Department of Internal Medicine, Yale University, New Haven, Connecticut 06520
Submitted 20 December 2002 ; accepted in final form 16 July 2003
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ABSTRACT |
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butyrate-dependent Na absorption; HCO3-dependent Na absorption; specific NHE isoform inhibitors; NHE-2 isoform; NHE-3 isoform
Colonic Na and Cl absorption is also enhanced by short-chain fatty acids (SCFA), especially by butyrate (2). Detailed studies of SCFA-dependent electroneutral Na-Cl absorption have proposed a model that is similar to, but differs from, that of HCO3-dependent electroneutral Na-Cl absorption (1, 31). The model of SCFA-dependent electroneutral Na-Cl absorption includes SCFA uptake across the apical membrane via an SCFA-HCO3 exchange (or alternatively via nonionic diffusion) that is coupled to both NHE and Cl-SCFA exchange. Central to both HCO3- and SCFA-dependent electroneutral Na-Cl absorption are apical membrane Na-H and Cl-anion exchanges. Lumen amiloride, an inhibitor of NHE, inhibits SCFA-dependent electroneutral Na-Cl absorption (2), similar to its effect on HCO3-dependent Na-Cl absorption.
Both cAMP and aldosterone inhibit HCO3-dependent Na-Cl absorption as a result, at least in part, of their inhibition of apical membrane NHE (3, 15, 27, 32). Although aldosterone inhibited butyrate-dependent electroneutral Na-Cl absorption, butyrate-dependent Na-Cl absorption was not altered by increases in mucosal cAMP (3, 33). An adequate explanation for this differential effect of cAMP on HCO3-dependent and butyrate-dependent Na-Cl absorption has not been advanced despite the involvement of NHE in both transport processes. Because at least two different NHE, NHE-2 and NHE-3, isoforms are present in the apical membrane of surface epithelial cells of the rat distal colon (7, 9, 18), we speculated that these two NHE isoforms might be coupled to HCO3-dependent and butyrate-dependent Na absorption differently and/or that cAMP might have different effects on NHE-2 and NHE-3 isoforms. NHE-2 and NHE-3 isoforms can be distinguished by specific different NHE isoform inhibitors (e.g., S3226), as well as by dose-dependent inhibition by HOE694 and by molecular characterization (13, 36, 43). Such approaches have demonstrated that aldosterone inhibits both NHE-2 and NHE-3 isoform activity, message, and protein (18). In contrast, molecular methods indicate that cAMP inhibits NHE-3 isoform, but its effect on NHE-2 isoform has been inconstant with prior studies providing evidence that cAMP may decrease, increase, or not alter NHE-2 isoform in different cells and tissues (8, 10, 19, 45).
The present study was designed to test the possibility that the failure of cAMP to inhibit butyrate-dependent Na-Cl absorption was a consequence of the role of NHE-2 isoform in butyrate-dependent Na-Cl absorption. In these studies of HCO3-dependent and butyrate-dependent Na transport, we demonstrate that 1) HCO3-dependent Na absorption requires NHE-3 and not NHE-2 isoform, 2) NHE-2 or NHE-3 isoforms are required for butyrate-dependent Na absorption, and 3) cAMP inhibits NHE-3 isoform but appears to increase NHE-2 isoform function. As a result, cAMP inhibits HCO3-stimulated Na absorption, which has an obligate requirement for NHE-3 isoform, but not butyrate-stimulated Na absorption.
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MATERIALS AND METHODS |
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The composition of the nominally HCO3-free Ringer solution in mM was 140 Na, 5.2 K, 1.2 Mg, 1.2 Ca, 119.8 Cl, 4.8 PO4, and 25 isethionate. The composition of the nominally HCO3-free Ringer solution was modified for the HCO3-Ringer and butyrate Ringer solutions. Isethionate was replaced by HCO3 and by butyrate in the HCO3-Ringer and butyrate-Ringer solutions, respectively. HCO3-free and butyrate-Ringer solutions were gassed with 100% O2, whereas the HCO3-Ringer solution was gassed with a 95%O2-5%CO2 mixture. All solutions were adjusted to pH 7.4 before use.
