Liver regeneration is transiently impaired in urokinase-deficient mice

Heather T. Roselli1, Ming Su2, Kay Washington3, David M. Kerins2, Douglas E. Vaughan1,2, and William E. Russell4

Departments of 1 Pharmacology, 2 Medicine, 3 Pathology, and 4 Pediatrics and Cell Biology, Vanderbilt University Medical Center and Veterans Affairs Medical Center, Nashville, Tennessee 37232

    ABSTRACT
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Abstract
Introduction
Methods
Results
Discussion
References

To test the hypothesis that urokinase-type plasminogen activator (uPA) plays an important role in liver regeneration in vivo, partial hepatectomy was performed on wild-type and uPA-deficient (uPA-/-) mice. Mice were studied at 24, 44, and 96 h and at 8 days and 4 wk post-partial hepatectomy for evidence of regeneration, as measured by mitotic indexes and [3H]thymidine incorporation. In wild-type mice, thymidine incorporation peaked at 44 h and this index was reduced by 47% in uPA-/- mice (P = 0.02). By 8 days, however, liver mass was comparable in both groups. Histological analysis revealed the presence of focal areas of fibrin deposition and cellular loss by 24 h that were more severe and prevalent in uPA-/- mice than in wild-type mice (62 and 23%, respectively; chi 2 = 3.939, P = 0.047). In contrast, regeneration was not impaired in uPA receptor (uPAR)-deficient mice at 24 and 44 h. Taken together, these data indicate that uPA, independent of its interaction with the uPAR, plays an important role in liver regeneration in vivo.

proteolysis; apoptosis; extracellular matrix; partial hepatectomy; urokinase-type plasminogen activator receptor; DNA synthesis

    INTRODUCTION
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Abstract
Introduction
Methods
Results
Discussion
References

LIVER REGENERATION IS a complex phenomenon that involves multiple parallel and sequential processes that are normally integrated to provide for the timely and complete restoration of liver mass following injury (7, 18, 35, 42). In addition to the elaboration of signals to initiate the coordinated proliferation of the various cellular compartments within the liver, there must be extensive restructuring of the extracellular matrix (ECM), maintenance of complex differentiated functions, and a timely restraint of the proliferative process to prevent overgrowth or neoplasia. Early studies had suggested the existence of circulating hepatic growth regulators acting through classic endocrine pathways (19, 37), whereas more recent studies have proposed that growth factors and cytokines produced within the liver and acting through paracrine pathways (reviewed in Refs. 7, 18, 35, and 42), are important regulators of liver regeneration. However, these two modes of regulation are not mutually exclusive: the nonproliferating liver is a repository of significant amounts of many of these growth-regulating agents in membrane- or matrix-anchored latent forms. Proteolytic activation of these proteins to release both endocrine and paracrine factors may be a significant mode of growth regulation during regeneration. In particular, latent or membrane-anchored forms of hepatocyte growth factor (HGF), transforming growth factor-alpha (TGF-alpha ), and TGF-beta have been shown to be concentrated in the liver (23, 33, 41).

Considerable circumstantial evidence has suggested that urokinase-type plasminogen activator (uPA) may play a key role in liver regeneration. uPA converts plasminogen to plasmin, which in turn promotes matrix degradation by activating matrix metalloproteinases (MMPs). Urokinase has the capacity to activate HGF (32, 38) and, via the generation of plasmin, also activates latent TGF-beta (20). Therefore, uPA activity may be required not only to initiate the breakdown of ECM but also to cleave inactive HGF to initiate hepatocellular proliferation (35) and, perhaps, to activate TGF-beta to restrain proliferation. In support of this model, uPA activity and the expression of urokinase-type plasminogen activator receptor (uPAR) increase within 5 min of partial hepatectomy (PH) in the rat (31). In addition, there is extensive degradation of ECM (24), generation of plasmin (24), and increased plasma levels of HGF (29) and TGF-beta (34) within minutes of PH, even though levels of HGF (53) and TGF-beta (5) mRNA do not change in the liver or other tissues for several hours. Direct evidence that uPA is critical for liver regeneration has not been reported, however. In this study, we test the hypothesis that uPA is necessary for liver regeneration in vivo by studying hepatic proliferation after PH in wild-type and uPA-deficient (uPA-/-) mice.

