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

Alan N. Charney1, Richard W. Egnor1, Jesline Alexander-Chacko1, Nicholas Cassai2, and Gurdip S. Sidhu2

1 Nephrology Section and 2 Department of Pathology, Veterans Affairs Medical Center, New York University School of Medicine, New York, New York 10010


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

We examined for vesicular trafficking of the Na+/H+ exchanger (NHE) in pH-stimulated ileal and CO2-stimulated colonic Na+ absorption. Subapical vesicles in rat distal ileum were quantified by transmission electron microscopy at ×27,500 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<UP><SUB>3</SUB><SUP>−</SUP></UP>/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 PCO2 value of 70 than 21 mmHg, was internalized when PCO2 was reduced, and was exocytosed when PCO2 was increased. We conclude that vesicle trafficking plays no part in pH-stimulated ileal Na+ absorption but is important in CO2-stimulated colonic Na+ absorption.

rat; ileum; colon; electrolyte transport


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

CO2 tension affects electroneutral colonic NaCl absorption by altering the activities of the Na+/H+ and Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchangers located along the apical membranes of epithelial cells (4, 6, 16, 25). This effect requires carbonic anhydrase and during the first several hours is mediated by a change in the supply of H+ and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> to these exchangers (10, 14). Because other means of reducing bulk cytosolic pH (pHi) did not stimulate Na+ absorption in these studies, we hypothesized that the effect of CO2 was restricted to a subapical region of the cell (3). Recently, we reported that CO2-stimulated colonic Na+ absorption primarily affects the Na+/H+ exhanger isoform 3 (NHE3) and involves the movement of NHE3-containing vesicles to and from the apical membrane (7). Increases in PCO2 decrease pHi, cause the exocytosis of such vesicles to the apical membrane, and stimulate Na+ absorption. Decreases in CO2 tension have the opposite effect.

Among the issues not addressed in this study of the colon were the relative effects of pH and CO2 on apical membrane NHE3 abundance. Such information could relate the quantity of apical membrane NHE3 to the steady-state level of colonic Na+ absorption. Because changes in PCO2 rather than pH affect colonic Na+ absorption, such data also would confirm that CO2 is the specific acid-base variable that affects vesicle movement. Finally, changes in the quantity of apical and subapical NHE3 protein after a change in CO2 tension would bear on the relative importance of H+ supply and vesicle movement to changes in colonic Na+ absorption.

Also not addressed was whether vesicle trafficking played an important role in the rat distal ileum. This is of particular interest because Na+ absorption in this tissue is specifically responsive to changes in Ringer solution pH rather than to PCO2 (8, 30). Changes in pH in the presence or absence of CO2 have an inverse effect on ileal Na+ absorption, whereas changes in PCO2 in the absence of a change in pH have no effect. An intraspecies comparison of rat ileum and colon would suggest whether acid-base-induced changes in intestinal Na+ absorption characteristically involve vesicular trafficking. Some evidence supports this possibility. Phosphatidylinositol 3-kinase (PI3-K), which modulates NHE3 activity and endosome recycling (24), mediates epidermal growth factor-stimulation of NaCl absorption in rabbit ileum (21), a tissue that also is specifically responsive to pH (5, 15). In addition, cultured opossum kidney clone P (OKP) cells that have been incubated for 6 h in a CO2-free medium at pH 6.8 exhibit increases in the exocytic insertion of NHE3 into the apical membrane as compared to incubation at pH 7.4 (31).

For these reasons, we measured the acute effects of acid-base variables on vesicular trafficking in the ileum. Our hypothesis was that because changes in pH rather than PCO2 affected ileal Na+ absorption, vesicular trafficking was not involved. We used a variety of techniques including counting the numbers of subapical vesicles in ileal epithelial cells, measuring the effect of pH on the internalization of apical membrane NHE, and measuring the effect of wortmannin on pH-stimulated ileal Na+ absorption. We also measured the effects of pH and PCO2 on the levels of apical membrane NHE2 and NHE3 proteins in ileal epithelial cells. In every case, a comparison of these segments suggested that pH-stimulated Na+ absorption in ileal epithelial cells, in contrast to CO2-stimulated colonic absorption, does not involve trafficking of NHE-containing vesicles to and from the apical membrane.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Approvals of the Dept. of Veterans Affairs (VA) Subcommittee for Animal Studies and the VA Research and Development Committee were obtained. Male Sprague-Dawley rats (Rattus norvegicus, 250-350 g) were maintained on a standard chow diet with free access to water. Pentobarbital sodium (5 mg/100 g of body wt) was used to achieve surgical anesthesia. The distal 10 cm of ileum ending 7 cm from the ileocecal valve or the distal 10 cm of colon was removed and rinsed with 0.9% saline. Euthanasia was by exsanguination under surgical anesthesia.

Chemicals and solutions. Reagent-grade chemicals were obtained from Sigma Chemical (St. Louis, MO) unless otherwise indicated. Rabbit anti-NHE3 and anti-NHE2 sera were gifts of Eugene B. Chang, University of Chicago. As described by Bookstein et al. (1), the NHE3 antibody was developed by constructing a glutathione-S-transferase (GST) fusion protein that included NHE3 amino acids 528-648. The BstYI1815-BstYI2181 fragment was ligated into BamHI-cut pGEX-3X, thereby generating an in-frame fusion to GST. Sanger dideoxy DNA sequencing confirmed that the fusion was in the correct reading frame (28). The NHE2 antibody was developed by constructing a GST fusion protein to NHE2 cDNA corresponding to amino acids 260-280 (2). Sanger dideoxy sequencing confirmed an in-frame correctly copied sequence. Both antibodies localize to the apical membranes of epithelial cells of rat small and large intestine (1, 2).