Experiments were designed to determine the role of NHE-2 and NHE-3 isoforms in mediating the stimulation of electroneutral Na absorption by HCO3 and by butyrate, used as a model SCFA. In one series of experiments, initial Na fluxes were determined in either HCO3- or butyrate-Ringer solution, and then the effect of amiloride or dibutyryl cAMP to alter Na absorption was assessed. In all other experiments, Na fluxes were initially determined in HCO3-free Ringer solution and the effect of amiloride (or the specific NHE inhibitors) to prevent the stimulation of Na absorption that is observed after the addition of either HCO3 or butyrate to the bathing solution was determined. In one series of these studies, the rate of HCO3-dependent Na absorption was modified by the addition of a carbonic anhydrase inhibitor, acetazolamide. Prior studies have shown that acetazolamide inhibits HCO3-dependent Na absorption (5) but does not inhibit butyrate-dependent Na absorption (2), NHE, Cl-HCO3 exchange, Cl-OH exchange, or butyrate-HCO3 exchange in the apical membrane of rat distal colon (Ref. 23 and Rajendran VM, unpublished observations). Thus the role of NHE isoforms in regulating butyrate-stimulated Na absorption was determined both in the presence and absence of HCO3-dependent Na absorption by the addition of acetazolamide and/or CO2/HCO3.
To distinguish the role of NHE-2 and NHE-3 isoforms, different NHE isoform inhibitors were employed. Experiments were performed with ethylisopropyl amiloride (EIPA), (methyl sulfonyl 4-piperidinoebenzoyl) guanidine methane sulfonate (HOE 694), and (3-[2-(3-guanidino-2 methyl-3-oxopropeny1)-5-methyl-pheny1]-N-isopropylidene-2-methylacrylamide dihydrochloride) (S3226) (36, 43). Each of these compounds has different NHE isoform specificity. EIPA inhibits all NHE isoforms. HOE 694 has been demonstrated to distinguish NHE isoforms on a dose-dependent basis (18, 43). Prior studies have established the following inhibitor constant (Ki) values for NHE-1, NHE-2, and NHE-3 isoforms: 0.16, 5, and 650 µM, respectively (43). Previous studies with apical membrane vesicles (AMV) from surface cells of rat distal colon established that 25 µM HOE 694 distinguishes NHE-2 and NHE-3 isoforms, which are both present in these apical membranes (18). Twenty percent of total [H] gradient-driven 22Na uptake was inhibited by 25 µM HOE 694 (18). Thus 50 µM HOE 694 was used in these present studies; inhibition of NHE activity by 50 µM HOE 694 indicated NHE-2 isoform activity, whereas NHE activity that was insensitive to 50 µM HOE 694 was considered to represent NHE-3 isoform activity (18).
S3226 has recently been reported as being highly specific as an NHE-3 isoform inhibitor (36). Therefore, a series of studies was designed to determine whether HOE 694 and S3226 inhibit the same and/or different fractions of [H] gradient-driven 22Na uptake. As shown in Fig. 1A, [H] gradient driven 22Na uptake was inhibited by 12 and 78% by the addition of 50 µM HOE 694 and 2 µM S3226, respectively. The presence of HOE 694 and S3226 together almost completely inhibited [H] gradient-driven 22Na uptake. These results indicate that [H] gradient-driven 22Na uptake is mediated by NHE-2 and NHE-3 isoforms in the apical membranes of rat distal colon and that 50 µM HOE 694 and 2 µM S3226 inhibit distinct and separate NHE isoforms, providing additional evidence of the specificity of these two amiloride analogs.
