Age-related changes in the mRNA levels of CYP1A1, CYP2B1/2 and CYP3A1 isoforms in rat small intestine

It has been established beyond doubt that, as well as the liver, the small intestine is an important site of first-pass metabolism of numerous drugs, food components and toxic xenobiotics. However, there is not much information available about age-dependent changes of intestinal biotransformation pathways. In the present paper, we evaluated the relationships between intestinal cytochrome P450 complex activity and the age of animals. The study was carried out on male Sprague–Dawley rats (n = 5) from 5 age series: 0.5-, 2-, 4-, 20-, and 28 months old. Animals at every age series were divided into 4 groups: control and three groups of rats treated with the CYP450 specific inducers: phenobarbital, β-naphtoflavone and dexamethasone, respectively. RNA was isolated from intestinal mucosa, and then standard RT-PCR was used for the analysis of CYP1A1, CYP2B1/2 and CYP3A1 mRNA expression. Additionally, the activities of NADPH-cytochrome P450 and NADH-cytochrome b₅ reductases in the microsomal fraction were biochemically estimated. The constitutive intestinal CYP1A1 mRNA expression changes during maturation and aging. Inducibility of CYP1A1 gene was evident in intestinal mucosa at 2-, 4- and 20-month-old rats. A similar pattern of changes was observed for CYP2B1/2 isoforms. CYP3A1 mRNA expression was not detected in small intestine of 2-week-old rats. In matured rats, constitutive intestinal CYP3A1 expression was low, although after induction, significant increases in CYP3A1 mRNA amount were noted in aged individuals. Intestinal activity of both analyzed reductases was lowest in immature rats and highest in 28-month-old animals. In conclusion, the activity of cytochrome P450 complex in rat small intestine was not decreased by the aging processes, so the high rate of oxidative metabolic reactions in intestinal mucosa can be maintained till the advanced life stage.     

The ability of scavengers to distinguish OH. production in the iron-catalyzed Haber-Weiss reaction: comparison of four assays for OH

Kinetic analysis has been used to access how well scavenger inhibition can characterize the reactivity of oxidants produced in the iron-catalyzed reaction of H2O2 with xanthine oxidase-derived O2-.. Formate oxidation to CO2, deoxyribose oxidation, benzoate hydroxylation, and ethylene production from alpha-keto-gamma-methiolbutyric acid (KMB) were measured. With Fe(EDTA) as catalyst, inhibition by most scavengers was quantitatively as expected for OH. involvement. Exceptions were urate and thiourea, which inhibited excessively and appeared to scavenge intermediates of the detection reactions. With nonchelated iron, there was minimal formate oxidation, but benzoate, KMB, and deoxyribose gave, respectively, 17%, 25%, and approximately the same product yield as with Fe(EDTA). Deoxyribose oxidation was not inhibited by some scavengers and excessively inhibited by others. However, scavengers that did not inhibit deoxyribose oxidation did inhibit with KMB and benzoate, and differences in scavenger effects in the presence and absence of EDTA in these assays were relatively minor. The results with formate and deoxyribose, but not KMB and benzoate, can therefore exclude free OH. as a significant oxidant product of the nonchelated iron-catalyzed Haber-Weiss reaction. It is proposed that the different patterns of scavenger inhibition arise in the different assays because scavengers can react with intermediates in the detection reactions, all of which are multistep chains. Thus, inhibition may not signify OH. involvement, and similarities with inhibition expected for OH. my be fortuitous.     

State of the liver antioxidant system and content of matrix metalloproteinase-2 of the large intestine under the effect of maleimide derivative in experimental colorectal carcinogenesis in rats