Bathing solutions included HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>/Ringer and HEPES/Ringer solutions that contained (in mM) 140.2 Na+, 4 K+, 1.2 Mg2+, 1 Ca2+, 100 Cl-, 2.4 HPO<UP><SUB>4</SUB><SUP>2−</SUP></UP>, 0.4 H2PO<UP><SUB>4</SUB><SUP>−</SUP></UP>, 2.2 SO<UP><SUB>4</SUB><SUP>2−</SUP></UP>, 18 gluconate, 10 glucose, and either 21 HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> or 21 HEPES. In all protocols in which the ileum was incubated (in the Ussing chamber), mannitol was substituted for glucose in the mucosal bathing solution. The HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>/Ringer solution was gassed with 3% CO2-97% O2 (PCO2 = 21 mmHg, pH 7.6) or 11% CO2-89% O2 (PCO2 = 70 mmHg, pH 7.1) to obtain the various pH values. The HEPES/Ringer solution was gassed with 100% O2 and titrated to pH 7.1 or 7.6 using 2 M H2SO4 or 1 M NaOH, respectively. All solutions were maintained at 37°C. The pH and PCO2 values of the Ringer solution were measured with a Radiometer BMS 3 Mk 2 system with a PHM 73 acid-base analyzer (London Company, Cleveland, OH).

Na+ transport. Ion fluxes were measured to determine whether the effect of pH on ileal Na+ absorption (in HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>/Ringer solution) was altered by wortmannin, an inhibitor of PI3-K and vesicle movement (24). As previously described (30), pairs of unstripped ileal segments were studied under short-circuit conditions in modified Ussing half-chambers that exposed 0.62 cm2 of surface area. Tissue conductance (G) was calculated from periodic bipolar pulses of 0.5 mV and was the basis for pairing tissues when G was no greater than 25%. The short-circuit potential difference (PD) was calculated from the short-circuit current (Isc) divided by G and was referenced to the mucosal side. Unidirectional fluxes of Na+ were measured by adding 2 µCi of 22Na+ (sp. act., 100 Ci/g; New England Nuclear, Boston, MA) to the mucosal side of one member of each tissue pair and the serosal side of the other.

Mucosal to serosal (J<UP><SUB>ms</SUB><SUP>Na<SUP>+</SUP></SUP></UP>) and serosal to mucosal (J<UP><SUB>sm</SUB><SUP>Na<SUP>+</SUP></SUP></UP>) fluxes (expressed as µeq · cm-2 · h-1) were measured for 16-30 min at a PCO2 value of 21 mmHg (pH 7.6) and then 70 mmHg (pH 7.1) after an initial 30-min equilibration. Aliquots of 500 µl were replaced with identical Ringer solution including wortmannin where appropriate. Net flux (J<UP><SUB>net</SUB><SUP>Na<SUP>+</SUP></SUP></UP>) was calculated as (J<UP><SUB>ms</SUB><SUP>Na<SUP>+</SUP></SUP></UP> - J<UP><SUB>sm</SUB><SUP>Na<SUP>+</SUP></SUP></UP>). In some experiments, wortmannin was added to the mucosal bathing solution at a final concentration of 0.75µM. Tissues were exposed to wortmannin for 36 min before the first flux period was performed and for an additional 90 min including the flux periods at each pH measurement.

Morphometry. Tissues were prepared and examined as previously described (7). Unstripped distal ileal segments were mounted and incubated in the Ussing chamber in either HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>/Ringer or HEPES/Ringer solution at pH 7.6 or 7.1 for 1 h. Tissues were then fixed by adding 3% glutaraldehyde (buffered in 0.1 M Sorensen's phosphate buffer, pH 7.3) to both reservoirs to a final concentration of 1.5% glutaraldehyde. After 10 min, the tissue was removed from the Ussing chamber and fixed in the undiluted 3% glutaraldehyde overnight at 4°C. Samples were cut into elongated strips (1 × 3 mm) using a no. 11 scalpel and then postfixed in 2% osmium tetroxide. The strips were dehydrated in a graded series of ethanols and propylene oxide and embedded in Eponate 812 (Ted Pella, Redding, CA) in flat embedding plates to facilitate orientation of the tissue. Thin sections were cut perpendicular to the cell-surface plane at 70 nm, mounted on copper grids, and then stained with uranyl acetate and Reynold's lead citrate. Sections were viewed in a JEM 1010 transmission electron microscope.

The number of vesicles in randomly selected areas was counted by an observer without knowledge of the experimental condition. A clear plastic grid was placed over conventional photomicrographs at ×27,500 magnification, and vesicles <= 0.3 µm in diameter were counted within the 4.9 × 1.2-µm area defined by the grid. The long axis of the grid was placed at the base of the microvilli parallel to the mucosal surface. Numbers of vesicles for each acid-base condition were expressed per grid (5.88 µm2). When coated and uncoated vesicles were differentiated, the same photographs were examined with the aid of a ×8 loupe.

