Intestinal adaptation and enterocyte apoptosis following small bowel resection is p53 independent

Cathy E. Shin1, Richard A. Falcone Jr.1, Christopher J. Kemp1, Christopher R. Erwin1, David A. Litvak2, B. Mark Evers2, and Brad W. Warner1

1 Division of Pediatric Surgery, Children's Hospital Medical Center, Department of Surgery, University of Cincinnati College of Medicine, Cincinnati, Ohio 45229-3039; and 2 Department of Surgery, University of Texas Medical Branch, Galveston, Texas 77555-0533


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Adaptation following small bowel resection (SBR) signals enterocyte proliferation and apoptosis. Because p53-induced p21waf1/cip1 may be important for apoptosis in many cells, we hypothesized that these genes are required for increased enterocyte apoptosis during adaptation. Male C57BL/6 (wild-type) or p53-null mice underwent 50% proximal SBR or sham operation (bowel transection-reanastomosis). Adaptation (DNA-protein content, villus height-crypt depth, enterocyte proliferation), appearance of apoptotic bodies, and p53 and p21waf1/cip1 protein expression were measured in the ileum after 5 days. Adaptation was equivalent after SBR in both wild-type and p53-null mice as monitored by significantly increased ileal DNA-protein content, villus height, and enterocyte proliferation. The number of crypt apoptotic bodies increased significantly after SBR evenly in both wild-type and p53-null mice. In the p53-null mice, SBR substantially induced the expression of p21waf1/cip1 protein in villus enterocytes. The p53-independent induction of p21waf1/cip1 may account for the similar intestinal response to SBR between wild-type and p53-null mice. Intestinal adaptation and increased enterocyte apoptosis following intestinal resection occur via a p53-independent mechanism.

enterectomy; mice; intestinal resection; short-gut syndrome; programmed cell death


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

EPITHELIAL CELLS THAT LINE the intestine are in a continuous state of turnover as multipotent crypt stem cells generate enterocytes, which ascend to the top of the villus and are ultimately extruded into the intestinal lumen. For maintenance of homeostasis, the balance between the rate of enterocyte production and loss must be tightly regulated. Neoplasia may result from situations in which this balance is shifted, as may occur when rates of cell production exceed disposal or rates of cell elimination fall below rates of production. Alternatively, mucosal atrophy may occur if the rate of cell production is reduced below the rate of loss or the rate of cell loss is increased beyond the rate of production.

The rate of enterocyte proliferation may be accelerated by a number of stimuli; however, the greatest provocation spawning the largest body of data is partial intestinal resection (33). The compensatory response of the remnant intestine to massive small bowel resection (SBR) is termed adaptation and morphologically consists of augmented mucosal surface area due to taller villi, deeper crypts, and increased intestinal caliber and length (13, 14, 33, 44). Incomplete intestinal adaptation results in the need for parenteral nutrition for the remainder of the patient's lifetime. Enhancing adaptation in these patients with what has been dubbed the short-gut syndrome may allow for earlier and more successful weaning from parenteral nutrition and its associated cost and morbidity. Although it is acknowledged that adaptation following massive SBR results in increased enterocyte proliferation, the rate or mechanism(s) for enterocyte loss during this process is not entirely clear. A thorough understanding of this side of the balance is requisite to the development of useful therapeutic strategies designed to amplify the adaptive response.

There is growing evidence to suggest that under normal conditions, apoptosis and not simple exfoliation of enterocytes from the villus tips accounts for the majority of cell loss into the gut lumen (10, 12, 29, 30). After a 50% SBR in mice, our laboratory has recently reported increased numbers of apoptotic bodies in both crypts and villi, suggesting increased rates of apoptosis (16). Although extremely complex and not well understood, the p53 tumor suppressor gene has been implicated as a principal mediator of apoptosis in many cell types under various conditions of cellular stress (reviewed in Ref. 2). Indeed, the increased apoptosis in the gastrointestinal tract following irradiation is believed to occur at least in part via a p53-dependent mechanism (23, 24). One mechanism for the effect of p53 on the cell cycle is via the induction of p21waf1/cip1, a G1 cyclin-dependent kinase inhibitor (7, 15). At this point, cells presumably repair damaged DNA prior to progressing to replication or undergo apoptosis if the DNA damage cannot be repaired. The role that these genes play during the adaptive response of the gut to massive intestinal resection has not previously been investigated.

