EGF promotes gastric mucosal restitution by activating Na+/H+ exchange of epithelial cells

Akinori Yanaka, Hideo Suzuki, Takeshi Shibahara, Hirofumi Matsui, Akira Nakahara, and Naomi Tanaka

Departments of Gastroenterology and Endoscopy, Institute of Clinical Medicine, University of Tsukuba, Tsukuba, Ibaraki 305-8575, Japan


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

This study was conducted to determine whether the contributions of epidermal growth factor (EGF) to gastric mucosal restitution after injury are mediated by stimulation of Na+/H+ exchangers in surface mucous cells (SMC). Intact sheets of guinea pig gastric mucosae were incubated in vitro. Intracellular pH (pHi) in SMC was measured fluorometrically, using 2',7'- bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein. Restitution after Triton X-100-induced injury was evaluated by recovery of electrical resistance. At neutral luminal pH, exogenous EGF (ex-EGF) increased pHi and enhanced restitution in the absence but not in the presence of serosal HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>. During exposure to luminal acid, ex-EGF not only prevented intracellular acidosis but also promoted restitution. These effects of ex-EGF were blocked by serosal amiloride or anti-EGF-receptor antibody. In the absence of ex-EGF, restitution was inhibited by replacement of luminal and serosal solutions with fresh solutions and was blocked more completely by serosal anti-EGF-receptor antibody. These results suggest that both endogenous and ex-EGF contribute to restitution via basolateral EGF receptors, with effects mediated, at least in part, by stimulation of basolateral Na+/H+ exchangers.

epidermal growth factor receptor; electrical resistance; 3H-labeled mannitol flux; intracellular pH; luminal acid


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

IT HAS BEEN GENERALLY ACCEPTED that epidermal growth factor (EGF) plays an important role in protection and repair of gastrointestinal mucosae (3, 9, 12, 19, 23, 29, 30). EGF is produced not only by salivary glands (23) but also by gastric mucosa (44). EGF receptors are present on the epithelial cells in the gastrointestinal tract (7, 18, 21, 27, 34, 40). Recent studies have shown that the production of EGF (15, 44) and expression of EGF receptors (39) increase after injury, suggesting an important role of endogenous EGF in mucosal repair after injury. The precise distribution of EGF receptors, however, is controversial, because some investigators have shown that EGF receptors are preferentially located on the basolateral membrane (18, 21, 27, 34), whereas others have reported that EGF receptors are present on the apical membrane of surface mucous cells (SMC) (7, 40).

Exfoliation of damaged and nonviable aged epithelial cells from the luminal surface of gastrointestinal mucosae is a daily event. Excessive loss of damaged epithelial cells from the luminal surface may result in mucosal erosions or ulcers. However, superficial wounds of the gastrointestinal mucosa undergo rapid repair, a process termed restitution (11). Restitution is characterized by rapid migration of surviving epithelial cells to restore epithelial continuity within minutes to hours without cell proliferation (11). EGF has been shown to enhance mucosal restitution in rabbit esophagus (12), rat stomach (19), rabbit duodenum (29), and human colon (30). The precise mechanism by which EGF contributes to gastric mucosal restitution, however, has not been well characterized.

One of the major actions of EGF, which appears to be related to mucosal protection or repair, is stimulation of the Na+/H+ exchanger (NHE) (10, 32, 33). Recent studies in gastric mucosae have demonstrated that NHEs are preferentially located on the basolateral but not on the apical membrane of gastric epithelial cells (14, 25).

It has been reported that EGF stimulates activity of NHE partially through increased phosphorylation of the NHE domain (32, 33). Other mechanisms have also been implicated (8, 42, 43). For example, EGF increases the activity of the NHE present on the plasma membrane by stimulating vesicle transport (8) and by interacting with the regulatory domain of the NHE, thus upregulating the H+ sensor of the NHE (42, 43).

We propose that growth factor-induced activation of NHE is particularly important in the protection and repair of gastric mucosa both in the presence and absence of luminal acid for several reasons. First, in the presence of luminal acid, EGF-induced activation of NHE contributes to protection of gastric mucosa by prevention of intracellular acidosis of gastric epithelial cells (9), whereas in the absence of exogenous EGF, gastric mucosal restitution is inhibited by relatively low concentrations of luminal acid (38). On the basis of these findings, we hypothesize that EGF-induced activation of NHE prevents intracellular acidosis of the migrating epithelial cells, thereby preserving the ability of these cells to restore epithelial continuity. Second, activation of NHE by EGF has been shown to enhance cell motility in the absence of acid (2, 31), and blockade of NHE by amiloride inhibits gastric mucosal restitution even in the absence of luminal acid (13). Although these prior studies have not specifically addressed the interaction among EGF, NHE, and cell migration, they suggest that EGF-induced activation of NHE plays an important role in epithelial cell migration during gastric mucosal restitution both in the presence or absence of acid.

