Secretin regulates paracellular permeability in canine gastric monolayers by a Src kinase-dependent pathway

Monica C. Chen1, Travis E. Solomon1, Eduardo Perez Salazar2, Robert Kui1, Enrique Rozengurt1,2, and Andrew H. Soll1

1 CURE/Division of Digestive Diseases and 2 Molecular Biology Institute, Department of Medicine, School of Medicine, University of California Los Angeles and The Medical and Research Services, Greater Los Angeles Veterans Affairs Health Care System, Los Angeles, California 70073


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Previous studies found that epidermal growth factor (EGF) decreased paracellular permeability in gastric mucosa, but the other physiological regulators and the molecular mechanisms mediating these responses remain undefined. We investigated the role of secretin and Src in regulating paracellular permeability because secretin regulates gastric chief cell function and Src mediates events involving the cytoskeletal-membrane interface, respectively. Confluent monolayers were formed from canine gastric epithelial cells in short-term culture on Transwell filter inserts. Resistance was monitored in the presence of secretin with or without specific kinase inhibitors. Tyrosine phosphorylation of Src at Tyr416 was measured with a site-specific phosphotyrosine antibody. Basolateral, but not apical, secretin at concentrations from 1 to 100 nM dose dependently increased resistance; this response was rapid and sustained over hours. PP2 (10 µM), a selective Src tyrosine kinase inhibitor, but not the inactive isomer PP3, abolished the increase in resistance by secretin but only modestly attenuated apical EGF effects. AG-1478 (100 nM), a specific EGF receptor tyrosine kinase inhibitor, attenuated the resistance increase to EGF but not secretin. Secretin, but not EGF, induced tyrosine phosphorylation of Src at Tyr416 in a dose-dependent fashion, with the maximal response observed at 1 min. PP2, but not PP3, dramatically inhibited this tyrosine phosphorylation. Secretin increases paracellular resistance in gastric mucosa through a Src-mediated pathway, while the effect of EGF is Src independent. Src appears to mediate the physiological effects of this Gs-coupled receptor in primary epithelial cells.

secretin receptors; epidermal growth factor receptors; gastric mucosal defense; paracellular pathway; regulation of paracellular pathway; apical epidermal growth factor receptors; PP2; tyrosine kinases


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

THE GASTRIC MUCOSA has a remarkable ability to defend itself against injury from the acid/peptic activity of gastric juice and to undergo rapid repair when injury does occur. Mucosal defense and repair mechanisms are multifactorial. Primary lines of defense involve the preepithelial mucous and bicarbonate barrier, epithelial cell mechanisms, and subepithelial blood flow, while critical repair mechanisms include restitution (lateral migration of cells), replication, and wound healing (1, 9, 21, 27, 36). Three categories of epithelial cell function are involved in mucosal defense: 1) the apical membrane, which is a component of the gastric barrier to acid diffusion (3, 4, 26); 2) basolateral Na+/H+ and Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchange, which permits mucosal cells to extrude "back-diffused" acid; and 3) intrinsic epithelial cell mechanisms, which defend against oxidative and other insults (15, 17). The pH gradient established by HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> secretion into a mucous layer protects the intracellular pH of surface mucosal cells (6). However, cells lining the gastric glands are not protected by a mucous layer and yet withstand pepsin at a pH <1.5. By studying monolayers formed from enzyme-dispersed canine oxyntic mucosal cells in primary culture, we found that gastric epithelial cells displayed marked resistance to apical but not basolateral acidification. This indicated that the apical membrane was a critical component of the gastric barrier to acid (26) and that the paracellular pathway was the first point of injury caused by extreme or prolonged apical acidification (3).

In studies with primary canine gastric monolayers, we also found that apical junctional permeability was regulated by epidermal growth factor (EGF; Ref. 4) and that this regulation had a major impact on the resistance of gastric monolayers to apical H+ (M. C. Chen and A. H. Soll, unpublished observations). These findings suggest that regulation of paracellular permeability is critical for maintenance of mucosal integrity in the face of luminal acid and pepsin. We also found consistent evidence that EGF increases transepithelial resistance (TER) by activating apical as well as basolateral receptors (4).

