Departments of Internal Medicine, Surgery, and Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut 06520-8019
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ABSTRACT |
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A novel Na/H exchange activity that requires Cl was recently identified in the apical membrane of crypt cells of the rat distal colon. This study explores the nature of the coupling of Cl and Na/H exchange. A concentration of 100 µM 5-nitro-2-(3-phenylpropylamino)benzoic acid, a Cl channel blocker, inhibited the Cl dependence of both proton gradient-driven 22Na uptake from crypt cell apical membrane vesicles and Na-dependent intracellular pH recovery from an acid load during microperfusion of the crypt lumen. Cl-dependent proton gradient-driven 22Na uptake was inhibited by 94% by 500 µM DIDS but only by 1% by 10 µM DIDS, an anion exchange inhibitor at low concentrations but a Cl channel blocker at high concentrations. In addition, a polyclonal antibody to the cystic fibrosis transmembrane conductance regulator (CFTR) inhibited Cl-dependent proton gradient-driven 22Na uptake by 38%. These results indicate that the Cl dependence of Na/H exchange in the colonic crypt apical membrane involves a Cl channel and not a Cl/anion exchange and permit the speculation that this Cl channel activity represents both CFTR and the outward rectifying Cl conductance.
colonic crypt glands; apical membrane vesicles; cystic fibrosis transmembrane conductance regulator; outward rectifying chloride conductance
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INTRODUCTION |
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SODIUM AND CHLORIDE absorptive and secretory processes have been extensively studied in several epithelia, including the small and large intestine. More than one mechanism can account for the close interrelationship between Na and Cl transport, including coupled cotransport (e.g., Na-K-2Cl cotransport), electrical or pH-coupled Na and Cl transporters (e.g., Na/H and Cl/HCO3 exchanges), and Na-dependent Cl/HCO3 exchange (4, 9, 16, 23).
An additional mechanism of Cl and Na interdependence was recently described in the rat distal colon (19). Cl-dependent Na/H exchange was identified in the apical membrane of colonic crypt cells using studies of 22Na uptake by isolated apical membrane vesicles (AMV) and intracellular pH (pHi) determinations in response to an acid load (19). Whereas minimal Cl-dependent Na/H exchange activity was noted in apical membranes of surface cells, Na/H exchange in apical membranes of crypt cells was almost exclusively Cl dependent (19). Distinct differences exist in the characteristics of Cl-dependent and -independent Na/H exchanges in the rat distal colon in that Cl-dependent Na/H exchange in crypt cells is relatively amiloride resistant compared with the amiloride sensitivity of Cl-independent Na/H exchange in surface cells (19).
The nature of the Cl dependence of Na/H exchange in the apical membrane of crypt epithelial cells is not known. It has not been established whether the Cl dependence of Na/H exchange represents 1) the coupling of a Cl transport protein with a known Na/H exchange isoform or with a previously unidentified Na/H exchange isoform or 2) the presence of a novel transport protein with both Cl transport and Na/H exchange activities. Because several mechanisms of Cl transport are present in the mammalian colon, including crypt epithelial cells, the Cl transport mechanism linked to Na/H exchange in the colonic crypt could represent either a Cl/anion exchange or a Cl channel [e.g., cystic fibrosis transmembrane conductance regulator (CFTR) or outward rectifying Cl conductance (ORCC)]. Presently, available information does not distinguish between these possibilities.
This study was designed to determine whether Cl-dependent Na/H exchange was associated with a Cl/anion exchange or a Cl channel by determining the effect of various inhibitors of both anion exchange and Cl channels on the Cl dependence of both proton gradient-driven 22Na uptake by AMV and Na-dependent pHi recovery to an acid load in microperfusion experiments. The results of this study indicate that the Cl dependence of Na/H exchange in the apical membrane of colonic crypt cells represents a Cl channel, not a Cl/anion exchange, that is linked to Na/H exchange function.
