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Changes in redox status of gastric mucosa cells are the main pathogenic factor of gastric erosion and gastric ulcer development. Pro-oxidants can affect cell transcription activity via changes in redox-sensitive transcription factors. Egr-1 and Sp-1 may regulate the transcription of genes that are associated with the pathogenesis of gastric ulcer (growthfactors, cell cycle regulators, etc.). The aim of the present study was to reveal the possible involvement of zinc-finger transcriptionfactors Egr-1 & Sp-1 in the molecular mechanisms underlying gastric lesions caused by aspirin administration and stress. Gastric ulcer was induced in male rats (180-220 g) by immobilization stress combined with water-immersion (IMO-WI) or aspirin gavage (10 mg/100 g). The rats were euthanized 20 min, 1 hour, or 3 hours following the ulcerogenic factor exposure. Protein expression was determined by Western blot analysis and RT-PCR; levels of SH-groups of proteins were determined by method of Ellman et al. Development of gastric ulcer lesions was associated with twofold (P < 0.05) decrease in concentration of protein SH-groups in the rat gastric mucosa. These changes were accompanied by significant (P < 0.05) increase in the expression of Egr-1 mRNA and protein in both gastric ulcer models, and the changes in IMO-WI were more profound. Increased levels of Egr-1 were associated with the decrease in SpI protein levels. We showed for the first time the competitive interaction between redox-sensitive transcription factors Egr-1 and Sp1 in the early phases of gastric ulcer development, which might facilitate inducible transcriptional activity of Egr-1 at the expense of reduction in Sp1 activity.

Gastric mucosa of aging individuals exhibits increased susceptibility to injury and delayed healing. Our previous studies in young rats showed that healing of mucosal injury depends on and is critically dependent on VEGF and angiogenesis. Since angiogenesis in aging gastric mucosa has not been examined before, in this study we examined the extent to which angiogenesis is impaired in gastric mucosa of aging vs. young rats and determined the underlying mechanisms with a focus on mucosal expression of VEGF (proangiogenic factor) and endostatin (antiangiogenic factor). Aging rats had significantly impaired gastric angiogenesis by ~12-fold, 5-fold, 4-fold, and 3-fold, respectively (vs. young rats; all P < 0.001) at 24, 48, 72, and 120 h following ethanol-induced gastric injury and reduced and delayed healing of mucosal erosions. In gastric mucosa of aging (vs. young) rats at baseline, VEGF expression was significantly reduced, whereas endostatin levels were significantly increased (P < 0.05 and P < 0.01, respectively). In contrast to young rats, gastric mucosal VEGF levels did not increase following ethanol-induced injury in aging rats. MMP-9 enzyme activity was significantly higher in gastric mucosa of aging vs. young rats both at baseline (2.7-fold) and 24 h (3.8-fold) after ethanol injury (both P < 0.001). Since endostatin is generated from collagen XVIII by MMP-9, this finding can explain the mechanism of increased endostatin expression in aging gastric mucosa. The above findings demonstrate that reduced VEGF and increased endostatin result in the impaired angiogenesis and delayed injury healing in gastric mucosa of aging rats.

Basic fibroblast growth factor (bFGF or FGF-2) is a pleiotropic growth factor that promotes growth of mesenchymal and epithelial cells, stimulates angiogenesis and neuroprotection. Moreover, exogenous bFGF by stimulating angiogenesis promotes healing of gastroduodenal ulcers and cardiac and brain injury. All these actions were demonstrated in regard to 18kDa bFGF isoform that is secreted by cells via an ER/Golgi-independent pathway and activates FGF receptors. However in some transformed and stressed cells and in some tissues (e.g. brain) the single copy bFGF gene encodes multiple gene products: 18 kDa and also higher molecular weight (HMW) bFGF isoforms: ∼21 and ∼22 kDa in rodents, and ∼22, ∼23 and ∼24 kDa in humans. The biologic roles of these HMW bFGF isoforms in vivo remain unknown. In this study we demonstrated that in normal, uninjured gastric mucosa, bFGF is almost exclusively expressed as 18kDa isoform translated through a classical AUG (methionine) codon. In contrast, in injured gastric mucosa of rat, bFGF gene is preferentially translated to HMW bFGF isoforms through alternative CUG (leucine) initiation codon. Gastric mucosal injury caused in rats a significant increase in bFGF mRNA at 8 and 24h vs. normal mucosa and a significant increase in bFGF protein at 24-72h, mainly due to increased expression of ∼21 and ∼22 kDa HMW bFGF isoforms. This is first demonstration that gastric mucosal injury and repair triggers local activation of bFGF gene with preferential translation of HMW bFGF isoforms through a non-canonical CUG codon. This study uncovered CUG-initiated HMW bFGF translation as a novel regulatory mechanism operating in vivo during gastric injury repair., (Published by Elsevier Inc.)

Background & Aims: Aging gastric mucosa has impaired mucosal defense and increased susceptibility to injury. Our aims were to determine the mechanisms responsible for above abnormalities., Methods: We used Fisher F-344 rats, 3 and 24 months of age. We measured gastric mucosal blood flow; visualized mucosal hypoxia; examined expression of early growth response-1 transcription factor and phosphatase and tensin homologue deleted on chromosome 10 (PTEN); assessed apoptosis; and determined expression of caspase-3, caspase-9, and survivin. We also examined susceptibility of gastric mucosa of young and aging rats to ethanol injury and whether down-regulation of PTEN affects susceptibility of aging gastric mucosa to injury. To determine human relevance, we examined expression of PTEN and survivin in human gastric specimens of young and aging individuals., Results: Gastric mucosa of aging (vs young) rats has a 60% reduction in mucosal blood flow; prominent hypoxia; and increased early growth response-1 transcription factor and PTEN messenger RNAs, and proteins. It also has increased expression of proapoptotic proteins caspase-3 and capase-9, reduced survivin, and a 6-fold increased apoptosis vs mucosa of young rats. Ethanol-induced gastric mucosal injury in aging (vs young) rats was significantly increased. The down-regulation of PTEN in gastric mucosa of aging rats completely reversed its increased susceptibility to ethanol injury. In aging human gastric mucosa, PTEN expression was significantly increased, whereas survivin was significantly reduced., Conclusions: (1) Gastric mucosa of aging rats has significantly reduced blood flow, tissue hypoxia, activation of Egr-1, PTEN; increased caspases; and reduced survivin. (2) These changes increase susceptibility of aging gastric mucosa to injury.

Aim: To investigate the role of major non-protein and protein sulfhydryls and disulfides in chemically induced gastric hemorrhagic mucosal lesions (HML) and the mechanism of gastroprotective effect of sucralfate., Methods: Rats were given 1 mL of 75% ethanol, 25% NaCl, 0.6 mol/L HCl, 0.2 mol/L NaOH or 1% ammonia solutions intragastrically (i.g.) and sacrificed 1, 3, 6 or 12 min later. Total (reduced and oxidized) glutathione (GSH + GSSG), glutathione disulfide (GSSG), protein free sulfhydryls (PSH), protein-glutathione mixed disulfides (PSSG) and protein cystine disulfides (PSSP) were measured in gastric mucosa and liver., Results: Reduced glutathione (GSH) was depleted in the gastric mucosa after ethanol, HCl or NaCl exposure, while oxidized glutathione (GSSG) concentrations increased, except by HCl and NaOH exposure. Decreased levels of PSH after exposure to ethanol were observed, NaCl or NaOH while the total protein disulfides were increased. Ratios of reduced to oxidized glutathione or sulfhydrils to disulfides were decreased by all chemicals. No changes in thiol homeostasis were detected in the liver after i.g. abbreviation should be spelled out the first time here administration of ethanol. Sucralfate increased the concentrations of GSH and PSH and prevented the ethanol-induced changes in gastric mucosal thiol concentrations., Conclusion: Our modified methods are now suitable for direct measurements of major protein and non-protein thiols/disulfides in the gastric mucosa or liver. A common element in the pathogenesis of chemically induced HML and in the mechanism of gastroprotective drugs seems to be the decreased ratios of reduced and oxidized glutathione as well as protein sulfhydryls and disulfides.