    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

Mice. Homozygous uPA-deficient mice, homozygous uPAR-deficient mice, as well as littermate controls were kindly provided by P. Carmeliet (Univ. of Leuven, Belgium) and have been previously described (9, 14). Animals were maintained on a 12:12-h light-dark cycle and fed a normal lab chow diet with ad libitum access to water. All procedures involving animals were performed according to the guidelines set forth by the Animal Use and Care Committee of Vanderbilt University.

Partial hepatectomy. PH (70%) was performed on male mice (27.9 ± 5.2 g) under methoxyflurane anesthesia (Metofane; Pittman-Moore, Chicago, IL) by ligating and removing the left lateral and median lobes, including the gallbladder. Warm isotonic saline (0.5 ml) was deposited into the peritoneum before closure. To control for the diurnal variation in liver DNA synthesis (3), all livers were harvested between 10 AM and 2 PM. To measure hepatocyte labeling index, [3H]thymidine (1 µCi/g body wt; 6 Ci/mmol; NEN, Wilmington, DE) was injected into the peritoneum under light anesthesia 2 h before the animal was killed. Aliquots of liver tissue were then snap frozen in liquid nitrogen or placed in 10% Formalin for paraffin embedding.

[3H]thymidine incorporation. [3H]thymidine incorporation into liver DNA was measured as described previously (40). Briefly, 100 mg of liver tissue were homogenized in 19 volumes of ice-cold 5% TCA followed by three washes in ice-cold 5% TCA. Nucleic acids were hydrolyzed in 1.5 N perchloric acid for 15 min at 90°C. Cleared hydrolysate was then mixed with Bioflour purchased from NEN (Boston, MA), and incorporated [3H]thymidine was counted. DNA content was measured by diphenylamine assay as described by Richards (39). Results are reported as counts per minute (cpm) per milligram of DNA ± SE. All statistical analyses were carried out using GraphPad Prism. Student's t-test was applied, and significance was defined as P < 0.05.

Nuclear labeling index. A labeling index was generated from 6-µm Formalin-fixed, paraffin-embedded sections of liver tissue that were dipped in NTB2 photographic emulsion (Kodak, New Haven, CT) and exposed for at least 6 wk at 4°C. The sections were then developed, fixed, and counterstained with Mayer's hematoxylin. Hepatocyte labeling index (% of hepatocytes) was calculated by counting labeled and unlabeled hepatocyte nuclei in three high-power fields (~300 hepatocyte nuclei/field).

Histology. Formalin-fixed, paraffin-embedded 6-µm representative sections of liver tissue from wild-type, uPA-/-, and uPAR-/- mice were stained with hematoxylin and eosin for general morphology, with Oil Red O for fat, and with Lendrum's stain for fibrin.

Apoptosis assay. Apoptosis was measured on 6-µm Formalin-fixed, paraffin-embedded sections of liver tissue using the ApopTag Plus Fluorescein in situ detection kit obtained from Oncor (Gaithersburg, MD) according to the manufacturer's directions. Negative controls were performed by substituting water for the transferase enzyme on an adjacent section. Apoptotic nuclei were visualized by fluorescence microscopy.

    RESULTS
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Abstract
Introduction
Methods
Results
Discussion
References

