Departments of Medicine and Physiology, Medical College of Virginia Campus, Virginia Commonwealth University, Richmond, Virginia 23298-0711
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ABSTRACT |
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Human intestinal smooth muscle in culture produces insulin-like growth factor (IGF)-I and IGF binding protein (IGFBP)-3, IGFBP-4, and IGFBP-5, which modulate the effects of IGF-I. This study examined the regulation of IGFBP production by endogenous IGF-I. R3-IGF-I, an agonist unaffected by IGFBPs, elicited concentration-dependent increase in growth, measured by [3H]thymidine incorporation, and production of IGFBP-3, IGFBP-4, and IGFBP-5, measured by Western blot. Antagonists of the IGF-I receptor, IGF-I Analog or monoclonal antibody 1H7, elicited concentration-dependent inhibition of growth and decrease in IGFBP-3, IGFBP-4, and IGFBP-5 production, implying that endogenous IGF-I stimulated growth and IGFBP production. R3-IGF-I-induced increase in IGFBP-3, IGFBP-4, and IGFBP-5 production was partially inhibited by a mitogen-activated protein (MAP) kinase or a phosphatidylinositol-3-kinase (PI 3-kinase) inhibitor and abolished by the combination. We conclude that endogenous IGF-I stimulates growth and IGFBP-3, IGFBP-4, and IGFBP-5 production in human intestinal smooth muscle cells. Regulation of IGFBP production by IGF-I is mediated by activation of distinct MAP kinase and PI 3-kinase pathways, the same pathways through which IGF-I stimulates growth.
mitogen-activated protein kinase; mitogen-activated protein kinase kinase; phosphatidylinositol 3-kinase; cell culture
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INTRODUCTION |
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INSULIN-LIKE GROWTH FACTOR (IGF)-I stimulates both growth and differentiation of a variety of muscle cell types including skeletal (22), cardiac (7), vascular (4, 5), and visceral (12, 18) smooth muscle. These same cells can also express up to six IGF-I binding proteins (IGFBP-1-6) that modulate the responses to IGF-I by inhibiting or facilitating binding of IGF-I to the IGF-I receptor (11). The action of individual IGFBPs, to inhibit or facilitate the binding of IGF-I, is both tissue and binding protein specific. In addition to modulation of IGF-I-induced growth, IGF-I-independent effects have been identified for IGFBP-1, IGFBP-3, and IGFBP-5 (2, 9, 17). These effects parallel their IGF-I-dependent effects, also inhibiting or stimulating growth in a particular cell type.
The interplay between IGF-I and IGFBPs is bidirectional. Not only do IGFBPs modulate IGF-I-induced growth, two mechanisms by which IGF-I can regulate IGFBP levels have been identified. Specific proteolytic mechanisms for IGFBP-2, -3, -4, and -5 degradation have been identified that are regulated by IGF-I (5, 6, 21, 23). In addition, IGF-I can regulate directly the production of IGFBPs (6, 21). Regulation of IGFBP production by endogenous IGF-I has been examined in A549 adenocarcinoma cells (21), in aortic smooth muscle cells (5), and in osteoblasts (6). In A549 cells, endogenous IGF-I stimulated IGFBP-3 production. In porcine aortic smooth muscle cells, endogenous IGF-I increased IGFBP-5 production and decreased IGFBP-4 levels. The effect of IGF-I on growth, therefore, is determined by its direct effects and indirectly by its effects on IGFBP levels.
Within the gastrointestinal tract, IGF-I and IGFBP-4 and IGFBP-5 are expressed by smooth muscle cells of the muscularis propria in rat colon and IGF-I and IGFBP-3, IGFBP-4, and IGFBP-5 are expressed by smooth muscle cells cultured from the human intestine and rat colon (3, 12, 15a, 27-29). The importance of endogenous IGF-I in regulating smooth muscle cell growth is highlighted by the increase in size of the muscularis propria in transgenic mice overexpressing a human IGF-I cDNA (19). In cultures of human intestinal muscle cells, IGF-I-induced growth is inhibited by IGFBP-3 and IGFBP-4 and augmented by IGFBP-5 (3, 15). Growth of human intestinal muscle cells induced by IGF-I is mediated jointly by activation of two major intracellular signaling cascades, the Raf/mitogen-activated protein (MAP) kinase pathway and the phosphatidylinositol 3-kinase (PI 3-kinase) pathway (14).