To directly test the significance of various genes considered important in the initiation and/or progression of intestinal adaptation, our laboratory has developed a murine model for massive (50%) intestinal resection and adaptation (18). With this unique model, we have performed intestinal resections on normal (i.e., wild-type) and homozygous p53-null mice. These experiments were designed to test the hypothesis that the p53 gene plays a critical role during the increased enterocyte apoptosis and the overall response of the gut mucosa to massive intestinal loss.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Animals. Breeder pairs of heterozygous p53-null mice and the corresponding background strain (wild-type), C57BL/6, were obtained from Jackson Laboratory (Bar Harbor, ME). The offspring were earclipped and genotype was determined by PCR (20). Homozygous p53-null or wild-type male mice (wt range 25-29 g) were selected and housed separately in groups of four at 21°C on 12:12-h day-night cycles (6 AM to 6 PM). Two days before the operation, the diet was changed from regular chow to liquid rodent diet (Micro-Stabilized Rodent Liquid Diet LAD 101/101A, Purina Mills, St. Louis, MO). These studies were approved by the Children's Hospital Research Foundation Institutional Animal Care and Use Committee.

Experimental design. Wild-type and homozygous p53-null mice were randomized to undergo either a 50% proximal SBR or sham operation. Parameters of intestinal adaptation (DNA and protein content, villus height, crypt depth, enterocyte proliferation) and p53 and p21waf1/cip1 protein levels were analyzed in the remnant ileum, and immunohistochemistry was performed to localize the expression of p21waf1/cip1 on postoperative day 5.

Operative procedure. Details of this procedure have already been presented (18). Briefly, the mice were anesthetized using a continuous flow of 2% isoflurane, 90% oxygen, and 4% carbon dioxide. With the aid of an operating microscope (×10-15 magnification) and through a midline abdominal incision, the small bowel was transected 12 cm proximal to the ileocecal valve. Mice undergoing sham operation had division and reanastomosis of the bowel only. In mice undergoing SBR, ~12 cm of proximal intestine was resected (~50% enterectomy) and an anastomosis was performed. Water was provided ad libitum for the first 24 h, and mice from each group were subsequently pair fed with liquid diet.

Tissue harvest. Mice were killed with an intramuscular injection of ketamine, xylazine, and acepromazine (4:1:1) followed by cervical dislocation. After the mice were killed, the anastomosis was identified and the first 1-cm distal to the anastomosis was discarded. In the remaining ileum, the luminal contents were gently expressed with cotton swabs. The first centimeter of ileum was fixed with 10% neutral-buffered formalin for histology, and the remaining bowel was used for determination of DNA and protein content.

Ileal DNA and protein content. Individual samples of ileum, ~2 cm distal to the anastomosis, were immediately placed in liquid nitrogen at the time of harvest. The samples were later homogenized in saline (PowerGen; Fisher Scientific, Pittsburgh, PA) and DNA and protein content were determined and expressed per centimeter of ileum as previously described (18).

Histology. Villus height and crypt depth were measured in hematoxylin and eosin-stained tissue sections (5 µm), without the investigator's knowledge as to experimental group, using a video-assisted integrated computer program (Image 1.57 TV; National Institutes of Health, Bethesda, MD) (18). At least 15 villi and crypts were counted per sample. Villi were chosen that had complete visualization of the central lymphatic channel and crypts in which the crypt-villus junction on both sides of the crypt were identified.

Proliferative index. One hour before they were killed, mice were given an intraperitoneal injection of 5-bromo-2-deoxyuridine (BrdU; 1 ml/100 g body wt; Zymed Laboratories, San Francisco, CA). Incorporation of BrdU into proliferating crypt cells (S phase) in tissue sections was used to derive an index of the rate of crypt cell proliferation as we have previously described (18). The crypt cell proliferation index was derived from the ratio of crypt cells incorporating BrdU to the total number of crypt cells. Fifteen representative crypts were counted (ability to visualize the crypt-villus junction on both sides of the crypt) from each animal. The investigator was blinded as to the origin of the tissue section during the scoring procedure.

