Developmental changes in the activities of sialyl- and fucosyltransferases in rat small intestine

To study an enzymatic basis for the postnatal changes in intestinal glycosylation, the activities of sialyl- and fucosyltransferases were determined in the particulate fraction of mucosal cells prepared from rat small intestine of various ages. The results show that sialyltransferase activity was present in increased levels compared to adults during the preweaning period (1-2 weeks) and subsequently declined 5-fold to adult levels after weaning, while fucosyltransferase activity was decreased compared to adults in the first 3 weeks of life, rapidly increased at 4 weeks, and reached adult levels (10-fold) by 5 weeks. The changes in both sialyl- and fucosyltransferase activities were reflected by the membranous content of glycosidic-bound sialic acid and fucose, respectively. Cortisone injection precociously induced a decreased sialyltransferase activity and an increased fucosyltransferase activity in 2-week-old suckling rats. This study indicates that the activities of sialyl- and fucosyltransferases were reciprocally related and modulated by cortisone action in the developing intestine. These enzyme changes may be responsible for the previously noted shift from sialylation to fucosylation of the intestinal mucosa during maturation.     

Studies on the effect of experimental inflammation on sialyltransferase in the mouse and guinea pig

The effect of inflammation on sialyltransferase was studied in the mouse and guinea pig. There was a three-fold increase in mouse liver sialyltransferase activity reaching a maximum at 72 hr after inflammation; serum levels were increased by five-fold at 72 hr after inflammation. The response of guinea pig sialyltransferase was slower and of lower magnitude compared with the response of the mouse enzyme; liver and serum sialyltransferase increased by about 50% reaching a maximum at 96 hr after inflammation. The specificity of the enzyme that responded to inflammation in the mouse and guinea pig was found to be Gal beta 1----4GlcNAc alpha 2----6 sialyltransferase, the same enzyme activity that was shown to be an acute phase reactant in earlier studies in the rat (Kaplan et al., 1983).     

Studies on the effect of inflammation on rat liver and serum sialyltransferase. Evidence that inflammation causes release of Gal beta 1 leads to 4GlcNAc alpha 2 leads to 6 sialyltransferase from liver

Turpentine induced inflammation has been shown to elevate liver sialyl- and galactosyltransferase activities (Turchen, B., Jamieson, J.C., Huebner, E., and van Caeseele, L. (1977) Can. J. Zool. 55, 1567-1571; Lombart, C., Sturgess, J., and Schachter, H. (1980) Biochem. Biophys. Acta 629, 1-12). We now report that serum sialyl-, but not galactosyltransferase activities are significantly elevated in turpentine inflammation. A liver slice system is used to demonstrate that liver releases large amounts of sialyltransferase activity into medium after inflammation, whereas only a low level of galactosyltransferase activity is released. Studies with rat and human asialo-alpha 1-acid glycoprotein as acceptors, coupled with the use of lactose to confirm the nature of the linkages formed, showed that Gal beta 1 leads to 4GlcNAc alpha 2 leads to 6 sialyltransferase is released from liver in turpentine inflammation and is mainly responsible for the elevated sialyltransferase activity found in serum. The alpha 2 leads to 6 sialyltransferase is exhibiting the properties of a typical acute phase reactant.     

Activity and secretion of sialyltransferase in primary monolayer cultures of rat hepatocytes cultured with and without dexamethasone

