Department of Medicine, University of California, San Diego, School of Medicine, San Diego, California 92103
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ABSTRACT |
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D-Myo-inositol (3,4,5,6) tetrakisphosphate [Ins(3,4,5,6)P4] or phosphatidylinositol 3-kinase (PI 3-kinase) activity acts to inhibit calcium-dependent chloride secretion in T84 colonic epithelial cells. To further distinguish between the contributions of these two signaling pathways to the inhibition of secretion, we studied effects of insulin, because the insulin receptor links to PI 3-kinase but not to pathways postulated to generate Ins(3,4,5,6)P4. Chloride secretion across T84 cell monolayers was studied in Ussing chambers. Activation of PI 3-kinase was assessed by Western blotting. Basolateral, but not apical, addition of insulin inhibited carbachol- and thapsigargin-induced chloride secretion in a time- and concentration-dependent fashion. Insulin-like growth factor-I (IGF-I) had similar effects. Insulin had no effect on Ins(3,4,5,6)P4 levels, and the inhibitory effects of insulin and IGF-I on chloride secretion were fully reversed by the PI 3-kinase inhibitors wortmannin and LY-294002. Western blot analysis showed that both insulin and IGF-I recruited the 85-kDa regulatory and 110-kDa catalytic subunits of PI 3-kinase to anti-phosphotyrosine immunoprecipitates. In conclusion, insulin and IGF-I act to inhibit calcium-dependent chloride secretion through a PI 3-kinase-dependent pathway. Because insulin is released in a pulsatile fashion postprandially and IGF-I levels are elevated in pathological settings, our findings may have physiological and/or pathophysiological significance.
insulin-like growth factor I; diarrhea; diabetes
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INTRODUCTION |
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THE SECRETION OF CHLORIDE is an important process involved in a number of physiological functions. In the intestine, chloride secretion maintains the fluidity of the gastrointestinal lumen and provides a medium for diffusion of digestive enzymes and ingested nutrients (3). This transport mechanism is tightly regulated, and diseases can result from regulatory breakdown: cystic fibrosis in the case of undersecretion and secretory diarrhea from oversecretion of chloride ions (3).
Our laboratory has been investigating the mechanism whereby epidermal growth factor (EGF) and calcium-dependent agonists inhibit calcium-dependent chloride secretion in the T84 colonic epithelial cell line. We showed previously (15, 38) that pretreatment of T84 epithelial cells with the muscarinic agonist carbachol or with EGF can inhibit subsequent calcium-dependent chloride secretion. Both carbachol and EGF inhibit chloride secretion independent of the rise in intracellular calcium but through different mechanisms. The ability of carbachol to inhibit subsequent stimulation by other calcium-dependent secretagogues appears to require activation of phospholipase C (PLC) for generation of the negative messenger D-myo-inositol (3,4,5,6) tetrakisphosphate [Ins(3,4,5,6)P4], whereas the inhibitory effects of EGF are mediated by activation of phosphatidylinositol 3-kinase (PI 3-kinase) and generation of its lipid products (Refs. 13 and 39; J. M. Uribe, S. J. Keely, and K. E. Barrett, submitted for publication). However, EGF is also capable of activating PLC in T84 cells (J. M. Uribe, S. J. Keely, and K. E. Barrett, submitted for publication) and of increasing levels of Ins(3,4,5,6)P4 to some extent (38). Therefore, whether PI 3-kinase activity can inhibit chloride secretion in the absence of a simultaneous elevation in Ins(3,4,5,6)P4 remained unresolved. Our goal was thus to clarify further the relative roles of these distinct inhibitory signaling pathways through the use of insulin and insulin-like growth-I (IGF-I).
Insulin and IGF-I are peptide hormones that are homologous in primary
structure. Insulin is a 6-kDa protein produced by the -cells of the
pancreas, which is secreted in a pulsatile manner in response to
nutritional stimuli (19). Insulin is the primary mediator
of anabolism and important in controlling glucose homeostasis. By
translocating an insulin-sensitive glucose transporter from intracellular storage vesicles to the cell surface, insulin is able to
stimulate the clearance of glucose into skeletal muscle, liver, and
adipose tissue (14, 43). IGF-I, or somatomedin C, is a
7.6-kDa peptide hormone produced mainly in the liver but also found in
many other cell types, including fibroblasts and muscle. Among its
numerous functions, IGF-I is primarily responsible for chronic
mitogenic effects such as DNA synthesis, cell division, and cell
proliferation (11).
