Pig Enterocytes Research Articles

Lamina propria macrophages involved in cell death (apoptosis) of enterocytes in the small intestine of rats.

In the rat small intestine, apoptotic enterocytes are exfoliated at the villus tip as a whole cell, in contrast to guinea pig enterocytes which are phagocytosed by macrophages in their cell body and shed off only in their apical cortex. While macrophages gather in the lamina propria of the intestinal villi in both species, their functions seem to differ. Unlike the guinea pig, lamina propria macrophages observed in the rat small intestine did not show morphological signs of phagocytosis, revealing few cellular elements in their phagosomes. At the "shoulder" of the villus, i.e., a certain distance proximal to the villus tip, subepithelial macrophages extended a thick process deep into the epithelium; their branched terminals penetrated the cytoplasm of enterocytes, resulting in the formation of excavated spaces in the cell body. Processes of macrophages frequently reached close to the brush border. At the shoulder of the villus, a few effete cells showed typical apoptotic signs and appeared to be pushed out into the lumen; still, the shedding of apoptotic enterocytes was recognized mainly at the very top of the villus, where no intraepithelial processes of macrophages could be seen. The present findings indicate that in the rat, lamina propria macrophages do not engulf aged enterocytes, but are involved in inducing their apoptosis.

Characterization and ontogenesis of nitric oxide synthase activity in pig enterocytes

Nitric oxide has been implicated as a local modulator of several gastrointestinal functions. In this study, we have measured nitric oxide synthase activity in homogenates of enterocytes isolated from post-weaned pigs. The enzyme required the presence of NADPH and 6-(R,S)-5,6,7,8-tetrahydro- l-biopterin. Conversely exogenous FAD and FMN did not appear to be necessary for enzyme activity. The enzyme activity was not affected by added Ca 2+ or EGTA and was inhibited by the arginine analogs N G-monomethyl- l-arginine and N ω-nitro- l-arginine. NO synthase activity was not detectable in enterocytes isolated at birth and increased slightly in suckling animals. NO synthase activity was found to be present mostly in the cytosolic fraction isolated from post-weaned pigs enterocytes.

Intestinal arginine metabolism during development. Evidence for de novo synthesis of L-arginine in newborn pig enterocytes.

The capacity for L-arginine metabolism was studied in villus enterocytes isolated from pigs at birth, after 2-8 days suckling and after weaning. Immediately after birth, enterocytes were able to convert 1 mM L-citrulline, 2 mM L-glutamine or 1 mM L-ornithine to L-arginine. In 2-8-day-old animals, the net production of L-arginine from L-citrulline (2.00 +/- 0.45 nmol x 10(6) cells-1 x 30 min-1), or from L-ornithine (0.29 +/- 0.06 nmol x 10(6) cells-1 x 30 min-1) was similar to the values obtained at birth. Furthermore, 40% of L-arginine synthetized de novo from L-citrulline were released into the incubation medium. In 2-8-day-old animals, the production of L-arginine from L-glutamine represented only 5% of the production at birth (the latter being 0.73 +/- 0.15 nmol x 10(6) cells-1 x 30 min-1). In enterocytes isolated from post-weaned pigs, no significant production of L-arginine from either L-glutamine or L-ornithine was detected. In contrast, although the L-arginine production from L-citrulline was very low in post-weaned animals, it was significantly enhanced in the presence of L-glutamine, representing 23% of the production measured in suckling animals. The capacity of enterocytes to cleave L-arginine to L-ornithine and urea was very limited at birth, but was increased more than threefold in 2-day-old animals. This was concomitant with a marked increase in arginase activity. In post-weaned animals, the flux through arginase in intact enterocytes, and the arginase activity were both threefold higher than in 2-8-day-old animals. It is concluded that enterocytes isolated from neonatal pigs exhibit the capacity for a net production of L-arginine since the metabolism of this amino acid is oriented to anabolism rather than catabolism. The results are discussed in relation to L-arginine metabolism in the neonatal liver.

