W1648 Regulation of Apical Cl−/OH− Exchanger, SLC26A3, By Neuropeptide Y (NPY) Is Lipid Raft Dependent

Saksena, Seema; Tyagi, Sangeeta; Nayak, Shishira; Goyal, Sonia; Gill, Ravinder K.; Alrefai, Waddah A.; Ramaswamy, Krishnamurthy; Dudeja, Pradeep K.

doi:10.1016/s0016-5085(09)63272-8

Cited by 3 publications

(14 citation statements)

References 0 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Cell monolayers grown on Transwell inserts (5,000 cells/well) for 12 days were used for immunofluorescence staining. (31). For uptake studies with EPEC, cells were pretreated with specific inhibitors for 30 min and then infected with EPEC for 30 min in the presence or absence of the inhibitor.…”

Section: Methodsmentioning

confidence: 99%

“…Lipid rafts are cholesterol-and sphingolipid-rich membrane microdomains that serve as key docking complexes for signaling molecules and adaptor and membrane proteins influencing membrane protein trafficking (4). Previous studies from our group showed that DRA is associated with lipid rafts in IECs and that neuropeptide Y stimulated DRA function in Caco-2 cells by increasing its association with lipid raft fractions (31). Studies from Lissner et al (22) also indicate that DRA is associated with lipid rafts in an intracellular fraction, such as recycling vesicles, and that its exocytic insertion into the apical plasma membrane depends on raft integrity and phosphoinositide 3-kinase.…”

Section: C842 Dra Endocytosis/exocytosis and Epec Infectionmentioning

confidence: 99%

“…Previous studies showed that partitioning of DRA in the membrane lipid raft fraction increases DRA functional activity (22,31). We next examined if DRA is preferentially associated with lipid raft domains in response to treatment with dynasore.…”

Section: /Ohmentioning

confidence: 99%

“…The supernatant was referred to as the detergent-soluble fraction. The pellet was resuspended in a RIPA lysis buffer [5 mM HEPES (pH 7.4), 150 mM NaCl, 10 mM EDTA, 1 mM DTT, 1% Triton X-100, 0.1% SDS, and 1ϫ Complete protease inhibitor cocktail] and designated the detergent-insoluble fraction (1,31). The detergent-soluble and -insoluble fractions obtained after treatment of Caco-2 cells with dynasore (or infection with EPEC) were analyzed by Western blotting.…”

Section: Assessment Of CLmentioning

confidence: 99%

See 3 more Smart Citations

Mechanisms of DRA recycling in intestinal epithelial cells: effect of enteropathogenic E. coli