The regulation of IGFBP-5 production by IGF-I has been examined in cultures of muscle cells isolated from the rat colon. In these cells, addition of exogenous IGF-I increased IGFBP-5 mRNA and protein production (29). The role of endogenous IGF-I in regulating IGFBP-3, IGFBP-4, and IGFBP-5 production in human intestinal muscle and the intracellular mechanisms mediating these effects in human intestinal smooth muscle have not previously been examined.
In the present study, the role of endogenous IGF-I in regulation of
IGFBP-3, IGFBP-4, and IGFBP-5 production was examined in smooth muscle
cells cultured from the human intestine. The effects of endogenous
IGF-I were identified using the IGF-I receptor antagonist IGF-I
Analog and the inhibitory IGF-I receptor -subunit antibody 1H7. The
results show that endogenous IGF-I stimulates production of
IGFBP-3, IGFBP-4, and IGFBP-5. Activation of the IGF-I
receptor and the resulting stimulation of IGFBP production are mediated
by activation of distinct MAP kinase and PI 3-kinase pathways, the same
pathways by which IGF-I stimulates growth in these cells.
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METHODS |
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Preparation and culture of human intestinal muscle cells.
Muscle cells from normal human jejunum were isolated and cultured as
described previously (3, 12, 14) from surgical specimens obtained by a
protocol approved by the Institutional Committee on the Conduct of
Human Research. Tissue slices from the circular muscle layer were
digested in a medium consisting of (mM) 120 NaCl, 4 KCl, 2.6 KH2PO4, 2 CaCl2, 0.6 MgCl2, 25 HEPES, and 14 glucose and 2.1% Eagle's
essential amino acid mixture with 0.2% collagenase (CLS type II) and
0.1% soybean trypsin inhibitor. Partially digested muscle was
dispersed in enzyme-free medium. Muscle cells were harvested by
filtration (500-µm Nitex). The cells were washed twice by
centrifugation for 5 min at 150 g and resuspended in Hanks'
medium containing 200 U/ml penicillin, 200 µg/ml streptomycin, 100 µg/ml gentamicin, and 2 µg/ml amphotericin B. Muscle cells were
resuspended in Dulbecco's modified Eagle's medium containing 10%
fetal bovine serum (DMEM-10) and antibiotics and plated at a density of
5 × 105 cells/ml. The medium was replaced every 3 days. Once confluence was attained, primary cultures were passaged and
seeded at a density of 2.5 × 105 cells/ml. All
subsequent studies were performed on first-passage cultured cells on
day 7 of culture, at which time the cells attained confluence.
We have previously shown (12, 25) that these cells express a phenotype
characteristic of intestinal smooth muscle as determined by
immunostaining for smooth muscle markers and expression of -enteric
actin. Endothelial cells, neurons, and interstitial cells of Cajal are
not detected in these cultures (25).
Preparation of conditioned medium. Conditioned medium was prepared as described previously from confluent muscle cells for measurement of soluble IGFBPs and IGF-II (3). The cells were washed and incubated in serum-free DMEM for 48 h. Conditioned medium was used to measure IGF-II as described below. The effects of R3-IGF-I, an IGF-I receptor agonist (8), were examined by addition of increasing concentrations (1-10 nM) of the agonist during the 48-h period. The effects of an antagonist of the IGF-I receptor, IGF-I Analog (20), were examined by addition of increasing concentrations (1-1,000 pM) of the antagonist during the 48-h period. After treatment, the conditioned medium was removed, phenylmethylsulfonyl fluoride (PMSF, 0.1 mM), aprotinin (0.1 µg/ml) and EDTA (10mM) were added, and the medium was concentrated 10-fold in Centricon-10 tubes at 4°C for measurement of soluble IGFBP-3, IGFBP-4, and IGFBP-5 production.