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Experiments were also designed to establish half-maximal inhibitory concentration (IC50) for S3226 on NHE-3 function in colonic AMV that had been prepared from normal rat distal colon by the method of Stieger et al. (39), as described previously (32). 22Na (NEN, Boston, MA) uptake was performed by rapid filtration techniques, as described previously (32). In the present study, the effect of increasing concentrations of S3226 on HOE 694-insensitive [H] gradient-driven 22Na uptake was performed. As shown in Fig. 1B, increasing concentration of S3226 progressively inhibited [H] gradient-driven 22Na uptake with an ID50 for S3226 of 196.8 ± 22.6 nM. This observation is consistent with the earlier report performed in NHE-3 isoform-transfected cells (36). Therefore, in the present 22Na flux studies, we used 2 µM S3226 to inhibit NHE-3 function. As a result, inhibition of Na absorption in the presence of 50 µM HOE 694 without an effect by 2 µM S3226 in the lumen bath indicated a role for NHE-2 isoform and not NHE-3 isoform. Similarly, the failure of 50 µM HOE 694 to alter Na absorption, together with inhibition of Na absorption by 2 µM S3226, provided evidence that NHE-3 isoform was involved.
Data are expressed as means ± SE. Unpaired or paired Student t-tests or one-way analysis of variance were employed to determine statistical significance. One-way analysis of variance was used to determine the significance in Fig. 4. A two-sided P value < 0.05 was considered statistically significant.
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RESULTS |
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Several previous studies have demonstrated that increased mucosal cAMP and cGMP levels inhibit HCO3-dependent Na absorption in the rat distal colon (22, 27-29, 33). In contrast, the addition of dibutyryl cAMP did not inhibit butyrate-dependent Na absorption (Fig. 2). Thus, although both HCO3-dependent Na absorption and butyrate-dependent Na absorption require an apical membrane NHE and cAMP inhibits apical membrane NHEs, cAMP does not inhibit butyrate-dependent Na absorption. The failure of cAMP to alter SCFA-dependent Na absorption has not been explained and is the basis for the present experiments.
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We proposed that this apparent paradox could be explained if butyrate-dependent Na absorption and HCO3-dependent Na absorption were mediated by different apical membrane NHE (i.e., NHE-2 and NHE-3) isoforms with varying sensitivities to cAMP. To test this hypothesis, we used NHE isoform-specific inhibitors, HOE 694 and S3226, to inhibit selectively NHE-2 and NHE-3 isoforms, respectively.
Bicarbonate-dependent Na absorption. To evaluate the role of NHE isoforms in HCO3-stimulated electroneutral Na absorption, experiments were designed to determine whether different NHE isoform-specific inhibitors would alter the stimulation of active Na absorption when HCO3 was added to a nominally HCO3-free Ringer solution. Table 2 reveals that the addition of HCO3 to both mucosal and serosal bath resulted in a substantial increase in Jnet (3.7 ± 1.5 µeq · h-1 · cm-2) as a result of an increase in Jms. The presence of 10 µM EIPA, an amiloride analog that inhibits NHE activity (without isoform specificity) but not Na channel function (24), prevented the increase Jnet (0.7 ± 1.4 µEq · h-1 · cm-2) secondary to a decrease in Jms and an increase in Jsm. In contrast, the addition of 50 µM HOE 694 failed to prevent the increase in Jnet observed after the addition of HCO3 (5.0 ± 1.3 µEq · h-1 · cm-2), thus indicating that HCO3-dependent electroneutral Na absorption is HOE 694 insensitive and suggesting that NHE-2 isoform is not required for HCO3 stimulation of Na absorption.
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HCO3 is also a secondary product of cellular metabolism and, therefore, the nominal absence of HCO3/CO2 in the bath solutions does not eliminate cellular HCO3 production. To study butyrate-dependent absorption in the absence of HCO3-dependent Na absorption, we sought to minimize the contribution by endogenous HCO3. In these experiments, HCO3-dependent Na absorption that is secondary to endogenous HCO3 production was reduced by the addition of a carbonic anhydrase inhibitor, acetazolamide. Confirming previous observations (1, 5), the addition of 100 µM acetazolamide to the nominally HCO3-free Ringer solution markedly reduced from 5.6 ± 0.8 to 0.5 ± 0.3 µEq · h-1 · cm-2. Figure 3 demonstrates that the addition of HCO3 to the nominally HCO3 free-Ringer solution in the presence of 100 µM acetazolamide resulted in a significant increase in
. The presence of 1.0 mM dibutyryl cAMP completely prevented the increase in HCO3-stimulated Na absorption. The effect of both 50 µM HOE 694 and 2 µM S3226 on the stimulation of Na absorption by HCO3 was also examined. Figure 3 also demonstrates that 2 µM S3226 prevented HCO3-stimulated Na absorption, whereas 50 µM HOE 694 did not affect HCO3-stimulated Na absorption. Because 2 µM S3226, but not 50 µM HOE 694, inhibits NHE-3 isoform, these results indicate that cAMP inhibits NHE-3 isoform, which is most likely required for HCO3-stimulated Na absorption.