To test the hypothesis that uPA plays an important role in liver regeneration in vivo, PH was performed on wild-type and uPA-deficient (uPA-/-) mice. Thymidine incorporation into DNA was measured in groups of wild-type and uPA-/- mice (7 <=  n <=  14) at 24, 44, and 96 h and at 8 days and 4 wk following PH. Before PH was performed, the rate of [3H]thymidine incorporation did not differ between uPA-/- and wild-type mice (data not shown). Consistent with previous reports of liver regeneration in mice (10, 52), DNA synthesis in wild-type mice remained low at 24 h, peaked at 44 h, and then gradually declined to baseline between 8 days and 4 wk (Fig. 1A). uPA-/- mice showed a similar pattern of DNA synthesis. There was negligible DNA synthesis at 24 h, a peak at 44 h, and a less rapid decline over the subsequent time points. There was a significant difference in the rate of DNA synthesis in the two groups, with [3H]thymidine incorporation reduced by 47% (P = 0.02) in the uPA-/- mice vs. wild-type mice at 44 h. To confirm this early reduction in DNA synthesis, a nuclear labeling index was determined from liver sections exposed to photoemulsion (Fig. 1B). Labeled and unlabeled hepatocytes were counted, and the percentage of labeled nuclei was calculated. In wild-type mice, the nuclear labeling index was 1.05 ± 0.28% at 24 h, which peaked at 24.9 ± 2.02% at 44 h and declined to near baseline by 8 days following PH, paralleling the data on [3H]thymidine incorporation. In the uPA-/- mice, however, the peak of nuclear labeling was decreased by 31% (P = 0.02) at 44 h, was slightly decreased at 96 h, and was still elevated at 8 days (224%) compared with control mice.


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Fig. 1.   A: DNA synthesis following partial hepatectomy in wild-type and urokinase-type plasminogen activator-deficient (uPA-/-) mice. [3H]thymidine incorporation [measured in counts/min (cpm) per mg DNA] was measured 24, 44, and 96 h and at 8 days and 4 wk following partial hepatectomy in wild-type and uPA-/- mice. Solid line, wild-type mice; dashed line, uPA-/- mice. ** P = 0.02. B: nuclear labeling index following partial hepatectomy in wild-type and uPA-/- mice. A nuclear labeling index was calculated 24, 44, and 96 h and 8 days following partial hepatectomy in wild-type and uPA-/- mice. Solid line, wild-type mice; dashed line, uPA-/- mice. ** P = 0.02. C: autoradiograph of a 44-h regenerating wild-type liver following a 2-h pulse of [3H]thymidine. Tissue was counterstained with hematoxylin. D: autoradiograph of a 44-h regenerating uPA-/- liver following a 2-h pulse of [3H]thymidine. Tissue was counterstained with hematoxylin.

To compare the cumulative extent of liver restoration, liver mass, expressed as total DNA normalized for body weight, was compared at two time points (Fig. 2). After PH at 8 or 24 days, no differences were identified between uPA-/- and wild-type mice.


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Fig. 2.   Comparison of liver mass following partial hepatectomy. Liver mass, expressed as total DNA normalized for body wt, was compared 8 days and 4 wk following partial hepatectomy. Open bars, wild-type mice; solid bars , uPA-/- mice.

At baseline, livers from uPA-/- mice were histologically normal and indistinguishable from livers of wild-type mice (data not shown). In addition to the effects on proliferation, three histological differences between wild-type and uPA-/- livers were noted. First, 24 h after PH, all of the wild-type livers contained fine droplets of microvesicular fat within hepatocytes (Fig. 3, A and B). Fat was present in some of the uPA-/- livers at this time point but generally in trace amounts (Fig. 3, C and E). The accumulation of fat became prominent and universal in the uPA-/- mice at 44 h following PH compared with wild-type mice (Fig. 3G). By 96 h after PH, fat accumulation was generally resolved in wild-type livers (Fig. 4A); however, an accumulation of fat was still seen at 96 h post-PH in the uPA-/- mice (Fig. 4B). The presence of fat was confirmed by Oil Red O staining (data not shown). Second, patchy areas of cellular loss, which were not associated with particular zonal locations, were present in over one-half of the uPA-/- livers at 24 h (62%) but were seen in only 23% of wild-type livers at the same time point (chi 2 = 3.939, P = 0.047; Fig. 3, C, E, and H). There was no inflammatory response associated with the areas of cell loss, which were characterized by fibrin deposits (Fig. 3H) and cytolysis of cells within the lesion, as well as nuclear pyknosis in intact cells at the periphery. Third, a difference was noted in the disappearance of thickened hepatocyte plates due to regeneration. Thickened hepatocyte plates were seen in wild-type and uPA-/- livers at 44 h, although this evidence of hepatocyte regeneration was more prominent in wild-type livers (Fig. 3, D and F). At 8 days post-PH, the hepatic plate architecture was largely normal in wild-type mice (Fig. 4C), but hepatic plates were still thickened in the uPA-/- livers (Fig. 4D). By 4 wk, hepatic plate architecture was normal in both wild-type and uPA-/- mice (Fig. 4, E and F).