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METHODS |
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AMV Preparation
AMV were isolated from distal colonic crypt cells that were isolated by the divalent chelation and sequential separation of Lomax et al. (12) as previously described (19). In brief, segments of distal colon were removed from male Sprague-Dawley rats (200-250 g) and washed in ice-cold buffer medium that contained (in mM) 96 NaCl, 27 sodium citrate, 0.8 KH2PO4, 5.6 Na2HPO4, 1.5 glucose, and 0.5 dithiothreitol (buffer A, pH 7.4). After a 15-min incubation in buffer A at 37°C, everted colonic segments were transferred to a prewarmed cell isolation buffer that contained (in mM) 112 NaCl, 5 KCl, 30 EDTA, 29 HEPES, and 0.5 dithiothreitol (buffer B, pH 7.1). Surface-to-crypt cell fractions were sequentially isolated by shaking and incubation in buffer B at 37°C (6 complete exchanges of 10 min each). Crypt cells were sedimented by centrifugation (Beckman GS-6KR; GH-3.8 rotor) at 500 g for 2 min. Fractions 5 and 6 were used to isolate crypt cells and then to isolate AMV with the use of the method of Stieger et al. (26) as previously described (20). Purity of the AMV preparation was periodically verified by the 10- to 12-fold enrichment of ouabain-sensitive H-K-ATPase (21). Uptake studies were performed by rapid filtration techniques that have been previously described (20). Protein was measured by the method of Lowry et al. (13).All experiments were performed with at least three different membrane preparations. Data presented are means of triplicate assays of a typical experiment. Standard errors are not provided when they are <5% of the mean.
pHi Measurements
All pHi experiments were performed on isolated perfused colonic crypts. The methods for isolation and perfusion of colonic crypts have previously been described in detail (21, 24). Briefly, individual colonic crypts were harvested from distal colon that was isolated and hand dissected at 4°C in HEPES-based Ringer solution. After dissection, individual crypts were transferred to the stage of an inverted microscope and attached to a series of concentric glass pipettes, cannulated, and perfused in vitro. After perfusion was established, the crypt was warmed to 37°C in a perfusion chamber that completely exchanged the volume four to eight times per second. The crypt was loaded with the pH-sensitive 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein-AM (BCECF-AM) (10 µM). After dye uptake by crypt cells, intracellular esterases cleaved the dye molecules, leaving the fluorescent, cell-impermeant pH marker BCECF trapped within the cytosol. All pH measurements were carried out using a dual-excitation monochrometer system. Data were collected using an intensified charge-coupled device system. To record pHi in semireal time, pixel intensity values were collected from at least five areas of interest taken at random locations along the crypt axis. The intensity information as well as the images from each excitation wavelength were recorded to the hard disk of a personal computer and stored to either an optical hard drive or to the computer disk. This method of data storage permitted subsequent analysis of the data following the experiment and examination of additional areas of interest following completion of the experiments. The fluorescent data were calibrated in vitro for the pH experiments and were converted to pHi values using the nigericin calibration technique (25). Nigericin (1 µg/ml) was placed into buffered solutions of varying pH as previously described (14, 22, 27).Solutions
The composition of the standard Na-containing, Cl-containing perfusion solution was (in mM) 125 NaCl, 5 KCl, 1.2 CaCl2, 1.2 MgSO4, 2 Na2H2PO4, 10.2 glucose, and 32.2 HEPES. In the Na-free solutions, Na was replaced with equivalent amounts of N-methyl-D-glucamine; in the Cl-free solutions, Cl was replaced by equivalent amounts of cyclamate and Ca was increased to 4.0 mM. The solutions were adjusted to a final osmolality of 300-310 mosmol/kgH2O. pH was adjusted to 7.4 at 37°C. ![]() |
RESULTS |
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36Cl Uptake Studies
Effect of Cl concentration on Cl-dependent Na/H exchange.
Initial experiments were performed both to establish the optimal Cl
concentration required to stimulate Cl-dependent Na/H exchange and to
identify the sidedness of Cl interaction (i.e., an extravesicular vs.
an intravesicular site) with Cl-dependent Na/H exchange. In this series
of studies, Cl-dependent proton gradient-driven
22Na uptake was examined in the
presence of different Cl concentration gradients: inward
(Clout > Clin; Fig.