Monolayers of hepatocytes attached on collagen-coated dishes were cultured for 20-24 h and were found suitable to study the activity and secretion of CMP-N-acetylneuraminate:asialo-alpha 1-acid glycoprotein sialyltransferase. A progressive increase of sialyltransferase activity in the culture medium was observed during incubation of the hepatocytes. After 24 h 34-48% of the total sialyltransferase activity of the hepatocyte incubation system was present in the medium. The enzyme activity present in the medium was soluble in nature and could not be stimulated by Triton X-100. The secretion of the enzyme was stimulated about twofold by dexamethasone. The activity of sialyltransferase in the hepatocytes was also increased by dexamethasone. The Km of either hepatocyte or medium sialyltransferase for CMP-sialic acid was only slightly changed by dexamethasone, whereas the Vmax was increased about twofold. The secretion of sialyltransferase could be inhibited partially by the anti-microtubular agent colchicine. The dexamethasone-induced increase of the sialyltransferase activity in cells and media could be eliminated by inclusion of alpha-amanitin in the culture media at 0 h. The inhibiting effect of alpha-amanitin was only partially expressed when the drug was added 4 h after the addition of dexamethasone to the media. The results suggest that isolated rat hepatocytes actively secrete sialyltransferase and that the increase in the sialyltransferase activity in cells and media owing to the synthetic glucocorticosteroid dexamethasone results from increased synthesis of the enzyme molecule. It is supposed that in the intact rat the increased levels of the enzyme activity in serum observed in inflammation may originate from an induction of the synthesis of sialyltransferase in the hepatocytes of rat liver by the increased levels of circulating corticosteroids.     

Effect of hydrocortisone on sialyltransferase activity in the rat small intestine during maturation. Changes along the villus-crypt axis and in fetal organ culture

Sialyltransferase activity was assayed in rat intestinal cells isolated as fractions reflecting the villus-crypt axis of differentiation. In 13-day-old rats both endo- and exogenous sialyltransferase activity reached their maximum in undifferentiated crypt cells and their peaks overlapped. In contrast, sialyltransferase of the adult intestine was 4-fold lower than that of sucklings in the crypts, with slight tendency to be transferred to the villus cells. Hydrocortisone applied to 10-day-old rats caused three days later a precocious drop of sialyltransferase activity in the crypt cells. Unlike in vivo, glucocorticoid responsiveness was accompanied by increased sialyltransferase activity in fetal small intestine cultivated for 17 days.     

A possible mechanism for the changes in hepatic and intestinal alkaline phosphatase activities in bile-duct-ligated rats or guinea pigs

The effects of bile-duct ligation on hepatic and intestinal (jejunum) alkaline phosphatase activities were studied using rats and guinea pigs. In ligated rats, the enzyme activity was increased 4.1-fold in the liver after 24 h and 2.8-fold in the intestine after 12 h. In guinea pigs, the hepatic and intestinal enzyme activities were increased 2.3-fold and 1.5-fold after 100 and 24 h, respectively. The intestinal activity was induced sooner after ligation than hepatic activity. The induction of alkaline phosphatase was inhibited by prior treatment of animals with amanitin, an inhibitor of RNA polymerase activity. This result indicates that the induction is associated with de novo enzyme synthesis. The content of cyclic AMP in liver and intestine increased immediately after ligation. The increase in alkaline phosphatase activities was also inhibited by pretreatment with chlorpromazine, an inhibitor of adenylate cyclase activity. Hence, cellular cyclic AMP may be implicated in playing a role in the induction of alkaline phosphatase by bile-duct ligation.     

Quantification of gut injury with diamine oxidase activity: development of a fission neutron RBE and measurements with combined injury in mouse models

Plasma and small intestine diamine oxidase (DAO) activities were measured on Days 2, 4, and 6 following irradiation of mice with a range of doses of fission neutrons and 60Co. With increasing doses of radiation, plasma DAO activity increased on Day 2 and intestinal DAO activity decreased on Day 4; moreover, the approximate relative biological effectiveness values for these changes in activity were 5.81 for plasma DAO activity on Day 2 and 3.88 for intestinal DAO activity on Day 4. On Day 6 relatively high levels of radiation caused DAO activity in the small intestine to remain depressed whereas low levels resulted in recovery with activities at or near controls. In animals with combined injury (radiation plus 30% surface burn or wound), changes in DAO activity in the intestine were similar to those with radiation alone; plasma DAO activity, in contrast to radiation alone, did not show an increase at the 2-day mark. These dose-dependent relationships should provide a basis for using DAO as a potential indicator of biological damage from radiation exposure within the lethal range.     

