, may modify prostaglandin formation and exert gastroprotective effects. Since activation of proteinase-activated receptor-1 (PAR1) reveals endogenous prostanoid-dependent gastroprotection, we investigated if two thiazolidinediones, ciglitazone and troglitazone, modulate the prostaglandin E 2 (PGE 2) release caused by activation of PAR1 in normal rat gastric mucosal epithelial RGM1 cells. Ciglitazone dramatically facilitated the PAR1-triggered PGE 2 production and cyclooxygenase-2 (COX-2) upregulation, although it had no effect by itself. In contrast, troglitazone suppressed the PAR1-triggered PGE 2 production and COX-2 upregulation. Either effect of ciglitazone and troglitazone was resistant to GW9662, a PPARγ antagonist. The facilitation of the PGE 2 release by ciglitazone was blocked by inhibitors of MEK, p38 MAP kinase (p38MAPK) and PI3-kinase (PI3K), but not JNK. Nonetheless, ciglitazone failed to enhance the PAR1-triggered phosphorylation of ERK and p38MAPK. In conclusion, ciglitazone and troglitazone, exert opposite effects on the PAR1-triggered PGE 2 production and COX-2 upregulation by targeting molecules other than PPARγ.

Dexamethasone Inhibits Tumor Necrosis Factor-α-stimulated Gastric Epithelial Cell Migration

Cell migration (restitution) occurs in the early phase of gastric ulcer healing. Tumor necrosis factor (TNF)-alpha is overexpressed at the ulcer margin and plays a physiologic role in gastric ulcer healing. Dexamethasone, which is a potent corticosteroid, delays rat gastric ulcer healing. We evaluated whether dexamethasone inhibited TNF-alpha-stimulated gastric epithelial cell migration using a rat normal gastric epithelial cell line (RGM-1). An artificial wound model was employed to measure cell migration. Western blot was performed to evaluate the possible mechanisms. Intracellular prostaglandin E2 level was measured using an enzyme-linked immunosorbent assay. TNF-alpha treatment (10 ng/mL) for 12-48 hours significantly increased RGM-1 cell migration, and TNF-alpha treatment increased cyclooxygenase (COX)-2 protein expression 8 hours later and prostaglandin E2 (PGE2) synthesis 12 hours later compared with control (p < 0.05). Dexamethasone (10(-6) M) significantly inhibited the stimulatory effect of TNF-alpha on RGM-1 cell migration, which was associated with a significant decrease in COX-2 expression and PGE2 level in cells (p < 0.05). TNF-alpha plays a regulatory role in rat gastric epithelial cell migration and dexamethasone inhibited TNF-alpha-stimulated cell migration, which was associated with a decrease in COX-2 expression and PGE2 formation.

Lansoprazole, a Proton Pump Inhibitor, Mediates Anti-Inflammatory Effect in Gastric Mucosal Cells through the Induction of Heme Oxygenase-1 via Activation of NF-E2-Related Factor 2 and Oxidation of Kelch-Like ECH-Associating Protein 1