Similar to EGF, both insulin and IGF-I activate cellular tyrosine phosphorylation on binding to their respective tyrosine kinase receptors. They also stimulate the activation of PI 3-kinase in different cell types. Our laboratory previously demonstrated (17, 18) a relationship between tyrosine phosphorylation and inhibition of chloride secretion. In addition, other groups have shown that insulin and IGF-I are capable of increasing the transepithelial permeability of T84 cell monolayers over 3-4 days (22, 23) and that, as shown by radioligand binding studies and receptor cross-linking studies, T84 cells express receptors for these peptides (24). IGF-I has also been shown to have a variety of effects in the gastrointestinal tract, including stimulation of intestinal growth and absorptive function (2, 12, 26, 29). Therefore, our goal in this study was to determine whether insulin and/or IGF-I were capable of inhibiting calcium-dependent chloride secretion. At least in some cell types, neither insulin nor IGF-I activates PLC (6). If this was also the case in intestinal epithelial cells, these peptides would allow us to test whether the activation of PI 3-kinase-dependent pathways, in the absence of increases in Ins(3,4,5,6)P4, is sufficient to inhibit calcium-dependent chloride secretion. Furthermore, an effect of these peptides in regulating epithelial chloride secretion might have important physiological and/or pathophysiological implications and could provide insights into underlying mechanisms whereby these peptides modulate ion transport responses in intact intestinal tissues (2, 12).
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METHODS |
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Materials. The following materials were purchased from the sources indicated: carbachol, wortmannin, insulin, polyclonal biotin-conjugated anti-rabbit IgG, and polyclonal rabbit antibodies to the p85 subunit of PI 3-kinase (Sigma, St. Louis, MO); polyclonal antibodies to the p110 subunit of PI 3-kinase (Santa Cruz Biotechnology, Santa Cruz, CA); human recombinant IGF-I (Genzyme, Cambridge, MA); thapsigargin (LC Laboratories, Woburn, MA); protein A agarose, anti-phosphotyrosine antibodies, and antibodies to PI 3-kinase p85 subunit (Upstate Biotechnology, Lake Placid, NY); protein A Sepharose (Pierce, Rockford, IL); and LY-294002 (Biomol, Plymouth Meeting, PA).
Cell culture. Methods for the growth and maintenance of T84 cells for use in transepithelial electrolyte transport studies have been described previously (7). In brief, T84 cells were grown on collagen-coated polycarbonate filters (Nuclepore, Pleasanton, CA) or on 6-mm Millicell HA inserts (Millipore, Bedford, MA) for 7 days before experiments. For experiments performed using serum free-media, cells were maintained in DME-F12 medium (JRH, Lenexa, KS) with 5% newborn calf serum for 5 days as described above and then changed to bronchial epithelial growth medium serum-free medium (Clonetics, San Diego, CA) supplemented with 1 mM CaCl2 for 2 days before the experiment. For the Western blotting experiments, T84 cells were grown on 30-mm Millicell HA inserts for 10 days before experiments.
Chloride secretion.
Studies of transepithelial chloride secretion were performed using
monolayers of T84 cells mounted in Ussing chambers modified for use
with cultured cells. Short-circuit current (Isc)
was used to quantitate active transepithelial chloride secretion.
Previous studies showed that T84 cells secrete chloride in response to carbachol and thapsigargin and that the resulting changes in
Isc are wholly reflective of the amounts of
chloride secretion induced by the secretagogues.
