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Nonsteroidal anti-inflammatory drugs (NSAIDs) are used extensively in clinical medicine. One disadvantage of their use, however, is the occurrence of adverse effects in the kidneys. The side effects produced in this organ have been classically attributed to the inhibitory effect of these drugs on the activity of cyclooxygenase, a key enzyme in prostaglandin synthesis. Our earlier work with indomethacin, a commonly used NSAID, has shown that oxidative stress and mitochondrial dysfunction occur in the kidney in response to the drug. In view of this, this study looked into the effect of indomethacin on brush border membranes (BBM) from the kidney, as these biomembranes are prime targets of oxygen free radicals. Rats, fasted overnight, were dosed with indomethacin (20 mg/kg) by gavage and sacrificed 24 h later. BBM were isolated from the kidneys by polyethylene glycol precipitation. It was found that there was an increase in levels of products of peroxidation and a fall in the level of alpha-tocopherol in the BBM from indomethacin-dosed rats. These BBM also exhibited impaired glucose transport. The lipid composition of the membranes was also found to be altered. Alterations in lipids were associated with up-regulation of phospholipase A2. Pretreatment with L-arginine, a nitric oxide donor, protected against these effects of indomethacin. Thus, this study suggests that indomethacin induces impairment in structure and function of BBM in the kidney, with these effects possibly mediated by free radicals and activation of phospholipases. We postulate that such alterations may be important in the pathogenesis of NSAID-induced nephropathy.

Background: Our earlier work has shown that surgical manipulation of the intestine results in oxidative stress and mucosal damage along with alterations in the brush border membrane (BBM). Glutamine feeding is known to offer protection against damage to mucosa under various conditions and this study looked at the effect of oral supplementation of glutamine or glutamic acid in the intestinal BBM alterations after surgical manipulation., Materials and Methods: Control and rats pretreated for 7 days with 2% glutamine or glutamic acid or isonitrogenous amino acids, glycine, or alanine were subjected to surgical manipulation of the intestine. BBMs were isolated from the intestine and functional and structural alterations to these membranes were assessed and compared., Results: Surgical manipulation resulted in oxidative stress in the enterocyte BBM and these changes included a decrease in alkaline phosphatase activity and alpha-tocopherol content along with an increase in lipid peroxidation parameters. A decrease in glucose transport by the isolated BBM vesicles suggested functional impairment. Surgical manipulation also resulted in phospholipid degradation possibly mediated by PLA(2) and membrane protease activation. Glutamine or glutamic acid supplementation prevented these changes but not by glycine or alanine., Conclusion: This study suggests that oral glutamine or glutamic acid supplementation prior to surgery can offer protection to the intestine and this might prevent postsurgical complications.

Nonsteroidal anti-inflammatory drugs (NSAIDs) cause small intestinal damage but the pathogenesis of this toxicity is not well established. Our earlier work has shown that villus enterocytes are most susceptible to the effects of indomethacin, a commonly used NSAID. This study looked at the acute effect of indomethacin on brush border membranes (BBM), which are present mainly in the villus cells and are in immediate contact with the contents of the small intestinal lumen. Evidence of oxidative stress was found in the mucosa of the small intestine of rats dosed with indomethacin, as indicated by increased activity of xanthine oxidase with corresponding decrease in the levels of several free radical scavenging enzymes. These changes were associated with an increase in peroxidation parameters in the BBM and a fall in the level of alpha-tocopherol. These BBM also exhibited impairment in glucose transport. Significant changes were seen in the lipid composition of these membranes, with upregulation of an 85kDa isoform of phospholipase A(2). Pretreatment of animals with allopurinol, arginine or zinc protected against these effects of indomethacin. Thus this study suggests that in an acute model of indomethacin dosing there is impairment in structure and function of the BBM in enterocytes, with the effects possibly mediated by free radicals and phospholipases.

Background: The intestine is highly susceptible to free radical-induced damage and our earlier work has shown that surgical manipulation of the intestine results in generation of oxygen free radicals and mucosal damage along with alterations in the brush border membrane (BBM). Heat preconditioning is known to offer protection against various stresses including oxidative stress and this study looked at the effect of heat preconditioning on the intestinal BBM alterations following surgical manipulation., Methods: Control and rats heat preconditioned were subjected to surgical manipulation by opening the abdominal wall and handling the intestine. BBM were isolated from the intestine and structural and functional alterations to these membranes were assessed., Results: Surgical manipulation resulted in oxidative stress suggested by a decrease in alkaline phosphatase activity and alpha-tocopherol content, accompanied by an increase in lipid peroxidation. A decrease in glucose transport by the isolated BBM vesicles suggested functional impairment. Surgical manipulation resulted in phospholipid degradation with generation of arachidonic acid along with appearance of cPLA(2) in the membrane. These changes were prevented by heat preconditioning of the animal prior to surgical manipulation., Conclusion: These results suggest that heat preconditioning offers protection from damage to the intestinal BBM following surgical manipulation and mild whole body hyperthermia might prevent postsurgical complications.