Polyamine metabolism in enterocytes isolated from newborn pigs

In the pig, the growth of intestinal mucosa is very intense after birth. Since the polyamines are key elements affecting cell proliferation and differentiation, the present work was undertaken in order to know whether this hypertrophy is associated with an adaptation of polyamine metabolism. Villus enterocytes isolated from pig immediately after birth or 2 days later were found to contain similar amounts of putrescine, spermidine and spermine, i.e., 0.23; 0.41 and 1.24 nmol 10 6 cells , respectively. At birth, despite a relatively high ODC activity, putrescine synthesis from 1 mM l-arginine or 2 mM l-glutamine was very low in isolated enterocytes ( 6.4 ± 3.8 pmol 10 6 cells per 30 min), while spermidine and spermine production were not detectable. This could be explained by a very low l-ornithine generation from both amino acids and to an inhibitory effect of polyamines on ODC activity. Two days later, polyamine synthesis from l-arginine remained undetectable despite a higher l-ornithine generation. This was concomitant with a dramatic fall in ODC activity. At both stages, enterocytes were able to take up polyamines from the extracellular medium in a temperature-dependent manner. It is concluded that de-novo synthesis of polyamines from l-arginine or l-glutamine does not play a significant role in the control of polyamine content of pig enterocytes during the postnatal period. In contrast, polyamine uptake by enterocytes would contribute to maintain a steady-state polyamine content during this period.

Iron-transferrin binding to isolated guinea pig enterocytes and the regional localisation of intestinal iron transfer during ontogeny

Neonatal animals are iron replete but in comparison with adults they display increased intestinal iron absorption. In order to examine possible mechanisms for this developmental adaptation we have measured the appearance of iron in peripheral blood following 30 min exposure of duodenal and ileal segments of adult and neonatal guinea pigs in vivo to 59Fe-ascorbate. Parallel experiments have determined the kinetics of 125I-labelled diferric transferring binding to villus enterocytes isolated from duodenum and ileum. In adult animals the rate of appearance of 59Fe in peripheral blood was 11-fold greater following duodenal, compared to ileal exposure to the radioligand. No such regional difference was detected in the neonate. Isolated cells showed saturable binding of [ 125I]transferrin which was maximal between 30 and 60 min. The kinetics of specific transferrin binding by adult duodenal and ileal enterocytes were similar and were also not significantly different to respective values in neonatal duodenal and ileal cells. Thus, it is likely that increased iron absorption in the neonate is due in part to enhanced ileal iron transfer. The interaction of transferrin with its receptor, however, is not involved in this developmental change in uptake.

Bioadhesion by means of specific binding of tomato lectin.

The possibility of developing bioadhesive drug delivery systems on the basis of molecules which selectively bind to the small intestinal epithelium by specific, receptor-mediated mechanisms was investigated using a lectin isolated from tomato fruits (Lycopersicum esculentum). The tomato lectin (TL) was found to bind specifically onto both isolated, fixed pig enterocytes and monolayers of human Caco-2 cell cultures with a similar affinity. TL-coated polystyrene microspheres (0.98 micron) also showed specific binding to enterocytes in vitro. Lectin binding was found to be favored at neutral pH and to be reduced in an acidic environment. Crude pig gastric mucin, however showed a marked cross-reactivity in vitro, indicating that lectin binding to the cell surface in vivo might be inhibited by mucus.

Compensatory increase in lactase expression by enterocytes of neonatal pigs on a low energy intake

A restricted energy intake in the immediate postnatal period has been found to result in a significant increase in the maximal expression of lactase by enterocytes from 2-week-old pigs. Although villus size was significantly reduced on a low compared with a high food intake, total villus lactase activity was unaffected because of the compensatory increase in lactase expression by individual enterocytes.

