Role of gastric blood flow, neutrophil infiltration, and mucosal cell proliferation in gastric adaptation to aspirin in the rat.

Gastric mucosa exhibits the ability to adapt to ulcerogenic action of aspirin but the mechanism of this phenomenon is unknown. In this study, acute gastric lesions were produced by single or repeated doses of acidified aspirin in rats with intact or resected salivary glands and with intact or suppressed synthase of nitric oxide. A single oral dose of aspirin produced a dose dependent increase in gastric lesions accompanied by considerable blood neutrophilia and mucosal neutrophil infiltration, significant reduction in gastric blood flow, and almost complete suppression of biosynthesis of prostaglandins. After rechallenge with aspirin, the mucosal damage became smaller and progressively declined with repeated aspirin insults. Gastric adaptation to aspirin was accompanied by a significant rise in gastric blood flow, reduction in both blood neutrophilia and mucosal neutrophil infiltration, and a remarkable increase in mucosal cell regeneration and mucosal content of epidermal growth factor. Salivectomy, which reduced the mucosal content of epidermal growth factor, aggravated the initial mucosal damage induced by the first exposure to acidified aspirin but did not prevent the adaptation of this mucosa to repeated aspirin insults. Pretreatment with NG-nitro-L-arginine (L-NNA), a specific inhibitor of nitric oxide synthase, eliminated the hyperaemic response to repeated aspirin but did not abolish the development of adaptation to aspirin showing that the maintenance of the gastric blood flow plays little part in this adaptation. In conclusion, the stomach adapts readily to repeated aspirin insults and this is accompanied by a considerable reduction in blood neutrophilia and the severity of neutrophil infiltration and by an extensive proliferation of mucosal cells possibly involving epidermal growth factor.     

Superoxide Anion and Nitric Oxide in High-Grade Esophagitis Induced by Acid and Pepsin in Rabbits

It has been proposed that free radicals are involved in the pathogenesis of esophageal mucosal damage induced by acid and pepsin. Recent data have suggested that nitric oxide (NO) is involved in the mucosal defense of the esophagus and that superoxide anion plays a minor role in low-grade esophagitis. To study the role and potential interaction of NO and superoxide anion in an experimental model of high-grade esophagitis, acidified pepsin was perfused (45 min/12 hr) for five days in rabbits with different agents to modulate the generation of these radicals. Measurements included both macroscopic and microscopic mucosal damage, superoxide anion generation, NO synthase mucosal activity, and peroxynitrite formation. High-grade esophagitis was associated with mucosal superoxide anion generation. Treatment with exogenous superoxide dismutase completely prevented mucosal damage. The perfusion of acidified pepsin in the lumen of the esophagus was initially associated with increased NO synthase mucosal activity but decreased with the progression of damage. Generation of peroxynitrites was present in those cases with severe damage. Treatment with NO-modifying agents did not induce consistent modification of mucosal damage. It is concluded that superoxide anion is involved in the induction of high-grade esophagitis and that it interacts with nitric oxide to generate peroxynitrite radicals in this model. Superoxide dismutase but not NO-donor-modifying agents might have a therapeutic role in preventing severe esophageal mucosal damage induced by acid and pepsin.     

Salivary Bicarbonate as a Major Factor in the Prevention of Upper Esophageal Mucosal Injury in Gastroesophageal Reflux Disease

Background

It has been previously demonstrated that the exposure of the lower esophageal mucosa to acid and pepsin results in significant increase in salivary protective factors secretion, mediated by the esophago-salivary reflex. The impact of the upper esophageal mucosal exposure to acid and pepsin on salivary secretory response remains unknown.

Aims

To investigate the rate of salivary protective factors secretion during the upper esophageal mucosal exposure to acid and pepsin and to compare with the corresponding results recorded during the lower esophageal mucosal exposure, in the same group of asymptomatic volunteers.

Methods

The study was conducted in 10 asymptomatic volunteers. Salivary samples were collected during the esophageal mucosal exposure to saline, followed by acid/pepsin and the final saline, using the esophageal perfusion catheter. Salivary bicarbonate and non-bicarbonate buffers were analyzed using TitraLab. Salivary mucin and protein were quantified through PAS and Lowry methodologies, respectively, whereas PE₂ using radioimmunoassay. Statistical analysis was performed using Σ-Stat software.