We tested the hypothesis that recognized gastroprotective agents exert direct protection against ethanol-induced injury in isolated rat gastric mucosal cells in vitro. If protection exists, we also wanted to identify subcellular targets in the reversible and/or irreversible stages of cell injury. Ethanol-induced cell injury was quantified by measuring plasma membrane leakage (trypan blue exclusion and lactate dehydrogenase release), mitochondrial integrity (succinic dehydrogenase), and nuclear damage (ethidium bromide-DNA fluorescence). Initial cell viability and responsiveness were estimated by the effects of carbachol, carbachol + atropine, or 16,16-dimethyl-PGE(2) on chief cell pepsinogen secretion. Enriched parietal cells were stimulated by histamine, carbachol, or histamine + IBMX. Preincubation of cells with PG, sucrose octasulfate, or the sulfhydryl compounds N-acetylcysteine, taurine, or cysteamine increased cell resistance

Our previous studies demonstrated that ethanol injury triggers the angiogenic response in gastric mucosa bordering necrosis. The present study was aimed to determine whether vascular endothelial growth factor (VEGF) (a potent angiogenic peptide selectively acting on endothelial cells) and Ras (a mediator of cell proliferation and a putative regulator of VEGF expression) are involved in gastric angiogenesis after ethanol injury. We studied the angiogenic response and expression of VEGF and Ras in gastric mucosa after ethanol injury. Ethanol damage triggered 1) angiogenesis in the gastric mucosa bordering necrosis, 2) significant increases in VEGF mRNA and protein expression, and 3) significant increases in the expression of Ki-ras mRNA and Ras proteins. Neutralizing anti-VEGF antibody significantly reduced (by greater than threefold) the angiogenic response to ethanol-induced injury. Moreover, mevastatin, an inhibitor of Ras activation, completely blocked the induction of VEGF expression in cultured primary endothelial cells. Because, in other tissues, VEGF is one of the most potent angiogenic factors and VEGF expression is dependent on Ras, our data indicate that Ras and VEGF are involved in gastric mucosal angiogenesis after ethanol injury.

4-Methylpyrazole (4-MP), a specific inhibitor of alcohol dehydrogenase, exerts gastroprotection of unusually long duration in rats. We tested the hypothesis that pretreatment with 4-MP might protect the human gastric mucosa against alcohol-induced acute injury. Fourteen healthy volunteers received pretreatment with either 4-MP, 15 mg/kg body weight dissolved in 50 ml of orange juice, or placebo and 2 hr later 100 ml of 40% ethanol. The endoscopic appearance of the gastric mucosa was evaluated and scored (scale 0-5) and mucosal biopsies were obtained just before pretreatment and 30 min after ethanol for histologic examination and prostaglandin E2 measurement. In the 4-MP group the mean endoscopic injury score was significantly lower than that in placebo group, in both the body and the antrum. Histologically, 4-MP significantly reduced disruption of surface epithelium and completely prevented the deep hemorrhagic mucosal lesions. In the 4-MP group no changes in gastric mucosal PGE2 levels were detected. In rats, 4-MP did not inhibit gastric acid output, whereas it markedly increased the adherent gastric mucus evaluated by the alcian blue recovery method. When lipid peroxidation was induced by carbon tetrachloride in hepatic microsomes, 4-MP caused significant inhibition of malondialdehyde generation. We conclude that 4-MP provides significant protection of the human stomach against alcohol-induced acute mucosal injury. 4-MP, besides inhibiting the conversion of alcohol to acetaldehyde, might protect the gastric mucosa by increasing adherent gastric mucus and by scavenging free radicals.

Previously our laboratory reported increased activity of the thiol proteinase cathepsin B in gastric juice after ethanol-induced mucosal injury. In this study we measured proteinase activity (PA) and proteinase inhibitory activity (PIA) with the general substrates hemoglobin, azocasein, and bovine serum albumin (BSA) at optimal pH (2.0, 5.6, and 7.4) of aspartic, cysteine, and serine proteinases. Homogenates of glandular stomach mucosa and gastric juice from fasted rats were incubated in the presence or absence of specific inhibitors and sulfhydryl (SH) alkylators N-ethylmaleimide and iodoacetate. PIA was measured after acid and heat inactivation of endogenous proteinases and addition of 20 micrograms/ml pepsin, 20 or 100 micrograms/ml thiol proteinase papain, or 20 micrograms/ml trypsin for 5 min before digestion at 37 degrees C. The highest proteolytic activity was found at pH 2.0 (pepsin) in juice and mucosal homogenate, but proteases were also found at pH 5.6 and 7.4, where pepsin was inactive. Pepstatin inhibited most proteolytic activity at pH 2.0. The SH protease inhibitor leupeptin diminished PA mainly at pH 5.6. N-ethylmaleimide or iodoacetate substantially reduced the PA in acidic milieu, with maximum effect at pH 5.6. Endogenous PIA, expressed as inhibition of the effect of 1 microgram of pepsin, papain, and trypsin on BSA, was 13.1, 1.4, and 9.2% in gastric mucosa and 15.3, 22.5, and 6.2% in gastric juice at pH 2.0, 5.6, and 7.4, respectively. We have concluded that 1) endogenous proteinases and inhibitors in rat stomach can be measured using BSA and hemoglobin as substrates, 2) of the proteinases found in the stomach, 98% was pepsin at pH 2.0 and up to 27% or 17% was SH sensitive at pH 5.6 or 7.4, respectively, and 3) proteinases and their specific endogenous inhibitors may play a role in gastric mucosal injury and protection.

Recently, it has been pointed out that growth factors play an important role in the healing of gastrointestinal ulcers. In the present study, we examined the role of endogenous basic fibroblast growth factor (bFGF) in the healing of gastric ulcers in the rat. In male SD rats, gastric ulcers were induced in the antrum by injection of acetic acid. Time-dependent changes in the area and bFGF content in the ulcerated area and distribution of bFGF in the ulcerated mucosa were examined. Effects of bFGF mutein CS23 (TGP-580) and a monoclonal antibody for bFGF (MAb 3H3) on the healing of the gastric ulcers and angiogenesis in the ulcer bed were also examined. The content of bFGF in the ulcerated area increased with time as the ulcer healed and reached a maximum 7 days after ulcer formation. In the gastric ulcer bed, many cells such as fibroblasts and macrophages were positively stained immunohistochemically by anti-bFGF antiserum. MAb 3H3 (0.1 mg/rat/day, i.v.) inhibited angiogenesis in the ulcer bed and significantly delayed ulcer healing, while TGP-580 (0.001-0.1 mg/kg x 2/day, p.o.) increased the number of microvessels in the ulcer bed and accelerated the healing. These results suggest that endogenous bFGF may play an important role in the healing of gastric ulcers in the rat and that the angiogenic properties of bFGF (TGP-580) may be involved in its effect on ulcer healing.

Since recent studies suggest an imbalance between cathepsin B and its tissue protease inhibitors (PI) in the pathogenesis of acute and chronic diseases, we tested the hypothesis that release of activated cysteine proteases (P) such as cathepsins B, H, and L might play a role in the pathogenesis of gastric hemorrhagic mucosal lesions (HML) induced by ethanol (E) or ammonia (A). Anesthetized rats received 1 ml of 50% E or 1% A solution intragastrically for 1 min during in situ gastric luminal perfusion. Rapid activation and release of cathepsins B, L, and H into the luminal perfusate preceded the formation of HML quantified by planimetry. Mucosal presence and activity of cysteine PI and cathepsin B have also been investigated in the pathogenesis of chemically induced HML. We extracted and partially isolated acid and thermostable inhibitors of cathepsin B in the gastric mucosa, and found rapid inactivation of PI and activation of cathepsin B in the early phase of E- or A-induced HML. Negative correlations were found between P and PI activities by E or A solutions. Both the activation of cathepsins B, L, and H and the development of E-induced HML were prevented by pretreatment with the sulfhydryl alkylator N-ethylmaleimide. These results suggest that cysteine P may be activated in the rat stomach after E or A exposure, and cysteine P may have a role in the pathogenesis of E- or A-induced gastric HML. Endogenous PI may also participate in the mechanisms of gastric mucosal lesions and gastroprotection.

