Insulinlike growth factor I receptors in rabbit gastrointestinal tract

GAS‘l’ROENTEROLOGY

1990;99:51-60

Insulinlike Growth Factor I Receptors in Rabbit Gastrointestinal Tract Characterization Localization

and Autoradiographic

BASEL TERMANINI, RONALD V. NARDI, INDU PARIKH, and LOUIS Y. KORMAN

THERESE

M. FINAN,

Medical and Surgical Service, Veterans Administration Medical Center, Washington, DC.; Departments of Medicine and Physiology, George Washington University School of Medicine, Washington, DC.; and Glaxo Research Inc., Research Triangle, North Carolina

Insulinlike growth factor I is a potent mitogen with insulinlike metabolic effects. Insulinlike growth factor I is synthesized in the liver, intestine, and other organs. Insulinlike growth factor I receptors are widely distributed and structurally similar to insulin receptors. Frozen sections of rabbit gastrointestinal tract were incubated in buffer containing 40 pmol/L [1251]insulinlike growth factor I. Binding was saturable, temperature- and time-dependent, and reversible. Saturation binding experiments showed a single class of high-affinity receptors (& = 0.9 nmol/L, B = 0.36 pmol/mg protein). The IC,, for insulinliF:growth factor I and insulinlike growth factor II were 3 nmol/L and 90 nmol/L, respectively; whereas insulin at 1-3 pmol/L displaced 50% of specific binding. Autoradiography of insulinlike growth factor I binding demonstrated significant differences in receptor density in gastrointestinal smooth muscle, epithelium of the esophagus, stomach, small intestine, and colon. These results indicate that a single class of specific, high-affinity insulinlike growth factor I receptors were distributed in muscular and mucosal layers of the entire rabbit gastrointestinal tract. Insulinlike growth factor I is likely to be an important local mediator of intestinal growth and metabolism.

I

nsulinlike growth factor I (IGF-I) is a peptide hormone similar in structure to insulin with A and B domains that have marked sequence homology with the A and B chains of insulin (l-3). Insulinlike growth factor I is synthesized and secreted primarily by the liver via a growth hormone-dependent process and circulates in the blood complexed to specific carrier

proteins (1); IGF-I stimulates glucose and amino acid transport, promotes DNA synthesis (with or without cell replication), and produces differentiation in some cell types (3-7). The effects of IGF-I are mediated by a tetrameric transmembrane glycoprotein receptor consisting of two extracellular (Ysubunits that bind the hormone and two fl subunits with tyrosine kinase activity on the cytoplasmic domain (4,B). Structural characterization of the IGF-I receptors has showed a high degree of sequence homology with insulin receptors (9). This structural similarity accounts for the ability of IGF-I to bind to the insulin receptor and the ability of insulin to bind to the IGF-I receptor (1,B). Insulinlike growth factor I mRNA, IGF-I immunoreactivity, and IGF-I receptors have been found in the gastrointestinal tract, suggesting that IGF-I may act as a paracrine or autocrine mediator of gastrointestinal metabolism and growth (7,1O-13). The present studies were designed to determine the binding characteristics of IGF-I receptors and to characterize the anatomic distribution of IGF-I receptors in all layers of the rabbit gastrointestinal tract by autoradiography. Methods Materials Iodinated ([3-‘Z51]iodotyrosyl)IGFFI with a specific activity of 2000 Ci/mmol was purchased from Amersham

Abbreviation used in this paper: HPLC, high-performance liquid chromatography. This is a U.S. government work. There are no restrictions on its use. 0018-5085/90/$0.00

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TIME (hours)

Figure 1. Time course of binding of [1261]IGF-I to sections of rabbit colon. Cryostat sections (20 pm) were mounted on ghtss slides and incubated in 2 mL of buffer with 40 pmol/L [1”61]IGF-Iat the temperatures indicated. Binding of [lUI]IGF-I was determined at the times specified, and results are expressed as percent of radioactivity maximally bound at 4OCat 24 hours. Each value was determined in quadruplicate, and the results shown are the mean * SEM of three separate experiments.

