The morphometry and biomechanical properties of the rat small intestine after systemic treatment with epidermal growth factor

Morphometric and passive biomechanical properties were studied in isolated segments of the duodenum, jejunum and ileum in 22 EGF-treated rats and 12 control rats. The rats were allocated to groups with EGF treatment for 2, 4, 7, and 14 days (n = 6 for each EGF treatment group except n = 4 for the 14 days group) or saline treatment (n = 3 for each group). The intestinal segments were pressurized with Krebs solution from 0 to 8 cmH2O for duodenum and 0 to 6 cmH2O for jejunum and ileum using a ramp distension protocol. The diameter and length were recorded at different pressure levels. Circumferential and longitudinal stresses (force per area) and strains (deformation) were computed from the length, diameter, pressure and the zero-stress state data. EGF treatment was associated with pronounced morphometric changes, e.g., the wall thickness, wall area, and the circumferential lengths significantly increased during EGF treatment in all intestinal segments (P < 0.05). Histological analysis showed that the thickness and area of the layers increased after EGF treatment. With respect to the biomechanical data, the opening angle increased in all segments during EGF treatment with the highest value in the 14 days EGF treatment group (P < 0.05). The same result was found for residual strain and the residual strain gradient through the intestinal wall. Linear regression analysis demonstrated that the opening angle mainly depended on the mucosa thickness and area. Furthermore, the circumferential stiffness increased in the duodenum and decreased in the jejunum and ileum during EGF treatment. A plateau was reached after 7 days where after it started to normalize (P < 0.01). In the longitudinal direction, all intestinal segments became stiffer after EGF treatment for 7 days. After 14 days the curve started to normalize in duodenum and jejunum but not in the ileum.     

Effects of enterally fed epidermal growth factor on the small and large intestine of the suckling rat

Epidermal growth factor (EGF) has been shown to be present in the milk of several species, including the rat, and to have gastrointestinal effects when given parenterally or orally in pharmacologic doses. We investigated the effect of enteral EGF in physiologic doses on the small intestine and colon of suckling rats. Serum thyroxine (T4) levels were also measured. Rats were gavage-fed by hand with an artificial formula with or without added EGF every 3 h from 11 to 14 days of age. Intake was adjusted to deliver 30 kcal/100 g b.wt./day of formula and 16 micrograms/kg/day of EGF approximating the daily caloric intake, and about twice the estimated daily EGF intake for suckling rats. Weight gain did not differ between groups (fed EGF: 3.8 + 0.2 g; not fed EGF: 3.7 + 0.1 g). The protein content of the whole colon of rats fed an EGF-containing formula was significantly lower and the DNA content significantly higher, than in rats fed formula without added EGF. The protein/DNA ratio was therefore markedly higher in the animals fed formula without added EGF; these effects were most evident in the distal colon. In contrast, there was no effect of EGF on small intestinal protein and DNA content; lactase, sucrase, and maltase activities were likewise unaffected, as was serum T4. These data suggest a physiologic role for breast milk EGF in the development of the suckling rat colon.     

Characterization of epidermal growth factor receptor in rat buccal mucosal cells

1. Receptor binding for epidermal growth factor (EGF) in rat buccal mucosa was characterized. Binding of [125I]EGF to rat buccal mucosa was time, temperature, cell number and [125I]EGF concentration dependent. 2. The [125I]EGF binding was reversible and specific. Unlabeled EGF competed for binding to buccal mucosal cells with an IC50 of 1.25 nM, whereas insulin failed to compete. 3. Scatchard analysis of the binding data revealed a curvilinear plot with dissociation constants of 3.39 nM and 2.14 microM, and binding capacities of 1.23 x 10(4) and 3.38 x 10(5) receptors per cell for high and low affinity sites, respectively. 4. Crosslinking of [125I]EGF to buccal mucosa followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed one major protein with Mw 170,000 which shares similar molecular weight with other known EGF receptors from different tissues and species. 5. The study is the first report to provide biochemical parameters of the specific EGF receptors in rat buccal mucosa.     