Preparation of whole cell lysates. After removal of the conditioned medium, the cell layer was washed three times with PBS at 4°C. The cells were lysed in PBS (pH 7.4) containing 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 0.1 µg/ml PMSF, 1% aprotinin, and 1 mM sodium orthovanadate as described previously (3). Lysates were then centrifuged at 12,000 g for 20 min at 4°C, and the supernatants were used for measurement of cell-associated IGFBP-3 and IGFBP-5 production.
Measurement of IGFBP production by Western blot analysis.
IGFBP production was measured by Western blot analysis as described
previously (3). Concentrated conditioned medium or whole cell lysates
were added to loading buffer [62.5 mM Tris (pH 6.8) with 2% SDS,
25% glycerol, 0.01% bromophenol blue and 5%
-mercaptoethanol] to provide samples derived from equal
amounts of total cellular protein (conditioned medium: 200 µg/30
µl, cell lysates: 30 µg/µl). Proteins separated on 12%
polyacrylamide gels were electrotransferred to nitrocellulose membranes
in 25 mM Tris (pH 8.3), 192 mM glycine, with added 20% methanol and
0.1% SDS. Membranes were incubated overnight at 4°C with a 1:1,000
dilution of an antibody to IGFBP-3, IGFBP-4, or IGFBP-5, each
displaying <1% cross-reactivity with other IGFBPs. Membranes were
incubated in a 1:1,000 dilution of goat anti-rabbit IgG-horseradish
peroxidase conjugate. Bands were visualized with chemiluminescence and
quantitated by densitometry.
[3H]thymidine incorporation assay. Growth of cultured muscle cells was measured by [3H]thymidine incorporation as described previously (12, 14). Cells were incubated for 48 h in serum-free DMEM with R3-IGF-I (0.10-100 nM) in the presence or absence of the IGF-I receptor antagonist (1 µM) or for 24 h with antagonist alone (1-1,000 pM). [3H]thymidine (1 µCi/ml) was added for the final 4 h, and its incorporation was measured in the perchloric acid-extractable pools using 2 N perchloric acid (30 min at 60°C). After solubilization, protein in the cell residue was measured using the BioRad protein reagent. [3H]thymidine incorporation was expressed as counts per minute per microgram of protein.
Measurement of IGF-II. IGF-II was measured in supernatants after acid-ethanol extraction to remove bound IGFBPs as previously described (12). Briefly, aliquots of conditioned medium were added to an acid-ethanol mixture [87.5% ethanol-12.5% (vol/vol) hydrochloric acid], vortexed, and centrifuged. A 100-µl aliquot of the resulting supernatant was neutralized with 40 µl of Tris (0.855 M), incubated at 4°C for 2 h, and recentrifuged. IGF-II was measured in the resulting supernatant by radioimmunoassay using a polyclonal antibody raised in rabbits against human IGF-II (33-40). This antibody reacts fully with human IGF-II but has no cross-reactivity with IGF-I or insulin. The limit of detection was 1 pg/tube, and the IC50 was 22 pg/tube.
Statistical analysis. Values represent means ± SE of n experiments, where n represents the number of experiments on cells derived from separate primary cultures. Statistical significance was tested by Student's t-test for either paired or unpaired data. Protein bands were measured with computerized densitometry, and results are reported in arbitrary units above background.
Materials.
Collagenase and soybean trypsin inhibitor were obtained from
Worthington Biochemical (Freehold, NJ);
[3H]thymidine (6 Ci/mmol) was obtained from NEN
(Boston, MA); DMEM and Hanks' medium were obtained from
Mediatech (Herndon, VA); fetal bovine serum was obtained
from BioWhittaker (Walkersville, MD); protein assay reagent was
obtained from BioRad Laboratories (Hercules, CA); R3-IGF-I and rabbit
polyclonal antibodies to IGFBP-3, IGFBP-4, and IGFBP-5 were obtained
from Upstate Biotechnology (Lake Placid, NY); IGF-I Analog was obtained
from Bachem (Torrance, CA); mouse monoclonal antibody to the IGF-I
receptor -subunit, 1H7, was obtained from Santa Cruz Biotechnology
(Santa Cruz, CA); IGF-II radioimmunoassay kit was obtained from
Peninsula Laboratories (San Carlos, CA); and plastic cultureware was
obtained from Corning (Corning, NY). All other chemicals were obtained
from Sigma (St Louis, MO).
