Brush Border Of Epithelial Cells Research Articles

Identification of a Tyrosine-based Motif (YGSI) in the Amino Terminus of Nramp1 (Slc11a1) That Is Important for Lysosomal Targeting

Identification of a Tyrosine-based Motif (YGSI) in the Amino Terminus of Nramp1 (Slc11a1) That Is Important for Lysosomal Targeting

The Motogenic Effects of Cyclic Mechanical Strain on Intestinal Epithelial Monolayer Wound Closure Are Matrix Dependent

Complex deformation during normal digestion due to peristalsis or villous motility may be trophic for the intestinal mucosa. Because tissue fibronectin is increased in inflammatory states that may accompany mucosal injury, we evaluated the effects of cyclic mechanical strain and fibronectin on intestinal epithelial monolayer wound closure in Caco-2 and IEC-6 intestinal epithelial cells. Wounds created in intestinal epithelial monolayers were subjected to cyclic deformation. Wound closure was assessed by morphometry using microscopic imaging. Cell signals were assessed by Western blot and confocal microscopy. Mechanical strain stimulated wound closure on fibronectin but inhibited closure on collagen in Caco-2 and IEC-6 cells. The effect was independent of proliferation or cell spreading. Myosin light chain (MLC) and extracellular signal-regulated kinase (ERK) were phosphorylated in response to strain in confluent monolayers on both collagen and fibronectin. Blocking MLC or ERK phosphorylation inhibited the motogenic effect of strain on fibronectin. Although phosphorylated MLC was redistributed to the leading edge of migrating cells following 6 hours of strain on collagen and fibronectin, phosphorylated ERK was redistributed to the lamellipodial edge only on fibronectin. Strain promotes intestinal epithelial wound closure by a pathway requiring ERK and MLC kinase. Fibronectin-dependent ERK redistribution in response to strain in confluent migrating cells may explain the matrix dependence of the motogenic effect. Repetitive deformation stimulates intestinal epithelial proliferation on a collagen substrate, but not fibronectin. Deformation may exert matrix-dependent effects on intestinal epithelial cells, promoting epithelial restitution in fibronectin-rich tissue and proliferation in fibronectin-poor mucosa.

Lipid raft organization and function in brush borders of epithelial cells (Review)

Polarized epithelial cells of multicellular organisms confront the environment with a highly specialized apical cell membrane that differs in composition and function from that facing the internal milieu. In the case of absorptive cells, such as the small intestinal enterocyte and the kidney proximal tubule cell, the apical cell membrane is formed as a brush border, composed of regular, dense arrays of microvilli. Hydrolytic ectoenzymes make up the bulk of the microvillar membrane proteins, endowing the brush border with a huge digestive capacity. Several of the major enzymes are localized in lipid rafts, which, for the enterocyte in particular, are organized in a unique fashion. Glycolipids, rather than cholesterol, together with the divalent lectin galectin-4, define these rafts, which are stable and probably quite large. The architecture of these rafts supports a digestive/absorptive strategy for nutrient assimilation, but also serves as a portal for a large number of pathogens. Caveolae are well-known vehicles for internalization of lipid rafts, but in the enterocyte brush border, binding of cholera toxin is followed by uptake via a clathrin-dependent mechanism. Recently, 'anti-glycosyl' antibodies were shown to be deposited in the enterocyte brush border. When the antibodies were removed from the membrane, other carbohydrate-binding proteins, including cholera toxin, increased their binding to the brush border. Thus, anti-glycosyl antibodies may serve as guardians of glycolipid-based rafts, protecting them from lumenal pathogens and in this way be part of an ongoing 'cross-talk' between indigenous bacteria and the host.

PUTATIVE NITROXIDERGIC CELLS IN THE DIGESTIVE SYSTEM OF SOME MYTILIDS (MOLLUSCA: BIVALVIA: MYTILIDAE) REVEALED BY NADPH-DIAPHORASE HISTOCHEMISTRY

Distribution and morphology of putative nitroxidergic (NO-ergic) cells were studied in the labial palps, esophagus, stomach, digestive gland, and intestine of the bivalves Crenomytilus grayanus, Modiolus modiolus, and Mytilus coruscus using NADPH-histochemistry (Hope & Vincent, 1989). NO-producing elements were found in all examined organs and regions of the digestive system. NADPH-diaphorase-(d)-positive staining was readily detectable in neurons, secretory cells, brush border epithelial cells, and in the nervous plexuses. Intraepithelial nerve cells were the most common NADPH-d-positive cell type throughout the digestive tube. These cells had a fusiform perikaryon, which gave rise to a thin apical process that extended toward the gut lumen, whereas the basal process contacted the basiepithelial NADPH-d-positive plexus. In the major typhlosole of C. grayanus, these cells constituted up to 1.28% of the total number of epithelial cells. The bivalve species studied exhibit a similar distribution pattern of ...

