Kevin J. Ivey

Prostaglandin Protection of Carbon Tetrachloride-Induced Liver Cell Necrosis in the Rat

We studied whether 16,16--dimethyl prostaglandin E2 (dmPGE2) may prevent acute liver damage induced by carbon tetrachloride (CCl4) in the rat. One hundred thirty male rats were divided into the following groups: (1) controls, (2) rats given CCl4 6670 mg/kg body wt subcutaneously, (3) rats pretreated with 5 micrograms/kg dmPGE2 given subcutaneously 30 min before, and 8 and 24 h after CCl4 administration, and (4) animals given dmPGE2 only as in group 3. Liver damage was assessed by biochemical studies (SGPT, serum alkaline phosphatase, and bilirubin) and by histology. In rats receiving CCl4 alone, SGPT activities were significantly elevated to 1024 +/- 82 U/L, 1270 +/- 120 U/L, 386 +/- 48 U/L and 208 +/- 20 U/L at 24, 48, 96, and 120 h after CCl4 respectively. In animals pretreated with dmPGE2 before CCl4, SGPT activities were 201 +/- 24 U/L, 55 +/- 4.6 U/L, 28 +/- 4 U/L, and 24 +/- 4 U/L at 24, 48, 96, and 120 h after CCl4, respectively (p less than 0.01, versus animals receiving CCl4 only). Histologically, livers of rats treated with CCl4 alone showed severe centrilobular necrosis at 24 and 48 h. Livers of animals pretreated with dmPGE2 before CCl4 did not show necrosis. It is concluded that dmPGE2 protects the liver against cell necrosis induced by CCl4 in the rat.

Cell Culture of Rat Gastric Fundic Mucosa

The purpose of this study was to develop a primary cell culture system of rat gastric fundic epithelial cells. The cells, isolated enzymatically, were cultured in Coons modified Hams F-12 medium supplemented with 10% fetal bovine serum, 15 mM HEPES buffer, fibronectin, and antibiotics. The inoculated cells started to grow rapidly on day 1 (doubling time, 26 h). The cells reached confluency on day 3. On phase contrast microscopy, over 90% of cells possessed epithelial characteristics. Histochemical studies showed (a) 90% of the epithelial cells contained PAS positive granules, (b) 5% of the cells gave a strong reaction for succinic dehydrogenase activity (presumably parietal cells), and (c) immunohistochemical localization of pepsinogen was negative. Ultrastructurally, microvilluslike structures, junctional complexes, Golgi apparatus, mitochondria, rough-surfaced endoplasmic reticulum, and mucous granules were observed. Mitotic figures were clearly observed on Giemsa staining and the mitotic index was maximum on day 2. Autoradiographic and biochemical studies showed these cells possessed the capability to synthesize deoxyribonucleic acid and this ability was maximum on day 2. These cells were able to synthesize and to secrete glycoprotein and this function was significantly increased by 16,16-dimethyl prostaglandin E2. Cyclic adenosine monophosphate produced by the cultured cells was enhanced by addition of 16,16-dimethyl prostaglandin E2 (p less than 0.01). This in vitro system provides a valuable model for studies of cellular functions of gastric mucosa.

Protection of cultured rat gastric cells against oxidant-induced damage by exogenous glutathione

BACKGROUND/AIMS Reduced glutathione (GSH) is an intracellular protectant against oxidants. The present study determined whether extracellular GSH protects against oxidant damage or whether an uptake system of GSH is present in cultured gastric cells. METHODS Hydrogen peroxide was generated by glucose oxidase and glucose. Cytotoxicity was assessed by 51Cr release. Intracellular GSH was assayed by the method of Tietze. RESULTS Pretreatment with extracellular GSH decreased H2O2-induced 51Cr release. Treatment with GSH enhanced cellular GSH content. Protection by pretreatment with GSH was prevented by buthionine sulfoximine (an inhibitor of gamma-glutamylcysteine synthetase). Enhancement of intracellular GSH was also prevented by buthionine sulfoximine. Acivicin (an inhibitor of gamma-glutamyl transpeptidase) prevented intracellular accumulation of GSH from extracellular GSH. Cysteine was effective in preventing damage and enhancing intracellular GSH content, whereas both glutamine and glycine were not. CONCLUSIONS Extracellular GSH protects cultured gastric cells from H2O2 damage by accelerating intracellular GSH synthesis; this is mediated by membrane-bound gamma-glutamyl transpeptidase acting on extracellular GSH (which supplies these cells with cysteine) and then by intracellular gamma-glutamylcysteine synthetase.