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RESULTS |
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Characterization of IGF-I Analog as an IGF-I receptor antagonist in
human intestinal muscle.
IGF-I Analog was used as an IGF-I receptor antagonist (20). The ability
of IGF-I Analog to act as an antagonist of IGF-I receptor activation
was confirmed by its ability to inhibit growth stimulated by R3-IGF-I.
R3-IGF-I, an Arg3-substituted IGF-I derivative, was chosen
as an agonist because of its greatly reduced affinity, ~1,000-fold,
for IGFBPs compared with native IGF-I and also because it does not bind
to the IGF-II/mannose 6-phosphate receptor (8). This eliminated the
modulatory effects of IGFBPs on IGF-I-induced effects, the effects of
IGF-I on degradation of IGFBPs, and possible effects mediated by
activation of the IGF-II/mannose 6-phosphate receptor (8). Incubation
of confluent human intestinal muscle cells with R3-IGF-I (0.1-100
nM) increased [3H]thymidine incorporation in a
concentration-dependent fashion (Fig. 1).
The increase was significantly greater than that elicited by native
IGF-I (Fig. 1) at all concentrations tested, implying that IGFBPs
modulate the interaction of IGF-I with the IGF-I receptor. The increase
in [3H]thymidine incorporation elicited by
R3-IGF-I was significantly inhibited at all concentrations tested
(P < 0.05) in the presence of 1 µM IGF-I receptor
antagonist (Fig. 1).
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R3-IGF-I augments IGFBP production. The role of IGF-I in the regulation of IGFBP production was investigated by measurements of IGFBPs in both conditioned medium (soluble binding protein) and in whole cell lysates (cell-associated/extracellular matrix-associated binding protein). Cultures of confluent muscle cells were incubated for 48 h with R3-IGF-I (1 and 10 nM), and the levels of IGFBP-3, IGFBP-4, and IGFBP-5 production were measured by Western blotting with densitometric analysis.
The production of soluble IGFBP-3 was augmented by R3-IGF-I in a concentration-dependent fashion (162 ± 56% increase above basal at 10 nM, P < 0.01; Fig. 3). The production of cell-associated IGFBP-3 was also increased by R3-IGF-I in a concentration-dependent fashion (43 ± 14% increase above basal at 10 nM, P < 0.05; Fig. 3).
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Endogenous IGF-I regulates IGFBP production. A role for endogenous IGF-I in the regulation of IGFBP production was suggested by the ability of R3-IGF-I to augment the production of IGFBP-3, IGFBP-4, and IGFBP-5 and by the previous finding that IGF-I is produced by these cells. This notion was investigated by using the IGF-I antagonist to block the effects of endogenous IGF-I and measuring IGFBP production. Cultures of confluent muscle cells were incubated for 48 h with increasing concentrations of the IGF-I antagonist, and the production of IGFBP-3 and IGFBP-5 was measured in conditioned medium and in cell lysates by Western blotting with densitometric analysis.
The production of soluble IGFBP-3 was inhibited by the IGF-I receptor antagonist in a concentration-dependent fashion with 60 ± 2% inhibition of basal production at 1 nM (P < 0.05; Fig. 6). This was paralleled by a concomitant concentration-dependent decrease in soluble IGFBP-5 production. In the presence of 1 nM IGF-I receptor antagonist, basal IGFBP-5 production was inhibited 36 ± 14% (P < 0.05; Fig. 7).
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IGF-I-mediated IGFBP production is MAP kinase dependent and PI 3-kinase dependent. We have previously shown (14) that IGF-I activates distinct MAP kinase-dependent, PI 3-kinase-independent and MAP kinase-independent, PI 3-kinase-dependent pathways that are coupled to growth. To determine whether the effects of IGF-I on IGFBP production were mediated by similar intracellular mechanisms, confluent muscle cells were incubated with R3-IGF-I in the presence of either the MAP kinase kinase (MEK) inhibitor (1) PD-98059 (10 µM), the PI 3-kinase inhibitor (26) LY-294002 (10 µM), or a combination of the two inhibitors. At the concentrations used, these inhibitors specifically abolish the activation of MAP kinase or PI 3-kinase without affecting activation of the other kinase (1, 14, 26).