1, right), outward
(Clout < Clin; Fig. 1, middle),
and no (Clout = Clin; Fig. 1,
left) Cl gradients. Figure 1 shows
that the presence of Cl, but not a Cl concentration gradient,
stimulated proton gradient-driven
22Na uptake. The presence of 5 mM
Cl stimulated 58% of maximal 22Na
uptake, whereas 22Na uptake was
not stimulated significantly in the presence of 3 mM Cl (data not
shown). Maximal 22Na uptake was
observed in the presence of 25 mM Cl, which was not significantly
enhanced any further by increasing Cl concentrations up to 100 mM.
These results did not establish a preferential vesicular side for Cl
action. This failure to identify a preferential vesicular side for Cl
stimulation of Na/H exchange is more consistent with the movement of Cl
through a Cl channel than via a Cl/anion exchange.
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Effect of Cl channel blockers and anion exchange inhibitors on
Cl-dependent Na/H exchange.
To determine the possible role of a Cl channel and/or Cl/anion
exchange on Cl-dependent Na/H exchange, the effects of
5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB), a Cl channel
blocker (8), and DIDS, an anion exchanger inhibitor at low
concentration but a Cl channel blocker at high concentration (11, 18),
were examined on Cl-dependent Na/H exchange activity. As shown in Fig.
2A,
Cl-dependent Na/H exchange was inhibited 20, 96, and 100% by 10, 100, and 500 µM NPPB, respectively. Cl-dependent Na/H exchange was
inhibited 0.8, 8, and 94% by 10, 100, and 500 µM DIDS, respectively
(Fig. 2B). Inhibition of
Cl-dependent Na/H exchange function by NPPB and DIDS at high but not
low concentrations indicates that an apical Cl channel, not a Cl/anion
exchange, is responsible for the Cl dependence of Na/H exchange.
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Effect of Cl channel blocker and anion exchange inhibitor on Cl
channel.
Studies were designed to examine the presence of Cl channel activity in
AMV prepared from colonic crypt cells. The effects of an
intravesicular-positive potential, generated by an inward K gradient
and valinomycin, a K ionophore, on
36Cl uptake were examined to
establish whether functional Cl channels are present in these apical
membranes. As shown in Fig. 4,
36Cl uptake was substantially
stimulated by the imposition of an intravesicular-positive potential.
In addition, stimulation of 36Cl
uptake by an intravesicular-positive potential was inhibited 44, 99, and 97% by 10, 100, and 500 µM NPPB, respectively (Fig. 4A).
36Cl uptake was also inhibited 5, 18, and 68% by 10, 100, and 500 µM DIDS, respectively (Fig.
4B). Because the dose responses of both DIDS and NPPB on Cl-dependent proton gradient-driven
22Na uptake and potential-driven
36Cl uptake were similar, these
results suggest that a Cl channel is linked to Cl-dependent Na/H
exchange.
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pHi Regulation Studies
Effect of a Cl channel blocker on pHi
regulation.
The effect of the Cl channel blocker, NPPB, was examined to establish
whether the Cl dependence of Na-dependent
pHi regulation in isolated
perfused crypt cells represents a Cl channel. When both Na and Cl were
removed from the bath and lumen,
pHi was reduced and did not return
to baseline pHi (Fig.
5A).
Similar to our previous studies (19), addition of luminal Na, in the
presence of Cl, increased pHi. pHi recovered to
resting pHi values only when both Cl and Na were returned to the luminal perfusate. If Na was returned to
the luminal perfusate before the addition of Cl to the lumen, pHi recovery did not occur (Fig.
5B). To confirm that the Cl
dependence was a result of an apical Cl channel, the Cl channel
inhibitor, NPPB (10 µM), was added to the luminal perfusate following
the bilateral removal of Na and Cl. When Na and Cl were returned to the
apical perfusate, a 96% inhibition of the Cl- and Na-dependent pHi recovery was observed (Fig.
5C). Addition of NPPB to the lumen in an Na-containing perfusate showed a small but reproducible decrease
(0.03 ± 0.01 pH units; n = 25) in
pHi. In similar studies the apical
removal of Cl in the presence of luminal Na resulted in a modest
intracellular acidification (0.04 ± 0.01 pH units; n = 25). These results suggest that a
Cl channel is coupled to an apical membrane transport protein with Na/H
exchange function and that inhibition of this Cl channel prevents
activation of Cl-dependent Na/H exchange activity.