Confocal microscopy. As previously described for rat colon (7), segments of unstripped rat distal ileum were mounted in Ussing chambers in HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>/Ringer solution gassed with 11% CO2 (PCO2 = 70 mmHg, pH 7.1) at 37°C for 30 min. Phytohemagglutinin-E labeled with fluoresceine isothiocyanate (PHA/FITC) was then added to the mucosal reservoir of each tissue at a final concentration of 50 µg/ml. After 10 min, one tissue was exposed to 3% CO2 (PCO2 = 21 mmHg, pH 7.6) and both tissues were incubated for an additional 30 min. In other experiments, the segments were incubated in HEPES/Ringer solution at pH 7.1 and after exposure to PHA/FITC, the incubation pH of one segment was increased to 7.6.

The fluorescence intensity within a rectangular area including the apical surface and extending into the epithelial cell was displayed as a graph in a window above the image. The fluorescence intensity also was analyzed by normalizing the peak intensity and the length of the scan, integrating the area under the curve, and comparing the arbitrary area values for each condition. Graphs showing similar fluorescence distributions would be expected to have similar areas under the curve. A broader fluorescent signal, i.e., fluorescence extending into the cell, would be indicated by an increase in area.

Apical membrane protein biotinylation and Western blotting. Steady-state levels of apical membrane NHE2 and NHE3 protein were measured in pairs of intact unstripped ileal segments, and NHE3 protein was measured in unstripped colonic segments. These segments were rinsed well with ice-cold PBS that contained 0.1 mM CaCl2 and 1 mM MgCl2 (PBS/CaMg solution). One of the ileal or colonic segments was incubated in a flask at pH 7.6 (in HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>/Ringer or HEPES/Ringer solution) and the other at pH 7.1. After incubation for 30 min at 37°C, the segments were chilled by placing them in ice-cold 10 mM triethanolamine, 2 mM CaCl2, and 150 mM NaCl, pH 7.4 (TEA). Biotinylation was performed by a modification of a previously described method (17, 31). The lumen of each segment was filled with 2 ml of TEA that contained 3 mg of EZ-Link sulfo-NHS-SS-biotin (Pierce, Rockford, IL) and was incubated for 1 h at 0°C. Segment contents were emptied, and the lumen was flushed with several milliliters of PBS/CaMg solution that contained 100 mM glycine and was then incubated for 20 min in the same buffer at 0°C.

Cells were lysed by incubation with 2 ml of RIPA buffer (150 mM NaCl, 50 mM Tris · HCl, pH 7.4, 5 mM EDTA, 1% Triton X-100, 0.5% sodium deoxycholate, and 0.1% SDS) containing protease inhibitor cocktail (Sigma) for 30 min at 0°C. The contents were then centrifuged at 100,000 g for 10 min. The protein concentration in the supernatant was measured by the Lowry method (27) which, as shown in preliminary experiments, was not affected by the RIPA buffer. After adjusting the protein concentration to 3 mg/ml, 480 µl of the RIPA extract were mixed with 120 µl of streptavidin-agarose beads (Pierce). Beads were sedimented by centrifugation at 15,000 g for 1 min and then washed twice with 500 µl of RIPA buffer. The bound proteins were then extracted and subjected to SDS-PAGE, immunoblotting, and quantitation.

To quantify the effect of reducing CO2 on membrane endocytosis, internalization of apical membrane NHE3 was measured in pairs of intact unstripped ileal and colonic segments (31). Segments were rinsed well with ice-cold PBS-CaMg solution and incubated in a flask containing HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>/Ringer solution at a PCO2 of 70 mmHg (pH 7.1) for 30 min at 37°C. Segments were then placed in ice-cold TEA, and biotinylation was performed. After segments were quenched with glycine, one of each pair was rinsed well with the ice-cold incubation solution and then incubated in HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>/Ringer solution at a PCO2 of 21 (pH 7.6) or 70 mmHg (pH 7.1) at 37°C for 30 min. Segment lumens were then filled with 2 ml of 50 mM 2-mercaptoethanesulfonic acid, sodium salt (mesna, a membrane-impermeant reducing agent) in 50 mM Tris · HCl, 100 mM NaCl, 1 mM EDTA, and 0.2% BSA, pH 8.6, and were incubated at 0°C for 30 min. Lumens were emptied and refilled with 2 ml of 50 mM mesna and incubated for 30 min two additional times. The contents were emptied, and residual mesna was oxidized with 0.5 ml of 150 mM iodoacetic acid for 10 min before lumens were rinsed thoroughly with PBS/CaMg solution. Cells were then lysed, and the biotinylated proteins (which represented internalized apical membrane) were extracted. The proteins recovered from the streptavidin-agarose beads were subjected to SDS-PAGE, immunoblotting, and quantitation as described below.

We also quantified the effect of increasing CO2 on the exocytosis of membrane vesicles containing NHE3 (31). Pairs of intact, unstripped, colonic segments were rinsed well inside and outside with ice-cold PBS/CaMg solution. All pairs were incubated in a flask containing HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>/Ringer solution at a PCO2 of 21 mmHg (pH 7.6). After 30 min at 37°C, tissues were chilled in ice-cold TEA. The lumen of each segment was filled with 2 ml of TEA containing 3 mg of EZ-Link sulfo-NHS-acetate (Pierce) and was incubated for 1 h at 0°C to block surface proteins. Segments were emptied and after the lumen was flushed with several milliliters of PBS/CaMg solution that contained 100 mM glycine, segments were incubated for 20 min in the same buffer at 0°C. After quenching, one of each pair of segments was rinsed well with ice-cold incubation solution and incubated in HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>/Ringer solution at a PCO2 of 21 (pH 7.6) or 70 mmHg (pH 7.1) at 37°C for 30 min. Apical membrane proteins were then biotinylated with 3 mg of EZ-Link sulfo-NHS-SS-biotin. The proteins recovered from the streptavidin-agarose beads (which represented newly inserted vesicles) were subjected to SDS-PAGE, immunoblotting, and quantitation (described as follows).