Immunohistochemical detection of insulin-like growth factor-I, transforming growth factor-beta2, basic fibroblast growth factor and epidermal growth factor-receptor expression in developing rat ovary

The aim of this study was to determine the immunohistochemical expression and localization of insulin-like growth factor-I (IGF-I), transforming growth factor-β2 (TGF-β2), basic fibroblast growth factor (bFGF) and epidermal growth factor-receptor (EGF-R) in developing rat ovaries.Eighteen female Wistar rats were enrolled in this study; newborn (n = 6), one-month-old (n = 6) and adult (n = 6) rats. Formalin-fixed and parafin-embedded ovarian tissues were stained with antibodies against IGF-I, TGF-β2, bFGF and EGF-R, immunohistochemically. The ovarian cells were evaluated by semi-quantitative scoring system under light microscope.The staining of IGF-I, TGF-β2, bFGF and EGF-R were most intense in the oocytes and were heavily at one-month-old rats. A moderate immunostaining in theca cells and corpus luteii reacted with IGF-I in adult rats. Furthermore the staining intensity for IGF-I was moderate in granulosa cells of newborn rat ovaries. We detected also a moderate staining for TGF-β2 in corpus luteii of adult rats. In addition, we found a bFGF immunostaining mainly in oocytes of follicles of young and adult rats. Immunostaining for EGF-R was moderate in granulosa cells of one-month-old rats.In conclusion, this study suggests that growth factors play a pivotal role in ovarian function, especially in follicular development. The role of growth factor in controlling degeneration or growth (or both) of ovary follicles remain as explained.     

Interaction of epidermal growth factor with specific binding sites of enterocytes isolated from rat small intestine during development

Isolated enterocytes from rat small intestine were characterized for their specific binding of epidermal growth factor (EGF). Intestinal epithelial cells were isolated at 4 degrees C to minimize the loss of receptor sites during the isolation procedure. 125I-labelled EGF binding to enterocytes from adult rats was found to be specific, saturable, temperature dependent and trypsin sensitive. Binding performed in the presence of a lysosomotropic agent (NH4Cl) increased the time required to reach maximal binding at 25 degrees C. NH4Cl had no significant effect on the time-course of EGF binding at 4 degrees C and 37 degrees C. A Scatchard plot showed a curvilinear relationship indicating that EGF binds to enterocytes with more than one binding site. Developmentally, enterocytes from fetuses and pups showed characteristic temperature dependence and trypsin sensitivity, but with different levels of binding to EGF. Specific EGF binding was demonstrably higher in enterocytes from small intestine of term fetuses. EGF binding to isolated enterocytes declined rapidly after birth, and the level stayed fairly constant thereafter. Pretreatment of enterocytes from fetal intestine with mature rat milk led to a dose-dependent decrease in EGF binding. These results suggest the presence of endogenous milk factors that modify EGF binding and account for, at least partly, the observed rapid decrease of EGF binding after birth.     

Renal origin of rat urinary epidermal growth factor

The origin of rat urinary epidermal growth factor (EGF) has been investigated. Unilateral nephrectomy decreased the concentration, total output of EGF and EGF/creatinine ratio by approximately 50%, while the output of creatinine was unchanged. Removal of the submandibular glands and duodenal Brunner's glands, organs known to produce EGF, had no influence on the output of EGF in urine. Renal clearance of EGF exceeded that of creatinine, and after bilateral nephrectomy or bilateral ligation of the ureters, the concentration of creatinine in serum increased, while the concentration of EGF was below the detection limit of the assay. Renal production of EGF was confirmed by immunohistochemistry demonstrating EGF immunoreactivity in the afferent arteriole of the juxtaglomerular apparatus. EGF in the submandibular glands and in urine was found to differ with chromatofocusing and reverse-phase HPLC. At isoelectric focusing the pI of submandibular EGF was 4.8 and 5.4 while that of urinary EGF was 5.3 and 6.4.In conclusion, this study demonstrates that urinary EGF mainly originates from the kidneys and is localized to the renal juxtaglomerular apparatus.     