Protective Role of Intracellular Glutathione Against Ethanol-Induced Damage in Cultured Rat Gastric Mucosal Cells

This study investigated whether intracellular glutathione is cytoprotective against ethanol-induced injury to cultured rat gastric mucosal cells in vitro. Secondly, it investigated whether reduced glutathione or oxidized glutathione is responsible for this cytoprotection. Cytolysis was quantified by measuring 51Cr release from prelabeled cells. Concentrations of ethanol greater than 12% caused cell damage and increased 51Cr release in a dose-dependent and time-related fashion. When a substrate for glutathione synthesis, N-acetyl-L-cysteine, was provided to cultured cells for 4 h before challenge with ethanol, cytolysis was significantly decreased corresponding with an increase in cellular glutathione content. Pretreatment with diethyl maleate, which depletes reduced glutathione without forming oxidized glutathione, potentiated ethanol-induced cell damage in a dose-dependent manner with the decrease of cellular glutathione content. The administration of tert-butyl hydroperoxide (which is specifically reduced by glutathione peroxidase to generate oxidized glutathione from reduced glutathione) or diamide (which nonenzymatically oxidizes reduced glutathione to oxidized glutathione) enhanced ethanol injury. We conclude that in cultured gastric mucosal cells, (a) intracellular glutathione maintains integrity of gastric mucosal cells against ethanol in vitro; and (b) reduced glutathione rather than oxidized glutathione is responsible for this cytoprotection. We postulate that the presence of reduced glutathione is essential to allow glutathione peroxidase to catalyze the ethanol-generated toxic oxygen radical, hydrogen peroxide.

A monolayer culture of human gastric epithelial cells.

Our aim was to develop a fibroblast-free monolayer culture of human gastric mucosal cells, using the specimens obtained by routine endoscopic biopsy. Human gastric mucosa obtained from normal volunteers by endoscopic biopsy was dissociated from collagenase and hyaluronidase. Dissociated cells were cultured in supplemented Coons modified Hams F-12 medium. Within 24 hr of inoculation, the cells were attached to the culture dishes. This was followed by cellular outgrowth. On phase-contrast microscopy, all cells had epithelial characteristics and fibroblasts were not observed. Ninety percent of cells contained periodic acid Schiff reaction-positive mucous granules after diastase digestion consistent with mucous epithelial cells. Two percent of the cells gave a strong reaction for succinic dehydrogenase activity (parietal cells). Immunohistochemical staining for pepsinogen in cultured cells was negative. On EM, microvilli-like projections, junctional complexes, Golgi apparatus, and mucous granules were apparent in the majority of cells. Mitotic figures were observed by day 3 with Giemsa staining. Autoradiographically, these cells were able to incorporate [3H] TdR into the nuclei. Cells were capable of synthesizing DNA, and this function was inhibited by cycloheximide. Cells could be cultured for up to two weeks without fibroblast contamination. A method of primary monolayer culture of human gastric mucosa obtained by a routine endoscopic biopsy has been successfully developed.

Cytotoxicity of Helicobacter pylori on human gastric epithelial cells in vitro: role of cytotoxin(s) and ammonia

Objective To evaluate the respective roles of vacuolating cytotoxin(s) and urease-mediated ammonia production in Helicobacter pylori-induced cytotoxicity on cultured human gastric epithelial cells. Methods Monolayers of MKN 28 cell line were incubated with broth culture filtrates of two urease-positive H. pylori strains: CCUG 17874, cytotoxin-producing and C21, non-cytotoxin-producing. After incubation, highly sensitive quantitative assays were used to measure the ammonia levels in the cell medium, cell vacuolation and cell death. In order to evaluate the cytotoxic role of ammonia more accurately, we performed an ‘ammonia simulation’ by measuring cell vacuolation and cytolethality induced by incubation with definite concentrations of ammonium chloride. Cell damage was also studied morphologically. Results Both H. pylori strains produced ammonia and induced cell vacuolation and cytolethality. With the G21 strain, both cell vacuolation and cytolethality appeared virtually identical to ammonia simulation. With the CCUG 17874 strain, which induced a higher degree of cell vacuolation and cell death compared with the G21 strain, cell vacuolation and cytolethality appeared largely ammonia-independent. The vacuoles induced by each H. pylori strain and by ammonia simulation were similar to each other. In less severely affected cells, vacuole formation and ammonia simulation were regularly limited by smooth membranes and morphologically and topographically resembled dilated endosomes or trans Golgi vesicles. In more severely affected cells, large autophagic-like vacuoles containing cell debris were also prominent. Conclusions The vacuolating cytotoxin(s) and ammonia are major H. pylori virulence factors. The cytotoxin(s) seem to play a more prominent role than ammonia in cell vacuolation. Nevertheless, ammonia seems to damage cells by other mechanisms in addition to vacuolation. A common pathway for cytotoxin- and ammonia-induced cell vacuolation seems likely.

Ethanol induced duodenal lesions in man. Protective effect of prostaglandin

This study aimed: 1. to investigate quantitatively mucosal changes in the human duodenum after ethanol instillation, 2. to determine the effect of 16,16 dimethyl prostaglandin E2 (dmPGE2) pretreatment on these changes. In 11 healthy subjects were instilled into the postbulbar duodenum solutions of normal saline or dmPGE2 and 15 minutes later 20 ml 40% ethanol. Mucosal changes were evaluated endoscopically and histologically. Thirty minutes after ethanol and saline, hemorrhagic changes involved the entire mucosa (endoscopic lesion index: 4.8 +/- 0.2) and all histological specimens showed erosions. Pretreatment with dmPGE2 significantly prevented ethanol-induced mucosal changes (endoscopic lesion index: 2.17 +/- 0.17 p less than 0.01) and mucosal biopsies did not show erosions outside endoscopically abnormal areas. In conclusion, direct instillation of ethanol induced consistent damage to duodenal mucosa in man. This damage was significantly reduced by dmPGE2 pretreatment.