Western blots were performed on proteins recovered from the streptavidin-agarose beads with loading buffer (1 mM Tris · HCl, pH 6.8, 1% SDS, 10% glycerol, and 1% beta -mercaptoethanol). Samples were size-fractionated by SDS-PAGE (10-15% gradient gel) and transferred to nitrocellulose electrophoretically using the Pharmacia PhastSystem (Amersham Pharmacia Biotech, Piscataway, NJ). Blots were probed with rabbit anti-NHE2 or -NHE3 at a 1:500 dilution followed by horseradish peroxidase-labeled goat anti-rabbit IgG at a 1:2,000 dilution. Labeling was visualized by chemiluminescence using Lumiglo (Cell Signaling, Beverly, MA) with exposure to Kodak BioMax MR film. Bands were quantified using a model GS-710 calibrated imaging densitometer and Quantity One image-analysis software (Bio-Rad, Hercules, CA). In preliminary experiments, NHE2 and NHE3 were purified by immunoprecipitation, and equal amounts were subjected to Western blotting. These experiments indicated that NHE2 was somewhat underestimated in relation to NHE3 by this procedure.

Statistics. Data were expressed as means ± SE and were compared by ANOVA or paired or unpaired Student's t-tests. Two-tailed P values <0.05 were considered significant.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Effect of pH on number of ileal subapical vesicles. We initially determined whether the number of subapical vesicles in ileal epithelial cells correlated with the level of Na+ absorption. We counted vesicles in a defined grid area as depicted in Fig. 1, top. Measurements were made in tissues exposed to HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>/Ringer or HEPES/Ringer solution at pH levels (7.6 and 7.1) associated with markedly different levels of ileal Na+ absorption. As shown in Fig. 1, bottom, the total number of vesicles per grid was not affected by the Ringer solution pH or PCO2 or the presence or absence of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>. The difference in the number of vesicles at PCO2 values of 21 and 70 mmHg was not statistically significant (NS; 9.1 ± 0.7, n = 38 vs. 7.8 ± 0.6, n = 35; P = NS) nor were the number of vesicles different at pH values of 7.6 and 7.1 in HEPES/Ringer solution (7.1 ± 1.2, n = 11 vs. 7.3 ± 0.9, n = 11; P = NS)


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Fig. 1.   Effect of pH on the number of ileal subapical vesicles and Na+ absorption. Segments of rat distal ileum were exposed to 21 mM HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>/Ringer solution at a PCO2 of 21 mmHg (pH 7.6) or 70 mmHg (pH 7.1) or 21 mM HEPES/Ringer solution at pH 7.6 or 7.1. After fixation, epithelial cells were examined by transmission electron microscopy at ×27,500 magnification. Grid (4.9 × 1.2 µm) was positioned parallel to the mucosal surface at the base of the microvilli of randomly chosen longitudinal sections of photomicrographs. In the sample photomicrographs shown (top), an uncoated vesicle (arrowhead) and a coated vesicle (arrow) are indicated. Small black extramitochondrial bodies are glycogen granules. Number of (coated and uncoated) vesicles expressed per grid (5.88 µm2) were not different at pH values of 7.6 and 7.1 in 21 mM HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>/Ringer or HEPES/Ringer (bottom). Values for net ileal Na+ absorption were obtained from a previous study under identical acid-base conditions (30). Net Na+ absorption increased when pH was reduced from 7.6 to 7.1 in the presence and absence of CO2 and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>. J<UP><SUB>net</SUB><SUP>Na<SUP>+</SUP></SUP></UP>, net Na+ flux. Values are means ± SE; dagger P < 0.01.

When coated and uncoated vesicles were enumerated separately, neither coated nor uncoated vesicle numbers were affected by the acid-base conditions. In HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>/Ringer solution at pH 7.6, coated vesicles numbered 1.8 ± 0.3 (n = 38) and uncoated vesicles numbered 7.3 ± 0.7 (n = 38); at pH 7.1, coated vesicles numbered 1.6 ± 0.2 (n = 35) and uncoated vesicles numbered 6.2 ± 0.5 (n = 35); P = NS. In HEPES/Ringer solution at pH 7.6, coated vesicles were 1.7 ± 0.5 (n = 10) in number and uncoated vesicles were 5.4 ± 1.0 (n = 10); at pH 7.1, coated vesicles were 1.1 ± 0.3 (n = 10) in number and uncoated vesicles were 6.2 ± 0.8 (n = 10); P = NS.

Also shown in Fig. 1, bottom, are the rates of net Na+ absorption under the same acid-base conditions. These transport data were derived from studies of ileal tissues in our laboratory under identical experimental conditions (30). Decreases in bathing solution pH from 7.6 to 7.1 in HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>/Ringer or HEPES/Ringer solution stimulated J<UP><SUB>net</SUB><SUP>Na<SUP>+</SUP></SUP></UP> more than twofold.

Effect of pH on endocytosis of ileal apical membrane. The effect of acid-base conditions on vesicular trafficking in the ileum also was studied by confocal microscopy. In these experiments, the apical membrane of ileal epithelial cells was labeled with PHA/FITC after exposure to HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>/Ringer or HEPES/Ringer solution at pH 7.1. This acid-base condition is associated with a relatively increased rate of Na+ absorption (Fig. 1). The pH of the bathing solution was then increased to 7.6, a condition that predictably lowers Na+ absorption, to determine whether PHA/FITC-labeled apical membrane moved into the cell. Internalization of apical membrane indicates the presence of endocytosis and the creation of subapical vesicles.