Duodenal Carbonic Anhydrase: Mucosal Protection, Luminal Chemosensing, and Gastric Acid Disposal

The duodenum serves as a buffer zone between the stomach and jejunum. Over a length of only 25 cm, large volumes of strong acid secreted by the stomach must be converted to the neutral-alkaline chyme of the hindgut lumen, generating large volumes of CO2, which the duodenum then absorbs. The duodenal mucosa consists of epithelial cells connected by low-resistance tight junctions, forming a leaky epithelial barrier. Despite this high permeability, the epithelial cells, under intense stress from luminal mineral acid and highly elevated P(CO2), maintain normal functioning. Furthermore, the duodenum plays an active role in foregut acid-base homeostasis, absorbing large amounts of H+ and CO2 that are recycled by the gastric parietal cells. Prompted by the high expression of cytosolic and membrane carbonic anhydrase (CAs) in duodenal epithelial cells, and the intriguing observation that CA activity appears to augment cellular acid stress, we formulated a novel hypothesis regarding the role of CA in duodenal acid absorption, epithelial protection, and chemosensing. In this review, we will describe how luminal CO2/H+ traverses the duodenal epithelial cell brush border membrane, acidifies the cytoplasm, and is sensed in the subepithelium.

Mannan-Binding Protein Blocks the Activation of Metalloproteases Meprin α and β

Mannan-binding protein (MBP) is a C-type serum lectin that is known to be a host defense factor involved in innate immunity, and recognizes mannose, fucose, and N-acetylglucosamine residues. Although some exogenous MBP ligands have been reported, little is known about its endogenous ligands. In the present study, we found that endogenous MBP ligands are highly expressed in the brush border epithelial cells of kidney-proximal tubules by immunohistochemistry, and both meprin alpha and beta (meprins), as novel endogenous MBP ligands, have been identified through affinity chromatography and mass spectrometry. Meprins are membrane-bound and secreted zinc metalloproteases extensively glycosylated and highly expressed in kidney and small intestinal epithelial cells, leukocytes, and certain cancer cells. Meprins are capable of cleaving growth factors, extracellular matrix proteins, and biologically active peptides. Deglycosylation experiments indicated that the MBP ligands on meprins are high mannose- or complex-type N-glycans. The interaction of MBP with meprins resulted in significant decreases in the proteolytic activity and matrix-degrading ability of meprins. Our results suggest that core N-linked oligosaccharides on meprins are associated with the optimal enzymatic activity and that MBP is an important regulator for modulation of the localized meprin proteolytic activity via N-glycan binding. Because meprins are known to be some of the major matrix-degrading metalloproteases in the kidney and intestine, MBP, which functions as a natural and effective inhibitor of meprins, may contribute, as a potential therapeutic target, to tumor progression by facilitating the migration, intravasation, and metastasis of carcinoma cells, and to acute renal failure and inflammatory bowel diseases.

Low-dose cyclosporine A in a patient with X-linked immune dysregulation, polyendocrinopathy and enteropathy