Controversial data have been obtained with direct cellular protection by prostaglandins and sulfhydryls. In the present studies, we compared the merits and liabilities of currently available cell viability assays, some of which have not been previously employed in studies of the gastric mucosa. We also tested the hypothesis that length of incubation of isolated cells with protective agents might influence the degree of cellular damage. Gastric mucosal cells were isolated from nonfasted rats and digested with various concentrations of pronase and/or EGTA. Cell viability was assessed by trypan blue and fast green exclusion, fluorescein diacetate hydrolysis, chromium release, LDH release, mitochondrial succinate dehydrogenase activity and nuclear fluorescence induced by ethidium bromide. The experiments revealed that both pronase and EGTA are needed to obtain mucosal cells with optimal yield and initial viability and that sequential additions of pronase (30 min) and EGTA (30 min), rather than their combination (60 min), increased viability without decreasing yield. Cell viability and yield were better when pronase was used in the first incubation. For LD50 determinations, a cell suspension was incubated with ethanol (0%-15%) for 5 min. For studying the effects of protective agents, the cells were pretreated with 16,16-dmPGE2 or cysteamine HCl at 37 degrees C for 30 min before a 5-min exposure to 7.5% or 15% ethanol. The LD50 value for ethanol injury was approximately 15% for all assays except LDH, where the LD50 value was 8.5%. Preincubating gastric mucosal cells for 30 min with 16,16-dmPGE2 or cysteamine resulted in no preservation of cell viability. However, when cells were preincubated with one of the protective agents for 60 min and then exposed to 8% or 10% ethanol for 5 min, partial protection was observed when assessed by succinate dehydrogenase activity and, in certain cases, by LDH release. We conclude that all seven cell viability assays yield a measurable LD50 value for ethanol-induced cell injury, but the results may vary by as much as 82%. Low concentrations of both pronase and EGTA are needed to obtain isolated mucosal cells with both high yield and initial viability. Biochemical measures of mitochondrial activity and nuclear damage provide reliable evidence of cell viability and should be used to complement membrane permeability assays. Long preincubation of cells (60 min) with protective agents resulted in only slight protection of mitochondrial function, in contrast to the rapid induction of gastroprotection seen with these compounds in vivo. We therefore surmise that processes that contribute to organ protection occur faster and more efficiently than those that control direct cell injury and protection.

Objective: To test the hypothesis that an early increase in vascular permeability is correlated with later gastric mucosal protection in the rat., Methods: Histamine, its agonists (H1, H2, H3) and bradykinin, were either given subcutaneously or intragastrically before the intragastric administration of ethanol. The extravasation of intravenously injected 99mTc-glucoheptonate into the gastric wall and into the gastric contents was used as an indicator of increased permeability. Gastric haemorrhagic lesions where measured by computerized planimetry and ethanol absorption was determined by an ACA Clinical Analyzer., Results: Histamine and bradykinin increased vascular permeability in the glandular stomach and provided significant gastroprotection, similar to H1-, H2- and H3-agonists, against ethanol-induced gastric haemorrhagic lesions. This gastroprotection was accompanied by low blood levels of ethanol, probably indicating decreased ethanol absorption and the creation of a histodilutional barrier in the stomach by histamine., Conclusions: These data indicate that an increase in vascular permeability dissipates the concentration, and may delay the absorption, of ethanol in gastric mucosa by creating a perivascular histodilutional barrier. Vascular injury, which is an early pathogenetic factor in the development of ethanol-induced gastric haemorrhagic erosions, may thus be prevented.

Background: Among the endogenous mediators (e.g., PG, SH) of gastroprotection, so far only PG was implicated in the mechanism of acute gastroprotection by sucralfate. Angiogenesis, which is stimulated by bFGF, is a recently recognized element in ulcer healing, and sucralfate binds bFGF in vitro and in vivo., Methods: In fasted rats the gastric mucosal concentration of GSH and protein SH was measured at 30, 60, 120, and 240 min after a single gastroprotective dose of sucralfate. In normally fed rats, angiogenesis was measured in the subcutaneous sponge assay 7 days after the implantation of sponges containing sucralfate and/or bFGF., Results: A gastroprotective dose of sucralfate time-dependently increased GSH concentration in the gastric mucosa. Sucralfate alone accelerated angiogenesis, which was significantly enhanced by combination with an ineffective dose of bFGF in the subcutaneous sponge assay. The same dose of sucralfate combined with an angiogenic dose of bFGF also resulted in synergistic stimulation (e.g., more than fivefold) of angiogenesis., Conclusions: It appears that elevated mucosal GSH concentration may represent a new factor in the mechanism of acute gastroprotection by sucralfate, and stimulation of angiogenesis is one of the mechanisms of ulcer healing by sucralfate.

Background/aims: The mechanisms of chemically induced injury are poorly understood. The purpose of this study was to develop a rapid method for isolation of gastric mucosal cells (GMCs) and sensitive tests for investigating cell damage and protection., Methods: GMCs were prepared from rat glandular stomach with pronase and ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) and kept in albumin-containing, albumin-free, or Hank's solutions. Initial cell viability and responsiveness were tested with trypan blue exclusion, and stimulation or inhibition of pepsinogen secretion were tested with carbachol, 16,16-dimethyl prostaglandin E2, or carbachol plus atropine. Plasma membrane damage was assessed by trypan blue exclusion, lactate dehydrogenase or cell protein leakage, mitochondrial integrity by succinic dehydrogenase activity, and nuclear lesions by ethidium bromide DNA fluorescence., Results: Incubation of GMC with ethanol resulted in 50% lethal dose for ethanol values of 12.3%, 7.4%, and 10.9% by trypan blue exclusion; 10.1%, 9.4%, and 9.1% by lactate dehydrogenase leakage; 12.8%, 10.0%, and 10.4% by succinic dehydrogenase; and 14.1%, 13.0%, and 11.5% by ethidium bromide DNA by binding using albumin-containing, albumin-free, and Hank's solutions, respectively., Conclusions: Rat GMC with high and long viability can be isolated by low concentrations of pronase and EGTA. The adapted biochemical and microscopic methods are suitable for parallel examination of chemical injury of plasma membrane, mitochondria, and nuclei. Albumin increased ethanol 50% effective concentration to GMC. A combination of biochemical and morphological methods with ethanol-induced injury to GMC is proposed for direct cytoprotection investigations.

Ca2+ modulates gastric function and dysfunction as well as the release of cysteine proteases and metalloproteinases which have been implicated in the pathogenesis of gastric mucosal lesions. We thus tested the hypothesis that pretreatment with the Ca2+ chelators, ethylene diamine tetraacetic acid (EDTA) and ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) might reduce the experimental gastric mucosal damage induced by restraint cold stress or 1.0 ml of 75% ethanol. Other rats were prepared with chronic indwelling gastric cannulas and the effects of EDTA and EGTA on conscious basal gastric acid output were assessed. In addition, rats were pretreated with EGTA or EDTA prior to pylorus ligation and their effects on acid and pepsin output assessed. Both EDTA and EGTA reduced significantly the extent of ethanol-induced gastric mucosal damage as well as the degree of stress-induced gastric lesions. To further characterize the mechanism of Ca2+ chelator protection against ethanol-induced gastric lesions, some rats were vagotomized or adrenalectomized prior to treatment with EGTA or EDTA, followed by ethanol or stress. Both adrenalectomy and vagotomy abolished gastroprotection by EGTA and slightly reduced that induced by EDTA in both models of experimental gastric mucosal injury. Both EDTA and EGTA reduced significantly basal gastric acid output, an effect which persisted for at least 2 h following their administration. Both compounds also decreased significantly acid and pepsin output in pylorus-ligated rats. We conclude that Ca2+ chelators attenuate both acid-dependent and acid-independent gastric lesions.(ABSTRACT TRUNCATED AT 250 WORDS)