Estrogen prevents intestinal inflammation after trauma-hemorrhage via downregulation of angiotensin II and angiotensin II subtype I receptor

Although angiotensin II (Ang II) plays a key role in development of organ ischemia-reperfusion injury, it remains unclear whether it is involved in development of intestinal injury following trauma-hemorrhage (T-H). Studies have shown that 17beta-estradiol (E2) administration following T-H improves small intestinal blood flow; however, it is unclear whether Ang II plays a role in this E2-mediated salutary effect. Male Sprague-Dawley rats underwent laparotomy and hemorrhagic shock (removal of 60% total blood volume, fluid resuscitation after 90 min). At onset of resuscitation, rats were treated with vehicle, E2, or E2 and estrogen receptor antagonist ICI 182,780 (ICI). A separate group of rats was treated with Ang II subtype I receptor (AT1R) antagonist losartan. At 24 h after T-H, plasma Ang II, IL-6, TNF-alpha, intercellular adhesion molecule (ICAM)-1, cytokine-induced neutrophil chemoattractant (CINC)-1 and CINC-3 levels, myeloperoxidase (MPO) activity, and AT1R expression were determined. T-H significantly increased plasma and intestinal Ang II, IL-6, TNF-alpha levels, intestinal ICAM-1, CINC-1, CINC-3 levels, MPO activity, and AT1R protein compared with shams. E2 treatment following T-H attenuated increased intestinal MPO activity, Ang II level, and AT1R protein expression. ICI administration abolished the salutary effects of E2. In contrast, losartan administration attenuated increased MPO activity without affecting Ang II and AT1R levels. Thus Ang II plays a role in producing small intestine inflammation following T-H, and the salutary effects of E2 on intestinal inflammation are mediated in part by Ang II and AT1R downregulation.     

Inhibition of in vivo DNA synthesis in regenerating rat liver following thermal injury

Hepatic repair and regeneration which is extremely important after thermal injuries can be inhibited by the acute inflammatory reaction. Since thermal injury initiates this acute inflammatory reaction, DNA synthesis was studied in the regenerating liver following this injury. In vivo incorporation of [3H]-thymidine into hepatic DNA, autoradiographic determination of a labeling index, and thymidine kinase activity were determined. Incorporation of [3H]-thymidine into hepatic DNA and labeling indices were markedly diminished at 24 hours if partial hepatectomy and thermal injury were carried out concurrently. After partial hepatectomy, the expected elevations in thymidine kinase activity were inhibited by the thermal injury (p less than 0.01) and elevation of serum fibrinogen, a marker of the acute phase reaction that normally follows thermal injury, was blunted by the partial hepatectomy (p less than 0.05). The combination of thermal injury and partial hepatectomy resulted in a greatly diminished DNA replicative response as compared to partial hepatectomy alone and suggests that multiplicative injury is more likely to result in multi-system failure.     

Post-Golgi apparatus localization and regional expression of rat intestinal sialyltransferase detected by immunoelectron microscopy with polypeptide epitope-purified antibody

During studies on the Golgi apparatus immunolocalization of beta-galactoside alpha 2,6-sialyltransferase in intestinal cells, immunostaining of a number of post-Golgi apparatus structures including mucus droplets and plasma membrane were observed. In order to determine if this labeling was in fact due to sialyltransferase and not carbohydrate-specific antibodies in the polyclonal antiserum preparation, fusion protein to sialyltransferase was used to epitope purify polypeptide-specific antibodies. The affinity purification was performed on a column containing a beta-galactosidase-sialyltransferase fusion protein expressed in Escherichia coli. Using such antibodies we present evidence that in intestinal cells sialyltransferase is not only present in the Golgi apparatus cisternal stack but also its transtubular network and various post-Golgi apparatus structures. In absorptive enterocytes, post-Golgi apparatus vesicles, the brush border and basolateral plasma membrane, multivesicular bodies, and lysosome-like structures were labeled. In goblet cells the limiting membrane and lumen of forming and mature mucus droplets as well as the plasma membrane exhibited label for sialyltransferase. The results provide evidence for "ecto-sialyltransferase" in the plasma membranes of these cells, and suggest that most of the sialyltransferase is released from the Golgi membranes and becomes secreted with the goblet cell mucus. In addition, the polypeptide epitope-purified antibody was also used to examine regional expression of sialyltransferase in the rat intestinal epithelium. Immunolabel was restricted to the large intestine and not found in duodenum, jejunum, and ileum. Direct measurement of the enzyme activity was found to correlate with the immunoelectron microscopic data. This observation suggests that there is regional specific expression of the beta-galactoside alpha 2,6-sialyltransferase.     

