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Results for “Intestinal Barrier Function”

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PubMed1999

Activation and peripheral expansion of murine T-cell receptor gamma delta intraepithelial lymphocytes.

BACKGROUND & AIMS: The intestinal epithelial compartment is populated by CD8(+) alpha beta and gamma delta intraepithelial lymphocytes (IELs), which monitor the integrity of the epithelial barrier. alpha beta IELs are activated by peptide antigens presented by class I major histocompatibility complex (MHC) molecules, but it is unclear how gamma delta IELs are activated. METHODS: G8 T-cell receptor (TCR) gamma delta transgenic (Tg) mice (specific for the class I MHC alloantigen, T22/10(b)) were crossed to class I MHC-deficient beta2-microglobulin-knockout (beta2m degrees) mice, and Tg+ IELs were examined for relative yields and surface and functional phenotype. RESULTS: Evidence for class I MHC-induced activation of Tg+ IELs was supported by the detection of 4-fold greater numbers of Tg+ IELs in G8 x beta2m+ mice that proliferated at 15-fold higher levels than IELs from G8 x beta2m degrees mice. However, expression of CD69, production of cytokine (interleukin 2 and interferon gamma), and detection of cytolytic function for IELs in G8 x beta2m degrees mice suggested that class I MHC was not required for gamma delta IEL development or maturation. CONCLUSIONS: These results suggest that CD8(+) TCR gamma delta IELs do not require class I MHC for development but support the notion that antigens presented by class I MHC molecules are involved in the peripheral expansion and differentiation of this subset.

Animals↗

PubMed2000

Expression from the human occludin promoter is affected by tumor necrosis factor alpha and interferon gamma.

The 65 kDa protein occludin is a membrane-spanning part of the epithelial tight junction, which is the main barrier of the paracellular pathway. The function of occludin as part of tight junctions is still poorly understood and even less is known about the regulatory mechanisms that influence occludin gene expression. This study aimed to identify the sequences essential in cis for genomic regulation of tight junction formation and to investigate their funcional role in cytokine-dependent tight junction regulation. Using genome walking cloning of occludin-specific human genomic DNA sequences, a 1853 bp DNA fragment containing the transcription start point of occludin cDNA sequences was amplified and sequenced. Subcloning of this fragment in front of the luciferase reporter gene revealed strong expression of enzymatic activity after transfection of the human intestinal cell line HT-29/B6. With subsequent deletions of parts of the promoter fragment, its size was reduced to 280 bp that are necessary and sufficient to mediate promoter activity. Tumor necrosis factor alpha and another cytokine involved in inflammation, interferon gamma, reduced transepithelial resistance in HT-29/B6 cells, which was preceded by a decrease in occludin mRNA expression as revealed by northern blot analysis. Tumor necrosis factor alpha and interferon gamma diminished occludin promoter activity alone and even synergistically, suggesting a genomic regulation of alterations of the paracellular barrier. In conclusion, proinflammatory cytokines such as tumor necrosis factor alpha and interferon gamma can downregulate the expression of the transmembrane tight junction strand protein occludin, paralleling the barrier disturbance detected electrophysiologically. This could be an important mechanism in gastrointestinal diseases accompanied by barrier defects, for example inflammatory bowel diseases.

Base Sequence↗

PubMed2004

High-molecular-weight polyethylene glycol prevents lethal sepsis due to intestinal Pseudomonas aeruginosa.

