AJP - GI AJP: Heart and Circulatory Physiology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Gastrointest Liver Physiol 293: G857-G863, 2007. First published August 9, 2007; doi:10.1152/ajpgi.00462.2006
0193-1857/07 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
293/4/G857    most recent
00462.2006v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Subramanya, S. B.
Right arrow Articles by Binder, H. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Subramanya, S. B.
Right arrow Articles by Binder, H. J.

MUCOSAL BIOLOGY

Differential regulation of cholera toxin-inhibited Na-H exchange isoforms by butyrate in rat ileum

Sandeep B. Subramanya,1,2 Vazhaikkurichi M. Rajendran,1 Pugazhendhi Srinivasan,1,2 Navalpur S. Nanda Kumar,2 Balakrishnan S. Ramakrishna,2 and Henry J. Binder1

1Department of Internal Medicine, Yale University School of Medicine, New Haven, Connecticut and 2Department of Gastrointestinal Sciences, Christian Medical College, Vellore, India

Submitted 5 October 2006 ; accepted in final form 9 August 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Electroneutral Na absorption occurs in the intestine via sodium-hydrogen exchanger (NHE) isoforms NHE2 and NHE3. Bicarbonate and butyrate both stimulate electroneutral Na absorption through NHE. Bicarbonate- but not butyrate-dependent Na absorption is inhibited by cholera toxin (CT). Long-term exposure to butyrate also influences expression of apical membrane proteins in epithelial cells. These studies investigated the effects of short- and long-term in vivo exposure to butyrate on apical membrane NHE and mRNA, protein expression, and activity in rat ileal epithelium that had been exposed to CT. Ileal loops were exposed to CT in vivo for 5 h and apical membrane vesicles were isolated. 22Na uptake was measured by using the inhibitor HOE694 to identify NHE2 and NHE3 activity, and Western blot analyses were performed. CT reduced total NHE activity by 70% in apical membrane vesicles with inhibition of both NHE2 and NHE3. Reduced NHE3 activity and protein expression remained low following removal of CT but increased to control values following incubation of the ileal loop with butyrate for 2 h. In parallel there was a 40% decrease in CT-induced increase in cAMP content. In contrast, NHE2 activity partially increased following removal of CT and was further increased to control levels by butyrate. NHE2 protein expression did not parallel its activity. Neither NHE2 nor NHE3 mRNA content were affected by CT or butyrate. These results indicate that CT has varying effects on the two apical NHE isoforms, inhibiting NHE2 activity without altering its protein expression and reducing both NHE3 activity and protein expression. Butyrate restores both CT-inhibited NHE2 and NHE3 activities to normal levels but via different mechanisms.

ileal mucosa; apical membrane vesicles; 22Na uptake; NHE2 and NHE3 isoforms; protein expression; differential regulation

SHORT CHAIN FATTY ACIDS (SCFA), acetate, propionate, and butyrate, are absorbed in the colon and ileum. In the colon, SCFA stimulate Na and fluid absorption secondary to enhancement of electroneutral Na-Cl absorption (3). The model of SCFA stimulation of Na-Cl absorption is primarily based on in vitro studies using apical membrane vesicles and ion fluxes across isolated colonic mucosa that include an apical membrane mechanism of SCFA uptake (i.e., SCFA-HCO3 exchange) coupled to parallel Na-H and Cl-butyrate exchanges (3, 23). Although the mechanism of SCFA absorption is controversial with evidence for both apical membrane SCFA-HCO3 exchange and nonionic diffusion (29), both of these apical membrane processes for SCFA uptake will result in a relative intracellular acidosis, which is required for maximal function of Na-H exchange that is coupled to Cl-SCFA exchange (23).

Increases in mucosal cAMP inhibit intestinal HCO3-dependent Na-Cl absorption as a result of its inhibition of apical membrane Na-H exchange (2, 21). In contrast to these observations with HCO3-dependent Na-Cl absorption, in vitro and in vivo experiments in large intestine have established that cAMP does not alter SCFA-dependent Na-Cl absorption (2, 24). To explore the explanation for this apparent paradox of the failure of cAMP to inhibit SCFA-dependent Na-Cl absorption, prior studies addressed the possibility that the two different colonic apical membrane NHE isoforms, NHE2 and NHE3, had both varying responses to cAMP and different regulation of HCO3-dependent and butyrate-dependent Na-Cl absorption (14, 32). These Na flux studies, in which dibutyryl cAMP (dbcAMP) was added directly to isolated colonic mucosa, made three principal observations: 1) HCO3-dependent Na absorption was linked only to NHE3 isoform and not to NHE2 isoform; 2) butyrate-dependent Na absorption, in contrast, was associated with both NHE3 and NHE2 isoforms; and 3) dbcAMP inhibited NHE3 isoform, as expected, but increased NHE2 isoform (13). As a result, these observations are consistent with the central role of Na-H exchange with SCFA-dependent Na absorption and the apparent failure of cAMP to inhibit SCFA-dependent Na-Cl absorption.