A cytotoxic haemolysin from Treponema hyodysenteriae--a probable virulence determinant in swine dysentery

The haemolysin from a virulent strain of Treponema hyodysenteriae was extracted and injected into ligated loops of the ileum and colon of germ-free pigs. It caused severe epithelial damage, especially to the differentiated cells at the tips of the villi in the ileum and the cells in the intercrypt zones of the colon; goblet cells were less affected. The changes in the colon were similar to those seen in natural cases of swine dysentery. The ligated loop offers a means of investigating pathogenic mechanisms and the mode of action of the toxin. This study demonstrated that the haemolysin was a potent cytotoxin for pig enterocytes, and a probable virulence determinant in swine dysentery.

Chicken Egg Antibodies for Prophylaxis and Therapy of Infectious Intestinal Diseases

The pathogenic effect of enterotoxogenic E. coli is mainly to be found in the jejunum. Hence, an effective immunoprophylaxis should be focussed at this site. This can be done effectively by an oral application of specific antibodies. The present study describes the effectiveness of specific antibodies against E. coli. The antibodies were gained from the yolk of E. coli immunized hens and tested for their in vitro activity. The tests showed a significantly reduced adhesion of enterotoxogenic E. coli onto isolated pig enterocytes as long as the bacteria had been incubated with the specific yolk antibodies beforehand. For this reason, yolk immunoglobulins from immunized hens prove to be an effective and handy source of specific antibodies which offers good protection against adhesion of E. coli onto pig enterocytes.

Oxidation of hemoglobin by hydrogen peroxide

Spin trapping of free radicals during the exposure of guinea pig enterocytes to bleomycin (BLM) was investigated using an in vitro cell suspension. The spin traps employed in this study were 5,5-dimethyl-1-pyrroline-1-oxide (DMPO) and 3,3-diethyl-5,5-dimethyl-1-pyrroline-1-oxide (DEDMPO). The hydroxyl radical spin-trapped adduct 2-hydroxy-5,5-dimethyl-1-pyrrolidinyloxyl (DMPO-OH) was observed with DMPO. In the presence of dimethyl sulfoxide (DMSO), the only 2,2,5-trimethyl-1-pyrrolidinyloxyl (DMPO-CH,) observed was that expected from hydroxyl radical formation by the decomposition of the Superoxide spin-trapped adduct 2-hydroperoxy-5,5-dimethyl-1-pyrrolidinyloxyl (DMPO-OOH). Production of hydroxyl radical was not detected in the presence of DEDMPO, which is a nitrone that will spin trap hydroxyl radical, but not Superoxide, at cellular concentrations. Thus, these data indicate that Superoxide was produced during the exposure of guinea pig enterocytes to BLM and that DMPO-OH resulted from the cellular bioreduction of DMPO-OOH by glutathione peroxidase. Addition of Superoxide dismutase to the in vitro reaction mixture indicated that Superoxide production was intracellular.

Detection of free radicals during the cellular metabolism of adriamycin

Experiments were conducted to determine which free radicals are generated during the metabolism of adriamycin (ADM) by canine tracheal epithelial (CTE) cells, guinea pig enterocytes, and rat hepatocytes. The technique employed in this study was spin trapping; the spin trap utilized was 5,5-dimethyl-1-pyrroline-1-oxide (DMPO). The spin adduct 2-hydroxy-5,5-dimethyl-1-pyrrolidinyloxyl (DMPO-OH) was observed during the metabolism of ADM by CTE cells. However, the addition of dimethyl sulfoxide to the in vitro system suggested that superoxide is initially spin trapped by nitrone, and that the adduct 2-hydroperoxy-5, 5-dimethyl-1-pyrrolidinyloxyl (DMPO-OOH) is rapidly bioreduced to afford DMPO-OH. The addition of superoxide dismutase to the system indicated that superoxide generation was primarily intracellular. The adriamycin semiquinone free radical (ADM-SQ) was produced during the metabolism by enterocytes and hepatocytes. The rate of the production of ADM-SQ was enhanced under anaerobic conditions, suggesting that molecular oxygen was responsible for the degradation of this carbon-centered free radical. However, spin trapping of oxygen radicals was not observed; this observation suggests that these reactive intermediates are not produced at concentrations sufficient for detection by spin-trapping experiments.