Results

The rate of salivary bicarbonate secretion was significantly higher (3.1-fold) during the upper versus the lower esophageal mucosal exposure to acid and pepsin (87.5 ± 14.4 vs. 28.0 ± 7.70 μEq/min, p < 0.05). The volumes of saliva, pH, salivary protein, mucin and PE₂ were similar in both esophageal perfusions.

Conclusions

Threefold stronger secretion of salivary bicarbonate could be a major factor protecting the upper esophageal mucosa. This phenomenon may represent an ultimate defense mechanism potentially preventing further complications within the upper esophageal mucosa; however, it needs to be confirmed in patients of gastroesophageal reflux disease.     

Nitric Oxide and Superoxide Anion in Low-Grade Esophagitis Induced by Acid and Pepsin in Rabbits

It has been suggested that free radicals are involved in esophagitis. To study the role and potential interaction of superoxide anion and nitric oxide (NO) in low-grade esophagitis, we perfused acidified pepsin (30 min every 12 hr) for seven days in rabbits treated with different agents to modulate the generation of these radicals. Measurements included macroscopic and microscopic damage, superoxide anion generation, mucosal nitric oxide synthase activity, and peroxynitrite formation. Low-grade esophagitis was associated with increased nitric oxide synthase mucosal activity and mucosal damage was dose-dependently increased by treatment with the NO synthase inhibitor N ^G-nitro-l-arginine. Superoxide anion was scarcely generated in the mucosa, but this was not accompanied by any change in the activity of mucosal superoxide dismutase. Treatment with superoxide dismutase did not improve mucosal damage. Generation of peroxynitrites was not detected. In conclusion, nitric oxide is involved in the mucosal defense of the esophagus against acid- and pepsin-induced damage. Superoxide anion generation seems irrelevant in the induction of low-grade esophagitis and not sufficient to interact with nitric oxide to generate measurable mucosal peroxynitrite radicals.     

Bismuth subsalicylate reduces peptic injury of the oesophagus in rabbits.

Bismuth subsalicylate was tested in an in vivo perfused rabbit model of oesophagitis for its ability to prevent the mucosal injury caused by pepsin. Treatment efficacy was assessed under both a treatment-before-injury protocol and a treatment-after-injury protocol. Oesophageal mucosal barrier function was evaluated by measuring flux rates of H+, K+, and glucose. The degree of oesophagitis was determined by gross and microscopic examination of the mucosa by several independent observers. Results showed that under both treatment protocols, bismuth subsalicylate significantly reduced the pepsin induced disruption of the mucosal barrier, as well as the morphologic changes. Bismuth subsalicylate when given after exposure to pepsin was also found to protect against the morphologic injury in a dose dependent manner. Experiments in vitro suggested that bismuth subsalicylate inhibits the proteolytic action of pepsin by interacting with pepsin, rather than with the pepsin substrate. We conclude that bismuth subsalicylate can protect the oesophageal mucosa against peptic injury, probably through inactivation of pepsin.     

Hydrochloric acid

The effect of a single instillation of acid and pepsin on the cell proliferation in the distal esophageal mucosa was investigated in four dogs. The doses of acid and pepsin used were lower than those provoking acute esophagitis and erosions. Usingin vitro labeling with [³H]thymidine and autoradiography, the epithelial mitotic and DNA synthesis indices were determined at 0, 4, 8, 12, 16, 20, and 24 hr after instillation of saline, of acid alone, or of acid with pepsin. Instillation of acid alone was followed, 16 hr later, by an increase (P<0.01) in DNA synthetic activity in the proliferative area. A mitotic peak (P<0.01) started from the 20th hour. After instillation of acid with pepsin in the same animals, a similar sequence of kinetic phenomena was observed, suggesting that the concentration of pepsin used did not potentiate the stimulating effect of acid on the cell proliferation in this epithelium. Our data indicate that hydrochloric acid stimulates the proliferative activity in the normal esophageal epithelium.This work was supported in part by a grant for cancer research from the Algemene Spaar-en Lijfrentekas and by the Fonds voor Geneeskundig Wetenschappelijk Onderzoek.     