Cysteamine, a potent duodenal ulcerogen, stimulates gastric acid and gastrin secretion and decreases immunoreactive somatostatin (IRS) from the gut and hypothalamus of the rat. To elucidate the structural requirements for this effect, we tested a series of cysteamine analogs for their IRS decreasing activity in comparison with their nucleophilic and reducing potencies. Adult female rats were sacrificed 4 hr after p.o. administration of the test chemicals given in molar equivalents to 30 mg/100 g of cysteamine-HCl. IRS decreasing activity in gastric mucosa, expressed as percentage of controls is listed in descending order: cystamine (55%), cysteamine (59%), 2-dimethylaminethanethiol (59%), ethylamine (66%), 1,3-propanedithiol (70%), propylamine (75%) and 3-aminothiophenol (79%). The following thiols and amines had no IRS decreasing effect (80% of controls): L-cysteine, ethanethiol, 1-propanethiol, penicillamine, dimercaprol, 1-4-dithiothreitol, ethanolamine, propionitrile, n-butyronitrile, o-, m- or p-aminophenol. The aryl 2-, 3- or 4-aminothiophenols, unlike most of their aminophenol analogs also decreased immunoreactive prolactin in the pituitary by 38 to 78%. IRS decreasing activity was independent of the reducing potency of cysteamine derivatives but was correlated significantly (r = 0.793, P < .01) with electron affinity of -SH, -NH2, -OH and -CN radicals in terminal alkyl chemicals. The structural requirement for decreasing activity is the presence of either -SH and -NH2 on a 2 to 3 carbon alkyl or aryl molecule. Both radicals when present together increase potency.(ABSTRACT TRUNCATED AT 250 WORDS)

Glutathione is one of the endogenous protective chemicals, like prostaglandins, in the gastric mucosa. Depletion of these agents aggravate the chemical- or stress-induced gastric erosions and ulcers. However, gastroprotection can be achieved even in the presence of low mucosal concentration of glutathione and prostaglandins, indicating the presence of other protective chemicals (e.g. polyamines, growth factors, neurotransmitters, steroids) in the stomach. Protein sulfhydryls were also implicated in the mechanism of action of gastroprotective drugs. We recently tested the hypothesis that cysteine proteases might be a target of gastroprotective and antiulcer agents, and decided to look for the presence of proteases and protease inhibitors (PI) in the gastric mucosa and juice. Protease activity and PI were measured with general substrates hemoglobin, azocasein and albumin at optimal pH (2.0, 5.6, 7.4) of aspartic, cysteine and serine proteases. Homogenates of glandular stomach mucosa and gastric juice from fasted rats were incubated in the presence or absence of specific inhibitors and gastroprotective SH alkylators such as NEM or iodoacetate. PI was measured after acid and heat inactivation of endogenous proteinases and addition of pepsin, cysteine proteinase papain, or trypsin. Our results indicate that of the proteases found in the stomach 98% was pepsin at pH 2.0, and up to 56% or 24% was SH-sensitive at pH 5.6 or 7.4, respectively. Intragastric administration of SH alkylators such as NEM or iodoacetate exerted a dose- and time-dependent gastroprotection against chemically induced acute erosions and ulcers. Thus, in addition to glutathione, proteinases and their specific endogenous inhibitors may also be involved in gastric mucosal injury and protection.

Vascular factors play an important role in the pathogenesis and prevention of acute gastric mucosal lesions. Endothelin-3 (ET-3), a potent vasoactive peptide, was infused intra-arterially to induce gastric microvascular and hemorrhagic mucosal lesions, and to enhance the damaging effects of dilute HCl and ethanol. ET-3 antibody was injected intravenously to decrease hemorrhagic mucosal lesions induced by ethanol. Locally infused ET (0.01, 0.1, and 1.0 nmol.100 g-1.min-1 for up to 15 min) was followed in some cases by intragastric dilute ethanol or HCl, which alone caused no or only mild vascular and mucosal lesions. Monastral blue was used to visualize and quantify vascular injury. ET-3 produced dose-dependent vascular lesions that affected the walls of mucosal capillaries and venules and induced mucosal congestion and focal endothelial labeling in vessels of the gastric muscular layers. The highest dose of ET induced hemorrhagic gastric mucosal lesions, mortality, and periods of hyper- and hypotension in the rat. Medium and low doses of ET-3 caused vascular injury, and dose-dependently potentiated the vascular and hemorrhagic mucosal lesions caused by dilute HCl and ethanol. Indomethacin slightly enhanced damage induced by ET and 50% ethanol, suggesting a limited mediatory role of prostaglandins in the ET-induced mucosal lesions. Anti-ET-3 serum dose-dependently decreased but did not abolish the hemorrhagic gastric mucosal lesions induced by 75% ethanol. Thus, ET-3 causes endothelial damage in capillaries and venules of rat stomach and predisposes to mucosal damage even after exposure to dilute ethanol or HCl. ET is more potent than leukotrienes and histamine and thus may play an important role in the mechanisms of acute gastric mucosal injury and protection where the vascular network appears to be a major target.

This review provides evidence that gastroduodenal mucosal injury is a complex process because of the heterogeneous structure and multiple functions of the gut. The action of exogenous etiologic agents is usually mediated in part, or amplified by endogenous mediators which very often exert biphasic, i.e., damaging and protective effects. The pathogenetic pathways involve direct/indirect chemical injury, vascular damage and its consequences, and acute or chronic inflammatory processes following infectious, chemical or ischemic injury. The role of oxygen, free radicals, calcium and proteases as well as the components and forms of gastroduodenal injury, e.g., reversible and irreversible cell injury, tissue necrosis, acute and chronic inflammation are also briefly discussed. Only a slight or moderate direct cytoprotection was demonstrated in vitro using isolated, mixed rat gastric mucosal cells by the known gastroprotective drugs including PG and SH compounds. Thus, the terms of organo or gastroprotection are more descriptive then the misleading "gastric cytoprotection".

The sulphydryl containing drug cysteamine protects gastric mucosa in vivo against acute injury. It is not known whether this protection includes a direct effect on gastric cells. Using gastric epithelial cell monolayers derived from a well differentiated human cell line, we evaluated whether cysteamine protects against taurocholate or indomethacin induced damage in conditions which completely exclude the influence of vascular, hormonal, and neural factors. The effect of cysteamine on prostaglandin production by monolayer cells in vitro was also assessed. Cysteamine decreased damage brought about by sodium taurocholate and indomethacin by 40% (p less than 0.01) and 50% (p less than 0.01) respectively. The sulphydryl blocker iodoacetamide prevented the protective effect of cysteamine. Pretreatment with indomethacin, which inhibited prostaglandin E2 output by 60%, did not prevent protection by cysteamine; incubation with cysteamine decreased prostaglandin E2 production by cultured cells. We conclude that (i) cysteamine directly protected gastric epithelial cells in vitro (ii) this protection occurred with indomethacin, which interferes with cellular metabolism of prostaglandins, and taurocholate, whose damaging action at neutral pH is unrelated to interference with prostanoid metabolism, (iii) cysteamine protection in vitro is unrelated to endogenous prostaglandins and is probably mediated by endogenous sulphydryl compounds.

Acetaminophen protects gastric mucosa in vivo against acute drug-induced damage and has also been shown to protect gastric epithelial cells in vitro. Protection afforded by acetaminophen in vitro is not associated with increased prostaglandin output. The present study evaluated whether protection of gastric epithelial cells by acetaminophen in vitro may be mediated by endogenous sulphydryls. Monolayers from a well-differentiated human gastric epithelial cell line were studied. Sodium taurocholate was used as a damaging agent and cell damage was assessed by the 51Cr release assay. Acetaminophen dose-dependently protected gastric cell monolayers against damage induced by sodium taurocholate. The sulphydryl blocker iodoacetamide dose-dependently decreased the concentration of nonprotein glutathione and cysteine and counteracted the protection afforded by acetaminophen. This suggests that sulphydryl compounds may mediate the protection of gastric epithelial cells by acetaminophen in vitro.

