Departments of 1 Internal Medicine and 2 Surgery, Yale University, New Haven, Connecticut 06520
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ABSTRACT |
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A novel Cl-dependent Na/H exchange (Cl-NHE) has been identified in apical membranes of crypt cells of rat distal colon. The presence of Cl is required for both outward proton gradient-driven Na uptake in apical membrane vesicles (AMV) and Na-dependent intracellular pH recovery from an acid load in the crypt gland. The present study establishes that Cl-dependent outward proton gradient-driven 22Na uptake 1) is saturated with increasing extravesicular Na concentration with a Michaelis constant (Km) for Na of ~24.2 mM; 2) is saturated with increasing outward H concentration gradient with a hyperbolic curve and a Km for H of ~1.5 µM; 3) is inhibited by the Na/H exchange (NHE) inhibitors amiloride, ethylisopropylamiloride, and HOE-694 with an inhibitory constant (Ki) of ~480.2, 1.1, and 9.5 µM, respectively; 4) is inhibited by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, an anion exchange inhibitor at low concentration and a Cl channel blocker at high dose, and by 5-nitro-2(3-phenylpropylamino)benzoic acid, a Cl channel blocker, with a Ki of ~280.6 and 18.3 µM, respectively; and 5) substantially stimulated Cl-NHE activity by dietary Na depletion, which increases plasma aldosterone and inhibits NHE in surface cell AMV. These properties of Cl-NHE are distinct from those of NHE1, NHE2, and NHE3 isoforms that are present in colonic epithelial cells; thus these results suggest that the colonic crypt cell Cl-NHE is a novel NHE isoform.
sodium depletion; aldosterone; regulation; kinetics
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INTRODUCTION |
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ELECTRONEUTRAL NA/H EXCHANGE (NHE) that extrudes intracellular H and absorbs extracellular Na is responsible for several physiological cell functions, including intracellular pH (pHi) regulation, volume regulation, and transepithelial Na absorption (1, 6, 8, 16, 26). NHEs with distinct kinetic and pharmacological properties have been localized in apical and basolateral membranes of epithelial cells (5, 9). Recent molecular studies have identified the expression of at least three NHE isoforms (NHE1, NHE2, and NHE3 isoforms) in colonic epithelial cells (2, 3, 11, 26).
Colonic NHE isoforms have different cell and tissue distribution as
well as transport characteristics. NHE2 and NHE3 isoforms are present
in apical membranes, whereas the NHE1 isoform is expressed in
basolateral membranes (2, 11, 26). The NHE3 isoform is
expressed only in surface cells, but NHE1 and NHE2 isoforms are
localized to both surface and crypt cells (Refs. 2, 3; see Table
1). Studies of 22Na uptake
that demonstrated NHE activity in apical membrane vesicles (AMV)
isolated from surface cells established that this NHE function is Cl
independent (17). In contrast, recent studies identified a
novel Cl-dependent Na/H exchange (Cl-NHE) in apical membranes of crypt
epithelial cells of rat distal colon by demonstrating that both H
concentration ([H]) gradient-driven 22Na uptake and
Na-dependent pHi recovery from an acid load required the
presence of Cl (18). The Cl dependence of Cl-NHE
appears to represent a Cl channel, as Cl-NHE function is inhibited by both NHE inhibitors and Cl channel blockers (19). It is
not known whether Cl-NHE is an existing NHE isoform that in the
presence of one or more Cl channels manifests Cl dependence or is a
novel NHE isoform. The present study, therefore, was initiated to
identify the kinetic properties of Cl-NHE. The observed results
establish that the properties of Cl-NHE are distinct and differ from
those of the NHE isoforms that have previously been identified in
colonic epithelial cells and suggest that Cl-NHE is a novel isoform of the existing gene family of NHE isoforms.
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METHODS |
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Vesicle preparation. Crypt cells were isolated from distal colon of both normal and Na-depleted diet-fed rats (Sprague-Dawley, 200-250 g) by the method of Lomax et al. (14), as described previously (18). Na-depleted animals were fed a Na-free diet (20 g/day), prepared in our laboratory, as described previously (10), for 6-7 days. AMV were prepared from crypt colonocytes by the modified method of Stieger et al. (23), as described earlier (18, 20). Protein was assayed by the method of Lowry et al. (15). Purity of the crypt cell apical membrane was assessed by ouabain-sensitive H-K-ATPase enrichment (10- to 12-fold), as described earlier (20, 21).