(Arlington Heights, IL); recombinant IGF-I was purchased from Bachem (Torrance, CA); IGF-II was purchased from Collaborative Research (Bedford, CA); porcine insulin was purchased from Lilly (Indianapolis, IN); aprotinin was purchased from FBA Pharmaceuticals (New York, NY]; and LKB Ultrofilm was purchased from LKB (Gaithersburg, MD). All other chemicals were reagent grade, obtained from Sigma Chemical Co. [St. Louis, MO).

Tissue

Preparation

New Zealand rabbits were killed and segments of the gastrointestinal tract were processed for autoradiography as described previously (14). In brief, segments were removed, rinsed in iced (4“C) saline, and frozen immediately on dry ice. After freezing, the tissue was oriented and embedded in gelatin to facilitate sectioning. Using a cryostat set at -20’C, 20-pm sections were cut, thaw mounted to gelatin-coated slides, and dried in a dessicator overnight. Segments not used immediately were stored at -7O’C and processed within 2 weeks. No differences in binding were noted between specimens used immediately and those stored.

Binding

Studies

Frozen sections of rabbit colon were affixed to slides and placed in polypropylene slide mailers with 3 ml of buffer containing 10 mmol/L HEPES, pH 7.6, 130 mmol/L NaCl, 4.7 mmol/L KCl, 5 mmol/L MgCl,, 1 mmol/L EGTA, 0.5% bovine serum albumin, 0.025% bacitracin, 0.0125% N-ethylmaleimide, 1000 KIU/mL aprotinin, and 40 pmol/L [‘2SI]IGF-I. Nonspecific binding was determined by incubating sequential sections in the same buffer but with 0.1 wmol/L IGF-I. Incubation was terminated by two sequential 2-minute washes in 100 mL of 10 mmol/L phosphate-

buffered saline (PBS], pH 7.4, at 4’C to remove unbound radiolabeled peptide, and sections were wiped off and counted in a gamma scintillation counter. Preliminary studies showed that total binding of [‘2SI]IGF-I to colon, small intestine, stomach, and esophagus was linear over the range of one to three sections per slide. Specific binding to the four sections in each mailer was 5.1% f 1.0% of the total counts added, or 5.7 f 1.2 fmol. In addition, preincubation of the sections at 20’C or 37°C did not alter the subsequent time course of binding. For this reason, sections were not preincubated in buffer before binding studies were performed. Binding constants were calculated using regression analysis. Protein content of sections was determined spectrophotometrically using the Bio-Rad (Richmond, CA) protein macroassay. To examine whether there was significant degradation of [1Z51]IGF-Iduring the 4°C incubation, sections were placed in 3 mL of buffer containing [‘251]IGF-I for 24 hours then, after the specimens were removed, freshly cut sections were placed in the buffer and incubated for another 24 hours. Sections from the first and second incubations were wiped off the slide and counted. There was no significant difference in specific binding between the two incubations. To examine further the possibility that degradation of tracer occurred during the 4’C incubation, additional studies were performed using differential binding to talc (15) and highperformance liquid chromatography (HPLC) analysis of preincubation and postincubation aliquots. During the 24hour 4’C incubation, 25-PL aliquots of buffer were removed, added to 2 mL of a 0.5% slurry of talc, and centrifuged at 3000 rpm, and the pellets were counted. The amount of radioactivity bound to talc was the same at the beginning and end of the incubation. In addition, 25-PL aliquots taken from the beginning and end of the incubation were applied to an Axxi-Chrom (Thomson Instruments, Springfield, VA) 4.6 x 100 mm Cl8 reverse-phase column with a multiseg

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TIME (minutes)

Figure 2. Time course for loss of bound [‘2SI]IGF-Ifrom sections of rabbit colon. Cryostat sections (20 am) were mounted on glass slides, incubated in 2 mL of buffer with 40 pmol/L [‘261)IGF-I for 24 hours at 4°C and resuspended in 50 volumes of excess buifer at the temperatures indicated. Binding of [lzSI]IGF-Iwas determined at the times specified and is expressed as percent of the radioactivity bound at the end of the first incubation. Each value was determined in quadruplicate, and the results are the mean + SEM of three separate experiments

July 1990

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o--o

IGF-I

?? -•IGF-II A-A

CONCENTRATION

(-log

Insulin

M)