Induction of the 2-5A System by Interferon and Transmissible Gastroenteritis Virus

Interferon (IFN) treatment increased the level of 2',5'-oligoadenylate (2-5A) synthetase and inhibited replication of transmissible gastroenteritis virus (TGEV) in cultured swine testicular cells. Despite a minimal increase in TGEV titer in IFN-treated swine testicular cells, there was rapid cellular destruction. IFN-treated swine testicular cells had detectable levels of 2-5A (3.6 nM 6 h post-infection) after infection with 30 pfu TGEV/cell. Infection of neonatal pigs with a virulent strain of TGEV caused a significant increase in serum IFN and enterocyte 2-5A synthetase activity, as compared to control pigs. The level of enterocyte 2-5A synthetase in TGEV-infected pigs was increased 25-fold before viral-induced damage of the intestine consistently was present. 2-5A was not detected in enterocytes of either TGEV-infected or control pigs (less than 0.5 nM). Preparations of enterocytes contained two subpopulations of cells, one of which does not support replication of the virus. The considerable dilution of TGEV-infected cells (villous enterocytes) with uninfected cells (crypt cells) may be responsible for our inability to detect 2-5A in enterocytes from TGEV-infected pigs. These results indicate that the 2-5A system in porcine enterocytes and cultured testicular cells is modulated by TGEV infection and/or interaction with IFN.

Conformational change of rabbit aminopeptidase N into enterocyte plasma membrane domains analyzed by flow cytometry fluorescence energy transfer.

Membrane vesicle preparations are very appropriate material for studying the topology of glycoproteins integrated into specialized plasma membrane domains of polarized cells. Here we show that the flow cytometric measurement of fluorescence energy transfer used previously to study the relationship between surface components of isolated cells can be applied to membrane vesicles. The fluorescein and rhodamine derivatives of a monoclonal antibody (4H7.1) that recognized one common epitope of the rabbit and pig aminopeptidase N were used for probing the oligomerization and conformational states of the enzyme integrated into the brush border and basolateral membrane vesicles prepared from rabbit and pig enterocytes. The high fluorescent energy transfer observed in the case of pig enzyme integrated into both types of vesicles and in the case of the rabbit enzyme integrated into basolateral membrane vesicles agreed very well with the existence of a dimeric organization, which was directly demonstrated by cross-linking experiments. Although with the latter technique we observed that the rabbit aminopeptidase was also dimerized in the brush border membrane, no energy transfer was detected with the corresponding vesicles. This indicates that the relative positions of two associated monomers differ depending on whether the rabbit aminopeptidase is transiently integrated into the basolateral membrane or permanently integrated into the brush border membrane. Cross-linking of aminopeptidases solubilized by detergent and of their ectodomains liberated by trypsin showed that only interactions between anchor domains maintained the dimeric structure of rabbit enzyme whereas interactions between ectodomains also exist in the pig enzyme. This might explain why the noticeable change in the organization of the two ectodomains observed in the case of rabbit aminopeptidase N does not occur in the case of pig enzyme.

The toxicity of flavonoids to guinea pig enterocytes

Flavonoids, a group of compounds found primarily in vegetables and fruits, are generally believed to be beneficial to biological systems. Isolated guinea pig enterocytes were exposed to three of these compounds (kaempferol, quercetin, and myricetin) in concentrations of 50–450 μ m. Toxicity was examined using trypan blue exclusion and lactic dehydrogenase (LDH) leakage. All three flavonoids produced cellular damage at 450 μ m: compared with a control incubation, cellular viability was 12–60% lower and LDH leakage 28–41% greater after a 3-hr incubation. In addition, as assessed by trypan blue exclusion, quercetin and myricetin, both of which produce superoxide on autoxidation, appeared to be more toxic than kaempferol. These results suggest that dietary flavonoids may have the potential for producing intestinal injury.