Amiloride inhibits constitutive internalization and increases the surface number of epidermal growth factor receptors in intact rat hepatocytes

In previous experiments the surface expression of epidermal growth factor (EGF) receptors in freshly isolated rat hepatocytes varied temperature- and time-dependently and was depleted by monensin and cycloheximide in a way suggesting that a subpopulation of these receptors are subject to constitutive cycling (Gladhaug and Christoffersen; 1988). We here report the finding that pretreatment of the hepatocytes with amiloride exerts marked effects on cellular EGF receptor movements. After 2 h incubation with 1 mM amiloride, the receptor level was approximately 270,000 sites/cell surface vs. 140,000 in the untreated cell, with no change in receptor affinity. Amiloride thus stabilized the surface EGF receptor pool at an elevated level. In cells pretreated with amiloride for 60 min, the relative endocytosis decreased from about 2.6 EGF molecules internalized per receptor during 15 min endocytosis in untreated cells to about 1.5 molecules/receptor in amiloride-treated cells. These results suggest that amiloride causes an accumulation of EGF receptors at the hepatocyte surface due to inhibition of constitutive receptor internalization. In addition, it was found that in amiloride-treated hepatocytes the phorbol ester TPA strongly inhibited high-affinity EGF binding without affecting the total surface receptor number. In control cells, TPA did not consistently affect binding. Pretreatment with amiloride prevented surface EGF receptor depletion induced by cycloheximide and puromycin, but it did not significantly inhibit surface receptor depletion caused by monensin. Although the underlying mechanism of the amiloride effect on intracellular receptor trafficking is not clear, the results provide further evidence for a continuous, ligand-independent EGF receptor cycling pathway in hepatocytes.     

Renal content and output of epidermal growth factor in long-term adrenergic agonist-treated rats

This study investigates the renal and urinary levels of epidermal growth factor (EGF) in rats under long-term treatment with - or β-adrenergic agonists. Urine samples were obtained on days 7, 14 and 21, and renal tissue samples on day 21. EGF was quantified by ELISA and tissue sections were used for immunohistochemistry and in situ hybridization. Fractional kidney weight was increased in the -adrenergic agonist-treated group by 35% when compared with controls. Histological examination of the kidney revealed well-defined wedge-shaped areas of tubular dilatations and luminal amorphous material in the distal tubules. Concomitantly, reduced levels of EGF and EGF mRNA were observed, and also the urinary levels of EGF were reduced. Together, these observations indicate -adrenergic treatment to affect the distal tubules. Treatment with the β-adrenergic agonist did not change fractional kidney weight, but initially the urinary excretion of EGF was reduced. The data add further evidence to the suggestion that activity of the sympathetic nervous system influences renal homeostasis of EGF, either directly or indirectly through renal histopathological changes.     

Effect of orally-administered epidermal growth factor on intestinal iron absorption and mucosal permeability

A progressive increase in intestinal 59Fe3+ absorption was observed on oral feeding of mice with physiological doses of EGF/UGO. Maximal changes were apparent after 3d and appeared to be dose-dependent. In addition to a small increase in intestinal cell proliferation, as reflected by increased ornithine decarboxylase activity, EGF/UGO-feeding increased mucosal permeability (evaluated with [51Cr]-EDTA): the latter could account for the increase in iron absorption. Sialoadenectomy, to remove the major source of endogenous EGF/UGO, had no appreciable effect on the intestinal absorption of iron.     

Rapid increases in the transglutaminase activity of A431 cells following treatment with epidermal growth factor

Transglutaminase activity was detected in lysates of A431 cells, a human epidermal carcinoma cell line. Enzyme activity was increased 1.5-2.5-fold in lysates prepared from cells pretreated with epidermal growth factor (EGF) relative to untreated control cells. Half-maximal activation of the transglutaminase activity occurred at 3-5 nM EGF, a concentration in good agreement with the Kd for EGF binding to its receptor in these cells. The increase in transglutaminase activity could be detected as early as 2 min after the addition of EGF, with the maximal response attained by 30 min. The activation was not blocked by pretreatment of the cells with cycloheximide, suggesting that the increased activity was not the result of an induction of transglutaminase synthesis. Fractionation of A431 cell lysates by centrifugation at 100000g for 30 min demonstrated that 90% of the transglutaminase activity was present in the soluble fraction and that this soluble transglutaminase activity was increased after treatment of the cells with EGF. The demonstration that EGF acutely increases the activity of a soluble, intracellular transglutaminase defines a novel pathway of growth factor action and provides a useful model system for identifying and comparing the mechanism(s) by which growth factors activate soluble enzymes.     