Whereas the earlier studies investigated the effects of dbcAMP and butyrate on SCFA-dependent Na transport processes in vitro, prolonged in vivo exposure to butyrate may have effects on these transport processes that will likely be relevant to clinical situations in which such changes can be therapeutically exploited. The present experiments were designed to explore further the role of butyrate in the regulation of Na-H exchange by cAMP by performing experiments in which endogenously increased mucosal cAMP levels were maintained over several hours, in contrast to the experimental design of the prior studies in which dbcAMP was acutely added directly to intestinal mucosa in vitro (14). In these present experiments we observed that, as expected, cholera toxin (CT) increased mucosal cAMP levels and decreased H concentration gradient-dependent 22Na uptake but that subsequent exposure of intact intestinal mucosa to butyrate in vivo reduced mucosal cAMP levels and increased NHE activity.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Sprague-Dawley rats weighing 200–250 g were used in all the experiments. These experiments were approved by the Yale University Animal Care and Use Committee.

In vivo incubation of CT in ileal loops. Rats fasted for 24 h were anesthetized with pentobarbital sodium (40 mg/kg body wt). At laparotomy a 10-cm ileal loop was constructed 5 cm proximal to the ileocecal junction. Ileal loops were flushed with warm HCO3-Ringer solution (in mM: 140 Na, 5.2 K, 1.2 Mg, 1.2 Ca, 119.8 Cl, 25 HCO3, 2.4 PO4, and 1.2 HPO4) (37°C) and then instilled with either 2 ml Ringer containing 100 µg CT (experimental) or 2 ml Ringer alone (control). The abdomen was then closed, and the anesthetized rats were maintained under heating lamp to maintain the body temperature at 37°C. In additional experiments, following the 5-h CT incubation an additional 2 h incubation was performed with 2 ml of either butyrate-Ringer solution (in mM: 140 Na, 5.2 K, 1.2 Mg, 1.2 Ca, 40 butyrate, 104.8 Cl, 2.4 PO4, and 1.2 HPO4) or isethionate-Ringer solution (in mM: 140 Na, 5.2 K, 1.2 Mg, 1.2 Ca, 40 isethionate, 104.8 Cl, 2.4 PO4, and 1.2 HPO4) instilled in ileal loops that had previously been incubated with CT. Following these incubations, the ileal loop contents were aspirated and the lengths were recorded. Net fluid movement was determined by calculating the difference between the initially instilled fluid volume and the fluid volume collected following the experiment. The rate of fluid movement was expressed as microliters per 5 h per centimeter. Positive and negative fluid movement represents net absorption and net secretion, respectively.

In additional control studies, ileal loops (not previously exposed to either CT or Ringer) were instilled with either butyrate-Ringer or isethionate-Ringer solutions and were incubated for 2 h. Ringer solutions containing HCO3 were gassed with 95% O2-5% CO2, whereas Ringer solutions containing butyrate or isethionate were gassed with 100% O2. All solutions were adjusted to pH 7.4.

AMV. Mucosa was scraped from both control and experimental ileal loops, and apical membrane vesicle preparation (AMV) was isolated by the divalent cation chelation method, as described previously by Stieger et al. (30). AMVs suspended in appropriate volume of vesicle buffer (in mM: 50 Mes-Tris, 150 K-gluconate, pH 5.5) were stored at –80°C until use. AMVs were used for both uptake studies and Western blot analyses. Protein assays were performed by the method of Lowry et al. (18).

Uptake studies. Outward H concentration gradient-dependent 22Na (0.1 mCi/mmol, Amersham, Arlington Heights, IL) uptake, as a measure of Na-H exchange (NHE) activity, was performed by rapid filtration techniques, as described previously (30). AMVs were thawed at room temperature and passed through a 26-gauge needle. Uptake was measured for 6 s by diluting 10 µl of vesicles into 90 µl of uptake medium (in mM: 50 HEPES-Tris, pH 7.4, 150 K-gluconate, 1 µM HOE694, 0.1 Na-gluconate, and trace of 22Na). Uptake was arrested by the addition of 1 ml of ice-cold stop solution (in mM: 50 HEPES-Tris, 150 K-gluconate, pH 7.4) and the AMVs were collected on 0.45 µM filter (HAWP Millipore) that was under constant vacuum were solubilized in liquid scintillation fluid. 22Na activity was determined by liquid scintillation spectroscopy.

