Acid-base effects on intestinal Cl- absorption and vesicular trafficking

Alan N. Charney,1 Richard W. Egnor,2 David Henner,1 Haroon Rashid,1 Nicholas Cassai,3 and Gurdip S. Sidhu3

1Nephrology Section, 2Research Service, and 3Department of Pathology, Veterans Affairs Medical Center, New York University School of Medicine, New York, New York 10010

Submitted 20 October 2003 ; accepted in final form 23 December 2003


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In rat ileum and colon, apical membrane exchange and net Cl- absorption are stimulated by increases in PCO2 or . Because changes in PCO2 stimulate colonic Na+ absorption, in part, by modulating vesicular trafficking of the Na+/H+ exchanger type 3 isoform to and from the apical membrane, we examined whether changes in PCO2 affect net Cl- absorption by modulating vesicular trafficking of the exchanger anion exchanger (AE)1. Cl- transport across rat distal ileum and colon was measured in the Ussing chamber, and apical membrane protein biotinylation of these segments and Western blots of recovered proteins were performed. In colonic epithelial apical membranes, AE1 protein content was greater at PCO2 70 mmHg than at PCO2 21 mmHg but was not affected by pH changes in the absence of CO2. AE1 was internalized when PCO2 was reduced and exocytosed when PCO2 was increased, and both mucosal wortmannin and methazolamide inhibited exocytosis. Wortmannin also inhibited the increase in colonic Cl- absorption caused by an increase in PCO2. Increases in PCO2 stimulated ileal Cl- absorption, but wortmannin was without effect. Ileal epithelial apical membrane AE1 content was not affected by PCO2. We conclude that CO2 modulation of colonic, but not ileal, Cl- absorption involves effects on vesicular trafficking of AE1.

PCO2; ileum; colon; anion exchanger 1; Na+/H+ exchanger type 3


INTESTINAL CL- ABSORPTION is mediated by an electroneutral anion exchange process located on the apical membranes of ileal and colonic epithelial cells in many species. Under normal conditions, extracellular Cl- is exchanged for intracellular and/or OH- (36), but butyrate and other organic anions may substitute for Cl- or , depending on the experimental conditions (1, 17, 20, 30, 31, 35, 37). Evidence in several studies suggests that more than one apical anion exchanger is present (3437, 41). However, in every case, the rate of exchange (i.e., the activity of the exchanger) is primarily determined by the concentration gradients of the transported ions (12, 21). Indeed, no other mechanism controlling the rate of exchange has been described. For example, the presence of luminal Na+ is believed to increase Cl- absorption by alkalinizing the cell by means of increased apical membrane Na+/H+ exchange (15, 25). By a similar mechanism {i.e., increasing intracellular , increases in either PCO2 or the extracellular stimulate ileal and colonic Cl- absorption and secretion (12, 29, 48, 49).

We recently reported that CO2 stimulates rat colonic Na+ absorption both by providing H+ for Na+/H+ exchange across the apical membrane of epithelial cells and by stimulating the exocytotic movement of the Na+/H+ exchanger type 3 (NHE3) isoform from a subapical compartment to the apical membrane (4, 5). Reductions in PCO2 decrease colonic Na+ absorption by providing fewer H+ and stimulating the endocytosis of NHE3-containing vesicles. Neither changes in medium pH in the absence of CO2 nor changes in medium at constant PCO2 affect vesicular trafficking. This finding was particularly compelling because CO2 is the specific extracellular acid-base variable that affects colonic Na+ absorption in vitro and in vivo (7, 18). Moreover, vesicular trafficking of NHE3 (and NHE2) was not observed in rat ileum (4), a tissue in which Na+ absorption is specifically responsive to pH rather than CO2 (6, 45).

The presence of a exchanger along the apical membranes of ileal and colonic epithelial cells and its response to changes in PCO2 suggested the possibility that Cl- absorption may also be regulated by vesicular trafficking. The mRNAs of the exchangers AE1, AE2, and down-regulated in adenoma (DRA) have been identified in the small and large intestines of the rat (22, 26, 39). The location of AE1 along the apical membranes of epithelial cells in the colon and of DRA in the duodenum suggested functional roles for these exchangers in Cl- absorption and secretion, respectively (22, 39). In fact, colonic AE1 decreased in abundance in Na+-depleted rats (39), a condition that predictably reduces colonic Cl- absorption in the Ussing chamber, and exchange in apical membrane vesicles (38, 44). The availability of antibodies to AE1 provided the opportunity to confirm that this isoform is present in rat ileum and colon and to determine whether it is present along the apical membranes and in subapical vesicles of epithelial cells. We also examined whether CO2-sensitive trafficking of vesicles containing this exchanger was present in the ileum and colon and correlated with CO2-sensitive Cl- absorption.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Approvals of the 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 body wt) was used to achieve surgical anesthesia. The distal 10 cm of ileum ending 7 cm from the ileocecal valve, the proximal 10 cm of the proximal colon extending from the distal cecum, and the distal 10 cm of colon were removed and rinsed with 0.9% saline. Death was by exsanguination under surgical anesthesia.