Presence and characteristics of epidermal growth factor receptors in human fetal small intestine and colon

In the present study, we demonstrate for the first time the presence of important concentrations of EGF binding sites in isolated epithelial cells of both human fetal small intestine and colon as early as 12 weeks gestation. The pattern of EGF binding in the small intestine between 12 and 17 weeks show that binding was significantly higher (2.5-fold) in younger fetuses than in older fetuses. Moreover, the fetal colon exhibited a much higher binding capacity (1.5-2.5 times) than corresponding intestinal cells for all age groups studied. Analysis of Scatchard representations reveal that the concentration of high- and low-affinity binding sites in colonic epithelial cells are twice the values observed in corresponding intestinal cells. The present data raise interesting possibilities as to the role of this growth factor in human fetal gut development.     

Effect of epidermal growth factor on cultured adult rat hepatocytes

When adult rat hepatocytes were cultured in plastic Petri dishes in a medium containing insulin and glucagon, supplementation with epidermal growth factor (EGF) had a pronounced effect on their viability, morphology, and biochemical integrity. Transmission and scanning electron microscopic studies showed that after 1 week cells denied EGF accumulated numerous non-electron-dense bodies and filamentous whorls, had irregular nuclei, and exhibited atypical cell surfaces. In contrast, cells grown for 2-3 weeks in the presence of EGF had well-preserved cellular organelles and remained as an epithelial-like monolayer. After 3 weeks EGF-exposed cultures were still inducible for liver-specific tyrosine aminotransferase, and both rat albumin and rat transferrin were recoverable from the culture medium. Virtually no viable cells were present at 3 weeks in EGF-deprived cultures.     

The components of taurine transport across the rat small intestine A kinetic study

Summary The transport of taurine across adult Sprague-Dawley rat small intestine was studied in vitro using small intestinal strips. The kinetics of the transport mechanism were investigated under both steady-state and influx conditions. Our findings were compatible with the presence of two distinct transport mechanisms; a linear non-carrier mediated component and a saturable carrier mediated component, with almost equal contribution from each. The mediated component was found to be largely Na⁺-dependent and exhibited marked inhibition by B-alanine and structurally related sulfur amino acids.     

Endothelin-1 induces mucosal mast cell degranulation in the rat small intestine

The enhanced production of endothelial cell-derived vasoactive mediators and the activation of mast cells (MCs) have been implicated in the pathogenesis of mucosal damage during ischemia and reperfusion injuries. The first objective of our study was to define the in vivo relation between endothelin-1 (ET-1) and the MC system. Secondly, we determined whether pretreatment with ET receptor antagonists would attenuate MC responses to exogenous ET-1. In the first series of experiments, increasing doses of ET-1 (0. 1, 1 and 3 nmol/kg i.v.) were administered to anesthetized rats. In the second series, the animals were pretreated with equimolar doses of the ET-A receptor antagonist BQ-610 or ETR-P1/fl peptide, and the ET-B receptor antagonist IRL-1038. Intestinal perfusion changes and macrohemodynamics were recorded, and the proportion of degranulated MCs was determined in ileal biopsies. The average mucosal thickness was recorded with an image analysis system. ET-1 induced dose-dependent alterations in the hemodynamic and morphological parameters and caused pronounced mucosal injury, with a significant reduction in villus height. The ratio of degranulated MCs was similar in all ET-treated groups (77%, 82% and 86%) to that observed in animals subjected to 15-min ischemia and 60-min reperfusion (85% degranulation). Pretreatment with BQ-610 and ETR-P1/fl peptide attenuated the ET-1 induced alterations in the hemodynamic parameters and decreased structural injury to the mucosa. ET-induced MC degranulation was significantly inhibited by the ET-A receptor antagonists, but not by IRL-1038. These results indicate that elevated levels of circulating ET-1 might induce intestinal mucosal tissue injury and MC degranulation via activation of ET-A receptors, and raise the possibility that ET-A receptor antagonist administration could exert a potentially beneficial effect through a mechanism other than the blockade of vasoconstriction in pathologies associated with an increased ET-1 release.     

Combined effects of somatomedin-like growth factors with fibroblast growth factor or epidermal growth factor in DNA synthesis in rabbit chondrocytes

