Rabbit Intestinal Tissues Research Articles

Inhibitory effect of TNF‐α on the intestinal absorption of galactose

Sepsis is a systemic response to infection in which toxins, such as bacterial lipopolysaccharide (LPS), stimulate the production of inflammatory mediators like the cytokine tumor necrosis factor alpha (TNF-alpha). Previous studies from our laboratory have revealed that LPS inhibits the intestinal absorption of L-leucine and D-fructose in rabbit when it was intravenously administered, and that TNF-alpha seems to mediate this effect on amino acid absorption. To extend this work, the present study was designed to evaluate the possible effect of TNF-alpha on D-galactose intestinal absorption, identify the intracellular mechanisms involved and establish whether this cytokine mediates possible LPS effects. Our findings indicate that TNF-alpha decreases D-galactose absorption both in rabbit intestinal tissue preparations and brush-border membrane vesicles. Western blot analysis revealed reduced amounts of the Na+/glucose cotransporter (SGLT1) protein in the plasma membrane attributable to the cytokine. On the contrary, TNF-alpha increased SGLT1 mRNA levels. Specific inhibitors of the secondary messengers PKC, PKA, the MAP kinases p38 MAP, JNK, MEK1/2 as well as the proteasome, diminished the TNF-alpha-evoked inhibitory effect. LPS inhibition of the uptake of the sugar was blocked by a TNF-alpha antagonist. In conclusion, TNF-alpha inhibits D-galactose intestinal absorption by decreasing the number of SGLT1 molecules at the enterocyte plasma membrane through a mechanism in which several protein-like kinases are involved.

Carrier-enhanced human growth hormone absorption across isolated rabbit intestinal tissue

Small molecular weight alpha acid derivatives are able to enhance the intestinal absorption of human growth hormone through isolated rabbit intestinal tissue. The enhancement is not through the usual tissue modification associated with traditional penetration enhancers nor is it through an active transport process. Rather these small molecules associate with human growth hormone in solution to make it more transportable through intestinal tissue. It is shown that the enhancer has specificity for a particular protein and the enhancer and human growth hormone must be in solution together to be effective, i.e. pretreating the tissue with enhancer and then adding the protein does not increase tissue permeability. Moreover, the enhancer does not increase the permeability of mannitol or progesterone, thus providing additional evidence of specificity and establishing that these agents are not classical penetration enhancers.

Active efflux kinetics of etoposide from rabbit small intestine and colon.

The aim of the present study was to investigate the directional transport kinetics of etoposide in rabbit intestinal tissues using side-by-side diffusion chambers. Etoposide is a routinely used mixed-mechanism 'efflux' inhibitor; however, its absorptive and secretory transport kinetics in rabbit intestinal tissues, a commonly used animal model, have not yet been reported. Kinetic studies revealed that the apical (AP) to basolateral (BL) (i.e. absorptive) transport of etoposide was not apparently mediated by specialized transporters, whereas secretion (i.e. BL to AP transport) by intestinal tissues was concentration dependent and saturable. Half-saturation constant values (K(m), mean+/-standard deviation (S.D.)) ranged from 53.6+/-35.8 microM to 168.7+/-127.3 microM, consistent with previous results from our group in intestinal tissues from other species and Caco-2 cell monolayers. Secretory permeability was greatest in the ileum, whereas values in the upper small intestine and colon were approximately equal, and represented only 50% of the value in the ileum. The ileal secretory transport of etoposide was temperature dependent, with the activation energy (E(a)) >4 kCal/mole at 5 microM, suggesting the involvement of the active, energy dependent mechanism. Etoposide inhibition by verapamil and saquinavir, known inhibitors of intestinal secretion, was characterized as competitive with K(i)'s equal to 193.0+/-164.4 microM and 72.6+/-53.5 microM, respectively. The current results demonstrate that the absorptive transport of etoposide in rabbit tissue was not mediated by specialized carriers, and that secretory transport was regionally dependent, mediated by a transporter or transporters, the K(m)'s were in the micromolar range, and involved the energy dependent mechanism(s). The relatively low k(m) of etoposide compared with its aqueous solubility (0.25-0.34 mM, pH 5-6.5, 25 degrees C) makes it the excellent mixed-mechanism competitive inhibitor for determining the secretory transport properties of putative drug substrates. Understanding the in vitro secretory transport kinetics of etoposide provides a mechanistic basis for ongoing studies exploring the functional role of 'efflux' in vivo.

Functional characterization of type IV pili expressed on diarrhea-associated isolates of Aeromonas species.