Apical membrane-specific NHE activities were measured in the presence of either 1 µM or 50 µM HOE694 (kindly provided by Dr. Hans J. Lang, Aventis, Frankfurt, Germany), which inhibits different NHE isoforms in a concentration-dependent manner (7, 13, 34). NHE activity measured in the presence of 1 µM HOE694 represents total apical membrane NHE activity as 1 µM HOE694 inhibits basolateral membrane NHE-1 isoform (34); NHE activity in the presence of 50 µM HOE694 represents the NHE3 fraction, whereas the NHE2 fraction was derived by subtracting NHE3 activity from total NHE activity.

RNA isolation and quantitative PCR. Total RNA was extracted using Trizol reagent (Sigma) and converted to cDNA in a final volume of 20 µl containing 0.5 mM of each nucleotide triphosphate, 40 units of RNAase inhibitor (Ambion), 50–100 ng of random hexamers (Amersham Pharmacia), 200 units of Moloney Murine Leukemia virus RT (Finnzymes), and 1 µg of the extracted RNA and incubated at 42°C for 1 h. Diluted cDNA sample (1 µl) was amplified in a thermal cycler (Chromo4, Bio-Rad) in 20 µl of 1x PCR buffer containing 200 µM of each nucleotide triphosphate (Finnzymes), 2.5 U Hot Gold Star polymerase (Eurogentec), 250 nM forward and reverse primers (IDT), and 1:50,000 diluted SYBRgreen dye (Amersham). Thermal cycling was carried out with initial denaturation at 95°C for 10 min, followed by 40 cycles of denaturation at 94°C for 20 s, annealing (at 59°C for NHE2 and NHE3 and at 56°C for GAPDH), and extension at 72°C for 30 s, then followed by measurement of fluorescence. Product specificity was confirmed by the presence of a single peak in the melting curve analysis. The primer sequences used for these studies are listed in Table 1. The concentration of mRNA for NHE2 and NHE3 was expressed relative to that of the house keeping gene GAPDH by the 2–{Delta}{Delta} CT method (17).


View this table:
[in this window]
[in a new window]

  		Table 1. Primer sequences used in RT-PCR studies

 
Western blot. Twenty-five micrograms of apical membrane protein electrophoresed on SDS-PAGE (7.5%) transferred to nitrocellulose membrane was blocked with 5% Blotto overnight at 4°C. The blot was exposed to primary antibody (NHE3 1:2,000; NHE2 1:2,000; beta-actin 1:5,000) at room temperature for 1 h. After the primary antibody was washed off, the blots exposed to NHE3 and NHE2 antibodies were exposed to horseradish peroxidase-conjugated goat anti-mouse and goat anti-rabbit, respectively. For internal control, blots were also probed with beta-actin (Sigma, St. Louis, MO). Immunoreactive bands were visualized by enhanced chemiluminescence (Amersham Bioscience, Piscataway, NJ). Monoclonal NHE3 antibody (1) was generously provided by Dr. Daniel Beimersderfer (Yale University, New Haven, CT). Polyclonal NHE2 antibody (12) was a generous gift from Dr. Mark Donowitz (Johns Hopkins School of Medicine, Baltimore, MD). Secondary antibodies and anti-beta-actin were obtained from Sigma. Protein expressions were quantitated by use of personal densitometer SI ImageQuant software.

cAMP assay. Cellular cAMP contents of scraped ileal mucosa was assayed using a nonacetylation enzyme immunoassay kit (Amersham Biosciences, cat. no. RPN225). Mucosal scrapings were obtained from ileal loops that were instilled with Ringer and CT-containing Ringer solutions for 5 h, and isethionate-Ringer and butyrate-Ringer solutions for 2 h following the 5-h experiment with CT.

Statistics. Unless otherwise indicated in the legends, all the results presented represent means ± SE from 4 different AMV preparations. Unpaired Student's t-test was performed to test significance of differences between groups. Two-tailed P < 0.05 was considered statistically significant. Statistical analyses were performed using SPSS software. In the mRNA studies statistical analyses were performed by Mann-Whitney test.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Effect of CT on fluid secretion and mucosal cAMP level in ileum. Studies were designed to maintain elevated mucosal cAMP levels over prolonged periods that are generally required to affect transport properties at translational and/or transcriptional levels. In these initial studies rat ileal loops were exposed to CT-Ringer for 5 h. Net fluid absorption was present in the control ileal loops. As predicted, there was both a fourfold increase in mucosal cAMP content (P < 0.001) (Fig. 1) and the induction of net fluid secretion in ileal loops exposed to CT for 5 h compared with ileal loops instilled with normal Ringer for 5 h (70.0 ± 5.5 vs. –113.0 ± 11.4 µl/ 5 h; P < 0.001). These observations establish that this CT-Ringer-exposed ileal loop could be used to assess the long-term effect of increased cAMP exposure on ileal electrolyte transport.