Isc measurements were carried out in oxygenated Ringer solution containing (in mM) 140 Na+, 5.2 K+, 1.2 Ca2+, 0.8 Mg2+, 119.8 Cl, 25 HCO
Immunoprecipitation of tyrosine-phosphorylated proteins. T84 cells were grown to confluence on 30-mm culture inserts. After being rinsed with Ringer solution, cell monolayers were stimulated with insulin or IGF-1 and incubated at 37°C according to the experimental design. Stimulation was terminated by washing the cell monolayer rapidly with ice-cold PBS. Lysis buffer [1% Nonidet P-40 detergent, 100 mM Tris (pH 7.4), 150 mM NaCl, protease inhibitors (1.0 µg/ml leupeptin, 1.0 µg/ml antipain, 1.0 µg/ml pepstatin, 100 µg/ml phenylmethylsulfonyl fluoride, 1.0 mM EDTA), and phosphatase inhibitors (1.0 mM sodium vanadate, 1.0 mM sodium fluoride)] was added apically, and the cells were incubated at 4°C for 1 h. Cells were then scraped into microfuge tubes and centrifuged for 15 min at 15,000 rpm, and the supernatants were removed to new tubes. After the samples were adjusted so that each contained equal amounts of protein using a Bio-Rad (Hercules, CA) protein assay kit, 5 µg of anti-phosphotyrosine was added to each sample and they were incubated for 60 min on a rotating platform at 4°C. This was followed by the addition of 50 µl of protein A sepharose (or protein A agarose in later experiments, with equivalent results), and the samples were incubated with gentle agitation for a further 60 min at 4°C. The sepharose beads were then spun down and washed three times with lysis buffer and three times with PBS. The beads were then resuspended in loading buffer (50 mM Tris pH 6.8, 2% SDS, 100 mM dithiothreitol, 0.2% bromophenol blue, 20% glycerol) just before electrophoresis.
Immunodetection of PI 3-kinase. PI 3-kinase was detected in immunoprecipitates using Western blotting kits from Boehringer Mannheim (Indianapolis, IN). To prepare samples for Western immunoblotting, we boiled the immunoprecipitates, prepared as described above, at 100°C for 5 min. The protein mixture was electrophoretically separated according to molecular weight using SDS-PAGE through a 7.5% Tris-glycine gel (Bio-Rad). Separated proteins were then transferred overnight using a Bio-Rad Mini Trans-Blot cell and subsequently immobilized on a polyvinylidene difluoride (PVDF) membrane (DuPont, Boston, MA). Nonspecific binding sites on the PVDF membrane were blocked for 30 min using 2% skim milk in PBS. The membrane was then incubated for 1 h using 5 µl of polyclonal rabbit antibodies to the p85 subunit (or Upstate Biotechnology anti-p85 antibodies in later experiments, with equivalent results) or 10 µl of polyclonal rabbit antibodies to the p110 subunit of PI 3-kinase and then washed with Tris-buffered saline (pH 7.4) with 1% Tween. The membrane was then incubated with horseradish peroxidase-conjugated anti-rabbit/anti-mouse IgG for 30 min and washed. The signal was detected using the detection reagent obtained from Boehringer Mannheim, and the luminescent protein bands were exposed onto X-ray film. In some experiments, ECL Plus reagents (Amersham, Piscataway, NJ) were used for detection, with equivalent results.
Determination of inositol phosphates. T84 cells, grown as monolayers in six-well dishes, were labeled with myo-[2-3H]inositol at a concentration of 50 µCi/ml as previously described (15). After labeling, the medium containing unincorporated radioisotope was removed and the cells were washed four times with Ringer solution. The cells were then incubated with the agents under study in Ringer solution at 37°C. Incubations were terminated by the addition of 1 ml of ice-cold methanol. Labeled inositol phosphates were extracted and quantitated by HPLC as previously described (37).
Data and statistical analysis. Results are expressed as means ± SE. Data were analyzed by Student's t-test or ANOVA as appropriate. Differences with an associated probability of <0.05 were considered to be statistically significant.
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RESULTS |
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Effect of insulin and IGF-I on carbachol-induced
chloride secretion.
T84 cells grown in serum-supplemented media were mounted in modified
Ussing chambers and pretreated with basolateral insulin (33 nM) or
IGF-I (13 nM) 15 min before basolateral addition of carbachol (100 µM). These doses were chosen on the basis of prior studies of the
effects of these hormones on T84 cell function (22-24). Neither insulin nor IGF-I alone had any
effect on chloride secretion. However, Fig.