Irritant-induced cyclooxygenase-2 is involved in the defense mechanism of the gastric mucosa in mice

Background. Endogenous and exogenous prostaglandins (PGs) have been shown to contribute to reducing the gastric injury caused by irritants given subse-quently. The aim of this study was to clarify whether cyclooxygenase-2 (COX-2) protein induced by pretreatment was involved in the prevention of subsequent ethanol-caused gastric injury in mice. Methods. Mice were pretreated with acidified ethanol or saline and then COX-2 protein expression in the stomach was immunohistochemically determined every 8 h. Mice were administered 95% ethanol 24 h after the acidified ethanol pretreatment, and gastric mucosal damage was evaluated macroscopically and histologically. The effects of NS-398 or indomethacin on the 95% ethanol-caused damage were also examined. Results. Acidified ethanol pretreatment induced COX-2 protein expression in lamina propria macrophages of the gastric mucosa, with a peak level 24 h after the pretreatment. The 95% ethanol treatment caused gastric mucosal damage. The degree of the damage was not different between mice pretreated with acidified ethanol and those pretreated with saline. However, NS-398 aggravated the ethanol-caused damage only in mice pretreated with acidified ethanol, while indomethacin aggravated the damage, evaluated histologically, irrespective of the pretreatment. Conclusions. Pretreatment-induced COX-2, in addition to COX-1, seemed to be involved in the defense mechanism through minimizing the damage caused by a subsequent irritant. Received: October 16, 2000 / Accepted: September 14, 2001     

Declined human esophageal mucin secretion in patients with severe reflux esophagitis

It has been recently demonstrated that human esophageal submucosal mucous glands exhibit the ability to secrete copious amounts of mucin, well known within the gastrointestinal tract for its protective quality against hydrogen ion and pepsin. Since mucin may also play a protective role within the esophageal compartment, we have studied the rate of secretion of esophageal mucin in patients with RE. Mucin was assessed by periodic acid-Schiff methodology in esophageal secretion collected during continuous perfusion with saline (period I) followed by HCl (period II), HCl/pepsin (period III), and final saline (period IV), mimicking the natural gastroesophageal scenario. The basal rate of the luminal release of mucin in patients with grade II RE was 18% lower as compared with controls. During exposure of the esophageal mucosa to an HCl/pepsin solution, esophageal mucin output in the RE group was 52% lower than in the control group (0.154 ± 0.027 vs 0.320 ± 0.049 mg/cm²/min;P=0.025). Furthermore, the rates of esophageal mucin output in patients with grade III RE during esophageal perfusion with saline and HCl/pepsin were 62% (0.090 ± 0.021 vs 0.239 ± 0.036 mg/cm²/min;P=0.016) and 86% (0.048 ± 0.010 vs 0.320 ± 0.049 mg/cm²/min;P=0.001) lower when compared with corresponding values in controls. After endoscopic healing of RE, the overall impairment in the rate of esophageal mucin secretion in patients with grade II improved from 31% to 17% at the end of therapy, whereas in patients with grade III the impairment in mucin secretion improved only marginally from 71% to 69%. Significant impairment in the rate of esophageal mucin output in patients with severe esophagitis (grade III), prevailing despite healing of endoscopic changes, indicates that a low rate of esophageal mucin secretion may be a potential contributing factor in the development of severe mucosal damage in patients with excessive gastroesophageal reflux.This project had been partly supported by AGA Foundation/SmithKline Beecham Clinical Research Award and Jeffress Trust grant J-240.     

Parenterally administered salivary tissue extracts reduce outputs of Na+ and K+ associated with bile salt-induced damage of rat oxyntic mucosa

Extirpation of the major salivary glands in rats has been shown to exacerbate the ionic fluxes associated with pharmacologically-induced damage to the gastric mucosal barrier. In the present study we have investigated the effect of the administration of a crude extract of submandibular-sublingual salivary glands on gastric secretion and gastric mucosal barrier integrity in the rat. The extract was administered in a dose (4 mg protein) which would reduce established acid secretion by 30%. Chronic parenteral (intraperitoneal) administration of salivary gland extract but not phosphate buffer resulted in a reduction in the luminal appearance of Na⁺ and K⁺ associated with bile salt-induced damage (5 mM sodium taurocholate in 150 mM HCl) to the gastric mucosa of sialoadenectomized animals. Oral administration of the extract depressed its protective action. Boiling the extract prior to parenteral administration also abolished its ability to protect the mucosa. Acute administration of the extract by intraperitoneal injection only 30 min prior to bile salt treatment was also ineffective. These results suggest that chronic parenteral but not enteral treatment with a crude salivary gland extract aids in the maintenance of the gastric mucosal barrier in sialoadenectomized rats. The inability of acute administration of the extract to reduce the net fluxes of Na⁺ and K⁺ suggests that its mode of action is not entirely dependent upon an inhibition of gastric acid secretion.This study was funded by a grant from the Medical Research Council of Canada, Grant no. MA-6426     