Several types of NHE isoforms, such as NHE-1, -2, -3, and -4, are present in the gastrointestinal tract (24). It has been suggested that NHE-1, known to be present in virtually all of the cell types in gastrointestinal mucosa, including those of the gastric fundus and antrum, contributes to maintenance of neutral intracellular pH (pHi) when the cells are exposed to an acidic ambient pH. In contrast, NHE-2, identified only in parietal cells (1), contributes to acid secretion (35). NHE-3, which plays a role in absorption of NaCl (47), is expressed mainly in intestine but less in stomach. NHE-4, known to be expressed only in stomach and kidney, regulates cell volume when the cells are exposed to hyperosmolarity (26). At present, the effect of EGF on the different isoforms of NHE in gastric mucosa is not clear. Some studies have shown that EGF stimulates NHE-1 (32, 33), whereas others have reported that EGF mainly stimulates NHE-2 in rat gastric surface mucous cells (9).

On the basis of this information, we hypothesize that in injured gastric mucosa, EGF in the gastric lumen gains access to the EGF receptors on the basolateral membrane of the gastric epithelial cells and stimulates the basolateral NHE of these cells. We propose that stimulation of the NHE by EGF not only enhances cell motility but also prevents intracellular acidosis of gastric epithelial cells during exposure to luminal acid, thereby promoting gastric mucosal restitution. The purposes of the present study were 1) to determine whether the EGF receptors are located on the basolateral or the apical membrane of the gastric epithelial cells in guinea pig gastric mucosa and 2) to determine whether EGF contributes to mucosal restitution by stimulation of NHE.

Our results clearly show that in injured gastric mucosae, EGF contributes to restitution by binding with the basolateral EGF receptors of the gastric epithelial cells and that the effects of EGF on restitution are mediated, at least in part, by stimulation of the basolateral NHE in the gastric epithelial cells both in the presence or absence of luminal acid.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Preparation of Intact Sheets of Guinea Pig Gastric Fundic Mucosa

Male guinea pigs (250-300 g, Hartley strain) fasted for 24 h were killed under anesthesia induced by intraperitoneal injection of ketamine hydrochloride (Ketalar, Sankyo Yell Pharmaceutical Products, Tokyo, Japan). The stomachs were removed and divided into paired halves by incising the greater and lesser curvatures. The muscularis propria was stripped from the mucosa under a dissecting microscope using small scissors and fine forceps. These protocols have been approved by the Committee on Care and Use of Laboratory Animals at the Univ. of Tsukuba, and all of the animals used for this study were treated in accordance with the international guidelines for animal use.

Electrophysiology

The mucosae were mounted in lucite Ussing chambers with an exposed area of 1.15 cm2. The chambers were connected to water-jacketed, gas-lift reservoirs maintained at 37°C, as described previously (46). The serosal side of the mucosae was bathed with a Ringer solution buffered with HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>/95% O2-5% CO2 (pH 7.4) or with HEPES/100% O2 (see Solutions). The luminal side was bathed with 150 mM NaCl/100% O2 (see Solutions). Transmucosal potential difference (PD) and electrical resistance (R) were monitored. R was calculated from the change in PD induced by passing 100 µA of electrical current from the luminal to the serosal side for 0.5 s every 5 min. H+ secretion of the tissue was inhibited in all tissues by pretreatment on the serosal side with 0.1 mM omeprazole for 1 h before the experiment, which resulted in total inhibition of H+ secretion for 6 h after removal of the omeprazole.

3H-Labeled Mannitol Flux Study

Mucosal permeability was assessed by measuring 3H-labeled mannitol flux (MF) from the serosal to the luminal solution, because the recovery of MF after injury has been shown to correlate well with the degree of restitution (20). In brief, omeprazole-treated fundic mucosae were incubated in Ussing chambers under open-circuited conditions. 3H-labeled mannitol (5 µCi) was added to the serosal solution. One-milliliter aliquots were collected from the luminal bath at 15-min intervals throughout the experiments. The entire luminal solution was replaced with well-oxygenated fresh luminal solutions at each 15-min interval immediately after collecting the 1-ml aliquot. Unlabeled 5 mM mannitol was present in both the serosal and the luminal solutions throughout the experiment to eliminate a solute gradient between the two sides. 3H-labeled MF at each 15-min period was evaluated from the radioactivity of 3H detected in each sample.

Morphology

Gastric mucosal specimens for light microscopy were fixed after the electrophysiological experiments by immersion in 4% buffered formalin for ~3-4 h at room temperature, washed in phosphate-buffered saline overnight, and stored in 70% ethanol. Fixed tissues were embedded in paraffin and sectioned at 4 µm. Sections were stained with hematoxilin and eosin for analysis of histology. These samples were coded, and the morphology was evaluated in a blind fashion. (see Experimental Protocols for Restitution Study).

Measurement of pHi in Surface Mucous Cells in Intact Sheets of Gastric Mucosa

pHi in SMCs in intact sheets of gastric mucosae was measured fluorometrically using the pH-sensitive fluorescent dye 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF), as described by Carter et al (5). In brief, perfusion of the luminal surface of the guinea pig gastric mucosae with 10 µM BCECF-AM for 1 h resulted in selective staining of SMC with BCECF in antral mucosa. Because BCECF loaded mainly in the oxyntic cells in fundic mucosae, we used antral mucosae to measure pHi in SMC in this study. Measurement of pHi was performed using a fluorometer after mounting the BCECF-loaded antral mucosa in a specially designed cuvette that enables separate perfusion of the luminal and the serosal sides of the mucosa. Careful examination of frozen sections of the BCECF-loaded mucosae showed that the SMC had a strong fluorescent signal, whereas the mucus gel just adjacent to the luminal surface of the mucosae had a weak signal. To exclude this extracellular fluorescent signal, we kept luminal pH (pHL) constant at 5.0 during measurement of pHi, because the BCECF-derived signal has been shown to become negligible at pH <5.5 (25). We confirmed that pHi in SMC in our mucosal preparations does not change by decreasing pHL from 7.0 to 5.0 (data not shown).