This brief overview of our recent results implicates vascular factors in the mechanisms of acute and chronic actions of sucralfate. Pretreatment of rats with sucralfate and its components, such as SOS and sodium sulfate, prevented the ethanol-induced microvascular injury and maintained blood flow in the gastric mucosa. Thus, preservation of microvascular integrity seems to be one of the mechanisms of acute gastroprotection by sucralfate. Chronic experiments with subcutaneously implanted sponges containing sucralfate or SOS revealed that both compounds stimulated angiogenesis, whereas only sucralfate enhanced the area of granulation tissue. These processes may have a role in the ulcer-healing action of sucralfate.

We tested the hypothesis that the gastrotoxicity of ethanol and other damaging agents is influenced through the modulation of alcohol dehydrogenase (ADH) by using either the ADH-inhibitor pyrazole or the noninhibitor derivatives of pyrazole. In time course experiments, the protection by both compounds was evident up to 48 hr before ethanol administration. Both drugs were also protected, from about 24 hr, from gastric mucosal damage induced by aspirin and hydrochloric acid. In order to examine the role of endogenous prostaglandins and sulfhydryls in this protection, indomethacin and N-ethylmaleimide were used, of which only the sulfhydryl alkylator antagonized (by about 50%) the protection by pyrazole and 3-methylpyrazole. Studies with monastral blue B revealed the protective role of both pyrazole and 3-methylpyrazole against early vascular injury in the gastric mucosa. We conclude that because both the ADH-inhibitor pyrazole and the noninhibitor derivatives of pyrazole exert gastro-protection, and because both compounds protect against aspirin and HCI, ADH inhibition is not involved in this protection. We also suggest that although prostaglandins appear to have minimal involvement in the mechanism of protection, endogenous sulfhydryls may be important mediators. Furthermore, the functional and structural mechanism of this protection seems to be the prevention of acute vascular injury.

Of the multiple actions of sucralfate, not all are equally relevant. In fact, certain actions of the drug contribute to healing of acute ulcers or to ulcer prevention, whereas other actions are more important in accelerating the healing of gastric and duodenal ulcers. The action of sucralfate can now be defined by the '1 x 1 x 1' mechanism of action--that is, one action of sucralfate is important for acute prevention, one is relevant to both acute and chronic protection, and one is important for chronic ulcer healing. Maintenance of mucosal vascular integrity and of blood flow, which ensures rapid epithelial restitution to repair superficial defects, are the most important acute actions of sucralfate. On the other hand, increased bicarbonate and mucus secretion seem to be relevant in both acute and chronic protection of the gastroduodenal mucosa. Finally, the enhanced binding by sucralfate of fibroblast growth factor and epidermal growth factor stimulates angiogenesis, granulation tissue, and epithelization for ulcer healing. This 1 x 1 x 1 theory of the mechanism of action of sucralfate concentrates on the relevant effects of this drug (which has more than a dozen actions) and may help to elucidate the molecular mechanism of action.

This article emphasizes and reviews the premise that because the pathogenesis of acute gastric mucosal injury is multifactorial, several protective mechanisms should also be considered in analyzing gastric mucosal defense. The first part of the article reviews the pathogenesis of acute gastric mucosal injury by major etiologic factors such as hypoxia and chemical and biological agents, and emphasizes the common endogenous mediators of damage (e.g, endothelins, leukotrienes, thromboxane, platelet-activating factor, monoamines, free radicals, proteases, ammonia, hydrochloric acid, and bile acids--in decreasing potency). The second part of the review is devoted to the gastroprotective mechanisms that are analyzed by anatomical (histologic) location and biochemical processes. The endogenous mediators of acute gastroprotection include prostaglandins, sulfhydryl (SH) compounds, non-SH antioxidants, polyamines, and epidermal growth factor, whereas the protective and mediatory role of glucocorticoids, somatostatin, pentagastrin, histamine, gangliosides, and calcitonin gene-related peptide need further studies. A list of endogenous and exogenous chemicals that exert biphasic, damaging, and protective effects on the gastric mucosa in also included. The final common pathway of acute gastroprotection at the structural and functional level seems to be the preservation of subepithelial microvascular integrity leading to maintenance of mucosal blood flow that allows the energy-dependent rapid restitution (cell migration) from surviving gastric neck cells to repair the superficial epithelial defect. Very new data on the contribution of a histodilutional barrier and release of proteases to gastroprotective processes are also discussed.

This review provides evidence that gastroduodenal mucosal injury is a complex process because of the heterogenous structure and multiple functions of the gut. The action of exogenous etiologic agents is usually mediated in part or amplified by endogenous mediators that very often exert biphasic, i.e., damaging and protective, effects. The pathogenetic pathways involved are direct/indirect chemical injury, vascular damage and its consequences, and acute or chronic inflammatory processes following infectious, chemical, or ischemic injury. The role of oxygen, free radicals, calcium, and proteases as well as the components and forms of gastroduodenal injury, e.g., reversible and irreversible cell injury, tissue necrosis, and acute and chronic inflammation, are also briefly discussed.

The goals of this article are to review the similarities and differences in the pathogenesis of acute gastric mucosal injury induced by alcohol, exemplified mostly by ethanol, and aspirin, as a representative of nonsteroidal anti-inflammatory drugs, and to deduce implications from pathogenetic studies for a better understanding of the concept of gastric cytoprotection. The main similarity between the hemorrhagic erosions caused by ethanol and aspirin is their localization in the acid-producing glandular stomach, the rate-limiting step in their pathogenesis being the extent of microvascular injury in the gastric mucosa. The major differences include the fast healing and low probability of transition into chronic gastritis after a single exposure to aspirin. On the other hand, perforated ulcer may develop, especially in the elderly, after chronic aspirin but not ethanol consumption. The main implications of pathogenetic investigations include the relative nature of gastroprotection: that is, initially, the superficial epithelial layer is not protected against concentrated luminal solutions, but it is rapidly replaced by migrating, adjacent, surviving cells if blood flow is maintained and the basement membrane is relatively intact. Vascular changes thus seem to be the rate-limiting step both in the pathogenesis and prevention of chemically induced acute gastric mucosal injury. The ultimate biochemical mechanisms of gastroprotection seem to include an effect on structural and enzymic proteins, and vascular mediators which influence vascular permeability and, indirectly, the extent of tissue injury.

We used in vivo microscopy and laser-Doppler velocimetry to examine the effects on the gastric mucosal microcirculation and in gastric mucosal blood flow of agents that induce acute gastric mucosal damage. In vivo microscopic observation of superficial mucosal capillaries revealed vascular stasis within a mean of 54, 81, or 61 s after 100% ethanol, 0.6 N HCl, or 0.2 N NaOH, with the subsequent development of hemorrhagic mucosal lesions. Mucosal blood flow estimated by laser-Doppler velocimetry decreased by 30% at 5 min after luminal application of 100% ethanol, and decreased further to about 40% of basal levels by 15 min. The decreased mucosal blood flow 15 min after application of 50% ethanol correlated with the extent of hemorrhagic mucosal lesions. Examination of the submucosal vessels that supply and drain the mucosa showed moderate dilation of small arterioles 1, 3, and 6 min after exposure to 100% ethanol but there were no consistent changes in venules. Mild vasoconstriction of small- and medium-sized venules could be detected 6, 10, and 15 min after NaOH but not after exposure to HCl. Pretreatment with 16,16-dimethyl prostaglandin E2 or sodium thiosulfate before exposure of the mucosa to ethanol prevented capillary stasis, maintained mucosal blood flow, and prevented the development of hemorrhagic gastric mucosal lesions. Topical mucosal application of 16,16-dimethyl prostaglandin E2 decreased, whereas topical exposure to sodium thiosulfate increased gastric mucosal blood flow, indicating that change in blood flow per se is an unlikely mediator of protection.

