1 Nephrology Section and 2 Department of Pathology, Veterans Affairs Medical Center, New York University School of Medicine, New York, New York 10010
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ABSTRACT |
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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
rat; ileum; colon; electrolyte transport
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INTRODUCTION |
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CO2 tension
affects electroneutral colonic NaCl absorption by altering the
activities of the Na+/H+ and
Cl/HCO
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.
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METHODS |
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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 HCONa+ transport.
Ion fluxes were measured to determine whether the effect of pH on ileal
Na+ absorption (in HCO
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
Confocal microscopy.
As previously described for rat colon (7), segments of
unstripped rat distal ileum were mounted in Ussing chambers in
HCO
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
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.
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RESULTS |
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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
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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
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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 HCOJ
2 · h
1, and
J
2 · h
1,
n = 11, whereas in the presence of wortmannin,
J
2 · h
1 and
J
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
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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
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DISCUSSION |
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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
J2 · 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--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
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
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.
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ACKNOWLEDGEMENTS |
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We thank Drs. Manuela Varzescu and Ute Frevert for assistance in the performance of these studies.
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FOOTNOTES |
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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.
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