1, A and B, shows
that insulin and IGF-1, respectively, significantly inhibited
carbachol-stimulated chloride secretion. The ability of insulin to
decrease carbachol-stimulated chloride secretion increased
somewhat as the length of preincubation with the hormone
increased from 1 to 15 min (Fig. 2),
although this trend did not achieve statistical significance. Control
experiments were performed with T84 cells grown in serum-free medium to
assess any possible effects of prior serum exposure. These studies
demonstrated that serum had no effect on the ability of insulin or
IGF-I to inhibit carbachol-induced chloride secretion (data not shown). Hence, all subsequent experiments were performed using
serum-supplemented media.
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Effect of insulin on thapsigargin-induced chloride secretion.
Previous studies showed that carbachol is able to induce a rapid and
transient response of chloride secretion through a calcium-dependent pathway (8). Carbachol is thought to activate PLC via its
G protein-linked receptor, thereby increasing intracellular levels of
inositol (1,4,5) trisphosphate and mobilizing
intracellular calcium pools. Thus it was possible that insulin might
alter calcium mobilization or, as we have reported previously
(38) for EGF, might "uncouple" a calcium signal from
the downstream response of chloride secretion. To distinguish between
these possibilities, we performed studies with thapsigargin. This agent
increases intracellular calcium via effects on the endoplasmic
reticulum calcium ATPase and thus bypasses receptor-dependent steps
involved in calcium mobilization (16). Pretreatment with
insulin was shown to significantly inhibit chloride secretion induced
by thapsigargin (Fig. 5). Thapsigargin alone caused an increase in Isc of 10.5 ± 1.8 µA/cm2, whereas pretreatment with insulin reduced the increase in
Isc to 5.1 ± 1.5 µA/cm2
(P < 0.05). These results suggest that insulin is
likely able to inhibit chloride secretion even in the presence of
elevated levels of intracellular calcium, similar to results we have
reported with EGF (38).
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Wortmannin and LY-294002 reverse inhibitory effect
of insulin and IGF-1 on carbachol-induced chloride
secretion.
Our laboratory has demonstrated (39) that the enzyme PI
3-kinase likely mediates the majority, if not all, of the
inhibitory effect of EGF on calcium-dependent chloride secretion. PI
3-kinase can be recruited by many receptors using tyrosine kinase
signaling pathways, including the insulin receptor. We therefore
speculated that insulin and IGF-I might mediate their inhibitory
effects on chloride secretion via a PI 3-kinase-dependent pathway.
Figure 6, A and B,
shows the effect of the PI 3-kinase inhibitor wortmannin on the
inhibitory effects of insulin and IGF-I, respectively, on
carbachol-induced chloride secretion. Addition of wortmannin alone had
no effect on either basal or carbachol-stimulated chloride secretion.
In contrast, pretreatment with wortmannin completely reversed the
inhibitory effects of insulin or IGF-I on chloride secretion.
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Insulin and IGF-I recruit 85-kDa
regulatory subunit and 110-kDa catalytic subunit of
PI 3-kinase to tyrosine-phosphorylated proteins.
Having shown that the PI 3-kinase inhibitors wortmannin and LY-294002
were able to reverse the inhibitory effects of insulin and IGF-I, we
next sought to determine whether insulin and IGF-I were indeed
activating PI 3-kinase. PI 3-kinase is a heterodimeric protein that
consists of two subunits, an 85-kDa regulatory subunit and a 110-kDa
catalytic subunit. Figure 9 shows the
levels of the 85-kDa subunit of PI 3-kinase in anti-phosphotyrosine
immunoprecipitates of T84 cells, measured as an indirect index of PI
3-kinase activation. Both insulin and IGF-I induced a substantial
increase in the levels of the 85-kDa subunit of PI 3-kinase present in
such immunoprecipitates at 1 min, an effect maintained for at least 15 min. At least for insulin, this effect was dependent on the
concentration of the hormone, with a maximal effect at 33 nM when the 1 min time point was considered (Fig.
10). This concentration dependence is
largely comparable with that observed for the inhibitory effect of
insulin on chloride secretion (Fig. 4). The concentration dependence of the effect of IGF-1 on this parameter was not assessed.