Recent investigations indicate that microvascular injury, leading to increased vascular permeability and capillary stasis, precede the development of chemically induced hemorrhagic mucosal lesions in the stomach. The vascular damage is more amenable to protection by prostaglandins and sulfhydryls than the diffuse surface mucosal cell injury. The vascular and mucosal lesions may be the result of direct toxicity of damaging agents (eg, ethanol, HCl, NaOH) and the release of vasoactive amines and leukotrienes. We review here our recent studies performed in rats indicating that intraarterial infusion of LTC4 or LTD4 in the stomach caused vascular injury as revealed by monastral blue. Infusion of leukotrienes alone caused no hemorrhagic mucosal lesions but aggravated the damage caused by 25, 50, or 100% ethanol and 0.2 N HCl given intragastrically. The ethanol-induced mucosal lesions were slightly diminished by the lipoxygenase inhibitor L-651,392 and markedly decreased by eicosapentaenoic acid, which competes with arachiconic acid as a substrate for 5-lipoxygenase. These results are discussed and correlated with biochemical results from other laboratories demonstrating increased levels of leukotrienes in the gastric mucosa after administration of ethanol and decreased release following pretreatment with prostaglandins or sulfhydryl-related agents. New data thus support a mediatory role for leukotrienes in the pathogenesis of vascular injury and mucosal lesions in the stomach.

Previous studies showed a rapid decrease of somatostatin concentration in the gut and an increase in serum gastrin levels after a single dose of the duodenal ulcerogen cysteamine. An attempt was made to identify morphologic changes that would correlate with these functional changes. Rats were killed 1, 4, 8, or 24 hr after a single dose of cysteamine and sections of gastric mucosa and pancreas were processed for electron and light microscopy. Subtle ultrastructural alterations were seen in D cells of the stomach (e.g., dilation of mitochondrial cristae and endoplasmic reticulum, and apparent increase in electron density of secretory granules) after cysteamine administration. The number of somatostatin-positive cells visualized by the immunoperoxidase technique using light microscopy was decreased in 1-4 hr but returned to normal by 24 hr. The alterations observed in the G cells after cysteamine administration are consistent with release of gastrin from mature granules and increased synthesis of the hormone. The lack of major morphologic changes in the D cells suggests that cysteamine affects somatostatin without causing cell necrosis or alteration in lysosome formation. The effect of the drug may thus be mediated at the biochemical level without marked morphologic alterations.

Ethanol induces hemorrhagic gastric erosions and causes a dose-dependent decrease in the concentration of nonprotein sulfhydryl compounds in rat gastric mucosa. Sulfhydryl-containing drugs protect rats from ethanol-induced gastric erosions, whereas sulfhydryl blocking agents counteract the mucosal cytoprotective effect of prostaglandin F2 beta. These observations suggest that endogenous nonprotein sulfhydryls may mediate prostaglandin-induced gastric cytoprotection and that sulfhydryl drugs may have potential for preventing or treating hemorrhagic gastric erosions.

Intragastric administration of cysteamine or diethyl maleate caused a dose-dependent reduction in the mean area of gastric lesions induced by absolute ethanol but failed to affect gastric ulcerations produced by acidified aspirin. Mucosal levels of glutathione were increased with cysteamine and reduced with diethyl maleate and did not correlate with the protective action of these agents on ethanol-induced mucosal lesions. The protective effects of these agents probably involved mucosal generation of prostaglandins, because pretreatment with indomethacin or aspirin, potent inhibitors of cyclooxygenase, partially prevented this protection. This study indicates that the mucosal generation of prostaglandins contributed, at least in part, to the gastric cytoprotection by agents altering mucosal contents of sulfhydryl compounds.

Administration of cysteamine (mercaptoethylamine) induces in rats severe perforating duodenal ulcers. Because the ulcerogenic properties of cysteamine are markedly reduced by treatment with somatostatin, we considered the possibility that cysteamine-induced duodenal ulcer might be mediated by depletion of tissue somatostatin, and thereby of its paracrine influences on gastrin and gastric acid secretion. To test this hypothesis, we measured the concentration of immunoreactive somatostatin (IR-somatostatin) in stomach and duodenal mucosa at intervals after administration of a single ulcerogenic dose (30 mg/kg by stomach tube). IR-somatostatin in these tissues fell rapidly to reach a minimum at 4 h (stomach 31%, duodenum 60% of control respectively). IR-somatostatin in hypothalamus and pancreas decreased gradually to a minimum at 7 h. Another duodenal ulcerogen, propionitrile (10 mg/100 g bw, s.c.) which is more toxic than cysteamine, and several stressful procedures including ether anesthesia, restraint and s.c. formalin did not lower stomach or duodenal IR-somatostatin. Gut, pancreas and hypothalamic VIP levels were not influenced by cysteamine. These findings suggest that cysteamine is a relatively specific depletor of tissue somatostatin. Because blood levels of somatostatin fell, and only trivial amounts of the peptide were found in the stomach lumen after cysteamine administration, it appears likely that this agent acts at the cellular level to cause breakdown of preformed somatostatin and/or to acutely reduce its synthesis.

To investigate the possible role of hormones in gastric mucosal protection, the effect of prostaglandin F2 beta, dimercaprol, or cysteamine on ethanol-induced gastric erosions, and of cimetidine on gastric erosions caused by aspirin was studied in intact, adrenalectomized, medullectomized, ovariectomized, or thyroidectomized rats. Cimetidine was administered at a low dose that did not inhibit hydrogen ion secretion. Adrenalectomized animals failed to exhibit the usual mucosal protective response to prostaglandin F2 beta, sulfhydryls, or cimetidine. Ovariectomy or thyroidectomy did not influence mucosal protection with these agents. The inhibition by total adrenalectomy of mucosal protection was not reversed by large intragastric doses or by parenteral administration of prostaglandin F2 beta. Adrenal medullectomy alone significantly diminished (by approximately one-third) ethanol-induced gastric mucosal injury; prostaglandin F2 beta or sulfhydryl drugs produced significant additional protection. Replacement therapy with glucocorticoids (triamcinolone, corticosterone) but not with mineralocorticoids (deoxycorticosterone, 9 alpha-fluorocortisol) restored the cytoprotective effect of prostaglandin F2 beta and sulfhydryls in adrenalectomized rats. The generation of prostaglandin E2- and prostaglandin I2-like activity in the gastric mucosa was unaltered by adrenalectomy. These studies suggest a permissive role for glucocorticoids in gastric mucosal protection induced by prostaglandins, sulfhydryls, and cimetidine.

Gastric cytoprotection is a real but relative phenomenon that is induced by both endogenous and exogenous compounds. The key element in acute mucosal protection is the preservation of vascular integrity and the resulting maintenance of blood flow. The critical factors in chronic mucosal protection include the stimulation of bicarbonate and mucus secretion, and the proliferation of epithelial, endothelial, and mesenchymal cells. Investigations are currently underway to identify the biochemical targets of cytoprotection.

Alkyl sulfhydryl drugs protect against acute gastric hemorrhagic mucosal lesions. We tested the protective effect of cyclic drugs containing oxidized (KT1-32, KT1-39, KT1-94), or reduced (KT1-66, KT1-109, KT1-293, KT1-720, KT1-756) sulfhydryls. The most potent protective agents (KT1-32, KT1-109, KT1-720, KT1-756) were investigated in detail. Drugs were administered intragastrically to fasted rats 30 min before 100% ethanol (1 ml) or acidified aspirin (10 mg/100 g), and mucosal lesions were measured planimetrically 1 hr later. Control rats receiving only ethanol had lesions involving 14.5% of the glandular mucosa. KT1-32, KT1-109, KT1-720, or KT1-756 (10 mg/100 g) reduced lesions to 0.7, 2.7, 1.8, or 0.7% of glandular stomach respectively. Aspirin-induced lesions involved 1.52% of the glandular mucosa and 10 mg/100 g of KT1-32, KT1-109, or KT1-720, or 2 mg/100 g of KT1-756 diminished the damage to 0.13, 0.02, or 0.04, or 0.00%, respectively. Indomethacin interfered with protection against ethanol by KT1-109, while the sulfhydryl alkylator N-ethylmaleimide abolished protection by both KT1-32 and KT1-109. Among the drugs investigated in detail, KT1-756 increased gastric acid output, while KT1-720 and KT1-756 significantly enhanced pepsin secretion. All four compounds studied in detail (ie, KT1-32, KT1-109, KT1-720, KT1-756) decreased the extent of vascular lesions in the gastric mucosa as revealed by monastral blue 1 min after ethanol. Thus, the mechanism of gastric mucosal protection by these novel aryl sulfhydryl compounds cannot be ascribed to an antisecretory effect, but may be related to prevention of vascular injury.