Secretin has well-defined effects on chief cells, stimulating pepsinogen secretion by canine chief cells, rat gastric glands, and purified guinea pig chief cells in vitro (20, 25, 28). Because chief cells are the major component of canine gastric epithelial monolayer cultures (3) and secretin induces pepsinogen release from these cultures (25), secretin is a candidate to regulate other actions in these gastric cells.

Numerous lines of evidence indicate that the paracellular pathway is regulated (16, 33). However, despite exciting recent progress in the molecular structure of the junction (2, 14, 35), the physiology, pathophysiology, and molecular mechanisms of the regulatory process governing paracellular permeability remain largely undefined. In particular, a role for Src in regulation of the apical junctional complex has not been defined. However, Src has been localized to the adherens junction [the zonula adherens (ZA)] of the apical junctional complex of epithelial cells (31). Furthermore, Src has been found to induce the tyrosine phosphorylation of beta -catenin and p120 (8), two members of the ZA. Therefore, the Src-family kinases are candidates that warrant investigation.

The rapid regulation of paracellular permeability by several chemotransmitters that we have found in our canine gastric monolayers provides a useful model for studying the molecular mechanisms underlying the regulation of paracellular permeability. In the studies presented here, we examined the effects and mechanism of action of secretin on paracellular permeability in cultured gastric mucosal cell monolayers. To define the molecular mechanisms mediating the action of secretin on paracellular permeability, we also studied the role of Src both using PP2, the highly selective inhibitor of the Src kinase family, and assessing auto-tyrosine-phosphorylation of Src kinase at Tyr416. We now report that secretin increases the resistance of the paracellular pathway of canine gastric cell monolayers in a fashion that is dependent on Src kinase family activity.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Tissue dispersion, cell separation, and culture. Enzyme-dispersed canine oxyntic mucosal cells were separated by elutriation and cultured on collagen-coated Transwell filter inserts (6 or 12 wells, Costar), as described previously (3, 4). The composition of the epithelial cells was about 70% chief cells and 20% parietal cells, with <10% surface and mucous neck cells (4, 10). Cells were cultured in DMEM-Ham's F-12 (1:1) plus 20 mM HEPES, 100 µg/ml amikacin, 100 U/ml penicillin, 100 µg/ml streptomycin, and 2% calf serum. Cultures were fed with the same medium every 48 h until confluent (~72 h). Monolayers were switched to serum-free medium (R0) for 6-16 h before experiments were performed.

Measurement of TER and mannitol flux. Resistance was monitored using an epithelial voltohmmeter (EVOM) with chopstick electrodes. This technique allows repeated measurements of monolayer resistance under sterile conditions. In all experiments, resistance data are calculated as the mean of 3 or 4 separate Transwell inserts and are expressed as a percent increase over the basal resistance. Apical-to-basolateral mannitol flux was determined by adding [3H]mannitol to the apical solution as previously described (3).

Western blot analysis. Monolayers were treated with or without various concentrations of kinase inhibitor for 15 min and followed by stimulation with growth factor or secretin. Cells were washed with ice-cold PBS containing PMSF (1 mM) and sodium vanadate (100 µM). Cells were lysed in 2× SDS-PAGE sample buffer (200 mM Tris · HCl, pH 6.8, 1 mM EDTA, 6% SDS, 2 mM EDTA, 4% 2-mercaptoethanol, 10% glycerol), followed by boiling for 10 min, and resolved by SDS-PAGE. After SDS-PAGE, proteins were transferred to Immobilon membranes. After transfer, membranes were blocked using 5% nonfat dried milk in PBS, pH 7.2, and incubated for 2 h at room temperature with rabbit anti- Src(P)Tyr416 Ab. The membranes were washed three times with PBS, pH 7.2, 0.1% Tween 20, and then incubated with secondary antibodies (horseradish peroxidase-conjugated donkey antibody to rabbit, NA 934) (1:5,000) for 1 h at room temperature. After washing three times with PBS, pH 7.2, 0.1% Tween 20, the immunoreactive bands were visualized using enhanced chemiluminescence (ECL) detection reagents. Autoradiograms were scanned, the labeled bands were quantified using the NIH Image program, and the intensities of specific bands were expressed in density units (% of basal).