Gujral

Kumar

Priyamvada

et al. 2015

American Journal of Physiology-Cell Physiology

Self Cite

View full text Add to dashboard Cite

opathogenic Escherichia coli (EPEC) is a food-borne pathogen that causes infantile diarrhea worldwide. EPEC decreases the activity and surface expression of the key intestinal Cl Ϫ /HCO 3 Ϫ exchanger SLC26A3 [downregulated in adenoma (DRA)], contributing to the pathophysiology of early diarrhea. Little is known about the mechanisms governing membrane recycling of DRA. In the current study, Caco-2 cells were used to investigate DRA trafficking under basal conditions and in response to EPEC. Apical Cl Ϫ /HCO 3 Ϫ exchange activity was measured as DIDS-sensitive 125 I Ϫ uptake. Cell surface biotinylation was performed to assess DRA endocytosis and exocytosis. Inhibition of clathrin-mediated endocytosis by chlorpromazine (60 M) increased apical Cl Ϫ /HCO 3 Ϫ exchange activity. Dynasore, a dynamin inhibitor, also increased function and surface levels of DRA via decreased endocytosis. Perturbation of microtubules by nocodazole revealed that intact microtubules are essential for basal exocytic (but not endocytic) DRA recycling. Mice treated with colchicine showed a decrease in DRA surface levels as visualized by confocal microscopy. In response to EPEC infection, DRA surface expression was reduced partly via an increase in DRA endocytosis and a decrease in exocytosis. These effects were dependent on the EPEC virulence genes espG1 and espG2. Intriguingly, the EPEC-induced decrease in DRA function was unaltered in the presence of dynasore, suggesting a clathrin-independent internalization of surface DRA. In conclusion, these studies establish the role of clathrin-mediated endocytosis and microtubules in the basal surface expression of DRA and demonstrate that the EPEC-mediated decrease in DRA function and apical expression in Caco-2 cells involves decreased exocytosis. DRA recycling; EPEC infection; endocytosis; exocytosis; clathrin; microtubules DOWNREGULATED IN ADENOMA (DRA), or SLC26A3, is an integral membrane transporter expressed on the apical membrane of epithelial cells, including the small intestine and colon. Mutations in the SLC26A3 gene are associated with congenital chloride diarrhea (CLD), characterized by voluminous diarrhea with high fecal chloride content (28). DRA knockout mice exhibit severe diarrhea and a phenotype similar to that of CLD patients (32). Emerging studies indicate that posttranslational modifications of DRA protein may underlie the pathophysiology of diarrheal diseases. For example, CLD-causing mutations induce diarrhea by causing loss of functional DRA protein at the plasma membrane, possibly via its misfolding and mistrafficking (25,36). Studies from our laboratory have shown that enteropathogenic Escherichia coli (EPEC) infection decreases DRA function and surface levels (7), which may underlie early diarrhea; however, the cellular recycling mechanisms of DRA under normal or diseased conditions are not well understood. Thus, investigation of the cellular pathways responsible for maintaining appropriate DRA levels on the apical membrane is important for understanding disease pathogenesis...

show abstract

Section: Methodsmentioning

confidence: 99%

Section: C842 Dra Endocytosis/exocytosis and Epec Infectionmentioning

confidence: 99%

Section: /Ohmentioning

confidence: 99%

Section: Assessment Of CLmentioning

confidence: 99%

See 2 more Smart Citations

Mechanisms of DRA recycling in intestinal epithelial cells: effect of enteropathogenic E. coli

Gujral

Kumar

Priyamvada

et al. 2015

American Journal of Physiology-Cell Physiology

Self Cite

View full text Add to dashboard Cite

show abstract

“…Quite large and highly stably organized super-lipid rafts are present at the membrane of the brush border of enterocytes (79)(80)(81). Brush border membrane-associated lipid rafts contain transporters involved in the transcellular passage of fluid and electrolytes, such as NHEs 1 to 3 (82,83) and Cl Ϫ /HCO 3 Ϫ /OH Ϫ exchangers (84). The antagonistic effect of peptide AF-16 against the AAEC1845 psat -induced increase in fluid dome formation was abolished in cells pretreated with MBCD.…”

Section: Discussionmentioning

confidence: 99%

Antisecretory Factor Peptide AF-16 Inhibits the Secreted Autotransporter Toxin-Stimulated Transcellular and Paracellular Passages of Fluid in Cultured Human Enterocyte-Like Cells