The maleimide derivative--1-(4-Cl-benzyl)-3-Cl-4-(CF3-phenylamino)-1H-pyrrol-2.5-dione (MI-1) with cytostatic activity did not cause substantial changes of liver antioxidant system and level of matrix metalloproteinase-2 in intestinal mucosa after chronic treatment (for 20 weeks). MI-1 did not cause significant changes in the content of thiobarbituric-active products and plasma membrane protein carbonyl groups in the rat liver. However activities of superoxide dismutase, glutathione peroxidase, and content of reduced glutathione were decreased in both doses--0.027 and 2.7 mg/kg. The level of matrix metalloproteinase-2 in intestinal mucosa was decreased just in maximum dose--2.7 mg/kg. The contents of thiobarbituric-active products, protein carbonyl groups, reduced glutathione, matrix metalloproteinase-2, activities of glutathione peroxidase and glutathione-S-transferase in the liver cells have increased in 1.2-dimethylhydrazine-induced colon cancer in rats. The activities of enzymes of the first line of antioxidant defense--superoxide dismutase and catalase were decreased to 40%. The maleimide derivative prevents development of oxidation stress and partially reduce them to control level.     

Monoacylglycerol acyltransferase. Evidence that the activities from rat intestine and suckling liver are tissue-specific isoenzymes

The monoglycerol acyltransferase (EC 2.3.1.22) (recommended name acylglycerol palmitoltransferase) activities from rat intestinal mucosa and suckling liver microsomes were compared in order to determine why substrate specificities differed in the two tissues. Suckling liver monoacylglycerol acyltransferase activity was highly specific for sn-2-mono-C18:1 glycerol and acylated rac-1-mono-C18:1 glycerol and 1- and 2-mono-C18:1 glycerol ethers poorly. In contrast, the substrate specificity of intestinal monoacylglycerol acyltransferase activity was broad. 1-Acyl- and 1- and 2-alkylglycerols were acylated at rates that were 45-78% of the rate observed with the preferred substrate sn-2-mono-C18:1 glycerol. Partial heat inactivation did not alter these relative specific activities, making it unlikely that intestinal microsomes contained a second acyltransferase capable of acylating the alternate substrates. The hypothesis that intestine and liver contain non-identical monoacylglycerol acyltransferase activities was further tested. Intestinal mucosa monoacylglycerol acyltransferase was much more thermolabile than the liver activity. Incubation with 50 microM diethylpyrocarbonate inactivated liver monoacylglycerol acyltransferase activity 84% but had little effect on the intestinal activity. Hydroxylamine completely reversed diethylpyrocarbonate inactivation, suggesting that critical histidine residues were more accessible in liver monoacylglycerol acyltransferase. 2,4,6-Trinitrobenzene sulfonic acid inactivated hepatic monoacylglycerol acyltransferase more than the intestinal activity, suggesting that critical lysine residues were more accessible. The intestinal and liver activities were also differently affected by acetone, detergents, MgCl2, phospholipids, and bovine serum albumin. Taken as a whole, the data strongly suggest that rat intestinal mucosa and suckling liver contain tissue-specific monoacylglycerol acyltransferase isoenzymes.     

Further studies on the effect of aldosterone on Mg2+-HCO3(-)-ATPase and carbonic anhydrase from rat intestinal mucosa

The main purpose of this study is to elucidate the effect of adrenocorticoids on Mg2+-HCO3(-)-ATPase and carbonic anhydrase which are thought to be related to anion transport in mammalian intestinal mucosa and renal tubulus. Rat duodenal mucosa, large intestinal mucosa and kidney cortex were excised and homogenized with mannitol-Tris buffer (pH 7.1) and brush border fraction and cytosol were obtained by a differential fractionation procedure. Brush border Mg2+-HCO3(-)-ATPase and cytosol carbonic anhydrase activities in the duodenal mucosa decreased to 70% and 37% of normal values, respectively 5-11 days after adrenalectomy. Adrenalectomy also decreased significantly both enzyme activities in large intestinal mucosa; on the other hand, renal enzyme activities did not change. Four hours after a single injection of 20-80 micrograms/kg of aldosterone, ip, to adrenalectomized rats, Mg2+-HCO3(-)-ATPase and carbonic anhydrase activities in duodenal mucosa increased gradually to normal or near normal in dose-dependent fashion. Both enzyme activities in large intestinal mucosa were also increased by a larger dose of aldosterone. Again, renal enzyme activities were not affected by any dose of aldosterone. In contrast, corticosterone (1 mg and 4 mg/kg) and dexamethasone (50 micrograms 200 micrograms/kg) had no replacement effect on enzyme activities in all organs. These results showed that the mineralocorticoid, but not glucocorticoids, is a regulator of the enzyme activity of Mg2+-HCO3(-)-ATPase and carbonic anhydrase from intestinal mucosa. The true mechanisms by which both enzymes are activated by aldosterone are not clear at present.     