Induction of heme oxygenase-1 (HO-1) expression has been associated with cytoprotective and anti-inflammatory actions of lansoprazole, a proton pump inhibitor, but the underlying molecular mechanisms remain largely unresolved. In this study, we investigate the role of transcriptional NF-E2-related factor 2 (Nrf2), its phosphorylation/activation, and oxidation of Kelch-like ECH-associating protein 1 (Keap1) in lansoprazole-induced HO-1 up-regulation using cultured gastric epithelial cells (rat gastric mucosal cell line, RGM-1). HO-1 expression of RGM-1 cells was markedly enhanced in a time- and dose-dependent manner by the treatment with lansoprazole, and this up-regulation of HO-1 contributed to the inhibition of chemokine production from stimulated RGM-1 cells. Transfection of Nrf2-siRNA suppressed the lansoprazole-induced HO-1. An electrophoretic mobility shift assay showed increases in the nuclear translocation and stress-response elements (StRE) binding activity of Nrf2 proteins in RGM-1 cells treated with lansoprazole. Furthermore, in RGM-1 cells transfected with HO-1 enhancer luciferase reporter plasmid containing mutant StRE, lansoprazole-induced HO-1 reporter gene activity was diminished. Lansoprazole promoted the phosphorylation of extracellular signal-regulated kinase (ERK), and lansoprazole-induced HO-1 up-regulation was suppressed by U0126, an ERK-specific inhibitor. Phosphorylated Nrf2 protein was detected in the phosphoprotein fraction purified by a Pro-Q Diamond Phosphoprotein Enrichment kit. Finally, an oxidative form of the Keap1 protein was detected in lansoprazole-treated RGM-1 cells by analyzing S-oxidized proteins using biotinylated cysteine as a molecular probe. These results indicate that lansoprazole up-regulates HO-1 expression in rat gastric epithelial cells, and the up-regulated HO-1 contributes to the anti-inflammatory effects of the drug. Phosphorylation of ERK and Nrf2, activation and nuclear translocation of Nrf2, and oxidation of Keap1 are all involved in the lansoprazole-induced HO-1 up-regulation.

The expression of heme oxygenase-1 induced by lansoprazole.

Our previous studies have demonstrated that lansoprazole inhibits acute inflammatory reactions as well as intestinal mucosal injuries induced by ischemia-reperfusion or indomethacin administration in rats. Thus, proton pump inhibitors such as lansoprazole have been demonstrated to prevent gastrointestinal mucosal injury by mechanisms independent of acid inhibition. In our in vitro study, lansoprazole induced the expression of heme oxygenase-1 (HO-1) on rat gastric epithelial cells (RGM-1 cells), and exerted anti-inflammatory effect on the dependent of HO-1 expression. Furthermore, NF-E2-related factor-2 (Nrf2) played an important role in HO-1 expression induced by lansoprazole. In this review, we focused on lansoprazole-induced HO-1 expression, its anti-inflammatory action, and the role of Nrf2 in its expression.

Involvement of aquaporin-1 in gastric epithelial cell migration during wound repair

We examined the expression of aquaporin (AQP)-1 in monolayers of the rat gastric epithelial cell line RGM1 and investigated the roles AQP1 plays in the epithelial restitution during wound healing. A round wound of constant size was produced in the center of a confluent cell monolayer using a rotating silicon tip. The RGM1 cells expressed AQP1 mRNA. Hg 2+ (HgCl 2), an inhibitor of AQPs, suppressed cell migration during wound repair in a concentration-dependent manner. Likewise, cell migration was also impaired in cells in which AQP1 was knocked down by RNA interference, resulting in a marked delay of wound healing. The AQP1 knockdown RGM1 cells showed a decrease in the formation of membrane protrusions (lamellipodia) at the leading edge of the wound as compared with control RGM1 cells. These results suggest for the first time that AQP1 plays a crucial role in gastric epithelial cell migration during wound healing.

W1552 Impairment By Allyl Isothiocyanate of Gastric Epithelial Wound Repair Through Inhibition of Ion Transporters

co- transporter (NBC). We examined the expression of TRPA1 and ion transporters in monolayers of the rat gastric epithelial cell line RGM1 and investigated the involvement of these factors in the inhibitory action of isothiocyanate on epithelial wound healing. After obtaining a confluent monolayer, a round artificial wound of constant size was induced in the center of the cell monolayer using a pencil-type mixer with a rotating silicon tip. Immediately after the wound induction, cells at the edge of the wound started to form lamellipodia, migrating towards the center of wound, and the cell-free area decreased with time. Addition of allyl isothiocyanate to standard buffer suppressed the recovery of the wound in a concentration- dependent manner without affecting the viability of the RGM1 cells. Icilin, another TRPA1 agonist, dose-dependently inhibited wound repair. Likewise, 4,4'-diisothio- cyanatostilbene-2,2'-disulfonic acid (DIDS), a stilbene compound containing an isothiocyanate group, also inhibited the recovery of epithelial wounds. In addition, the repair of epithelial wounds was suppressed when the cells were incubated in Na + , Cl- or HCO3 - free buffer. The RGM1 cells expressed the mRNAs of AE2a and NBC1 but not TRPA1. These results suggested that isothiocyanate impairs the repair of epithelial wounds in RGM1 cells, probably through the inhibition of ion transporters such as AE2a and NBC1 and not the activation of the TRPA1 channel. It is assumed that the process of epithelial repair is associated with the regulation of cell volume and intracellular pH (pHi) by these ion transporters.