BACKGROUND & AIMS: During stress, erosion of protective intestinal mucus occurs in association with adherence to and disruption of the intestinal epithelial barrier by invading opportunistic microbial pathogens. The aims of this study were to test the ability of a high-molecular-weight polyethylene glycol compound, polyethylene glycol 15-20, to protect the intestinal epithelium against microbial invasion during stress. METHODS: The ability of polyethylene glycol 15-20 to protect the intestinal epithelium against the opportunistic pathogen Pseudomonas aeruginosa was tested in cultured Caco-2 cells. Bacterial virulence gene expression, bacterial adherence, and transepithelial electrical resistance were examined in response to apical inoculation of P. aeruginosa onto Caco-2 cells. Complementary in vivo studies were performed in a murine model of lethal sepsis due to intestinal P. aeruginosa in which surgical stress (30% hepatectomy) was combined with direct inoculation of P. aeruginosa into the cecum. RESULTS: High-molecular-weight polyethylene glycol (polyethylene glycol 15-20) conferred complete protection against the barrier-dysregulating effects of P. aeruginosa in Caco-2 cells. Intestinal application of polyethylene glycol 15-20 in stressed mice protected against the lethal effects of intestinal P. aeruginosa. Mechanisms of this effect seem to involve the ability of polyethylene glycol 15-20 to distance P. aeruginosa from the intestinal epithelium and render it completely insensate to key environmental stimuli that activate its virulence. CONCLUSIONS: High-molecular-weight polyethylene glycol has the potential to function as a surrogate mucin within the intestinal tract of a stressed host by inhibiting key interactive events between colonizing microbes and their epithelial cell targets.

Animals↗

PubMed2002

Role of VASP in reestablishment of epithelial tight junction assembly after Ca2+ switch.

Epithelial permeability is tightly regulated by intracellular messengers. Critical to maintaining barrier integrity is the formation of tight junction complexes. A number of signaling pathways have been implicated in tight junction biogenesis; however, the precise molecular mechanisms are not fully understood. A growing body of evidence suggests a role for intracellular cAMP in tight junction assembly. Using an epithelial model, we investigated the role of cAMP signal transduction in barrier recovery after Ca2+ switch. Our data demonstrate that elevation of intracellular cAMP levels significantly enhanced barrier recovery after Ca2+ switch. Parallel experiments revealed that epithelial barrier recovery is diminished by H-89, a specific and potent inhibitor of cAMP-dependent protein kinase (protein kinase A) activity. Of the possible PKA effector proteins, the vasodilator-stimulated phosphoprotein (VASP) is an attractive candidate, since it has been implicated in actin-binding and cross-linking functions. We therefore hypothesized that VASP may play a role in the cAMP-mediated regulation of epithelial junctional reassembly after Ca2+ switch. We demonstrate here that VASP is phosphorylated via a PKA-dependent process under conditions that enhance barrier recovery. Confocal laser scanning microscopy studies revealed that VASP localizes with ZO-1 at the tight junction and at cell-cell borders and that phospho-VASP appears at the junction after Ca2+ switch. Subsequent transfection studies utilizing epithelial cells expressing truncated forms of VASP abnormal in oligomerization or actin-binding activity revealed a functional diminution of barrier recovery after Ca2+ chelation. Our present studies suggest that VASP may provide a link between cAMP signal transduction and epithelial permeability.

Blotting, Western↗

PubMed2000

Superantigen immune stimulation activates epithelial STAT-1 and PI 3-K: PI 3-K regulation of permeability.

Signal transducers and activators of transcription (STATs) are critical intracellular signaling molecules for many cytokines. We compared the ability of T84 epithelial cells to activate STATs in response to cytokines [interferon-gamma (IFN-gamma), interleukin (IL)-4, IL-10, and tumor necrosis factor-alpha (10 ng/ml)] and conditioned medium from superantigen [Staphylococcus aureus enterotoxin B (SEB)]-activated peripheral blood mononuclear cells (PBMC) using electrophoretic mobility shift assays (EMSA). Of the cytokines tested, only IFN-gamma caused a STAT-1 response. Exposure to SEB-PBMC-conditioned medium resulted in STAT-1 or STAT-1/3 activation, and inclusion of anti-IFN-gamma antibodies in the conditioned medium abolished the STAT-1 signal. Cells treated with transcription factor decoys, DNA oligonucleotides bearing the STAT-1 recognition motif, and then SEB-PBMC-conditioned medium displayed a reduced STAT-1 signal on EMSA, yet this treatment did not prevent the drop in transepithelial resistance (measured in Ussing chambers) caused by SEB-PBMC-conditioned medium. In contrast, the phosphatidylinositol 3'-kinase (PI 3-K) inhibitor LY-294002 significantly reduced the drop in transepithelial resistance caused by SEB-PBMC-conditioned medium. Thus data are presented showing STAT-1 (+/-STAT-3) and PI 3-K activation in epithelial cells in response to immune mediators released by superantigen immune activation. Although the involvement of STAT-1/-3 in the control of barrier function remains a possibility, PI-3K has been identified as a regulator of T84 paracellular permeability.