Materials. Materials for cell cultures were obtained from sources outlined previously (4, 5). Transwell inserts were from Costar (Cambridge, MA), and human recombinant EGF was from R&D Systems (Minneapolis, MN). Rat secretin was obtained from the University of California Los Angeles Peptide Synthesis Core Facility. The EVOM Millicell-ERS was from Millipore (Bedford, MA). ECL reagents were from Amersham Pharmacia Biotech. Rabbit anti-Src(P)Tyr416 Ab was from Biosource (Camarillo, CA). AG-1478, Genestein, wortmannin, PP2, and PP3 were obtained from Calbiochem. Other chemicals were from Sigma Chemical (St. Louis, MO).


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Secretin increases TER of gastric monolayers. Dispersed gastric mucosal cells plated in Transwell inserts form monolayers after 72 h of incubation. Baseline TER of these monolayers, monitored with chopstick electrodes positioned using a derrick, was 1,635 ± 425 Omega  · cm2 (mean ± SE; n = 8 separate preparations). Increases in resistance are a reliable measure of decreased paracellular permeability, as reflected by decreases in the flux of mannitol (4). Because secretin regulates gastric chief cell function and since these monolayers have a predominant chief cell population, we investigated the effect of secretin on the TER of these monolayers.

Secretin applied basolaterally increased TER; in contrast, application of secretin at the apical surface was without effect (Fig. 1). The increase in resistance in response to secretin was rapid, being evident at 10 min, and stable, persisting for a 2- to 3-h period (Fig. 1A). This response to secretin was dose-dependent over a secretin concentration range from 0.1 to 50 nM (Fig. 1). Statistically significant responses were found at the 0.1 nM concentrations (P < 0.05, n = 4). The response to higher doses of secretin is more biphasic than that of the lower doses. Mannitol flux experiments were performed on a parallel set of cell cultures to further characterize secretin-dependent changes in paracellular permeability (Fig. 1C). Basolateral secretin (10 nM) significantly decreases mannitol flux, and the effect lasts ~3 h. These data suggest a role for secretin in a multifactorial modulation of the function of the gastric barrier.


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Fig. 1.   Basolateral secretin increases transepithelial resistance (TER) and decreases mannitol flux. Cells were plated on Transwell inserts and reached confluence after 72 h. TER was monitored by epithelial voltohmmeter (EVOM) to confirm confluence; all studies were performed after the TER was >1,000 Omega  · cm2. Data are means ± SE from 3 separated preparations. A: various doses of rat secretin were added to monolayers basolaterally (bl), and the TER was measured. Secretin at 0.1 to 50 nM dose dependently increased TER. B: secretin was added apically (ap) at the indicated doses. C: basolateral secretin and apical [3H]mannitol were added at t = 0. Aliquots from the basolateral medium at t = 30 min were sampled to determine the radioactivity as the initial flux (100%). R0, serum-free medium. * P < 0.05, ** P < 0.01, control vs. secretin.

Src mediates secretin but not EGF actions on TER. Src tyrosine kinase family is known to modulate interactions at the cytoskeletal-membrane interface. Recently, Gs has been shown to promote Src activation (12). Given that the secretin receptor couples to Gs, we examined whether PP2, a specific inhibitor of Src-family tyrosine kinases, prevented the increase in TER induced by secretin. In initial studies, PP2 alone at a concentration of 10 µM caused up to a 20% transient decrease in resistance compared with untreated controls. However, concentrations of 5 µM and below had no significant effect on baseline resistance (data not shown, P > 0.2; n = 4). Subsequent studies were performed using these lower concentrations. PP2 caused a dose-dependent inhibition of secretin effects on TER (Fig. 2A). Inhibition by PP2 was detectable at 0.1 µM and reached maximal at 5 µM. PP2 at 5 µM decreased secretin-stimulated TER from 24 ± 2.5 to 1.7 ± 2%, n = 4, at t = 2 h. Inhibition was comparable when PP2 was added to either the apical or basolateral solutions (P > 0.2; n = 3; data not illustrated). A 5 µM concentration of PP3, a structurally related but biologically inactive isomer, did not prevent the increase in TER induced by secretin (Fig. 2B).