The delayed release of PGE2 mediated by PAR1 involves slowly developing production of arachidonic acid in rat gastric mucosal epithelial cells

We previously showed that activation of proteinase‐activated receptor‐1 (PAR1) caused delayed (18 h later) PGE2 release in rat gastric mucosal epithelial RGM1 cells, although the maximal up‐regulation of COX‐2 protein was observed much earlier (6 h later). In this study, we analyzed mechanisms for the delay of PGE2 release caused by PAR1 stimulation in RGM1 cells. Stimulation with a PAR1‐activating peptide, TFLLR‐NH2 (TF), in combination with arachidonic acid (AA) or linoleic acid (LA) induced rapid and synergistic increase in PGE2 release within 3 h. CP‐24879, a Δ5/Δ6‐desaturase inhibitor, suppressed the PGE2 release induced by TF plus LA, but not by TF plus AA or TF alone, at 18 h. The TF‐induced delayed PGE2 release was suppressed by combined application of inhibitors of cPLA2, iPLA2 and sPLA2, but not by each of them. Protein levels of microsomal PGE synthase (mPGES)‐1, mPGES‐2 and cytosolic PGES were unchanged by stimulation with TF. These data suggest that the delay of PAR1‐triggered PGE2 release is due to slowly developing AA production in RGM1 cells, and that three isozymes of PLA2 may complementarily contribute to the AA production.

AHNAK expression and subcellular localization in gastric surface epithelial cells

We have previously demonstrated the expression of AHNAK protein in normal human organ donor stomach tissues. In order to identify the specific cells that express AHNAK in human stomach, we performed immunohistochemistry. We evaluated the subcellular localization and movement of AHNAK protein by immunohistochemistry, sucrose density gradient centrifugation, and Western blot analysis using specific AHNAK and subcellular marker antibodies. AHNAK immunoreactivity was localized to PAS, Alcian Blue, MUC5A positive gastric surface epithelial cells in normal human organ donor stomachs. Normal rat gastric mucosa (RGM1) cells demonstrated strong cytoplasmic staining for AHNAK. AHNAK protein was found predominately in the cytoplasmic fraction under standard culture conditions of RGM1 cells. AHNAK is often described as a nuclear phosphoprotein that translocates to the plasma membrane where it is involved in signal transduction, cell membrane differentiation, and repair. Further investigation and understanding of the potential recruitment of AHNAK to the plasma membrane may allow for novel strategies to regulate specific signaling cascades and induce membrane repair in gastric surface epithelial cells. This research was supported by an American Heart Association Predoctoral Fellowship and a Veterans Affairs Merit grant.

Bile Acids Induce Cdx2 Expression Through the Farnesoid X Receptor in Gastric Epithelial Cells

Clinical and experimental studies showed that the reflux of bile into the stomach contributes to the induction of intestinal metaplasia of the stomach and gastric carcinogenesis. Caudal-type homeobox 2 (Cdx2) plays a key role in the exhibition of intestinal phenotypes by regulating the expression of intestine-specific genes such as goblet-specific gene mucin 2 (MUC2). We investigated the involvement of the farnesoid X receptor (FXR), a nuclear receptor for bile acids, in the chenodeoxycholic acid (CDCA)-induced expression of Cdx2 and MUC2 in normal rat gastric epithelial cells (RGM-1 cells). RGM-1 cells were treated with CDCA or GW4064, an FXR agonist, in the presence or absence of guggulsterone, an FXR antagonist. CDCA induced dose-dependent expression of Cdx2 and MUC2 at both the mRNA and protein levels. The maximum stimulation of Cdx2 and MUC2 mRNA induced by CDCA was observed at 3 h and by 6 h, respectively. GW4064 also induced expression of these molecules. The effects of CDCA and GW4064 on expression of Cdx2 and MUC2 were abolished by guggulsterone. These findings suggest that bile acids may induce gastric intestinal metaplasia and carcinogenesis through the FXR.