Antineoplastic Agents↗

PubMed1995

[Dendritic resident cells and their immunohistologic determination].

Resident dendritic cells exist in various tissues. They belong to RES according to determining marks and their transitions. The main marks represent phagocytosis, fixed and mobile scavenging, endothelial and stroma-parenchyma barrier function and collagens production. Suppression of some extreme marks stimulates prevalence of remaining marks. An interesting topic concerns immune engaged dendritic cells. Their main mark is inability of transporting antigens and of presenting them to lymphatic tissue so that they start an immune reaction. Differences among dendritic cells can be detected by immunohistology. Immunopositivity of resident dendritic cells was studied in 20 tissues (lymphatic, skin, bone, soft, nervous, myocardial, lung, oesophagus, stomach, intestinal and others) by using antibodies: CD 34, F XIIIa, F VIII, actin, CD 68, S-100 protein, HLA-DR, CD3, and OPD4. Because a simple comparison of detected immunophenotypes was often ambiguous, positivity of dendritic cells was compared with positivity level of neighbouring tissues in 16 cases. Nevertheless, any single marker was not relevant in all cases. But decisive positive combinations did exist (CD 34 for non-X cells, CD 68 for phagocytes, S-100 for X cells). Dendritic cells in different and often opposite relations were found in a group of 36 cases. Diversity of tissue representation of "immunosupervising" dendritic cells reflected probably a frequence and quality of antigen stimulation in locally specific immune conditions. A surprising presence of CD 34 positive dendritic cells in border-line and stromal structures of nerves and in their tumours contributed to differential diagnosis of neurofibromas and some storiform tumours. CD 34 positivity alone does not solve problems sufficiently and applied methods will not do for determination of the share of "immunosupervising" cells in stromal reaction of malignant tumours.

Antigens, CD↗

PubMed1996

[The effect of a protein concentrate from milk whey and its fractions on the macromolecular permeability of the intestinal barrier in rats with experimental food anaphylaxis].

Rats received dietary supplement of milk whey concentrate (KSP) being manufactured from skimmed milk by addition of apple pectin (Pec), KSP ultrafiltrated fractions and pure Pec during 21 day. A part of animals were sensitized i/p with chicken ovalbumin and finally subjected to systemic anaphylaxis (SA) by i/v antigen challenge. Gastrointestinal barrier macromolecular permeability (MP) for polyethylene glycol 4000 (PEG-4000) was measured by its urinary excretion after an intragastric load. MP increased significantly in nonsensitized rats after feeding with KSP and its high molecular (HM) fraction but not after feeding with low molecular (LM) KSP fraction compared to NaCl fed group. Feeding with Pec dramatically (5-fold) increased permeability. On the contrary the permeability decreased in sensitized rats subjected to SA when fed with KSP, its LM and particularly HM fraction the later group permeability being normalized down to normal value found in nonsensitized animals. It's concluded that KSP whey protein may be recommended for incorporation into specialized foods for patients with impaired gastrointestinal function.

Anaphylaxis↗

PubMed1978

Absence of Na+--H+ barrier function in mucosa of canine small bowel.