The increase in soluble IGFBP-3 production elicited by 10 nM R3-IGF-I was partially inhibited by either the PI 3-kinase inhibitor (48 ± 9% inhibition, P < 0.01) or the MEK inhibitor (57 ± 7% inhibition, P < 0.01) and abolished by the combination of the two inhibitors (92 ± 12% inhibition, P < 0.01) (Fig. 9). The production of soluble IGFBP-4 elicited by 10 nM R3-IGF-I was also partially inhibited by either the PI 3-kinase inhibitor (51 ± 7% inhibition, P < 0.01) or the MEK inhibitor (48 ± 9% inhibition, P < 0.01) and was strongly inhibited by the combination of the two inhibitors (76 ± 9% inhibition, P < 0.01) (Fig. 8). A pattern similar to that of IGFBP-3 was observed for the production of soluble IGFBP-5 elicited by 10 nM R3-IGF-I; it was partially inhibited by either the PI 3-kinase inhibitor (77 ± 11% inhibition, P < 0.01) or the MEK inhibitor (68 ± 18% inhibition, P < 0.01) and was abolished by the combination of the two inhibitors (93 ± 5% inhibition, P < 0.01) (Fig. 8). The pattern of inhibition of IGFBP-3, IGFBP-4, and IGFBP-5 production by the selective PI 3-kinase and MEK inhibitors and the additive effects of the two inhibitors implied that the increase in IGFBP production was mediated by activation of two distinct pathways, a MAP kinase-dependent, PI 3-kinase-independent pathway and a MAP kinase-independent, PI 3-kinase-dependent pathway.
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DISCUSSION |
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This study shows that both growth and the production of IGFBP-3, IGFBP-4, and IGFBP-5 are regulated by endogenous IGF-I in confluent cultures of human intestinal muscle cells. The regulation of IGFBP production by IGF-I occurs by activation of the same intracellular mechanisms previously shown to mediate IGF-I-induced growth and activation of distinct MAP kinase-dependent, PI 3-kinase-independent and MAP kinase-independent, PI 3-kinase-dependent pathways (14).
The evidence supporting a direct role for IGF-I in growth of confluent cultures of human intestinal muscle cells can be summarized as follows: 1) R3-IGF-I elicited concentration-dependent proliferation; 2) proliferation induced by R3-IGF-I was inhibited by the IGF-I receptor antagonist; and 3) IGF-I receptor antagonist inhibited basal proliferation in a concentration-dependent fashion. These results implied that endogenous IGF-I stimulates growth in confluent human intestinal muscle cells.
The evidence supporting a direct role for IGF-I in the regulation of IGFBP production by human intestinal smooth muscle cells in culture can be summarized as follows: 1) R3-IGF-I elicited a concentration-dependent increase in both soluble and cell-associated IGFBP-3 and IGFBP-5 production and in soluble IGFBP-4 production; 2) the IGF-I receptor antagonist elicited a concentration-dependent decrease in basal soluble and cell-associated IGFBP-3 and IGFBP-5 production; and 3) an antibody inhibiting IGF-I receptor activation elicited a concentration-dependent decrease in basal soluble IGFBP-4 production. The results implied the presence of an autocrine mechanism whereby endogenous IGF-I regulates IGFBP levels.
The regulation of IGFBP production by IGF-I has been investigated in a number of smooth muscle cell types including vascular and visceral smooth muscle (5, 10, 21, 29). Mechanisms of IGF-I-dependent regulation of IGFBP gene transcription, IGFBP mRNA stability, translation, and posttranslational processing have been identified depending on the specific IGFBP. In rat aortic smooth muscle, activation of the IGF-I receptor elicits an increase in IGFBP-3 and IGFBP-4 protein levels without concomitant changes in either IGFBP-3 or IGFBP-4 mRNA levels (10). In rat colonic smooth muscle IGF-I increases IGFBP-5 expression and production (29). In many cell types, IGFBP-4 levels are influenced by proteolysis. In previous studies we have shown that in human intestinal muscle IGF-I regulates IGFBP-4 levels through an IGF-I-activated proteolytic mechanism. Changes in IGFBP-4 levels were not caused by IGF-I-induced changes in IGFBP-4 mRNA levels (15). The focus of the current study is on the regulation of IGFBP production. Transcriptional activity and mRNA stability were therefore not examined. We have previously shown (14), however, that maintenance of basal IGFBP levels was dependent on continual new protein synthesis.