Uptake studies.
The initial rate of Cl-dependent proton gradient-driven
22Na (NEN, Boston, MA) uptake in AMV was performed for
6 s using a "rapid uptake" machine, as described previously
(19). In brief, AMV preloaded with 150 mM KCl and 50 mM
MES-Tris (pH 5.5) were incubated in medium containing 50 mM HEPES-Tris
(pH 7.5), 150 mM KCl, 0.1 mM 22Na, 25 µM valinomycin, and
either 0 or 10 µM 5-ethylisopropylamiloride (EIPA). EIPA-sensitive
proton gradient-driven 22Na uptake presented was calculated
by subtracting the uptake in the presence of 10 µM EIPA from that in
its absence. Some of the experiments were also performed in the
presence of 25 mM Cl, as previous studies established that maximal
Cl-NHE activity was observed in the presence of 25 mM Cl
(19). Specific details of each experiment are given in the
legends for Figs. 1-7. All of the experiments were repeated at
least three times with different membrane preparations. Results
presented represent means ± SE of triplicate assays of a typical
experiment.
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RESULTS |
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Figure 1 presents the initial rate of 22Na uptake in AMV isolated from normal rat distal colonic crypt cells that was measured in the presence of either Cl or gluconate. As shown in Fig. 1, 22Na uptake was almost identical both in the presence [extracellular pH (pHo)/pHi: 7.5/5.5] and absence (pHo/pHi: 5.5/5.5) of an outward proton gradient in medium with gluconate. In contrast, 22Na uptake was substantially stimulated by an outward proton gradient in medium that contained Cl (Fig. 1). These results are consistent with previous observations (18).
Kinetic studies were performed to establish the characteristics of
Cl-NHE. As shown in Fig. 2, increasing
extravesicular Na concentration ([Na]) from 3 to 100 mM stimulated
and saturated an outward-directed [H] gradient-driven
22Na uptake. A Lineweaver-Burke plot of these data yielded
a Michaelis constant (Km) for Na of ~24.2 ± 2.9 mM and a maximal velocity (Vmax) of
309.6 ± 21.3 pmol · mg protein1 · 6 s
1.
Experiments were also designed to determine whether Cl-NHE is responsible for vectorial Na absorption and/or pHi regulation by assessing the presence of a proton modifier site. Thus the effect of increasing outward-directed [H] gradient on Cl-NHE activity was measured. With a fixed pHo of 7.5, decreasing pHi from 7.5 to 5.5 resulted in an increase in the initial rate of 22Na uptake with a hyperbolic curve (Fig. 3). An apparent Km for a proton of outward-directed [H] gradient-driven 22Na uptake was 1.5 ± 0.3 µM (pH 5.8). In additional studies Cl-NHE activity was measured at different [H] but with identical pH gradients (pHo/pHi: 7.5/6.5 vs. 6.5/5.5). As shown in Fig. 4A, the initial rate of Cl-NHE activity was almost similar at both pH gradient conditions. Previous studies have demonstrated that Cl-NHE also functions as an Na/Na exchanger (18). Therefore, Na/Na exchange was measured as outward Na gradient-stimulated 22Na uptake at different proton concentrations. As shown in Fig. 4B, Na/Na exchange was also identical at both low and high proton concentrations. These observations are not consistent with the presence of a proton modifier site and suggest that the function of Cl-NHE is not associated with pHi regulation but is linked to vectorial Na absorption.
Previous studies have demonstrated that inhibitors both of NHE function and of Cl channel activity inhibit Cl-NHE activity (18, 19). Therefore, the kinetics of these inhibitors on Cl-NHE function was also determined. Increasing concentrations of amiloride resulted in progressive inhibition of the initial rate of outward-directed [H] gradient-driven 22Na uptake with an apparent inhibitory constant (Ki) of 480.2 ± 8.6 µM (Fig. 5). This observation is consistent with our initial observation that suggested that Cl-NHE in AMV from crypt cells is relatively resistant to amiloride (19). Experiments were also performed with two specific inhibitors of NHE, EIPA and 3-methyl sulfonyl-4-piperidinoebenzoyl guanidine (HOE-694). The apparent Ki of EIPA of Cl-NHE was 1.1 ± 0.3 µM (Fig. 5). Similarly, Cl-NHE activity was moderately sensitive to HOE-694 with an apparent Ki of 9.5 ± 2.2 µM (Fig. 5).