Evidence for intracellular superoxide formation following the exposure of guinea pig enterocytes to bleomycin

Spin trapping of free radicals during the exposure of guinea pig enterocytes to bleomycin (BLM) was investigated using an in vitro cell suspension. The spin traps employed in this study were 5,5-dimethyl-1-pyrroline-1-oxide (DMPO) and 3,3-diethyl-5,5-dimethyl-1-pyrroline-1-oxide (DEDMPO). The hydroxyl radical spin-trapped adduct 2-hydroxy-5,5-dimethyl-1-pyrrolidinyloxyl (DMPO-OH) was observed with DMPO. In the presence of dimethyl sulfoxide (DMSO), the only 2,2,5-trimethyl-1-pyrrolidinyloxyl (DMPO-CH,) observed was that expected from hydroxyl radical formation by the decomposition of the Superoxide spin-trapped adduct 2-hydroperoxy-5,5-dimethyl-1-pyrrolidinyloxyl (DMPO-OOH). Production of hydroxyl radical was not detected in the presence of DEDMPO, which is a nitrone that will spin trap hydroxyl radical, but not Superoxide, at cellular concentrations. Thus, these data indicate that Superoxide was produced during the exposure of guinea pig enterocytes to BLM and that DMPO-OH resulted from the cellular bioreduction of DMPO-OOH by glutathione peroxidase. Addition of Superoxide dismutase to the in vitro reaction mixture indicated that Superoxide production was intracellular.

Dexamethasone selectively increases sodium-dependent alanine transport across neonatal piglet intestine.

1. Lysine and alanine uptake by pig enterocytes has been measured in piglet mid intestine both during normal development and 3 days after injection of dexamethasone and epidermal growth factor (EGF) into 3-day-old animals. 2. Alanine uptake measured in the presence of sodium increased markedly during the first 4 weeks of post-natal life. Similar effects on alanine uptake could be produced through injection of dexamethasone, but not EGF, into 3-day-old piglets. Alanine uptake measured in the absence of sodium and lysine uptake measured in the presence of sodium remained unchanged during development and unaffected by injection of dexamethasone or EGF. 3. Enterocytes capable of transporting alanine in the presence of sodium were found, by quantitative autoradiography, to cover the top 400 micron of the villus in 6-day-old and 3-4-week-old control pigs. Alanine concentrations in villus tip enterocytes in 3-4-week-old pigs were four times those found in 6-day-old animals. Qualitative examination of selected villi, however, showed alanine uptake taking place over a considerably greater area of villus surface in 6-day-old compared with 3-4-week-old animals. 4. Injection of dexamethasone and EGF into 3-day-old piglets caused an increase in crypt depth without apparent change in crypt cell proliferation. The rate at which enterocytes migrated out of the crypt and the length of individual villi also remained unchanged by dexamethasone or EGF injection. 5. Dexamethasone produces its effect on alanine uptake by acting on older enterocytes present on the upper part of the villus. These enterocytes can be shown, by calculations based on enterocyte migration rate, to have already been present on the villus at the time the pig was born. 6. The above findings are discussed in relation to the ability of villus as well as crypt enterocytes to change their programme of differentiation in response to external stimuli. The particular ability of dexamethasone to induce system A type carrier function is further discussed in relation to normal changes found to occur during neonatal development. It is finally suggested, as a working hypothesis, that endogenous glucagon might act as the final mediator of both developmentally controlled and dexamethasone-induced changes in amino acid transport.

Alteration of lactase and aminopeptidase N expression in porcine enterocytes by energy intake and environment.

The influence of nutrition, environmental temperature and time of feeding on lactase and aminopeptidase N activities in enterocytes of young pigs has been investigated. Animals were kept in the cold (10 degrees C) or warm (35 degrees C) and given either a high or low energy intake. In animals from the cold, lactase activity as determined by scanning microdensitometry was significantly lower at 4 h after a meal than it was in animals from the warm. At 6 h, the activities were similar in all groups, while at 20-24 h after feeding, lactase activity was much greater in those kept at 10 degrees C than in those at 35 degrees C. Level of energy intake had no statistically significant effect and no differences were found with respect to aminopeptidase N. The possibility that these differences in lactase activity are related to thyroid hormone metabolism and energy supply in relation to demand is discussed.