Expression of nuclear genes encoding the urea cycle enzymes, carbamoyl-phosphate synthetase I and ornithine carbamoyl transferase, in rat liver and intestinal mucosa

RNA dot-blot, quantitative electron microscope immunocytochemistry, and electrophoretic immunoblotting techniques were employed to investigate the expression of carbamoyl-phosphate synthetase I (CPS) and ornithine carbamoyl transferase (OCT) genes in rat liver and intestinal mucosa. Comparing only those cell types in the two tissues which express these enzymes, we show that the concentration of CPS and OCT in hepatocyte mitochondria is 2.3-times and 1.2-times greater, respectively, than in intestinal epithelial cell mitochondria. As a percentage of total tissue protein, however, liver homogenates contain 10-20 times more CPS and 5-10 times more OCT than is found in intestinal mucosa. These relatively large differences in enzyme protein levels between the two tissues are not reflected by differences in their mRNA levels. As a percentage of total translational activity in vitro (based on incorporation of [35S]methionine), total liver mRNA directed synthesis of about twice as much precursor CPS (pCPS) and precursor OCT (pOCT) than did equivalent amounts of mRNA from intestinal mucosa. The ratio of pCPS and pOCT mRNA levels between the two tissues (2:1, liver:intestinal mucosa) was confirmed by dot-blot and Northern hybridizations employing specific cDNA probes. The sizes of the respective mRNAs were the same for the two tissues: about 6000 residues for pCPS mRNA and about 1700 residues for pOCT mRNA.     

Isolation and identification of 1,25-dihydroxy-24-oxo-vitamin D3 and 1,23,25-trihydroxy-24-oxo-vitamin D3. New metabolites of vitamin D3 produced by a C-24 oxidation pathway of metabolism for 1,25-dihydroxyvitamin D3 present in intestine and kidney

Two new vitamin D metabolites were isolated in pure form from separate incubations of homogenates of chick small intestinal mucosa or rat kidney employing either 1 alpha,25-dihydroxyvitamin D3 (28 microM) or 1 alpha,24R,25-trihydroxyvitamin D3 as substrate (0.17-1.3 microM). The newly characterized compounds and the amounts isolated in pure form from separate isolations are, respectively: 1 alpha,25-dihydroxy-24-oxo-vitamin D3 (1,25(OH)2-24-oxo-D3), 147 micrograms from kidney and 4.2 and 40 micrograms from intestine; 1 alpha,23,25-trihydroxy-24-oxo-vitamin D3 (1,23,25(OH)3-24-oxo-D3), 155 micrograms from kidney and 5.9 and 34 micrograms from intestine. Their structures were identified after extensive high pressure liquid chromatography by means of ultraviolet absorption spectrometry, mass spectrometry of the free compounds and their trimethylsilylated derivatives, proton nuclear magnetic resonance spectrometry, specific chemical reduction of the 24-oxo functionality with sodium borohydride, as well as direct comparison with synthetic 1,25(OH)2-24-oxo-D3. These structural assignments for both compounds correct previous determinations which had been proposed (Ohnuma, N., Kruse, J. R., Popjak, G., and Norman, A. W. (1982) J. Biol. Chem. 257, 5097-5102). The activity of the C-24 oxidation pathway used for the production of the 1,25(OH)2-24-oxo-D3 and 1,23,25(OH)3-24-oxo-D3 can be enhanced 10-fold by prior priming of the chicks or rats with a single intravenous dose of 1,25(OH)2D3 (1-12 nmol/100 g body weight); the induction of the enzyme activity is maximal by 3-6 h and returns to basal levels within 12 h. Further, 1,25(OH)2D3, 1,24,25(OH)3D3, and 1,25(OH)2-24-oxo-D3 all were found to be capable of serving as a precursor with chick intestine and rat kidney homogenates of 1,23,25(OH)3-24-oxo-D3. Collectively these results suggest the existence of a C-24 oxidation pathway for metabolism of 1,25(OH)2D3 by the target intestinal mucosa and kidney to 1,23,25(OH)3-24-oxo-D3. The pathway may play an important role in controlling the tissue levels of this hormonally active form of vitamin D3.     