With the use of this system, we examined the effects of exogenous EGF on pHi. Human recombinant EGF (~0.1-100 ng/ml) was placed in either the luminal or the serosal solution. Anti-EGF receptor antibody (10 µg/ml) and amiloride (1 mM) were added to the serosal solution. We found that addition of EGF (~0.1-100 ng/ml), anti-EGF receptor antibody (~0.1-10 µg/ml), amiloride (~0.03-3 mM), or Hoechst 642 (~0.03-3 µM) to the serosal solution did not affect PD, R, or lactate dehydrogenase (LDH) release into the solution (data not shown).

Measurement of EGF Release and EGF Content in the Gastric Mucosa

Release of EGF into the luminal or the serosal solutions was evaluated as follows. The gastric mucosa was incubated in Ussing chambers under standard conditions for 2 h before injury and for 6 h after injury. The luminal and the serosal solutions were replaced with fresh solutions every 2 h. One-milliliter aliquots of the luminal and the serosal solutions were collected before replacement of the solutions with the fresh solution to determine the amounts of the endogenous EGF released from the mucosae. Levels of EGF were measured by an EGF ELISA kit (R&D Systems, Cambridge, MA).

Experimental Protocols for Restitution Study

All of the tissues mounted in Ussing chambers were incubated for 2 h until PD and R were stable. Then, the tissues were incubated for 2 h without treatment or with the following agents: human recombinant EGF, anti-EGF receptor antibody, or amiloride. These agents were placed in either the luminal or the serosal solution or both, depending on the experimental conditions, and were present throughout the remaining period of the experiments. After 1 h with or without treatment, the luminal side of the mucosa was exposed to 0.5% Triton X-100 for 5 min. This treatment induced rapid and marked decreases in PD and R accompanied by a reciprocal increase in MF. Histology of the Triton X-100-treated mucosae showed superficial injury of gastric mucosa (see Fig. 5A). After the luminal solution was replaced with fresh luminal solution, the tissues were incubated for 6 h to monitor the PD, R, and MF in the continuing presence of the experimental agents. Restitution was quantitatively evaluated by the recovery of PD and R and/or the changes in MF after the injury, as described in several reports (13, 20, 30, 38, 46). Restitution was also evaluated morphologically using photomicrographs of the mucosae. Morphometrical analysis was conducted by two investigators who were not aware of the experimental conditions. The surface areas with or without epithelial cells in each mucosa were measured using an image processing and analysis program (NIH Image). Quantitative estimation of the degree of restitution was expressed as the percentage of the surface area covered with epithelial cells. We examined the effects of EGF on restitution in the presence or absence of serosal HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>. pHL was kept constant at either 7.0 or 3.0, because our previous studies showed that restitution in guinea pig gastric mucosae is inhibited at pHL 3.0 (46). Exogenous EGF was added to either the luminal or the serosal solution. Anti-EGF receptor antibody, amiloride, and Hoechst 642 were placed in the serosal solution.

Chemicals

Human recombinant EGF was purchased from Biological Research (Lake Placid, NY). Mouse anti-human EGF receptor antibody, purchased from Upstate Biotechnology (Lake Placid, NY), was used to inhibit the EGF receptor, because this agent has been shown to inhibit a variety of biological effects of EGF on human endothelial cells (36). 4-Isopropyl-3-methylsulfonylbenzoyl-guanidine methanesulfonate (Hoechst 642) was obtained from Hoechst Marrion Roussel (Tokyo, Japan). Omeprazole and amiloride were from Sigma Chemical (St. Louis, MO). All other reagents were from Wako Pure Chemical (Osaka, Japan).

Solutions

The serosal solution contained (in mM) 122 Na+, 5.0 K+, 1.8 Ca2+, 0.8 Mg2+, 130 Cl-, 25 HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>, 0.8 H2PO<UP><SUB>4</SUB><SUP>−</SUP></UP>, 10.0 glucose (total calculated osmolarity was 300 mosM); pH in the serosal solution was kept between 7.35 and 7.45 by gassing with 95% O2 and 5% CO2 throughout the experiment. In some experiments, 25 mM HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> in the serosal solution was replaced with 25 mM HEPES gassed with 100% O2. pH in the HEPES-buffered solution was adjusted to 7.40 by addition of HCl and/or NaOH. The luminal solution contained 150 mM Na+ and 150 mM Cl- (total calculated osmolarity was 300 mosM) and was gassed with 100% O2.

Statistical Analysis

Student's paired or unpaired t-tests were used for the comparison of paired or unpaired values, respectively. P < 0.05 was considered to be significant. Results were expressed as means ± SE.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Changes in Release and Content of Gastric Mucosal Endogenous EGF After Injury

This series of experiments was conducted to determine whether endogenous EGF exists in intact sheets of mucosal preparations and to examine whether the generation of EGF increases after injury. Endogenous EGF (~0.3-0.7 ng/ml) was detected in both the luminal and the serosal solutions during the 2-h incubation period before the injury (Fig. 1). The release of endogenous EGF into both the luminal and the serosal solutions increased immediately after the injury and decreased gradually over 4-6 h (Fig. 1). Hereafter, EGF released from exfoliated cells and from the tissue is referred to as endogenous EGF.