The study was designed to determine whether the special Na+--H+ barrier function of the gastric mucosa is present in the mucosa of the small bowel and whether a gastric mucosal barrier breaker (hexanoic acid) would accelerate the fluxes of sodium in duodenum-jejunum and ileum as in the stomach. The observations were made in healthy conscious dogs with Thiry-Vella fistulae of the small bowel or Heidenhain pouches of the gastric corpus. These barrier characteristics of the stomach were completely absent in the small intestine where bidirectional Na fluxes were 5--10 times greater than in the stomach and were not accelerated by hexanoic acid as they were in the stomach. A comparison was made between the rates of absorption of hexanoic acid, sodium hexanoate, and HCl from the pouches and fistulae. The lipid-soluble fatty acid was transported at all sites more rapidly than its water-soluble sodium salt. In the stomach and ileum the H+ of HCl and sodium hexanoate were absorbed at similar slow rates. The duodenal-jejunal mucosa, however, transported H+ at rates nearly identical to those of hexanoic acid. In our tests HCl was not neutralized in duodenal contents while large quantities were neutralized in the contents of ileum.

Animals↗

PubMed1998

Interactions between bacterial toxins and intestinal cells.

Bacterial toxins which act on intestinal cells display a great diversity of size, structure and mode of action. Some toxins interact with the cell by transducing a signal across the membrane leading to stimulation of intracellular second messenger (E. coli heat stable enterotoxin), others form pores (C. perfringens enterotoxin, ...) permitting the leakage of cellular components and cell lysis. The most sophisticated toxins comprise at least two functional domains or components, one being a binding domain permitting the internalization into the cell of an enzymatic domain which modifies an intracellular target. The enzymatic modification (ADP-ribosylation, UDP-glucosylation, glycohydrolysis, proteolysis, ...) of a specific target (heterotrimeric G-protein, small G-protein, monomeric actin, ribosomal RNA, ...) alters the cell physiology (increase of ions and water secretion, cytoskeleton rearrangement, protein synthesis inhibition, apoptosis, ...) and tissue organization (modification of barrier permeability, necrosis, ...). The study of bacterial toxins leads to the understanding of the interactions between pathogenic bacteria and their hosts and constitutes also a new approach in cell biology, by facilitating the exploration of certain regulatory pathways such as that controlling actin polymerization.

Animals↗

PubMed1990

Characterization of the barrier properties of mucosal membranes.

The barrier properties of the nasal, rectal, and vaginal membranes were investigated in the rabbit by comparing physical characteristics and permeability to model hydrophilic and lipophilic compounds. It was found that the nasal, rectal, and vaginal mucosae differ in their physical characteristics and interactions. The nasal mucosa had the highest in vitro transport of both the model hydrophilic compound, mannitol, and the model lipophilic compound, progesterone. Although the nasal mucosa was found to have some hydrophilic character, it appears that these mucosae are primarily lipophilic in nature. The role of the lipid domain in the barrier properties of these mucosal membranes was investigated using progesterone and a series of its hydroxyprogesterone analogues. Membrane permeability was found to decrease as the order of hydrophilicity of the progestins increased, indicating that the lipid domain plays an important role in the barrier function of these membranes. The changes observed in the physical properties and permeability of the mucosal membranes after extraction of the lipids also demonstrated the importance of the lipid domain in the maintenance of the mucosal barrier.

Animals↗

PubMed1999

Typhlitis.

OBJECTIVE: To provide an overview of the pathophysiological features and management of the clinical entity typhlitis. DATA SOURCES AND STUDY SELECTION: The data presented are derived from a review of the English-language literature on typhlitis. The majority of papers analysed were small clinical series. DATA EXTRACTION AND SYNTHESIS: Data derived from the literature review were collated. The major finding was that typhlitis comprises a number of different diseases characterized by the presence of right lower quadrant pain, an immunocompromised host and altered function of the mucosal barrier of the right colon. CONCLUSIONS: Typhlitis should be suspected in any immunocompromised patient presenting with right lower quadrant pain with compatible radiographic findings. Most patients can be treated conservatively with intravenously administered fluids and antibiotics, although surgery may be necessary if complications arise.