Biochemical changes in the intestine associated with anoxia and reoxygenation: In vivo and in vitro studies

In ischemia/reperfusion injury, it is hypothesized that superoxide is responsible for the component of injury due to reperfusion. The superoxide is hypothesized to result from the aerobic oxidation of purines produced by the ischemia-mediated breakdown of high-energy phosphates. This oxidation is catalyzed by xanthine oxidase proposed to be rapidly formed as a result of ischemia-mediated protease conversion from xanthine dehydrogenase. In vivo experiments with the intestine of either rats or guinea pigs were unable to confirm the rapid conversion of xanthine dehydrogenase to xanthine oxidase as a result of ischemia. In vitro experiments with isolated guinea pig enterocytes did show a significant increase in xanthine oxidase activity after these cells were first placed in an anaerobic environment for 60 min and then reoxygenated; however, the magnitude of the increase is such that the biological importance of this finding remains uncertain. Using a variety of techniques, including spin trapping, hydroxylamine oxidation, and vanadate NADPH oxidation, we explored the possibility that superoxide was produced as a result of anoxia followed by reoxygenation in the in vitro enterocyte system. From these experiments, we determined that superoxide is generated as a result of anoxia/reoxygenation. However, from xanthine oxidase inhibition experiments using pterinaldehyde, only a small percentage of the total superoxide produced comes from the action of this enzyme on purines.

The effect of postnatal age on the adherence of Shigella flexneri, Escherichia coli 0124, and E. coli 0128 to guinea pig intestinal cells.

Since postnatal development of the gastrointestinal tract has an important effect on its microbial flora and may influence the types of intestinal infections, we examined the effect of age on bacterial adherence to intestinal epithelial cells. Radiolabeled bacteria were incubated with guinea pig enterocytes released by treating loops of the intestine with solutions containing EDTA, dithiothreitol, and citrate. Nonbound bacteria were separated from intestinal cells by sedimentation on a Percoll gradient. The colonic cells avidly bound Shigella flexneri (64 bacteria per cell), Escherichia coli 0124 (59), and E. coli 0128 (53). The adherence process was Ca2+ and temperature dependent, was inhibited by fucose, glucose, and mannose, and was shown to be mediated by a carbohydrate-binding protein (lectin) on the colonic cells. Adherence of these bacteria to intestinal cells of newborn animals was only 15-25% of the adherence to adult animal cells and increased gradually, reaching adult values at about 2 weeks of age. The lectin activity, which was determined by agglutination of bacteria, was secreted with the colonic mucus. It was undetectable in the newborn animal, appeared gradually with age, and its titer correlated with the adherent capability of the colonic cells. E. coli 0128 was the only one of the bacteria tested which significantly adhered to the ileum (19 bacteria per cell) in a process inhibited by mannose. This adherence was mediated by a mannose-sensitive lectin in the bacterial pili, and not on the intestinal cells. The postnatal age had no effect on the adherence to the ileum; the newborn animal had the same adherence capability as the adult one.(ABSTRACT TRUNCATED AT 250 WORDS)

Mucosal surface lesions in young protein-deficient pigs infected with Ascaris suum (Nematoda)

The morphology of the mucosal surface of samples from the small intestine of young protein-deficient pigs was examined by scanning electron microscopy. Three pigs had been infected with Ascaris suum for at least 58 days while 1 had remained uninfected; all pigs were housed and maintained under the same conditions, at the same time. The tissue samples collected at post mortem examination from pigs which contained A. suum showed varying degrees of villous atrophy and fusion. In addition, unusual small craters and as yet unidentified objects were observed at the surfaces of the enterocytes of Ascaris-infected pigs. These changes in mucosal morphology were not seen in the tissue taken from the uninfected pig. The possible association between the mucosal lesions and lactose maldigestion in young, protein-deficient pigs infected with A. suum is discussed.