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Acid-base effects on intestinal Na(+) absorption and vesicular trafficking

Charney, Alan N; Egnor, Richard W; Alexander-Chacko, Jesline; Cassai, Nicholas; Sidhu, Gurdip S
We examined for vesicular trafficking of the Na(+)/H(+) exchanger (NHE) in pH-stimulated ileal and CO(2)-stimulated colonic Na(+) absorption. Subapical vesicles in rat distal ileum were quantified by transmission electron microscopy at x27,500 magnification. Internalization of ileal apical membranes labeled with FITC-phytohemagglutinin was assessed using confocal microscopy, and pH-stimulated ileal Na(+) absorption was measured after exposure to wortmannin. Apical membrane protein biotinylation of ileal and colonic segments and Western blots of recovered proteins were performed. In ileal epithelial cells incubated in HCO/Ringer or HEPES/Ringer solution, the number of subapical vesicles, the relative quantity of apical membrane NHE isoforms 2 and 3 (NHE2 and NHE3, respectively), and apical membrane fluorescence under the confocal microscope were not affected by pH values between 7.1 and 7.6. Wortmannin did not inhibit pH-stimulated ileal Na(+) absorption. In colonic epithelial apical membranes, NHE3 protein content was greater at a PCO(2) value of 70 than 21 mmHg, was internalized when PCO(2) was reduced, and was exocytosed when PCO(2) was increased. We conclude that vesicle trafficking plays no part in pH-stimulated ileal Na(+) absorption but is important in CO(2)-stimulated colonic Na(+) absorption
PMID: 12176753
ISSN: 0363-6143
CID: 39608

Carbon dioxide affects rat colonic Na+ absorption by modulating vesicular traffic

Charney, Alan N; Egnor, Richard W; Cassai, Nicholas; Sidhu, Gurdip S
BACKGROUND & AIMS: We examined whether CO2 affects colonic Na+ absorption by endosome recycling of the Na+/H+ exchanger NHE3. METHODS: Rat distal colon segments exposed to various acid-base conditions were examined by transmission electron microscopy at 27,500x magnification and subapical vesicles quantified. Immunocytochemistry was used to identify vesicular NHE3. Endocytosis was tested for by observing internalization of apical membrane labeled with fluorescein isothiocyanate-phytohemagglutinin and Cy-3-NHE3 antibody using confocal microscopy. The effects of mucosal 5-(N,N-dimethyl)-amiloride (DMA), which inhibits NHE2 and/or NHE3, and wortmannin, which inhibits phosphatidylinositol 3-kinase, on CO2-stimulated Na+ absorption were measured in the Ussing chamber. RESULTS: The number of (coated and uncoated) subapical vesicles in epithelial cells was specifically and inversely related to net colonic Na+ absorption and PCO2. Immunoperoxidase labeling localized NHE3 on microvilli and vesicle membranes. Under the confocal microscope, a fluorescent band along apical membranes at PCO2 70 mm Hg became a subapical haze at PCO2 21 mm Hg. This pattern was not affected by carbonic anhydrase inhibition or when pH or [HCO3-] was changed, but PCO2 was held constant. DMA inhibition indicated that NHE3 mediates CO2-stimulated Na+ absorption. Wortmannin inhibited CO2-stimulated vesicle movement (exocytosis) and Na+ absorption. CONCLUSIONS: CO2 affects Na+ absorption in rat distal colon epithelium in part by modulating the movement of NHE3-containing vesicles to and from the apical membrane
PMID: 11832447
ISSN: 0016-5085
CID: 39717

pH stimulation of ileal Na+ absorption does not involve membrane trafficking [Meeting Abstract]

Charney, AN; Egnor, RW; Gummakonda, RV
ISSN: 0016-5085
CID: 2450432

A new model of CO2 regulation of colonic Na+ absorption [Meeting Abstract]

