Ethanol-induced changes in lipid peroxidation of enterocytes across the crypt–villus axis in rats.

Background Reactive oxygen species (ROS) have been implicated in the turnover of epithelial cells in the rat intestine. The metabolism of ethanol generates ROS, which are implicated in cellular injury, but the levels of lipid peroxidation in intestine in chronic alcoholism are unknown. Aim To investigate the effects of ethanol ingestion on lipid peroxidation, and anti- and pro-oxidant enzyme systems in enterocytes across the crypt–villus axis in intestine. Methods Wistar rats (90–100 g) were administered 1 mL of 30% ethanol daily for 39 days. Intestinal epithelial cells were isolated in fractions. Malondialdehyde levels, and activities of glutathione-S-transferase (GST), glutathione reductase (GR), superoxide dismutase (SOD) and catalase were determined in various cell fractions. Incorporation of H3- thymidine into DNA of enterocytes was also determined. Results Lipid peroxidation was elevated by two- to threefolds in both villus and crypt cells in ethanol-fed animals compared to controls. The activities of GST and GR were four- to six-folds higher in villus tip cells compared to crypt base cells. The activities of SOD and catalase were five- to seven-fold higher in crypt base cells compared to villus tip cells. Ethanol feeding elevated the activities of SOD (76-190%) and catalase (20-150%) in enterocytes all along the crypt–villus axis compared to the controls. H3 thymidine incorporation into DNA of enterocytes was reduced by half in ethanol-fed rats compared to controls. Conclusions There is a gradient in the concentration of lipid peroxides in enterocytes across the crypt–villus axis, being high at the villus tip and low at the crypt base in the rat intestine. Ethanol feeding enhanced lipid peroxidation in both villus and crypt cells.

Effect of Na+ ions on pH-dependent conformational changes in brush border sucrase-isomaltase in mice intestine.

Intestinal brush border sucrase-isomaltase (sucrose D-glucosidase E.C. 3.2.1.48, E.C. 3.2.1.10) exhibits pH-dependent stimulatory or inhibitory effects in response to Na+ ions. However, whether the enzyme undergoes conformational modulations as a function of pH and in the presence of alkali metal ions is not known. In this paper, we investigated the structural and functional relationship of purified murine sucrase in response to pH and Na+ ions using UV-CD fluorescence and spectroscopic studies. Kinetic studies revealed that at pH 5.0, the enzyme activation by Na+ ions was V-type, which changed to K-type at pH 7.2, whereas at alkaline pH (8.5), Na+ ions inhibited the enzyme activity and inhibition was uncompetitive in nature, affecting both the Km and Vmax components. Far UV-CD spectra of protein at pH 7.2 in the absence and presence of Na+ were almost overlapping, suggesting that secondary structure of protein was not affected upon addition of the salt. However, near UV-CD spectra indicated marked alterations in the tertiary structure of protein in presence of 50 mM Na+ ions. Increase in pH from 7.2 to 8.5 resulted in a marked rise in fluorescence intensity and red shift in lambda max due to tryptophan residues in the enzyme molecule. These findings suggested that alterations in enzyme activity as a function of pH and Na+ ions was associated with ionization of key amino acid residues together with structural modifications in the enzyme conformation around neutral or alkaline pH.

Effect of tannic acid on brush border disaccharidases in mammalian intestine.

Tannic acid is a glucoside (penta-m-digallolyl-glucose), which exhibits a wide variety of physiological functions. Around neutral pH, 0.4 mM tannic acid produced 84% inhibition of rat brush border sucrase activity, but 35-40% enzyme inhibition was observed in the rabbit intestine at 0.08 mM concentration. In the mice, 74-77% enzyme inhibition was observed at 0.05 mM concentration of tannic acid. The observed inhibition was reversible in rat intestine. Tannic acid (0.2 mM) also inhibited lactase (18% in adult and 71% in suckling animals), maltase (76%) and trehalase (88%) activities in rat intestine. pH versus activity curves showed that 0.2 mM tannic acid inhibited enzyme activity in rat by 91% at pH 5.5 which was reduced to 14% at pH 8.5 compared to the respective controls. In the rabbit 18-60% enzyme inhibition was noticed below pH 7.0, however at pH 8.5, it was of the order of 38%. Kinetic analysis revealed that tannic acid is a competitive inhibitor of rat brush border sucrase at pH 6.8. Effect of tannic acid together with various -SH group reacting reagents revealed that the enzyme inhibition is additive in nature, suggesting the distinct nature of binding sites on the enzyme for these compounds. The results suggest that tannic acid is a potent inhibitor of intestinal brush border disaccharidases, and could modulate the intestinal functions.

Characteristics and physiological role of surfactant-like particles secreted by entrocytes.

Intestinal epithelium secretes novel unilamellar membranes having characteristics similar to lung surfactants and thus has been named Surfactant-like particles (SLP). The chemical analysis of the membranes revealed cholesterol/phospholipid molar ratio of 0.68-0.78, which is much distinct from that of the underlying microvillus membranes (1.34-1.49). The membrane contains 4-6 proteins with a molar weight of 30-120 kDa and is enriched with alkaline phosphatase, contains low amounts of disaccharidases but no Na+, K(+)-ATPase activity. The secretion of SLP is stimulated by fat feeding. Chronic ethanol ingestion also induces the formation of SLP in rat intestine. A number of physiological functions have been attributed to SLP, which include: (i) as a protective lubricant in intestinal lumen, (ii) a role in triacylglycerol transport, (iii) as a vehicle for the transport of luminal proteins into blood, (iv) as a stratum for the adhesion of microorganisms in intestinal lumen, and (v) a role in trans-signalling mechanism across the basolateral surface of enterocytes.