Regulation of beta-galactoside alpha 2,6-sialyltransferase gene expression by dexamethasone

The hepatic acute phase response is accompanied by increased levels of Gal beta 1-4GlcNAc alpha 2,6-sialyltransferase activity in liver and in circulation. Previous studies suggested that cytokines and glucocorticoids mediate the induction of this sialyltransferase activity. In this study the regulation of sialyltransferase expression by dexamethasone in H35 rat hepatoma cells is assessed by Northern hybridization and enzyme activity assays. Exposure of H35 cells to 1 microM dexamethasone for 24 h causes a 3-4-fold enrichment of sialyltransferase mRNA and a corresponding increase in enzymatic activity. The induction of sialyltransferase mRNA begins within 3 h of dexamethasone treatment and reaches a plateau within 24 h. Sialyltransferase mRNA induction is dose dependent; the minimum concentration of dexamethasone necessary for induction is 10(-8) M, and induction was maximal at 10(-6) M. Induction is sensitive to actinomycin D, suggesting that regulation may be exerted by altering the rate of mRNA synthesis. Puromycin and cycloheximide are ineffective in blocking induction, suggesting that de novo protein synthesis is not required for induction. Finally, dexamethasone alone is sufficient for maximum induction of sialyltransferase mRNA. In contrast, maximal induction of alpha 1-acid glycoprotein, a well studied hepatic acute phase reactant, requires both dexamethasone and cytokines, implying that different pathways exist for the induction of participants in the acute phase response.     

Antioxidant enzyme levels in intestinal and renal tissues after a 60-minute exercise in untrained mice.

The present study was designed to determine the effects of exercise on the antioxidant enzymatic system and lipid peroxidation in small intestine and kidney, during the post-exercise period in untrained mice. Two days after the last adaptation running exercise, animals were ran on the treadmill for 60 min at 18 m/min. 5 degrees slope. After the acute exercise the animals were killed by cervical dislocation, immediately (0 h), 3 hours (3 h) and 24 hours (24 h) after the exercise. Control animals were killed without running exercise. Their proximal small intestinal and renal tissues were quickly removed. Changes in the concentration of thiobarbituric acid reactive substance (TBARS), as an index of lipid peroxidation, in intestine and kidney were studied in mice after the running exercise and in unexercised control group. The activities of superoxide dismutase (SOD) and glutathione peroxidase (GPx) were determined in these tissues. Tissue SOD, GPx activities and TBARS level were not increase by the exercise in kidney. Intestinal SOD activity decreased after exercise (0 h and 3 h respectively, p<0.05, p<0.01) and retumed to control levels. Intestinal GPx activity increased after exercise (0 h, p<0.05) and returned to control levels. There was no significant difference among groups in intestinal tissue TBARS levels. These findings could suggest that submaximal exercise may not cause oxidative stress in proximal small intestinal tissue and kidney.     

Activities of conjugating and antioxidant enzymes following endotoxin exposure

Endotoxin exposure elicits various responses in mammals including the acute phase response that has been shown to cause changes in the activity of several forms of cytochrome P450s and other enzymes. Therefore, the hepatic conjugating enzyme, glutathione S‐transferase (GST), and UDP‐glucuronosyltransferase (UDPGT), the antioxidant enzymes, glutathione peroxidase (GSHPx), catalase, and superoxide dismutase (SOD), as well as lipid peroxidation were investigated following the administration of endotoxin to male Sprague–Dawley rats (8 mg/kg body weight). Rats were euthanized at various times following endotoxin administration and the livers removed and processed to assess various enzyme activities. Glutathione S‐transferase, UDPGT, and GSHPx activity showed statistically significant decreases after 24 hours and remained lower than controls for the duration of the study. Decreases in total SOD and catalase activities were seen at 24, 48, and 72 hours following endotoxin administration; however, only catalase activity showed statistically significant differences between control and treated samples at those time points, and total SOD activity showed a statistically significant decrease at 24 hours. No statistically significant changes were seen in the level of lipid peroxidation in the liver microsomes from endotoxin‐treated animals. Changes in the conjugative enzymes and the free‐radical scavenging enzymes following endotoxin exposure may alter the host's metabolism and response to free radicals. © 1998 John Wiley & Sons, Inc. J Biochem Toxicol 13: 63–69, 1999     