Successful treatment with oral low-dose cyclosporine A given as a single daily dose is reported in a Japanese patient with immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome. Immune dysregulation, polyendocrinopathy and enteropathy is a rare and potentially fatal X-linked disease (IPEX, OMIM 304790). It is characterised by multiple autoimmune disorders in infancy, that warrants long-term aggressive immunosuppressive therapy [2, 3,6]; however, prolonged use of immunosuppressants may lead to direct toxicity [3]. We previously reported a Japanese infant with IPEX who presented with a novel mutation of the FOXP3gene [5]. The patient was successfully treated by early initiation of oral low-dose cyclosporine A (CsA) given as a single daily dose combined with low-dose prednisolone; for over 3 years he remained free of the both IPEX signs and therapyrelated toxicity. The patient reported here presented with severe secretory diarrhoea at the age of 2 months, and was diagnosed as having IPEX, based on the duodenal biopsy findings (total villous atrophy with crypt hyperplasia), presence of circulating anti-enterocyte antibody (by the immunohistochemical method using the patient’s serum and normal human ileal tissue: when the patient’s serum or control sera were applied to normal mucosa, only the patient’s serum stained the brush border of ileum epithelial cells) and results of sequence analysis of the FOXP3gene: a novel mutation of T1117G substitution in exon 10 [5]. Complete remission was achieved within a short time following prompt initiation of low-dose CsA (2 mg/kg, single daily dose, initially administered intravenously and later switched to oral administration of Neoral before breakfast), combined with prednisolone (0.5 mg/kg, daily). The peak blood levels of oral CsA (1 h post dosing) were maintained at around 700–800 ng/ml, while the trough blood levels of the drug were less than 25 ng/ml. No extra-intestinal signs were encountered, such as type 1 diabetes mellitus, pancreatitis, thyroiditis, haemolytic anaemia, glomerulonephritis or atopic dermatitis. The patient remained reasonably healthy thereafter and developed well under treatment with low-dose oral CsA given as a single daily dose and prophylactic intravenous gamma-globulin administration (200 mg/kg, administered monthly). The prednisolone dose could eventually be tapered to 0.25 mg/kg, daily.

Coming out of the NHERF family

In this issue of The Journal of Physiology, four individual reviews are presented that together made up a symposium entitled, ‘PDZ Domain Scaffolding Proteins and their Functions in Polarized Cells’, presented in San Diego, CA, USA on 4 April 2005 (Donowitz et al. 2005; Hernando et al. 2005; Thelin et al. 2005; Weinman et al. 2005). All presentations dealt with four multi PDZ domain containing proteins that are present in the brush border of mammalian small intestine, colon and renal proximal tubule. These proteins have between two and four PDZ domains each, and while all occur in epithelial cell brush borders, they have a widely divergent organ, cell and membrane, as well as species expression. The four proteins were cloned separately by multiple groups of scientists and consequently have multiple names, the most widely used of each being NHERF (Na/H exchanger regulatory factor), E3KARP (NHE3 kinase A regulatory protein), PDZK1 (PDZ domain protein from kidney 1) and IKEPP (intestinal and kidney epithelial PDZ domain protein). The authors of these symposium reviews include several of those who were responsible for cloning each of the four NHERF family members. As was reported at this meeting, these four proteins are all closely related. This relationship was determined from BLAST searches which demonstrated that the most closely homologous mammalian proteins for each of these proteins were the other three members of this family (Donowitz et al. 2005). Eleven of the twelve total PDZ domains present in these four proteins could be divided into three groups based on having high identity with a threshold of 20% (Donowitz et al. 2005; Thelin et al. 2005). Each of the 12 PDZ domains could have its origin traced to the same two PDZ domain containing proteins in C. elegans and/or either of two PDZ domain containing proteins in D. melanogaster (Donowitz et al. 2005). Thus we concluded that these four proteins are members of the same gene family. The original names of the first two cloned members of this family, NHERF and E3KARP, were meant to recognize the interactions of these proteins with the brush border Na/H exchanger isoform 3. However, since then ∼60 additional binding substrates of NHERF1 and NHERF2 have been recognized, with the number rapidly increasing and likely to be much larger. Nonetheless, given the widespread use of the NHERF nomenclature in the literature we suggest naming each protein as a member of the NHERF gene family. Moreover, we proposed that they be named to recognize this association in the order of their cloning, rather than using their many current designations, which are shown in parentheses: NHERF1 (NHERF, EBP50) (Donowitz et al. 2005; Hernando et al. 2005; Thelin et al. 2005; Weinman et al. 2005), NHERF2 (E3KARP, TKY-1, SIP-1) (Donowitz et al. 2005; Hernando et al. 2005; Thelin et al. 2005; Weinman et al. 2005), NHERF3 (PDZK1, CLAMP, CAP70, DIPHOR-1, NaPi-CaP1) (Donowitz et al. 2005; Thelin et al. 2005; Weinman et al. 2005) NHERF4 (IKEPP, DIPHOR-2, NaPiCaP2) (Donowitz et al. 2005; Hernando et al. 2005; Thelin et al. 2005; Weinman et al. 2005). A common name is proposed to allow recognition of their relationships in their primary structures and their similar expression pattern in many cell types. Given these similarities, scientists need to establish the specificity of effects attributed to one protein and to show that effects are not compensated by the presence of other NHERF family proteins. Moreover, more than one may be involved in any given regulatory process given their propensity to homo or heteromultimerize with each other. The symposium reviewed progress in understanding the functions of each of the four NHERF family members, with some emphasis on their roles in regulation of NHE3 and NaPiIIa activity, their ligands which have been studied in most detail. Given the importance of their many other ligands, an understanding of the role of this family of proteins is just beginning to be attained and these four reviews should be considered as early progress reports.