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Fig. 1.   Release of endogenous epidermal growth factor (EGF) by gastric mucosae into luminal and serosal solutions after Triton X-100 injury. The serosal and the luminal sides of the omeprazole-pretreated gastric fundic mucosae were incubated with 25 mM HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>/95% O2-5% CO2 (pH 7.4) and with 150 mM NaCl/100% O2 (pH ~7.0), respectively. Release of endogenous EGF into luminal and serosal solutions was estimated by measuring EGF concentration in each solution. Luminal pH (pHL) was kept constant at 7.0 throughout. Data are expressed as means ± SE; n, no. of experiments; * P < 0.05; ** P < 0.01, significantly greater than the corresponding values before injury.

Effects of Endogenous or Exogenous EGF on pHi in SMC in Uninjured Mucosa

Human recombinant EGF used in all of the present studies and added to our systems is hereafter referred to as exogenous EGF. This series of experiments was conducted 1) to examine the effects of endogenous and/or exogenous EGF on pHi in SMC and 2) to determine whether the effects of EGF on pHi are related to stimulation of the NHE. In all of the experiments, the serosal side was buffered with HEPES/100% O2.

Effects of exogenous EGF, anti-EGF-receptor antibody, and inhibitors of NHE on baseline pHi in SMC. Exogenous EGF in the serosal solution (~0.3-10 ng/ml) dose dependently increased pHi in SMC at pHL 5.0, whereas exogenous EGF in the luminal solution had no effect on pHi in SMC at all concentrations examined (Fig. 2A), indicating that EGF affects pHi via the basolateral surface of SMC.


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Fig. 2.   Effects of exogenous EGF, anti-EGF receptor antibody (Ant-EGF-R-Ab), amiloride, or Hoechst 642 on intracellular pH (pHi) in surface mucous cells in intact sheets of uninjured antral mucosae. pHi in surface mucous cells in intact sheets of gastric antral mucosa was measured fluorometrically after staining the cells with a pH-sensitive fluorescein 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein. Omeprazole-pretreated gastric fundic mucosae were incubated on the serosal side with 25 mM HEPES/100% O2 (pH 7.4) and on the luminal side with 150 mM NaCl/100% O2 (pH ~5.0). pHL was kept constant at 5.0 throughout. Exogenous EGF (~0.03-100 ng/ml), Ant-EGF-R-Ab (0.3~30 µg/ml), and/or amiloride (~0.03-3 mM) were added to the serosal side. Data are expressed as means ± SE; n, no. of experiments. A: effect of graded doses of exogenous EGF on pHi. * P < 0.05; ** P < 0.01, significant difference from the corresponding values in the absence of exogenous EGF. B: effect of graded doses of Ant-EGF-R-Ab on pHi. * P < 0.05; ** P < 0.01, significant difference from the corresponding values in the absence of Ant-EGF-R-Ab. C: effect of graded doses of amiloride or Hoechst 642 on pHi. * P < 0.05; ** P < 0.01, significant difference from the corresponding values in the absence of amiloride or Hoechst 642. D: effect of serosal amiloride (1 mM) on pHi at different serosal pH. * P < 0.05, significant difference from the corresponding values in the absence of amiloride.

In the absence of exogenous EGF, presence of anti-EGF receptor antibody (~1-10 µg/ml) in the serosal but not in the luminal solution dose dependently decreased baseline pHi in SMC at pHL 5.0 (Fig. 2B), indicating that endogenous EGF contributes to maintenance of neutral pHi, presumably by activating basolateral EGF receptors.

In the absence of exogenous EGF, amiloride (~0.1-1 mM) or Hoechst 642 (~0.1-1 µM), an agent known to induce specific inhibition of NHE-1 at these concentrations (1), on the serosal but not on the luminal side dose dependently decreased baseline pHi in SMC at pHL 5.0 (Fig. 2C). This indicates that basolateral NHE, most likely NHE-1, contributes to maintenance of neutral pHi in SMC.

The effects of amiloride on pHi at different serosal pH (pHs) were also examined to evaluate activity of NHE at different ambient pH. Baseline pHi was adjusted by altering the pH in the serosal solution, as described previously (45). Amiloride (1 mM) in the serosal solution significantly decreased baseline pHi at pHs between 6.8 and 7.4 but not at pHs 7.6 (Fig. 2D). The effects of amiloride on pHi were greater at the lower serosal pH than at the higher serosal pH (Fig. 2D).

Effects of anti-EGF receptor antibody and/or inhibitors of NHE on EGF-induced changes in pHi in the absence or presence of luminal acid. At pHL 5.0, anti-EGF receptor antibody (10 µg/ml) or amiloride (1 mM) singly or together in the serosal solution similarly abolished the increase in pHi induced by exogenous EGF (Fig. 3A). Hoechst 642 (1 µM) in the serosal solution completely prevented the increase in pHi induced by exogenous EGF in the serosal solution, effects almost identical to those induced by 1 mM amiloride (data not shown). These results indicate that in the absence of luminal acid, EGF contributes to maintenance of neutral pHi by stimulating NHE, most likely NHE-1.


