1 Digestive System Research Unit, Hospital General Vall d'Hebron, Barcelona 08035; and 2 Department of Pathology, Hospital Mutua, Terrassa 08221, Spain
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ABSTRACT |
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Epidermal growth factor (EGF) is produced in Brunner's glands and plays a role in healing and repair of duodenal ulcers. We examined the participation of zwitterionic phospholipids of mucus in the effects of EGF. Under anesthesia, groups of rats received an intraduodenal bolus of either saline or EGF. Some rats received subcutaneous indomethacin followed by EGF or EGF followed by a detergent (5% Brij 35, a nonionic detergent that solubilizes luminal phospholipids). Thirty minutes after treatment, mucosal surface hydrophobicity and phospholipid concentration in the mucus layer were measured. Matched groups of rats were challenged with 0.5 M HCl, instilled intraduodenally 30 min after treatment, and mucosal damage was assessed 1 h after acid challenge. Exogenous EGF significantly increased surface hydrophobicity and phosphatidylcholine concentration in the mucus layer. EGF treatment also reduced mucosal damage induced by acid. However, indomethacin pretreatment or detergent administration after EGF abolished both protection against acid and changes in the mucus layer. These data suggest that EGF increases duodenal resistance to luminal acid via stimulation of mucosal zwitterionic phospholipids.
indomethacin; mucosal barrier; phospholipids; surfactant
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INTRODUCTION |
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EPIDERMAL GROWTH FACTOR (EGF) is a 6-kDa polypeptide (3) that is secreted into the gastrointestinal lumen by salivary glands, Brunner's glands of the duodenum, and pancreas (28). Secretion of EGF is under cholinergic, adrenergic, and humoral control (20, 21). In addition to the well-recognized effects of EGF on cell proliferation and tissue repair, cumulative evidence indicates that EGF and EGF-like peptides are active mediators of mucosal protection and adaptation to injury (28). The mechanism or mechanisms by which EGF exerts protective actions on the mucosa are not fully understood but certainly involve several factors: inhibition of gastric acid secretion (29), changes in mucosal blood flow (8, 27), and secretion of mucus (11).
Localization of EGF in Brunner's glands of the proximal duodenum suggests a physiological role of EGF in local defense and repair mechanisms. The duodenal mucosa is normally challenged by intermittent exposure to acid because of periodic emptying of gastric juice into the duodenum (18). We have previously shown (17) in the rat that pulses of acid entering the duodenum trigger a response of adaptation, which renders the mucosa more resistant to subsequent acid challenge. The hydrophobic character of the mucosa seems to be a critical factor in the adaptation and protection of the gastroduodenal mucosa against luminal acid (13, 16). Surface hydrophobicity is attributable to a monolayer of surface-active phospholipids adsorbed directly on glycoproteins of the mucus that covers the surface epithelium (6, 9). Mild acid and prostaglandins, which offer gastroduodenal protection against injury, induce secretion of surface-active phosphatidylcholines by gastric mucus cells and submucosal glands of the proximal duodenum (12, 16). On the other hand, mucus phospholipids and hydrophobicity are attenuated by several injurious agents, including aspirin, nonsteroidal anti-inflammatory drugs, bile salts, or restraint stress (12, 15).
Our hypothesis was that EGF plays a physiological role in the adaptation of the duodenal mucosa to gastric acid. In the present study we have investigated the effects of EGF in preventing duodenal mucosal injury by acid as well as the participation of surface hydrophobicity and zwitterionic phospholipids of the mucus layer in this process.
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MATERIALS AND METHODS |
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Animals and experimental design. Studies were performed on male Sprague-Dawley rats weighing 200-250 g (Centre d'Elevage R. Janvier, Le Genest, France). Animals were fed standard rodent chow (Letica, Barcelona, Spain) and tap water ad libitum. All experimental procedures were approved by the local research committee (Comite Etic d'Experimentacio Animal, Hospital Vall d'Hebron).