A novel method of brush border membrane vesicle (BBMV) preparation from the small intestinal mucosa using polyethylene glycol (PEG) precipitation has been presented. This preparation is compared with calcium-precipitated BBMVs in marker enzyme enrichment, contamination by other subcellular membranes, transport of glucose, and lipid composition. PEG-precipitated BBMVs are comparable with calcium-precipitated membranes in all parameters except lipid composition and thiol content. PEG-precipitated membranes have more phosphatidylcholine and phosphatidylethanolamine and less lysophosphatidylcholine and lysophosphatidylethanolamine as compared to calcium-precipitated membranes. Diacylglycerol and triacylglycerol content are also high in PEG-precipitated membranes. Alteration in lipid composition indicate the possible activation of lipase and phospholipase by calcium during BBMV preparation, which is not seen in PEG precipitation. Thiol content is almost double in PEG-precipitated membranes as compared to calcium-precipitated membranes. These results indicate that PEG can be used for the preparation of BBMVs in native form from the intestine without any alteration in their structural components, and these membranes show comparable transport activity., (Copyright 2001 Academic Press.)

Background: Surgical stress can lead to translocation of bacteria from the intestine into the systemic circulation. The intestinal brush border membrane (BBM) plays an important role in defense against such invasion by luminal bacteria and endotoxin. Our earlier work has shown the development of oxidative stress in the intestine after surgical stress and since the BBM is sensitive to free radical attack, this study examined the effect of surgical stress on the structure and function of intestinal BBM., Methods: Intestinal BBM were isolated from control and after surgical stress and compared for structural and functional alterations. Surgical stress was also carried out following pretreatment with the xanthine oxidase inhibitor allopurinol or the nitric oxide donor l-arginine, to study the protection offered by these compounds., Results: Surgical stress affected intestinal BBM structure as well as function. A decrease in alkaline phosphatase activity and alpha-tocopherol content, accompanied by an increase in lipid peroxidation, was seen. Surgical stress induced phospholipid degradation with generation of arachidonic acid. Functional impairment with a decrease in glucose transport ability was also seen. These changes are prevented by inhibition of xanthine oxidase by allopurinol pretreatment but not by NO., Conclusion: Surgical stress in the small intestine causes structural and functional alterations in the BBM through oxidative stress. This damage could affect gut barrier integrity and generation of arachidonic acid might mediate distal organ dysfunction., (Copyright 2000 Academic Press.)

Oxygen-derived free radicals are known to be generated during ischemia/reperfusion injury and biomembranes are the prime target of these active species. In order to study the effect of in vivo generated free radicals on intestinal mucosal membrane, brush border membranes (BBM) were isolated from rat small intestine after subjecting to ischemia (I) and ischemia/reperfusion (I/R) injury and their lipid composition and marker enzyme activity were compared with BBM prepared from control animals. No significant alteration in the lipid composition of BBM was observed after I or I/R as compared to control. Membrane fluidity measurements showed that I/R increased the fluidity of BBM. Activity of alkaline phosphatase, one of the marker enzymes for BBM was reduced by I or I/R whereas activity of another BBM enzyme, sucrase was not altered. The decrease in alkaline phosphatase activity was more after reperfusion. In vitro fluidization of BBM using benzyl alcohol indicated that the inactivation of alkaline phosphatase was not due to change in fluidity. These results suggest that free radicals generated during I/R inactivate BBM alkaline phosphatase partially without altering the membrane lipid composition.

Brush border membranes isolated from monkey intestinal mucosa was found to contain considerable amount of nonesterified fatty acids. Incubation of brush border membranes with fatty acid free albumin selectively removed the free fatty acids more than 80% without altering the level of phospholipids or cholesterol. The sodium dependent D-glucose transport was stimulated by the albumin treatment. Kinetic study showed that albumin treatment did not alter the apparent affinity (Km) of the transporter for glucose whereas the maximal velocity (Vmax) was increased significantly. The sodium dependent D-glucose transport was inhibited by the exogenously added unsaturated fatty acids. Saturated fatty acids and methyl esters of unsaturated fatty acids showed no inhibition. Based on these results, it may be concluded that free fatty acids inhibit the sodium dependent intestinal D-glucose transport either by directly interacting with the transport protein or by abolishing the sodium gradient.