Apoptotic index. Apoptosis was quantitated by scoring the number of apoptotic bodies identified in the crypts of hematoxylin and eosin-stained histological sections (16) by criteria that included the presence of pyknotic nuclei, condensed chromatin, and nuclear fragmentation. Counting the number of apoptotic bodies per crypt derived an apoptotic index. This method is different from a previous study in which we used immunohistochemical labeling of DNA strand breaks and confirmed morphologically using propidium iodide staining (16). We chose the current method based on its simplicity, the high frequency of nonspecific staining associated with the labeling of DNA strand breaks (25), as well as the suggestion of greater reliability using morphological criteria (22). Blinded scoring of 50-100 crypts per mouse was performed in duplicate by two separate investigators.

p53 and p21waf1/cip1 Protein expression. Frozen ileal segments were ground with mortar and pestle under liquid nitrogen and then homogenized in lysis buffer [PBS with 1% nonylphenoxy polyethoxy ethanol (NP-40), 0.5% sodium deoxycholate, 0.1% SDS, complete protease inhibitor cocktail (Boehringer Mannheim, Indianapolis, IN), and 2 mM phenylmethylsulfonyl fluoride at 4°C]. Samples were transferred to microfuge tubes and centrifuged at 14,000 rpm for 30 min at 4°C. The supernatant was collected, and the samples were recentrifuged at 14,000 rpm for 30 min at 4°C. The supernatant was collected and stored at -70°C. Samples were thawed just prior to use, and protein concentrations were determined. Samples (200 µg/lane) were resolved by 10% SDS-PAGE and transferred to Immobilon-P membranes (Millipore, Bedford, MA) as described previously . Membranes were blocked (5% nonfat milk, 80 mM Tris, pH 8.0, 50 mM NaCl, 2 mM calcium chloride) with 0.2% Tween 20 overnight, and then incubated with rabbit polyclonal p21 (Oncogene Research, Cambridge, MA) or polyclonal goat p53 (Santa Cruz Biotechnology, Santa Cruz, CA) primary antibody. After the membranes were washed three times in Tris-buffered saline (80 mM Tris, pH 7.4, and 50 mM NaCl) with 0.05% Tween 20, they were incubated with horseradish peroxidase-conjugated anti-rabbit (p21) or anti-goat (p53) secondary antibody (Santa Cruz) for 45 min and then washed. Membranes were visualized by using enhanced chemiluminescence detection. The blots were then stripped and incubated with primary antibody for the constitutively expressed protein beta -actin (Santa Cruz) to ensure equal loading of intact protein for both p21 and p53 blots.

Immunohistochemistry. Immunolocalization of p21waf1/cip1 protein was done on formaldehyde-fixed paraffin-embedded tissue after the method of Wilson et al. (35). A rabbit polyclonal anti-p21waf1/cip1 IgG antibody (Oncogene Research) was used as the primary antibody and a Vector Laboratories ABC Staining Kit (Burlingame, CA) for secondary antibody and detection using the following modifications. Before exposure to the primary antibody, antigen retrieval by the heat-treatment method was done on slide sections by microwaving for 5 min in 10 mM sodium citrate two times, cooling for 20 min, rinsing with deionized water three times, and then placing in 70% EtOH for 5 min. To inactivate endogenous peroxidase, slides were covered in methanol containing 0.5% H2O2 for 30 min and washed two times for 5 min with distilled water. Staining without the primary antibody was performed and served as a negative control.