The study included three experimental protocols. The first protocol investigated the effect of EGF on the hydrophobic surface properties of the duodenal mucosa. The second protocol studied changes in phospholipid concentration in the duodenal mucous layer induced by EGF. Finally, the third protocol investigated the role of EGF in resistance of the duodenal mucosa to intraluminal acid. Surgical procedures were similar in the three protocols. Following overnight fast, rats were anesthetized with urethan (1.25 g/kg ip; Fluka Chemie, Buchs, Switzerland). A tracheostomy was performed, and an indwelling catheter of polyethylene tubing (Clay Adams, Parsippany, NJ) was placed in the carotid artery for measurement of arterial blood pressure. The abdomen of each rat was incised, and a double-lumen tube, prepared with polyvinyl tubing (ID 1.40 mm, OD 1.90 mm; Abbot, Sligo, Ireland), was inserted through the forestomach into the gastric lumen and passed through the pylorus. The tube was held in place by ligatures around the forestomach and the pylorus. One of the lumens was used to drain the gastric contents by siphonage. The second lumen opened just beyond the pylorus and was used for pulse instillations of normal saline, acid, EGF, or other drugs into the duodenal lumen. The abdominal wall was sutured, and the rats received 1-ml saline pulses through the stomach and the duodenal line every 30 min for 2 h to achieve full equilibration (17). Thereafter, additional duodenal pulses were instilled as follows: Saline group, 1 ml saline and 20 min later 2 ml saline; EGF group, 50 µg/kg mouse EGF (receptor grade; Sigma, St. Louis, MO) in 1 ml saline and 20 min later 2 ml saline; Brij group, saline and 20 min later 2 ml of a nonionic detergent (Brij 35, 5% polyoxyethylene 23 lauryl ether; Sigma); EGF-Brij group, 50 µg/kg EGF and 20 min later 2 ml of the detergent Brij 35; Indo group, a subcutaneous injection of 5 mg/kg indomethacin (Sigma) 30 min before the intraduodenal pulse of saline, and Indo-EGF group, a subcutaneous injection of 5 mg/kg indomethacin 30 min before the intraduodenal pulse of EGF. Instillation of detergent and administration of indomethacin was aimed at reducing the phospholipid layer from the luminal surface of the duodenal mucosa (15, 16). Rats remained under general anesthesia for the full duration of the experiments.Hydrophobicity of the duodenal mucosa. Thirty minutes after saline or EGF administration, animals were killed by cervical dislocation (5-7/group). To quantify mucosal hydrophobicity, the duodenum was removed, opened along the mesenteric side, and rinsed with 0.9% saline at room temperature. Duodenal specimens were carefully placed on a glass microscope slide, mucosal side up, and prepared for measurement of surface hydrophobicity by determination of the contact angle that conforms a drop of saline (5 µl) deposited on the mucosal surface, as described by Goddard et al. (5). Adherent fluid was removed gently from the mucosa with soft tissue, and the surface was allowed to dry for 1 h at room temperature. The specimen was transferred to the stage of a goniometer (100-00 Ramé-Hart, Mountain Lakes, NJ). Four to five contact angle readings (in units of degrees) were made on each duodenum and averaged for 1 value/duodenum.
Phospholipid concentration in the mucous layer. The same groups of rats described above were studied (n = 13-15). All animals were killed 30 min after the last pulse of saline or EGF. The duodenum was removed, opened along the mesenteric side, and laid on a flat surface. The mucus on the mucosal surface up to a distance of 1 cm caudal to the pylorus was gently scraped using filter paper (Whatman no. 3; Whatman International, Maidstone, UK). As shown previously, histological examination disclosed no damage to the epithelium attributable to the scraping procedure (16).
