PMCC PMCC

Search tips
Search criteria

Advanced
Results 1-7 (7)
 

Clipboard (0)
None

Select a Filter Below

Journals
Year of Publication
Document Types
1.  Functional Activity of Pat-1 (Slc26a6) Cl−/HCO3− Exchange in the Lower Villus Epithelium of Murine Duodenum 
Aims
The apical membrane anion exchanger Pat-1 is expressed at significant levels in the lower villus epithelium of murine duodenum. However, previous studies of Cl−/HCO3− exchange in the lower villus have failed to demonstrate Pat-1 function. Those studies routinely included luminal glucose which induces Na+-coupled glucose transport and acidifies the villus epithelium. Since Pat-1 has been proposed to be an electrogenic 1Cl−/2HCO3− exchanger, membrane depolarization or cell acidification during glucose transport may obscure Pat-1 activity. Therefore, we investigated the effects of luminal glucose on Cl−IN/HCO3−OUT exchange activity in the lower villus epithelium.
Methods
Cl−IN/HCO3−OUT exchange of villus epithelium in duodenal mucosa from Pat-1 knockout (KO), Slc26a3 (Dra) KO, cystic fibrosis transmembrane conductance regulator (Cftr) KO and wild-type (WT) littermate mice was measured using the pH-sensitive dye BCECF. Short-circuit current (Isc) was measured in Ussing chambers.
Results
During glucose absorption, Cl−IN/HCO3−OUT exchange in the lower villus epithelium was abolished in the Dra KO and unaffected in the Pat-1 KO relative to WT. However, during electroneutral mannose absorption or electrogenic α-D-methyl glucoside absorption, Cl−IN/HCO3−OUT exchange was reduced in both Pat-1 KO and Dra KO villi. Exposure to high [K+] abolished Cl−IN/HCO3−OUT exchange in the Dra KO but not the Dra/Cftr double KO epithelium, suggesting that Pat-1 activity is little affected by membrane depolarization except in the presence of Cftr.
Conclusions
The metabolic and electrogenic activity of glucose transport obscures Cl−IN/HCO3−OUT exchange activity of Pat-1 in the lower villus. The inhibitory effects of membrane depolarization on Pat-1 Cl−IN/HCO3−OUT exchange may require concurrent membrane association with Cftr.
doi:10.1111/j.1748-1716.2010.02210.x
PMCID: PMC3005274  PMID: 20969732
anion exchange; Cftr; down regulated in adenoma; Dra; facilitated diffusion
2.  Role of Down-Regulated in Adenoma Anion Exchanger in HCO3- Secretion across Murine Duodenum 
Gastroenterology  2008;136(3):893-901.
Background & Aims
The current model of duodenal HCO3- secretion proposes that basal secretion results from Cl-/HCO3- exchange whereas cAMP-stimulated secretion depends on a cystic fibrosis transmembrane conductance regulator channel (Cftr)-mediated HCO3- conductance. However, discrepancies in applying the model suggest that Cl-/HCO3- exchange also contributes to cAMP-stimulated secretion. Of two candidate Cl-/HCO3- exchangers, studies of putative anion transporter-1 (Pat-1) knockout (KO) mice find little contribution of Pat-1 to basal or cAMP-stimulated secretion. Therefore, the role of down-regulated in adenoma (Dra) in duodenal HCO3- secretion was investigated using DraKO mice.
Methods
Duodenal HCO3- secretion was measured by pH stat in Ussing chambers. Apical membrane Cl-/HCO3- exchange was measured by microfluorometry of intracellular pH (pHi) in intact villous epithelium. Dra expression was assessed by immunofluorescence.
Results
Basal HCO3- secretion was reduced ~55-60% in the DraKO duodenum. cAMP-stimulated HCO3- secretion was reduced ~50% but short-circuit current (Isc) was unchanged, indicating normal Cftr activity. Microfluorimetry of villi demonstrated that Dra is the dominant Cl-/HCO3- exchanger in the lower villous epithelium. Dra expression increased from villous tip to crypt. DraKO and WT villi also demonstrated regulation of apical Na+/H+ exchange by Cftr-dependent cell shrinkage during luminal Cl- substitution.
Conclusions
In murine duodenum, Dra Cl-/HCO3- exchange is concentrated in the lower crypt-villus axis where it is subject to Cftr regulation. Dra activity contributes most basal HCO3- secretion and ~50% of cAMP-stimulated HCO3- secretion. Dra Cl-/HCO3- exchange should be considered in efforts to normalize HCO3- secretion in duodenal disorders such as ulcer disease and cystic fibrosis.
doi:10.1053/j.gastro.2008.11.016
PMCID: PMC2694732  PMID: 19121635
3.  Down-regulated in Adenoma Cl/HCO3 Exchanger Couples With Na/H Exchanger 3 for NaCl Absorption in Murine Small Intestine 
Gastroenterology  2008;135(5):1645-1653.e3.
Background & Aims