Some properties of the fatty alcohol oxidation system and reconstitution of microsomal oxidation activity in intestinal mucosa

This paper describes the metabolism of fatty alcohols by microsomal and cytosolic fractions from intestinal mucosa. Microsomes of rabbit intestinal mucosa had a high activity of [1-14C]dodecanol oxidation as did those of liver. The intestinal cytosolic fraction also exhibited oxidation activity to a lesser extent than the microsomes did. The reaction product was determined as lauric acid using thin-layer chromatography. Laurylaldehyde was detected as another product, when semicarbazide was added to the incubation system. Cyclodextrins exhibited a stimulation effect similarly to bovine serum albumin on the microsomal activity. We have compared the stimulatory effects of dimethyl-beta-cyclodextrin, beta-cyclodextrin, gamma-cyclodextrin and alpha-cyclodextrin, which decrease in that order. Effects of NAD+ and dodecanol concentrations, pH and pyrazole on microsomal activity were compared with those on cytosolic activity. Dodecanol oxidation activity was solubilized and reconstituted with a fatty alcohol dehydrogenase and a fatty aldehyde dehydrogenase separated from the intestinal microsomes. These findings indicate that both the dehydrogenases participate in microsomal oxidation of fatty alcohols to fatty acids with fatty aldehydes as intermediates in the reaction.     

Inhibition of the mutagenic activity of some heterocyclic dietary carcinogens and other mutagenic/carcinogenic compounds by rat organ preparations

The mutagenic activity of some dietary mutagens, 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-6-methyldipyrido[1,2-a:3',2'-d]imidazole (Glu-P-1) and 2-amino-dipyrido[1,2-a:3',2'-d]imidazole (Glu-P-2), was inhibited in the Salmonella-plate test preincubated with heat-inactivated rat intestinal preparations. A similar inhibition was observed by preincubating intestinal preparations with 2-acetylaminofluorene (AAF) and benzo[a]pyrene (B[a]P). The effect was not specific for small intestine and was also obtained with spleen, liver, lung, colon and stomach preparations. Mutagenic activity was not inhibited by beef muscle proteins. Lipids extracted from intestinal mucosa preparations were equally effective as inhibitors of the mutagenic activity. Lipid fractions from intestinal mucosa were capable of inhibiting the formation of activated IQ by mammalian S9, and other components of the intestinal preparations were able to bind the promutagens and their active metabolites. The mutagenic activity of 1-(2-hydroxyethyl)-2-methyl-5-nitroimidazole (metronidazole) and of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) was also inhibited by intestinal preparations, but not by their lipid fractions. A binding of IQ to intestinal preparations was also demonstrated with HPLC techniques. The data indicate that tissue components may reduce the mutagenic activity of chemicals by interfering with the activation process and by reducing the concentration of the promutagens and their active metabolites at target sites.     

Metabolism of 5-methylthioribose to methionine

During ethylene biosynthesis, the H₃CS- group of S-adenosylmethionine is released as 5′-methylthioadenosine, which is recycled to methionine via 5-methylthioribose (MTR). In mungbean hypocotyls and cell-free extracts of avocado, [¹⁴C]MTR was converted into labeled methionine via 2-keto-4-methylthiobutyric acid (KMB) and 2-hydroxy-4-methylthiobutyric acid (HMB), as intermediates. Incubation of [ribose-U-¹⁴C]MTR with avocado extract resulted in the production of [¹⁴C]formate, indicating the conversion of MTR to KMB involves a loss of formate, presumably from C-1 of MTR. Tracer studies showed that KMB was converted readily in vivo and in vitro to methionine, while HMB was converted much more slowly. The conversion of KMB to methionine by dialyzed avocado extract requires an amino donor. Among several potential donors examined, l-glutamine was the most efficient. Anaerobiosis inhibited only partially the oxidation of MTR to formate, KMB/HMB, and methionine by avocado extract. The role of O₂ in the conversion of MTR to methionine is discussed.     