Figure 3. Effect of IGF-I, IGF-II, and insulin on binding of [lzSI]IGFI to rabbit colon. Cryostat sections (20 pm) were mounted on glass

slides and incubated in 3 mL of buffer with 40 pmol/L [12SI)IGF-I plus the peptides indicated for 24 hours at 4% Specificbinding was expressed as a percentage of the value for bound [lBI)IGF-Iin the absence of additions. Bach value was determined in quadruplicate, and the results are the mean + SEM of three separate experiments.

ment gradient (16). Both single peaks of radioactivity

HPLC elution at 38 minutes.

profiles

showed

Autoradiography Frozen sections of different segments of the rabbit gastrointestinal tract were incubated under the same conditions as for binding, i.e., 4’C for 24 hours in 3 mL buffer in the absence and presence of 0.1 Nmol/L IGF-I. At the end of the incubation, sections were washed, air dried, and tightly juxtaposed to an LKB Ultrofilm for 3 days. After exposure, the film was developed in Kodak D19 developer (Rochester, NY] for 3 minutes and fixed in Kodak rapidfix for 5 minutes. Sequential sections corresponding to those used for binding were mounted on slides, fixed, and stained with H&E. @

a

53

Stained slides and their corresponding autoradiograms were studied and compared under light or phase microscopy. The duration of exposure was adjusted so that the optical density of the exposed segments was in the linear range of the film. To measure autoradiographic binding, the optical density of tissue sections on exposed films was analyzed by computerized densitometry using the RAS 1000 system (Loats Associates, Westminster, MD). Polygonal areas of the mucosa and muscular layers were outlined on digitized autoradiograms, and the mean optical density for those areas was obtained. All optical density measurements were made using tissue prepared from the same experiment and apposed to the same film. To measure inhibition of binding in different layers, sections were incubated with increasing concentrations of unlabeled ligand, washed, air dried, apposed to the same film, then analyzed. Results [1251/Insulinlike Growth Factor I Binding The kinetics of association of [1251]IGF-I to rabbit colon frozen sections are shown in Figure 1. At 4”C, specific binding increased slowly and progressively over 24 hours. Nonspecific binding was
T

IGF-I RECEPTORS

60 i

I

20 IGF-I

BOUND

(PM)

30 IGF-I

40 (nM)

50

60

70

Figure 4. Binding of [‘z51]IGF-Ias a function of radiolabeled peptide concentration. A. Scatchard

plot. Cryostat sections (20 rm] were mounted on glass slides and incubated in 3 mL of buffer with 40 pmol/L [‘251JIGF-Iand the increasing concentrations of IGF-I for 24 hours at 4°C. Calculated Kd = 0.9 nmol/L and B,,, = 0.36pmol/mg protein.

B. Saturation binding curve corresponding separate experiments.

to A. Each value was determined

in quadruplicate,

and the results are the mean +

SEM of

five

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bation at 37°C for 75 minutes resulted in a 70% reduction of bound ligand. To determine the specificity of [‘251]IGF-I binding, the ability of increasing concentrations of IGF-I and related compounds IGF-II and insulin to displace bound [‘251]IGF-I was measured (Figure 3). The most potent inhibitor was IGF-I, with an IC,, of 3 nmol/L. With an IC,, of 90 nmol/L, IGF-II was approximately 30 times less potent. Insulin was not only less potent, reducing [1251]IGF-I binding by 50% at a dose of l-3 pmol/L, but increasing the concentration of insulin above this range did not decrease [1251]IGF-Ibinding. Saturation binding studies using [1251]IGF-Ishowed a single class of saturable binding sites with a K, of 0.9 nmol/L and a B,, of 0.36 pmol/mg protein (Figure 4). Insulinlike Growth Factor 1 Receptor Distribution by Autoradiography Studies of anatomic distribution of IGF-I receptors in rabbit cecum, colon, small intestine, stomach, and esophagus were performed using autoradiographic techniques. Figure 5 illustrates a typical section of rabbit colon and its corresponding autoradiograph. In the cecum and colon, the density of ]1251]IGF-I binding was similar in the mucosa and muscularis: 0.47 + 0.10 and 0.54 f 0.10, respectively. There was no difference in grain density between the crypts and the superficial layer of the epithelium: 0.47 + 0.90 vs. 0.48 + 0.09 [Figure 5). To characterize further the [1251]IGF-I binding to colonic mucosa and smooth muscle layers, autoradiographs were obtained after incubation of sections with [1251]IGF-Iand increasing concentrations of nonradioactive IGF-I, IGF-II, and insulin. The films were analyzed by determining the optical density over the mucosal and muscular layers (Figure 6 and 7). Analysis of the change in optical density as a measure of displacement of [1251]IGF-I showed that the mucosal and muscular IC,, values for IGF-I, IGF-II, and insulin were equivalent to those observed in the