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DISCUSSION |
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The goal of this study was to determine whether insulin and IGF-I are capable of inhibiting calcium-dependent chloride secretion and, if so, whether this occurs through a PI 3-kinase-dependent pathway. We have shown that both insulin and IGF-I inhibit chloride secretion induced by the prototypic calcium-dependent agonist carbachol and by thapsigargin. Because these agents both induce chloride secretion by increasing intracellular calcium, but through different mechanisms, we suspect that insulin and IGF-I exert their inhibitory effects by uncoupling the rise in intracellular calcium from its consequent effect on chloride secretion. These findings are comparable to those in previous reports showing that carbachol and EGF are also capable of eliciting this uncoupling effect (38, 40).
The present study has also shown that the inhibitory effects of insulin and IGF-I on calcium-dependent chloride secretion by T84 epithelial cells likely involve activation of PI 3-kinase. Previously, we demonstrated that both carbachol and EGF exert inhibitory effects on chloride secretion through different mechanisms: carbachol through the activation of PLC and production of Ins(3,4,5,6)P4 (Ref. 39; J. M. Uribe, S. J. Keely, and K. E. Barrett, submitted for publication) and EGF predominantly through the activation of PI 3-kinase (39). However, we were unable to distinguish whether activation of a PI 3-kinase signaling pathway alone could lead to an inhibition of chloride secretion. This ambiguity arose because EGF has also been shown to elevate the levels of Ins(3,4,5,6)P4 in T84 cells, albeit to a much lesser extent than observed with carbachol (35, 37). The fact that insulin is unable to generate Ins(3,4,5,6)P4 in T84 cells (Fig. 8), yet is capable of inhibiting calcium-dependent chloride secretion, provides evidence that activation of PI 3-kinase can inhibit chloride secretion independent of the activation of PLC-dependent signaling pathways. It should be acknowledged that IGF-I, at least, has been shown to activate PLC isoforms and/or to elevate intracellular calcium concentrations in some cell systems (10, 21, 28), although in some cases PLC activation appears to be restricted to the nucleus and thereby linked to mitogenic signaling (21). However, this is unlikely to have occurred in the current study because IGF-I alone had no effect on Isc across T84 cells, whereas PLC activation and consequent calcium mobilization would be expected to evoke chloride secretion. Moreover, neither insulin nor IGF-I had any effect on Ins(3,4,5,6)P4 levels.
Peptide growth factors that bind to tyrosine kinase receptors stimulate the kinase activity of the receptor and autophosphorylation of receptor dimers. In the case of growth factors such as EGF and platelet-derived growth factor, this then allows for either direct or indirect high-affinity "docking" of other substrates containing Src homology 2 domains, including PI 3-kinase, Ras GTPase-activating protein, and PLC (9). However, the insulin and IGF-I receptors depart slightly from this paradigm in that they tyrosine phosphorylate a major substrate, the insulin receptor substrate-1 (IRS-1). It is IRS-1, and not the receptors themselves, that in turn binds effector molecules such as PI 3-kinase (35, 42).
Our laboratory has shown (39) that both carbachol and EGF
recruit the 85-kDa regulatory subunit but only EGF markedly recruits the 110-kDa catalytic subunit of PI 3-kinase and increases PI 3-kinase
activity in an in vitro kinase assay (39). Working with
rat HTC hepatoma cells, other investigators (31)
demonstrated that both insulin and IGF-I induce the formation of
multimolecular signaling complexes involving the p85 subunit of PI
3-kinase. In some of these complexes, p85 likely serves in a
p110-independent fashion as an adaptor molecule (linking the receptors
to IRS-1 among other proteins). However, complexes including the
catalytically active PI 3-kinase heterodimer are also formed (31,
36). In keeping with this, we show in this study that insulin
and IGF-I are capable of recruiting both the p85 and p110 subunits of
PI 3-kinase to tyrosine-phosphorylated residues in T84 cells. Moreover, two distinct PI 3-kinase inhibitors were able to reverse the inhibitory effects of insulin and IGF-I on calcium-dependent chloride secretion, thereby implicating PI 3-kinase activity in the inhibitory effect. The
mechanism whereby PI 3-kinase exerts this inhibitory effect has not
been elucidated fully. However, it likely involves the recruitment of
protein kinase C-, as we showed recently (5) for the
inhibitory effect of EGF on calcium-dependent chloride secretion,
although this remains to be tested directly for insulin or IGF-I.