Chemicals and solutions. Reagent grade chemicals were obtained from Sigma (St. Louis, MO) unless otherwise indicated. Rabbit anti-AE1 was obtained from Alpha Diagnostics (San Antonio, TX) (27). This antiserum was generated from a 20-amino acid peptide sequence (control peptide) near the NH2 terminus of the cytoplasmic domain of rat AE1. The control peptide was coupled to keyhole limpet hemocyanin, and antibodies were generated in rabbits. The antiserum was affinity purified. This peptide sequence does not have significant sequence homology to other AEs or proteins. Rabbit anti-NHE3 was a gift from E. B. Chang (University of Chicago) (2).

Ringer solutions contained (in mM): 140.2 Na+, 4 K+, 1.2 Mg2+, 1 Ca2+, 100 Cl-, , , , 18 glucose, and either 21 or 21 HEPES Na+ gluconate, 10 salt. In all protocols in which the ileum was incubated in the Ussing chamber, mannitol was substituted for glucose in the mucosal bathing solution. The Ringer was gassed with 3% CO2 (PCO2 21 mmHg, pH 7.6) or 11% CO2 (PCO2 70 mmHg, pH 7.1) (balance O2) to obtain the indicated pH values. The HEPES Ringer 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 of the Ringer solution were measured with a Radiometer BMS 3 Mk 2 system with a PHM 73 acid-base analyzer (The London Company, Cleveland, OH).

Immunoperoxidase labeling. As previously described (5), two segments of unstripped distal ileum or stripped distal colon were mounted in Ussing chambers in 6 ml of Ringer at 37°C and exposed to 3% CO2 for 30 min. Six milliliters of 10% formalin containing 1% ZnSO4 were added to each reservoir and incubated for 5 min. Tissues were then removed from the chambers and fixed in full-strength fixative for 30 min. All subsequent procedures were carried out at room temperature unless otherwise noted. Tissues were permeabilized and blocked with a mixture of PBS/5% Triton X-100/1% BSA/15% normal goat serum (blocking solution) for 4 h. Experimental samples were exposed overnight to rabbit anti-AE1 serum (1:10). Control samples were incubated overnight in blocking solution containing nonimmune serum or serum that had been preincubated with excess control peptide, and immune complexes were removed by centrifugation. All tissues were then washed 3 x 0.5 h with blocking solution. Samples were reacted with secondary antibody conjugated to peroxidase (goat anti-rabbit IgG, 1:50; Cell Signaling, Beverly, MA) for 4 h and washed 3 x 0.5 h in blocking solution. Peroxidase was visualized using the Sigma FAST 3,3'-diaminobenzidine tablet set for 60 min. Samples were washed in 50 mM Tris·HCl (pH 7.6) 3 x 0.5 h and then held in 3% glutaraldehyde in 0.1 mM Na2HPO4 buffer (pH 7.4) before embedding and sectioning for examination by transmission electron microscopy at x110,000 magnification. The tissues were processed with uranyl acetate staining so that the tissue ultrastructure would be more evident.

Apical membrane protein biotinylation. Levels of apical membrane AE1 were measured in pairs of intact unstripped distal ileal and whole colonic segments under various acid-base conditions. The use of whole colons in these studies was based on the qualitatively similar effect of PCO2 and wortmannin on Cl- fluxes in the proximal and distal colon (see RESULTS). These ileal and colonic segments were rinsed well with ice-cold PBS containing 0.1 mM CaCl2 and 1 mM MgCl2 (PBS-CaMg). One of the ileal or colonic segments was incubated in a flask at pH 7.6 (in or HEPES Ringer) 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 previously described method (19, 50) as modified in our laboratory (4, 19). The lumen of each segment was filled with 2 ml of TEA containing 3 mg EZ-Link sulfo-NHS-SS-biotin (Pierce, Rockford, IL) and incubated for 1 h at 0°C. Segment contents were emptied, and the lumen was flushed with several milliliters of PBS-CaMg containing 100 mM glycine and 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% NaDOC, 0.1% SDS) containing protease inhibitor cocktail (Sigma) for 30 min at 0°C. The lysate was then centrifuged at 100,000 g for 10 min. The protein concentration in the supernatant was measured by the Lowry method (33), which, as shown in preliminary experiments, was not affected by the RIPA buffer. After the protein concentration was adjusted 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 1 ml of RIPA. The bound proteins were then extracted and subjected to SDS-PAGE, immunoblotting, and quantification as described in Western blots.