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Butyrate-dependent Na absorption. Parallel experiments were designed to assess the effect of cAMP on butyrate-stimulated Na absorption, and the results are presented in Table 3. The addition of butyrate to the nominally HCO3-free solution (and in the presence of acetazolamide) resulted in a substantial increase in solely as a result of an increase in Jms (Table 3A). The increment in
induced by the addition of butyrate to the mucosal bath in this series of studies is shown in Fig. 4. Similar to prior studies in which cAMP, theophylline, and cholera enterotoxin did not inhibit butyrate-stimulated Na absorption (3, 22), butyrate-stimulated Na absorption was also not altered by the presence of 1.0 mM dibutyryl cAMP (5.0 ± 0.4 vs. 4.8 ± 0.8 µeq · h-1 · cm-2) (Table 3B), suggesting that in contrast to HCO3-stimulated Na absorption, a cAMP-insensitive NHE is required for butyrate-stimulated Na absorption. The presence of 50 µM HOE 694 (in the absence of dibutyryl cAMP) also did not alter butyrate stimulation of
(7.0 ± 0.8 vs. 4.8 ± 0.8 µE · h-1 · cm-2) (Table 3C); however, the simultaneous presence of both 50 µM HOE 694 and dibutyryl cAMP significantly reduced butyrate stimulation of
(1.7 ± 0.4 vs. 4.6 ± 0.8 µeq·h-1·cm-2; P < 0.005) (Table 3D and Fig. 4). Thus, in the absence of cAMP, butyrate stimulation of Na absorption is HOE 694 insensitive, but when dibutyryl cAMP is present, butyrate stimulation of Na absorption is HOE 694 sensitive.
This observation that HOE 694, a selective NHE-2 inhibitor, inhibited butyrate stimulation of Na absorption in the presence, but not in the absence, of dibutyryl cAMP could be explained by cAMP activating NHE-2 isoform function while simultaneously inhibiting NHE-3 isoform function. Therefore, experiments were also performed with a selective NHE-3 isoform inhibitor, S3226. In the absence of dibutyryl cAMP, the addition of 2 µM S3226 in the mucosal bath inhibited butyrate stimulation of Na absorption (1.7 ± 0.3 vs. 4.6 ± 0.8 µeq · h-1 · cm-2; P < 0.002) (Table 3E and Fig. 4), indicating a role for NHE-3 isoform. In additional experiments, we also observed that the presence of both S3226 and HOE 694 resulted in an identical rate of butyrate-stimulated net Na absorption (compared to S3226 alone) (3.0 ± 0.3 vs. 3.0 ± 0.5 µeq · h-1 · cm-2), indicating that in the absence cAMP, NHE-2 isoform did not contribute to net Na absorption. In contrast, in the presence of both 1.0 mM dibutyryl cAMP and 2 µM S3226 butyrate-stimulated Na absorption was not altered (5.2 ± 0.6 vs. 4.3 ± 0.6 µeq · h-1 · cm-2), suggesting the involvement of a non-NHE-3 isoform (Table 3F). Therefore, although both cAMP and S3226 inhibit NHE-3 isoform, their effects on butyrate-stimulation of Na absorption are not identical. The inhibition of butyrate stimulation of Na absorption by the addition of HOE 694 in the presence of dibutyryl cAMP, combined with the failure of S3226 to inhibit butyrate stimulation of Na absorption in the presence of cAMP, provide strong evidence that cAMP, in addition to its inhibition of NHE-3 isoform activity, activates NHE-2 isoform function.