Our past work has shown that long, flexible type IV pili (single or in bundles) are the predominant pili expressed on fecal isolates of diarrhea-associated species of Aeromonas (Aeromonas veronii biovar sobria and A. caviae). They represent a family of type IV pili which we have designated Bfp (for bundle-forming pili). Reports from Japan suggest that Bfp are intestinal colonization factors. This study presents compelling evidence to support this conclusion. Aeromonas bacteria and/or Bfp purified from a strain of A. veronii biovar sobria were shown to adhere to epithelial and intestinal cell lines, freshly isolated human enterocytes, and fresh and fixed human and rabbit intestinal tissues, as determined by light and electron microscopy and immunohistochemical detection. Removal of Bfp by mechanical means decreased adhesion to cell lines by up to 80%. Purified Bfp blocked adhesion of the test strain to intestinal cells in a dose-dependent manner. Adhesion was also blocked by the Fab fraction of anti-Bfp immunoglobulin G. Moreover, ultrastructural studies (ruthenium red staining and transmission and scanning electron microscopy) demonstrated for the first time that Aeromonas adhesion to human enterocytes is pilus mediated and suggested that Bfp may also promote colonization by forming bacterium-to-bacterium linkages. Bfp-positive isolates examined for type IV pilus-mediated twitching motility in agar and slide culture assays developed for Pseudomonas aeruginosa did not, however, exhibit this function.

Degradation and aggregation of human calcitonin in vitro.

To investigate the degradation of human calcitonin (hCT) by enzymes or mucosa from different gastrointestinal (GI) compartments and evaluate the stabilization effect of a synthetic ionizable copolymer on the stability of hCT in an aqueous solution. These data are a prerequisite for the development of a hydrogel based colon-specific hCT delivery system. Luminal and brush border membrane (BBM) enzymes from the colon and small intestine (SI) of the rabbit were isolated and their enzymatic activity toward hCT in vitro was evaluated. Human fecalase was used to mimic the luminal enzymatic activity in the human colon and its degradation ability was assessed. Excised intact rabbit intestinal tissues from both the colon and the SI were used to study the degradation patterns of hCT by intact mucosa. Detection of intact human calcitonin was performed using gradient elution, reverse phase high-pressure liquid chromatography (RP-HPLC). The structure of the hCT fragments was determined by Matrix Assisted Laser Desorption/lonization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) analysis. UV/VIS and fluorescence spectroscopy methods were used to evaluate the influence of a copolymer, possessing the same structure as the primary chains in hydrogels degradable in the colon, on the fibrillation process of hCT. In vitro results showed that isolated luminal enzymes and BBM enzymes from the SI were more potent in degrading intact hCT, as expected. Moreover, BBM enzymes were far more abundant in the SI than in the colon. Compared with rabbit colonic luminal enzymes, the degradation potency of human fecalase was further abated. Intact mucosal studies revealed extensive degradation by the SI mucosa but not by the colonic mucosa. The primary structures of the peptide fragments were identified by MALDI-TOF MS analysis. Fibrillation studies of hCT indicated that acrylic acid-containing polymeric materials were able to decrease the aggregation of hCT in aqueous solutions. Reduced proteolytic activity suggests that the colon is an advantageous site for peptide delivery. The structures of hCT degradation products were identified and the participation of particular enzymes in the degradation process was suggested. In addition, it was determined that an acrylic acid-containing copolymer improved the physical stability of hCT in aqueous solution.

OPENING INTESTINAL TIGHT JUNCTIONS: A NOVEL MECHANISM OF INTESTINAL SECRETION.

Zonula occludens toxin (Zot) is a novel toxin elaborated by Vibrio cholerae that modulates intestinal tight junctions. Aim of the study was to establish whether the permeabilizing effect of the toxin leads to intestinal secretion. Rabbit intestine was mounted in Ussing chambers and exposed to increasing concentrations of purified toxin. The tissues were also fixed, exposed to Zot, and then processed for fluorescence microscopy to determine the distribution of the toxin receptor within the intestine. Finally, purified toxin was simultaneously perfused in three distinct rabbit intestinal segments in vivo and water and electrolytes absorption measured. Zot induced a time- and dose-dependent decrease of rabbit intestinal tissue resistance in vitro starting at a toxin concentration of 1.1×10-13M. When tested in vivo, the toxin induced a jejunal secretion of water (18.2±3.8 vs -28.1±7.5 μl/min.cm, control period vs Zot-exposed period, respectively, p=0.003) and chloride (10.8±3.2 vs -2.53±0.06 μEq/min.cm, p=0.01). Similar changes were observed in the ileum (water 39.3±7.1 vs -18.7±4.8 μl/min.cm, p=0.003; chloride 9.6±1.2 vs -2.5±0.3 μEq/min.cm), but not in the colon (water 11.5.3±4.7 vs 17.5±5.0 μl/min.cm; chloride 6.0±1.3 vs 8.6±2.3 μEq/min.cm). The secretory effect of Zot in the small intestine was associated with an increased passage of polyethylene glycole 4000 in the bloodstream. Both the in vitro and in vivo effects of the toxin were reversible and paralleled the distribution of the toxin receptor within the intestine. In conclusion, the intestinal secretion induced by Zot follows the opening of tight junctions caused by the toxin and may represent a novel mechanism of intestinal secretion.