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Fig. 2.   A selective Src family tyrosine kinase inhibitor, PP2, dose dependently blocks the secretin effect on TER. Data are means of 4 Transwells from a representative of 3 identical preparations that showed similar findings. A: increasing concentrations of PP2 (0.1-10 µM) were added basolaterally 30 min before secretin (5 nM). * P < 0.05, # P > 0.05, PP2 + secretin (Sec) vs. DMSO + Sec. B: In contrast to PP2, PP3 at 5 µM did not attenuate secretin stimulation. ** P < 0.05, PP2 + Sec vs. DMSO + Sec.

In contrast to the dramatic inhibition of secretin action (Fig. 3C), PP2 did not block the effects of apical EGF on resistance even at a concentration of 10 µM (Fig. 3B). In contrast, the EGF receptor (EGFR) tyrosine kinase inhibitor AG-1478 (100 nM) markedly attenuated the increase in resistance in response to apical EGF (Fig. 3A), but did not alter the response to basolateral secretin administration (Fig. 3D). Because the inhibitory effects of PP2 were observed over a concentration range in which PP2 is a selective inhibitor of Src-family kinases, these data indicate that the effect of secretin, but not EGF, on TER is dependent on the activity of the Src kinase family.


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Fig. 3.   Effects of AG-1478 and PP2 on epidermal growth factor (EGF) regulation of TER. The selective EGF receptor (EGFR) tyrosine kinase inhibitor AG-1478 (100 nM) was added apically 30 min before apical EGF (10 nM; A) or basolateral secretin (1 nM; D) was added. Comparable effects were observed adding AG-1478 apically, basolaterally, or to both bathing solutions. Similarly, PP2 (10 µM) was added apically 30 min before EGF (B) and secretin (C) were added. AG-1478 blocks EGF stimulation of TER but minimally attenuates secretin stimulation. In contrast, PP2 inhibits secretin effect but does not attenuate EGF activation in TER. Data are means of 4 separate Transwells from a single preparation and are representative of 3 other identical preparations that showed similar findings. * P < 0.05, AG-1478 + EGF vs. EGF in A; * P < 0.05, PP2 + Sec vs. Sec in C.

Secretin induces autophosphorylation of Src at Tyr416. A site-specific anti-phosphotyrosine antibody to Tyr416 of Src was used to assess the activation of Src via this known autophosphorylation event. Western blots, performed on lysates run on PAGE and probed with antibody to Src(P)Tyr416, revealed two bands at estimated molecular masses of 62 and 58 kDa (Fig. 4). PP2, in concentrations under 5 µM, selectively blocked Src(P)Tyr416 phosphorylation of the 58-kDa band (Fig. 4B). Quantification of this band using scanning densitometry demonstrated that PP2 inhibited the phosphorylation of Tyr416 of Src in a dose-dependent fashion (Fig. 4D). Half-maximal effect was obtained at a concentration of ~1 µM. The results indicate the expression and phosphorylation at Tyr416 of PP2-sensitive Src-family kinases in canine gastric mucosa.


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Fig. 4.   Secretin-induced autophosphorylation of a Src kinase at Tyr416. A: time course. Confluent monolayers were cultured in R0 media (no serum or growth factors) for at least 12-16 h. Secretin (1 nM) was added basolaterally and incubated for the indicated times. Various concentrations of kinase inhibitor were added for 15 min, followed by treatment with secretin (1 nM) for the indicated times (B). Monolayers were washed with ice-cold PBS containing PMSF (1 mM) and sodium vanadate (100 µM) and lysed in 2× SDS-PAGE sample buffer, boiled for 10 min, and the supernatants were resolved by SDS-PAGE. Proteins were transferred to Immobilon membranes, blocked, and probed with the Src(P)Tyr416 antibody. The immunoreactive bands were visualized using ECL detection reagents. The autoradiograms showed two bands at estimated molecular masses of 62 and 58 kDa. Quantification of this 58-kDa band was performed by scanning densitometry (C and D). Secretin stimulated the tyrosine phosphorylation of Src-family kinases rapidly and reached the maximal level at 1 min. PP2, but not PP3, inhibited Src(P)Tyr416 autophosphorylation in a dose-dependent manner. Data are means ± SE from 3 separate preparations. Cont, control.