Membrane endocytosis. To quantify the effect of reducing CO2 on membrane endocytosis, internalization of apical membrane AE1 was measured in pairs of intact unstripped whole colonic segments (4). After being rinsed well with ice-cold PBS-CaMg, the segments were incubated in a flask containing Ringer at PCO2 70 mmHg (pH 7.1) for 30 min at 37°C. Segments were then placed in ice-cold TEA, and biotinylation was performed as described above. After being quenched with PBS-glycine, one of each pair of segments was rinsed well with the ice-cold incubation solution and then incubated in Ringer at PCO2 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, 0.2% BSA, pH 8.6), and 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 being rinsed thoroughly with PBS-CaMg. Cells were then lysed, and the biotinylated proteins (representing internalized apical membrane) were extracted as described above. The proteins recovered from the streptavidin-agarose beads were subjected to SDS-PAGE, immunoblotting, and quantification as described in Western blots.

Membrane exocytosis. We also quantified the effect of increasing CO2 on the exocytosis of membrane vesicles containing AE1 (4). Intact unstripped whole colonic segments were rinsed well inside and out with ice-cold PBS-CaMg. All pairs were incubated in a flask containing Ringer at PCO2 21 mmHg (pH 7.6) in the presence and absence of luminal 0.75 µM wortmannin or luminal and serosal 0.1 mM methazolamide for 30 min at 37°C. Tissues were then chilled in ice-cold TEA. The lumen of each segment was emptied and then filled with 2 ml of TEA containing 3 mg EZ-Link sulfo-NHS-acetate (Pierce) and 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 containing 100 mM glycine, segments were incubated for 20 min in the same buffer at 0°C. After being quenched, each segment was rinsed well with ice-cold incubation solution and incubated in Ringer (containing when specified 0.75 µM wortmannin or 0.1 mM methazolamide) at PCO2 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 EZ-Link sulfo-NHS-SS-biotin as described above. The proteins recovered from the streptavidin-agarose beads (representing newly inserted vesicles) were subjected to SDS-PAGE, immunoblotting, and quantitation as described in Western blots.

Western blots. 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, 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-AE1 at 1:250 or NHE3 at 1:500 followed by horseradish peroxidase-labeled goat anti-rabbit IgG at 1:2,000. Labeling was visualized by chemiluminescence using Lumiglo (Cell Signaling) 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). The linearity of Western blots was ensured by using serial dilutions of the recovered biotinylated proteins and by doing exposures of varying duration. Only blots falling within the linear range of the detection system were considered. Results were expressed in arbitrary optical density units (OD·mm2).

Cl- transport. Ion fluxes were measured to determine whether the effect of CO2 on ileal and colonic Cl- absorption was altered by wortmannin, an inhibitor of phosphatidylinositol 3-kinase and vesicle movement (28). As previously described (5, 18, 45), pairs of unstripped ileal and stripped proximal and distal segments were studied under short-circuit conditions in modified Ussing half-chambers exposing 0.62-cm2 (ileum) or 1.12-cm2 (colon) surface area. Tissue conductance (G) was calculated from periodic bipolar pulses of 0.5 mV and was the basis for pairing tissues when the difference in 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 Cl- were measured by adding 1 µCi 36Cl- (100 Ci/g specific activity; 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 (Jms) and serosal-to-mucosal (Jsm) fluxes (expressed as µeq·cm-2·h-1) were measured for 30 min at PCO2 21 mmHg (pH 7.6) and then 70 mmHg (pH 7.1) or the reverse. During a third flux period at the same CO2 tension, 0.75 µM wortmannin was present in the mucosal bathing solution. The PCO2 was then returned to the initial level and a fourth flux period was performed. All gas change periods were preceded by a 16-min equilibration; wortmannin addition was followed by a 30-min equilibration before flux was measured. Aliquots of 500 µl were replaced with identical Ringer including wortmannin where appropriate. Net flux (Jnet) was calculated as Jms - Jsm.