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Fig. 3.   Effects of Ant-EGF-R-Ab and/or amiloride on pHi in surface mucous cells in intact sheets of uninjured antral mucosae. Omeprazole-pretreated gastric fundic mucosae were incubated on the serosal side with 25 mM HEPES/100% O2 (pH 7.4) and on the luminal side with 150 mM NaCl/100% O2. Exogenous EGF (10 ng/ml), Ant-EGF-R-Ab (10 µg/ml), and/or amiloride (1 mM) were added to the serosal side. Data are expressed as means ± SE; n, no. of experiments. aP < 0.05, significant difference from the corresponding values in the absence of exogenous EGF. bP < 0.05, significant difference from the corresponding values in the presence of exogenous EGF. cP < 0.05, significant difference from the corresponding values in the presence of Ant-EGF-R-Ab alone. A: effects of Ant-EGF-R-Ab and/or amiloride on basal and EGF-induced changes in pHi at pHL 5.0. B: effects of Ant-EGF-R-Ab and/or amiloride on basal and EGF-induced changes in pHi during exposure to luminal acid (pHL 1.5).

During exposure to luminal acid (pHL 1.5), the decrease in pHi was mitigated by exogenous EGF in the serosal solution (Fig. 3B). Anti-EGF receptor antibody in the serosal solution not only abolished the effect of exogenous EGF but also caused a more prominent decrease in pHi to a lower level than that in the absence of exogenous EGF (Fig. 3B). This is consistent with the proposal that both endogenous and exogenous EGF contribute to prevention of intracellular acidosis of SMC during exposure to luminal acid. Because amiloride caused a more prominent decrease in pHi than anti-EGF receptor antibody (Fig. 3B), an effect not augmented by combining both agents, pHi in SMC is likely maintained by both EGF-dependent and -independent (i.e., acid-stimulated) activation of the NHE.

Role of Endogenous and/or Exogenous EGF in Restitution of Injured Gastric Mucosa

This series of experiments was conducted to determine whether endogenous and/or exogenous EGF contributes to gastric mucosal restitution of injured guinea pig gastric mucosa and, if so, to define the mechanisms by which EGF promotes restitution.

Effects of exogenous EGF on recovery of R and MF after injury. In the absence of serosal HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> at pHL 7.0, exogenous EGF in either the luminal or the serosal solution, at ~1-10 ng/ml, dose dependently enhanced recovery of R (Fig. 4A).


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Fig. 4.   Effects of exogenous EGF on recovery of electrical resistance (R) and 3H-labeled mannitol flux (MF) after injury. Recovery of R and 3H-labeled MF, expressed as percentages of the corresponding basal values, at 2 h after Triton X-100 injury in the presence or absence of exogenous EGF (10 ng/ml) in the luminal or the serosal solution are shown as means ± SE. n, no. of experiments. aP < 0.05, significant difference from the corresponding values in the absence of exogenous EGF. bP < 0.05, significant difference from the corresponding values in the presence of exogenous EGF on the luminal side. A: dose-response studies on the effects of exogenous EGF on recovery of R at pHL 7.0. Serosal side was buffered with 25 mM HEPES/100% O2. B: effects of exogenous EGF on recovery of R at 2 h after injury in the presence or absence of luminal acid. Serosal side was buffered with 25 mM HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>/95% O2. pHL was kept constant at either 7.0 or 3.0. C: effects of exogenous EGF on MF after injury. Serosal side was buffered with either 25 mM HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>/95% O2-5% CO2 or HEPES/100% O2. pHL was kept constant at either 7.0 or 3.0.

In the presence of serosal HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>, at pHL 7.0, neither luminal nor serosal exogenous EGF (10 ng/ml) affected recovery of R or MF after injury (Fig. 4, B and C). In contrast, at pHL 3.0, exogenous EGF either luminally or serosally significantly enhanced recovery of R and MF, effects more prominent when the exogenous EGF was placed on the serosal side (Fig. 4, B and C).

Effects of exogenous EGF on changes in morphology after injury. Exposure of the luminal surface to 2% Triton X-100 for 5 min induced marked damage to surface and gastric pit cells, characterized by exfoliation of extensive areas of SMC, without causing apparent injury of the cells in gastric glands (Fig. 5A). Typical morphology of the mucosae incubated in the presence of serosal HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> at pHL 3.0 with or without addition of exogenous EGF for 2 h after injury is shown in Fig. 5, B-D. The degree of restitution by morphometric analysis at 1 and 2 h after injury was significantly greater in the presence than in the absence of exogenous EGF (Fig. 5E). The enhancement of restitution by exogenous EGF (10 ng/ml) at pHL 3.0 was greater when EGF was placed in the serosal than in the luminal solution (Fig. 5E).


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Fig. 5.   Effects of exogenous EGF on morphology of gastric mucosae after injury. Effects of exogenous EGF (10 ng/ml) on morphology after Triton X-100-induced injury were examined in omeprazole-pretreated gastric fundic mucosae. In A-D, the serosal side was buffered with 25 mM HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>/95% O2-5% CO2, and luminal pH was kept constant at 3.0. Original magnification ×720, bar = 10 µm. A: mucosa fixed immediately after the injury showed that large numbers of the surface mucous cells were exfoliated, whereas gastric glands were not severely injured. B: mucosa incubated without exogenous EGF showed poor restitution at 2 h after injury. C and D: mucosa incubated with luminal (C) or serosal (D) exogenous EGF showed almost complete restitution at 2 h after injury. E: effects of exogenous EGF on degree of restitution in the presence or absence of serosal HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> at pHL 3.0 or 7.0. Degrees of restitution are expressed as the percentages of the total mucosal surface area covered with epithelial cells at 1 and 2 h after injury. aP < 0.05, significant difference from the corresponding values in the absence of exogenous EGF. bP < 0.05, significant difference from the corresponding values in the presence of exogenous EGF on the luminal side.