Figure 1
View larger version (11K):
[in this window]
[in a new window]

  		Fig. 1. Effect of butyrate on cholera toxin-elevated cAMP level in ileal mucosal cells. cAMP content was determined in mucosal cells scraped from ileum instilled with either Ringer alone (Control), Ringer containing cholera toxin (CT) for 5 h, CT-Ringer followed by 2 h 40 mM isethionate (CT/Ise) or CT-Ringer followed by 2 h 40 mM butyrate (CT/But). *P < 0.001 compared with control. **P < 0.001 compared with CT/Ise.

 
Effect of CT on NHE activities in AMV. Experiments were performed to identify whether CT-Ringer-induced fluid secretion is accompanied by concomitant inhibition of Na absorption (NHE activities) in apical membranes of ileum. As shown in Fig. 2A, total NHE activity was reduced by 70% in AMV from ileum instilled with CT-Ringer compared with that present in AMV from ileum instilled with normal Ringer. Additional experiments were performed to identify whether CT inhibited specific NHE isoform function, because both NHE2 and NHE3 isoforms are present in AMV from human, rabbit, and rat ileum (12, 33). In these studies NHE2 and NHE3 isoform-specific activities were distinguished by use of HOE694, a NHE inhibitor that alters specific NHE isoform in a concentration-dependent manner (7). As shown in Fig. 2, B and C, ~30 and 70% of the total NHE activities were contributed by NHE2 and NHE3 isoform-specific functions in normal ileal AMV, respectively. This observation is consistent with observations in apical membranes of rabbit and rat ileum and in rat colon (6, 33). Parallel to total NHE activities, both NHE2 and NHE3 activities were also inhibited in AMV from CT-Ringer incubated ileum (Fig. 2, B and C). These observations indicate that increased mucosal cAMP inhibits both NHE2 and NHE3 isoform-specific activities.


Figure 2
View larger version (13K):
[in this window]
[in a new window]

  		Fig. 2. Effect of butyrate on cholera toxin-inhibited sodium-hydrogen exchanger (NHE) activities in ileal apical membrane vesicles. A: total NHE activity, in apical membrane vesicle preparations (AMV) isolated from ileal loops instilled with either Ringer alone for 5 h, Ringer containing 100 µg cholera toxin for 5 h, 5 h CT-Ringer followed by 2 h with 40 mM isethionate-Ringer, or 5 h CT-Ringer followed by 2 h with 40 mM butyrate-Ringer. NHE activities were measured in the presence and absence of 50 µM HOE694. HOE-sensitive NHE activity represents the NHE2 fraction (B), and HOE-insensitive component represents the NHE3 fraction (C). Total NHE and NHE isoform-specific activities were calculated, as described in MATERIALS AND METHODS. *P < 0.001 compared with control. #P < 0.001 compared with CT. $P < 0.001 compared with CT/Ise. @P < 0.001 compared with CT/Ise. NS, not significant, compared with CT.

 
Effect of butyrate on NHE activities in AMV. Butyrate has been shown to block cAMP inhibition of electroneutral Na absorption in rat colon (14). Therefore, studies were designed to identify whether butyrate could reverse CT-Ringer-inhibited NHE activities. In these studies NHE activities were measured in AMV isolated from ileal loops that were incubated for 2 h with either 40 mM butyrate-Ringer or 40 mM isethionate-Ringer following an initial 5 h CT-Ringer instillation. As shown in Fig. 2, incubation with isethionate, following removal of luminal CT, resulted in a slight increase in total NHE activity without an increase in NHE3 activity. In contrast, NHE2 activity in the presence of isethionate (i.e., following withdrawal of CT) was significantly increased. Incubation with butyrate (again following withdrawal of CT) resulted in substantially increased total NHE activity; NHE activity returned to values comparable to controls that were never exposed to CT. Increase in total NHE activity after butyrate incubation was due to increases in both NHE3 and NHE2 activities (Fig. 2, B and C) which were essentially similar in magnitude to that in control loops never exposed to CT. Thus 2-h incubation with butyrate following CT exposure completely reversed the inhibitory effect of CT (i.e., cAMP) on NHE activity. These observations suggest that the inhibition of NHE2 activity by CT was rapidly reversible and transient, since this activity rapidly increased in the presence of isethionate-containing Ringer. On the other hand, removal of CT did not modify NHE3 activity, whereas the presence of butyrate resulted in a return in NHE3 activity to normal following CT exposure. These observations suggest that the modes of CT inhibition of NHE2 and NHE3 activities differ.