The total amount of mucus recovered was pooled, weighed, and extracted with chloroform and methanol following the method of Folch et al. (4). The lipid phase was evaporated to dryness under a stream of nitrogen and weighed to estimate total lipid concentration in mucus. Dried samples were then resuspended in benzene, and concentration of inorganic phosphorus was determined using the method described by Ames (1) to estimate total phospholipids in mucus. Phospholipid species in the extracts were analyzed by thin-layer chromatography on plates coated with silica gel G-60 (Merck, Darmstadt, Germany). The developing solvent mixture used was chloroform-methanol-acetic acid-water, 170:40:18:10 (vol/vol). All solvents were HPLC grade (Merck). Spots on all chromatograms were visualized by spraying the plates with sulfuric acid and heating them at 180°C for 30 min. Identification of each phospholipid species was confirmed by comparison with pure standards (Sigma) of sphingomyelin, phosphatidylserine, phosphatidylinositol, phosphatidylcholine, and phosphatidylethanolamine chromatographed in parallel. The silica of each spot was scraped off for quantitation of inorganic phosphorus by the Ames method (1).Duodenal resistance to acid. Studies were performed in the same groups of animals as indicated above (Saline, EGF, Brij, EGF-Brij, Indo, and Indo-EGF groups; n = 5-6). Thirty minutes after the last intraduodenal pulse of saline or EGF, all animals received an intraduodenal pulse of 500 µmol HCl (in 1 ml saline). The acid load in the 500-µmol HCl pulse was far beyond the physiological range and was aimed at inducing duodenal mucosal lesions.
Animals were killed by cervical dislocation 1 h after acid administration. The stomach and the duodenum were removed and immediately fixed by intraluminal perfusion of a neutral buffered 10% formalin solution for 30 min via the double-lumen tube used in the experiments. Thereafter, the specimens were opened along the greater curvature and laid on a flat surface. All samples were coded for macroscopic and histological scoring of the lesions by two pathologists (A. Domingo and A. Salas) unaware of the treatment administered to each rat. A global score of duodenal damage was obtained by summation of macroscopic findings (mucosal appearance and length of the damaged area) and histological findings (depth of mucosal necrosis and vascular thrombosis) (Table 1).
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Statistical analysis. All results are expressed as means ± SE. Statistical analysis of the data was performed using the SPSS package. Homogeneity of variance was confirmed by the Bartlett method. Overall differences between means were tested with ordinary ANOVA. If the analysis of variance was significant, then Fisher's least significant difference method was used as a post hoc test for comparison between group means. Correlation coefficients were calculated by the Pearson correlation procedure.
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RESULTS |
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Hydrophobicity of the duodenal mucosa.
As shown in Fig. 1, intraduodenal
administration of 50 µg/kg EGF significantly increased surface
hydrophobicity of the duodenal mucosa compared with saline-treated
rats. A bolus of 2 ml of 5% Brij 35, administered through the duodenal
line 20 min after the saline pulse, significantly reduced duodenal
hydrophobicity. Moreover, detergent flushing abolished the effect of
EGF on surface hydrophobicity. On the other hand, rats pretreated with
a single parenteral dose of 5 mg/kg indomethacin showed reduced contact
angle values in the surface duodenum compared with saline-treated rats.
In rats pretreated with indomethacin, no changes in surface
hydrophobicity were observed after EGF administration.
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Phospholipid concentration in the mucous layer.
Table 2 shows the amount of mucus
recovered by scraping the duodenal surface and the lipid extract from
the mucus layer. Intraduodenal administration of 50 µg/kg EGF
significantly increased the release of mucus, lipids, and phospholipids
by the mucosa. The effect of EGF on the release of mucus and lipids was
also evident after flushing Brij 35 through the duodenal line, but the
amount of phospholipids recovered in the mucus scrapings diminished to
the control value (i.e., Saline group), suggesting chemical association
between detergent and phospholipids. In rats pretreated with
indomethacin, the administration of EGF also enhanced the release of
mucus and phospholipids compared with saline-treated rats but did not
modify the total lipid extract of the mucus layer.