Enterocolitis↗

PubMed2000

Gastroprotection of DNA with a synthetic cholic acid analog.

The oral delivery of functional DNA to the gastrointestinal system would constitute a desirable, noninvasive method for potentially treating a variety of diseases. The digestive process, however, remains a formidable barrier. This dilemma may be addressed by using targeted liposomes both to protect the polynucleotide and to deliver the therapeutic DNA with high tissue specificity. The present study represents the initial steps toward developing a novel gene delivery system designed to interact with the enterohepatic receptors of the small intestine. Two cholic acid esters were synthetically modified at position C(3) to incorporate a DNA-binding domain. These novel compounds were evaluated for their ability to protect DNA from the nucleases found in gastrointestinal segments. Additionally, the compounds were screened as a component of a gene delivery vector. Formulations containing the new bile salt derivatives protected DNA from degradation for more than 2 h and were capable of transfecting cultured NIH 3T3 cells.

3T3 Cells↗

PubMed2005

Spondyloarthritis: update on pathogenesis and management.

A great deal of progress has occurred in the past few years in elucidating the causes and designing new treatments for ankylosing spondylitis and other types of spondyloarthritis. In addition to the human leukocyte antigen (HLA)-B27 and other major histocompatibility complex (MHC) genes, chromosomal regions and genes elsewhere in the genome are being implicated both in disease susceptibility and severity. The various ways HLA-B27 may function in causing spondyloarthritis now are better understood to encompass not only antigen presentation but also other mechanisms, possibly all being operative in pathogenesis (misfolding of the HLA-B27 molecule, impaired intracellular killing of bacteria, and HLA-B27 itself serving as an autoantigen). Specific enteric and sexually acquired infections can trigger reactive arthritis, though no specific microbe has been identified in other forms of spondyloarthritis. Intestinal inflammation with impairment of the gut:blood barrier may be operative in driving ankylosing spondylitis and enteropathic arthritis. A number of treatments have been tried in spondyloarthritis, including older agents such as methotrexate and sulfasalazine but also newer drugs such as pamindronate. The recent introduction of tumor necrosis factor (TNF) blockers in the treatment of spondyloarthritis has offered the most hope in not only relieving symptoms and signs of both peripheral arthritis and enthesitis but also spinal disease, which often has been refractory to other agents. Their high cost and considerable side effect profile, however, have necessitated the establishment of guidelines for their use in these diseases in order to target the patient in whom they are likely to have the most benefit.

Anti-Bacterial Agents↗

PubMed2026

Bacterial Outer Membrane Vesicles in Colorectal Cancer: Interdomain Communication Hubs in Pathogenesis and Immunotherapy.

The complex interaction between the intestinal microbiota and host mucosal immunity plays a defining role in colorectal cancer (CRC) development and therapeutic outcomes. Recently, bacterial outer membrane vesicles (OMVs)-nano-sized, lipid-bilayered extracellular particulates released by both commensal and pathogenic microorganisms-have emerged as critical long-range signaling vehicles within the gut. This review provides a comprehensive synthesis of the dual functionalities of OMVs in CRC pathogenesis and oncology. Mechanistically, pathogenic OMVs cross compromised mucosal barriers to drive horizontal gene transfer (HGT) of chimeric episomes, such as SPHINX DNAs and Bovine Meat and Milk Factors (BMMFs), thereby promoting genomic instability and neoplastic transformation. Conversely, there is a paradigm shift toward exploiting next-generation probiotic and engineered OMVs as highly tunable therapeutic platforms. By combining cutting-edge bioengineering strategies-such as biomimetic mineralization to neutralize local tissue acidity and chemotherapeutic packaging-these nanovectors effectively reprogram the immunosuppressive tumor microenvironment (TME). Specifically, optimized OMVs modulate macrophage polarization from an M2 to an M1 phenotype and stimulate CXCL10-mediated CD8+ T-cell infiltration, effectively turning immunologically "cold" tumors "hot." Finally, the great translational challenges regarding systemic endotoxicity, scalability, and target delivery, providing a strategic approach for the integration of OMV-based platforms into synergistic immune checkpoint inhibition regimens.