Inhibitory effect ofl-glutamine on gastric irritation and back diffusion of gastric acid in response to aspirin in the rat

l-Glutamine given orally at 750 mg/kg significantly reduced the formation of gastric mucosal lesions induced by aspirin at 100 mg/kg at 1, 3, and 7 hours in pylorus-ligated rats.l-Glutamine markedly inhibited the loss of H⁺ and a corresponding increment of Na⁺ through the aspirin-damaged mucosal barrier, which suggests inhibition of acid back diffusion as the mechanism of action. In vagotomized rats, the diffusion of H⁺ from the instilled acid solution into the gastric mucosa and outflux of Na⁺ from the mucosa into the lumen was so strong that aspirin could not show any acceleration of a back diffusion of H⁺ in contrast to the aspirin-free group. However,l-Glutamine given with or without aspirin inhibited the back diffusion of H⁺ instilled into the vagotomized rat stomach.     

Gastric adaptation to injury by repeated doses of aspirin strengthens mucosal defence against subsequent exposure to various strong irritants in rats.

Gastric adaptation to injury during repeated doses of acetyl salicylic acid (ASA) is a well documented finding but it is not known whether this adaptation affects the tolerance of the mucosa to other strong irritants. Gastric adaptation was induced by repeated daily doses of acidified ASA (100 mg/kg in 1.5 ml of 0.2 N HCl) given intragastrically (series A rats). Control rats with an intact stomach were given daily intragastric vehicle only (1.5 ml of 0.2 N HCl) (series B). After full adaptation to ASA (5 days), rats were challenged again with acidified ASA or, for comparison, with strong irritants such as 100% ethanol, 200 mM acidified taurocholate, or 25% NaCl for 1 hour or with water immersion and restraint for 3.5 hours. The first dose of ASA produced numerous gastric lesions and deep histological necrosis accompanied by a fall in the gastric blood flow, negligible expression of epidermal growth factor (EGF) and transforming growth factor alpha (TGF alpha) or their receptors, and no evidence of mucosal proliferation. As adaptation to ASA developed, however, the areas of gastric lesions were reduced by more than 80% and there was a noticeable decrease in deep necrosis, a partial restoration of gastric blood flow, an approximately four-fold increase in EGF expression (but not in TGF alpha) and its receptors, and an appreciable increase in mucosal cell proliferation compared with vehicle treated rats. Increases in the mucosal expression of EGF receptors and the luminal content of EGF were also found in ASA adapted animals. In ASA adapted rats subsequently challenged with 100% ethanol, 200 mM TC, 25% NaCl, or stress, the area of the gastric lesions and deep histological necrosis were appreciably reduced compared with values in vehicle treated rats. This increased mucosal tolerance to strong irritants was also accompanied by the return of the gastric blood flow towards control levels and further significant increases in the mucosal expression of EGF receptors and mucosal cell proliferation. Gastric adaptation to ASA enhances the mucosal resistance to injury by strong irritants probably as a result of the restoration of the gastric blood flow and increased cell proliferation that may result from increased mucosal expression of EGF and its receptors.     

Esophageal mucosal resistance. A factor in esophagitis

The development of esophageal damage depends on a number of factors. The components in the refluxate, including H+ ion, pepsin, bile salts, and pancreatic enzymes, are able to permeate the mucosa and cause injury. These agents may act individually or in combination. Balancing the effects of these damaging agents is the "esophageal mucosal barrier." This barrier is an integrated complex of anatomic and physiologic components that acts to maintain the integrity of the mucosa. Although the relative efficacy of the various components in developing an effective barrier is not understood completely, their physiologic and clinical importance in the face of "noxious" luminal contents remains critical. Understanding the interplay between the injurious agents in the refluxate and the esophageal mucosal barrier may allow for the development of new therapeutic measures in the treatment and prevention of gastroesophageal reflux disease.     

