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GASTROINTESTINAL, HEPATIC, PULMONARY, AND RENAL
Department of Surgery, Beth Israel Deaconess Medical Center, Boston, Massachusetts (R.R., E.N., L.M., S.Y., S.J.H.); and Department of Pharmacology and Experimental Therapeutics, Kyoto Pharmaceutical University, Kyoto, Japan (E.N., S.H., K.T.)
Received February 21, 2007; accepted July 2, 2007.
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Abstract |
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In a variety of diverse cell systems, such as Caco-2 cells, skin, endothelial cells, and epithelial cells of the gastrointestinal tract, including the stomach (Lacy and Ito, 1984
; Critchlow et al., 1985; Ito and Lacy, 1985), small intestine (Donowitz and Madara, 1982; Feil et al., 1987; Moore et al., 1989; Riegler et al., 1991), and colon (Feil et al., 1989; Prasad et al., 1997), repair of superficial wounds occurs rapidly and with similar characteristics, whereby viable cells on either side of an injury flatten, spread thin actin-containing lamellipodia and filopodia along the basement membrane, migrate, and ultimately cover the denuded surface with polarized epithelial cells. At least five factors are known to influence restitution after injury, including growth factors (Paimela et al., 1993; Yanaka et al., 1996), Ca2+ (Critchlow et al., 1985), actin (Critchlow et al., 1985), glycolysis (Cheng et al., 2001), and ion transport (Joutsi et al., 1996; Yanaka et al., 2002; Hagen et al., 2004). Ion transport is required to regulate intracellular pH (pHi) in migrating cells after injury, a process that is particularly important in the stomach due to the acidic luminal environment.
It was recently reported that both amiloride, an Na+/H+ exchange blocker, and 4-acetamido-4-isothiocyanatostilbene-2,2'-disulfonic acid (SITS), a bicarbonate transport antagonist, inhibit restitution of the guinea pig gastric mucosa (Joutsi et al., 1996). In the frog, restitution is inhibited by a similar bicarbonate transport antagonist, 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), but ion substitution experiments showed that bicarbonate transport from the nutrient solution per se is not required for restitution to occur (Hagen et al., 2004). These results suggest that SITS and DIDS, in addition to blocking bicarbonate transport, inhibit other pathways important to restitution. To date, the pathway blocked by SITS and DIDS during restitution in the ex vivo gastric mucosa is unknown as is whether it is the isothiocyanate (ITC) group or another aspect of these compounds that inhibits restitution. In addition, it is not known whether blockade of wound repair occurs by SITS and DIDS in cultured gastric cells, results that would implicate the action of SITS and DIDS on epithelial cells rather than on other cells in the tissue.
In the present study, we examined the role of SITS and DIDS in restitution of the mammalian gastric mucosa, ex vivo, and in an in vitro cell wounding model using rat gastric mucosal-1 (RGM1) cells. Because many cellular functions, in addition to bicarbonate transport by the sodium bicarbonate cotransporter (NBC), are blocked by the ITC group on stilbenes such as SITS and DIDS, we explored the role of three important DIDS-inhibitable pathways, including purino (ATP) receptors, barium-sensitive KATP channels such as Kir6.1/SUR6A, and the monocarboxylate transporter MCT-1. Our data suggest that MCT-1, along with NBC, may be the relevant binding sites for DIDS/ITC in gastric surface epithelial cells after injury, and they support the notion that MCT-1 plays an important role in restitution and the maintenance of barrier integrity after injury.
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Materials and Methods |
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).