To demonstrate that the conclusions of the experiments presented in Table 3 were not related either to the effects of acetazolamide or to the absence of HCO3-dependent Na absorption, parallel studies were designed with HOE 694 and butyrate-Ringer solution in the absence of acetazolamide. Table 4 presents the results of these experiments that were performed in the presence of dibutyryl cAMP. These results confirm previous data (3, 22) that butyrate-stimulated active Na absorption is not inhibited by cAMP. In contrast, 50 µM HOE 694 completely inhibited butyrate-stimulated Na absorption (0.8 ± 0.9 vs. 5.0 ± 0.8 µeq · h-1 · cm-2). Thus butyrate stimulation of electroneutral Na absorption was inhibited by HOE 694 only when NHE-3 isoform function was inhibited by cAMP. These observations provide evidence that 1) cAMP upregulates NHE-2 isoform function and 2) either cAMP-sensitive, HOE 694-insensitive (i.e., NHE-3 isoform), or cAMP-insensitive, HOE 694-sensitive (i.e., NHE-2 isoform) NHE isoforms are associated with the stimulation of electroneutral Na absorption by SCFA.
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DISCUSSION |
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In contrast, bicarbonate-dependent electroneutral Na-Cl absorption that represents the coupling of parallel Na-H and Cl-HCO3 exchanges is inhibited by increases in mucosal cAMP, intracellular Ca, and aldosterone in rat distal colon (4, 21, 27, 32, 44). This inhibition is a result of an inhibition of apical membrane NHE (18, 32, 35). Although two NHE isoforms, NHE-2 and NHE-3 isoforms, are present in the apical membrane of colonic epithelial cells and prior studies of glucocorticoid stimulation of Na absorption in the rabbit ileum indicated the importance of NHE-3 isoform (42), the role of these two NHE isoforms in HCO3-dependent and butyrate-dependent Na absorption has not previously been addressed. The present studies provide experimental observations consistent with the thesis that the NHE isoform required for butyrate-dependent electroneutral Na absorption is either NHE-2 or NHE-3 isoform, whereas NHE-3 isoform is the NHE isoform that is solely responsible for HCO3-stimulated electroneutral Na absorption. However, the present results are also consistent with the closely related conclusion that cAMP not only inhibits NHE-3 isoform but stimulates NHE-2 isoform function or another NHE isoform that is inhibited by 50 µM HOE 694. This conclusion is based on the observations that 50 µM HOE 694, a concentration of this NHE isoformspecific inhibitor that inhibits NHE-2 isoform, but not NHE-3 isoform (18), prevented butyrate-stimulated Na absorption, only in the presence of dibutyryl cAMP (Tables 3D and 4).
The present data show two distinct but complementary observations. 1) In the experiments presented in Table 3 (performed in the presence of acetazolamide) and in Table 4 (performed in the absence of acetazolamide), butyrate stimulation of Na absorption was not altered by the presence of either dibutyryl cAMP alone (NHE-3 inhibition) or 50 µM HOE 694 alone (NHE-2 inhibition). However, when dibutyryl cAMP and 50 µM HOE 694 were present together, butyrate-dependent Na absorption was markedly reduced. These observations suggest that in presence of cAMP, a HOE 694-sensitive NHE isoform (e.g., NHE-2 isoform) (that is not active in the basal state) is upregulated. 2) S3226, a NHE-3 isoform inhibitor, reduced butyrate-stimulated Na absorption when present alone (Table 3E), indicating that NHE-3 isoform is associated with butyrate-stimulated Na absorption basally. However, in the presence of dibutyryl cAMP, butyrate-stimulated Na absorption was not inhibited by S3226 but was inhibited by 50 µM HOE 694 at a concentration that inhibits NHE-2, not NHE-3 isoform. These observations confirm that in the presence of cAMP, NHE-3 activity is inhibited, whereas NHE-2 activity is upregulated, and that butyrate-stimulated Na absorption can be mediated by either NHE-2 or NHE-3 isoforms. These experiments also indicate that although both S3226 and dibutyryl cAMP are inhibitors of NHE-3 isoform, they do not have identical effects on butyrate stimulation of Na absorption, suggesting that cAMP has an effect(s) on NHE isoforms other than its inhibition of NHE-3 isoform.