Ornithine decarboxylase induction and polyamine levels in the kidney of estradiol-treated castrated male rats

Ornithine decarboxylase (ODC), S-adenosylmethionine decarboxylase (SAMDC), and thymidine kinase (TK) activities and polyamine concentrations on the kidneys of male castrated rats were studied following sc injection of estradiol. Estradiol caused an 11-fold increase in ODC activity 24 hours after administration. SAMDC activity doubled but TK activity decreased by two-thirds 2 days after estradiol treatment. The concentrations of polyamines, especially putrescine, showed sharp elevations 2 days following estradiol treatment, 1 day after the peak of ODC activity. The increase in ODC activity was suppressed by cycloheximide and by actinomycin D. Estradiol and diethylstilbestrol (DES), but not progesterone increased ODC activity. Estradiol suppressed ODC activities of liver, thymus, adrenal glands, testes and prostate. A specific estradiol-binding protein was demonstrated in the rat kidney. The dissociation constant (Kd) was 1.64 × 10^?10 M and numbers of binding sites were 31 fmoles/mg protein. Correlation between the binding of estradiol to the cytosol protein and elevation of ODC by estradiol was observed.     

The effect of selenium and/or vitamin E treatments on radiation-induced intestinal injury in rats

Cytotoxic effects of ionizing radiation on gastrointestinal epithelium may be related to oxidative stress. In this study, we wanted to investigate the effects of selenium, vitamin E and selenium plus vitamin E pretreatments prior to whole abdominal irradiation on intestinal injury. Irradiation caused increased lipid peroxide and decreased GSH levels in the intestine. Intestinal superoxide dismutase and glutathione peroxidase activities were increased, but glutathione transferase activity decreased following irradiation. Selenium and/or vitamin E pretreatments ameliorated these disturbances in prooxidant-antioxidant balance. This amelioriation has been verified with histopathological findings. These results indicate that antioxidant pretreatments prior to irradiation may have some beneficial effects against irradiation-induced intestinal injury.     

Protease activity increases in plasma, peritoneal fluid, and vital organs after hemorrhagic shock in rats

Hemorrhagic shock (HS) is associated with high mortality. A severe decrease in blood pressure causes the intestine, a major site of digestive enzymes, to become permeable - possibly releasing those enzymes into the circulation and peritoneal space, where they may in turn activate other enzymes, e.g. matrix metalloproteinases (MMPs). If uncontrolled, these enzymes may result in pathophysiologic cleavage of receptors or plasma proteins. Our first objective was to determine, in compartments outside of the intestine (plasma, peritoneal fluid, brain, heart, liver, and lung) protease activities and select protease concentrations after hemorrhagic shock (2 hours ischemia, 2 hours reperfusion). Our second objective was to determine whether inhibition of proteases in the intestinal lumen with a serine protease inhibitor (ANGD), a process that improves survival after shock in rats, reduces the protease activities distant from the intestine. To determine the protease activity, plasma and peritoneal fluid were incubated with small peptide substrates for trypsin-, chymotrypsin-, and elastase-like activities or with casein, a substrate cleaved by multiple proteases. Gelatinase activities were determined by gelatin gel zymography and a specific MMP-9 substrate. Immunoblotting was used to confirm elevated pancreatic trypsin in plasma, peritoneal fluid, and lung and MMP-9 concentrations in all samples after hemorrhagic shock. Caseinolytic, trypsin-, chymotrypsin-, elastase-like, and MMP-9 activities were all significantly (p<0.05) upregulated after hemorrhagic shock regardless of enteral pretreatment with ANGD. Pancreatic trypsin was detected by immunoblot in the plasma, peritoneal space, and lungs after hemorrhagic shock. MMP-9 concentrations and activities were significantly upregulated after hemorrhagic shock in plasma, peritoneal fluid, heart, liver, and lung. These results indicate that protease activities, including that of trypsin, increase in sites distant from the intestine after hemorrhagic shock. Proteases, including pancreatic proteases, may be shock mediators and potential targets for therapy in shock.     