Serotonin (5-HT) release and uptake measured by real-time electrochemical techniques in the rat ileum

Serotonin (5-HT) is released from the enterochromaffin cells and plays an important role in regulating intestinal function. Although the release of 5-HT is well documented, the contribution of the serotonin reuptake transporter (SERT) to the levels and actions of 5-HT in the intestine is unclear. This study aimed to demonstrate real-time SERT activity in ileal mucosa and to assess the effects of SERT inhibition using fluoxetine. Electrochemical recordings were made from the mucosa in full-thickness preparations of rat ileum using a carbon fiber electrode to measure 5-HT oxidation current and a force transducer to record circular muscle (CM) tension. Compression of the mucosa stimulated a peak 5-HT release of 12 +/- 6 microM, which decayed to 7 +/- 4 microM. Blockade of SERT with fluoxetine (1 microM) increased the peak compression-evoked release to 19 +/- 9 microM, and the background levels of 5-HT increased to 11 +/- 7 microM (P < 0.05, n = 7). When 5-HT was exogenously applied to the mucosa, fluoxetine caused a significant increase in the time to 50% and 80% decay of the oxidation current. Fluoxetine also increased the spontaneous CM motility (P < 0.05; n = 7) but did not increase the CM contraction-evoked 5-HT release (P > 0.05, n = 5). In conclusion, this is the first characterization of the real-time uptake of 5-HT into the rat intestine. These data suggest that SERT plays an important role in the modulation of 5-HT concentrations that reach intestinal 5-HT receptors.     

Analysis of gangliosides from carp intestinal mucosa

The gangliosides of carp intestinal mucosa were isolated and analysed by thin-layer chromatography (TLC), TLC immunostaining test, and TLC/secondary ion mass spectrometry (TLC/SIMS). Four species of gangliosides, designated as G-1, G-2, G-3 and G-4, were separated on TLC. The TLC/SIMS analysis of the G-1 ganglioside of carp intestinal mucosa revealed a series of [M-H](-)ions from m/z 1061 to m/z 1131 representing the molecular mass range of GM4-like ganglioside with NeuAc. G-2, G-3 and G-4 gangliosides were analysed by the TLC immunostaining test. G-2 ganglioside was recognised by the monoclonal antibody specific for ganglioside GM1 (AGM-1 monoclonal antibody). However, G-3 ganglioside migrating on TLC between GM3 and GM1 ganglioside was not recognised by anti-GM3 monoclonal antibody and by AGM-1 monoclonal antibody. Furthermore, G-4 ganglioside with a similar TLC mobility as GD1a ganglioside did not show the reactivity to the anti-GD1a monoclonal antibody. In addition using the AGM-1 monoclonal antibody, the expression of GM1 ganglioside in the carp intestinal tissue was studied. GM1 ganglioside was detected on the epithelial cell surface of carp intestinal mucosa.     

Localization of UDP-GalNAc:NeuAc alpha 2,3Gal-R beta 1,4(GalNAc to Gal)N-acetylgalactosaminyltransferase in human stomach. Enzymatic synthesis of a fundic gland-specific ganglioside and GM2.