As shown in Fig. 2A, in HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>/Ringer solution at a PCO2 of 70 mmHg (pH 7.1), a band of fluorescence marks the apical brush-border membrane of the mucosa as indicated by the drawn outline of an epithelial cell. A graph of the fluorescence intensity is indicated above the photomicrograph. When the pH of the bathing solution was increased to 7.6 by reducing the PCO2 (to 21 mmHg), the band of fluorescence (and the width of the graph) remained nearly unchanged (Fig. 2B). Tissues exposed to HEPES/Ringer solution at pH 7.1 and 7.6 are shown in Fig. 2, C and D, respectively. The band of fluorescence at the apical brush-border membrane also did not change when bathing solution pH was increased. The mean values for the areas under the curves that describe the fluorescence distribution were 15% greater at a PCO2 of 21 mmHg (pH 7.6) (146 ± 6 arbitrary area units, n = 5) than at a PCO2 of 70 mmHg (pH 7.1) (124 ± 6, n = 7; P < 0.05). However, in HEPES/Ringer solution, the areas were equivalent at pH 7.6 (116 ± 11 arbitrary area units, n = 5) and pH 7.1 (119 ± 8, n = 5; P = NS).


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Fig. 2.   Effect of pH on the localization of phytohemagglutinin-E labeled with fluoresceine isothiocyanate (PHA/FITC) by confocal microscopy. Segments of rat distal ileum were incubated in 21 mM HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>/Ringer solution at PCO2 of 70 mmHg (pH 7.1) or in 21 mM HEPES/Ringer solution at pH 7.1 before exposure to PHA/FITC. pH was then increased to 7.6 in HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>/Ringer solution by reducing the PCO2 to 21 mmHg and in HEPES/Ringer solution by titration. Tissues were fixed and examined by confocal microscopy, examples of which are depicted. Graphs above each image depict the relative fluorescence intensity along the longitudinal axis of a superimposed rectangle which straddles one epithelial cell (drawn for comparison). The y-axis is the fluorescence intensity in arbitrary units and the x-axis is the distance in pixels along the longitudinal axis of the rectangle. Similar bands of fluorescence were present in HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>/Ringer solution at PCO2 of 70 mmHg (A) and 21 mmHg (B), and in HEPES/Ringer solution at pH values of 7.1 (C) and 7.6 (D).

Effect of wortmannin on pH-stimulated ileal Na+ absorption. We then examined the role of vesicular trafficking in ileal Na+ absorption by measuring the effect of wortmannin at concentrations reported to inhibit exocytosis (20, 24). Unidirectional Na+ fluxes were measured across ileal tissues bathed in HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>/Ringer solution at a PCO2 of 21 mmHg (pH 7.6) in the presence or absence of mucosal 0.75 µM wortmannin. Bathing solution PCO2 was then increased to 70 mmHg (pH 7.1), and the measurements were repeated. We found that wortmannin did not affect the increments in J<UP><SUB>ms</SUB><SUP>Na<SUP>+</SUP></SUP></UP> and J<UP><SUB>net</SUB><SUP>Na<SUP>+</SUP></SUP></UP> caused by a decrease in pH. In the absence of wortmannin, Delta J<UP><SUB>ms</SUB><SUP>Na<SUP>+</SUP></SUP></UP> was 4.1 ± 0.6 µeq · cm-2 · h-1, and Delta J<UP><SUB>net</SUB><SUP>Na<SUP>+</SUP></SUP></UP> was 2.2 ± 0.2 µeq · cm-2 · h-1, n = 11, whereas in the presence of wortmannin, Delta J<UP><SUB>ms</SUB><SUP>Na<SUP>+</SUP></SUP></UP> was 3.5 ± 0.5 µeq · cm-2 · h-1 and Delta J<UP><SUB>net</SUB><SUP>Na<SUP>+</SUP></SUP></UP> was 2.1 ± 0.6 µeq · cm-2 · h-1, n = 6; P = NS.

Effect of pH on ileal apical membrane NHE2 and NHE3 proteins. The effects of CO2 and pH on the NHE protein content of ileal apical membranes was then examined. In these experiments, the steady-state levels of NHE2 and NHE3 were measured at pH 7.6 and 7.1 in HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>/Ringer or HEPES/Ringer solution. As shown in Fig. 3A, in HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>/Ringer solution at a PCO2 of 21 mmHg (pH 7.6), NHE3 protein content was 6.3 ± 1.9 OD · mm2, n = 6. At a PCO2 of 70 mmHg (pH 7.1), this value was similar: 6.7 ± 1.7 OD · mm2, n = 6; P = NS. NHE2 protein content also was not different at PCO2 values of 21 and 70 mmHg in HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>/Ringer solution: 2.6 ± 1.3, n = 4, vs. 1.6 ± 0.2 OD · mm2, n = 4; P = NS. NHE3 and NHE2 content also were not different in HEPES/Ringer solution at pH 7.6 and 7.1 (Fig. 3B). At pH 7.6, NHE3 content was 9.1 ± 2.1 OD · mm2, n = 5, and at pH 7.1 it was 8.3 ± 1.8 OD · mm2, n = 5; P = NS. At pH 7.6, NHE2 content was 3.6 ± 1.5 OD · mm2, n = 5, and at pH 7.1 it was 4.0 ± 1.6 OD · mm2, n = 5; P = NS.