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


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Immunolocalization of AE1. Figure 1 depicts electron micrographs of epithelial cells of ileum and colon exposed to Ringer at PCO2 21 mmHg (pH 7.6). Peroxidase reaction product was deposited along the microvilli and, as shown in the ileal specimen, on some subapical vesicular membranes. Reaction product was not observed on mitochondrial membranes or on basolateral cell membranes. Tissues incubated with nonimmune serum were devoid of reaction product.



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Fig. 1. Immunolocalization of anion exchanger (AE)1 in ileal and colonic microvilli and subapical vesicles. Segments were incubated in Ringer at PCO2 21 mmHg, fixed, permeablized, and exposed to rabbit anti-AE1 serum and goat antirabbit IgG conjugated to peroxidase or to nonimmune or pre-absorbed serum. Segments were prepared for examination by transmission electron microscopy. In the ileum (A) and colon (B), the peroxidase reaction product was deposited on microvilli (M; arrows) and on vesicular membranes (V; arrowhead). No reaction product was observed on mitochondrial membranes or on basolateral cell membranes (not shown). Ileal (C) and colonic (D) tissues exposed to nonimmune serum (shown) or preabsorbed serum (not shown) were devoid of reaction product. Note the 40% difference in the size reference bar between the ileal and colonic photomicrographs.

 

Effect of CO2 and pH on apical membrane AE1 protein content. The effect of CO2 and pH on the AE1 protein content of epithelial apical membranes was then examined. In these experiments, the level of AE1 was measured in separate distal ileal and whole colon segments in Ringer at PCO2 21 mmHg (pH 7.6) and PCO2 70 mmHg (pH 7.1). As shown in Fig. 2, at PCO2 21 mmHg, AE1 protein content of ileal apical membranes was 1.4 ± 0.3 OD/mm2 (n = 6). At PCO2 70 mmHg, this value was similar: 1.4 ± 0.2 OD/mm2 [n = 6, P = not significant (NS)]. In the colon, AE1 protein content was 1.1 ± 0.2 (n = 6) at PCO2 21 mmHg and was significantly greater at PCO2 70 mmHg: 2.5 ± 0.5 OD/mm2 (n = 6, P < 0.05).



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Fig. 2. Effect of CO2 and pH on AE1 protein content of apical membranes of ileal and colonic epithelial cells. Segments of rat distal ileum and whole colon were incubated in Ringer at PCO2 21 mmHg (pH 7.6) or 70 mmHg (pH 7.1) or in HEPES Ringer at pH 7.6 or 7.1. Apical membrane proteins of the segments were then biotinylated, and Western blots of the recovered biotinylated proteins were performed and AE1 was quantified. The levels of AE1 on ileal apical membranes were similar at pH 7.6 and 7.1. The levels of AE1 on colonic apical membranes were higher at PCO2 70 than 21 mmHg but similar at pH 7.6 and 7.1 in HEPES Ringer ( 0 mM, PCO2 0 mmHg). OD, optical density. Values are means ± SE; n = 6 for each condition. *P < 0.05 when values were compared by paired Student's t-test.

 

We then examined whether the effect on colonic apical membrane AE1 was due to the difference in PCO2 or pH. As shown in Fig. 2, AE1 protein content was similar at pH 7.6 and 7.1 in CO2-free HEPES Ringer. Ileal AE1 protein content was also similar at pH 7.6 and 7.1 in HEPES buffer (data not shown).

Effect of CO2 on AE1 endocytosis and exocytosis in the colon. The different levels of apical membrane AE1 at PCO2 21 and 70 mmHg in the colon suggested that AE1-containing apical membrane may recycle in response to a change in CO2 tension. To examine whether PCO2 affected endocytosis of AE1, we tested the effect of decreasing PCO2 on biotinylated apical membrane. As shown in Fig. 3, when apical membrane was biotinylated after 30-min incubation in Ringer at PCO2 70 mmHg and then incubated for an additional 30 min at PCO2 70 mmHg, AE1 protein in internalized apical membrane was 1.1 ± 0.3 OD/mm2 (n = 6). In tissues similarly biotinylated, 30 min after the PCO2 was decreased from 70 to 21 mmHg, internalized biotinylated apical membrane AE1 protein almost doubled to 2.1 ± 0.3 OD/mm2 (n = 6, P < 0.05).



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Fig. 3. Effect of CO2 on endocytosis of AE1 protein in colonic epithelial cells. Segments of whole colon were incubated in Ringer at PCO2 70 mmHg (pH 7.1), and the internalization (endocytosis) of AE1 in biotinylated apical membranes was measured by Western blotting 30 min after leaving the PCO2 unchanged or decreasing it to 21 mmHg (pH 7.6). A greater quantity of AE1 internalized at PCO2 21 than 70 mmHg. Values are means ± SE; n = 6 for each condition. *P < 0.05 when values were compared by paired Student's t-test.