Intermediate Filaments Interact with Dormant Ezrin in Intestinal Epithelial Cells

Ezrin connects the apical F-actin scaffold to membrane proteins in the apical brush border of intestinal epithelial cells. Yet, the mechanisms that recruit ezrin to the apical domain remain obscure. Using stable CACO-2 transfectants expressing keratin 8 (K8) antisense RNA under a tetracycline-responsive element, we showed that the actin-ezrin scaffold cannot assemble in the absence of intermediate filaments (IFs). Overexpression of ezrin partially rescued this phenotype. Overexpression of K8 in mice also disrupted the assembly of the brush border, but ezrin distributed away from the apical membrane in spots along supernumerary IFs. In cytochalasin D-treated cells ezrin localized to a subapical compartment and coimmunoprecipitated with IFs. Overexpression of ezrin in undifferentiated cells showed a Triton-insoluble ezrin compartment negative for phospho-T567 (dormant) ezrin visualized as spots along IFs. Pulse-chase analysis showed that Triton-insoluble, newly synthesized ezrin transiently coimmunoprecipitates with IFs during the first 30 min of the chase. Dormant, but not active (p-T567), ezrin bound in vitro to isolated denatured keratins in Far-Western analysis and to native IFs in pull-down assays. We conclude that a transient association to IFs is an early step in the polarized assembly of apical ezrin in intestinal epithelial cells.

A Two-Photon FRAP Analysis of the Cytoskeleton Dynamics in the Microvilli of Intestinal Cells

The molecular structure of the brush-border of enterocytes has been investigated since the 1980s, but the dynamics of this highly specialized subcellular domain have been difficult to study due to its small size. To perform a detailed analysis of the dynamics of cytoskeleton proteins in this domain, we developed two-photon fluorescence recovery after photobleaching and a theoretical framework for data analysis. With this method, fast dynamics of proteins in the microvilli of the brush border of epithelial intestinal cells can be measured on the millisecond timescale in volumes smaller than 1μm3. Two major proteins of the cytoskeleton of the microvilli, actin and myosin 1a (Myo1a; formerly named brush border myosin I), are mobile in the brush-border of Caco-2 cells, an enterocyte-like cellular model. However, the mobility of actin is very different from that of Myo1a and they appear to be unrelated (diffusion coefficient of 15μm2 s−1 with a mobile fraction of 60% for actin, and 4μm2 s−1 with a mobile fraction of 90% for Myo1a). Furthermore, we show for the first time, in vivo, that the dynamics of Myo1a in microvilli reflect its motor activity.

Detection of Helicobacter pylori in gastric biopsy and resection specimens

To compare the sensitivity of detecting H. pylori in gastric biopsy and resection specimens using modified Giemsa stain and immunohistochemistry, using a commercially available anti-H. pylori antibody (Dako, Denmark). Gastric antral biopsy specimens showing chronic gastritis (28 cases) together with tissue blocks from gastrectomy specimens for duodenal ulcer (2 cases) were stained with modified Giemsa and immunoenzymatic alkaline phosphatase - anti-alkaline phosphatase (APAAP) method, and were carefully examined for the presence of H. pylori. Using a modified Giemsa stain, the spiral shaped bacteria of H. pylori stained blue, were attached to the brush border of the gastric foveolar epithelial cells. However, the specificity of modified Giemsa stain depended on the morphological appearance of H. pylori. The specificity of immunostaining permitted detection of low numbers or even single organisms. In all cases bacteria were more prominent and easier to detect in immunostained preparations. H. pylori was identified in 22 (73.3%) of 30 sections stained with modified Giemsa stain, but it could be identified with greater frequency in sections stained with APAAP, in 27 (90%) of 30 sections. Immunohistochemical identification of H. pylori was better than Giemsa stain for detecting that organism.