The Expression of Heme Oxygenase-1 Induced by Lansoprazole

Our previous studies have demonstrated that lansoprazole inhibits acute inflammatory reactions as well as intestinal mucosal injuries induced by ischemia-reperfusion or indomethacin administration in rats. Thus, proton pump inhibitors such as lansoprazole have been demonstrated to prevent gastrointestinal mucosal injury by mechanisms independent of acid inhibition. In our in vitro study, lansoprazole induced the expression of heme oxygenase-1 (HO-1) on rat gastric epithelial cells (RGM-1 cells), and exerted anti-inflammatory effect on the dependent of HO-1 expression. Furthermore, NF-E2-related factor-2 (Nrf2) played an important role in HO-1 expression induced by lansoprazole. In this review, we focused on lansoprazole-induced HO-1 expression, its anti-inflammatory action, and the role of Nrf2 in its expression.

Dexamethasone inhibits basic fibroblast growth factor-stimulated gastric epithelial cell proliferation

Basic fibroblast growth factor (bFGF) is essential for gastric ulcer healing, whereas glucocorticoids delay gastric ulcer healing. We found that dexamethasone inhibited bFGF-stimulated rat gastric epithelial RGM-1 cells proliferation and attempted to elucidate the possible mechanistic pathway. Flowcytometry was used to determine cell proliferation. Western blot and RT-PCR were performed to evaluate changes in signaling pathways. Results showed that bFGF significantly increased mRNA expression of FGF receptor (FGFR)1 and FGFR2 at 10 min and increased expression of phosphorylated extracellular signal-regulated kinase (pERK1/pERK2) but not phosphorylated p38 mitogen-activated protein kinase (MAPK) or phosphorylated phosphatidylinositol 3-kinase (PI3K) within 30 min. This was followed by an increase of cyclooxygenase (COX)-2 mRNA and protein expression at 30 and 240 min, respectively. Mitogen-activated protein kinase kinase (MEK) inhibitor-PD98059 (10 −5 M) markedly suppressed bFGF-stimulated COX-2 expression and cell proliferation, but neither p38 MAPK inhibitor-SB203580 nor PI3K inhibitor-Wortmannin had any effect. Dexamethasone (10 −6 M) substantially reduced bFGF-stimulated ERK activation at 10 min, COX-2 mRNA and protein expression at 30 and 240 min, respectively, and prostaglandin E 2 synthesis at 8 h. Dexamethasone (10 −6 M) also significantly decreased mRNA expression of FGFR1 and FGFR2 at basal and bFGF-stimulated conditions at 10 min. This study indicated that bFGF-stimulated gastric epithelial RGM-1 cells proliferation via up-regulating FGFR1 and FGFR2, activating ERK1/ERK2 signal transduction pathway and COX-2 pathway. Dexamethasone significantly suppresses bFGF-stimulated RGM-1 cells proliferation in part via down-regulation of FGFR1/FGFR2, then decreasing bFGF-stimulated activation of ERK1/ERK2, followed by inhibition of COX-2 activation, and finally DNA synthesis.

W1982 Malignant Transformation of Rat Gastric Epithelial RGM-1 Cell Line Induced By Carcinogen N-Methyl-N'-Nitro-N-Nitrosoguanidine (Mnng) Treatment Was Accompanied with the Ectopic Expression of H+,K+-Adenosine Triphosphatase (ATPase)

W1982 Malignant Transformation of Rat Gastric Epithelial RGM-1 Cell Line Induced By Carcinogen N-Methyl-N'-Nitro-N-Nitrosoguanidine (Mnng) Treatment Was Accompanied with the Ectopic Expression of H+,K+-Adenosine Triphosphatase (ATPase)

Cellular membrane fluidity measurement by fluorescence polarization in indomethacin-induced gastric cellular injury in vitro