Statistical analysis. Results are presented as means ± SE. Statistical differences were determined using ANOVA followed by a pairwise multiple comparison Student-Newman-Keuls method using the SigmaStat statistical package (Jandel Scientific, San Rafael, CA). P < 0.05 was considered significant.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Intestinal adaptation is not affected after SBR in p53-null mice. Both wild-type and p53-null mice underwent either a 50% proximal SBR or sham operation using our previously described technique (18). Five days after operation, mice were killed and the remnant ileum was removed for analysis. Survival was not significantly different between any groups of the p53-null or wild-type mice when they were killed. The survival for background mice that underwent sham operation was 17 of 18 (94%) and 19 of 20 (95%) following SBR. The survival for homozygous p53-null mice was 6 of 7 (86%) after sham-operation and 12 of 13 (92%) after SBR. The surviving mice appeared healthy, were eating liquid diet, and showed no evidence for operative complication (e.g., obstruction).

We first determined whether there were differences in intestinal adaptation between wild-type and p53-null mice after SBR by measuring changes in DNA and protein content in the remnant ileum. The expected significant increases in DNA (Fig. 1) and protein content (Fig. 2) were observed after SBR in the background mice. A similar increase in both of these parameters occurred after SBR in the p53-null group. There were no differences in these parameters after SBR between wild-type and p53-null mice, suggesting that p53 expression is not required for intestinal adaptation to occur after SBR.


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Fig. 1.   DNA content (means ± SE) in remnant ileum 5 days following either 50% proximal small bowel resection (SBR) or sham operation (transection of intestine with reanastomosis alone). Mice were either background wild-type strain or homozygous p53-null genotype. NS, not significant. * P < 0.05 vs. sham groups.



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Fig. 2.   Protein content (means ± SE) in remnant ileum 5 days following either 50% proximal SBR or sham operation. * P < 0.05 vs. sham groups.

To assess whether there were histological differences in the ileum of the two groups of mice, morphometric measurements of villus height and crypt depth were performed. Consistent with the changes in ileal DNA and protein content, SBR in both the wild-type and p53-null mice resulted in significant increases in villus height compared with the corresponding sham-operated controls; there were no differences between either the wild-type or p53-null mice in their response to SBR (Fig. 3). Although the mean values for crypt depth were increased after SBR, they did not achieve statistical significance in either the wild-type or p53-null mice (Fig. 4). Enterocyte proliferation was significantly increased in the ileum following SBR in both background and p53-null groups (Fig. 5). There were no statistical differences between background or p53-null mice after either sham operation or SBR.


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Fig. 3.   Villus height (means ± SE) measurements in remnant ileum 5 days following either 50% proximal SBR or sham operation. Minimum of 15 villi was measured per sample. * P < 0.05 vs. sham saline groups.



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Fig. 4.   Crypt depth (means ± SE) measurements in remnant ileum 5 days following either 50% proximal SBR or sham operation. Minimum of 15 crypts was measured per sample.



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Fig. 5.   Enterocyte proliferation at 5 days following either 50% proximal SBR or sham operation. Proliferative index (means ± SE) was derived by blinded counting the number of enterocytes per crypt that incorporate 5-bromo-2-deoxyuridine divided by total number of cells in crypt. * P < 0.05 vs. sham saline groups.

These findings demonstrate that knockout of the p53 gene does not affect the adaptive response that occurs in the remnant mucosa following massive intestinal resection.

Apoptosis increased after SBR in p53-null mice. Because p53 has been implicated in the apoptosis of various cells and tissues, we next determined whether apoptosis was altered in the p53-null mice after SBR. The frequency of apoptotic bodies was increased in both wild-type mice and p53-null mice after SBR (Fig. 6). The differences were statistically significant in the p53-null group. Although not statistically different in the wild-type group, the trend was the same and we have previously demonstrated significantly increased rates of apoptosis in both crypts and villi of the remnant ileum after SBR in wild-type mice (16). Overall, these results indicate that apoptosis is not affected in the p53-null mice after SBR, thus suggesting that this process is occurring by a p53-independent mechanism.


