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.

Hypolactasia as a molecular basis of lactose intolerance.

Lactase-phlorizin hydrolase (LPH), a membrane-bound glycoprotein present in the luminal surface of enterocytes in the intestine is responsible for lactose intolerance, a phenomenon prevalent in humans worldwide. In the rodent intestine, the post-natal development of the LPH follows a specific pattern, such that the enzyme levels are high in the peri-natal period, but declines considerably upon maturation. The observed maturational decline in the LPH activity is very similar to adult-type hypolactasia observed in humans. Majority of the studies have been carried out using animal models or cell lines and a number of hypotheses have been put forward to explain the maturational decline of lactase activity such as: (a) decreased amount of lactase protein, (b) defect in post-translational modification of precursor lactase to the mature enzyme, and (c) synthesis of an inactive, high molecular weight lactase with altered glycosylation, however, the precise underlying mechanism of adult-type hypolactasia remains undefined. The present review describes the recent developments in understanding the regulation of lactase expression and the possible mechanism of adult-type hypolactasia, as a cause of lactose intolerance.

Susceptibility of lactase to luminal proteases in developing rat intestine.

BACKGROUND: Postnatal development of rat intestine is associated with a decline in brush-border lactase activity. This phenomenon is similar to the adulthood hypolactasia in humans. However, the mechanism underlying this process is not understood. METHODS: The effect of luminal proteases from adult rat intestine on the intestinal lactase activity in animals aged 7, 14, 21 and 30 days was studied in in vitro experiments. Lactase levels were estimated using enzyme assays and Western blot analysis. RESULTS: Incubation of purified brush borders with increasing concentrations of luminal proteases reduced the lactase activity in intestine of 7-day-old rats, but not in that of adult animals. Western blot analysis revealed low signal of the 220-kDa lactase protein in 7-day-old animals, but not that of older weaned animals. CONCLUSIONS: Our findings suggest that luminal proteases may be responsible for the maturational decline in intestinal lactase activity.

In vitro translation of RNA to lactase during postnatal development of rat intestine.

mRNA levels encoding lactase were detected by Northern blot analysis using two different probes in developing rat intestine. Probe I and probe II corresponding to second half of prolactase gene showed a 6.8 kb mRNA transcript in 7, 14, 21 and 30 day old rat intestine. There was no change in quantity of lactase mRNA detected using probe II, but hybridization with probe I showed a progressive decrease in mRNA transcript encoding lactase with age. At day 7 and 14 of postnatal development, the lactase mRNA was quite high, but it reduced upon weaning. The in vitro translation products of RNA detected by Western blot analysis using brush border lactase antibodies showed several isoforms of lactase antigen with molecular weight ranging from 100-220 kDa. Analysed at days 7 and 30 of postnatal development, lactase isoforms of molecular weight 130 kDa and 220 kDa were similar to those found in purified brush border membranes. The translation of RNA to 220 kDa lactase protein was high in 7 and 14 day old pups, but it was markedly reduced in 30 day old animals. These findings support the contention that translation of mRNA to lactase is impaired in weaned animals, which may also be responsible for the maturational decline in lactase activity in adult rat intestine.

Postnatal development of alcohol dehydrogenase in liver and intestine of rats exposed to ethanol in utero.

BACKGROUND AND OBJECTIVES: Ethanol exposure during gestation induces marked aberrations in growth and development of offsprings collectively known as foetal alcohol syndrome (FAS);. However, its effects on the postnatal development of alcohol dehydrogenase (ADH) are not adequately investigated. Therefore, ADH activity in liver and intestine of rats exposed to ethanol during gestation was studied in relation to postnatal development. METHODS: Pregnant female rats beginning at day 1 of gestation were fed 1 ml of 30 per cent ethanol daily during the entire gestation period. ADH activity was determined in liver and intestine postnatally at day 4, 8, 14, 20 and 30. DNA and RNA contents and intestinal histology were also examined. RESULTS: During the first two weeks of postnatal life, there was no difference in ADH levels of rat liver and intestine in control and prenatally ethanol exposed pups but ADH levels were significantly reduced at 3-4 wk in ethanol fed group compared to control. A similar decrease in DNA and RNA contents of intestine and changes in tissue morphology were observed in ethanol exposed pups during postnatal development. INTERPRETATION AND CONCLUSION: The findings of our study suggested that prenatal ethanol exposure modified ADH activity in liver and intestine during postnatal development. This could affect ethanol metabolism under these conditions.

Effect of Salmonella typhimurium toxin on the expression of rabbit intestinal functions.

BACKGROUND & OBJECTIVES: Infection by Salmonella Typhimurium is one of the leading causes of intestinal dysfunction, however the underlying mechanism of this effect is largely unknown. Hence the effect of enterotoxin secreted by Salmonella Typhimurium-(S-LT) was studied on D-glucose absorption and brush border enzymes in rabbit ileum. mRNA levels encoding these proteins were also analysed. METHODS: Adult male New Zealand white rabbits were used. The polymyxine B extract of enterotoxin obtained from Salmonella Typhimurium was tested for the presence of enterotoxicity by rabbit ileal loop test. D-glucose uptake by ileal tissue was measured by the tissue accumulation method. Intestinal brush border membranes were isolated and the effect of S-LT on various brush border enzymes studied. RESULTS: S-LT significantly inhibited (P < 0.01) the absorption of Na+ dependent D-glucose uptake but had no effect on Na+ independent sugar uptake in rabbit ileum. The activities of brush border sucrase (72% P < 0.001) and lactase (47% P < 0.01) and alkaline phosphatase (43% P < 0.01) were also significantly reduced in infected animals as compared to the controls. Northern blot analysis revealed that mRNA levels encoding Na+ glucose co-transporter (SGLT1), brush border lactase and sucrase activities were unaffected in Salmonella infected rabbit ileal loops. INTERPRETATION & CONCLUSION: The findings suggest that the intestinal dysfunctions observed in Salmonella infection are unrelated to mRNA expression encoding Na+ glucose co-transporter and brush border enzyme proteins in rabbit ileum.