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Duodenal resistance to acid. As previously reported (17), an intraduodenal pulse of 500 µmol HCl induced severe duodenal lesions in saline-pretreated rats. After 1 h of the acid instillation, the mucosa showed large necrotic areas descending along the duodenum. By histological examination, the surface epithelial cells covering the villi showed signs of coagulation necrosis with lightly stained cytoplasm and indistinct nuclei. Damage was evident along the whole height of the villi. The lamina propria showed edema, congestion, some vessels occluded by fibrin thrombi, and hemorrhagic foci.
Pretreatment with 50 µg/kg EGF 30 min before acid instillation resulted in significantly lower mucosal injury than pretreatment with saline (Fig. 3). EGF-treated animals showed an intact or slightly damaged mucosa with only petechial lesions. In some rats, epithelial erosions appeared at apex of the villi but no other changes in the mucosa were observed. An intraduodenal bolus of 2 ml of 5% Brij 35 administered 20 min after EGF completely suppressed the protective effect of EGF. Ancillary experiments in rats not receiving acid showed that 5% Brij 35 did not induce any damage, indicating that the detergent by itself did not account for the mucosal damage.
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DISCUSSION |
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EGF-like peptides are a large family of ligands that bind to the EGF receptor. Several of these ligands have been identified in the gut, and they play an important role in the development and maintenance of gastrointestinal epithelial continuity. The aim of the present study was to investigate the effects of EGF on the acid-resistant barrier properties of the duodenal mucosa.
Our data indicate that EGF applied topically to the duodenum markedly increases the zwitterionic phospholipid concentration in the duodenal mucus layer and enhances surface mucosal hydrophobicity. Under our experimental conditions, EGF provides mucosal protection against acid challenge. On the other hand, duodenal instillation of the nonionic detergent Brij 35 after EGF treatment or indomethacin pretreatment precludes the effect of EGF on mucosal surface hydrophobicity and protection against acid. Thus our data suggest that EGF improves the mucosal barrier by increasing duodenal surface hydrophobicity.
The relevance of the mucus layer covering the gastrointestinal mucosa in the functional barrier against acid is well established. The hydrophobic character of this layer is one of the most important biophysical characteristics of mucus, and it is clearly related to the functional properties of the barrier (12). The hydrophobic character of the duodenal mucosa varies among species, but in the rat (15) and human (26), the proximal duodenum exhibits high contact angle values that are slightly lower than those observed on the gastric surface. Kao and Lichtenberger (10) localized surfactant-like reactive hot spots in the Brunner's glands of the rodent duodenal mucosa. In the present study, we report that intraluminal EGF administration increases the hydrophobicity of the duodenal surface, rendering the mucosa nonwettable. Surface hydrophobicity has been implicated in the protective mechanisms afforded by other gastroprotective agents (12).
The effect of EGF on surface hydrophobicity was associated with a significant increase in the concentration of phospholipids within the duodenal mucus layer and with a higher concentration of the phosphatidylcholine fraction. Phosphatidylcholines are a major constituent of the gastroduodenal surfactant (12, 13). Morphological evidence with different techniques indicates that mucous cells and submucosal glands located in different parts of the gut are able to store and secrete surfactant-like particles (10). A previous study in our laboratory with similar experimental conditions (16) showed that intraluminal administration of 16,16-dimethyl PGE2 and a low concentration of HCl protect duodenal mucosa against a subsequent injurious acid load. This protection was associated with a marked increase of phosphatidylcholines in the mucus layer. We also observed a high density of hydrophobic lipid spots in Brunner's glands. These results supported the notion that surfactant-rich particles are synthesized in Brunner's glands and secreted into the lumen. Our current data suggest that EGF would enhance phospholipid secretion by Brunner's glands. It is well known that EGF stimulates secretion of mucin along the gastrointestinal tract, but no previous data about the effect of EGF on phospholipid secretion have been reported. However, other studies have shown that EGF stimulates the secretion of pulmonary surfactant (7) and enhances the incorporation of choline into phosphatidylcholines (24).