 

The effect of increasing PCO2 on exocytosis of AE1 in colonic epithelial cells was examined in a similar way. After blockade of apical membrane biotinylation after 30-min incubation at PCO2 21 mmHg, the PCO2 was either left unchanged or increased to 70 mmHg for 30 min and the surface was then biotinylated. Thus only AE1 that had undergone exocytosis was measured at the apical membrane. As shown in Fig. 4, when the PCO2 of 21 mmHg remained unchanged, apical membrane AE1 protein resulting from exocytosis of vesicles was 0.7 ± 0.1 OD/mm2 (n = 14). When the PCO2 was increased to 70 mmHg, exocytic insertion of AE1 protein into the apical membrane increased twofold to 1.4 ± 0.2 OD/mm2 (n = 14, P < 0.001).



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Fig. 4. Effect of CO2, wortmannin, and methazolamide on exocytosis of AE1 protein in colonic epithelial cells. Segments of whole colon were incubated in Ringer at PCO2 21 mmHg. After the surface proteins were blocked, the exocytosis of AE1-containing vesicles in biotinylated apical membranes was measured by Western blotting 30 min after leaving the PCO2 unchanged or increasing it to 70 mmHg. In several exocytosis experiments, colonic segments were exposed to 0.75 µM wortmannin or 0.1 mM methazolamide before and during the increase in PCO2 to 70 mmHg. A greater quantity of AE1 exocytosed at PCO2 70 than 21 mmHg. Both wortmannin and methazolamide inhibited the exocytosis of AE1 in response to increases in PCO2. Values are means ± SE; n = 14 for experiments at PCO2 21 and 70 mmHg, and n = 7 when either wortmannin or methazolamide was present. *P < 0.001 when values were compared by paired Student's t-test.

 

Effect of wortmannin and methazolamide on AE1 exocytosis in the colon. To confirm that the effect of CO2 on apical membrane AE1 protein content in colonic epithelial cells in fact represented an effect on AE1 exocytosis, we tested for an effect of wortmannin. Apical membrane biotinylation was blocked after a 30-min incubation at PCO2 21 mmHg in the presence of 0.75 µM wortmannin, a wortmannin concentration reported to inhibit exocytosis (4, 5, 23, 28). The PCO2 was then left unchanged or increased to 70 mmHg for 30 min and the surface was biotinylated. As shown in Fig. 4, the presence of wortmannin prevented the CO2-stimulated exocytotic increment in AE1 so that the level of exocytosis (0.7 ± 0.2 OD/mm2, n = 7) was similar to the level observed when the PCO2 was not increased.

Inhibition of carbonic anhydrase (CA) with methazolamide decreases the stimulatory effect of CO2 on colonic Cl- absorption (12). To examine whether this effect was mediated, in part, by inhibition of exocytosis, methazolamide was added to both bathing solutions at the same step that wortmannin was added at a concentration known to inhibit Cl- absorption (12). As described above, the PCO2 was then left unchanged or increased to 70 mmHg for 30 min and the surface was biotinylated. As shown in Fig. 4, methazolamide reduced the stimulatory effect of CO2 on AE1 exocytosis so that the level of AE1 at 70 mmHg PCO2 in the presence of methazolamide (0.7 ± 0.2 OD/mm2, n = 7) was not different from the level at 21 mmHg PCO2 in its absence.

Effect of wortmannin and methazolamide on NHE3 exocytosis in the colon. Because in a previous study we demonstrated CO2-stimulated exocytosis of NHE3 (4, 5), it was of interest to determine whether wortmannin and methazolamide would have similar effects on the exocytosis of NHE3 and AE1. As shown in Fig. 5, an increase in PCO2 from 21 to 70 mmHg increased NHE3 exocytosis and the presence of wortmannin prevented this increase. Also shown in Fig. 5, methazolamide also prevented the stimulatory effect of CO2 on NHE3 exocytosis. These effects of wortmannin and methazolamide on NHE3 exocytosis were qualitatively and quantitatively similar to their effects on the exocytosis of AE1 (compare with Fig. 4).