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Fig. 3.   Effect of pH on Na+/H+ exchanger isoforms 2 and 3 (NHE2 and NHE3, respectively) protein content of apical membranes of ileal epithelial cells. Segments of rat distal ileum were incubated in 21 mM HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>/Ringer solution at a PCO2 of 21 mmHg (pH 7.6) or 70 mmHg (pH 7.1) or in 21 mM HEPES/Ringer solution at pH values of 7.6 or 7.1. Apical membrane proteins of the segments were then biotinylated, Western blots of the recovered biotinylated proteins were performed, and NHE2 and NHE3 were quantified. Steady-state levels of NHE3 on ileal apical membranes was greater than NHE2, but there was no difference in the quantity of NHE3 or NHE2 at pH values of 7.6 and 7.1 in either HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>/Ringer (A) or HEPES/Ringer solutions (B). Effect of increasing pH from 7.1 to 7.6 by reducing PCO2 from 70 to 21 mmHg on the internalization (endocytosis) of biotinylated apical membrane at 30 min also is shown (C). A similar quantity of NHE3 entered the cell at pH 7.1 and 7.6. Values are means ± SE.

To examine the degree to which endocytosis of ileal apical membrane NHE3 was affected by a change in pH, we examined the effect of increasing pH (in HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>/Ringer solution) on biotinylated apical membrane. As shown in Fig. 3C, when apical membrane was biotinylated at pH 7.1 (PCO2 = 70 mmHg) and then incubated for 30 min, NHE3 protein in internalized apical membrane was 6.1 ± 1.5 OD · mm2, n = 6. Thirty minutes after the pH was increased to 7.6 (by decreasing PCO2 from 70 to 21 mmHg), internalized biotinylated apical membrane NHE3 protein was unchanged at 6.1 ± 1.2 OD · mm2, n = 6; P = NS.

Effect of CO2 on colonic apical membrane NHE3 protein. The steady-state level of apical membrane NHE3 protein was affected by CO2 in the colon. As shown in Fig. 4A, in HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>/Ringer solution at a PCO2 of 21 mmHg (pH 7.6), NHE3 protein content was 3.9 ± 1.3 OD · mm2, n = 6 and at a PCO2 of 70 mmHg (pH 7.1), protein content was almost twice as great, 7.4 ± 1.1 OD · mm2, n = 6; P < 0.05. By contrast, NHE3 protein was not affected by a similar pH difference in HEPES/Ringer solution: 5.0 ± 0.9, n = 6, vs. 4.6 ± 0.5 OD · mm2, n = 6; P = NS (Fig. 4B).


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Fig. 4.   Effect of pH on NHE3 protein content of apical membranes of colonic epithelial cells. Segments of rat distal colon were incubated in 21 mM HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>/Ringer solution at a PCO2 of 21 mmHg (pH 7.6) or 70 mmHg (pH 7.1) or in 21 mM HEPES/Ringer solution at pH values of 7.6 or 7.1. Apical membrane proteins of the segments were then biotinylated, Western blots of the recovered biotinylated proteins were performed, and NHE3 was quantified. Steady-state levels of NHE3 were greater at PCO2 of 70 mmHg than at PCO2 of 21 mmHg (A) and a similar pH change in the absence of CO2 had no effect (B). Effect of reducing PCO2 from 70 to 21 mmHg on the internalization (endocytosis) of biotinylated apical membrane at 30 min also is shown (C). A greater quantity of NHE3 entered the cell at a PCO2 of 21 mmHg than 70 mmHg. Effect of increasing the PCO2 from 21 to 70 mmHg on the exocytosis of NHE3-containing vesicles at 30 min is indicated in D. A greater quantity of NHE3-containing vesicles insert into the apical membrane at a PCO2 of 70 mmHg than 21 mmHg. Values are means ± SE; * P < 0.05; dagger P < 0.01.

To examine the degree to which endocytosis of colonic apical membrane NHE3 was affected by a change in CO2 tension, we examined the effect of decreasing PCO2 on biotinylated apical membrane. As shown in Fig. 4C, when apical membrane was biotinylated at a PCO2 of 70 mmHg (in HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>/Ringer solution) and then incubated for 30 min, NHE3 protein in internalized apical membrane was 4.5 ± 1.2 OD · mm2, n = 5. Thirty minutes after the PCO2 was decreased from 70 to 21 mmHg, internalized biotinylated apical membrane NHE3 protein doubled to 9.6 ± 1.5 OD · mm2, n = 5; P < 0.05.

The effect of increasing PCO2 on exocytosis in colonic epithelial cells was examined in a similar way. After blockade of apical membrane biotinylation at a PCO2 of 21 mmHg, the PCO2 was either unchanged or increased to 70 mmHg, and the surface was then biotinylated. Thus only NHE3 that had undergone exocytosis was measured at the apical membrane. As shown in Fig. 4D, when the PCO2 of 21 mmHg remained unchanged, apical membrane NHE3 protein that resulted from exocytosis of vesicles was 7.2 ± 0.4 OD · mm2, n = 4. When the PCO2 was increased to 70 mmHg, exocytic insertion of NHE3 protein into the apical membrane increased more than twofold to 17.7 ± 2.7 OD · mm2, n = 4; P < 0.01.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Studies of the effects of acid-base variables on intestinal Na+ absorption have revealed important differences between the ileum and colon. The most obvious difference is the specific response of the ileum and colon to changes in ambient pH and PCO2, respectively (8, 9, 16, 30). In the colon, ion flux studies [using dimethylamiloride (DMA)], and immunocytochemistry and confocal microscopy (using polyclonal antibodies) demonstrated that an increase in PCO2 provides H+ for apical membrane NHE3 and stimulates its exocytosis (7). Wortmannin, an inhibitor of PI3-K and exocytosis (24), inhibited 75% of the increment in Na+ absorption caused by an increase in PCO2 (from 21 to 70 mmHg). If in fact wortmannin completely inhibited CO2-stimulated exocytosis of NHE3 and did not affect NHE activity, then this finding suggests the relative importance of the two mechanisms of CO2 action. That is, colonic Na+ absorption was stimulated much more by CO2-stimulated exocytosis of NHE3-containing vesicles than by CO2 provision of H+ for apical membrane Na+/H+ exchange.