Many intestinal disorders result from breakdown in the secretion, absorption, and/or production of insulin and/or IGF-I. For example, although many patients with diabetes mellitus suffer from constipation, likely secondary to neuropathy and disordered motility (1), a distinct subset of diabetic patients have severe and/or chronic diarrhea (20, 33, 40a, 41). Likewise, neonatal insulin-dependent diabetes mellitus has been associated with severe diarrhea and infant death (30). These clinical findings would be consistent, at least in part, with a failure in normal regulatory mechanisms that serve to limit intestinal secretion appropriately. Moreover, the concentrations of insulin and IGF-I found to be active in reducing chloride secretion are within the physiological range. For example, IGF-I levels measured by radioimmunoassay in a number of human exocrine secretions of the gastrointestinal tract include (in nM) saliva 0.9, gastric juice 3.5, jejunal chyme 24.6, pancreatic juice 3.6, and bile 0.9 (4).
Insulin is secreted in a pulsatile manner by the pancreas in response to elevated blood glucose or signals from the intestine released in response to ingestion of a meal. Because the inhibitory effect that insulin exerts on calcium-dependent chloride secretion is rapid in onset, we can speculate that this inhibitory effect might serve as a physiological brake on intestinal secretory function during the postprandial period. Conversely, IGF-I is produced mainly in the liver and circulates at fairly constant levels in association with various protein carriers (32). However, IGF-I levels can change dramatically in the setting of intestinal inflammation, and the gastrointestinal tract is considered one of the major targets of IGF-I action (42). Likewise, IGF-I has mitogenic effects in gut-resected, dexamethasone-treated, or normal rats (12, 34) and may promote repair in a rat model of intestinal injury associated with total parenteral nutrition (28). Of interest, intestinal tissues obtained from IGF-I-treated animals in this latter study showed markedly diminished responses to the calcium-dependent agonist of chloride secretion, carbachol, when examined in vitro. Similarly, oral IGF-I enhanced nutrient and electrolyte absorption, but not ion secretory processes, in neonatal piglets deprived of colostrum, independent of changes in mucosal mass or surface area (2). Thus we speculate that the ability of IGF-I to inhibit secretion, thereby conserving cellular energy, may be important in the setting of epithelial repair after an inflammatory reaction. The findings described here may also provide a mechanistic basis for the previously demonstrated rapid effects of this growth factor in vivo, as discussed above.
In summary, we have shown that both insulin and IGF-I are capable of inhibiting calcium-dependent chloride secretion and that they do so at concentrations that are physiologically relevant. The inhibitory mechanism involves the activity of PI 3-kinase and occurs independently of Ins(3,4,5,6)P4. The data likewise provide evidence that PI 3-kinase is able to inhibit calcium-dependent chloride secretion independent of concomitant activation of PLC. Finally, we propose that the data presented here likely imply important physiological and/or pathophysiological roles for insulin and IGF-I in regulating chloride transport by the mammalian intestine and perhaps also in other tissues.
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ACKNOWLEDGEMENTS |
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We thank Ginger Westbrook and Glenda Wheeler for help with manuscript preparation and Sharon Okonkwo for skilled technical assistance.
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FOOTNOTES |
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These studies were supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-28305 to K. E. Barrett and by an institutional Minority Biomedical Research Support Grant (GM-47165) in which K. E. Barrett is a participating investigator. J. M. Uribe was the recipient of a predoctoral fellowship from an institutional training grant in digestive diseases (DK-07202). S. J. Keely is the recipient of a Career Development Award from the Crohn's and Colitis Foundation of America. J. M. Uribe was a graduate student in the Biomedical Sciences Ph.D. Program, UCSD School of Medicine, at the time these studies were conducted.
A preliminary account of some portions of this work was presented at the Annual Meeting of the American Gastroenterological Society and has been published in abstract form (Gastroenterology 110: A317, 1996).
Present address of J. M. Uribe: Dept. of Internal Medicine, University of Maryland Hospital, Baltimore, MD 21201.
Address for reprint requests and other correspondence: K. E. Barrett, UCSD Medical Center, 8414, 200 W. Arbor Dr., San Diego, CA 92103-8414 (E-mail: kbarrett{at}ucsd.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.
Received 17 July 2000; accepted in final form 1 February 2001.
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