Charney, AN; Egnor, RW; Zaharia, V; Gummakonda, RV
ISSN: 0016-5085
CID: 55037

Effects of short chain fatty acids on colonic Na+ absorption and enzyme activity

Zaharia V; Varzescu M; Djavadi I; Newman E; Egnor RW; Alexander-Chacko J; Charney AN
Short chain fatty acids (SCFA) stimulate colonic Na+ absorption and inhibit cAMP and cGMP-mediated Cl- secretion. It is uncertain whether SCFA have equivalent effects on absorption and whether SCFA inhibition of Cl- secretion involves effects on mucosal enzymes. Unidirectional Na+ fluxes were measured across stripped colonic segments in the Ussing chamber. Enzyme activity was measured in cell fractions of scraped colonic mucosa. Mucosal 50 mM acetate, propionate, butyrate and poorly metabolized isobutyrate stimulated proximal colon Na+ absorption equally (300%). Neither 2-bromo-octanoate, an inhibitor of beta-oxidation, nor carbonic anhydrase inhibition affected this stimulation. All SCFA except acetate stimulated distal colon Na+ absorption 200%. Only one SCFA affected proximal colon cGMP phosphodiesterase (PDE) (18% inhibition by 50 mM butyrate). All SCFA at 50 mM stimulated distal colon cAMP PDE (24-43%) and decreased forskolin-stimulated mucosal cAMP content. None of the SCFA affected forskolin-stimulated adenylyl cyclase in distal colon or ST(a)-stimulated guanylyl cyclase in proximal colon. Na+-K+-ATPase in distal colon was inhibited 23-51% by the SCFA at 50 mM. We conclude that all SCFA (except acetate in distal colon) stimulate colonic Na+ absorption equally, and the mechanism does not involve mucosal SCFA metabolism or carbonic anhydrase. SCFA inhibition of cAMP-mediated secretion may involve SCFA stimulation of PDE and inhibition of Na+-K+-ATPase
PMID: 11223395
ISSN: 1095-6433
CID: 21243

Effect of E. coli heat-stable enterotoxin on colonic transport in guanylyl cyclase C receptor-deficient mice

Charney AN; Egnor RW; Alexander-Chacko JT; Zaharia V; Mann EA; Giannella RA
We studied the functional importance of the colonic guanylyl cyclase C (GCC) receptor in GCC receptor-deficient mice. Mice were anesthetized with pentobarbital sodium, and colon segments were studied in Ussing chambers in HCO3- Ringer under short-circuit conditions. Receptor-deficient mouse proximal colon exhibited similar net Na+ absorption, lower net Cl- absorption, and a negative residual ion flux (J(R)), indicating net HCO3- absorption compared with that in normal mice. In normal mouse proximal colon, mucosal addition of 50 nM Escherichia coli heat-stable enterotoxin (STa) increased the serosal-to-mucosal flux of Cl- (J(s-->m)(Cl)) and decreased net Cl- flux (J(net)(Cl)) accompanied by increases in short-circuit current (I(sc)), potential difference (PD), and tissue conductance (G). Serosal STa had no effect. In distal colon neither mucosal nor serosal STa affected ion transport. In receptor-deficient mice, neither mucosal nor serosal 500 nM STa affected electrolyte transport in proximal or distal colon. In these mice, 1 mM 8-bromo-cGMP produced changes in proximal colon J(s-->m)(Cl) and J(net)(Cl), I(sc), PD, G, and J(R) similar to mucosal STa addition in normal mice. We conclude that the GCC receptor is necessary in the mouse proximal colon for a secretory response to mucosal STa
PMID: 11208543
ISSN: 0193-1857
CID: 26788

Action of E. coli STa on colonic ion transport in guanylate cyclase C deficient mice [Meeting Abstract]

Charney, AN; Egnor, RW; Zaharia, V; Mann, EA; Giannella, RA
ISSN: 0016-5085
CID: 54592

CO2 stimulates colonic Na+ absorption by affecting NHE-3 vesicular traffic [Meeting Abstract]

Charney, AN; Egnor, RW; Cassai, ND; Sidhu, GS
ISSN: 0016-5085
CID: 54593

Effects of short chain fatty acids on colonic mucosal adenylate cyclase, guanylate cyclase and phosphodiesterase activities [Meeting Abstract]

Varzescu, M; Frisch, DR; Egnor, RW; Zaharia, V; Charney, AN
ISSN: 0016-5085
CID: 54594

Relative effects of short chain fatty acids on colonic Na+ absorption [Meeting Abstract]

Zaharia, V; Egnor, RW; Djavadi, I; Charney, AN
ISSN: 0016-5085
CID: 54590