A glycolipid detected in human gastric mucosa with anti-GM2 monoclonal antibody was characterized to be GalNAc beta 1-4[NeuAc alpha 2-3]Gal beta 1-4GlcNAc beta 1-3Gal 1-4Glc-Cer (NGM-1), which was lost in gastric cancer tissue with complementary increase of GM2 sharing the same terminal carbohydrate structure as NGM-1 (Dohi, T., Ohta, S., Hanai, N., Yamaguchi, K., and Oshima, M. (1990) J. Biol. Chem. 265, 7880-7885). The study on differential expression of NGM-1 in gastric fundic mucosa, pyloric mucosa, gastric cancer, and various other tissues indicated that NGM-1 existed specifically in fundic mucosa. The content of GM3 and sialylparagloboside (SPG), which are the substrates for the synthesis of GM2 and NGM-1, respectively, were not significantly different in these tissues. Therefore, the presence of two kinds of beta 1,4GalNAc transferases having different substrate specificity was considered to be critical for the expression of NGM-1 and GM2. The activity of beta 1,4GalNAc transferase which synthesizes GM2 or NGM-1 was determined by detecting the products with specific monoclonal antibodies. The activity of beta 1,4GalNAc transfer to SPG was high in fundic mucosa, while it was absent in pyloric mucosa or cancer. On the other hand, the increased activity of beta 1,4GalNAc transfer to GM3 was observed in cancer tissues and cancer cell lines which were rich in GM2. Our conclusion is that the limited expression of NGM-1 in fundic mucosa and the increase of GM2 in cancer are attributed to two types of beta 1,4GalNAc transferases localized in each region with different substrate specificity; the one in fundic mucosa transfers GalNAc to SPG but not to GM3, and the other one enhanced in cancer transfers GalNAc to GM3 but not to SPG.     

Acylation of lysolecithin in the intestinal mucosa of rats

1. The presence of an active acyl-CoA-lysolecithin (1-acylglycerophosphorylcholine) acyltransferase was demonstrated in rat intestinal mucosa. 2. ATP and CoA were necessary for the incorporation of free [1-(14)C]oleic acid into lecithin (phosphatidylcholine). 3. The reaction was about 20 times as fast with [1-(14)C]oleoyl-CoA as with free oleic acid, CoA and ATP. 4. With 1-acylglycerophosphorylcholine as the acceptor, both oleic acid and palmitic acid were incorporated into the beta-position of lecithin; the incorporation of palmitic acid was 60% of that of oleic acid. 5. Of the various analogues of lysolecithin tested as acyl acceptors from [1-(14)C]oleoyl CoA, a lysolecithin with a long-chain fatty acid at the 1-position was most efficient. 6. The enzyme was mostly present in the brush-border-free particulate fraction of the intestinal mucosa. 7. Of the various tissues of rats tested for the activity, intestinal mucosa was found to be the most active, with testes, liver, kidneys and spleen following it in decreasing order.     

A comparison of the conversion of 1-amino-2-ethylcyclopropane-1-carboxylic acid stereoisomers to 1-butene by pea epicotyls and by a cell-free system

The characteristics of the conversion of 1-aminocyclopropane-1-carboxylic acid (ACC) to ethylene by pea (Pisum sativum L.) epicotyls and by pea epicotyl enzyme are compared. Of the four stereoisomers of 1-amino-2-ethylcyclopropane-1-carboxylic acid (AEC), only (1R,2S)-AEC is preferentially converted to 1-butene in pea epicotyls. This conversion is inhibited by ACC, indicating that butene production from (1R,2S)-AEC and ethylene production from ACC are catalyzed by the same enzyme. Furthermore, pea epicotyls efficiently convert ACC to ethylene with a low K _m (66 M) for ACC and do not convert 4-methylthio-2-oxo-butanoic acid (KMB) to ethylene, thus demonstrating high specificity for its substrate. In contrast, the reported pea epicotyl enzyme which catalyzes the conversion of ACC to ethylene had a high K _m (389 mM) for ACC and readily converted KMB to ethylene. We show, moreover, that the pea enzyme catalyzes the conversion of AEC isomers to butene without stereodiscrimination. Because of its lack of stereospecificity, its low affinity for ACC and its utilization of KMB as a substrate, we conclude that the reported pea enzyme system is not related to the in-vivo ethylene-forming enzyme.Abbreviations ACC 1-Amino cyclopropane-1-carboxylic acid - AEC 1-amino-2-ethylcyclopropane-1-carboxylic acid - EFE ethylene-forming enzyme - KMB 4-methylthio-2-oxobutanoic acid     

TLR2, TLR3, and TLR4 activation specifically alters the oxidative status of intestinal epithelial cells