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DISCUSSION |
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Colonic epithelial cells arise from progenitor cells in the base of the crypt and migrate along the crypt to become mature surface epithelial cells that, over a 3- to 6-day period, are sloughed into the colonic lumen. Despite this close relationship of crypt and surface cells, a separation of surface and crypt cell epithelial function has been a long-established concept of intestinal epithelial cell fluid and electrolyte transport (1). Until recently, absorptive processes have been generally considered to be localized solely to surface epithelial cells and secretory processes to crypt epithelial cells. Recent studies, however, provide abundant evidence that absorptive and secretory processes are present both in surface and in crypt epithelial cells (10, 24).
Electroneutral Na-Cl absorption is the primary transport mechanism responsible for Na absorption in the rat distal colon and is the result of the pH coupling of Na/H exchange and Cl/HCO3 exchanges (1, 9). To date, five distinct Na/H exchange isoforms have been cloned and sequenced (15), and recent studies established that the Na/H exchange isoform NHE3 message and protein are located exclusively in surface and not in crypt epithelial cells of the rat distal colon (2). Because studies of fluid absorption in isolated colonic crypts demonstrated Na-dependent fluid absorption (24), it is unlikely that the NHE3 isoform is the mechanism of Na-dependent fluid absorption in isolated colonic crypt cells. As a result, the identity of the Na transport process responsible for the Na-dependent fluid absorptive process in the crypt had to differ from that in surface cells. Subsequent studies designed to establish the nature of Na uptake across the apical membrane of crypt epithelial cells in the rat distal colon established the presence of a novel Na/H exchange, Cl-dependent Na/H exchange, in the luminal membrane of crypt cells but not in surface epithelial cells of the distal colon (19).
The present studies were designed to characterize the nature of the Cl dependence of Na/H exchange in the colonic crypt. Because both Cl/anion exchange1 and Cl channels are present in the rat distal colon (1), the experimental strategy was to determine the effect of inhibitors of Cl/anion exchange and Cl channels on Cl-dependent Na/H exchange function. Our results suggest that the Cl dependence of Na/H exchange in the apical membrane in the colonic crypt represents a Cl channel, not a Cl/anion exchange. This conclusion is based primarily on the effects of NPPB, a Cl channel blocker, and DIDS, an inhibitor of Cl/anion exchanges at low concentrations but of Cl channels at high concentrations (8, 11). First, Cl-dependent proton gradient-driven 22Na uptake was not significantly inhibited by 10 µM DIDS but was inhibited by 500 µM DIDS (Fig. 2B). Second, 100 µM NPPB inhibited Cl-dependent proton gradient-driven 22Na uptake (Fig. 2A). Third, 36Cl uptake by crypt cell AMV induced by a positive electrical potential was also inhibited by 100 µM NPPB (Fig. 4). Finally, fourth, 10 µM NPPB also inhibited the Cl-dependent component of Na-dependent recovery of pHi to an acid load (Fig. 5C). These observations provide compelling evidence that a Cl channel is responsible for Cl-dependent Na/H exchange in the apical membrane of the colonic crypt cell.
At least two different Cl channels are present in apical membranes of the epithelial cells: CFTR and ORCC (6, 7). Although these two Cl channels are best characterized by patch-clamp studies, differences in the specificity of some Cl channel blockers can provide information suggesting the nature of apical Cl channels. NPPB inhibits both CFTR and ORCC (6, 7), whereas DIDS, at high concentrations, primarily inhibits ORCC but may also inhibit CFTR (6, 11). A partial role for CFTR in Cl-dependent Na/H exchange is confirmed by the 38% inhibition of Cl-dependent Na/H exchange by a CFTR polyclonal antibody (Fig. 3) (3). Taken together, these observations indicate that two distinct apical Cl channels, CFTR and ORCC, are linked to Na/H exchange in the colonic crypt, as Cl-dependent proton gradient-driven 22Na uptake was almost completely inhibited by 500 µM DIDS and 100 µM NPPB and partially inhibited by a CFTR antibody.