Addition of secretin at 1 nM induced a marked increase in tyrosine phosphorylation of Src-family kinases. Secretin-stimulated Src(P)Tyr416 phosphorylation was very rapid, achieving a maximal response at 1-2.5 min (Fig. 4A). This secretin-induced tyrosine phosphorylation of Src was blocked by addition of PP2, but not of PP3 (Fig. 4B). Taken together, these findings indicate that the secretin stimulates activation of Src-family kinases in gastric epithelial cells.


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
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The findings presented in this study establish three points: 1) secretin at nanomolar concentrations increases TER in canine gastric monolayer cultures; 2) the specific Src inhibitor PP2 blocks the increase in TER induced by secretin; and 3) secretin induces rapid autophosphorylation of a Src kinase, and PP2 attenuates this activation. These findings indicate that secretin regulates paracellular permeability and that Src kinase plays a critical role in mediating this effect. These results not only throw light on a potential physiological action of secretin in regulating mucosal resistance to injury, but they also highlight potential physiological roles for the Src kinase family both in the action of a receptor coupled to Gs and in regulating paracellular permeability.

Similar to EGF, basolateral secretin caused rapid, concentration-dependent increases in TER in our mixed-epithelial cell model of the gastric mucosa. Secretin increased TER within 5 min of treatment. However, the effects of secretin and EGF on TER differed in several ways. Secretin was effective only when applied to the basolateral surface of the epithelial monolayer, while EGF increased TER, acting at both apical and basolateral receptors. The action of secretin was blocked by the Src kinase inhibitor PP2, but not by the specific EGFR tyrosine kinase inhibitor AG-1478. In contrast, we found that the action of EGF on TER was blocked by AG-1478, but not PP2. We have also found that combined treatment with secretin and EGF produces a potentiating effect wherein the combined response is greater than the sum of the individual responses, a further indication that secretin and EGF act via different mechanisms to increase TER (Chen, Solomon, and Soll, unpublished observations).

Secretin has been reported by Rotoli et al. (22) to also modulate paracellular permeability in CAPAN-1 pancreatic cancer cells grown to confluence. However, in this system secretin was only studied at very high concentrations (1 µM) and produced opposite effects, decreasing TER and increasing paracellular permeability. It is unclear whether the opposite effects in this system reflect tissue differences (stomach vs. pancreas), neoplastic vs. normal cells, or the much higher concentrations studied.

The potency of secretin for altering TER in these gastric epithelial monolayers indicates activation at a specific secretin receptor. Indirect evidence supports the existence of functional secretin receptors on gastric chief and mucous cells, but not parietal cells (11, 20, 25, 28). To date, only one secretin receptor has been cloned and characterized (34). It is a G protein-coupled receptor (GPCR) with a very long amino terminal extracellular region, a structural characteristic that has been used to define a large subfamily of GPCRs. This receptor appears to be widely distributed in many organs (including the stomach), based on Northern blot analysis of tissue extracts (34). However, the specific cell types expressing the receptor have only been defined in the pancreas and liver.

The secretin receptor is generally considered to be coupled to Gs and thus to activate cells via adenylyl cyclase and protein kinase A (PKA). In every secretin-responsive cell type that has been studied, secretin has been found to activate adenylyl cyclase and/or increase cellular cAMP concentration (34). However, at high concentrations, secretin also increases cellular Ca2+ levels (18, 30), indicating that coupling to Gq can occur under certain circumstances. At the concentrations used in our experiments, we anticipate that secretin receptor action is primarily coupled to Gs.