胃黏膜对阿司匹林适应性的实验研究

目的 探讨胃黏膜对阿司匹林的适应性及其发生机制。方法 连续数天经胃管给大鼠灌入酸化阿司区林（ａｃｉｄｉｆｉｅｄ ａｓｐｉｒｉｎ,ＡＳＡ）,计算胃黏膜损伤面积和损伤深度。用ＲＩＡ法检测胃黏膜组织内ＥＧＦ的含量。为观察一氧化氮（ＮＯ）在胃黏膜对ＡＳＡ适应性中的 应用ＡＳＡ前３０ｍｉｎ经大鼠性静脉分另注入Ｌ－精氨酸（Ａｒｇ）、Ｌ－ＮＡＭＥ及Ｌ－Ａｒｇ＋ＬＮＡＭＥ,观察胃黏膜损伤面积及深度。结果 初次应用     

Re-thinking the functions of IgA+ plasma cells

The intestinal mucosa harbors the largest population of antibody (Ab)-secreting plasma cells (PC) in the human body, producing daily several grams of immunoglobulin A (IgA). IgA has many functions, serving as a first-line barrier that protects the mucosal epithelium from pathogens, toxins and food antigens (Ag), shaping the intestinal microbiota, and regulating host–commensal homeostasis. Signals induced by commensal colonization are central for regulating IgA induction, maintenance, positioning and function and the number of IgA⁺ PC is dramatically reduced in neonates and germ-free (GF) animals. Recent evidence demonstrates that the innate immune effector molecules tumor necrosis factor α (TNFα) and inducible nitric oxide synthase (iNOS) are required for IgA⁺ PC homeostasis during the steady state and infection. Moreover, new functions ascribed to PC independent of Ab secretion continue to emerge, suggesting that PC, including IgA⁺ PC, should be re-examined in the context of inflammation and infection. Here, we outline mechanisms of IgA⁺ PC generation and survival, reviewing their functions in health and disease.     

The role of pepsin in acid injury to esophageal epithelium

OBJECTIVES: The development of reflux esophagitis in humans is a process resulting from esophageal exposure to refluxed gastric contents. There is no doubt that damage to the esophageal epithelium requires exposure to gastric acid; however, the role of refluxed pepsin as contributor to this damage seems to be underappreciated. METHODS: The role of physiological concentrations of pepsin was examined in Ussing chambered rabbit esophageal epithelium and in cultured esophageal epithelial cells. RESULTS: The results of this investigation reaffirmed the ability of pepsin to increase the rate and degree of esophageal cell and tissue damage at acidic pH, although the range of activity was limited to pH < 3.0. Moreover, the increased rate of tissue damage by acidified pepsin rapidly (within 15 min) produced a lesion that was irreversible, whereas, in a similar time frame, acid alone produced a lesion that was completely reversible. This early lesion by acidified pepsin was localized by performance of mannitol fluxes in apparently undamaged esophageal epithelium on light microscopy to the intercellular junctional complex. Further acid produced similar degrees of cell killing as acidified pepsin at pH < 3.0 in rabbit esophageal epithelial cells in suspension but not when growing on coverslips or present within intact epithelium. CONCLUSIONS: These studies suggest that acidified pepsin plays a key role in the development of reflux esophagitis by producing an early irreversible lesion that results in an increase in paracellular permeability, which indirect evidence suggests is due to damage to the junctional complex. The irreversibility of the increase in paracellular permeability is likely to aid conversion of nonerosive to erosive damage to the epithelium by permitting luminal acid greater access to the basolateral membrane of esophageal epithelial cells, which is known to be acid permeable.     

