iNOS expression in human intestinal microvascular endothelial cells inhibits leukocyte adhesion.

Increased nitric oxide (NO) production by inducible nitric oxide synthase (iNOS) has been associated with intestinal inflammation, including human inflammatory bowel disease. However, NO can downregulate endothelial activation and leukocyte adhesion, critical steps in the inflammatory response. Using primary cultures of human intestinal microvascular endothelial cells (HIMEC), we determined the role of NO in the regulation of HIMEC activation and interaction with leukocytes. Both nonselective (NG-monomethyl-L-arginine) and specific (N-iminoethyl-L-lysine) competitive inhibitors of iNOS significantly increased binding of leukocytes by HIMEC activated with cytokines and lipopolysaccharide. Increased adhesion was reversible with the NOS substrate L-arginine and was not observed in human umbilical vein endothelial cells (HUVEC). Activation of HIMEC significantly upregulated HIMEC iNOS expression and NO production. NOS inhibitors did not augment cell adhesion molecule levels in activated HIMEC but did result in sustained increases in intracellular reactive oxygen species. In addition, antioxidant compounds reversed the effect of NOS inhibitors on HIMEC-leukocyte interaction. Taken together, these data suggest that after HIMEC activation, iNOS-derived NO is an endogenous antioxidant, downregulating leukocyte binding and potentially downregulating intestinal inflammation.

Increased expression of inducible nitric oxide synthase and cyclooxygenase-2 in Barrett's esophagus and associated adenocarcinomas.

Barrett's esophagus is a premalignant condition arising in response to chronic reflux esophagitis. Inducible nitric oxide synthase (iNOS; NOS-2) and cyclooxygenase-2 (COX-2) are mediators of inflammation and regulators of epithelial cell growth. Expression levels of iNOS and COX-2 are high in colorectal adenomas and carcinomas, and COX-2 expression is elevated in gastric cancers. To determine the involvement of iNOS and COX-2 in Barrett's-associated neoplasia, we measured expression of these genes in metaplastic Barrett's and esophageal adenocarcinomas. We detected elevated iNOS and COX-2 mRNA levels in Barrett's mucosa compared with paired gastric control tissues in 16 of 21 (76%) and 17 of 21 (80%) patients, respectively (P < 0.001 for both genes). In esophageal adenocarcinomas, iNOS and COX-2 mRNA levels were increased in four of five and five of five cases, respectively. Furthermore, in 10 of 10 Barrett's patients, immunohistochemical staining for iNOS and COX-2 expression was strongly positive and higher than in matched gastric controls. Increased COX-2 expression was confirmed by Western blotting. These findings support the hypothesis that iNOS and COX-2 are involved early and often in Barrett's-associated neoplastic progression.

Helicobacter pylori stimulates inducible nitric oxide synthase expression and activity in a murine macrophage cell line.

BACKGROUND & AIMS: Helicobacter pylori uniquely colonizes the human stomach and produces gastric mucosal inflammation. High-output nitric oxide production by inducible nitric oxide synthase (iNOS) is associated with immune activation and tissue injury. Because mononuclear cells comprise a major part of the cellular inflammatory response to H. pylori infection, the ability of H. pylori to induce iNOS in macrophages was assessed. METHODS: H. pylori preparations were added to RAW 264.7 murine macrophages, and iNOS expression was assessed by Northern blot analysis, enzyme activity assay, and NO2- release. RESULTS: Both whole H. pylori and French press lysates induced concentration-dependent NO2- production, with peak levels 20-fold above control. These findings were paralleled by marked increases in iNOS messenger RNA and enzyme activity levels. iNOS expression was synergistically increased with interferon gamma, indicating that the H. pylori effect can be amplified by other macrophage-activating factors. Studies of lipopolysaccharide (LPS) content and polymyxin B inhibition of LPS suggested that the H. pylori effect was attributable to both LPS-dependent and -independent mechanisms. CONCLUSIONS: iNOS expression in macrophages is activated by highly stable H. pylori products and may play an important role in the pathogenesis of H. pylori-associated gastric mucosal disease.

Synthesis and brush border expression of intrinsic factor-cobalamin receptor from rat renal cortex.