The intracellular signal transduction cascades that are activated by IGF-I and mediate IGF-I-induced regulation of IGFBP production have not previously been identified in intestinal smooth muscle. Two signaling cascades activated by IGF-I and coupled to stimulation of growth of human intestinal muscle cells are the Raf/MAP kinase and the PI 3-kinase pathways (14). Both pathways contribute additively to IGF-I-induced growth. Similar mechanisms are involved also for the regulation of IGFBP-3, IGFBP-4, and IGFBP-5 production. IGF-I activates both the MAP kinase pathway and the PI 3-kinase pathway, which contribute additively to increased production of IGFBP-3, IGFBP-4, and IGFBP-5 in these cells.
Growth of human intestinal muscle in culture is determined by the
interplay of at least two autocrine growth factors, IGF-I and
transforming growth factor-1 (TGF-
1), and is modulated by IGFBP-3, IGFBP-4, and IGFBP-5, which are also produced by the cells (3, 12-14). The production of IGF-I and TGF-
1 is time dependent and reciprocal in culture. IGF-I levels are high during the
initial period of rapid growth and decrease as cells attain confluence.
In contrast, TGF-
1 levels are low initially and increase when the
cells reach confluence. IGFBP-5, which attains its highest levels
during the initial period of rapid growth concurrently with IGF-I,
augments IGF-I-induced growth, whereas IGFBP-3, which attains its
highest levels concurrently with TGF-
1, inhibits IGF-I-induced
growth. IGFBP-4 inhibits IGF-I-induced growth. IGFBP levels,
therefore, are regulated by the interplay of several simultaneous processes that vary during growth in culture, IGF-I- and
TGF-
1-regulated production, and IGF-I-regulated proteolysis.
Integration of these regulatory stimuli determines the high levels of
growth-promoting IGFBP-5 and low levels of growth-inhibiting IGFBP-3
during the period of rapid growth and the progressively diminishing
levels of IGFBP-5 and increasing levels of IGFBP-3 as cells attain
confluence. The interplay between IGF-I, TGF-
1, and IGFBP production
regulates, in part, growth of human intestinal muscle from a
proliferating to a confluent contractile state.
The importance of IGF-I in development of the intestine is exemplified by the increased size of the muscularis propria in transgenic mice overexpressing a human IGF-I cDNA (19) and in suckling rats infused with either IGF-I or R3-IGF-I (24). The observations that IGF-I and IGFBP-5 expression is increased in the intestine of patients with Crohn's disease (15a) and in rat models of enterocolitis (27-29) suggest that the IGF-I system may participate in the response of the intestine to inflammation. In both cases, IGF-I mRNA expression was increased in the intestine and IGFBP-5 expression was increased specifically within the muscularis propria (28, 29).
In summary, this study shows that endogenous IGF-I stimulates the production of IGFBP-3, IGFBP-4, and IGFBP-5 in cultures of human intestinal smooth muscle cells. We have previously shown (12) that ambient endogenous IGF-I levels in confluent muscle cells are 29.7 ± 7.7 nM, which is within the range examined in these studies. This process is regulated by activation of distinct MAP kinase-dependent and PI 3-kinase-dependent pathways, the same pathways mediating the growth effects of IGF-I in human intestinal smooth muscle cells.
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ACKNOWLEDGEMENTS |
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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-49691.
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FOOTNOTES |
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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: J. F. Kuemmerle, Div. of Gastroenterology, Medical College of Virginia Campus, Virginia Commonwealth Univ., PO Box 980711, Richmond, VA 23298-0711 (E-mail: jkuemmerle{at}hsc.vcu.edu).
Received 8 August 1999; accepted in final form 6 November 1999.
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