Colon cancer↗

PubMed1986

Simulation of the metabolism and enterohepatic circulation of endogenous chenodeoxycholic acid in man using a physiological pharmacokinetic model.

The metabolism and enterohepatic circulation of chenodeoxycholic acid (CDC), a major primary bile acid in man, has been stimulated using a multicompartmental physiological pharmacokinetic model which was previously reported and used to simulate the metabolism of cholic acid. The model features compartments and linear transfer coefficients. Compartments, which are defined as the pools of single chemical species in well defined anatomical volumes, are aggregated into nine 'spaces' based on anatomical and physiological considerations (liver, gall-bladder, bile ducts, duodeno-jejunum, ileum, colon, portal blood, sinusoidal blood, and general circulation). Each space contains several compartments which correspond to the compounds present in that space, for example, the compound in question and its biotransformation products. For CDC (as for cholic acid in the previous simulation) each space contains three compartments corresponding to the unconjugated bile acid, its glycine amidate, and its taurine amidate. Transfer coefficients, which denote the fractional amount of the compartment's contents exiting per unit time, are categorized according to function: flow, for example gall-bladder contraction (which involves transfer of all substances contained in the space at the same fractional rate); biotransformation (which transfers the substrate from one compartment to another within the same space); or transport (which denotes movements between contiguous compartments, belonging to different spaces across a diffusion membrane or a cellular barrier). The model is made time-dependent by incorporating meals which trigger gall-bladder emptying and modify intestinal flow. The transfer coefficients in the cholic acid model were modified for the CDC model since there is indirect evidence that CDC amidates (probably chenodeoxycholylglycine) are absorbed from the duodeno-jejunum and the first pass hepatic clearance of CDC species differs from that of cholyl species. The model was then used with all existing experimental data to simulate CDC metabolism in healthy humans over a 24-h period during which three meals were ingested. Satisfactory agreement was obtained between simulated and experimental data indicating that this model continues to be useful for describing the metabolism of bile acids and may also be of value for describing the metabolism of drugs whose metabolism is similar to that of bile acids.

Bile Acids and Salts↗

PubMed2004

Salmonella typhimurium SipA-induced neutrophil transepithelial migration: involvement of a PKC-alpha-dependent signal transduction pathway.

Salmonella typhimurium elicits an intense proinflammatory response characterized by movement of polymorphonuclear neutrophils (PMN) across the epithelial barrier to the intestinal lumen. We previously showed that S. typhimurium, via the type III secretion system effector protein SipA, initiates an ADP-ribosylation factor-6- and phospholipase D-dependent lipid-signaling cascade that directs activation of protein kinase C (PKC) and subsequent transepithelial movement of PMN. Here we sought to determine the specific PKC isoforms that are induced by the S. typhimurium effector SipA in model intestinal epithelia and to link the functional consequences of these isoforms in the promotion of PMN transepithelial migration. In vitro kinase PKC activation assays performed on polarized monolayers of T84 cells revealed that S. typhimurium and recombinant SipA induced activation of PKC-alpha, -delta, and -epsilon. To elucidate which of these isoforms play a key role in mediating epithelial cell responses that lead to the observed PMN transepithelial migration, we used a variety of PKC inhibitors with different isoform selectivity profiles. Inhibitors selective for PKC-alpha (Gö-6976 and 2,2',3,3',4,4'-hexahydroxyl-1,1'-biphenyl-6,6'-dimethanoldimethyl ether) markedly reduced S. typhimurium- and recombinant SipA-induced PMN transepithelial migration, whereas inhibitors to PKC-delta (rottlerin) or PKC-epsilon (V1-2) failed to exhibit a significant decrease in transepithelial movement of PMN. These results were confirmed biochemically and by immunofluorescence coupled to confocal microscopy. Our results are the first to show that the S. typhimurium effector protein SipA can activate multiple PKC isoforms, but only PKC-alpha is involved in the signal transduction cascade leading to PMN transepithelial migration.

