Volume 123, Issue 1, Pages 173-186 (July 2002) Gastric nitric oxide synthase expression during endotoxemia: Implications in mucosal defense in rats  Kenneth S. Helmer, Sonlee D. West, Lily Chang, Yan Cui, David W. Mercer  Gastroenterology  Volume 123, Issue 1, Pages 173-186 (July 2002) DOI: 10.1053/gast.2002.34178 Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 1 Inducible nitric oxide synthase (iNOS) immunoreactivity in gastric fundic sections from rats receiving saline (top) or LPS (20 mg/kg) for 5 hours (bottom). Very little iNOS immunoreactivity (red) is seen in saline-treated rats. A 5-hour treatment with LPS resulted in a marked increase in iNOS immunoreactivity in the remaining mucus neck cells of the fundic gastric pit regions. Actin is green and nuclei are blue. Gastroenterology 2002 123, 173-186DOI: (10.1053/gast.2002.34178) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 2 Effect of intraperitoneal saline or lipopolysaccharide (20 mg/kg) on gastric mucosal calcium-dependent and calcium-independent NOS activity at 1, 3, and 5 hours in crude homogenates of gastric mucosa containing both cytosolic and membrane-bound proteins. Gastric mucosa was not exposed to a mild irritant. Data are means ± SE; n ≥ 5 for all groups. *P ≤ 0.02 vs. saline. Gastroenterology 2002 123, 173-186DOI: (10.1053/gast.2002.34178) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 3 Effect of intraperitoneal saline or lipopolysaccharide (20 mg/kg) on gastric mucosal calcium-dependent and calcium-independent NOS activity at 1, 3, and 5 hours in gastric mucosal homogenates consisting of the membranous fraction of gastric mucosal proteins. Gastric mucosa was not exposed to a mild irritant. Data are means ± SE; n ≥ 5 for all groups. *P ≤ 0.02 vs. saline. Gastroenterology 2002 123, 173-186DOI: (10.1053/gast.2002.34178) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 4 Effect of intraperitoneal lipopolysaccharide (20 mg/kg) and saline on gastric luminal nitrate and nitrite (NOX) accumulation at 1, 3, and 5 hours. Gastric mucosa was not exposed to a mild irritant. Data are means ± SE; n ≥ 5 for all groups. *P ≤ 0.02 vs. saline. Gastroenterology 2002 123, 173-186DOI: (10.1053/gast.2002.34178) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 5 Morphologic injury in gastric sections from rats receiving saline or LPS (20 mg/kg) for 5 hours, followed by intraluminal instillation of either distilled water or 5 mmol/L ATC for 10 minutes. (A) Light micrograph (×20) of gastric mucosa from anesthetized rat given 5-hour treatment with 0.9% saline and then 3 mL of intraluminal distilled water for 10 minutes shows intact surface mucosal cells aligning lumen of the stomach and intact gastric pits. (B) Light micrograph (×20) of gastric mucosa 5 hours after LPS (20 mg/kg), followed by 3 mL of intraluminal distilled water for 10 minutes, shows some loss of surface epithelial cells, disruption of normally organized gastric pits, and vacuolization of surface mucosal cells. Glands are intact. (C) Light micrograph (×20) of gastric mucosa given 5-hour treatment with 0.9% saline and then 3 mL of intraluminal 5 mmol/L ATC for 10 minutes shows damage to the surface epithelium and upper gastric pits, with exfoliation of surface mucosal cells into the gastric lumen. (D) Light micrograph (×20) of gastric mucosa 5 hours after LPS (20 mg/kg), followed by 3 mL of intraluminal 5 mmol/L ATC for 10 minutes, shows massive hemorrhage into the lamina propria and necrosis and disruption of entire gastric pits and upper glands. Gastroenterology 2002 123, 173-186DOI: (10.1053/gast.2002.34178) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 6 F-Actin immunofluorescence (green) in gastric sections from rats receiving saline or LPS (20 mg/kg) for 5 hours, followed by intraluminal instillation of either distilled water or 5 mmol/L ATC for 10 minutes. Nuclei stain blue. (A) Light micrograph (×60) of gastric mucosa from anesthetized rat given 5-hour treatment with 0.9% saline and then 3 mL of intraluminal distilled water for 10 minutes, shows intact, discrete linear staining of F-actin representing intact cellular membranes of surface mucosal cells aligning the lumen of the stomach and dipping into intact gastric pits. (B) Light micrograph (×60) of gastric mucosa 5 hours after saline, followed by 3 mL of intraluminal 5 mmol/L ATC for 10 minutes, shows some loss of surface epithelial cells with minimal disruption of normally organized gastric pits but with intact, discrete staining of F-actin. Glands are intact. (C) Light micrograph (×60) of gastric mucosa given 5-hour treatment with LPS (20 mg/kg) and then 3 mL of intraluminal distilled water for 10 minutes shows disrupted actin filaments in the surface mucosal cells as shown by smearing and punctuate formation of F-actin as well as disrupted and discontinuous cellular membranes. (D) Light micrograph (×60) of gastric mucosa 5 hours after LPS (20 mg/kg), followed by 3 mL of intraluminal 5 mmol/L acidified taurocholate for 10 minutes, shows disruption of F-actin and cellular membranes as well as exfoliation of nuclei into the gastric lumen. Gastroenterology 2002 123, 173-186DOI: (10.1053/gast.2002.34178) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 7 Effect of local intraarterial infusion (15 minutes) of S-nitroso-N-acetyl-penicillamine (SNAP; 20 μg/kg/min) or saline on rat macroscopic gastric injury caused by exposure of the stomach to 3 mL of 5 mmol/L acidified taurocholate or 20% ethanol while control stomachs were exposed to distilled water. SNAP was given 5 minutes before exposing the stomach to an irritant. Data are means ± SE; n ≥ 5 for all groups. *P ≤ 0.001 vs. saline counterpart; **P ≤ 0.004 vs. SNAP/water. Gastroenterology 2002 123, 173-186DOI: (10.1053/gast.2002.34178) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 8 Effect of NOS inhibitors on macroscopic gastric injury in rats treated with saline or LPS (20 mg/kg) for 5 hours, followed by 3 mL of intraluminal 5 mmol/L ATC for 10 minutes. Both aminoguanidine (AG) and L-NAME were given concurrently with saline or LPS. Data are means ± SE; n ≥ 5 for all groups. *P ≤ 0.001 vs. saline/saline; **P ≤ 0.05 vs. saline/LPS. Gastroenterology 2002 123, 173-186DOI: (10.1053/gast.2002.34178) Copyright © 2002 American Gastroenterological Association Terms and Conditions