The results of the Western blots reported here extend these findings by demonstrating that in the steady state, there was almost twice as much NHE3 protein on the apical membrane of colonic epithelial cells at a PCO2 of 70 mmHg than at a PCO2 of 21 mmHg. This is approximately the difference observed in colonic net Na+ absorption at these CO2 tensions both in vitro and in vivo (10, 14, 16). The same pH difference in the absence of CO2 had no effect on apical membrane NHE3 protein content. In previous studies in our laboratory, changes in pH in the absence of CO2 or in the absence of a change in PCO2 had little or no effect on colonic Na+ absorption (9, 14, 16).

We also measured the effects of changing the CO2 tension on the endocytosis and exocytosis of apical membrane NHE3 protein in colonic epithelial cells. When PCO2 was reduced from 70 to 21 mmHg, the apical membrane NHE3 protein that internalized (by endocytosis) increased approximately twofold at 30 min. In a similar way, when PCO2 was increased from 21 to 70 mmHg, NHE3 appearance on the apical membrane (by exocytosis) at 30 min increased slightly more than twofold. The similarity of these fractional changes may mean that a much greater quantity of NHE3 protein internalized when PCO2 was reduced than joined the apical membrane when PCO2 was increased. This conjecture is based on our finding that the steady-state quantity of NHE3 protein on the apical membrane was relatively greater before endocytosis was stimulated by lowering the PCO2 than before exocytosis was stimulated by raising the PCO2, and the assumption that there were no changes in other modulators of membrane trafficking. The functional consequence of this quantitative difference may be the greater reduction in colonic J<UP><SUB>ms</SUB><SUP>Na<SUP>+</SUP></SUP></UP> and J<UP><SUB>net</SUB><SUP>Na<SUP>+</SUP></SUP></UP> when PCO2 is decreased (~8 µeq · cm-2 · h-1) than the increase in these parameters when PCO2 is increased (~5 µeq · cm-2 · h-1) (7, 9, 14).

The mechanism of the effect of changing CO2 tension on vesicle movement is uncertain. It is unlikely that carbonic anhydrase (CA) activity is involved. CA inhibition by methazolamide did not cause NHE3 endocytosis (at PCO2 70 mmHg) or inhibit endocytosis in colonic cells caused by a decrease in PCO2 (7). It is possible that CA is required for NHE3 exocytosis in conjunction with CO2 stimulation of colonic Na+ absorption, but this has not been studied. CO2-stimulated vesicle trafficking also did not depend on changes in pHi. Changes in pHi in the absence of changes in PCO2 do not affect colonic Na+ absorption, and isohydric changes in PCO2 stimulate Na+ absorption (13, 14). Endocytosis of NHE3 could involve clathrin-coated pits and vesicles as appears to be true in rabbit ileal villus cells, and a subapical compartment of recycling endosomes as described in NHE3-transfected adaptor protein-1 cells (11, 12). However, nothing about clathrin, the clathrin coat lattice, or the formation or uncoating of clathrin-coated vesicles suggests the CO2 sensitivity or specificity observed here (22). In our previous study in which subapical vesicles were enumerated in colonic epithelial cells (7), both coated and uncoated vesicles varied with PCO2.

The effect of CO2 on NHE3 activity and trafficking also may involve apical membrane lipid rafts and the actin cytoskeleton. The latter was shown to have a specific role in clathrin-mediated apical membrane endocytosis in Madin-Darby canine kidney cells (18). In rabbit ileum, stimulation of NHE3 activity by epidermal growth factor and clonidine was associated with a marked increase in brush-border raft and cytoskeletal pools of NHE3 (26). Disruption of lipid rafts with methyl-beta -cyclodextrin or destabilization of the actin cytoskeleton with cytochalasin D decreased early endosome-associated NHE3. However, regulated NHE3 exocytosis and endocytosis were not directly measured in this study. Furthermore, the fact that Na+ absorption in rabbit ileum is pH rather than CO2 sensitive (5) suggests that lipid rafts may have more to do with the effect of pH in rat ileum than CO2 in rat colon.