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Fig. 5. Effect of CO2, wortmannin, and methazolamide on exocytosis of Na+/H+ exchanger (NHE)3 protein in colonic epithelial cells. Segments of whole colon were incubated in Ringer at PCO2 21 mmHg. After the surface proteins were blocked, the exocytosis of NHE3-containing vesicles in biotinylated apical membranes was measured by Western blotting 30 min after leaving the PCO2 unchanged or increasing it to 70 mmHg. In several exocytosis experiments, colonic segments were exposed to 0.75 µM wortmannin or 0.1 mM methazolamide before and during the increase in PCO2 to 70 mmHg. A greater quantity of NHE3 exocytosed at PCO2 70 than 21 mmHg. Both wortmannin and methazolamide inhibited the exocytosis of NHE3 in response to increases in PCO2. Values are means ± SE; n = 10 for experiments at PCO2 21 and 70 mmHg; n = 4 when wortmannin was present and n = 6 when methazolamide was present. *P < 0.05 when values were compared by paired Student's t-test.

 

Effect of wortmannin on CO2-stimulated ileal Cl- absorption. The similarity of AE1 protein content on the apical membranes of ileal epithelial cells at 21 and 70 mmHg PCO2 suggested that AE1 trafficking in this tissue, if it occurs, is not sensitive to CO2. To confirm this conclusion, we measured unidirectional Cl- fluxes across ileal tissues bathed in Ringer at 21 (pH 7.6) and 70 mmHg (pH 7.1) PCO2 in the absence and then in the presence of mucosal 0.75 µM wortmannin. As shown in Table 1, in ileal tissues initially exposed to 21 mmHg PCO2, an increase in PCO2 to 70 mmHg stimulated Cl- absorption. This increase in Jnet was primarily due to an increase in Jms and was accompanied by decreases in Isc and PD as have been described previously (45). Wortmannin addition at 70 mmHg PCO2 did not significantly affect Cl- fluxes and did not affect the reduction in Cl- absorption caused by a decrease in PCO2 to 21 mmHg.


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Table 1. Effect of CO2 and wortmannin on ileal Cl- absorption

 

Also shown in Table 1 are ileal tissues initially exposed to 70 mmHg PCO2 in which a decrease in PCO2 to 21 mmHg diminished net Cl- absorption. Wortmannin addition at 21 mmHg PCO2 did not significantly affect Cl- fluxes and did not affect the increase in Cl- absorption caused by an increase in PCO2 to 70 mmHg. A direct comparison of the CO2-stimulated increments (2.6 ± 0.7, n = 8 vs. 2.5 ± 1.2 µeq·cm-2·h-1, n = 9, P = NS) and decrements (-3.3 ± 0.4, n = 9 vs. -3.6 ± 0.9 µeq·cm-2·h-1, n = 8, P = NS) in net Cl- absorption in the absence and presence of wortmannin confirmed the lack of effect of wortmannin.

In both protocols, decreases in PCO2 from 70 to 21 mmHg caused an increase in Jsm. In addition, a small, progressive increase in Jsm occurred during the course of the 3-h experiment accompanied by an increase in G. These findings have been observed previously in ileal flux experiments (11, 45) and are not believed to reflect changes in active Cl- secretion (11). Neither finding was affected by the presence of wortmannin.

Effect of wortmannin on CO2-stimulated colonic Cl- absorption. Colonic Cl- fluxes were measured to show that wortmannin inhibition of AE1 exocytosis in both proximal and distal colonic epithelial cells affected Cl- absorption. As described above, unidirectional Cl- fluxes were measured in colonic tissues bathed in Ringer at PCO2 21 (pH 7.6) and 70 mmHg (pH 7.1) in the absence and then in the presence of mucosal 0.75 µM wortmannin. As shown in Table 2, the proximal colon responded to increases in PCO2 from 21 to 70 mmHg with increases in Jms and Jnet. These changes were accompanied by decreases in Isc and PD. The presence of wortmannin did not affect Cl- fluxes at PCO2 70 mmHg or the reduction in Cl- absorptive fluxes when the PCO2 was decreased to 21 mmHg.


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Table 2. Effect of CO2 and wortmannin on proximal colonic Cl- absorption

 

In a similar way, decreases in PCO2 from 70 to 21 mmHg caused decreases in Jnet and the addition of wortmannin did not affect Cl- fluxes at PCO2 21 mmHg. A comparison of the CO2-induced decrement in net Cl- fluxes in the absence and presence of wortmannin was not quite statistically significant (-2.4 ± 1.8, n = 6 vs. -7.4 ± 1.7 µeq·cm-2·h-1, n = 6, P = 0.07). However, wortmannin reduced the increment in Cl- absorptive fluxes when the PCO2 was increased to 70 mmHg as indicated by a comparison of the increments in net Cl- fluxes in the absence and presence of wortmannin (5.7 ± 1.3, n = 6 vs. 2.6 ± 0.6 µeq·cm-2·h-1, n = 6, P < 0.05).