Indomethacin decreases resistance of gastric barrier to disruption by alcohol

Using a canine chambered stomach preparation, the effects of three 30-min exposures of the gastric mucosa to 20% ethanol in 100 mN HCl on gastric mucosal barrier disruption and ulcer formation were assessed. The interval between exposures was 30 min. Following an initial exposure to 20% ethanol, the net fluxes of H⁺, Na⁺, and K⁺ ions and perfusate volume induced by a second and third exposure of the gastric epithelium to this damaging agent were significantly reduced. Only minimal ulceration was observed following the first exposure which did not worsen with subsequent exposure to ethanol. If indomethacin was given intravenously either before or immediately after the first ethanol exposure, recovery of barrier function was significantly lessened after this challenge, and the resistance to barrier disruption was significantly decreased during the two subsequent exposures to ethanol when compared to experiments in which mucosa was exposed to 20% ethanol without concomitant administration of indomethacin. In addition, marked mucosal ulceration was observed during the second and third ethanol exposures if indomethacin was given. these studies suggest that the first alcohol challenge may have elicited the synthesis and release of tissue prostaglandins and thereby enhanced resistance of the gastric mucosa to subsequent challenge by this damaging agent. When prostaglandin synthesis was blocked by indomethacin, the increased resistance to gastric injury did not occur.This work was supported by research grant AM 25838 from the National Institute of Arthritis, Metabolism and Digestive Diseases.     

Re-thinking the functions of IgA+ plasma cells

The intestinal mucosa harbors the largest population of antibody (Ab)-secreting plasma cells (PC) in the human body, producing daily several grams of immunoglobulin A (IgA). IgA has many functions, serving as a first-line barrier that protects the mucosal epithelium from pathogens, toxins and food antigens (Ag), shaping the intestinal microbiota, and regulating host–commensal homeostasis. Signals induced by commensal colonization are central for regulating IgA induction, maintenance, positioning and function and the number of IgA⁺ PC is dramatically reduced in neonates and germ-free (GF) animals. Recent evidence demonstrates that the innate immune effector molecules tumor necrosis factor α (TNFα) and inducible nitric oxide synthase (iNOS) are required for IgA⁺ PC homeostasis during the steady state and infection. Moreover, new functions ascribed to PC independent of Ab secretion continue to emerge, suggesting that PC, including IgA⁺ PC, should be re-examined in the context of inflammation and infection. Here, we outline mechanisms of IgA⁺ PC generation and survival, reviewing their functions in health and disease.     

The role of mucin in GERD and its complications

Acid, pepsin and other noxious material reach the esophageal mucosa and interact with the luminal aspect of the squamous epithelium. The first protective barrier to these potentially injurious substances is the mucus buffer layer that covers the mucosa. In healthy people, the esophagus has a protective surface adherent mucus gel barrier. Levels of mucin glycoprotein are considerably increased in response to acid and pepsin. A wide spectrum of mucin genes are expressed in normal esophageal mucosa, squamous cell carcinoma of the esophagus, Barrett epithelium and esophageal adenocarcinoma. The mucins MUC5AC and MUC6 are expressed to a similar degree in Barrett metaplasia and gastric mucosa, as is MUC2 in Barrett intestinal metaplasia and small bowel mucosa. Increased expression of MUC1 is associated with progression from dysplasia to adenocarcinoma of the esophagus. Thus, mucins have an important role in the defense of esophageal mucosa against the acid, pepsin and bile that are present in the refluxate. Changes in the expression of mucins occur in patients with GERD, and might lead to the development of new drugs.     

Role of arachidonic acid metabolites in acid-pepsin injury to rabbit esophagus

The role of cyclooxygenase and lipoxygenase metabolites of arachidonic acid in experimental esophageal were lumenally perfused for 1 h with acidified saline (pH 2.0) with or without pepsin followed by a second hour with acidified saline. Separate groups of pepsin-perfused animals were pretreated with indomethacin, a cyclooxygenase inhibitor, or BW755C, a lipoxygenase-cyclooxygenase inhibitor. Esophageal injury was graded grossly. H+ and hemoglobin fluxes were determined. Acidified saline caused no significant damage. Pepsin induced moderate injury. Indomethacin decreased pepsin-induced H+ flux by 55% without affecting the other indices. BW755C, by all measurements, dramatically increased pepsin-induced injury. In separate experiments, cyclooxygenase activity was decreased by indomethacin and BW755C by 62% and 49%, respectively. Lipoxygenase activity was decreased 74% by BW755C and was not significantly affected by indomethacin. These results suggest that esophageal cytoprotection is mediated by endogenous lipoxygenase metabolites.