Ussing Chamber Studies. After anesthesia, the stomach was removed, and it was divided into paired halves by incision of the greater and lesser curvature. The muscularis propria was stripped from corpus mucosa with small scissors and fine forceps under a dissecting microscope. Stripped mucosae were mounted in Lucite Ussing chambers (exposed area of 0.636 cm2) connected to water-jacketed gas-lift reservoirs maintained at 37°C. The serosal side was bathed with buffer containing 147 mM Na+, 5.0 mM K+, 131 mM Cl–, 1.3 mM Mg2+, 1.3 mM 
, 2 mM Ca2+, 25 mM 
, 15 mM HEPES, and 20 mM D-glucose, and it was continuously gassed with 95% O2, 5% CO2. The luminal side was bathed with 150 mM NaCl, gassed with 100% O2, and kept at pH 4.0 with pH-stat device (Radiometer Copenhagen GmbH, Copenhagen, Denmark). Transmucosal electrical resistance (TER) and potential difference were monitored throughout the experiment. After potential difference reached a stable value, injury was induced by exposing the luminal side of tissues to 150 mM NaCl containing 0.5% Triton X-100 for 5 min. Subsequently, the mucosa was washed three times in buffer, and the serosal and luminal solutions were replaced. To determine whether it is the ITC group or other aspects of stilbenes (Fig. 1) that block restitution after injury, tissues were incubated for 3 h in the absence or presence of DIDS, 4,4-diisothiocyanatodihydrostilbene-2,2'-disulfonic acid (H2DIDS), SITS, or 4,4-diinitrostilbene-2,2'-disulfonic acid (DNDS), which were added from a concentrated stock in DMSO. Control tissues were incubated with DMSO alone, and the concentration of DMSO was 0.1% in all experiments. Luminal pH was maintained at 4.0 throughout the experiment by titration with 10 mM HCl or 10 mM NaOH. Restitution was evaluated quantitatively by the recovery of TER after injury. At the end of the experiment, tissues were fixed for light microscopy for 10 min at room temperature in the chamber and then overnight at 4°C in 4.0% phosphate-buffered formalin. Fixed tissues were embedded in paraffin, sectioned, and then stained with hematoxylin and eosin for light microscopy to evaluate restitution in each experiment. All samples were coded, and morphological evaluation was conducted without foreknowledge of their source by four investigators. Each tissue was evaluated from 0 to 5, as follows. A score of 0 indicated that the apical surface of the mucosa was denuded without cell migration from the gastric pits (no restitution). A score of 1 indicated that less than 25% of the apical surface was covered with flattened surface epithelial cells. A score of 2 indicated that 25 to 50% of the apical surface was covered with flattened surface epithelial cells. A score of 3 indicated that 50 to 75% of the apical surface was covered with flattened epithelial cells. A score of 4 indicated that 100% of the apical surface was covered with flattened epithelial cells. Finally, a score of 5 indicated that 100% of the apical surface was covered by cuboidal or columnar, rather than flattened, epithelial cells. After analysis by each investigator, the code was broken, and the results were analyzed as described below.
RGM1 Cell Culture. RGM1 cells, established by Dr. H. Matsui [Institute of Physical and Chemical Science (RIKEN) Cell Bank and Institute of Clinical Medicine, University of Tsukuba, Tsukuba, Japan] (Kobayashi et al., 1996), are nontransformed gastric surface epithelial cells. RGM1 cells were cultured in DMEM/F-12 (1:1) supplemented with heat-inactivated 10% fetal bovine serum (Invitrogen, Carlsbad, CA), 100 U/ml penicillin, 100 U/ml streptomycin, and 0.25 µg/ml amphotericin B. Confluent monolayers of RGM1 cells were starved for 24 h in culture medium without fetal bovine serum (DMEM/F-12 containing 15 mM HEPES, pH 7.4) at 37°C under 5% CO2 in air, and then they were used as a confluent monolayer for experiments. All experiments were performed at pH 7.4 in the presence or absence of reagent(s).