To establish whether the reversal of NHE activities following CT withdrawal and butyrate exposure was immediate or required a period of time, the following experiment was performed. NHE activity was first inhibited by incubation for 5 h with CT-Ringer and then exposed to butyrate-Ringer or isethionate-Ringer for 10 min, following which AMVs were prepared from the ileal loops and used for transport studies. The removal of CT in the presence of isethionate (9.9 ± 4.2 vs. 29.2 ± 6.3 pmol/mg protein; 6 s; P < 0.001) or butyrate (9.9 ± 4.2 vs. 30.2 ± 5.4 pmol/mg protein; 6 s; P < 0.001) resulted in a threefold increase in NHE2 activity, which is identical to that observed in the presence of isethionate during a 2-h incubation (Fig. 2B). In contrast, NHE3 activity was not altered by the removal of CT in the presence of either isethionate or butyrate (data not shown). These observations establish that the effect of butyrate on both NHE2 and NHE3 activities requires more than 10 min.

To establish whether butyrate increases only CT-inhibitable NHE activity or whether it also activates basal NHE activity, total NHE activity was measured in AMV isolated from normal ileal loops that were instilled with either butyrate-Ringer or isethionate-Ringer for 2 h. Butyrate did not alter total NHE activities in AMV of normal ileal loops compared with isethionate group (104.2 ± 12.4 vs. 101.4 ± 13.5 pmol/mg protein; 6 s). These results indicate that butyrate reversed CT-inhibition of NHE activity but did not augment basal NHE activity.

Effect of CT and butyrate on NHE2- and NHE3-specific protein expression in AMV. To establish whether CT inhibition of NHE2 and NHE3 activity and their reversal by CT removal of butyrate occurred as a result of functional changes in these transporters or whether this was due to deletion and insertion of membrane proteins, Western blot analyses were performed with apical membranes using NHE3 and NHE2 isoform-specific antibodies. As shown in Fig. 3, NHE3 protein expression was decreased approximately by 60% in apical membranes from ileal loops that were instilled with CT-Ringer. NHE3 protein expression was not altered in apical membranes from ileal loops exposed to isethionate-Ringer following the removal of CT. NHE3 protein expression increased by threefold in apical membranes from ileal loops that were exposed to butyrate-Ringer following CT-Ringer (Fig. 3). In contrast to reduced NHE3 protein expression, CT-Ringer instillation did not reduce, but modestly increased NHE2 protein expression (Fig. 4). Following the removal of CT, butyrate, but not isethionate, enhanced NHE2 protein expression in CT-Ringer-exposed ileum (Fig. 4). Neither CT, isethionate, nor butyrate affected the level of expression of the housekeeping protein beta-actin. These observations establish that CT reduced NHE2 and NHE3 activities by different mechanisms. CT reduced NHE3 activity by decreasing NHE3 protein expression in apical membranes, whereas butyrate reversed CT-reduced NHE activity by enhancing NHE3 protein expression in apical membranes. In contrast, CT reduced NHE2 activity without altering NHE2 protein expression.


Figure 3
View larger version (47K):
[in this window]
[in a new window]

  		Fig. 3. Effect of butyrate on CT-reduced NHE3 isoform-specific protein expression in ileal apical membranes. A: Western blot analyses of apical membranes isolated from ileum incubated with either Ringer alone, Ringer containing cholera toxin, CT-Ringer followed by 2 h isethionate, or CT-Ringer followed by 2 h butyrate. Immunoblots were performed using NHE3 isoform-specific monoclonal antibody (1). Western blot was also performed using beta-actin antibody as an internal control. B: relative expression of NHE3-specific protein in apical membrane of Control, CT, CT/Ise, and CT/But were quantitated by densitometry from 3 different membrane preparations. *P < 0.005 compared with control. $P < 0.001 compared with CT.

 

Figure 4
View larger version (55K):
[in this window]
[in a new window]

  		Fig. 4. Effect of butyrate on NHE2 isoform-specific protein expression in CT-treated ileal apical membranes. A: Western blot analyses of apical membranes isolated from ileum incubated with either Ringer alone, Ringer containing cholera toxin, CT-Ringer followed by 2 h isethionate, or CT-Ringer followed by 2 h butyrate. Immunoblots were performed using NHE2 isoform-specific polyclonal antibody (12). Western blot was also performed using beta-actin antibody as an internal control. B: relative expression of NHE2 specific protein in apical membrane of Control, CT, CT/Ise, and CT/But were quantitated by densitometry from 3 different membrane preparations (12). *P < 0.001 compared with control.

 
Effect of CT and butyrate on NHE2 and NHE3 mRNA. NHE2 and NHE3 mRNA levels were determined using quantitative real-time PCR and expressed relative to that of GAPDH mRNA. Minor changes were present between the groups, but none were significantly different from their corresponding control.

Effect of butyrate on cAMP levels. The above results establish that CT inhibited NHE3 activity and protein expression, which was reversed by in vivo exposure to butyrate. Experiments were designed to establish whether the effects produced by butyrate were mediated by changes in mucosal cAMP content. Figure 1 presents the results of these experiments in which CT-increased mucosal cAMP content following incubation with butyrate-Ringer for 2 h resulted in an approximate 40% reduction in mucosal cAMP levels. In contrast, incubation with isethionate-Ringer did not affect mucosal cAMP levels.