Previous structure-activity and pharmacologic studies with duodenal ulcerogens cysteamine and propionitrile implicating catecholamines in the pathogenesis of duodenal ulceration have now been followed up by dose- and time-response biochemical investigations to assess the importance of monoamines in the development of duodenal ulcers. The concentrations of norepinephrine (noradrenaline), dopamine, serotonin and their metabolites were measured in total brain, brain regions, stomach, duodenum, pancreas and adrenals in the rat. Turnover of catecholamines was determined in rats pretreated with the inhibitor of tyrosine hydroxylase alpha-methyl-p-tyrosine. The duodenal ulcerogens caused a dose- and time-dependent depletion of norepinephrine in virtually all the tissues examined. The effect was maximal 4 or 7 hr after cysteamine or propionitrile, and norepinephrine levels returned to normal in 24 hr. Dopamine changes were selective and often biphasic, e.g., elevation in adrenals, biphasic in brain cortex, hippocampus and midbrain, but uniformly decreasing in glandular stomach and duodenum. In the median eminence dopamine levels decreased by 181 and 324% at 15 and 30 min, respectively, after cysteamine, but neither dopamine nor 3,4-dihydroxyphenylacetic acid was modified in the periventricular nucleus. Serotonin levels were relatively stable, revealing slight elevations or no changes in most of the tissues. The turnover of norepinephrine was accelerated by both chemicals in virtually all brain regions, but dopamine turnover was affected only in a few areas, e.g., in the corpus striatum and medulla oblongata cysteamine decreased dopamine turnover, whereas propionitrile first (at 1 hr) accelerated then (at 8 hr) significantly suppressed it.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetazolamide, a carbonic anhydrase inhibitor, prevents acute gastric hemorrhagic lesions induced by ethanol. We used acetazolamide and other carbonic anhydrase inhibitors to correlate their gastroprotective effects with the degree of inhibition of carbonic anhydrase. Since acetazolamide is a thiadiazole, we also investigated structurally related thiadiazoles that contain sulfhydryls to test the hypothesis that the protection against ethanol-induced gastric erosions is related to the presence of sulfhydryls. Dose-response studies with acetazolamide revealed that the protection did not correlate with the inhibition of carbonic anhydrase in the rat gastric mucosa. The carbonic anhydrase inhibitors sulfanilamide and ethoxzolamide, did not offer protection. Bismuthiol I, a thiadiazole with two sulfhydryls, was twice as protective as 2-amino-5-mercapto-1,3,4-thiadiazole with only one sulfhydryl group. We conclude that the protection by acetazolamide against ethanol-induced lesions is not related to the inhibition of carbonic anhydrase in the gastric mucosa. The gastroprotective effect of acetazolamide and its derivatives may be related to their content of sulfhydryls in an oxidized or reduced state.

Recent investigations suggest that the mucosal vascular endothelium is not a passive bystander, and that alterations within the blood vessel wall actively participate in the pathogenesis of gastric mucosal injury. We review here our data on rapidly developing vascular injury as detected by monastral blue deposition and increased vascular permeability measured by Evan's blue extravasation in dose- and time-dependent experiments with ethanol, HCl, and NaOH in the rat. In addition, using in vivo microscopy and laser-Doppler velocimetry, we demonstrate circulatory stasis in the superficial capillaries within about 1 min after topical application of damaging agents, and a gradual decrease in blood flow that correlates with the extent of hemorrhagic erosions. Prostaglandins or sulfhydryl agents prevented the circulatory standstill and the development of hemorrhagic mucosal lesions. We conclude that microvascular damage, increased vascular permeability, and capillary stasis precede the development of hemorrhagic mucosal lesions.

We investigated whether lipid peroxidation is an important biochemical mechanism in acute gastric mucosal injury induced by acid, base, or postischemic reperfusion. Lipid peroxidation products, i.e., the concentration of conjugated dienes (absorbance at 242 nm), products absorbing at 270 nm, and malondialdehyde were measured in the gastric mucosa of rats killed 30 s or 1, 3, or 6 min after intragastric (ig) administration of 0.6 N HCl or 0.2 N NaOH. No increase in any of the lipid peroxidation products was detected at any of the time intervals. Mucosal lesions, however, were already visible at 30 s. Cumene hydroperoxide (1 ml, 100 mM ig) induced neither mucosal lesions nor lipid peroxidation. In vitro incubation of mucosal homogenates with cumene hydroperoxide (1 or 10 mM) however, rapidly induced elevation of the three parameters of lipid peroxidation. Ischemia induced by clamping the blood vessels of the stomach for 20, 30, and 40 min followed by reperfusion for 10, 30, and 40 min did not induce lipid peroxidation, despite the development of hemorrhagic mucosal lesions. Similarly, in gastric mucosal lesions induced by hemorrhagic shock no lipid peroxidation products were detected. In the small intestine, however, prolonged ischemia (2 h) followed by reperfusion (30 min) resulted in mucosal necrosis and elevation of malondialdehyde levels. These results suggest that lipid peroxidation is not a major pathogenetic mechanism in rapidly developing acute gastric mucosal injury caused by 0.6 N HCl or 0.2 N NaOH, or in the more slowly developing mucosal injury caused by postischemic reperfusion.

Gastric mucin glycoproteins form an adherent gel over the surface epithelium that is thought to protect the stomach against chemical and physical damage. The purpose of this study was to measure the release of mucin glycoproteins from rat stomach after treatment with cysteamine and prostaglandin F2 beta, two structurally unrelated drugs that have been shown to protect the stomach against the noxious effects of alcohol and other damaging agents. Gastric mucin was separated into soluble (washout) and insoluble (adherent) phases before colorimetric quantitation of total mucin, protein-bound hexose, and sialic acid. Cysteamine produced a dose-dependent increase in release of soluble and gel mucin. Prostaglandin F2 beta caused a dose-dependent release of hexose-containing mucin but had no effect on sialic acid-containing glycoproteins. Sepharose 4B chromatography of both the soluble and adherent mucus revealed that greater than 90% was a high molecular weight glycoprotein fraction. N-Ethylmaleimide, a known inhibitor of cytoprotection by cysteamine, had no effect on mucin secretion. Similarly, indomethacin inhibited mucin secretion by cysteamine but did not significantly influence cytoprotection. Thus the secretion of mucin by cytoprotective agents is unlikely by itself to explain the ability of the stomach to resist chemical or physical damage.

In this study the role of free radicals and lipid peroxidation as mediators of chemically induced mucosal damage was investigated. Two enzymatic antioxidants, superoxide dismutase or catalase injected intravenously, reduced mucosal damage either by ethanol or aspirin. Of six nonenzymatic antioxidants, given in a wide dose range subcutaneously 30 min before intragastric administration of absolute ethanol, only propyl gallate decreased mucosal damage, while four of the antioxidants tested against aspirin were protective. These nonenzymatic antioxidants were antisecretory in the pylorus-ligated rat. The concentration of conjugated dienes and malondialdehyde measured in the gastric mucosa shortly after ethanol or aspirin administration remained unchanged or slightly decreased. These results indicate that free radicals may be involved in the pathogenesis of acute gastric mucosal injury caused by chemicals, but their mechanisms of action probably does not involve lipid peroxidation.