Modulation of Intestinal Tight Junctions: A Novel Mechanism of Intestinal Secretion † 482

Zonula occludens toxin (Zot) is a novel toxin elaborated by Vibrio cholerae that modulates intestinal tight junctions. Aim of the study was to establish whether the permeabilizing effect of the toxin leads to intestinal secretion. Rabbit intestine was mounted in Ussing chambers and exposed to increasing concentrations of purified toxin. The tissues were also fixed, exposed to Zot, and then processed for fluorescence microscopy to determine the distribution of the toxin receptor within the intestine. Finally, purified toxin was simultaneously perfused in three distinct rabbit intestinal segments in vivo and water and electrolytes absorption measured. Zot induced a time- and dose-dependent decrease of rabbit intestinal tissue resistance in vitro, starting at a toxin concentration of 1.1×10-13M. When tested in vivo, the toxin induced a secretion of water and chloride and the passage of polyethylene glycole 4000 in the bloodstream. Both the in vitro and in vivo effects of the toxin were reversible, were observed only in the small intestine but not in the colon, and paralleled the distribution of the toxin receptor within the intestine. In conclusion, the intestinal secretion induced by Zot follows the opening of tight junctions caused by the toxin and may represent a novel mechanism of intestinal secretion.

Cellular and regional expression of transcripts of the plasma membrane calcium pump PMCA1 in rabbit intestine.

The plasma membrane Ca(2+)-pumping adenosinetriphosphatase (PMCA) is the energy-dependent step in the active vitamin D-dependent absorption of dietary Ca2+ by the enterocyte. Studies of the various PMCA genes and splicing variants in humans and rats have indicated that the isoform known as PMCA1b is the predominant form expressed in small intestine. Using an oligonucleotide probe, we have studied the regional and cellular distribution of PMCA1 transcripts in rabbit intestinal tissues by in situ hybridization. On small intestinal RNA blots, this hybridized to species similar in size to those detected by PMCA1-specific cDNA probes; an additional larger transcript was present in rabbit than in rat or human. In situ hybridization signals were principally in the enterocyte population of the mucosa and were maximal in differentiating enterocytes on the lower part of the villus, a pattern similar to that previously demonstrated for other nutrient transporters. Reflecting the capacity of the different small intestinal segments to transport Ca2+, much higher levels of transcript were detected by both methods proximally (in duodenum) than distally (in jejunum and ileum) and were also higher in cecum and ascending colon mucosa than in descending colon. We conclude that as enterocytes differentiate in regions that absorb Ca2+, they express high levels of mRNA for PMCA1. These results confirm the importance of transcriptional regulation of this gene for active Ca2+ absorption.

Mechanism of dextran transport across rabbit intestinal tissue and a human colon cell-line (CACO-2).

The in vitro permeabilities of 14C labeled dextrans (10, 40, and 70 kD) were calculated from mass transport across Peyer's patches and non-patch tissues derived from rabbit jejunum, and a human colon cell line (Caco-2) grown as a monolayer on polycarbonate filters. Size distribution of dextrans did not change upon transport as judged from size exclusion chromatography. Permeabilities decreased in a size-dependent manner. Ranking of permeabilities for dextran 10 and 40 kD were: Caco-2 > non-patch tissue > Peyer's patches; while dextran 70 kD demonstrated no difference among the barriers. Tissue resistance, expressed as 1/(permeability.tissue thickness) was virtually the same in Peyer's patches and non-patch tissue, suggesting that tissue thickness and not interaction determines the difference in permeability. ATP depletion with ouabain, Na(+)-azide and 2-deoxy-D-glucose, and low temperature (4 degrees C) did not result in reduced permeabilities suggesting passive transport. The results suggest that the investigated intestinal barriers transport dextrans in a similar fashion independent of their source. However, comparison of the ratios dextran 10 kD/mannitol and PEG 900/mannitol between rabbit tissue and Caco-2 monolayers suggests Caco-2 monolayers may serve as a model to study absorption potential of potentially harmful compounds in coeliac disease, gastroenteritis, and colon carcinoma.