Recovery of a Round Wound Induced in RGM1 Cell Monolayers. A round artificial wound was induced in the center of confluent RGM1 cell monolayers by using a rotating silicon tip driven by a pencil-type mixer (Iuchi, Osaka, Japan), resulting in a cell-free area of uniform size after wounding. The monolayer was then washed with standard (STD) buffer, containing 147 mM Na+, 5.0 mM K+, 131 mM Cl–, 1.3 mM Mg2+, 1.3 mM , 2 mM Ca2+, 25 mM , 15 mM HEPES, and 20 mM D-glucose, and reagents were then added in STD buffer or in ion-substituted buffer to the wells. To determine whether the ITC group or other aspects of stilbenes block the recovery of wounds, 3 to 300 µM DIDS or H2DIDS or 3 to 500 µM SITS or DNDS was solubilized in DMSO and added so that the final concentration of DMSO did not exceed 0.1%. To determine the role of bicarbonate in the recovery of wounds, mannitol was substituted for bicarbonate in the STD buffer. To evaluate the role of purinoreceptors in wound repair, wounded RGM1 cell monolayers were incubated with 1 mM ATP or 1 mM of the nonhydrolyzable analogs of ATP 
,
-methyl-ATP (,-meATP) or 2-methylthio-ATP (2-meSATP) to determine whether ATP accelerates wound repair. Furthermore, 10 to 300 µM suramin was used to determine whether blockade of purinoreceptors inhibits wound repair. To determine whether barium-sensitive KATP channels such as Kir6.1/SUR6A are involved in wound repair, injured monolayers were incubated with 0.1 to 5 mM barium chloride (BaCl2) in sulfate-free buffer that contained 160 mM Na+, 5.0 mM K+, 143 mM Cl–, 1.3 mM Mg2+, 2 mM Ca2+, 25 mM , 15 mM HEPES, and 20 mM D-glucose. Barium chloride precipitated in STD buffer, so sulfate-free buffer was used to ensure that the barium remained in solution. Control STD buffer, to balance osmolarity, contained an additional 23.7 mM mannitol in experiments with barium. To evaluate the role of MCT in wound recovery, phloretin was used at 30 and 60 µM. Phloretin also blocks glucose transport; so, to determine the role of glucose transport in wound repair, phloretin was added in both STD buffer and in glucose-free buffer, where mannitol was substituted for glucose. Closure of the wound was monitored at 4 and 8 h with a Nikon TE300 microscope (MicroVideo Instruments, Avon, MA) outfitted with an Orca charge-coupled device camera (Hamamatsu Photonics, Hamamatsu City, Japan). Change in the cell-free area was quantified using IPLab software (Scanalytics, Fairfax, VA).
Determination of Cell Viability. The viability of RGM1 cells was evaluated by a colorimetric assay using crystal violet, a cytochemical stain that binds to chromatin. For this assay, RGM1 cells were washed with phosphate-buffered saline, fixed with methanol, and then air-dried. The dried cells were stained with 1% crystal violet, washed in tap water, and then air-dried. Stained cells were solubilized with 0.5% SDS, and the absorbance was measured at 590 nm using a kinetic microplate reader (Molecular Devices, Sunnyvale, CA).
Analysis of MCT Expression by Reverse Transcription-Polymerase Chain Reaction. RGM1 cells were grown to confluence, and then they were isolated from the culture plate by using 0.1% trypsin in 0.5 mM EDTA at 37°C for 10 min. Isolated cells were pelleted and frozen at –80°C before use. Total RNA from the thawed cells was extracted with the single-step acid phenol-chloroform extraction procedure using TRIzol (Invitrogen). Total RNA was reverse-transcribed with SuperScript preamplification (Invitrogen). The sequences for sense and antisense primers, obtained from GenBank, for 1) rat MCT1 (D63834 [GenBank] ) were 5'-GTCTACGACCTATGTTGGG-3' and 5'-CCTCCGCTTTCTGTTCT-3' to obtain a 394-bp product; 2) rat MCT2 (X97445 [GenBank] ) were 5'-GGGGCTGGGTTGTAGT-3' and 5'-GACGGTGAGGTAAGTTCTA-3' to obtain a 367-bp product; 3) rat MCT3 (AF059258 [GenBank] ) were 5'-CGCTGCTCTAAGAACATCTCATC-3' and 5'-TCTGGCCTCGTGCCTCAT-3' to obtain a 427-bp product; and 4) rat MCT4 (U87627 [GenBank] ) were 5'-GGCAGTCCCGTGTTCCTTT-3' and 5'-GCACCTTCTTGAGCCCTGTTAT-3' to obtain a 369-bp product.
Reagents. H2DIDS was purchased from Invitrogen. All other chemicals, including DIDS, SITS, and DNDS, were purchased from Sigma-Aldrich (St. Louis, MO), unless noted otherwise.
Statistical Analysis. In RGM1 cell experiments, the data represent means ± S.E. for two wells from three different experiments. In Ussing chamber experiments, the data represent means ± S.E. obtained from at least six experiments. Statistical differences were evaluated by analysis of variance, Dunnett's multiple comparison test, and Student's t test using SigmaPlot (SPSS Inc., Chicago, IL). Significance of the data is denoted as *, P < 0.05, **, P < 0.01, and ***, P < 0.001 compared with tim-ematched control samples or as 
, P < 0.05, , P < 0.01, and , P < 0.001 compared with time-matched samples from another treatment.