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Fig. 6.   Enterocyte apoptosis at 5 days following either 50% proximal SBR or sham operation. Apoptotic index (means ± SE) was determined by counting the number of apoptotic bodies per crypt. Blinded scoring of 50-100 crypts per mouse was performed in duplicate by 2 independent investigators. * P < 0.05 SBR vs. sham.

p21waf1/cip1 Protein levels are induced in p53-null mice after SBR. Because the cyclin-dependent kinase inhibitor p21waf1/cip1 is considered to be one important downstream mediator of p53-induced apoptosis and integral in the control of proliferation in a number of cells, we next determined the effect of SBR on p21waf1/cip1 protein levels in the ileum of p53-null mice. High levels of p21waf1/cip1 protein were noted in the remnant ileum of sham-operated wild-type mice; these levels did not significantly change with SBR (Fig. 7). In striking contrast, minimal expression of p21waf1/cip1 protein was noted in the ileum of sham-operated p53-null mice. These levels markedly increased after SBR to reach similar levels as noted in the wild-type mice. The expression of this protein was immunolocalized to villus enterocytes (Fig. 8).


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Fig. 7.   Western blot for p21waf1/cip1 and p53 protein expression in ileum of either wild-type (C57BL/6) or p53-null mice at 5 days following either 50% proximal SBR or sham operation. Expression of beta -actin protein was determined to normalize for protein loading.



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Fig. 8.   Expression of p21waf1/cip1 in ileum of either wild-type (C57BL/6) or p53-null mice at 5 days following 50% proximal SBR. p21 expression was localized to villus enterocytes taken from the p53-null mice (arrows) but not identified in the wild-type mice.

As a control, the blot was stripped and reprobed with the antibody to p53. As expected, no p53 protein was detected after either sham operation or SBR in the ileum of the p53-null mice. Levels of p53 protein were detected in the ileum of the wild-type mice after sham operation; these levels actually decreased after SBR. As a control for protein loading, the blot was then stripped and reprobed with an antibody to beta -actin, which demonstrated relatively equal protein loading in all lanes.

Taken together, these findings demonstrate for the first time that despite no apparent expression of p21waf1/cip1 protein in the ileum after sham operation in p53-null mice, induction of p21waf1/cip1 occurs after SBR in differentiating villus enterocytes. The induction of this protein may explain why there were no discernible differences in parameters of adaptation (DNA or protein content), morphology (villus height or crypt depth), enterocyte proliferation, or apoptosis between p53-null and wild-type mice following SBR.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In this study, we analyzed intestinal adaptation and apoptosis in a murine model of intestinal resection using either wild-type or p53-null mice. We have first shown that the adaptive response of the intestine to SBR (increases in DNA and protein content, villus height and crypt depth measurements, and enterocyte proliferation) is preserved in p53-null mice. Second, the increase in apoptosis associated with intestinal adaptation appears to occur despite absent p53 expression. Finally, we have identified induced expression of p21waf1/cip1 protein in differentiating enterocytes of the p53-null mice following SBR. Taken together, these results suggest that intestinal adaptation after massive enterectomy and the increased apoptosis that occurs during this response takes place via a p53-independent mechanism.

Inasmuch as apoptosis was significantly increased after SBR in the p53-null mice to the same extent as we have previously described in wild-type p53-expressing mice (16), it would appear that the apoptosis during intestinal adaptation occurs by a mechanism that is p53 independent. Apoptosis has been induced via p53-independent pathways in varied cells and tissues using several stimuli, including glucocorticoids, calcium activation, and in vitro cellular aging (3), mutations of Ras (4), treatment with TGF-beta (19) or interferon-gamma (27), and as a delayed response to high-dose irradiation (23). Specifically with regard to the intestine, other conditions of p53-independent induction of apoptosis include hyperthermia (11) and ischemia-reperfusion (5). The specific cellular pathway for p53-independent apoptosis following SBR is presently unclear but may involve Rho proteins (8) or various members of the caspase family (36). It will be important in future studies of intestinal adaptation to consider these other mechanisms.

The significance of the magnitude of increase in the appearance of apoptotic bodies in the crypts after SBR is presently unclear. Although the increase in apoptotic index was modest (~25%), this closely matched the degree of increase in proliferation (~23%) that we observed after SBR. Thus this correlation would support an important physiological role for the rate of apoptosis to counterbalance the rate of proliferation during the mitotic stimulus of adaptation. Furthermore, the increased appearance of apoptotic bodies is primarily observed in the stem-cell region of the crypt (28). Since a single crypt stem cell may give rise to 60-120 cells (30), only modest changes in rates of apoptosis in crypts would be necessary to result in dramatic changes in crypt morphology. These factors all suggest that the increased apoptosis that occurs during intestinal adaptation is a critical response.