This review is based on results from our laboratory and those published by others, and is focused on the early stages of pathogenesis that can be studied mostly in animals. Gastric mucosal injury is analysed on the examples of ethanol- and aspirin-induced lesions. Ethanol (50-100%) rapidly penetrates the mucosa, causes directly and/or indirectly (e.g., release of vasoactive products) endothelial damage in superficial and deep capillaries and venules. The vascular damage results in increased vascular permeability and decrease in blood flow leading to complete circulatory standstill in superficial capillaries 1-2 min after intragastric administration of concentrated ethanol. The direct chemical damage to surface mucosal epithelium is then followed by hypoxia and deep hemorrhagic necrosis in 1-5 min (erosion or ulcer). Unionized aspirin initiates a similar and complex yet slower progressing and less extensive erosion than alcohol. Duodenal erosion and ulcer produced by cysteamine, mepirizole or MPTP are preceded by excess acid in the proximal duodenum. This could be due to increased gastric acid output (1-4 hr), decreased bicarbonate secretion or duodenal dysmotility (0.5-8 hr) preventing the proper mix of acid and base in duodenal bulb. Necrosis and desquamation of absorptive cells in duodenal villi are evident 2-4 hr, followed by villus amputation (4-8 hr), erosion and ulcer (8-24 hr). The pathogenesis of gastroduodenal mucosal injury can thus be reconstructed from results obtained with animal models and from human studies. The results should serve as a basis to design protective drugs that are active on the basis of pathogenetic events.

Dopamine and norepinephrine concentrations in the stomach, duodenum, and brain were measured after subcutaneous injection of tyrosine methyl ester in the rat. Tyrosine significantly increased dopamine concentrations in each tissue without altering norepinephrine levels. The increase in dopamine concentration in the stomach or duodenum was observed 1 h after tyrosine methyl ester injection, but it returned to normal 4 h after the administration of the chemical.

Protection of the gastric mucosa may be the result of either increased cellular resistance to injury (cytoprotection) or, alternatively, decreased exposure of mucosal cells to the damaging agent. To determine whether decreased exposure of mucosal cells to damaging agents plays a role in mucosal protection by 16,16-dm PGE2 or sodium thiosulfate, we estimated the intramucosal concentration of 22NaCl and measured its absorption from the gastric lumen into the systemic circulation 1 and 5 min after intragastric administration of hypertonic (25% w/v) 22NaCl. In an attempt to explain the differences observed, we also measured the net transmucosal water flux in control animals and rats pretreated with the protective agents. Administration of hypertonic NaCl rapidly (within 1 min) induced extensive hemorrhagic mucosal lesions that were significantly reduced by pretreatment with 16,16-dm PGE2 or sodium thiosulfate. Ultra-low temperature autoradiography indicated that luminal hypertonic 22NaCl penetrates the upper layers of the mucosa in relatively high concentrations (12.5% w/v) within 1 min but its concentration decreases rapidly and reached low levels (3.12% w/v) by 5 min. Absorption of NaCl from the gastric lumen into the systemic circulation 1 and 5 min after hypertonic NaCl was lower in both pretreatment groups than in the control. Net gastric transmucosal water flux (from serosa to mucosa) increased (P less than 0.05) from 100 +/- 2 in controls, to 1470 +/- 8 and 715 +/- 9 microliters in rats pretreated with 16,16-dm PGE2 and sodium thiosulfate, respectively. We conclude that 16,16-dm PGE2 and sodium thiosulfate protect the gastric mucosa against hypertonic NaCl, diminish mucosal penetration of NaCl, decrease mucosal absorption of NaCl, and significantly increase serosal to mucosal transmucosal water flux.(ABSTRACT TRUNCATED AT 250 WORDS)

The hypothesis that vascular injury contributes to the development of hemorrhagic erosions after intragastric administration of ethanol has been examined in the rat using vascular tracers. Extravasation of intravenously injected Evans blue into the gastric wall and into gastric contents was used as an indicator of vascular permeability. India ink and monastral blue, which label damaged blood vessels, were used to demonstrate vascular injury morphologically. Intragastric instillation of 75% and 100% ethanol induced increased vascular permeability within 1-3 min and resulted in monastral blue labeling of vessels in 13% and 17%, respectively, of the glandular mucosa within 1 min. After 1 h of 100% ethanol exposure, the areal density of monastral blue-stained blood vessels did not increase compared with that seen at 1 min, but the areal density of grossly visible hemorrhagic lesions increased strikingly and approximated that of vessel staining. The hemorrhagic erosions consistently occurred in regions of glandular mucosa where vessels were stained with monastral blue. Pretreatment with prostaglandin F2 beta or cysteamine reduced ethanol-induced Evans blue extravasation and monastral blue staining of mucosal blood vessels but did not reduce histologic evidence of gastric surface cell damage in the glandular mucosa. As increased vascular permeability and morphologically detectable vascular lesions consistently preceded the development of grossly visible hemorrhagic erosions in the glandular mucosa, we suggest that vascular injury is an early pathogenetic factor in the development of ethanol-induced gastric hemorrhagic erosions. The data also indicate that the degree of vascular damage, unlike the injury to surface epithelial cells, is reduced by pretreatment with prostaglandin F2 beta or the sulfhydryl cysteamine.

Impairment of the mucosal microcirculation may contribute to ethanol-induced gastric mucosal damage. In this report, we describe diffuse and severe ultrastructural damage to the capillaries of the gastric glandular mucosa of the rat that occurred within 1 min after intragastric instillation of 100% ethanol. There was a gradient of damage in that endothelial cell structure was most severely disrupted in profiles of capillaries located close to the luminal surface but some morphologic evidence of damage was evident in the wall of capillary profiles to a mean depth of 256 micron. Capillary structure was generally normal in the deeper regions of the mucosa. Pretreatment with intragastric cysteamine, 30 mg/100 g, or intragastric prostaglandin F2 beta, 0.5 mg/100 g, significantly reduced the depth in the mucosa to which damage to capillaries extended. Pretreatment with intragastric prostaglandin F2 beta, 0.2 mg/100 g, afforded no significant protection. We conclude that a 1-min exposure to 100% ethanol induces striking damage to the microcirculation of glandular mucosa of the rat stomach with severe damage to capillary profiles near the lumen and sparing of capillary profiles near the muscularis mucosa, and pretreatment with the sulfhydryl agent, cysteamine, or with a large dose of prostaglandin F2 beta reduces the extent of but does not abolish ethanol-induced damage to gastric mucosal capillaries.

Somatostatin and somatostatin derivatives were tested for their ability to prevent gastric hemorrhagic erosions induced by ethanol. The somatostatin analogues were cyclohexapeptides with the rearranged amino acids 7-14 of somatostatin (S-14): Phe-Thr-Lys(Z)-Trp-Phe-D-Pro(I), Phe-Thr-Lys-Trp-Phe-D-Pro(II), Phe-Thr-Lys-D-Trp-Phe-Tyr(III) and Tyr-Phe-D-Trp-Lys-Thr-Phe(IV). In Spraque-Dawley rats receiving ethanol alone, the lesions involved 18.1 +/- 3.2% of the glandular stomach while after S- 14 (10(-7) mol/rat) the lesioned area was reduced to 6.3 +/- 1.1% (p less than 0.05). Peptide I and peptide II (doses 10(-7) -10(-9) mol/rat) decreased the area of erosions to less than 5%. Peptide III was less active and peptide IV was inactive. In rats with chronic gastric fistula S- 14 and peptide II decreased the cysteamine-stimulated acid secretion without affecting the pepsin output. We also continuously measured the intraluminal pH in the stomach of Wistar rats which develop gastric erosions after subcutaneous injection of cysteamine. The erosions were reduced by S- 14 or SMS while the intraluminal pH did not change under the influence of cysteamine or the combination of cysteamine plus S- 14 or SMS. Thus some of the peptides derived from S- 14 exert prominent gastric mucosal protection without influencing gastric secretion.