Mechanism for enterohepatic injury caused by circulatory disturbance of hepatic vessels in the rat

We reported previously that a transient occlusion followed by reperfusion of the portal vein and the hepatic artery of the rat significantly decreased the transhepatic transport of a cholephilic compound, and that this decrease was prevented by pretreating animals with poly(styrene co-maleic acid butyl ester)-conjugated superoxide dismutase (SM-SOD). To elucidate the mechanism for oxidative injury of the liver and the site for the generation of superoxide radicals, the effect of a portosystemic bypass on the liver function was examined in the rat whose hepatic vessels were temporarily occluded. A portosystemic bypass inhibited the reperfusion-induced decrease in hepatic transport of bromosulfophthalein as effectively as did SM-SOD. Kinetic analysis using 125I-labeled albumin revealed that the permeability of the small intestine markedly increased after a transient occlusion. The increase in intestinal permeability was also inhibited either by SM-SOD or by the portosystemic bypass. Xanthine oxidase activity in portal plasma markedly increased during occlusion and reperfusion, while it remained within normal ranges in the bypassed group. Thus, superoxide radical, and/or its metabolite(s), might play a critical role in increasing the intestinal permeability and in the pathogenesis of reperfusion-induced liver injury.     

The acute phase response to inflammation: the role of monokines in changes in liver glycoproteins and enzymes of glycoprotein metabolism

The role of monocyte derived factors in the acute phase response to inflammation is discussed. The kinetics of response of alpha 1-acid glycoprotein, sialyltransferase and albumin to a rat monokine preparation is described. There was an increase in synthesis of alpha 1-acid glycoprotein and sialyltransferase and a decrease in albumin synthesis following administration. However, the kinetics of response of sialyltransferase to the monokine was much slower than was found for the other two proteins. The possibility that sialyltransferase responds to a different monokine compared to the other acute phase proteins is discussed.     

IL-10 deficiency augments acute lung but not liver injury in hemorrhagic shock

In hemorrhagic shock and trauma, patients are prone to develop systemic inflammation with remote organ dysfunction, which is thought to be caused by pro-inflammatory mediators. This study investigates the role of the immuno-modulatory cytokine IL-10 in the development of organ dysfunction following hemorrhagic shock. Male C57/BL6 and IL-10 KO mice were subjected to volume controlled hemorrhagic shock for 3 h followed by resuscitation. Animals were either sacrificed 3 or 24 h after resuscitation. To assess systemic inflammation, serum IL-6, IL-10, KC, and MCP-1 concentrations were measured with the Luminex? multiplexing platform; acute lung injury (ALI) was assessed by pulmonary myeloperoxidase (MPO) activity and lung histology and acute liver injury was assessed by hepatic MPO activity, hepatic IL-6 levels, and serum ALT levels. There was a trend towards increased IL-6 and KC serum levels 3 h after resuscitation in IL-10 KO as compared to C57/BL6 mice; however this did not reach statistical significance. Serum MCP-1 levels were significantly increased in IL-10 KO mice 3 and 24 h following resuscitation as compared to C57/BL6 mice. In IL-10 KO mice, pulmonary MPO activity was significantly increased 3 h following resuscitation and after 24 h histological signs of acute lung injury were more apparent than in C57/BL6 mice. In contrast, no significant differences in any liver parameters were detected between IL-10 KO and C57/BL6 mice. Our data indicate that an endogenous IL-10 deficiency augments acute lung but not liver injury following hemorrhagic shock.