Nicolas

Moal

2015

Infect Immun

View full text Add to dashboard Cite

Diarrheal diseases affect millions of people, and 2.5 million children under the age of 5 years die from these diseases every year (1). Diarrheal disease develops through a multifactorial process that counteracts the net absorption of water as the result of an increase in the secretion or a decrease in the absorption of water. In the intestinal tract, the passage of water across the epithelial barrier is tightly regulated by both the transcellular and paracellular movements of fluid and electrolytes. Transcellular passage develops through an asymmetric intracellular distribution of membrane-associated pumps and channels, whereas paracellular permeability is regulated by structural and functional proteins located at the tight junctions (TJs) (2). Enteric bacterial pathogens have developed sophisticated strategies to manipulate the host's normal water balance by producing both structural and functional changes in the epithelial barrier (3). In particular, enterovirulent Escherichia coli strains alter the structural organization of polarized epithelial cells and/or deregulate the functional systems involved in the regulation of the transcellular or paracellular passage of fluids and electrolytes in the intestinal epithelial barrier by the production of deleterious cytotoxic or cytotonic toxins (3).The gastrointestinal system uses a variety of antisecretory or proabsorptive hormonal and protein agonists to balance the outflow of fluid and electrolytes. Those that have been more extensively studied are neuropeptide Y/peptide YY (NPY/PYY) (4) and antisecretory factor (AF) (5). AF is a 41-kDa endogenous protein which was originally purified from the pig pituitary gland by Lön-nroth and Lange (6). Its gene has been cloned and sequenced (7). AF is phylogenetically well preserved, since it appears to be a single protein with several conformational variants (8), and no AF-like proteins have been reported. AF is present in most tissues, including the nasal, respiratory, urinary, and gastrointestinal mucosae (9, 10), and is secreted into plasma and other tissue fluids in mammals (11-13). AF appears in rat tissues after a challenge with cholera toxin (CT) or Clostridium difficile toxin (CDT) (14-16). A region of AF that supports its antisecretory activity has been identified between residues 36 and 51, located in the N-terminal part of the full-length protein (14,(17)(18)(19)(20). Experimentally, AF inhibits the intestinal secretion of fluids induced by a variety of toxins, including CT (6,7,14,17,[21][22][23][24][25][26], Campylobacter toxin (24, 27), Escherichia coli heat-labile enterotoxin (LT) (18, 23) and heatstable enterotoxins (ST) (23,25,28), CDT (14,15,21), and Dinophysis toxin (24). Moreover, AF and the AF peptide containing the active peptide sequence from residues 36 to 51 have been shown to block the out-in permeation of 36 Cl in nerve cell membranes isolated from rabbit Dieter cells (20,29,30). Clinically, AF appears to be effective, since administration of a medicinal food containing AF-rich egg yolk powder (B2...

show abstract

Air-Liquid-Interface Reorganizes Membrane Lipid and Enhance Recruitment of Slc26a3 to Lipid-Rich Domains in Human Colon

Tse

Rong

Zhang

et al. 2022

Preprint

View full text Add to dashboard Cite

Background and Aims Cholesterol-rich membrane domains, also called lipid rafts (LR), are specialized membrane domains that provide a platform for intracellular signal transduction. Membrane proteins often cluster in LR that further aggregate into larger platform-like structures that are enriched in ceramide and are called ceramide-rich platforms (CRPs). The role of CRPs in the regulation of intestinal epithelial functions remains unknown. Down Regulated in Adenoma (DRA) is an intestinal Cl-/HCO3- antiporter which is enriched in LR. However, little is known regarding the mechanisms involved in the regulation of DRA activity. Methods Air liquid interface (ALI) was created by removing apical media for a specified number of days from 12-14 days post confluency Caco-2/BBe cells or confluent colonoid monolayer grown as submerged cultures. Confocal imaging was used to examine the dimensions of membrane microdomains that contain DRA. Results DRA expression and activity were enhanced by culturing Caco-2/BBe cells and human colonoids using an ALI culture method. ALI causes an increase in acid sphingomyelinase (ASMase) activity, an enzyme responsible for enhancing ceramide content in the plasma membrane. ALI cultures expressed a larger number of DRA-containing platforms with dimensions >2 um compared to cells grown as submerged cultures. ASMase inhibitor, desipramine disrupted CRPs and reduced the ALI-induced increase in DRA expression in the apical membrane. Exposing normal human colonoid monolayers to ALI increased the ASMase activity and enhanced differentiation of colonoids along with enhancing basal and forskolin-stimulated DRA activity. Conclusions ALI increases DRA activity and expression by increasing ASMase activity and platform formation in Caco-2/BBe cells and by enhancing the differentiation of normal human colonoids.

show abstract

W1648 Regulation of Apical Cl−/OH− Exchanger, SLC26A3, By Neuropeptide Y (NPY) Is Lipid Raft Dependent

Cited by 3 publications

References 0 publications

Mechanisms of DRA recycling in intestinal epithelial cells: effect of enteropathogenic E. coli

Mechanisms of DRA recycling in intestinal epithelial cells: effect of enteropathogenic E. coli

Antisecretory Factor Peptide AF-16 Inhibits the Secreted Autotransporter Toxin-Stimulated Transcellular and Paracellular Passages of Fluid in Cultured Human Enterocyte-Like Cells

Air-Liquid-Interface Reorganizes Membrane Lipid and Enhance Recruitment of Slc26a3 to Lipid-Rich Domains in Human Colon

Contact Info

Product

Resources

About