Protective role of intracellular superoxide dismutase against extracellular oxidants in cultured rat gastric cells.

We examined the role of intracellular superoxide dismutase (SOD) as an antioxidant by studying the effect of diethyldithiocarbamate (DDC) on extracellular H2O2-induced damage in cultured rat gastric mucosal cells. 51Cr-labeled monolayers from rat stomachs were exposed to glucose oxidase-generated H2O2 or reagent H2O2, which both caused a dose-dependent increase in 51Cr release. DDC dose-dependently enhanced 51Cr release by hydrogen peroxide, corresponding with inhibition of endogenous SOD activity. This inhibition was not associated either with modulation of other antioxidant defenses, or with potentiation of injury by nonoxidant toxic agents. Enhanced hydrogen peroxide damage by DDC was significantly prevented by chelating cellular iron with deferoxamine or phenanthroline. Inhibition of cellular xanthine oxidase (possible source of superoxide production) by oxypurinol neither prevented lysis by hydrogen peroxide nor diminished DDC-induced sensitization to H2O2. We conclude that (a) extracellular H2O2 induces dose dependent damage to cultured gastric mucosal cells; (b) intracellular SOD plays an important role in preventing H2O2 damage; (c) generation of superoxide seems to occur intracellularly after exposure to H2O2, but independent of cellular xanthine oxidase; and (d) cellular iron mediates the damage by catalyzing the production of more reactive species from superoxide and H2O2, the process which causes ultimate cell injury.

Effect of sucralfate and its components on taurocholate-induced damage to rat gastric mucosal cells in tissue culture

The present study evaluated the effect of sucralfate and its components, sucrose octasulfate and aluminum hydroxide, on: (1) damage to rat cultured gastric mucosal cells induced by sodium taurocholate in a neutral environment and in conditions independent of systemic factors, (2) prostaglandin E2 and on 6-keto prostaglandinF1α release by cultured cells, and (3) sulfhydryl content of cultured cells. Cell damage was quantitated by chromium-51 release assay. Prostaglandin E2 and 6-keto prostaglandinF1α were measured by radioimmunoassay. Total sulfhydryl content of cultured cells was determined calorimetrically. Microscopically, sucralfate was found to adhere tightly to epithelial cell surfaces despite frequent washings. Sucralfate 2 mg/ml and 5 mg/ml significantly decreased taurocholate-induced damage, reducing taurocholate-induced specific51Cr release by 11.8 points (equal to 29% decrease in cell damage, (P< 0.01) and 22.9 points (equal to 56% decrease in cell damage, (P<0.001), respectively. Sucrose octasulfate and aluminum hydroxide did not exert significant protection against damage induced by sodium taurocholate. The protective effect of sucralfate was not prevented by indomethacin, nor was it counteracted by the sulfhydryl blocker, iodoacetamide. Sucralfate, but not its components, significantly and dose-dependently stimulated prostaglandin E2 (r=0.94, P<0.05 and 6-keto prostaglandinF1α (r=0.89, P<0.05) production by cultured cells. Neither sucralfate nor its components affected sulfhydryl content of cultured cells. In conclusion, sucralfate, but not its components, (1) protects rat gastric mucosal cells against taurocholate-induced damage in conditions independent of systemic factors and in a neutral environment and (2) significantly stimulates prostaglandin production by cultured cells. (3) The protection by sucralfatein vitro does not seem to depend on its stimulatory effect on endogenous prostaglandin synthesis.

Propranolol reduces ethanol-induced gastric mucosal damage in portal hypertensive rats

In a standardized rat model of portal hypertension, we investigated the effects of propranolol on alcohol-induced gastric mucosal damage. Portal hypertensive rats pretreated with 2 mg propranolol, compared with those receiving saline, had significantly reduced portal pressures (24±1 vs 32±1 cm saline), macroscopic mucosal damage (24 ±1 vs 39±4% of mucosa), and histologic deep necrosis (36±2 vs 61±4% of mucosal length). Increased dosage of propranolol to 4 mg did not produce any further reduction of portal pressure or mucosal damage. Central venous and systemic arterial pressures were not significantly altered by propranolol. The extent of mucosal damage correlated with levels of portal pressure (P<0.01) in portal hypertensive rats. Sham-operated normotensive rats had less macroscopic mucosal damage (26±4%) than portal hypertensive rats, and propranolol did not affect the extent of ethanol-induced damage or portal pressures in these animals. We conclude: (1) Propranolol is effective in reducing extent of ethanol-induced gastric mucosal damage in portal hypertensive rats, but not in shamoperated controls; (2) this effect correlates with reduction of portal pressure; and (3) our study supports the clinical impression that reducing portal pressure may be one approach for the prevention and therapy of gastric mucosal damage in portal hypertension.