binding studies described above and in Figure 3. These studies demonstrated that there was no apparent difference in the affinity of IGF-I receptors in mucosal or muscular layers. Autoradiographic sections of the esophagus illustrate that [1251]IGF-Ibinding as measured by the grain density was present in the squamous mucosa and the muscular layers (Figure 5). The silver grain density was higher in the mucosal than the muscular layer: 0.20 + 0.07 and 0.07 + 0.02, respectively. Comparison with the stained section showed that the highest density of IGF-I binding within the mucosa occurred in the germinal (basal] layer of the squamous epithelium. In the gastric fundus, [1251]IGF-I grain density over the superficial portion of the gastric epithelium, which consists of surface and mucous cells, was greater than in the gastric pits (Figure 8): 0.39 k 0.09 and 0.27 c 0.06, respectively. This distinction between superficial and deep mucosa was not evident in the sections through the gastric antrum (figure not shown]. In the duodenum, jejunum, and ileum, mucosal binding was markedly less than in the muscular layer (Figure 8). In the mucosal layer, [1251]IGF-I grain density was highest in the mucosal crypts. Binding to the muscularis mucosae and to the muscularis propria was uniformly distributed, and there was no difference between circular and longitudinal smooth muscle. Densitometric measurements of specific binding of IGF-I to sections from various regions of the gastrointestinal tract showed that binding was not uniformly distributed along the length of the gastrointestinal tract. Furthermore, the relationship between muscular and mucosal binding was not the same at the anatomic sites examined (Figure 91. Muscular IGF-I binding was greater in the small and large intestine than in the esophagus or stomach. Mucosal IGF-I binding was very low in the duodenum and small intestine compared with the rest of the intestinal epithelium.

Figure 5. Autoradiographic distribution of [1251)IGF-I binding in rabbit gastrointestinal tract. Cryostat sections (20 pm) were mounted on glass slides and incubated in 3 mL of buffer with 40 pmol/L [1261)IGF-I for 24hours at 4’C, washed, dried, then apposed to LKB Ultrofihu for 3 days. Nonspecific binding was determined by incubating sections in the presence of 0.1rmol/L IGF-I.Bar = 0.1mm. A. H&E-stained section of rabbit colon with the mucosa (Mu), muscularis mucosa [MM), and muscularis propria (MP]. B. [‘ZSI]IGF-I autoradiographic binding. High grain density is uniformly distributed muscularis mucosae [MM) and muscularis propria.

along the epithelium

and is equivalent

to that in the

C. Nonspecific binding of [‘Z51]IGF-Ito an adjacent section. D. H&E-stained crosssection of rabbit esophagus with the mucosa (Mu), muscularis mucosa [MM), and muscularis propria (MP). E. [‘2sI]IGF-I autoradiographic [arrowhead).

binding. High grain density is present over the squamous epithelium,

F. Nonspecific binding of [‘251]IGF-Ito an adjacent section.

particularly

in the basal cell layer

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Figure 6. Effect of IGPI, IGF-II,and insulin on binding of (‘=I]IGF-Ito mucosa and muscularis proprla of rabbit colon. Autoradiogram of effect of increasing concentrations of IGF-I on [‘T)IGF-I binding: A, maximum binding: II, 0.1 nmoI/L; C, 1 nmoI& D, 10 xunoI& E, 100 nmol& F, 1 pmoI/L. Cryostat sections (20 run) were mounted on glass slides, incubated in buffer with 40 pmol/L [lasI]IGF-Iplus the peptides indicated for 24 hours at 4%. Each section was apposed to LKB Ukrofibn for 3 days.