The surfactant layer results from the balance between active release of phospholipids by mucous cells and continuous removal from the surface by friction with luminal contents. In our study, luminal application of the nonionic detergent Brij 35 significantly reduced mucosal hydrophobicity and solubilized phosphatidylcholines, as shown by a low concentration of this phospholipid species in rats treated with the detergent. Those changes were associated with a reduced mucosal protection against acid, even in rats pretreated with EGF. These findings suggest that the effect of EGF on the mucosal barrier is a critical mechanism for mucosal protection.
On the other hand, a subcutaneous injection of indomethacin before EGF administration abolished EGF-induced mucosal protection. A prostaglandin-dependent pathway might mediate protection by EGF. However, indomethacin produced by itself a disruption of the phospholipid layer on the mucus gel and a significant reduction in duodenal hydrophobicity. Lichtenberger and co-workers (14) have demonstrated that topical nonsteroidal anti-inflammatory drugs, including indomethacin, attenuate surface hydrophobicity by weakening lateral interaction between zwitterionic phospholipid molecules, rendering the surface mucus layer wettable. A previous study from our laboratory (15) showed that a single parenteral dose of indomethacin reduced duodenal mucosal hydrophobicity for up to 24 h after dosing. At the dose of indomethacin tested, prostaglandin synthesis by the mucosa was inhibited for only 1 h, but 24 h after dosing mucosal prostaglandin synthesis was at control levels. This finding suggested that reduction of surface hydrophobicity by indomethacin was independent of prostaglandin synthesis inhibition. In addition, we found that indomethacin had no effect on mucosal surface hydrophobicity in bile duct-ligated rats. Topical interaction of the drug or its metabolites, excreted through the biliary tract, with the duodenal surface would account for the effects observed on surface hydrophobicity. A recent study in the rat has shown that indomethacin may damage the mucosa not by a direct irritant action but by competing for the available protective phosphatidylcholine molecules (2). It is interesting to note that in our study indomethacin treatment reduced the concentration of phosphatidylcholines in the mucus layer and increased the proportion of phosphatidylethanolamines. A similar alteration of the phospholipid profile has been found in the gastric mucus layer of rats treated with intragastric ethanol at 40% (19). Phosphatidylethanolamine has been described as a rigidifying phospholipid because of the formation of hydrogen bonds between its primary amine and neighboring phospholipid head groups. Thus the strength of the hydrophobic barrier may not only be a consequence of high phosphatidylcholine concentration in mucus but also relates to lipid composition and molecular interactions between phospholipids and glycoproteins in the mucus layer.
Parenteral administration of EGF appears to be more effective in preventing drug-induced injury to the rat gastric mucosa than orogastric administration of the growth factor (25). This is probably due to the rapid degradation of intact EGF to less active forms in the presence of acid and pepsin, as previously shown by Playford et al. (23). In our study, EGF was administered through a polyvinyl tube directly into the duodenal lumen, thus avoiding gastric digestion of the compound. Most studies on the role of EGF in mucosal protection have used the topical approach (28), since EGF is naturally found in secretions of the salivary glands, duodenum (from Brunner's glands), and pancreas. In addition, recent studies suggest that growth factors present in colostrum, including EGF-like peptides, might be useful as an oral food supplement for the prevention and treatment of a wide variety of gastrointestinal conditions (22). Nevertheless, it must be emphasized that in the current experimental study we have used a high dose of EGF, beyond the physiological range, and a nonphysiological dose of hydrochloric acid to induce mucosal damage.
In conclusion, the gastroduodenal mucus layer constitutes an functional barrier against diffusion of acid. The ability of EGF to induce surface-active phospholipid secretion and improve the hydrophobic strength of the mucus layer may play an important physiological role in the maintenance of a mucosal defense line.
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FOOTNOTES |
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Address for reprint requests and other correspondence: F. Guarner, Digestive System Research Unit, Hospital General Vall d'Hebron, Barcelona 08035, Spain (E-mail: fguarnera{at}meditex.es).
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 27 July 2000; accepted in final form 21 November 2000.
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