Prior studies of Cl-NHE demonstrated that Cl-dependent outward-directed [H] gradient-driven 22Na uptake by crypt AMV was also inhibited by the Cl channel blockers 5-nitro-2(3-phenylpropylamino)benzoic acid (NPPB) and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS; see Ref. 19). Experiments were therefore performed to establish the kinetics of both DIDS and NPPB inhibition of Cl-NHE. The apparent Ki of DIDS was 280.6 ± 12.6 µM, whereas the Ki of NPPB for Cl-NHE was 18.3 ± 3.9 µM (Fig. 6). Other transport inhibitors, bumetanide (an Na-K-2Cl cotransport inhibitor), ouabain (an Na-K-ATPase inhibitor), benzamil (an Na channel blocker), and cimetidine [an inhibitor that inhibits basolateral NHE (12)], did not inhibit Cl-dependent proton gradient-driven 22Na uptake (data not shown).
Dietary Na depletion with resulting increased levels of serum
aldosterone inhibits electroneutral Na-Cl absorption and NHE in AMV
prepared from surface cells from the rat distal colon (10, 20). Thus the effect of dietary Na depletion on Cl-NHE activity was also determined. As shown in Fig. 7,
Cl-NHE activity in AMV from an Na-depleted animal was 46% higher than
that in AMV from normal animals. Kinetic studies were also performed to
establish whether the increased Cl-dependent NHE activity in
Na-depleted rats was a result of a change in affinity for Na or an
increased number/turnover rate. As shown in Fig. 2, similar to AMV
prepared from control animals, increasing [Na] also saturated
Cl-dependent NHE in AMV from Na-depleted animals. Linweaver-Burke
plot analyses yielded a Km for Na of
~21.4 ± 2.2 mM and a Vmax of 732.3 ± 29.3 pmol · mg protein1 · 6 s
1. These observations differ qualitatively from those of
AMV from surface cells of Na-depleted animals (11, 20).
These results establish that the characteristics of Cl-NHE are distinct
from those of other NHE isoforms that are present in colonic epithelial cells.
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DISCUSSION |
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Na absorption in the rat distal colon is electroneutral and is primarily a result of the NHE3 isoform, which is located in the apical membrane of surface epithelial cells (2). Because studies of fluid movement in the isolated microperfused colonic crypt revealed the presence of Na-dependent net fluid absorption (22), the mechanism of Na absorption in the colonic crypt was uncertain, since the NHE3 isoform is not present in the apical membrane of crypt cells (2). The demonstration that NHE in crypt AMV required the presence of Cl (Fig. 1) and that NHE in surface cell AMV was Cl independent (18) suggested that Na-dependent net fluid absorption in the isolated colonic crypt was secondary to Cl-NHE.
The initial studies of Cl-NHE established that Na-dependent recovery of pHi to an acid load also required lumen Cl, which was inhibited by amiloride (18). Additional studies revealed that [H] gradient-driven Na uptake by crypt AMV in the presence of Cl was relatively amiloride resistant in that 250 µM amiloride did not inhibit 50% of Cl-NHE activity (18). Furthermore, the requirement for Cl was not specific, as other halides could also stimulate [Na] gradient-driven 22Na uptake (Cl>Br>F>I). Subsequent studies examined the role of Cl and found that both intravesicular and extravesicular Cl were equally effective in stimulation of [H] gradient-driven 22Na uptake and that 5 mM Cl resulted in 58% of maximal Cl-NHE activity (19).
These previous studies also demonstrated that the Cl dependence of NHE function in colonic crypts is related to a Cl channel and not to Cl/anion exchange (19). NPPB (100 µM), a Cl channel inhibitor (7), and DIDS (500 µM) completely prevented Cl-NHE activity; DIDS is an anion exchange inhibitor at low concentrations but is an outward-rectifying Cl channel blocker at higher concentrations (24, 25). NPPB (10 µM) also blocked Na/Cl-dependent pHi recovery from an acid load. Although an antibody to CFTR partially inhibited [H] gradient-driven 22Na uptake (19), the nature of the interaction between one or more Cl channels and an apical membrane NHE is unknown.