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.------• IGF-I IGF-I A- -* IGF-II A-- -* IGF-II m-.-m insulin b---o insulin

0.6 nc

IGF-I RECEPTORS

a

57

mucosa muscularis mucosa muscular-is mucosa muscularis

Figure 7. Effect of IGF-I, IGFII, and insulin on binding of [‘2sIjIGF-I to mucosa and muscularis propria of rabbit colon. Binding was expressed as an optical density value for bound [12sIpGF-I over each layer in the presence of increasing concentrations of added ligand. Each value was

determined in triplicate, and the results are the mean 2 SEM of three separate experiments.

Discussion Insulinlike growth factor I is a polypeptide that affects the growth, development, and metabolism of many tissues (1,3,7,17,18). The action of IGF-I has been characterized in a variety of models, including intact animals, isolated cells, and cell cultures (17-24). Insulinlike growth factor I has been shown to mediate short-term metabolic and long-term growth effects by interacting with a unique class of cell surface receptors that possess tyrosine kinase activity (4,25). Although IGF-I is present in the gastrointestinal tract, the liver, and pancreas, little is known about either the distribution of IGF-I receptors or their physiological role (10,12,13,26). The present studies demonstrate that (a) a single class of specific, high-affinity IGF-I receptors accounts for IGF-I binding to colonic mucosa, muscularis mucosa, and muscle; (b) specific IGF-I receptors are present throughout the gastrointestinal tract in the mucosa, muscularis mucosa, and muscle; and (c) IGF-I receptors are not uniformly distributed transmurally or in a caudal-caudad direction. In rat gastrointestinal epithelial membranes, highaffinity binding sites for IGF-I (Kd = 7.2 nmol/L) and IGF-II (Kd = 9.5 nmol/L) have been demonstrated (13). Within the epithelium there was a gradient for IGF-I binding between tip and crypt cells and a doubling of binding between colon and small intestine (13). High-affinity insulin receptors have also been demonstrated in normal and malignant gastrointestinal tissue (27-31). The present studies in the rabbit indicate that both mucosa and smooth muscle possess

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CONCENTRATION (-log

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M)

a single class of specific, high-affinity IGF-I receptors. Furthermore, in rabbit colon these receptors have an affinity significantly higher than that reported for rat epithelium: K, = 0.9 and 7.2, respectively (13). This difference may be a function of the techniques used (membranes vs. slices) or of the species. These studies in the rabbit also indicate that the density of the IGF-I receptor can vary significantly in mucosal and muscular layers (Figure 9). Because autoradiographic studies indicated that displacement of binding between tissue layers of the colon was uniform [Figure 6), it is likely that differences in density were the result of differences in the number of available binding sites (B,,,) rather than affinity. Thus, IGF-I receptor expression in the gastrointestinal tract is dependent on cellular differentiation, as illustrated by the decrease in small intestinal epithelial binding from crypt to tip, and on tissue type (e.g., stomach vs. colon and muscle vs. epithelium). The localization of the IGF-I receptors to absorptive cells of the colon, surface and secretory cells of the stomach, and smooth muscle cells of the muscularis mucosae and muscularis propria suggests that IGF-I may have a maintenance function as well as an action on growth and differentiation. Receptors for IGF-I may mediate short-term metabolic response in these differentiated nondividing cells. For example, IGF-I has been shown to increase glucose and amino acid transport in several postmitotic cells via an IGF-I rather than an insulin receptor (3,23,25,32). This action was independent of its effect on cell growth and

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c I

0.6

0

MUCOsA

I

MUSCULARIS PROPRIA

I

ESOPHAGUS

CECUM

DUODENUM

FUNDUS IVilRUM

IlEUM

COLON

Figure 9. Regional distribution of specific [‘zJI]IGF-Ibinding to mucosa and muscularis propria. Cryostat sections (20 pm) were mounted on glass slides and incubated in 3 mL of buiTer with 40 pmol/L [‘261]IGF-Ifor 24 hours at 4% then apposed to LKB Ultrofllm for 3 days. Specific binding is determined by subtracting the optical density obtained in the presence of 0.1rrmol/L IGF-I from that obtained without addition of unlabeled ligand. For each experiment, all sections were incubated under the same conditions and apposed to the same film. Each value represents the mean k SEM of duplicate determinations from three separate experiments.

occurred in a to the affinity the hypothesis bile or locally and paracrine growth effects

concentration range that was equivalent of the receptor. These findings support that IGF-I transported via the blood or synthesized is an important endocrine modulator of both maintenance and in the gastrointestinal tract.