Effects of anti-EGF-receptor antibody and/or amiloride on basal and EGF-induced recovery of R after injury. This series of experiments was conducted first to determine whether the enhancement of restitution by EGF is mediated via the EGF receptors on the gastric epithelial cells and second to determine whether the enhancement of restitution by EGF is related to stimulation of the NHE.

In the absence of serosal HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> at pHL 7.0, basal (control) and EGF-induced recovery of R after injury was significantly inhibited by the anti-EGF receptor antibody (10 µg/ml), amiloride (1 mM), or by Hoechst 642 (1 µM; Fig. 6, A and B). The magnitudes of the inhibition of restitution induced by anti-EGF receptor antibody were greater than those caused by amiloride or by Hoechst 642. Combination of anti-EGF receptor antibody and amiloride caused more potent inhibition than amiloride alone but did not augment the inhibition induced by the antibody alone (Fig. 6B). These results indicate that at pHL 7.0, both endogenous and exogenous EGF promote restitution, in part by stimulation of the NHE and in part by other mechanisms not related to stimulation of the NHE.


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Fig. 6.   Effects of Ant-EGF-R-Ab and/or inhibitors of Na+/H+ exchange on basal (control) and EGF-induced restitution. Effects of Ant-EGF-R-Ab and/or amiloride on basal and EGF-induced recovery of R after Triton X-100 injury were examined in omeprazole-pretreated gastric fundic mucosae incubated with HEPES/100% O2 on the serosal side. pHL was kept constant at either 7.0 (A, B, and D) or 3.0 (C). Exogenous EGF (10 ng/ml) was added to the luminal side. Ant-EGF-R-Ab (10 µg/ml), amiloride (1 mM), and Hoechst 642 (1 µM) were added to the serosal solution. Data are expressed as means ± SE. n, no. of experiments. A: effects of graded doses of Ant-EGF-R-Ab, amiloride, or Hoechst 642 on basal recovery of R in the absence of exogenous EGF. * P < 0.05, significant difference from the corresponding values in the absence of Ant-EGF-R-Ab, amiloride, or Hoechst 642. B: effect of Ant-EGF-R-Ab and/or amiloride on basal and EGF-induced recovery of R at pHL 7.0. aP < 0.05, significant difference from the corresponding values in the absence of Ant-EGF-R-Ab, amiloride, or Hoechst 642. bP < 0.05, significant difference from the values in the presence of amiloride alone. C: effect of Ant-EGF-R-Ab and/or amiloride on basal and EGF-induced recovery of R at pHL 3.0. aP < 0.05, significant difference from the corresponding values in the absence of Ant-EGF-R-Ab, amiloride, or Hoechst 642. bP < 0.05, significant difference from the values in the presence of Ant-EGF-R-Ab alone. D: effect of serosal amiloride (1 mM) on recovery of R at different serosal pH. * P < 0.05, significant difference from the corresponding values in the absence of amiloride.

At pHL 3.0, basal and EGF-induced recovery of R were significantly inhibited by anti-EGF-receptor antibody or amiloride in the serosal solution (Fig. 6C). The magnitude of the inhibition of restitution induced by amiloride was greater than that caused by anti-EGF receptor antibody. Combination of amiloride and the antibody caused greater inhibition of the recovery than antibody alone but did not augment the inhibition induced by amiloride alone (Fig. 6C). These results indicate that at pHL 3.0, both endogenous and exogenous EGF promote restitution mainly by stimulating the NHE.

Effects of anti-EGF receptor antibody and/or amiloride on recovery of R at different serosal pH. Because the magnitude of the inhibition of restitution by amiloride was greater at pHL 3.0 than at 7.0, we hypothesized that retardation of restitution by amiloride may be caused by intracellular acidosis induced by the inhibition of the NHE. Thus we examined the effect of amiloride on restitution at different serosal pH, because pHi changes in parallel with the changes in the serosal pH (see Fig. 2D).

In the absence of serosal HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> at pHL 7.0, amiloride inhibited restitution at all examined pHs between 7.0 and 7.6, although the magnitude of the inhibition was greater at lower pHs than those at higher pHs (Fig. 6D).

Effect of removal of endogenous EGF on recovery of R after injury. In some tissues, endogenous EGF was removed from the chamber by replacement of both the luminal and the serosal solutions with fresh solutions at 15-min intervals after injury, whereas other tissues were incubated without replacement of the solutions throughout the entire period after injury.

Recovery of R in the absence of serosal HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> at pHL 7.0 was significantly greater in the mucosae incubated without replacement of the solutions than those incubated with replacement, an effect abolished by the presence of anti-EGF receptor antibody on the serosal side (Fig. 7). The recovery of R was greater in the absence than in the presence of anti-EGF receptor antibody, even after frequent replacement of the solutions. These results indicate that endogenous EGF, at least in part, does contribute to restitution.