These present studies were designed to characterize the properties of the novel Cl-dependent NHE that we have identified in apical membranes in crypt but not in surface epithelial cells of the rat distal colon (18, 19). To date, at least six NHE isoforms have been identified, and at least three are present in epithelial cells of rat distal colon (2, 3, 6, 11, 26). The NHE1 isoform has been identified on the basolateral membrane, is likely responsible for the regulation of one or more cell functions, e.g., volume and pHi, and has often been referred to as a "housekeeper." NHE function in the basolateral membranes is not affected by dietary Na depletion, and NHE1 message or protein is also not altered in dietary Na-depleted rats (l1). NHE2 and NHE3 isoforms are also present in the distal colon but are localized to the apical membrane. Because NHE2 and NHE3 isoforms when expressed in PS 120 cells do not require Cl (M. Donowitz, personal communication), the absence of any NHE activity in the absence of Cl (Fig. 1) established that Cl-NHE is the only NHE isoform present in crypt apical membranes.
The results presented in Figs. 2-4 indicate that the kinetic properties of Cl-NHE are distinct from those of other NHE isoforms that are present in colonic epithelial cells. The apparent Km for Na of NHE1, NHE2, and NHE3 isoforms are approximately between 15 and 18 mM (4, 13), which is not physiologically different from the apparent Km for Na of Cl-NHE (24.2 mM). The kinetics for intravesicular [H] for Cl-NHE yielded Michaelis-Menten kinetics with a Hill coefficient of ~1 (Fig. 3). In contrast, studies of NHE1, NHE2, and NHE-3 isoforms in transfected PS 120 cells did not show Michaelis-Menten kinetics and revealed Hill coefficients of ~2 (13). However, studies with AMV prepared from surface cells of rat distal colon in which both NHE2 and NHE3 isoforms are probably present also did not reveal evidence of a proton modifier site (17). The absence of a proton modifier site in Cl-NHE may also be explained by the following reasons: 1) a modifier site may require other intracellular signaling factors, which are absent in isolated vesicles; and 2) sensing capacity of the proton modifier site may be altered during membrane preparation.
Comparison of the kinetics of amiloride and the amiloride analogs that are more specific NHE inhibitors also reveals that the kinetic properties of Cl-NHE are dissimilar from those of the other NHE isoforms present in colonic epithelial cells (see Table 1). The Ki of Cl-NHE for amiloride was substantially greater than those of NHE1, NHE2, and NHE3 isoforms, whereas Ki values of Cl-NHE for both EIPA and HOE-694 were similar only to those of the NHE2 isoform (Fig. 4).
The effect of dietary Na depletion and aldosterone on apical membrane NHEs provides additional evidence that the properties of Cl-NHE are distinct from those of both NHE2 and NHE3 isoforms. Recent studies have demonstrated that NHE3 isoform-specific NHE activity is completely inhibited in AMV from dietary Na-depleted animals (11). Furthermore, dietary Na depletion results in a substantial decrease in NHE3 mRNA and protein in the rat distal colon (11). Parallel studies revealed that dietary Na depletion produced a moderate decrease in NHE2 transport function, mRNA abundance, and protein expression (11). In contrast, Fig. 7 provides evidence that dietary Na depletion increases Cl-NHE transport function, which is a result of an increase in Vmax without any change in Km (Fig. 2).
The molecular identification of Cl-NHE has not been established, but these present studies provide compelling evidence that the properties of Cl-NHE are distinct and dissimilar from other colonic NHE isoforms. It is possible that Cl-NHE might represent an unrelated transport protein with both NHE transport function and Cl channel activity. However, the inhibition of NHE transport function by amiloride, EIPA, and HOE-694 (Fig. 5) suggests that Cl-NHE likely represents a novel isoform that is related to the existing gene family of NHE isoforms and is closely linked to one or more Cl channels rather than an unrelated transport protein with both NHE and Cl channel function.
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ACKNOWLEDGEMENTS |
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We thank Ann Thompson for secretarial assistance.
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FOOTNOTES |
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This study was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-14669.
Address for reprint requests and other correspondence: H. J. Binder, Dept. of Internal Medicine, Yale Univ., P.O. Box 208019, New Haven, CT 06520-8019 (E-mail: Henry.Binder{at}yale.edu).
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.
Received 23 February 2000; accepted in final form 3 October 2000.
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