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The present experiments explored the apparent paradox that the model of butyrate-dependent Na absorption includes an apical membrane Na-H exchange; that cAMP is known to inhibit NHE3 isoform, an apical membrane Na-H exchange; but that cAMP does not inhibit butyrate-dependent Na absorption (2, 23). To address this important issue, experiments were designed that resulted in prolonged increases in mucosal cAMP in rat ileal mucosa following the instillation of CT. As expected in this in vivo model, enhanced levels of mucosal cAMP for up to 5 h were demonstrated together with reduced NHE2 and NHE3 activities, as determined by 22Na uptake by AMV (Fig. 2).

The novel observation of these present studies was that these two central findings, i.e., the increase in mucosal cAMP and the reduction in NHE activities, were partially or completely reversed by exposing the ileal mucosa in vivo to butyrate for 2 h (Figs. 1 and 2). Control experiments established that neither in vivo incubation of ileal mucosa with butyrate for 10 min nor in vitro incubation of AMV with butyrate for 2 h was sufficient to induce these changes in NHE activities (unpublished observations). The decrease in mucosal cAMP levels by butyrate could be due to either activation of phosphodiesterases or inhibition of adenylate cyclase by butyrate. Evidence for both of these possible mechanisms has been reported (26, 35). However, a definitive molecular understanding how butyrate reverses or prevents the effects of cAMP on NHE has not as yet been established.

Butyrate has multiple effects on intestinal epithelial function. Butyrate affects both cell differentiation and ion transport (5, 8, 14, 15). First, butyrate inhibits cAMP- and cGMP-stimulated Cl secretion but not Ca-mediated Cl secretion (26). Second, butyrate also inhibits both Cl-dependent HCO3 secretion and cAMP-stimulated HCO3 secretion (31). Third, NHE expression is upregulated in intestinal epithelial C2bbe cells by butyrate when incubated for 48 h (19). Fourth, butyrate enhances fluid absorption as a result of stimulating electroneutral Na-Cl absorption (1). Fifth, butyrate inhibits histone deacylation, which may be associated with its effect to increase cell differentiation.

It would be important to compare the present results that were performed in vivo with prior in vitro experiments that had also been designed to explain the failure of cAMP to inhibit butyrate-dependent Na absorption (13) as well as the two initial observations that cAMP did not affect SCFA-dependent Na and fluid absorption (24). In the previous experiments both dbcAMP and butyrate were added to intact colonic mucosa in vitro for no more than 20 min prior to the determination of Na transport. This experimental design parallels the demonstration that the acute addition of either dbcAMP or theophylline did not alter butyrate-dependent Na absorption in vitro. In contrast, the present experiments were performed in vivo in which mucosal cAMP levels were elevated secondary to CT for several hours prior to the transport studies.

The clinical application of this paradox that cAMP does not inhibit butyrate-dependent Na absorption has been the development of a modified oral rehydration solution (ORS) by the addition of amylase-resistant starch (RS), i.e., starch that is relatively resistant to amylase digestion, to ORS (25). The effectiveness of WHO-ORS, an isoosmolar glucose-electrolyte solution, is based on the physiological principles that absorptive and secretory processes are separate and distinct, that stimulation of Cl secretion does not affect glucose-stimulated Na absorption because cAMP that induces Cl secretion does not alter glucose-stimulated Na absorption, and that stimulation of glucose-stimulated Na absorption does not alter activated Cl secretion (10). As a consequence, WHO-ORS, which enhances fluid and Na absorption in the small intestine, has dramatic effects to correct the dehydration and metabolic acidosis associated with severe diarrhea especially in children but does not result in substantial reduction in diarrhea (9, 11). The initial proposal to employ RS-ORS was based on the expectation that RS would result in an increase in the delivery of nonabsorbed carbohydrate to the colon (4). As a consequence, there would be an increase in the production of SCFA, which in turn would result in an increase in colonic fluid and Na absorption in the colon. Clinical studies in both adults with acute cholera (25) and children with noncholera diarrhea (20, 22) have confirmed this hypothesis by the demonstration that RS-ORS is significantly better than WHO-ORS in shortening the time to the first formed stool.

These present studies were performed in ileal mucosa because preliminary experiments using rat colon failed to establish a model in which CT consistently induced fluid secretion in vivo. Prior studies have demonstrated that SCFA are absorbed in both small and large intestine by similar mechanisms (27, 28). Under normal conditions SCFA are almost exclusively present in the colon and are the predominant anion in stool because SCFA are not present in the diet and are produced (fermented) by colonic bacteria from nonabsorbed carbohydrate. However, in conditions in which bacteria are found in the terminal ileum as in many healthy residents of the tropics, SCFA may presumably be found in the lumen of the colon.