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Fig. 3.   Histological characterization of liver tissue from wild-type and uPA-deficient (uPA-/-) mice 24 and 44 h following partial hepatectomy. A-G were stained with hematoxylin and eosin, and H was stained for fibrin. A: wild-type liver at 24 h (×100). B: wild-type liver at 24 h (×400). C: uPA-/- liver at 24 h (×100). Outlined box indicates area enlarged in E. D: wild-type liver at 44 h (×400). E: uPA-/- liver at 24 h (×400). F: uPA-/- liver at 44 h (×400). G: uPA-/- liver at 44 h (×400). H: uPA-/- liver at 24 h with fibrin stain (×400). Open arrow indicates mitotic figures; areas between solid arrows indicate thickened hepatic plates; asterisk indicates region of fibrin deposition.


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Fig. 4.   Histological characterization of liver tissue from wild-type and uPA-/- mice 4 and 8 days and 4 wk following partial hepatectomy. Formalin-fixed, paraffin-embedded liver sections removed 4 and 8 days and 4 wk following partial hepatectomy were stained with hematoxylin and eosin. A: wild-type liver at 4 days. B: uPA-/- liver at 4 days. C: wild-type liver at 8 days. D: uPA-/- liver at 8 days. E: wild-type liver at 4 wk. F: uPA-/- liver at 4 wk. Open arrow indicates mitotic figure; areas between solid arrows indicate thickened hepatic plates; asterisks indicate areas of fat deposition. For A-F, magnification is ×400.

Because mitogens such as HGF have been shown to protect some cell types from undergoing programmed cell death (21) and because uPA has been reported to have mitogenic effects (1, 13), we investigated whether the areas of cellular loss were associated with apoptosis. Sections of liver tissue from wild-type and uPA-/- mice at different time points following PH were subjected to in situ labeling to identify nuclei containing fragmented DNA. Basal liver sections from both wild-type and uPA-/- mice displayed virtually no apoptotic cells (data not shown). However, by 24 h post-PH, uPA-/- liver sections contained substantially more apoptotic nuclei compared with control mice, especially within and surrounding the areas of patchy cellular loss (Fig. 5). At 44 h following PH, there were virtually no apoptotic hepatocytes in either wild-type or uPA-/- liver sections. However, at 96 h post-PH, apoptotic hepatocyte nuclei were increased in uPA-/- livers compared with controls. By 8 days following PH, both wild-type and uPA-/- livers contained virtually no apoptotic nuclei (data not shown).


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Fig. 5.   Apoptotic nuclei in liver sections following partial hepatectomy in wild-type and uPA-/- mice. An in situ fluorescein procedure was used to label apoptotic nuclei in liver sections from wild-type and uPA-/- mice 24 h after partial hepatectomy. A: uPA-/- liver (×100). B: uPA-/- liver (negative control; ×100). C: uPA-/- liver (×400). D: wild-type liver (×100). E: wild-type liver (negative control; ×100). Solid arrows indicate normal hepatocyte nuclei; open arrows indicate labeled hepatocyte nuclei undergoing DNA fragmentation.

In an attempt to determine the mechanism by which uPA contributes to hepatic regeneration, groups of wild-type and uPAR-/- mice were subjected to PH. DNA synthesis at 24 and 44 h post-PH was then determined in wild-type and uPAR-/- mice. No difference was found in [3H]thymidine incorporation between wild-type and uPAR-/- mice at 24 h (12.3 ± 2.5 and 9.9 ± 1.8 cpm/mg DNA, P = 0.4389) or 44 h following PH (203.1 ± 52.8 and 329.6 ± 94.9 cpm/mg DNA, P = 0.2192) (Fig. 6), suggesting that uPA-mediated signaling events via the uPAR or enhanced activity of uPA may not be important in the regeneration process. No obvious differences in histology were noted between wild-type and uPAR-/- mice (data not shown).