Proteomic analysis of plasma membrane vesicles isolated from the rat renal cortex

Polarized epithelial cells are responsible for the vectorial transport of solutes and have a key role in maintaining body fluid and electrolyte homeostasis. Such cells contain structurally and functionally distinct plasma membrane domains. Brush border and basolateral membranes of renal and intestinal epithelial cells can be separated using a number of different separation techniques, which allow their different transport functions and receptor expressions to be studied. In this communication, we report a proteomic analysis of these two membrane segments, apical and basolateral, obtained from the rat renal cortex isolated by two different methods: differential centrifugation and free-flow electrophoresis. The study was aimed at assessing the nature of the major proteins isolated by these two separation techniques. Two analytical strategies were used: separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) at the protein level or by cation-exchange high-performance liquid chromatography (HPLC) after proteolysis (i.e., at the peptide level). Proteolytic peptides derived from the proteins present in gel pieces or from HPLC fractions after proteolysis were sequenced by on-line liquid chromatography-tandem mass spectrometry (LC-MS/MS). Several hundred proteins were identified in each membrane section. In addition to proteins known to be located at the apical and basolateral membranes, several novel proteins were also identified. In particular, a number of proteins with putative roles in signal transduction were identified in both membranes. To our knowledge, this is the first reported study to try and characterize the membrane proteome of polarized epithelial cells and to provide a data set of the most abundant proteins present in renal proximal tubule cell membranes.

Characteristic expression of gamma-aminobutyric acid and glutamate decarboxylase in rat jejunum and its relation to differentiation of epithelial cells.

To investigate the expression between gamma-aminobutyric acid (GABA) and glutamate decarboxylase and its relation with differentiation and maturation of jejunal epithelial cells in rat jejunum. Immunohistochemical expression of GABA and glutamate decarboxylase (GAD, including two isoforms, GAD65 and GAD67) was investigated in rat jejunum. Meanwhile, double staining was performed with GAD65 immunohistochemistry, followed by lectin histochemistry of fluorescent wheat germ agglutinin. Furthermore, evaluation of cell kinetics in jejunum was conducted by (3)H-thymidine autoradiography and immunohistochemistry using a monoclonal antibody to proliferating cell nuclear antigen (PCNA). The cells showing positive immunoreactivity GABA and GAD65 were mainly distributed in the villi in rat jejunum, while jejunal epithelial cells were negative for GAD67. Positive GABA or GAD65 staining was mainly located in the cytoplasm and along the brush border of epithelial cells in the middle and upper portions. In addition, a few GABA and GAD65 strongly positive cells were scattered in the upper two thirds of jejunal villi. Double staining showed that GAD65 immunoreactivity was not found in goblet cells. (3)H-thymidine-labeled nuclei were found in the lower and middle portions of jejunal crypts, which was consistent with PCNA staining. Therefore, GABA and GAD65 were expressed in a maturation or functional zone. The characteristic expression of GABA and GAD suggests that GABA might be involved in regulation of differentiation and maturation of epithelial cells in rat jejunum.

Kidney injury molecule-1: a tissue and urinary biomarker for nephrotoxicant-induced renal injury.

Nephrotoxicity is a common side effect of therapeutic interventions, environmental insults, and exposure to toxicants in the workplace. Although biomarkers for nephrotoxicity are available, they often lack sensitivity and are not specific as indicators of epithelial cell injury. Kidney injury molecule-1 (Kim-1) is a type 1 membrane protein with extracellular immunoglobulin and mucin domains. The mRNA and protein for Kim-1 are expressed at very low levels in normal rodent kidney, but expression increases dramatically after injury in proximal tubule epithelial cells in postischemic rodent kidney and in humans during ischemic acute renal failure. To evaluate the utility of Kim-1 as a biomarker for other types of renal injury, we analyzed tissue and urinary expression in response to three different types of nephrotoxicants in the rat: S-(1,1,2,2-tetrafluoroethyl)-l-cysteine (TFEC), folic acid, and cisplatin. Marked increases in Kim-1 expression were confirmed by immunoblotting in all three models. The protein was shown to be localized to the proximal tubule epithelial cell by immunofluorescence. Furthermore, Kim-1 protein was detected in urine of toxicant-treated rats. The temporal pattern of expression in response to TFEC is similar to the Kim-1 expression pattern in the postischemic kidney. In folic acid-treated kidneys, Kim-1 is clearly localized to the apical brush border of the well-differentiated proximal tubular epithelial cells. After folic acid treatment, expression of Kim-1 is present in the urine despite no significant increase in serum creatinine. Cisplatin treatment results in early detection of urinary Kim-1 protein and diffuse Kim-1 expression in S3 cells of the proximal tubule. Kim-1 can be detected in the tissue and urine on days 1 and 2 after cisplatin administration, occurring before an increase in serum creatinine. The upregulation of expression of Kim-1 and its presence in the urine in response to exposure to various types of nephrotoxicants suggest that this protein may serve as a general biomarker for tubular injury and repair processes.