Summary The somatomedin-like growth factors cartilage-derived factor (CDF) and multiplication-stimulating activity (MSA) stimulate DNA synthesis and proliferation of rabbit costal chondrocytes under serum-free conditions. Previously, we suggeted that CDF and MSA act on chondrocytes in an early G₁ phase to stimulate DNA synthesis. CDF and MSA have synergistic effects with epidermal growth factor (EGF) or fibroblast growth factor (FGF) in stimulating DNA synthesis of the cells. The mode of combined action of CDF or MSA with EGF or FGF in chondrocytes was studied by sequential treatments with these agents. EGF or FGF had synergistic effects with CDF or MSA in stimulating DNA synthesis, even when added 10 h after the latter. Synergism was also observed in cells pretreated with CDF or MSA; That is, the cultures were treated for 5 h with CDF or MSA and then washed, and treated with FGF or EGF. However, when CDF or MSA was added more than 5 h after EGF or FGF, no synergism of effects was observed. These findings suggest that the cultured chondrocytes become activated to interact with FGF or EGF for commitment to DNA synthesis when they are exposed to somatomedin-like growth factors at an early stage in the G₁ phase. Thus chondrocytes are under a different mechanism of growth control from fibroblastic cells.Abbreviations CDF cartilage-derived factor - MSA multiplication-stimulating activity - EGF epidermal growth factor - FGF fibroblast growth factor     

Direct protective action of epidermal growth factor on isolated gastric mucosal surface epithelial cells.

Epidermal growth factor (EGF) protects gastric mucosa against acute injury produced by a variety of damaging agents, but the mechanism of its protective action is not clear. Since the surface epithelial cells (SEC) are important component of gastric mucosal defence, we studied whether EGF may directly protect isolated gastric SEC against ethanol injury in vitro, in condition independent of systemic factors and whether endogenous prostaglandins may play a role in EGF's protective action. The isolated SEC from rat gastric mucosa were preincubated in medium only, or medium containing 0.0001-10.0 micrograms/ml of h-rEGF for 15 minutes, and incubated with 8% ethanol for 1 hour. In another study the above experiment was repeated but cells were pretreated with 10(-4) or 10(-5) M indomethacin before EGF treatment. The cell viability was assessed by fast green exclusion test. Incubation of SEC with 8% ethanol significantly reduced SEC cell viability to 50 +/- 2%: EGF 0.1 or 1.0 microgram/ml significantly reduced ethanol induced damage (cell viability 59 +/- 3 and 62 +/- 3% respectively). Pretreatment with 10(-4) M indomethacin (the dose which does not affect SEC viability but inhibit PGE2 and PGI2 generation), significantly reduced protective action of EGF against 8% ethanol injury. EGF 1.0 and 10.0 micrograms/ml alone without ethanol increased PGE2 and 6 keto PGF1 alpha generation by SEC. These studies demonstrated: 1) EGF is able to protect gastric surface epithelial cells directly without mediation by systemic factors. 2) EGF induced protection of SEC may in part be mediated by prostaglandins.     

Stimulation by epidermal growth factor of phospholipid methyltransferase in isolated rat hepatocytes

Epidermal growth factor produces a time- and dose-dependent activation of phospholipid methyltransferase activity in hepatocytes isolated from juvenile and mature hepatectomized rats. This treatment however has no effect with hepatocytes isolated from mature or laparotomized rats. Dansylcadaverine (50μM), an inhibitor of receptor-mediated internalization of epidermal growth factor, has no effect on basal phospholipid methyltransferase but inhibits the stimulation of this enzyme by epidermal growth factor.

These results indicate a possible role of phospholipid methylation during liver proliferation.     

Expression of epidermal growth factor in the rat kidney

Summary The renal localization and the site of synthesis of epidermal growth factor (EGF) were investigated in the rat kidney by immunohistochemistry and in situ hybridization techniques. EGF was localized in the cells of the thick ascending limb of Henle (TAL) and distal convoluted tubule (DCT). At the ultrastructural level, EGF immunoreactivity was distributed on the apical membrane and trans-Golgi complex of the TAL and DCT cells. These segments of the rat nephron also hybridized to prepro-EGF cRNA probes in a specific manner, indicating that TAL and DCT are the sites of EGF synthesis in the rat kidney.     

Possible physiological role of epidermal growth factor in the development of the mouse mammary gland during pregnancy

Epidermal growth factor stimulated cell proliferation in a primary mammary epithelial cell culture derived from mice at different stages of pregnancy. Moreover, the peptide hormone inhibited casein production induced by the synergistic actions of insulin, cortisol and prolactin. The inhibitory effect of epidermal growth factor was influenced by the gestational stages of the mammary gland. These effects of epidermal growth factor were exerted at physiological concentrations. The dual actions of epidermal growth factor on mammary cells implicate its participation in regulation of the growth and differentiation of the mammary gland during pregnancy.