Identical experiments were performed in the distal colon and qualitatively similar results were obtained. As shown in Table 3, in the absence of wortmannin, increases and decreases in PCO2 caused increases and decreases, respectively, in Jms and Jnet. This effect has been described previously (12, 18). Furthermore, the addition of wortmannin did not affect Cl- fluxes when added at either CO2 tension and did not reduce the decrement caused by a reduction in PCO2 from 70 to 21 mmHg (-4.9 ± 1.1, n = 7 vs. -4.0 ± 1.0 µeq·cm-2·h-1, n = 7, P = NS). However, as was noted in the proximal colon, the presence of wortmannin reduced the stimulatory effect of increasing PCO2 from 21 to 70 mmHg on Cl- absorption (3.8 ± 0.7, n = 7 vs. 1.2 ± 0.8 µeq·cm-2·h-1, n = 7, P < 0.05).


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Table 3. Effect of CO2 and wortmannin on distal colonic Cl- absorption

 

In both proximal and distal colon, increases in PCO2 decreased Jsm, and decreases in PCO2 increased Jsm. This effect of CO2 apparently was mediated by the action of on a basal, active Cl- secretory process, as previously described (9). In addition, a small, progressive increase in Jsm was observed during the course of the experiment accompanied by an increase in G. Neither finding was affected by the presence of wortmannin.


    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The original observation that the number of subapical vesicles in colonic epithelial cells correlated with PCO2 suggested the possibility that CO2 stimulation of Na+ absorption may involve vesicular trafficking (5). Additional studies from our laboratory (4, 5) confirmed that this process is present in the rat colon, specifically affects the NHE3 isoform, and is specifically responsive to CO2. Wortmannin inhibition of 75% of the CO2-stimulated increment in colonic Na+ absorption suggested that membrane recycling of NHE3 may be an even more important mechanism of CO2 action than provision of H+ for apical membrane Na+/H+ exchange. Colonic Cl- absorption is also stimulated by increases in PCO2, although other means of alkalinizing epithelial cells also stimulate Cl- absorption (12, 48, 49). Because the vesicles observed by transmission electron microscopy in the above studies may have contained as well as Na+/H+ exchangers, we hypothesized that colonic Cl- absorption is also regulated by vesicular trafficking.

AE1 was investigated because AE1 mRNA has been found along the rat intestine (26, 27, 39) and antibody to this AE has recently become available. DRA is also distributed along the rat intestine, and when antibody becomes available to this AE, an examination of the roles and relative importance of these exchangers will be of great interest. For now, based on the results of the present study, we can conclude that AE1 is involved in CO2-stimulated colonic Cl- absorption and that its contribution is modulated, at least in part, by means of vesicular trafficking. Presumably, AE1 activity is also affected by the supply of provided by the CA-catalyzed hydration of CO2. This effect is mediated by changes in the gradient across the apical membrane of colonic epithelial cells, as previously described (12, 13).

The evidence that AE1 undergoes membrane trafficking includes its presence along the apical membranes of colonic epithelial cells, the correlation of AE1 protein content with CO2 tension, the presence of CO2-sensitive endocytosis and exocytosis of AE1, and the effect of wortmannin to inhibit both CO2-stimulated exocytosis of AE1 and CO2-stimulated Cl- absorption. Wortmannin, at the concentrations used in our study, inhibits exocytosis in various cell systems including the colon (4, 5, 23, 28). This was not a nonspecific or toxic effect of this chemical as wortmannin did not affect baseline fluxes at PCO2 21 or 70 mmHg or exaggerate the decrease in Cl- absorption caused by a reduction in PCO2. Remarkably, the inhibitory effects of wortmannin on CO2-stimulated AE1 exocytosis and Cl- absorption were analogous and quantitatively similar to the effects of wortmannin on CO2-stimulated colonic NHE3 exocytosis and Na+ absorption (5). Together, these findings suggest that CO2 modulation of vesicular trafficking of NHE3 and AE1 may be a very important means of regulating colonic Na+ and Cl- absorption.