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Effects of Various Stilbene Compounds and Bicarbonate Transport Inhibitors on Wound Repair in the in Vitro Rat Gastric RGM1 Cell Model. To determine whether the stilbene compounds DIDS, H2DIDS, and SITS inhibit wound repair in a reductionist model of injury and repair, RGM1 cells were used (Fig. 4). Consistent with results from the guinea pig ex vivo model in Fig. 2, DIDS (Fig. 4A) and H2DIDS (Fig. 4B), stilbene compounds with two ITC groups, dose-dependently inhibited wound repair with a maximal effect at 300 µM. Using the same concentration curve, from 3 to 300 µM, wound repair was dose-dependently inhibited by approximately 50% with SITS (Fig. 4C), a stilbene with one ITC group. Even at 500 µM SITS, there was not complete inhibition of wound repair (Fig. 4C). DNDS, a stilbene with no ITC groups (Fig. 4D), did not inhibit wound repair in a dose-dependent manner. Instead, there was some inhibition of wound repair at all doses tested, which reached significance at 300 to 500 µM compared with controls (Fig. 4D). As shown in Fig. 2B, the stilbene compounds tested did not reduce the viability of RGM1 cells when examined 8 h after wounding, so the results cannot be attributed to toxicity of drugs at the concentrations used for this study. In the RGM1 cell model, bicarbonate-free conditions resulted in a significant but modest inhibition of wound repair (See Fig. 6).
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Role of Purino (ATP) Receptors and KATP Channels on Wound Repair in the in Vitro RGM1 Cell Model. ATP binding to purinoreceptors and the activity of KATP channels such as Kir6.1/SUR6A are both known to be inhibited by DIDS (Furukawa et al., 1993; Bültmann and Starke, 1994; Proks et al., 2001). To determine whether these pathways are involved in wound repair in the RGM1 cell model, we incubated RGM1 cells with ATP and analogs of ATP, or with barium, a nonspecific antagonist of Kir6.1/SUR6A channels (Fig. 5). ATP and nonhydrolyzable analogs of ATP, such as ,-methyl-ATP and 2-methylthio-ATP would be expected to bind P2-type purinoreceptors on the cell surface (Bültmann and Starke, 1994; Suzuki et al., 2000) and to accelerate the rate of wound repair (Dignass et al., 1998; Chen et al., 2006). Figure 5A shows that wound repair in RGM1 cells is unaffected by ATP. Likewise, barium should inhibit wound repair if Kir6.1/SUR6A channels are required, even if many other K+ channels are also blocked by barium. Sulfate-free conditions were used to maintain the solubility of barium, and this condition did not reduce cell viability as long as the barium concentration was 5 mM or below (data not shown). Sulfate-free conditions alone did not inhibit wound repair nor did sulfate-free conditions with 0.1 to 0.5 mM barium (Fig. 5B). Higher doses of barium, including 1 to 5 mM, resulted in a modest but significant inhibition of wound repair (Fig. 5B).
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; Halestrap and Price, 1999; Halestrap and Meredith, 2004). To determine whether monocarboxylate transport pathways are involved in wound repair in the RGM1 cell model, we incubated RGM1 cells with phloretin, which potently and dose-dependently inhibited wound repair (Fig. 6A) without affecting the viability of RGM1 cells (Fig. 6B). Because DIDS would block both NBC and MCT in live cells, we also determined whether inhibition of wound repair would be greater if both transporters were blocked simultaneously by using phloretin and bicarbonate-free conditions (Fig. 6A). Phloretin (60 µM) in combination with bicarbonate-free buffer resulted in complete inhibition of wound repair as occurs with DIDS (compare Fig. 6A with Fig. 4A). Like with phloretin alone, there was no reduction in viability with phloretin in bicarbonate-free buffer (Fig. 6B). Because phloretin blocks not only MCT but also glucose transporters such as GLUT1, we tested whether inhibition of glucose transport in the RGM1 cell model would inhibit wound repair (Fig. 7). Although there was a significant but modest slowing of wound repair in glucose-free buffer, there was no difference in the rate of wound repair with or without phloretin that could account for the results with phloretin alone (Fig. 7A). Like before, phloretin in glucose-free buffer did not affect the viability of RGM1 cells (Fig. 7B).
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To determine whether RGM1 cells have MCT transporters, reverse transcription-polymerase chain reaction was performed (Fig. 8). Using primers specific for rat MCTs that are published in GenBank, our results show that the message for MCT-1 was expressed in RGM1 cells (Fig. 8).