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Fig. 7.   Effect of removal of endogenous EGF from the luminal and the serosal solutions on recovery of R. Recovery of R after injury was examined in the absence of exogenous EGF in the mucosae incubated with or without replacement of both the luminal and the serosal solutions with fresh solutions at 15-min intervals. The serosal side was buffered with HEPES/100% O2. pHL was kept constant at 7.0 throughout the experiment. Data are expressed as means ± SE. n, no. of experiments. aP < 0.05, significant difference from the corresponding values in the mucosae incubated without replacement of the luminal and the serosal solutions with fresh solutions. bP < 0.05, significant difference from the corresponding values in the presence of anti-EGF-receptor antibody (10 µg/ml) in the serosal solution.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The present study indicates that endogenous EGF plays an important role in restitution of injured mucosae, because in the absence of exogenous EGF, restitution was significantly inhibited by anti-EGF receptor antibody or by removal or reduction of endogenous EGF by changing the luminal and the serosal solutions. Other investigators have also shown that endogenous EGF within the gastric mucosa contributes to mucosal protection and repair after injury (15, 44). Our findings also suggest that other mechanisms may be involved in the EGF receptor-mediated enhancement of epithelial cell migration, because anti-EGF receptor antibody had an inhibitory effect on restitution even after elimination of endogenous EGF. We believe that the increased release of endogenous EGF after injury in our experiments was the result of disruption of injured cells rather than a rise in production, because the increase was demonstrated immediately (at 30 min) after injury and was not prevented by a protein synthesis inhibitor cycloheximide (unpublished observations).

We used antral mucosa in the measurement of pHi, because measurement of pHi in antral SMC is not confounded by BCECF loading in oxyntic cells of fundic mucosae. Unpublished experiments in our laboratory have shown that the effects of EGF or amiloride on restitution in antral mucosae are almost identical to those of fundic mucosae. Although no studies have compared the effects of EGF on NHE activity in antral SMC and fundic SMC, recent studies in rat gastric mucosa have shown that the distribution of NHE in antrum is almost identical to that of fundus (24). Thus we believe that the nature of EGF-induced cellular responses in antral SMC is identical to that of the SMC in fundic mucosa.

The present study provides several lines of evidence for the close relationship among EGF, NHE, and restitution. In the absence of luminal acid, exogenous EGF dose dependently increased pHi (~0.3-10 ng/ml) and stimulated restitution (~3-10 ng/ml), effects abolished or attenuated by either anti-EGF receptor antibody or amiloride. In the presence of luminal acid, exogenous EGF (10 ng/ml) in the serosal solution not only prevented decreases in pHi but also enhanced restitution. Furthermore, these effects were abolished or attenuated by either anti-EGF receptor antibody or amiloride. Thus, in both the presence and absence of luminal acid, the effect of exogenous EGF on restitution is closely related to its effect on NHE activity. To our knowledge, no studies have examined the direct interaction between EGF, NHE, and cell migration. Our results, however, are in agreement with the findings of others, who have shown that EGF enhances cell migration (2, 6) and promotes restitution (13, 31) and that blockade of NHE by amiloride inhibits cell migration and restitution (13, 16).

The present findings also suggest that the interaction among EGF, NHE, and restitution is slightly different in the absence or presence of luminal acid. In the absence of luminal acid, the increase in pHi induced by exogenous EGF was completely abolished by either amiloride or anti-EGF receptor antibody, whereas the enhancement of restitution by exogenous EGF was blocked partially by amiloride and almost completely by the antibody. Thus, in the absence of luminal acid, exogenous EGF stimulates restitution not only by activating NHE activity but also by enhancing some other mechanism not directly related to stimulation of NHE. On the other hand, in the presence of luminal acid, the effects of amiloride on pHi and restitution were greater than those of anti-EGF receptor antibody. In addition, the amiloride-induced inhibition of restitution was not accentuated by the anti-EGF receptor antibody. These results suggest that in the presence of luminal acid, EGF enhances restitution mainly by stimulating NHE activity and that during exposure to luminal acid, NHE is not only activated by EGF but is also stimulated by intracellular acidosis induced by luminal acid.

We propose that the potent inhibition of restitution by amiloride in the presence of luminal acid is caused by augmentation of intracellular acidosis, because amiloride caused more profound intracellular acidosis in the presence than in the of absence of luminal acid. It is possible that 1 mM amiloride per se could induce nonspecific toxic damage to the gastric epithelial cells, thereby causing more potent inhibition of restitution than the antibody. However, this is very unlikely, because amiloride, up to 3 mM, did not cause any cell damage, as measured by LDH release, electrophysiology, and MF. Studies in intact sheets of gastric mucosal preparations by others (13) and by us (45) have also shown that 1 mM amiloride does not cause any damage in SMC, as evaluated by electrophysiology and morphology.