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Fig. 6.   DNA synthesis following partial hepatectomy in wild-type and uPAR-/- mice. [3H]thymidine incorporation was measured 24 and 44 h following partial hepatectomy in wild-type and uPAR-/- mice.

    DISCUSSION
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Abstract
Introduction
Methods
Results
Discussion
References

Proteolysis of ECM and of cell surface proteins can elicit a variety of rapid and dramatic cellular responses (reviewed in Ref. 51). Among these responses are ECM remodeling and consequent loss of adhesion and viability signals, including apoptosis, the exposure of previously cryptic biological activities in ECM, the activation of immune pathways and the release of cytokines, the release of soluble or activated growth factors from latent or membrane-associated precursor forms, and the direct activation of growth factor receptors. uPA is a soluble serine protease whose activity is focused at the cell surface by its interactions with the uPAR (16). The uPAR, in turn, interacts with both vitronectin and with integrin beta -chains and has recently been shown to trigger intracellular signals (15, 25).

We found liver regeneration to be transiently impaired after PH in uPA-/- mice. Peak DNA synthesis, which occurred at 44 h after PH in wild-type mice, was significantly reduced in uPA-/- mice. Other evidence of delay was noted: the normal phase of fat accumulation (6), which occurred at 24 h in wild-type controls, was delayed until 44 h in the uPA-/- mice and was more prolonged. Despite this early impairment, the liver mass of uPA-/- mice was completely restored by 8 days, although the restoration of normal hepatic plate architecture was delayed until 4 wk. In addition to evidence of delayed proliferation, patchy areas of cellular loss associated with an increase in hepatocyte apoptosis and fibrin deposition were noted in uPA-/- mice 24 h after PH.

Although we have shown that the absence of uPA impairs hepatocellular proliferation, others have demonstrated that overexpression of uPA in the liver is toxic to hepatocytes and leads to subsequent regeneration (27, 28, 43). Both lines of evidence suggest that a local environment containing uPA is conducive to the proliferation of liver cells. Urokinase could contribute to the regenerative process by at least three different pathways: 1) uPA-mediated activation of latent growth factors such as HGF or TGF-beta , 2) uPA-mediated conversion of plasminogen to plasmin, leading to increased MMP activity and matrix remodeling, and 3) uPA-activated signaling via the uPAR, independent of the proteolytic activity of uPA. The availability of genetically engineered uPA and uPAR-/- mice provides a unique opportunity to determine whether uPA and its receptor are absolutely required for liver regeneration and to begin to study broader aspects of proteolysis during liver regeneration. uPA-/- mice exhibit normal fertility, growth, and life span but develop fibrin deposits in the skin and gastrointestinal tract as they age. In addition, occasional fibrin deposits have been noted in the hepatic sinusoids (9). These sinusoidal fibrin deposits may be related to the cellular loss we noted in regenerating liver from uPA-/- mice, as we rarely noted similar lesions in wild-type controls.

Urokinase is a protease that is known to cleave and activate growth factors sequestered in the liver. Urokinase activity increases within minutes of PH in rats (31), whereas the expression of plasminogen activator inhibitor type I mRNA increases within 2 h (46, 47). uPA has been shown to activate latent (single chain) HGF in vitro (38), and Lindroos et al. (29) reported a 17-fold increase in plasma HGF concentrations within 2 h of PH in the rat, consistent with a role for uPA in the release and possibly the activation of latent HGF. HGF is a potent mitogen that has been proposed to play a prominent role in the regulation of liver growth (45) and regeneration (35). Despite this circumstantial linkage of uPA activation and HGF release, Miyazawa et al. (36) failed to detect conversion of HGF to its two-chain form in the liver after PH, although they did detect activated HGF in liver lysates after CCl4 administration. Furthermore, exposure of liver to collagenase has been shown to increase its subsequent mitogenic responsiveness to HGF in the absence of PH, suggesting that proteolytic alterations in the ECM can also augment hepatocyte sensitivity to mitogens (30). Our efforts to directly measure HGF activation in uPA-/- mice after PH have thus far failed to show major differences in HGF activation due to poor cross-reactivity with mouse HGF (data not shown).