References 1. Nissley SP, Rechler MM. Insulin-like

growth factors: biqsynthesis, receptors and carrier proteins. In: Li CH, ed. Hormonal proteins and peptides. San Diego, CA: Academic, 1984:127-203. 2. Rinderknecht E, Humbel RE. The amino acid sequence of human insulin-like growth factor I and its structural homology to proinsulin. J Biol Chem 1978;253:2769-2776. 3. Froesch ER, Schmid C. Schwander J, Zapf J. Actions of insulinlike growth factors. Annu Rev Physiol1985:47:443-467. 4. Yarden Y, Ullrich A. Growth factor receptor tyrosine kinases. Annu Rev Biochem 1988;57:443-478. 5. Nissley SP, Haskell JF, Sasaki N, DeVroede MA, Rechler MM.

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Insulin-like growth factor receptors. J Cell Sci 1985;3(suppl):3951. 6. Rechler MM, Nissley SP. The nature and regulation of the receptors for insulin-like growth factors. Annu Rev Physiol 1985;47:425-442. 7. Underwood LE. D’Ercole AJ, Clemmons DR. Van Wyk JJ. Paracrine functions of somatomedins. Clin Endocrinol Metab 1986;15:59-77. a. Rechler MW, Nissley SP. The nature and regulation of the receptors for insulin-like growth factors. Annu Rev Physiol 1985;47:425-442. 9. Ulrich A, Gray A, Tam AW, Yang-Feng T, Tsubokawa M, Collins C, Henzel W, Le Bon T, Kathunia S, Chen E, Jacobs S, Francke LJ, Ramachandran J, Fujita-Yamaguchi Y. Insulin-like growth factor I receptor primary structure: comparison with insulin receptor suggests structural determinants that define functional specificity. EMBO J 1985;5:2503. 10.Lund PK, Moats-Staats BM, Hynes MA, Simmons JG, Jansen M, D’Ercole AJ, Van Wyk JJ. Somatomedin-C/insulin-like growth factor I and insulin-like growth factor-II mRNAs in rat fetal and adult tissues. J Biol Chem 1986;261:14539-14544. 11.D’Ercole AJ, Hill DJ, Strain AJ, Underwood LE. Tissue and plasma somatomedin-C/insulin-like growth factor I concentrations in the human fetus during the first half of gestation. Pediatr Res 1986;20:253-255. 12.D’Ercole AJ, Stiles AD, Underwood LE. Tissue concentrations of somatomedin C: further evidence for multiple sites of synthesis and paracrine or autocrine mechanisms of action. Proc Nat1 Acad Sci USA 1984;81:935-939. 13.Laburthe M, Rouyer-Fessard C, Gammeltoft S. Receptors for insulin-like growth factors I and II in rat gastrointestinal epithelium. Am J Physiol1988;254:G457-G462. 14.Sayadi H, Harmon JW, Moody TW, Korman LY. Autoradiographic distribution of vasoactive intestinal polypeptide receptors in rabbit and rat small intestine. Peptides 1988;9:23-30. 15.Keltz TN, Straus E, Yalow RS. Degradation of vasoactive intestinal polypeptide by tissue homogenates. Biochem Biophys Res Commun 1980;92:669-674. 16.Snider RH, Moore CF, Nylen ES, Becker KL. Prediction of peptide retention times on reversed-phase HPLC: prospects and limitations. Biochromatography 1988;3:100-104. 17.Smith BT. Post M, Stiles AD. Paracrine regulation of lung growth and maturation: the substrate of normal functional development. Prog Clin Biol Res 1983;140:135-141. 18.Perdue JF. The role of somatomedin/insulin-like growth factors and their receptors in skeletal growth and fetal development: a mini-review. Prog Clin Biol Res 1983;132:405-413. 19.Clemmons DR. Van Wyk JJ. Evidence for a functional role of endogenously produced somatomedinlike peptides in the regulation of DNA synthesis in cultured human fibroblasts and porcine smooth muscle cells. J Clin Invest 1985:75:1914-1918.