In the present study, the effects of endogenous and/or exogenous EGF on NHE were not completely in parallel with those on restitution. First, in the presence of serosal HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> at pHL 7.0, exogenous EGF (10 ng/ml) did not affect restitution but did cause a significant increase in pHi. It seems likely that the presence of serosal HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> masked the effects of exogenous EGF on restitution, because serosal HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> has been shown to enhance restitution (38). The finding that exogenous EGF significantly enhanced restitution at pHL 7.0 in the absence of serosal HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> strongly supports this possibility. Second, the minimal concentration of exogenous EGF (3 ng/ml) required for stimulation of restitution in injured mucosa was greater than that required to stimulate NHE in uninjured mucosae. It is conceivable that in injured mucosae, the minimal effect of lower concentrations of exogenous EGF (~0.3-3 ng/ml) on restitution was masked by the effects of relatively high concentrations of endogenous EGF that had been released from the mucosa immediately after injury. Third, at serosal pH 7.6, amiloride did not affect pHi but did cause significant inhibition of restitution. Thus we propose that NHE in the nonmigrating cells in uninjured mucosae is not activated at high pHs, whereas the NHE in migrating cells in injured mucosae is activated even at high pHs, because activation of the NHE has been shown to play an important role in epithelial cell migration (16) and mucosal restitution (13).

The minimal dose of exogenous EGF required to stimulate restitution (3 ng/ml) was significantly greater than the concentration of endogenous EGF detected in the luminal and the serosal solutions after injury (~1.5 ng/ml). This difference could be attributable to the presence of a diffusion barrier, which might attenuate diffusion of exogenous EGF in the luminal or the serosal solution and also block diffusion of endogenous EGF released from SMC into the luminal or the serosal solution. We often found that a thick mucus gel or mucoid caps remained attached to the luminal surface of the mucosae for several hours after injury. This mucus could reduce diffusion of exogenous EGF in the luminal solution to the SMC. On the serosal side of the preparations, the muscularis mucosae and connective tissues in the lamina propria could also decrease diffusion of exogenous EGF from the serosal solution to the SMC. These possibilities are supported by our previous observation that mechanical removal of the mucus gel or the muscularis mucosae enhances the efficacy of agents added to the luminal or the serosal solution (45). Thus we propose that the actual concentrations of endogenous EGF adjacent to the SMC beneath the mucoid cap are significantly greater than those detected in the luminal or the serosal solution and that the actual concentrations of exogenously added EGF at the surface of SMC could presumably be even less than the concentrations of exogenous EGF in the luminal or the serosal solutions.

On the basis of the present findings, we believe that there are several mechanisms by which EGF-induced stimulation of NHE enhances restitution. First, our data show that stimulation of NHE by exogenous EGF prevents intracellular acidosis of gastric epithelial cells during exposure to luminal acid. Second, it is also possible that stimulation of NHE by EGF activates Rho protein to reorganize actin filaments, thereby enhancing the rearrangement of the cytoskeleton in migrating epithelial cells. Other studies have shown that EGF enhances Rho-induced cell migration (31) and that activation of NHE is necessary to increase RhoA-induced stress fiber formation (41). Third, EGF-induced stimulation of NHE may enhance cell motility by modulating cell volume regulation in the migrating epithelial cells, because amiloride has been shown to block both recovery from cell shrinkage and cell migration (13, 17).

The present results on the effects of exogenous EGF and/or anti-EGF receptor antibody on pHi in SMC of uninjured mucosa suggest that EGF affects SMC preferentially through the basolateral rather than the apical side of uninjured mucosae. Our data on restitution in injured mucosae are consistent with the proposal that luminal EGF exerts its effect from the basolateral side of the mucosa, because the enhancement of restitution by luminal EGF was completely abolished by the presence of serosal anti-EGF receptor antibody. These results are in agreement with a number of recent studies that have found the presence of EGF receptors on the basolateral membrane of gastrointestinal epithelial cells (18, 21, 27, 34). Because EGF has a molecular weight of 6,045 (4), it is likely that luminal EGF has no access to the basolateral EGF receptors in normal uninjured intact gastric mucosae. In injured gastric mucosae, however, disruption of epithelial continuity enables EGF in the gastric lumen to gain direct access to the EGF receptor on the basolateral membrane of SMC. This possibility is supported by the findings of a number of other investigators that luminal EGF has no effects on uninjured gastric mucosae but does enhance mucosal repair after injury both in vivo (22, 37) and in vitro (12, 19, 29, 30).

In the present study, the enhancement of restitution by luminal exogenous EGF at pHL 3.0 was less than that induced by serosal exogenous EGF. Because biological activity of EGF has been shown to decrease at pH <3.0 in vitro (28), it is likely that at pHL 3.0, some exogenous EGF in the luminal solution is inactivated by luminal acid, thereby showing less effect on restitution than the serosal EGF.

In summary, the present study shows that both endogenous and exogenous EGF contribute to restitution of injured gastric mucosae. Effects appear to be mediated via EGF receptors and NHE on the basolateral membrane of migrating gastric epithelial cells.


    ACKNOWLEDGEMENTS

We gratefully thank Drs. W. Silen and S. Ito for critical and careful review of the manuscript.


    FOOTNOTES

This work was supported by a grant-in-aid for scientific research (10670450) and on priority areas from the Ministry of Education, Science, Sports, and Culture of Japan.

Address for reprint requests and other correspondence: A. Yanaka, Depts. of Gastroenterology and Endoscopy, Institute of Clinical Medicine, Univ. of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan (E-mail: ynk-aki{at}md.tsukuba.ac.jp).

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.

First published January 2, 2002;10.1152/ajpgi.00150.2001

Received 10 April 2001; accepted in final form 21 December 2001.


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