ADP-Ribosylation Factor 6↗

PubMed2006

In vivo tumor targeting using a novel intestinal pathogen-based delivery approach.

Efficient methods for tumor targeting are eagerly awaited and must satisfy several challenges: molecular specificity, transport through physiologic barriers, and capacity to withstand extracellular or intracellular degradation and inactivation by the immune system. Through interaction with its hosts, the intestinal pathogen-produced Shiga toxin has evolved molecular properties that are of interest in this context. Its nontoxic B-subunit binds to the cellular toxin receptor, glycosphingolipid Gb3, which is highly expressed on human cancers and has recently been reported to be involved in the formation of metastasis in colorectal cancers. Its function as a target for cancer therapy has already been addressed in xenograft experiments. We here show that after oral or i.v. injections in mice, the B-subunit targets spontaneous digestive Gb3-expressing adenocarcinomas. The nontumoral mucosa is devoid of labeling, with the exception of rare enteroendocrine and CD11b-positive cells. As opposed to other delivery tools that are often degraded or recycled on cancer cells, the B-subunit stably associates with these cells due to its trafficking via the retrograde transport route. This can be exploited for the in vivo delivery of contrast agents to tumors, as exemplified using fibered confocal fluorescence endoscopy and positron emission tomography (PET) imaging. In conclusion, the data presented in this manuscript lay the groundwork for a novel delivery technology that, in addition to its use for molecular imaging applications such as noninvasive PET, could also be exploited for targeted tumor therapies.

Adenocarcinoma↗

PubMed2004

Characteristics of cultured subepithelial fibroblasts in the rat small intestine. II. Localization and functional analysis of endothelin receptors and cell-shape-independent gap junction permeability.

Subepithelial fibroblasts form a cellular network with gap junctions under the epithelium of the gastrointestinal tract. Previously, we have reported their unique characteristics, such as reversible rapid cell-shape changes from a flat to a stellate configuration induced by dBcAMP and endothelins (ETs), and Ca2+ responses to, for example, ETs, ATP, and substance-P. We have now investigated the subtypes of ET receptors both in the rat small intestine and in primary cultured subepithelial fibroblasts isolated from rat duodenal villi. Their properties were compared between wild-type and endothelin-B-receptor-mutant sl/sl rats. Light- and electron-microscopic immunohistochemistry showed intense ETA immunoreactivity in the subepithelial fibroblasts from the small intestine and colon of both wild-type and sl/sl rats. In culture, immunocytochemistry, reverse transcription/polymerase chain reaction analysis, Ca2+ response measurements, and cell-shape change analysis indicated functional ETA and ETB receptors in the wild-type cells, but only ETA in the sl/sl cells. However, wild-type cells were more sensitive to ET-1 than to ET-3 by about one order of magnitude. ETA seemed to be dominant both in vivo and in vitro. The relationship between cell-shape change and gap junction permeability was examined by fluorescence recovery after photobleaching; the gap junctions were usually open but were blocked by carbenoxolone. Permeability did not change significantly with cell-shape change. This network of differentiated subepithelial fibroblasts may maintain intercellular communication via gap junctions to transduce signals evoked in the local network to the whole network. The cell-shape change of the cells through ETA activation may play an important role as a barrier and for intercellular signaling in the intestinal villi.

Animals↗