Expression of the iron transporter DMT1 in kidney from normal and anemic mk mice

DMT1 (Nramp2/DCT1) is the major apical iron transporter in absorptive cells of the duodenum, but also transports transferrin-iron across the membrane of acidified endosomes in peripheral tissues. DMT1 mRNA and protein expression has been detected in rat and mouse kidney, but its role at that site remains to be clarified. Immunoblotting and immunohistochemistry with specific affinity purified anti-DMT1 polyclonal antibodies were used to study expression and localization of DMT1 in mouse kidney. Possible regulation of DMT1 protein expression by the body iron stores also was examined in normal mice deprived of dietary iron, and in the genetically anemic mk mice that bear a loss of function mutation at DMT1 (G185R). In microsomal kidney fractions, DMT1 isoform I (encoded by the iron responsive element (IRE)-containing mRNA) is detected as an abundant 70 to 75 kD membrane protein. DMT1 is expressed in the cortex and not in the medulla, and is present at the brush border and apical pole of epithelial cells of proximal tubules. In contrast to the intestine, DMT1 protein expression in kidney is only slightly increased upon deprivation of dietary iron, suggesting different regulation at the two sites. In kidneys from mk/mk mice, the level of detectable DMT1(G185R) protein is drastically decreased compared to mk/+ controls. These results suggest that DMT1 may act as a re-uptake system for divalent cations at the brush border of kidney proximal tubules. A pathological mutation at DMT1 affects targeting/expression of the protein in the kidney.

Regulation of phospholipase C-gamma(1) by the actin-regulatory protein villin.

The actin-regulatory protein villin is tyrosine phosphorylated and associates with phospholipase C-gamma(1) (PLC-gamma(1)) in the brush border of intestinal epithelial cells. To study the mechanism of villin-associated PLC-gamma(1) activation, we reconstituted in vitro the tyrosine phosphorylation of villin and its association with PLC-gamma(1). Recombinant villin was phosphorylated in vitro by the nonreceptor tyrosine kinase c-src or by expression in the TKX1 competent cells that carry an inducible tyrosine kinase gene. Using in vitro binding assays, we demonstrated that tyrosine-phosphorylated villin associates with the COOH-terminal Src homology 2 (SH2) domain of PLC-gamma(1). The catalytic activity of PLC-gamma(1) was inhibited by villin in a dose-dependent manner with half-maximal inhibition at a concentration of 12.4 microM. Villin inhibited PLC-gamma(1) activity by sequestering the substrate phosphatidylinositol 4,5-bisphosphate (PIP(2)), since increasing concentrations of PIP(2) reversed the inhibitory effects of villin on PLC activity. The inhibition of PLC-gamma(1) activity by villin was reversed by the tyrosine phosphorylation of villin. Further, we demonstrated that tyrosine phosphorylation of villin abolished villin's ability to associate with PIP(2). In conclusion, tyrosine-phosphorylated villin associates with the COOH-terminal SH2 domain of PLC-gamma(1) and activates PLC-gamma(1) catalytic activity. Villin regulates PLC-gamma(1) activity by modifying its own ability to bind PIP(2). This study provides biochemical proof of the functional relevance of tyrosine phosphorylation of villin and identifies the molecular mechanisms involved in the activation of PLC-gamma(1) by villin.

Human duodenal mucosal brush border Na(+)/H(+) exchangers NHE2 and NHE3 alter net bicarbonate movement.