Intestinal inflammatory diseases are the result of multiple processes, including mucosal oxidative stress and perturbed homeostasis between commensal bacteria and mucosal immunity. Toll-like receptors (TLRs) recognize molecular-associated microorganisms' patterns and trigger innate immunity responses contributing to intestinal homeostasis and inflammatory responses. However, TLRs effects on redox balance in intestinal mucosa remain unknown. Therefore, the present study analyzes the effect of TLR2, TLR3, and TLR4 on both oxidative damage of lipids and proteins, and the activity of antioxidant enzymes in enterocyte-like Caco-2 cells. The results show that the activation of these TLRs increased lipid and protein oxidation levels; however, the effect on the antioxidant enzymes activity is different depending on the TLR activated. These results suggest that the activation of TLR2, TLR3, and TLR4 might affect intestinal inflammation by not only their inherent innate immunity responses, but also their pro-oxidative effects on intestinal epithelial cells.     

Glycerolipid biosynthesis in isolated rat intestinal epithelial cells.

Intestinal epithelial cells were prepared from fasted rats by dispersion with collagenase (EC 3.4.24.3). The structural and metabolic integrity of the cells was verified by electron microscopy, a high percentage of Trypan Blue exclusion, a low degree of release of lactate dehydrogenase (EC 1.1.1.27) in the medium, and by the retention of sensitivity to agents known to modify metabolic and transport activity in everted sacs of intestinal mucosa. The isolated intestinal epithelial cells were used to study glycerolipid biosynthesis from glucose, glycerol, 2-monoacylglycerol, and free fatty acids. The cells actively incorporated the labeled precursors into glycerolipids without specific cofactor requirements. Addition of fatty acids stimulated the incorporation of both glucose and glycerol into triacylglycerols and glycerophospholipids, the greatest effect being observed with palmitate. The stimulation of monoacylglycerol acylation appeared to depend on both the nature of the monoacylglycerol and fatty acid supplied. Stereospecific analyses of the diacylglycerols formed from 2-monoacylglycerols and free fatty acids showed that 1,2-diacyl-sn-glycerols (62-70%) were the major and that 2,3-diacyl-sn-glycerols (30-38%) the minor intermediates in triacylglycerol biosynthesis. The data indicate that isolated intestinal epithelial cells exhibit a total capacity of glycerolipid synthesis and a stereochemical course of reaction which is comparable to that observed for triacylglycerol formation in everted sacs of intestinal mucosa, but much less specific than that seen in microsomal preparations of intestinal mucosa.     

Enzyme activities of intestinal triacylglycerol and phosphatidylcholine biosynthesis in Atlantic salmon (Salmo salar L.)

The substitution of fish oil with plant-derived oil in diets for carnivorous fish, such as Atlantic salmon, has previously revealed the potentially deleterious supranuclear accumulation of lipid droplets in intestinal cells (enterocytes) which may compromise gut integrity, and consequently, fish health. This suggests that unfamiliar dietary lipid sources may have a significant impact on intestinal lipid metabolism, however, the mode of lipid resynthesis is largely unknown in teleost fish intestine. The present study aimed at characterising three key lipogenic enzymes involved in the biosynthesis of triacylglycerol (TAG) and phosphatidylcholine (PC) in Atlantic salmon enterocytes: monoacylglycerol acyltransferase (MGAT), diacylglycerol acyltransferase (DGAT), and diacylglycerol cholinephosphotransferase (CPT). Furthermore, to investigate the dietary effect of plant oils on these enzymes, two experimental groups of fish were fed a diet with either capelin (fish oil) or vegetable oil (rapeseed oil:palm oil:linseed oil, 55:30:15 w/w) as the lipid source. The monoacylglycerol (MAG) pathway was highly active in the intestinal mucosa of Atlantic salmon as demonstrated by MGAT activity (7 nmol [1-(14)C]palmitoyl-CoA incorporated min(-1) mg protein(-1)) and DGAT activity (4 nmol [1-(14)C]palmitoyl-CoA incorporated min(-1) mg protein(-1)), with MGAT appearing to also provide adequate production of sn-1,2-diacylglycerol for potential utilisation in PC synthesis via CPT activity (0.4 nmol CDP-[(14)C]choline incorporated min(-1) mg protein(-1)). Both DGAT and CPT specific activity values were comparable to reported mammalian equivalents, although MGAT activity was lower. Nevertheless, MGAT appeared not to be the rate-limiting step in salmon intestinal TAG synthesis. The homology between piscine and mammalian enzymes was established by similar stimulation and inhibition profiles by a variety of tested cofactors and isomeric substrates. The low dietary n-3/n-6 PUFA ratio presented in the vegetable oil diet did not significantly affect the activities of MGAT, DGAT, or CPT under optimised assay conditions, or in vivo intestinal mucosa lipid class composition, when compared to a standard fish oil diet.     