Determination of transport rates for arginine and acetaminophen in rabbit intestinal tissues in vitro.

The in vitro Ussing technique was employed to examine transport rates for acetaminophen and arginine across rabbit intestinal tissues. Mannitol and transepithelial conductance were used to monitor the integrity of rabbit intestinal tissues and the basal and stimulated short-circuit current were measured to assess functional viability. Transepithelial transport of acetaminophen, arginine, and mannitol was determined in rabbit jejunum, ileum, and distal colon. Transepithelial transport of arginine in the ileum and jejunum was composed of both passive (nonsaturable) (Pm = 0.06) and saturable components (KT = 0.6-0.7 mM; Jmax = 0.3-0.4 mumol/hr.cm2). The saturable component of arginine fluxes was abolished by pretreatment of the tissue with serosal ouabain (0.1 mM). In the distal colon, both unidirectional arginine fluxes were nonsaturable. In the segments examined, both unidirectional fluxes of acetaminophen were nonsaturable over the concentration range from 0.1 to 30 mM. These results provide values for maximal permeabilities attained by molecules traversing both the cellular and the paracellular pathways and, by comparison to their in vivo bioavailabilities, provide selection criteria for evaluating drug candidates for oral activity.

Multiple determinants of verotoxin-producing Escherichia coli O157:H7 attachment-effacement

Verotoxin-producing Escherichia coli strains of the serotype O157:H7 belong to a class of gastrointestinal pathogens that adhere to epithelial cells in a characteristic pattern known as attaching and effacing. Recent insight into the nature of E. coli O157:H7 adhesion was provided by the cloning and sequencing of the chromosomal eaeA (for E. coli attaching and effacing) gene homolog (G. Beebakhee, M. Louie, J. De Azavedo, and J. Brunton, FEMS Microbiol. Lett. 91:63-68, 1992, and J. Yu and J. B. Kaper, Mol. Microbiol. 6:411-417, 1992) and isolation of a 60-MDa plasmid referred to as pO157 (I. Toth, M. L. Cohen, H. S. Rumschlag, L. W. Riley, E. H. White, J. H. Carr, W. W. Bond, and I. K. Wachsmuth, Infect. Immun. 58:1223-1231, 1990, and S. Tzipori, H. Karch, K. I. Wachsmuth, R. M. Robins-Browne, A. D. O'Brien, H. Lior, M. L. Cohen, J. Smithers, and M. M. Levine, Infect. Immun. 55:3117-3125, 1987) and an approximately 94-kDa outer membrane protein (94-kDa OMP; P. Sherman, F. Cockerill III, R. Soni, and J. Brunton, Infect. Immun. 59:890-899, 1991). In this study, we examined the gene products of both eaeA and pO157 in relation to the 94-kDa OMP and as candidate effectors for O157:H7 attachment-effacement. Peptide sequencing and immunoassay demonstrated that the C. coli O157:H7 eaeA gene product is distinct from the 94-kDa OMP. Using ultrastructural analyses, we found that both parent and pO157 plasmid-cured O157:H7 strains demonstrated attaching and effacing adhesion to host epithelial cells and reacted equally well to rabbit antiserum raised against the 94-kDa OMP. By both transmission electron microscopy and light microscopy, E. coli HB101 transformed separately with the cloned eaeA gene and the pO157 plasmid did not form attaching and effacing lesions on cultured epithelial cells in vitro and rabbit intestinal tissues in vivo. Since additional determinants may mediate the attaching and effacing phenotype, we examined transposon TnphoA mutants constructed from E. coli O157:H7 strain CL8. Two TnphoA mutants were found deficient in bacterial factors that are necessary for O157:H7 attachment-effacement and likely distinct from the eaeA gene product.