Several lines of evidence indicate that Src kinase plays a physiological role in secretin action in this model system. First, secretin autophosphorylation of Src was demonstrated using site-specific antibodies for Tyr416. Second, autophosphorylation of Src was rapid (peak at 1 min) and occurred over the same nanomolar concentration range in which secretin regulated TER. Third, secretin action on both TER and autophosphorylation of Tyr416 was inhibited by the same low micromolar concentration range of PP2, whereas the inactive analog PP3 was without effect. PP2 in these concentrations is highly specific for the Src kinase family. Our observations that secretin acts in a Src-dependent fashion are novel.

Src kinases are an important family of nonreceptor protein tyrosine kinases that transduce multiple upstream signals into numerous downstream actions on cell growth, differentiation, migration, structure, and secretion (29). Although linked to the actions of growth factors such as EGF, Src kinases also participate in the actions of GPCRs. Most of the available evidence regarding GPCRs indicates Src activation by Gq-coupled receptors (13, 19, 23). However, recent studies with primary cultures of mouse brown adipocytes indicated that PP2 partially inhibited vascular endothelial growth factor gene expression in response to the Gs-coupled beta 3-adrenergic receptor (7). Our findings that secretin induces the autophosphorylation of Src and that PP2 completely inhibits secretin action indicate that Src-family kinases can mediate Gs-coupled receptor action.

The mechanism by which secretin activates Src kinases is unknown. Assuming that it involves primarily a Gs-coupled initiation site, there are at least two potential subsequent steps. The first would be the well-defined adenylyl cyclase-cAMP-PKA pathway initiated by Gs activation. Src has potential sites of PKA serine/threonine phosphorylation (29), but it is not known if such phosphorylation occurs or if it alters Src function. This mechanism is supported by the work of Fredriksson et al. (7) who found the action of beta 3-receptor activation was completely blocked by the PKA inhibitor H89 and, as described above, partially by the Src kinase inhibitor PP2. One interpretation of this pattern is the sequential activation of PKA and Src kinase. Another possible pathway has recently been described by Ma et al. (12), who demonstrated that both activated Gs and Gi, but not Gq or G12, bound to and activated Src. Furthermore, tyrosine phosphorylation in response to constitutively active Gs was markedly reduced in fibroblasts from knockout strains deficient in Src, Fyn, and Yes (12). Taken together, these studies support a link between Gs-coupled receptors and Src, although there is disagreement regarding whether PKA is involved in activating Src. The identity of the 58-kDa Src-family kinase involved in the dramatic secretin activation in our system is not resolved in the current study. Our preliminary data suspect that the Src family member may be Fyn, not Yes, but we have not yet been able to firmly establish this point.

The manner in which Src activation leads to changes in TER is not known. Src activates a variety of proteins involved in cytoskeletal-membrane function, such as FAK, paxillin, and Pyk2 (24, 32). Src has been localized to the ZA (31) and induces the tyrosine phosphorylation of beta -catenin and p120 (8), but no role in the function or structure of the apical junctional complex has been defined. It is likely that changes in the architecture and ionic characteristics of the apical junctional region are of paramount importance to controlling permeability, but these changes have not yet been defined.

Our model appears well suited to sort out both the physiological relevance and molecular mechanisms linking Gs-coupled receptors to Src and governing regulation of the paracellular permeability.


    ACKNOWLEDGEMENTS

This research was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-19984, the Gonzales Foundation, and the Medical and Research Services of the Department of Veterans Affairs.


    FOOTNOTES

Address for reprint requests and other correspondence: A. H. Soll, VA Wadsworth Hospital Center, Bldg. 115, Rm. 215, 11301 Wilshire Blvd., Los Angeles, CA 90073 (E-mail: asoll{at}ucla.edu).

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.

July 3, 2002;10.1152/ajpgi.00429.2001

Received 5 October 2001; accepted in final form 7 June 2002.


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ABSTRACT
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RESULTS
DISCUSSION
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Am J Physiol Gastrointest Liver Physiol 283(4):G893-G899




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