Electroneutral NaCl absorption across small intestine contributes importantly to systemic fluid balance. Disturbances in this process occur in both obstructive and diarrheal diseases, eg, cystic fibrosis, secretory diarrhea. NaCl absorption involves coupling of Cl−/HCO3− exchanger(s) primarily with Na+/H+ exchanger 3 (Nhe3) at the apical membrane of intestinal epithelia. Identity of the coupling Cl−/HCO3− exchanger(s) was investigated using mice with gene-targeted knockout (KO) of Cl−/HCO3− exchangers: Slc26a3, down-regulated in adenoma (Dra) or Slc26a6, putative anion transporter-1 (Pat-1).
Methods
Intracellular pH (pHi) of intact jejunal villous epithelium was measured by ratiometric microfluoroscopy. Ussing chambers were used to measure transepithelial 22Na36Cl flux across murine jejunum, a site of electroneutral NaCl absorption. Expression was estimated using immunofluorescence and quantitative polymerase chain reaction.
Results
Basal pHi of DraKO epithelium, but not Pat-1KO epithelium, was alkaline, whereas pHi in the Nhe3KO was acidic relative to wild-type. Altered pHi was associated with robust Na+/H+ and Cl−/HCO3− exchange activity in the DraKO and Nhe3KO villous epithelium, respectively. Contrary to genetic ablation, pharmacologic inhibition of Nhe3 in wild-type did not alter pHi but coordinately inhibited Dra. Flux studies revealed that Cl− absorption was essentially abolished (>80%) in the DraKO and little changed (<20%) in the Pat-1KO jejunum. Net Na+ absorption was unaffected. Immunofluorescence demonstrated modest Dra expression in the jejunum relative to large intestine. Functional and expression studies did not indicate compensatory changes in relevant transporters.
Conclusions
These studies provide functional evidence that Dra is the major Cl−/HCO3− exchanger coupled with Nhe3 for electroneutral NaCl absorption across mammalian small intestine.
doi:10.1053/j.gastro.2008.07.083
PMCID: PMC2673535  PMID: 18930060
4.  Effect of Polarized Release of CXC-Chemokines from Wild-Type and Cystic Fibrosis Murine Airway Epithelial Cells 
The respiratory epithelium lining the airway relies on mucociliary clearance and a complex network of inflammatory mediators to protect the lung. Alterations in the composition and volume of the periciliary liquid layer, as occur in cystic fibrosis (CF), lead to impaired mucociliary clearance and persistent airway infection. Moreover, the respiratory epithelium releases chemoattractants after infection, inciting airway inflammation. However, characterizing the inflammatory response of primary human airway epithelial cells to infection can be challenging because of genetic heterogeneity. Using well-characterized, differentiated, primary murine tracheal cells grown at an air–liquid interface, which provides an in vitro polarized epithelial model, we compared inflammatory gene expression and secretion in wild-type and ΔF508 CF airway cells after infection with Pseudomonas aeruginosa. The expression of several CXC-chemokines, including macrophage inflammatory protein–2, small inducible cytokine subfamily member 2, lipopolysaccharide-induced chemokine, and interferon-inducible cytokine–10, was markedly increased after infection, and these proinflammatory mediators were asymmetrically released from the airway epithelium, predominantly from the basolateral surface. Equal amounts of CXC-chemokines were released from wild-type and CF cells. Secreted mediators were concentrated in the thin, periciliary fluid layer, and the dehydrated apical microenvironment of CF airway epithelial cells amplified the inflammatory signal, potentially resulting in high chemokine concentration gradients across the epithelium. Consistent with this observation, the enhanced chemotaxis of wild-type neutrophils was detected in CF airway epithelial cultures, compared with wild-type cells. These data suggest that P. aeruginosa infection of the airway epithelium induces the expression and polarized secretion of CXC-chemokines, and the increased concentration gradient across the CF airway leads to an exaggerated inflammatory response.
doi:10.1165/rcmb.2009-0249OC
PMCID: PMC3266059  PMID: 20639462
inflammation; chemotaxis; chemokine; airway; epithelium
5.  Epithelial myosin light chain kinase–dependent barrier dysfunction mediates T cell activation–induced diarrhea in vivo 
Journal of Clinical Investigation  2005;115(10):2702-2715.