Gastric complications of indomethacin involve generation of reactive oxygen species, which induce gastric mucosal injury via lipid peroxidation of cell membranes. Peroxidation by reactive oxygen species alters the amounts of unsaturated fatty acids in the cell membrane and thus affects membrane fluidity. Indomethacin-induced lipid peroxidation can thus be detected by measuring cellular membrane fluidity by the fluorescence polarization (FP) method. The aim of this study was to elucidate the usefulness of the FP method for detecting indomethacin-induced gastric cellular injury in RGM-1 cells. Indomethacin-treated RGM-1 cells were investigated by conventional cytotoxicity assay, fluorometry of diphenyl-1-pyrenylphosphine (DPPP) to detect lipid peroxidation, and FP. The effects of both a radical scavenger and an initiator on membrane fluidity change (MFC) in RGM-1 cells were examined. The sensitivity of FP in detecting cellular injury was compared with those of DPPP fluorometry and conventional cytotoxicity measurements. Indomethacin caused an increase in MFC as determined by FP before cytotoxicity was detected by conventional methods. The increase in MFC was associated with increased membrane phospholipid peroxidation (MPP) but not with a prostaglandin deficiency, and the increases in both MFC and MPP were prevented by vitamin E. The FP method is potentially useful for detecting cellular injury in vitro.

Neoplastic transformation and induction of H+,K+-adenosine triphosphatase by N-methyl-N′-nitro-N-nitrosoguanidine in the gastric epithelial RGM-1 cell line

N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) induces gastric cancer in animal models. We established an MNNG-induced mutant of the rat murine RGM-1 gastric epithelial cell line, which we named RGK-1, that could be used as an in vitro model of gastric cancer. This cell line showed signs of neoplasia and transformation, in that it lost contact inhibition and formed tumors in nude mice. The mutant cells also expressed parietal cell-specific H(+),K(+)-adenosine triphosphatase (H(+),K(+)-ATPase), which parent RGM-1 did not. The results suggested that parent RGM-1 cells were gastric progenitor cells. This mutant RGK-1 cell line will contribute to future investigation on gastric carcinogenesis and to the development of other pathophysiologic fields.

Regulation of mammalian gastrin/CCK receptor (CCK2R) expression in vitro and in vivo

The gastrin/CCK receptor (CCK2R) mediates the physiological functions of gastrin in the stomach, including stimulation of acid secretion and cellular proliferation and migration, but little is known about the factors that regulate its expression. We identified endogenous CCK2R expression in several cell lines and used luciferase promoter–reporter constructs to define the minimal promoter required for transcription in human gastric adenocarcinoma, AGS, and rat gastric mucosa, RGM1, cells. Consensus binding sites for SP1, C/EBP and GATA were essential for activity. Following serum withdrawal from RGM1 and AR42J cells, endogenous CCK2R mRNA abundance and the activity of a CCK2R promoter–reporter construct were significantly elevated. Transcription of CCK2R was also increased in AGS-GR and RGM1 cells by gastrin through mechanisms partly dependent upon protein kinase C (PKC) and mitogen/extracellular signal-regulated kinase (MEK). Gastrin significantly increased endogenous CCK2R expression in RGM1 cells, and CCK2R protein expression was elevated in the stomach of hypergastrinaemic animals. In mice with cryoulcers in the acid-secreting mucosa, CCK2R expression increased progressively in the regenerating mucosa adjacent to the ulcer repair margin, evident at 6 days postinjury and maximal at 13 days. De novo expression of CCK2R was observed in the submucosa beneath the repairing ulcer crater 6–9 days postinjury. Many of the cells in mucosa and submucosa that expressed CCK2R in response to cryoinjury were identified as myofibroblasts, since they coexpressed vimentin and smooth muscle α-actin but not desmin. The data suggest that increased CCK2R expression might influence the outcome of epithelial inflammation or injury and that the response may be mediated in part by myofibroblasts.