Plasmin generated by uPA in the liver is also likely to result in the activation of MMPs and of latent TGF-beta , which increases in the plasma shortly after PH (34). Recent data from Kim and Michalopoulos (24) provide evidence of extensive ECM remodeling after PH in rats. They reported increases in the conversion of plasminogen to plasmin and the degradation of fibrinogen, fibronectin, entactin, and laminin within hours of PH, but they did not establish whether plasmin acted directly on matrix proteins or activated MMPs. Previous studies have shown that MMPs, perhaps of Ito cell origin (2), are upregulated after PH or CCl4 injury to the liver (22), whereas the Ca2+-activated neutral proteinase is also upregulated after CCl4 injection (54).

Association of uPA with its receptor, the uPAR, is instrumental in enhancing its enzymatic activity (17), and the uPAR itself has been shown to activate cellular signal transduction pathways involving changes in intracellular tyrosine phosphorylation (4), diacylglycerol formation (12), and activation of the Janus kinase/signal transducer and activator of transcription pathway (15, 25). The uPAR itself contains no tyrosine kinase or transmembrane domains and attaches to the cell membrane through a glycosyl phosphatidylinositol anchor (11). It is expressed in hepatocytes, Kupffer cells, and endothelial cells (26) and is upregulated within minutes of PH (31). In a variety of different cell types, uPA acts as a mitogen (1, 13) and, by virtue of its association with the uPAR, mediates cell adhesion and migration through its interactions with integrins and vitronectin (48-50). It has been proposed that the uPAR is likely to be responsible for many, if not all, of the actions of uPA, either by its direct signaling or its localization and augmentation of the catalytic activity of uPA. However, uPAR-/- mice have a surprisingly mild phenotype. Despite impaired plasminogen activation in vitro, they exhibit normal thrombolytic activity and ECM degradation in vivo (14). These results are mirrored in our observations that uPAR-/- mice do not exhibit the aberrant regeneration seen in the uPA-/- mice. Our findings suggest that soluble rather than uPAR-bound uPA plays a predominant role in regulating liver regeneration. Alternatively, other proteins may exist that bind, localize, or activate uPA.

Given the myriad of regulatory pathways potentially activated by proteases, the contributions of uPA to the control of liver regeneration are likely to be complex and multifactorial. Our results provide no clear evidence that uPA initiates critical signals that lead to cellular proliferation. Our finding of reduced or delayed proliferation in uPA-/- mice suggests that uPA may play a permissive but not essential role in normal liver regeneration, perhaps due to the existence of alternative mechanisms that can compensate in its absence. It is also unclear whether the effects we observed are specific for uPA or if activity from a related protease such as tissue-type plasminogen activator could also play a role. Furthermore, the regulatory actions of inflammatory cytokines on the uPA system (8, 44) suggest that other models of liver injury may utilize the uPA/uPAR system in different ways. It is important to note that previous studies implicating uPA and the uPAR in the control of regeneration have been carried out in the rat, whereas our studies were performed in mice. Critical differences between mouse and rat liver regeneration may complicate the interpretation of our current findings. Elucidation of the mechanisms by which uPA influences regeneration is likely to generate models of liver repair in which mechanisms of cellular proliferation are more closely linked to those of tissue and matrix remodeling. Moreover, the potential involvement of the plasminogen activator system in states of disordered repair such as fulminant hepatic failure, liver fibrosis, or hepatocarcinogenesis warrants further investigation.

    ACKNOWLEDGEMENTS

This work was supported in part by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-53804 (W. E. Russell) and National Heart, Lung, and Blood Institute Grant HL-50878 (D. E. Vaughan) and by training grants HL-07751 and HL-07411 (H. T. Roselli) and by a Merit Award from the Dept. of Veteran Affairs Research Service (D. E. Vaughan). D. E. Vaughan is a recipient of a Clinical Investigator Award from the Dept. of Veteran Affairs Research Service.

    FOOTNOTES

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: W. E. Russell, Vanderbilt Univ., Division of Pediatric Endocrinology, T-0107 MCN, Nashville, TN 37232-2579.

Received 3 June 1998; accepted in final form 10 September 1998.

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Abstract
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Discussion
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Am J Physiol Gastroint Liver Physiol 275(6):G1472-G1479
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