The effects of CT and cAMP on NHE2 and NHE3 protein levels and functional activities differ. Although cAMP inhibited both NHE3 and NHE2 function equally, only NHE3 protein expression was reduced by increased levels of mucosal cAMP during a 5-h period. This differential regulation of NHE isoforms by cAMP is consistent with prior studies (14). Furthermore, following the removal of CT and the addition of isethionate and butyrate, NHE3 and NHE2 functional activity and protein expression also differed. NHE3 protein and function parallel each other during the 2-h butyrate experiment, resulting in a significant increase in both NHE3 isoform activity and protein level. On the other hand, NHE2 function increased following CT withdrawal and was further enhanced by butyrate. In addition, NHE2 protein expression was not changed despite the decrease in NHE2 activity. Although a mechanism(s) for these disparate changes is not apparent as yet, these observations establish that CT inhibits both NHE2 and NHE3 activities by different and distinct mechanisms (Fig. 2B). This conclusion is consistent with and supplements our earlier demonstration that cAMP inhibits NHE3 but not NHE2 function in rat distal colon (14). In this earlier study we had shown that under basal conditions both HCO3-dependent and butyrate-dependent Na absorptions are mediated by NHE3 function, whereas in the presence of cAMP butyrate-dependent but not HCO3-dependent Na absorption is present, and that butyrate-dependent Na absorption is mediated by NHE2 isoform in rat distal colon (14). This differential effect of CT on NHE2 and NHE3 provides a molecular explanation for several clinical and experimental observations.

These present studies establish that the regulation of NHE2 and NHE3 by cAMP differs and are consistent with present concepts regarding the regulation of different NHEs (35). NHE3 protein is present both in apical membranes and in a subapical membrane endosomal compartment (16). Furthermore, NHE3 function is regulated by its trafficking between these two cellular compartments that likely requires phosphorylation. In contrast, NHE2 protein is generally believed to be present solely in the plasma membrane and its regulation is likely a result of changes in its turnover number without evidence of either membrane trafficking or phosphorylation (35). Thus, in the present study, although cAMP inhibited both NHE2 and NHE3 activity (Fig. 2), NHE3 but not NHE2 protein expression was altered by cAMP (Figs. 3 and 4). This observation is consistent with cAMP reducing NHE3 protein recycling to the apical membrane. Because cAMP did not reduce either NHE2 or NHE3 mRNA expression, cAMP's effects on NHE3 activity are at a posttranscriptional level whereas those on NHE2 activity are at a posttranslational level. Since butyrate partially reduced cAMP levels (Fig. 1), it is not known whether all of the increase in NHE2 and NHE3 activity in the presence of butyrate is due to this decrease in cAMP alone or whether butyrate increased NHE activity independent of the change in cAMP.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
Dr. Subramanya was supported by an Infectious Diseases Initiative grant no. 063150 from the Wellcome Trust, London, UK. This study was supported by the Burroughs Wellcome Fund and Wellcome Trust, London, UK, through international trilateral collaborative grant no. 063150 and by a research grant (DK-60069) from the National Institute of Diabetes and Digestive and Kidney Diseases.