In fact, in contrast to the colon, the rat ileum exhibited no evidence that vesicle trafficking plays a role in pH-modulated Na+ absorption. Under acid-base conditions associated with markedly different rates of Na+ absorption, the numbers of total, coated, and uncoated subapical vesicles counted in ileal epithelial cells remained unchanged. In colonic epithelial cells, subapical vesicle numbers decreased by 31% when PCO2 was increased from 21 to 70 mmHg and were not affected by changes in pH or Na+ absorption in the absence of a change in PCO2 (7). Evidence of pH- or CO2-stimulated endocytosis was not found in the ileum by confocal microscopy. Epithelial apical membranes labeled with FITC internalized when pH was increased by lowering the PCO2, but an identical pH increase in HEPES buffer that causes a similar reduction in ileal Na+ absorption had no effect. Exocytosis was examined for in ion-flux studies of the ileum. We found that the increments in ileal J<UP><SUB>ms</SUB><SUP>Na<SUP>+</SUP></SUP></UP> and J<UP><SUB>net</SUB><SUP>Na<SUP>+</SUP></SUP></UP> caused by a decrease in pH were unaffected by the presence of wortmannin at concentrations that inhibit exocytosis (7, 20, 24). As noted above, using identical protocols in the colon, we found that FITC-labeled apical membranes always internalized when Na+ absorption decreased, and wortmannin markedly inhibited the CO2-stimulated increase in colonic Na+ absorption (7).

These findings were confirmed by surface biotinylation and Western blots that showed similar steady-state levels of both NHE2 and NHE3 protein on the apical membrane of ileal epithelial cells at pH values of 7.6 and 7.1. Moreover, there was no difference in the quantity of NHE3 that internalized when pH was increased. This contrasts with the rat colon, which had a much greater steady-state quantity of apical membrane NHE3 at pH 7.1 (PCO2 = 70 mmHg) than at 7.6 (PCO2 = 21 mmHg) and internalized a markedly greater quantity of NHE3 when PCO2 was decreased from 70 to 21 mmHg (compare Figs. 3 and 4). Colonic NHE3 alone was examined because of the predominance of this isoform in mediating CO2-stimulated Na+ absorption as shown by inhibition studies using DMA (7). Whether the role of NHE3 in CO2-stimulated colonic Na+ absorption reflects its greater abundance or whether this isoform is specifically linked to CO2 stimulation is not clear. DMA cannot be used to determine the relative importance of the NHE2 and NHE3 isoforms in pH-stimulated Na+ absorption in the ileum because DMA does not inhibit these isoforms in this tissue (A. N. Charney and R. W. Egnor, unpublished observations). In any case, as noted above, the abundance of ileal NHE2 and NHE3 was not affected by pH levels that predictably alter Na+ absorption.

The lack of evidence for pH-related vesicle trafficking in the ileum is compelling because the pH range studied was physiological, the studies were performed in HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>/Ringer as well as HEPES/Ringer solutions, and a variety of studies examining exocytosis, endocytosis, or both were performed. Indeed, the finding that all of these experiments were positive in the colon (implying the presence of CO2-stimulated vesicle trafficking) and were negative in the ileum also supports the validity and interpretation of the results. We would suggest that the difference between the sensitivity of ileal and colonic Na+ absorption to pH and CO2, respectively, reflects this link to vesicular transport. Changes in PCO2 in colonic epithelial cells affect vesicle trafficking as well as the H+ supply. As a result, there is an extremely large difference in the colonic Na+ absorptive response to a change in PCO2 as compared to a change in pH (in the absence of a change in PCO2) (9, 16). Although changes in PCO2 affect ileal Na+ absorption, similar quantitative changes in J<UP><SUB>ms</SUB><SUP>Na<SUP>+</SUP></SUP></UP> and J<UP><SUB>net</SUB><SUP>Na<SUP>+</SUP></SUP></UP> are observed when pH is changed to a similar degree in CO2-free (HEPES)/Ringer solution (8, 30). It appears that this is because the mechanisms that link CO2 to the vesicle transport system are not present in this segment. Apparently, changes in Ringer solution (and cellular) pH affect ileal Na+ absorption by altering the supply of H+ to the apical membrane NHE and possibly by altering its allosteric properties (23, 29). In this regard in LLC-PK1 cells, pH activation of NHE3 (in the absence of CO2) was not accompanied by changes in the association of NHE3 with the actin cytoskeleton or lipid rafts or in the abundance of surface NHE3 but did involve a change in its conformational state (19).

In conclusion, we have shown that pH-stimulated ileal Na+ absorption does not involve changes in vesicular trafficking between the epithelial apical membrane and a subapical compartment. Under conditions in which ileal Na+ absorption is altered, no evidence of ileal NHE endocytosis or exocytosis could be found by electron and confocal microscopy, apical membrane biotinylation and Western blotting, or Ussing chamber ion fluxes in the presence of wortmannin. By comparison, CO2-stimulated Na+ absorption in the colon requires vesicular trafficking for a maximal response. NHE3 protein along the apical membrane of colonic epithelial cells was greater at a PCO2 of 70 than 21 mmHg, which is consistent with greater Na+ absorption at higher CO2 tensions. In addition, using all of the above techniques, endocytosis and exocytosis of NHE3-containing colonic vesicles were demonstrated when PCO2 was decreased and increased, respectively (7). These findings suggest that the presence or absence of CO2-responsive vesicular trafficking accounts in part, if not entirely, for the differing Na+-absorptive responses of the ileum and colon to pH and CO2, respectively.


    ACKNOWLEDGEMENTS

We thank Drs. Manuela Varzescu and Ute Frevert for assistance in the performance of these studies.


    FOOTNOTES

This work was supported by the Office of Research and Development, Medical Research Service, Department of Veterans Affairs.

Address for reprint requests and other correspondence: A. N. Charney, Nephrology Section, VA Medical Center, 423 East 23rd St., New York, NY 10010 (E-mail: alan.charney{at}med.va.gov).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

May 22, 2002;10.1152/ajpcell.00079.2002

Received 19 February 2002; accepted in final form 15 May 2002.


    REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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