A DNA polymorphism influencing α(1,2)fucosyltransferase activity of the pig FUT1 enzyme determines susceptibility of small intestinal epithelium to Escherichia coli F18 adhesion

The alpha(1,2)fucosyltransferases (FUT1 and FUT2) contribute to the formation of blood group antigen structures, which are present on cell membranes and in secretions. In the present study we demonstrate that both FUT1 and FUT2 are expressed in the pig small intestine. FUT1 polymorphisms influence adhesion of F18 fimbriated Escherichia coli (ECF18) to intestinal mucosa, and FUT2 is associated with expression of erythrocyte antigen 0. The FUT1 polymorphisms result in amino acid substitutions at positions 103 (Ala-->Thr) and 286 (Arg-->Glu). Tightly controlled expression of the FUT2 gene results in either an abundance or an absence of mRNA in small intestinal mucosa. ECF18-resistant animals were shown to be homozygous for threonine at amino acid 103 of the FUT1 enzyme. Susceptibility to ECF18 adhesion appeared to be solely dependent on the activity of FUT1 in intestinal epithelia. In intestinal mucosae of ECF18-resistant pigs which expressed FUT1 but not FUT2 RNA, the levels of alpha(1,2)fucosyltransferase activity were significantly lower (28- to 45-fold, P<0.001) than in susceptible pigs. Moreover, lysates of CHO cells transfected with FUT1 constructs encoding threonine at amino acid position 103 also showed significantly reduced enzyme activity compared with constructs encoding alanine at this position. Our genetic and enzymatic studies support the hypothesis that the FUT1 enzyme, and particularly the amino acid at position 103, is likely important in the synthesis of a structure that enables adhesion of ECF18 bacteria to small intestinal mucosa.     

Effect of aldosterone antagonists on aldosterone-induced activation of Mg2+ -HCO3- -ATPase and carbonic anhydrase in rat intestinal mucosa

In previous studies, Mg2+ -dependent, HCO3- -activated ATPase in the brush border and carbonic anhydrase in the cytoplasm of rat duodenal and jejunal mucosa decreased after adrenalectomy. Both enzyme activities increased to near normal levels 4 h after i.p. injection of aldosterone (40 micrograms/kg). These results suggest the possibility that both enzymes in the small intestinal mucosa may be mediators of the action of aldosterone. In the present studies, therefore, the effects of actinomycin D (500 micrograms/kg, i.p.), spironolactone (50 mg/kg, s.c.) and potassium canrenoate (50 mg/kg, s.c.) on aldosterone-induced activation of both enzymes in the upper small intestinal mucosa from adrenalectomized rats were examined to clarify the mechanism of action of aldosterone in enzyme levels. Actinomycin D inhibited carbonic anhydrase activity in small intestinal mucosa from normal rats 4 h after i.p. injection but had no effect on ATPase activity, while two other drugs had no effect on either enzyme activity in normal rats up to 4 h later. Pretreatment with these 3 drugs 1 h before aldosterone administration (40 micrograms/kg, i.p.) to adrenalectomized rats blocked the aldosterone-induced activation of ATPase and carbonic anhydrase in the upper small intestine. On the other hand, adrenalectomy and administration of aldosterone and its antagonists, alone or in combination, had no effect on kidney enzyme activities. These results confirm that Mg2+ -HCO3- -ATPase and carbonic anhydrase are mediators of the action of aldosterone in the upper small intestinal mucosa.