The present observations indicate that under basal conditions (i.e., in the absence of cAMP), butyrate-dependent Na absorption is mediated by NHE-3; in contrast, in the presence of cAMP, the butyrate-dependent Na absorption is mediated by NHE-2 isoform. Although in basal condition, NHE-2 isoform was nonfunctional, Musch et al. (25) had demonstrated a role for both NHE-2 and NHE-3 isoforms under basal conditions in studies performed in the rat transverse colon (25). In this prior study, Musch et al. demonstrated that long-term exposure of colonic C2/bbe cell lines to SCFA increased expression and activity of NHE-3, but not NHE-2 isoform, in a time-dependent manner. These investigators did not examine either HCO3-dependent or butyrate-dependent Na absorption.
The effect of cAMP on electroneutral Na absorption contrasts with the effect of aldosterone in that aldosterone inhibits both HCO3-stimulated and butyrate-stimulated Na absorption in the distal colon of dietary Na-depleted rats (3). On the basis of the results of the present studies, one would predict that aldosterone should inhibit both NHE-2 and NHE-3 isoforms. Such observations have recently been reported as aldosterone was demonstrated to inhibit NHE activity, message, and protein of both NHE-2 and NHE-3 isoforms in rat distal colon (18).
The experiments with butyrate-stimulated Na absorption were performed both in the presence and absence of HCO3-stimulated Na absorption (Tables 3 and 4). A reduction of HCO3-stimulated Na absorption was achieved by the combined absence of HCO3 in the bath solution (i.e., nominally HCO3-free) and the addition of acetazolamide, a carbonic anhydrase inhibitor. Prior studies have established that butyrate-stimulated Na absorption is not affected either by bath HCO3 or by the addition of acetazolamide to the bath solution (2). Furthermore, Na-H, Cl-OH, Cl-HCO3, or butyrate-HCO3 exchanges in rat colonic and rabbit ileal apical membranes are not altered by acetazolamide (Refs. 20, 21, 23, and Rajendran VM, unpublished observations). Fifty micromolars HOE 694, a concentration that inhibits NHE-2, but not NHE-3 isoform, prevented the stimulation of Na absorption by the addition of butyrate in the presence of dibutyryl cAMP both in the presence (Table 3D) and the absence (Table 4) of acetazolamide. Thus inhibition of carbonic anhydrase by acetazolamide did not affect the interpretation of the present experiments.
The present observations that apical membrane NHE isoforms in rat distal colon are differentially regulated by cAMP is consistent with some but not all previous experiments. Thus, in the present experiments, dibutyryl cAMP inhibits NHE-3 isoform while activating NHE-2 isoform in rat distal colon. Similarly, when NHE isoforms are expressed in AP1 cell, cAMP inhibited Na-dependent intracellular pH (pHi) recovery from an acid load in NHE-3 isoform-transfected cells while activating pHi recovery in NHE-2 isoformtransfected cells (10). In contrast, different observations were observed in NHE-2 and NHE-3 isoform transfected PS120 and C2/bbe cells. In C2/bbe cells both NHE-2 and NHE-3 isoform activity was inhibited by cAMP, whereas cAMP did not inhibit either NHE-2 or NHE-3 isoform activities when expressed in PS120 cells (8, 45). Because cAMP regulation of NHE-3 isoform is mediated by the regulatory protein NHERF, this lack of effect of cAMP in PS120 cells (45) may be a result of the absence of NHERF in these cells. Although NHE-2 isoform-specific accessory regulatory protein(s) have not been identified, the differential regulation of NHE-2 isoform function by cAMP in AP1 and C2/bbe cells is consistent with the presence of distinct regulatory proteins in different cells and/or species. We speculate that a regulatory protein that mediates cAMP activation of NHE-2 isoform function is present in rat distal colon and AP1 cells, but not in either C2/bbe or PS120 cells. Additional evidence has recently been presented that cGMP also differentially alters NHE-2 and NHE-3 isoforms as activation of nitric oxide inhibits NHE-3 isoform, but not NHE-2 isoform in Caco-2 cells (16).
