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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.

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.

Background/aims: Human basic fibroblast growth factor (bFGF) is an endothelial mitogen that stimulates angiogenesis and proliferation of other cells such as fibroblasts and smooth muscle cells. After this peptide was stabilized to acid and pepsin by site-specific mutagenesis, it was tested whether bFGF might accelerate the healing of experimental duodenal ulcers., Methods: This mutein peptide (bFGF-CS23) was administered orally in comparison with cimetidine to rats with chronic duodenal ulcers previously induced by cysteamine., Results: Oral bFGF-CS23 therapy maintained for 21 days at 100 ng/100 g twice daily resulted in (1) significant acceleration of healing of duodenal ulcers, i.e., reduction of mean ulcer area by 83% in the bFGF-CS23-treated rats compared with only 61% for cimetidine therapy and 40% for untreated controls; (2) complete healing with no residual ulcer in 62% of the bFGF-CS23-treated rats compared with only 7% of untreated rats; and (3) a ninefold increase in angiogenesis in the ulcer bed compared with untreated controls. A single dose of the bFGF-CS23 mutein had no effect on gastric output of hydrochloric acid or pepsin, but daily treatment for 2 or 3 weeks resulted in enhanced acid and pepsin outputs., Conclusions: Chronic duodenal ulcers can be healed rapidly by stimulating angiogenesis and other wound-healing processes in the ulcer bed without reduction of gastric acid.

Cysteamine and propionitrile are members of a family of compounds which induce the formation of acute duodenal ulcers in fasted and fed rats. Gastric acid secretion is increased by both agents, and acid hypersecretion appears to be required for ulcer formation. To determine the role of gastrin in the ulcerogenic mechanism, cysteamine and propionitrile were administered to fasted rats and their effect on fasting and food-stimulated serum gastrin levels was studied. Intragastric administration of cysteamine caused a 3- to 4-fold increase in fasting serum gastrin levels over the values of controls. Propionitrile was a less effective stimulant of gastrin release, causing a 1.5- to 2-fold increase in gastrin levels over matched control rats. The food-stimulated rise in serum gastrin levels after either a chow meal or intragastric instillation of a peptone solution was markedly enhanced by cysteamine pretreatment. Three hours after feeding the serum gastrin levels of cysteamine pretreated rats were 6 times higher than those of fed controls. The high serum gastrin levels of cysteamine-pretreated fed rats could not be explained solely by the additive effects of cysteamine and food, indicating that a potentiating interaction may exist between the two stimulants of gastrin release. The importance of this drug-induced stimulation of gastrin release, under both fasted and fed conditions, in the ulcerogenic process has yet to be ascertained.

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.

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.

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.

Recent evidence suggests a role of endogenous sulfhydryls (SHs) in gastric "cytoprotection." Because divalent metals bind to or oxidize SH groups, their effect on ethanol-induced gastric erosions was studied. For comparative biochemical studies the SH cysteamine, the glutathione depletor diethylmaleate. and SH alkylating agent N-ethylmaleimide (NEM) were also used. Rats pretreated with CdCl2, ZnCl2, or Cu(NO3)2 6 h before absolute ethanol showed a significant dose-dependent decrease in the mucosal lesions. Copper was effective in preventing the lesions up to 15 min before the ethanol. Iron and manganese were active at 30 min, but not at 6 h before the ethanol lesions. Indomethacin administration decreased the protection afforded by iron, manganese, and cadmium, but did not modify that by lead and copper. N-ethylmaleimide abolished the protection by iron, manganese, and cadmium, but did not affect the protection caused by lead and copper when given after the metals. However, when NEM was given before lead and copper, it diminished the protection. Secretory studies revealed that cadmium and zinc slightly inhibited gastric acid secretion, but a similar reduction of acid output by cimetidine did not decrease the ethanol-induced gastric erosions. Biochemical studies of endogenous SH showed that the protective metals and NEM decreased the glutathione concentration in the nonprotein fraction, whereas these metals diminished and NEM, which antagonizes mucosal protection, elevated the cysteine concentration in the protein fraction of the gastric mucosa. The common factor with the protective agents thus seems to be the blocking of protein SH by binding or oxidation by protective agents. These endogenous SHs may mediate cellular responses to injury.