Disruption of the intestinal epithelial barrier occurs in many intestinal diseases, but neither the mechanisms nor the contribution of barrier dysfunction to disease pathogenesis have been defined. We utilized a murine model of T cell–mediated acute diarrhea to investigate the role of the epithelial barrier in diarrheal disease. We show that epithelial barrier dysfunction is required for the development of diarrhea. This diarrhea is characterized by reversal of net water flux, from absorption to secretion; increased leak of serum protein into the intestinal lumen; and altered tight junction structure. Phosphorylation of epithelial myosin II regulatory light chain (MLC), which has been correlated with tight junction regulation in vitro, increased abruptly after T cell activation and coincided with the development of diarrhea. Genetic knockout of long myosin light chain kinase (MLCK) or treatment of wild-type mice with a highly specific peptide MLCK inhibitor prevented epithelial MLC phosphorylation, tight junction disruption, protein leak, and diarrhea following T cell activation. These data show that epithelial MLCK is essential for intestinal barrier dysfunction and that this barrier dysfunction is critical to pathogenesis of diarrheal disease. The data also indicate that inhibition of epithelial MLCK may be an effective non-immunosuppressive therapy for treatment of immune-mediated intestinal disease.
doi:10.1172/JCI24970
PMCID: PMC1224297  PMID: 16184195
6.  Uroguanylin knockout mice have increased blood pressure and impaired natriuretic response to enteral NaCl load 
Journal of Clinical Investigation  2003;112(8):1244-1254.
Guanylin and uroguanylin, peptides synthesized in the intestine and kidney, have been postulated to have both paracrine and endocrine functions, forming a potential enteric-renal link to coordinate salt ingestion with natriuresis. To explore the in vivo role of uroguanylin in the regulation of sodium excretion, we created gene-targeted mice in which uroguanylin gene expression had been ablated. Northern and Western analysis confirmed the absence of uroguanylin message and protein in knockout mice, and cGMP levels were decreased in the mucosa of the small intestine. Ussing chamber analysis of jejunum revealed that Na+/H+ exchanger–mediated Na+ absorption and tissue conductance was not altered in the knockout animals, but short-circuit current, an index of electrogenic anion secretion, was reduced. Renal clearance measurements showed that uroguanylin deficiency results in impaired ability to excrete an enteral load of NaCl, primarily due to an inappropriate increase in renal Na+ reabsorption. Finally, telemetric recordings of blood pressure demonstrated increased mean arterial pressure in uroguanylin knockout animals that was independent of the level of dietary salt intake. Together, these findings establish a role for uroguanylin in an enteric-renal communication axis as well as a fundamental principle of this axis in the maintenance of salt homeostasis in vivo.
doi:10.1172/JCI200318743
PMCID: PMC213491  PMID: 14561709
7.  T cell activation causes diarrhea by increasing intestinal permeability and inhibiting epithelial Na+/K+-ATPase 
The Journal of Clinical Investigation  2002;110(11):1739-1747.
Inflammatory bowel disease (IBD) is associated with mucosal T cell activation and diarrhea. We found that T cell activation with anti-CD3 mAb induces profound diarrhea in mice. Diarrhea was quantified by intestinal weight-to-length (wt/l) ratios, mucosal Na+/K+-ATPase activity was determined and ion transport changes were measured in Ussing chambers. Anti-CD3 mAb increased jejunal wt/l ratios by more than 50% at 3 hours, returning to base line after 6 hours. Fluid accumulation was significantly reduced in TNF receptor-1 (TNFR-1–/–), but not IFN-γ knockout mice. Anti-CD3 mAb decreased mucosal Na+/K+-ATPase activity, which was blocked by anti-TNF mAb and occurred to a lesser degree in TNFR-1–/– mice. Neither α nor β subunits of Na+/K+-ATPase decreased in abundance at 3 hours. Intestinal tissue from anti-CD3–treated mice exhibited increased permeability to mannitol at 1 hour and decreases in electroneutral Na+ absorption, Na+-dependent glucose absorption, and cAMP-stimulated anion secretion at 3 hours. Furthermore, enteral fluid accumulation was observed in CFTR–/– mice, indicating a minor role of active anion secretion. These data suggest that diarrhea in IBD is due to TNF-mediated malabsorption rather than to secretory processes. T cell activation induces luminal fluid accumulation by increasing mucosal permeability and reducing epithelial Na+/K+-ATPase activity leading to decreased intestinal Na+ and water absorption.
doi:10.1172/JCI15695
PMCID: PMC151630  PMID: 12464679

Results 1-7 (7)