The proximal duodenal mucosa secretes HCO that serves to protect the epithelium from injury. In isolated human duodenal enterocytes in vitro, multiple luminal membrane proteins are involved in acid/base transport. We postulated that one or more isoforms of the Na(+)/H(+) exchanger (NHE) family is located on the apical surface of human duodenal mucosal epithelial cells and thereby contributes to duodenal mucosal HCO transport. Duodenal biopsies were obtained from human volunteers, and the presence of NHE2 and NHE3 was determined by using previously characterized polyclonal antibodies (Ab 597 for NHE2 and Ab 1381 for NHE3). In addition, proximal duodenal mucosal HCO(3)(-) transport was measured in humans in vivo in response to luminal perfusion of graded doses of amiloride; 10(-5)--10(-4) M amiloride was used to inhibit NHE2 and 10(-3) M amiloride to inhibit NHE3. Both NHE2 and NHE3 were localized principally to the brush border of duodenal villus cells. Sequential doses of amiloride resulted in significant, step-wise increases in net duodenal HCO(3)(-) output. Inhibition of NHE2 with 10(-5) M and 10(-4) M amiloride significantly increased net HCO(3)(-) output. Moreover, there was an additional, equivalent increase (P < 0.05) in duodenal HCO(3)(-) output with 10(-3) M amiloride, which inhibited NHE3. We conclude that 1) NHE2 and NHE3 are localized principally to the brush border of human duodenal villus epithelial cells; 2) sequential inhibition of NHE2 and NHE3 isoforms resulted in step-wise increases in net HCO(3)(-) output; 3) NHE2 and NHE3 participate in human duodenal villus cell HCO(3)(-) transport; and 4) the contribution of NHE-related transport events should be considered when studying duodenal HCO(3)(-) transport processes.

Ontogeny of P-Glycoprotein in Mouse Intestine, Liver, and Kidney

BackgroundP-glycoprotein (Pgp) is an ATP-dependent, integral plasma-membrane efflux pump that is constitutively expressed on (i) adult apical brush-border epithelial cells of the intestine, (ii) the bile canalicular face of hepatocytes,...

Cystinuria type I: Identification of eight new mutations in SLC3A1

Cystinuria is a heritable disorder of amino acid transport characterized by the defective transport of cystine and the dibasic amino acids through the brush border epithelial cells of the renal tubule and intestine tract. Three types of cystinuria (I, II, and III) have been described based on the urinary excretion of cystine and dibasic amino acids in obligate heterozygotes. The SLC3A1 gene coding for an amino acid transporter named rBAT is responsible for type I cystinuria, whereas the SLC7A9 gene coding for a subunit (b0,+AT) of rBAT is involved in determining non-type I (types II and III) cystinuria. The SLC3A1 gene sequence was investigated in a sample of seven type I/type I, three type I/non-type I, six type I/untyped, and four untyped unrelated cystinuric patients by RNA single-strand conformation polymorphism (RNA-SSCP). Eight new point mutations (S168X, 765+1G>T, 766-2A>G, R452Q, Y461X, S547W, L564F, and C673W) and seven previously reported mutations were detected. These new mutations increase the number of mutated alleles so far characterized in SLC3A1 to 62. We have found SLC3A1 mutations in 0.739 of the type I chromosomes studied. The relatively high proportion of uncharacterized type I chromosomes suggests either that there may be mutations not yet found in SLC3A1 or that many of the assigned type I chromosomes in mixed type I/non-type I patients may have mutations in SLC7A9. If the hypothesis is excluded in the future, we believe that a third gene may be involved in cystinuria.

Distribution of type-1 and type-2 angiotensin receptors in the normal human lung and in lungs from patients with chronic obstructive pulmonary disease.

This study was designed to examine the cellular distribution of the angiotensin II type-1 (AT1) and type-2 (AT2) receptors in the normal human and pathological human lung. Riboprobes were prepared against specific portions of each receptor DNA and labelled with FITC for detection using an anti-FITC antibody in combination with the alkaline phosphatase-anti-alkaline phosphatase technique and new Fuchsin. These were used to detect the presence of receptor mRNA in the lung. Specific antibodies were used to detect receptor protein in cells by immunocytochemistry. Image analysis was used in order to semi-quantify receptor density. AT1 receptor mRNA and protein were localised on vascular smooth muscle cells, macrophages and in the stroma underlying the airways epithelium probably relating to underlying fibroblasts. The AT1 receptor protein was not expressed in the epithelium although there was a low level of mRNA. In contrast, AT2 receptor RNA and protein was observed in the epithelium, with strong staining on the bronchial epithelial cell brush border and also on many of the underlying mucous glands. The AT2 receptor was also present on some endothelial cells. These findings were supported by the presence of mRNA in each case. In patients with chronic obstructive pulmonary disease, there was a five- to sixfold increase in the ratio of AT1 to AT2 receptors in the regions of marked fibrosis surrounding the bronchioles. This correlated well with the reduced lung function as expressed by the forced expiratory volume.