Intestinal adaptation was associated with a significant increase in the expression of p21waf1/cip1 protein in the p53-null mice. Although p21waf1/cip1 gene activation is one downstream effector of p53 (7), other factors have been described to induce p21waf1/cip1 gene expression, which are p53 independent (9, 21, 26). It is possible that intestinal resection sets the stage for mitogen-activated cell-cycle entry due to the stimulus for increased proliferation, an event that has been shown to induce p21waf1/cip1 gene activation in the absence of p53 (26). Alternatively, p21waf1/cip1 may act as a brake to halt enterocyte proliferation beyond a specific level following SBR. This has been the proposed mechanism for increased p21waf1/cip1 protein levels measured in the regenerating liver following partial hepatectomy in rats (1, 6) and p53-null mice (1). It is also possible that p21waf1/cip1 is necessary for the newly produced enterocytes to arrest in G1 phase to differentiate (21, 31, 32) and may account for the localization of expression that we observed in differentiating villus enterocytes.

It must be considered that in the absence of p53, increased p21waf1/cip1 protein may be involved in the increased apoptosis that occurs following SBR. Indeed, in adriamycin-treated human T-cell leukemia virus type I-transformed cell lines with inactive p53 protein, increased expression of p21waf1/cip1 protein has been correlated with increased apoptosis (9). This may offer a partial explanation as to why we observed absent p21waf1/cip1 protein in the intestine of the p53-null mice following sham operation that was induced following SBR.

At present, we cannot account for why p53 protein disappeared after SBR in the wild-type mice. Because p53 does not appear to be required for adaptation or for the increase in apoptosis following SBR, it is possible that there is a reprioritization of the synthesis of specific proteins to mediate these responses. As a result, one of these expression responses could have involved attenuating p53 expression. Alternatively, with the greater rate of enterocyte turnover induced by intestinal resection, the cells undergoing apoptosis and expressing p53 may have been extruded into the lumen with a greater frequency, thus leaving less detectable p53 protein in the intestinal wall homogenates. Further studies will be required to explain these results.

Understanding the exact mechanism of apoptosis during the adaptive response of the intestine to SBR may pave the way toward novel and effective therapy as a means to amplify this important response. Our laboratory has previously observed enhanced adaptation and attenuated apoptosis in the intestine following SBR by the exogenous administration of epidermal growth factor (EGF) (17). In that study, rates of apoptosis were highest after SBR in mutant strain of mice with reduced EGF-receptor tyrosine kinase activity (waved-2 mice). These observations support an important relationship between EGF receptor signaling, enterocyte proliferation, apoptosis, and intestinal adaptation. Futures studies designed to elucidate the complex apoptosis systems in the intestine during adaptation may prove useful clinically to reduce parenteral nutrition requirements and improve survival of patients with the short-gut syndrome.


    ACKNOWLEDGEMENTS

We gratefully acknowledge the assistance of Dr. David P. Witte and Pamela Groen, Dept. of Pathology, Children's Hospital Medical Center, in the performance of the p21 immunohistochemistry.


    FOOTNOTES

This work was supported by a Trustees Grant from the Children's Hospital Research Foundation, Children's Hospital Medical Center, and the National Institute of Diabetes and Digestive and Kidney Diseases Grants RO1-DK-53234 (to B. W. Warner) and RO1-DK-48498 (to B. M. Evers).

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. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: B. W. Warner, Div. of Pediatric Surgery, Children's Hospital Medical Center, 3333 Burnet Ave., Cincinnati OH 45229-3039 (E-mail: brad.warner{at}uc.edu).

Received 11 August 1998; accepted in final form 9 June 1999.


    REFERENCES
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Gastroint Liver Physiol 277(3):G717-G724
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