4

Figure 8. Autoradiographic distribution of [‘TflGF-I binding in rabbit gastrointestinal tract. Cryostat sections (20 pm) were mounted on glass slides and incubated in 3 mL of buffer with 40 pmol/L [‘“qIGF-I for 24 hours at PC, washed, dried, then apposed to LKB Ultroilhn for 3 days. Nonspecific binding was determined by incubating sections in the presence of 0.1 pmol/L IGF-I. Bar = 0.1mm. A. H&E-stained section of rabbit gastric fundus with the mucosa [Mu), muscularis mucosa (MM), and muscularis propria (MP). B. [‘Z51JIGF-Iautoradiographic binding. High grain density is present over the superficial layer (S) of the epithelium. Grain density over the gastric pits (P) and muscularis propria (MP) are equivalent. C. Nonspecific binding of [‘Z51]IGF-Ito an adjacent section. D. H&E-stained section of rabbit small intestine with the mucosa (Mu), submucosa (SM), and muscularis propria (MP). E. High grain density is present over the crypts [C), muscularis mucosae [MM), and muscularis propria (MP). Grain density over the villi is low but greater than nonspecific binding.

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20. Burgess SK, Jacobs S, Cuatrecasas P, Sahyoun N. Characterization of a neuronal subtype of insulin-like growth factor I receptor. J Biol Chem 1987;262:1618-1622. 21. Prosser CG, Sankaran L, Hennighausen L, Topper YJ. Comparison of the roles of insulin and insulin-like growth factor I in casein gene expression and in the development of alphalactalbumin and glucose transport activities in the mouse mammary epithelial cell. Endocrinology 1987;120:1411-1416. of normal rat 22.Deeks S, Richards J, Nandi S. Maintenance mammary epithelial cells by insulin and insulin-like growth factor 1. Exp Cell Res 1988;174:448-460. 23. Arnqvist JJ, Ballerman BJ, King GL. Receptors for and effects of insulin and IGF-I in rat glomerular mesangial cells. Am J Physiol1988;254:C411-C416. 24. Caverzasio J, Bonjour JP. Influence of recombinant IGF-I (somatomedin C] on sodium-dependent phosphate transport in cultured renal epithelium. Prog Clin Biol Res 1988;252:385-386. 25. Ota A. Wilson GL, Spilberg 0. Pruss R, LeRoith D. Functional insulin-like growth factor I receptors are expressed by neuralderived continuous cell lines. Endocrinology 1988;122:145-152. 26. Kirschner BS, Sutton MM. Somatomedin-C levels in growthimpaired children and adolescents with chronic inflammatory bowel disease. Gastroenterology 1986;91:830-836. 27. Wong M, Holdaway IM. Insulin binding by normal and neoplastic colon tissue. Int J Cancer 1985:35:335-341. 28. Zimmerman TW, Reinprecht JT, Binder HJ. Peptide binding to

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30.

31.

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intestinal epithelium: distinct sites for insulin, EGF and VIP. Peptides 1985;6:229-235. Forgue-Lafitte ME, Horvat A, Rosselin G. Insulin binding by a cell line (HT29) derived from human colon cancer. Mol Cell Endocrinol1979;14:123-130. MountJoy KG, Holdaway IM, Finlay GJ. Insulin receptor regulation in cultured human tumor cells. Cancer Res 1983;43:45374542. Sodoyez-Goffaux F, Sodoyez JC, De Vos CJ. Insulin receptors in the gastrointestinal tract of the rat fetus: quantitative autoradiographic studies. Diabetologia 1985;28:45-50. Verspohl EJ, Maddux BA, Goldfine ID. Insulin and insulin-like growth factor I regulate the same biological functions in HEP-G2 cells via their own specific receptors. J Clin Endocrinol Metab 1988;67:169-174.

Reeeived July 10,1989. Accepted January 18,199O. Address requests for reprints to: Dr. Louis Y. Korman, Medical Service (151W), Veterans Administration Medical Center, 50 Irving Street NW, Washington, DC 20422. This work was supported by funds from the Medical Research Service of the Veterans Administration and Glaxo Research Inc. The authors thank Dr. Susan Burgess and Mr. Harry Loats for their help with the binding and autoradiographic studies.