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; Hagen et al., 2004), it was unclear whether blockade was by the ITC "R" group or due to other characteristics of the molecule. Here, we provide evidence that ITC groups dose-dependently block restitution and wound repair of the gastric mucosa with an efficacy of DIDS = H2DIDS (two ITC) > SITS (one ITC). DNDS, with no ITC groups but an otherwise similar structure, has little effect on wound repair or on restitution, suggesting that the backbone of stilbene compounds contributes little to their overall inhibitory properties. These results are important to consider when designing new therapies to treat diseases of the stomach and perhaps of the intestine and colon, where mechanisms that regulate restitution are similar. In particular, ITC-containing compounds from cruciferous vegetables, such as sulforaphane from broccoli and broccoli sprouts, have been proposed to treat H. pylori infection of the stomach (Fahey et al., 2002; Haristoy et al., 2003; Galan et al., 2004), or sinigrin from Brussels sprouts to inhibit carcinogenesis in the colon (Bonnesen et al., 2001; Smith et al., 2003, 2004). Although efficacious in vitro, ITC-containing compounds may inhibit important repair processes in vivo to affect mucosal integrity. To support this idea, chronic atrophic gastritis was shown to be twice as high in subjects who ate broccoli once or more weekly compared with those who consumed a negligible amount of this vegetable (Sato et al., 2004).
Elucidating the mechanism by which ITC-containing compounds inhibit wound repair is complicated by the fact that many transporters and receptors avidly bind ITC groups. In restitution, the first obvious ITC-binding transporter that may be affected is NBC (Fig. 9), which is known to regulate pHi and be potently inhibited by DIDS, H2DIDS, and SITS. However, previous studies in the frog gastric mucosa demonstrated that bicarbonate-free conditions, which would inhibit bicarbonate transport by NBC, did not affect restitution (Hagen et al., 2004). Using cultured RGM1 cells in the present study, this result was confirmed, suggesting that transporters other than or in addition to NBC are targets of the ITC group on DIDS, H2DIDS, or SITS during restitution and wound repair.
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). Purinoreceptors facilitate cell signaling after binding extracellular ATP, and they are known to accelerate wound repair in intestinal IEC-6 cells (Dignass et al., 1998) and chemotaxis in neutrophils (Chen et al., 2006). As such, we incubated RGM1 cells with either ATP or nonhydrolyzable analogs of ATP, each of which binds to purinoreceptors, and we found that exogenous ATP does not accelerate wound repair in RGM1 cells. To verify this negative finding, we also blocked purinoreceptors with suramin, and we saw no effect on the rate of wound repair, or on viability, in RGM1 cells (data not shown). Because ATP did not accelerate nor did blockade of purinoreceptors inhibit wound repair in our study, we conclude that P2-type purinoreceptors are probably not the target of DIDS, H2DIDS, or SITS. Likewise, KATP channels such as Kir6.1/SUR2A have been shown to regulate cell migration, and they are blocked by DIDS and by barium. Because our data showed that barium has only a minimal effect on wound repair, we conclude that Kir6.1/SUR2A K+ channels are also not the major target of DIDS, H2DIDS, or SITS in this study. However, our results suggest that one or more barium-sensitive K+ channels are involved in wound repair in RGM1 cells, and further studies would be required to investigate this interesting finding.
The remaining transport mechanism to test was MCT, which has been shown to be potently and irreversibly inhibited by the ITC group on DIDS. MCT is a protein catalyzed proton-linked pyruvate and lactate transporter that was initially called MEV because of its mevalonate transport properties, but it was cloned, sequenced, and renamed MCT-1 (Garcia et al., 1994; Wang et al., 1994). In the stomach, MCT-1 is localized exclusively at the basolateral surface of gastric surface epithelial cells (Garcia et al., 1994), but no function for it has been elucidated. MCT-1 is a potent H+ and monocarboxylate cotransporter that transports lactate and H+ against a concentration gradient, especially during glycolysis when lactate efflux is required (Poole et al., 1993; Halestrap and Meredith, 2004). MCT is blocked not only by 4,4'-substituted stilbene-2,2'-disulfonates such as DIDS but also by bulky or aromatic monocarboxylates such as -cyano-4-hydroxycinnimate and by amphiphilic compounds such as quercetin and phloretin (Poole et al., 1993; Halestrap and Meredith, 2004). We determined that phloretin is a good MCT antagonist for RGM1 cell studies, because it did not kill cells in STD buffer or in bicarbonate-free conditions. We also demonstrated that, like in native surface cells, MCT-1 is present in RGM1 cells.