Oral rehydration solution (ORS) was introduced into clinical medicine thirty-five years ago and represents an extremely important medical advance for the treatment of acute diarrhea (11). Despite the substantial intestinal fluid secretion and diarrhea observed in diarrheal diseases like cholera and other conditions associated with bacterial enterotoxins, ORS markedly enhances small intestinal fluid absorption. The physiological basis for ORS is that 1) absorptive and secretory processes are separate and distinct intestinal transport mechanisms that can function independent of each other, 2) glucose-stimulated fluid and solute absorption is normal despite enterotoxin-induced fluid secretion, and 3) mucosal cAMP does not alter glucose-stimulated Na absorption, although cAMP stimulates active Cl secretion and inhibits HCO3-dependent electroneutral Na-Cl absorption (11).
The failure of cAMP to alter butyrate-dependent electroneutral Na-Cl absorption in the colon is parallel to the normal rate of glucose-stimulated Na-Cl absorption in the presence of cAMP in the small intestine (40). As a result, the possibility was raised that an improved ORS could be developed on the basis of the normal rate of colonic butyrate-dependent Na-Cl absorption in the presence of an increase in mucosal cAMP. Recent studies confirmed this hypothesis in that the administration of ORS with an amylase-resistant starch, i.e., starch that is relatively resistant to amylase digestion and resulting in an increase in colonic SCFA production, to adult patients with acute cholera resulted in a decrease in stool weight and a decrease in the time to the first formed bowel movement in a prospective clinical trial that compared resistant starch-ORS to both standard ORS and an ORS formulation with added rice flour (34). These observations are consistent with the failure of cAMP to inhibit butyrate-dependent Na absorption and the upregulation of NHE-2 isoform mRNA and protein by SCFA (25).
The rate of net Na absorption in the HCO3-free Ringer solution in the presence of acetazolamide (Table 2) is considerably less than that observed in HCO3-Ringer solution in the presence of amiloride (Table 1). Although acetazolamide does not inhibit either Cl-HCO3 exchange or NHE in apical membrane vesicle experiments (Refs. 20, 21, and Rajendran VM, unpublished observations), recent studies have emphasized the probable importance of the effect of acetazolamide on Cl-HCO3 exchange function independent of its inhibition of carbonic anhydrases activity (12, 17, 26, 37, 38, 41). Evidence has been presented that both erythrocytes and HEK 293 cells that have no intrinsic Cl-HCO3 exchange activity when transfected with AE1 cDNA or DRA cDNA have enhanced Cl-HCO3 exchange and that acetazolamide inhibits Cl-HCO3 exchange function in these transfected HEK293 cells to a greater extent than could be predicted by its inhibition of carbonic anhydrases activity alone (26, 41). It is not unlikely that acetazolamide in rat distal colon has also inhibited Cl-HCO3 exchange function independent of action on carbonic anhydrase (Table 2 vs. Table 3).
In conclusion, these present experiments, together with prior studies with aldosterone (3, 18), provide evidence that butyrate- and HCO3-dependent Na absorption are associated with different NHE isoforms, with butyrate-dependent Na absorption being mediated by both NHE-2 and NHE-3 isoforms, whereas HCO3-dependent Na absorption has an obligate requirement for NHE-3 isoform. Further, aldosterone inhibits both NHE-2 and NHE-3 isoform activities, whereas cAMP selectively downregulates NHE-3 isoform activity but upregulates the activity of a NHE isoform that is inhibited by 50 µM HOE 694 (e.g., NHE-2 isoform).
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DISCLOSURES |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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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