It is unclear how this effect of CO2 is related to the dependence of CO2-stimulated colonic transport on CA activity. CA catalyzes the reaction CO2 + H2O -> H2CO3 and thereby supplies H+ and to apical membrane Na+/H+ and exchangers. Many findings support such a role including the high activity of CA in the mammalian colon (8), its presence at the apical membrane (as CA isozyme IV) and in cytoplasm (as CA isozymes I and II) (16, 32), and the finding that CA inhibition reduces CO2-stimulated colonic Na+ and Cl- absorption (8, 1214, 18). Nevertheless, recent evidence suggests that CA may play a noncatalytic role in colonic absorption as well. For example, inhibition of this enzyme reduces ileal and colonic absorption even in the nominal absence of CO2 (3).

In an earlier study, we examined the possibility that CA inhibition with methazolamide, a membrane-permeant CA inhibitor, affects CO2-stimulated endocytosis of NHE3. We found that it did not (5). However, in the present study, we found that methazolamide, at concentrations that inhibit CO2-stimulated Na+ and Cl- absorption (12), inhibited CO2-stimulated exocytosis of both AE1 and NHE3. This suggests that the well-described inhibitory effect of CA inhibitors on CO2-stimulated Na+ and Cl- absorption may be due as much (or more) to inhibition of exocytosis as to inhibition of the catalyzed production of H+ and . Presumably, methazolamide inhibits exocytosis after binding to CA, which itself is bound to or associated with apical membrane (and possibly vesicle membrane) AE1. Physical and functional links between CA II and CA IV with membrane-bound AE1 have been demonstrated, albeit not in intestinal tissues (24, 40, 42, 43, 46, 47). One possibility is that methazolamide's effects on exocytosis reflect a role for CA in vesicle trafficking perhaps independent of its catalytic hydration of CO2. Alternatively, methazolamide's effects on electrolyte transport may reflect, in part, inhibition of the exocytotic process via its attachment to CA, independent of its inhibition of CA catalytic activity.

The observation that changes in PCO2 affect the movement (in the same direction) of NHE3 and AE1-containing vesicles suggested the possibility that these vesicles may contain both exchangers. Although separate sets of NHE3 and AE1-containing vesicles are physiologically within reason, it seems likely that the correspondence between colonic Na+ and Cl- absorption in the rat under most acid-base conditions reflects a physical as well as functional association. However, this linkage between NHE3 and AE1 may not be invariant, because several examples of the dissociation of colonic Na+ and Cl- absorption have been described (10, 48). This suggests the presence of independent function and perhaps independent locations of at least some of these exchangers. In any case, the fact that methazolamide inhibited the exocytosis of both NHE3 and AE1 and that CA binds AE1 but not NHE3 strongly supports the possibility that AE1 and NHE3 are both present on the same subapical vesicles.

These findings in the colon may be contrasted with the lack of evidence for vesicular trafficking of AE1 in the ileum. Although AE1 was found on apical membranes of ileal epithelial cells by immunocytochemistry, apical membrane AE1 protein content did not vary with changes in PCO2 or pH. Moreover, wortmannin, at concentrations that inhibited CO2-stimulated colonic Cl- absorption, did not affect CO2-stimulated ileal Cl- absorption. The lack of effect of pH and CO2 to modulate vesicular trafficking of ileal AE1 is similar to the lack of effect of these acid-base variables on the trafficking of ileal NHE3 (and NHE2) (4). Because NHE3, NHE2, and AE1 are all present on ileal apical membranes, we may suppose that they have roles in Na+ and Cl- absorption. However, the stimulatory effects of pH on ileal Na+ absorption, and of CO2 and on ileal Cl- absorption, are apparently not mediated by effects on membrane trafficking.

In summary, our data suggest that CO2 modulation of colonic Cl- absorption involves effects on vesicular trafficking of AE1. Although AE1 is present along the apical membranes of both ileal and colonic epithelial cells, CO2-sensitive Cl- absorption in the ileum does not involve such trafficking. Remarkably, wortmannin inhibited the CO2-induced exocytosis of AE1 and 70% of the increment in net Cl- absorption in the colon. This suggests that CO2 stimulation of colonic Cl- absorption is mediated more by an effect on vesicular trafficking than by supplying ions for apical membrane exchange. A similar quantitative effect of wortmannin on CO2-induced exocytosis of NHE3 and colonic net Na+ absorption strongly supports the importance of this mechanism of CO2 action. In addition, the inhibitory effect of methazolamide on the exocytosis of colonic NHE3 and AE1 suggests that the catalyzed production of H+ and from CO2 and H2O may not be the primary way that CA mediates CO2-sensitive Na+ and Cl- absorption in rat colon.


    ACKNOWLEDGMENTS
 
GRANTS

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


    FOOTNOTES
 

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.


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