Involvement of voltage-gated K+ and Na+ channels in gastric epithelial cell migration

Epithelial cell migration plays an important role in gastrointestinal mucosal repair. We previously reported that multiple functional ion channels, including a Ba(2+)-sensitive K(+) inward rectifier K(ir)1.2, 4-aminopyridine (4-AP)-sensitive voltage-gated K(+) channels K(v)1.1, K(v)1.6 and K(v)2.1, and a nifedipine-sensitive, tetrodotoxin (TTX)-insensitive voltage-gated Na(+) channel Na(v)1.5 were expressed in a non-transformed rat gastric epithelial cell line (RGM-1). In the present study, we further investigated whether these ion channels are involved in the modulation of gastric epithelial cell migration. Cell migration was determined by monolayer wound healing assay. Results showed that blockade of K(v) with 4-AP or Na(v)1.5 with nifedipine inhibited RGM-1 cell migration in the absence or presence of epidermal growth factor (EGF), which effectively stimulated RGM-1 cell migration. Moreover, high concentration of TTX mimicked the action of nifedipine, suggesting that the action of nifedipine was mediated through specific blockade of Na(v)1.5. In contrast, inhibition of K(ir)1.2 with Ba(2+), either in basal or EGF-stimulated condition, had no effect on RGM-1 cell migration. In conclusion, the present study demonstrates for the first time that voltage-gated K(+) and Na(+) channels are involved in the modulation of gastric epithelial cell migration.

Impairment by activation of TRPA1 of gastric epithelial restitution in a wound model using RGM1 cell monolayer

We examined the influence of TRPA1 on the epithelial restitution using a rat gastric epithelial cell line RGM1 monolayer. RGM1 cells were inoculated in 24-well plates cultured for 24 hr, and then starved for 24 hr in a culture medium at 37 degrees C under 5 % CO(2) in air. After obtaining a confluent RGM1 cell monolayer, a round artificial wound of constant size was induced in the center of the cell monolayer using a pencil-type mixer with a rotating silicon tip. The repair process was monitored by quantitatively measuring the area of the epithelial wound (cell-free area). Immediately after the wound induction, cells at the edge of wound started to form lamellipodia, migrating toward the center of wound, and by so doing the cell-free area was decreased over time. The addition of icilin, the TRPA1 agonist, suppressed the recovery of the epithelial wound in a concentration-dependent manner. Likewise, another TRPA1 agonist, ally isothiocyanate, also significantly inhibited the wound repair. In addition, the recovery of the epithelial wound was potently inhibited when the ambient temperature was lowered to 17 degrees C, the threshold temperature where TRPA1 is known to be activated. By contrast, the wound healing was not affected by either menthol, the TRPM8 agonist, or capsaicin, the TRPV1 agonist. These results showed for the first time that the activation of TRPA1 inhibited the repair of the epithelial wound in the stomach, probably by the suppression of cell migration, and suggested the involvement of TRPA1 in the mechanism of gastric epithelial restitution.

A protective role of hydrogen sulfide against oxidative stress in rat gastric mucosal epithelium

We investigated effect of hydrogen sulfide (H 2S) on oxidative stress-caused cell death in gastric mucosal epithelial cells. In rat normal gastric epithelial RGM1 cells, NaHS, a H 2S donor, at 1.5 mM strongly suppressed hydrogen peroxide (H 2O 2)-caused cell death, while it slightly augmented the H 2O 2 toxicity at 0.5–1 mM. The protective effect of NaHS was abolished by inhibitors of MEK or JNK, but not of p38 MAP kinase. NaHS at 1.5 mM actually phosphorylated ERK and JNK in RGM1 cells. Glibenclamide, an ATP-sensitive K + ( K ATP + ) channel inhibitor, did not affect the protective effect of NaHS, although mRNAs for K ATP + channel subunits, Kir6.1 and SUR1, were detected in RGM1 cells. In anesthetized rats, oral administration of NaHS protected against gastric mucosal lesion caused by ischemia-reperfusion. These results suggest that NaHS/H 2S may protect gastric mucosal epithelial cells against oxidative stress through stimulation of MAP kinase pathways, a therapeutic dose range being very narrow.