In the present work, we showed that 60 µM phloretin inhibited wound repair in RGM1 cells by approximately 60%. In liver cells, 60 µM phloretin inhibited lactate/H+ transport by approximately 85% (Jackson and Halestrp, 1996), which may explain why wound repair was not blocked completely by phloretin in our experiments. Although ITC groups on DIDS, H2DIDS, and SITS can inhibit MCT-1 activity, they can also bind to NBC, suggesting that two pathways could be blocked simultaneously to affect wound repair and restitution in live cell experiments (Fig. 9). Our data suggest that this is the case, because blocking MCT-1 with phloretin and NBC with bicarbonate-free buffer completely inhibits wound repair in RGM1 cells, and these results are exactly the same as with DIDS. The mechanism by which this inhibition occurs is largely unknown, but it may be related to the role MCT-1 plays in H+/lactate transport (export) during glycolysis, which is suggested to be the main energy source for cell migration during restitution (Cheng et al., 2001). Alternatively, both NBC and MCT-1 transporters may be involved in the maintenance of pHi. Further experiments will be required to test these two interesting possibilities in the RGM1 cell-wounding model.
Although phloretin is commonly used to inhibit MCT-1 activity, it is a nonspecific drug, and it can block a number of other pathways that may be important for wound repair. First, phloretin is a potent inhibitor of protein kinase C (PKC), and it has been used to study PKC-dependent pathways. However, when used to block PKC activation, it was determined that 200 to 250 µM phloretin is required (Emeljanova et al., 2002), which is far above the highest concentration used in the current study. Although unlikely, we cannot exclude that phloretin inhibits PKC activation to some extent in our study, and further experiments would be required to test this condition in the RGM1 wounding model. Another target of phloretin is glucose transport, where small concentrations of phloretin inhibit GLUT1 and GLUT2 activity (Hersey et al., 1982; Kellett and Helliwell, 2000; Walker et al., 2005). Therefore, we tested the role of glucose transport in wound repair, and we showed that although glucose regulates the rate of wound repair in RGM1 cells, blockade of this process is not significant enough to explain the results obtained with phloretin. These results are consistent with studies in the ex vivo frog gastric mucosa where it was suggested that gastric cells can use internal glucose stores to drive glycolysis in the absence of extracellular glucose transport for at least 24 h (Hagen et al., 2004).
In conclusion, the results presented here demonstrate that the ITC group(s) on stilbenes such as DIDS, H2DIDS, and SITS block restitution and wound repair in a dose-dependent manner, and they suggest they do so by binding to both NBC and MCT-1 on gastric surface cells (Fig. 9). The precise mechanism of inhibition is yet to be defined, but it is worthy of further investigation in light of the importance of restitution for the maintenance of mucosal barrier integrity. The results presented here also suggest that new drug therapies that include ITC derivatives must take into account the important pathways they block in live cell systems, including mucosal repair after injury.
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Footnotes |
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: pHi, intracellular pH; SITS, 4-acetamido-4-isothiocyanatostilbene-2,2'-disulfonic acid; DIDS, 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid; ITC, isothiocyanate; RGM1, rat gastric mucosal-1; NBC, sodium bicarbonate cotransporter; MCT, monocarboxylate transporter; TER, transmucosal electrical resistance; H2DIDS, 4,4-diisothiocyanatodihydrostilbene-2,2'-disulfonic acid; DNDS, 4,4-diinitrostilbene-2,2'-disulfonic acid; DMSO, dimethyl sulfoxide; DMEM, Dulbecco's modified Eagle's medium; STD, standard buffer; ,-meATP, ,-methyl-ATP; 2-MeSATP, 2-methylthio-ATP; SUR, sulfonylurea receptor; bp, base pairs; GLUT, glucose transporter; PKC, protein kinase C.
Address correspondence to: Dr. Susan J. Hagen, Department of Surgery, E/DA-805, Beth Israel Deaconess Medical Center, 330 Brookline Ave., Boston, MA 02215. E-mail: shagen{at}bidmc.harvard.edu
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)-free. Values represent means ± S.E. of two wells from three different experiments, and they are expressed as percentage of the initial wounded area. **, P < 0.01 compared with the STD buffer control containing 