The main objective of the current study was to investigate the factors that affect brush border membrane expression of intrinsic factor-cobalamin receptor (IFCR). Because of high levels of IFCR expression (Seetharam, B., Levine, J. S., Ramasamy, M., and Alpers, D. H. (1988) J. Biol. Chem. 263, 4443-4449) in the rat kidney, we have studied the synthesis and expression of IFCR using rat cortical slices in culture. The IFCR activity in the renal apical brush border was maximum from rats between the age of 20-24 days and about 75% of the activity was lost from the isolated apical surface membranes following culture of cortical slices with nonradioactive intrinsic factor-cobalamin. However, the membrane IFCR activity recovered to 100 or 75%, respectively, when the slices were cultured with intrinsic factor-cobalamin mixed with either leupeptin or chloroquine. When these lysosomotropic agents were added during the metabolic labeling of the cortical slices with trans-35S-label neither the synthesis nor the amount of [35S]IFCR transported to the apical membrane was inhibited. However, with the addition of colchicine, the apical membrane expression of [35S]IFCR was inhibited by 75-80%. Metabolic labeling of cortical slices with trans-35S-label and immunoprecipitation of the Triton X-100 extract from the total, internal, and apical membranes revealed the presence of a 230-kDa band following sodium dodecyl sulfate-polyacrylamide gel electrophoresis. With either continuous or pulse-chase labeling of the cortical slices, the amount of 230-kDa [35S]IFCR recovered in the apical membrane did not exceed 10-15% of the total labeled receptor synthesized. Based on these and our recent studies (Seetharam, S., Dahms, N., Li, N., Ramanujam, K.S., and Seetharam, B. (1991) Biochem. Biophys. Res. Commun. 177, 751-756), we propose that rat renal IFCR is synthesized as a single polypeptide chain of 220 kDa and is transported slowly to the apical membrane during which four or five N-linked oligosaccharides are processed to the complex type. Moreover, the brush border expression of IFCR is regulated by the biosynthetic and not by the endocytic pathway.

Leupeptin and ammonium chloride inhibit intrinsic factor mediated transcytosis of [57Co]cobalamin across polarized renal epithelial cells.

The [125I] intrinsic factor (IF) mediated transcytosis of [57Co]Cyanocobalamin (Cbl) by polarized opossum kidney cells was inhibited (greater than 80%) by preincubation of the cells with lysosomotropic agents leupeptin or ammonium chloride. Inhibition of Cbl transcytosis resulted in the intracellular accumulation of both [125I]IF (48 kDa) and [57Co]Cbl. Intracellular degradation of [125I]IF occurred during normal cellular transcytosis of [57Co]Cbl and in one h following internalization the major intracellular degradation products of IF were two polypeptides of Mr 29 kDa and 19 kDa. The size of the major degradation product of IF in the basolateral media was 10 kDa. Based on these results, we suggest that IF is internalized by the renal epithelial cells and is degraded by leupeptin-sensitive acid proteases during Cbl transcytosis.

In vitro translation and expression of renal intrinsic factor-cobalamin receptor.

The primary translation product of intrinsic factor (IF)-cobalamin receptor (IFCR) mRNA from rat kidney is a single polypeptide chain of Mr = 215,000. When expressed in Xenopus laevis oocytes the IFCR binding activity is expressed with mRNA of a size between 5 to 7 kb. These results suggest that IFCR mRNA transcripts are present in the renal tissue and encode a single chain, large molecular weight precursor. Furthermore, Xenopus oocytes can be used as a screening system in the expression cloning of the renal IFCR.

Expression of cobalamin transport proteins and cobalamin transcytosis by colon adenocarcinoma cells.

Human colon adenocarcinoma (Caco-2) cells express both intrinsic factor-cobalamin receptor and transcobalamin II (TC II). The expression of these activities began to rise by day 6 and reached peak levels between 10 and 15 days in culture. The postconfluent Caco-2 cell membranes bound approximately 30-35 fmol of intrinsic factor (IF) [57Co]Cbl/mg protein. The size of the mature receptor expressed in the apical brush border had a relative molecular mass of 230 kDa. The intracellular form of TC II had a Mr of 43, 5 higher than the secreted form of TC II. TC II was secreted unidirectionally via the basolateral direction when Caco-2 cells were grown on culture inserts. When grown on culture inserts, the Caco-2 cells were polarized (electrical resistance greater than 200 omega/cm2) and transcytosed [57Co]Cbl bound to IF from apical-to-basal but not from basal-to-apical direction. Under these conditions, [57Co]Cbl complexed to haptocorrin was not transported. These cells also transcytosed free [57Co]Cbl, although less efficiently. The [57Co]Cbl transcytosed using either IF[57Co]Cbl or free [57Co]Cbl as ligands was bound exclusively to TC II. Intracellular [57Co]Cbl decreased during transcytosis with a slow (t1/2 = 4 h) transfer of [57Co]Cbl from IF to TC II. These results show that the transport of Cbl in Caco-2 cells is very similar to the human enterocyte system.

Control of alcohol addiction by SKV therapy--its action on water, food intake, brain function and cell membrane composition.

Alcohol being easily permeable through cell membrane causes toxic damage to many tissues. Rats drinking aqueous ethanol (25% v/v) for 120 days and 240 days showed an initial rise in body weight. The reduced rate in weight gain in chronic alcoholism is associated with a fall in food intake. Ethanol ingesting animals showed slow response to stimuli and increase in blood ethanol and serum GGTP levels. Liver plasma membrane, kidney brush-border membrane and pancreatic plasma membrane from alcoholic rats showed significant alterations in cholesterol/phospholipid molar ratio and membrane ATPases. Water retention with the enlargement of liver and kidney associated with increased fluid consumption are also seen during alcoholism. SKV by breaking alcohol dependence reduces drinking, lowers blood ethanol level and fluid intake without developing withdrawal symptoms. Restriction of ethanol intake by SKV therapy resulted in the reversal of organ enlargement and membrane composition in alcoholics.