Role of the Yersinia outer membrane protein YadA in adhesion to rabbit intestinal tissue and rabbit intestinal brush border membrane vesicles

The Yersinia virulence plasmid confers on strains of Yersinia pseudotuberculosis and Y. enterocolitica an adhesive potential superior to the one encoded by the chromosome alone. We have evaluated the role of the plasmid-encoded outer membrane protein YadA (formerly called Yopl) in adhesion. Insertional inactivation of the yadA gene (formerly called yopA), which encodes YadA, led to a reduction in the capacity of plasmid-carrying strains of Y. pseudotuberculosis 0:III and Y. enterocolitica 0:9 to adhere to intestinal tissue, brush border membranes and polystyrene surfaces. The adhesive characteristics of the mutants were comparable to those of their plasmid-cured counterparts. When the yadA gene from Y. pseudotuberculosis serotype 0:III or Y. enterocolitica serotype 0:3 or 0:8 was cloned into an Escherichia coli strain, increased ability to adhere to intestinal tissue, brush border membrane vesicles and polystyrene was transferred concomitantly. The introduction of the yadA gene from Y. pestis, which is unable to express YadA due to a one base pair deletion, did not change the adhesive characteristics of E. coli. Expression of YadA in the outer membrane may, therefore, make an important contribution to intestinal adherence of the two enteropathogenic members of the Yersinia species, Y. pseudotuberculosis and Y. enterocolitica.

Quantitation of the rabbit intestinal glycolipid receptor for Shiga toxin: Further evidence for the developmental regulation of globotriaosylceramide in microvillus membranes

Shiga toxin, produced by Shigella dysenteriae 1, causes enterotoxic, cytotoxic, and neurotoxic effects, which may be mediated by a glycolipid receptor, globotriaosylceramide, Gb3. To study the relationship of this receptor and toxin effects, globotriaosylceramide was quantitated and further characterized in rabbit small intestinal microvillus membranes at various ages. Glycolipids were extracted from rabbit microvillus membranes, purified on Unisil columns, and quantitated by highperformance liquid chromatography. The major glycolipid peaks were hydroxylated fatty acid-containing glucosylceramide, lactosylceramide, and globotriaosylceramide. There was a marked increase of globotriaosylceramide levels with age, ranging from 0.02 to 16.2 pmol/μg microvillus membrane protein in neonates and adults, respectively. The globotriaosylceramide peak was susceptible to α-galactosidase treatment, which produced an elevation in the lactosylceramide peak, but markedly reduced globotriaosylceramide content in 34-day-old rabbits. Binding of iodinated Shiga toxin to globotriaosylceramide was documented on highperformance thin-layer chromatography plates by autoradiography. The glycolipid receptor for Shiga toxin in rabbit microvillus membranes is thus a hydroxylated fatty acid-containing globotriaosylceramide. This moiety is virtually absent in neonates and gradually increases with age. Quantitative differences in globotriaosylceramide may be the underlying basis for the age-specific differences in functional responsiveness of rabbit intestinal tissue to Shiga toxin.

Role of cyclic GMP in the action of heat-stable enterotoxin of Escherichia coli

ENTEROTOXIGENIC strains of Escherichia coli elaborate two enterotoxins, a heat-labile toxin (LT) and a heat-stable toxin (ST), which cause diarrhoeal disease in humans1; ST-producing E. coli cause diarrhoea in adult volunteers2 and have been associated with epidemic diarrhoea in a nursery for the newborn3 and with sporadic adult diarrhoea among North American tourists to Latin America4 and the Navajo people in Arizona5. LT-producing strains are identified by the ability of culture filtrates to cause fluid accumulation in rabbit ileal loops at 18 h (ref. 6) or by morphological alteration of Chinese hamster ovary (CHO) cells7 or of Y-l adrenal cells8. ST-producing strains are identified by the ability of culture filtrates to cause earlier fluid accumulation in rabbit ileal loops (peak accumulation at 6 h (ref. 6), or by fluid accumulation in the gut of the suckling mouse at 3 h (refs 9, 10). LT acts in a manner similar to cholera toxin (CT) by activating adenylate cyclase11. The mechanism of action of ST is unknown, however. Culture filtrates of a strain of E. coli that produced both LT and ST caused immediate net fluid secretion in canine jejunal segments without the 1-h delay characteristic of the response to CT12,13. In addition, culture filtrates caused an immediate increase in canine jejunal adenylate cyclase activity as measured by enzymatic generation of P32-cyclic AMP from P32-labelled ATP13, also in contrast to the delay in appearance of increased adenylate cyclase activity following exposure to CT12. The possibility that the early effect of ST was mediated by changes in cyclic nucleotide concentrations was investigated; culture filtrates of ST-producing strains of E. coli caused increased cyclic GMP concentrations in rabbit intestinal tissue and the cyclic GMP analogue 8BrcGMP mimicked ST in magnitude and time course of intestinal fluid accumulation in both rabbits and suckling mice.