Cl-dependent Na/H exchange is most likely the only Na/H exchange in the apical membrane of crypt epithelial cells, as neither proton gradient-driven 22Na uptake nor Na-dependent pHi recovery from an acid load was identified in crypt cells in the absence of Cl. Although Cl-independent Na/H exchange is readily identified in AMV prepared from surface cells (17, 19, 20), it is uncertain whether Cl-dependent Na/H exchange is also present in surface cell apical membranes, despite the demonstration that a relatively small component of proton gradient-driven 22Na uptake in AMV prepared from surface cells was Cl dependent (19). This latter phenomenon could represent either contamination of the surface cell AMV preparation with crypt cell apical membranes or the presence of Cl-dependent Na/H exchange in surface cell apical membranes (19).
Sodium and chloride movement in the intestine is the primary driving force for fluid absorption and secretion in both small and large intestine, and several different mechanisms have been identified to explain the interrelationship of these two ions. Parallel absorption of sodium and chloride can be explained by both cotransport processes and the coupling of parallel and independent ion exchanges. Thus Na-K-2Cl cotransport is responsible for Na and Cl movement across the basolateral membrane and is critical for active Cl secretion (16). Although Na-Cl cotransport is present in nonepithelial and other epithelial cells (4), its presence has not been unequivocally established in the intestinal tract. In contrast, parallel Na/H and Cl/HCO3 exchanges that are coupled by pH can also explain electroneutral Na-Cl absorption in the small and large intestine (9). Na and Cl absorption can also be coupled by an electrical gradient. Thus both electrogenic Na absorption in the distal colon via apical amiloride-sensitive Na channels and glucose-stimulated Na absorption mediated by SGLT1 in the small intestine will result in an increase in lumen-negative electrical potential difference, which acts as the driving force for transepithelial Cl movement.
In the absence of parallel transepithelial movement of Na and Cl, other mechanisms of Na and Cl dependence of Cl and Na transport, respectively, have also been described. Na-dependent Cl/HCO3 exchange, which is distinct from Na-independent Cl/HCO3, regulates pHi at the basolateral membrane (23). Previous experiments on transepithelial 36Cl fluxes in the rat proximal colon established that electroneutral Na/H exchange in normal animals is Cl dependent in the absence of net Cl absorption (5). Because studies of proton gradient-driven 22Na uptake by AMV prepared from proximal colon did not indicate the presence of Cl-dependent Na/H exchange (17), the mechanism of Cl-dependent Na/H exchange in the apical membrane of the rat proximal colon is not yet available.
In conclusion, the present studies establish a Cl dependence of both proton gradient-driven 22Na uptake and Na-dependent pHi recovery to an acid load that is 1) largely inhibited by NPPB, a Cl channel blocker, 2) largely inhibited by DIDS only at high concentrations at which it is also a Cl channel blocker, and 3) partially inhibited by a CFTR polyclonal antibody. This pattern of inhibition of Cl-dependent Na/H exchange is consistent with the possibility that apical membrane Cl channels are coupled to either a known or to a previously unidentified Na/H exchange. It is not known whether these Cl channels represent an integral component of a transport protein with both Cl channel and Na/H exchange activities or the close coupling of a Cl channel with either a previously identified Na/H exchange isoform or a novel and as yet unidentified Na/H exchange isoform.
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ACKNOWLEDGEMENTS |
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Affinity-purified CFTR502-511 polyclonal antipeptide antibody and irrelevant antibody were kindly provided by Drs. John Dedman and Marcia Kneisel, University of Cincinnati.
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FOOTNOTES |
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This study was supported in part by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-14669.
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. §1734 solely to indicate this fact.
1 Both Cl/OH and Cl/HCO3 exchanges are present on apical membranes of surface cells of rat distal colon (18). Preliminary studies have established that Cl/HCO3 exchange is not present on apical membranes of crypt cells of rat distal colon (18a). Cl/OH exchange is present in both surface and crypt cells.
Address for reprint requests: H. J. Binder, Yale Univ. School of Medicine, PO Box 208019, 333 Cedar St., New Haven, CT 06520-8019.
Received 6 July 1998; accepted in final form 25 September 1998.
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