   FOOTNOTES
 

Address for reprint requests and other correspondence: H. J. Binder, Dept. of Internal Medicine, Yale Univ. School of Medicine, New Haven, CT 06520-8010 (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.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Biemesderfer D, Rutherford PA, Nagy T, Pizzonia JH, Abu-Alfa AK, Aronson PS. Monoclonal antibodies for high-resolution localization of NHE3 in adult and neonatal rat kidney. Am J Physiol Renal Physiol 273: F289–F299, 1997.[Abstract/Free Full Text]
  2. Binder HJ, Mehta P. Characterization of butyrate-dependent electroneutral Na-Cl absorption in the rat distal colon. Pflügers Arch 417: 365–369, 1990.[CrossRef][Web of Science][Medline]
  3. Binder HJ, Mehta P. Short-chain fatty acids stimulate active sodium and chloride absorption in vitro in the rat distal colon. Gastroenterology 96: 989–996, 1989.[Web of Science][Medline]
  4. Brown I. Complex carbohydrates and resistant starch. Nutr Rev 54: S115–S119, 1996.[Web of Science][Medline]
  5. Charney AN, Giannella RA, Egnor RW. Effect of short-chain fatty acids on cyclic 3',5'-guanosine monophosphate-mediated colonic secretion. Comp Biochem Physiol A Mol Integr Physiol 124: 169–178, 1999.[CrossRef][Medline]
  6. Collins JF, Kiela PR, Xu H, Zeng J, Ghishan FK. Increased NHE2 expression in rat intestinal epithelium during ontogeny is transcriptionally mediated. Am J Physiol Cell Physiol 275: C1143–C1150, 1998.[Abstract/Free Full Text]
  7. Counillon L, Scholz W, Lang HJ, Pouyssegur J. Pharmacological characterization of stably transfected Na+/H+ antiporter isoforms using amiloride analogs and a new inhibitor exhibiting anti-ischemic properties. Mol Pharmacol 44: 1041–1045, 1993.[Abstract]
  8. Dagher PC, Egnor RW, Taglietta-Kohlbrecher A, Charney AN. Short-chain fatty acids inhibit cAMP-mediated chloride secretion in rat colon. Am J Physiol Cell Physiol 271: C1853–C1860, 1996.[Abstract/Free Full Text]
  9. Desjeux JF, Briend A, Butzner J. Oral rehydration solution in year 2000: pathophysiology, efficacy and effectiveness. Baillieres Clin Gastroenterol 11: 509–527, 1997.[CrossRef][Web of Science][Medline]
  10. Field M. Ion transport in rabbit ileal mucosa. II. Effects of cyclic 3',5'-AMP. Am J Physiol 221: 992–997, 1971.[Free Full Text]
  11. Greenough WB III. A simple solution: curing diarrhea. J Diarrhoeal Dis Res 11: 1–5, 1993.[Web of Science][Medline]
  12. Hoogerwerf WA, Tsao SC, Devuyst O, Levine SA, Yun CH, Yip JW, Cohen ME, Wilson PD, Lazenby AJ, Tse CM, Donowitz M. NHE2 and NHE3 are human and rabbit intestinal brush-border proteins. Am J Physiol Gastrointest Liver Physiol 270: G29–G41, 1996.[Abstract/Free Full Text]
  13. Ikuma M, Kashgarian M, Binder HJ, Rajendran VM. Differential regulation of NHE-isoform by sodium depletion in proximal and distal segments of rat colon. Am J Physiol Gastrointest Liver Physiol 276: G539–G549, 1999.[Abstract/Free Full Text]
  14. Krishnan S, Rajendran VM, Binder HJ. Apical NHE isoforms differentially regulate butyrate-stimulated Na absorption in rat distal colon. Am J Physiol Cell Physiol 285: C1246–C1254, 2003.[Abstract/Free Full Text]
  15. Kunzelmann K, Mall M. Electrolyte transport in the mammalian colon: mechanisms and implications for disease. Physiol Rev 82: 245–289, 2002.[Abstract/Free Full Text]
  16. Kurashima K, Szabo EZ, Lukacs G, Orlowski J, Grinstein S. Endosomal recycling of the Na+/H+exchanger NHE3 isoform is regulated by the phosphatidylinositol 3-kinase pathway. J Biol Chem 33: 20828–20836, 1998.
  17. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods 25: 402–408.
  18. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem 193: 265–275, 1951.[Free Full Text]
  19. Musch MW, Bookstein C, Xie Y, Sellin JH, Chang EB. SCFA increase intestinal Na absorption by induction of NHE3 in rat colon and human c2/bbe cells. Am J Physiol Gastrointest Liver Physiol 280: G687–G693, 2001.[Abstract/Free Full Text]
  20. Rabbani GH, Teka T, Zaman B, Majid N, Khatun M, Fuchs GJ. Clinical studies in persistent diarrhea: dietary management with green banana or pectin in Bangladeshi children. Gastroenterology 121: 554–560, 2001.[CrossRef][Web of Science][Medline]
  21. Racusen LC, Binder HJ. Alteration of large intestinal electrolyte transport by vasoactive intestinal polypeptide in the rat. Gastroenterology 73: 790–796, 1977.[Web of Science][Medline]
  22. Raghupathy P, Ramakrishna BS, Oommen SP, Ahmed MS, Priyaa G, Dziura J, Young GP, Binder HJ. Amylase-resistant starch as adjunct to oral rehydration therapy in children with diarrhea. J Pediatr Gastroenterol Nutr 42: 362–368, 2006.[CrossRef][Web of Science][Medline]
  23. Rajendran VM, Binder HJ. Apical membrane Cl-butyrate exchange: mechanism of short chain fatty acid stimulation of active chloride absorption in rat distal colon. J Membr Biol 141: 51–58, 1994.[Web of Science][Medline]
  24. Ramakrishna BS, Nance SH, Roberts-Thomson IC, Roediger WE. The effects of enterotoxins and short-chain fatty acids on water and electrolyte fluxes in ileal and colonic loops in vivo in the rat. Digestion 45: 93–101, 1990.[CrossRef][Web of Science][Medline]
  25. Ramakrishna BS, Venkataraman S, Srinivasan P, Dash P, Young GP, Binder HJ. Amylase-resistant starch plus oral rehydration solution for cholera. N Engl J Med 342: 308–313, 2000.[Abstract/Free Full Text]
  26. Resta-Lenert S, Truong F, Barrett KE, Eckmann L. Inhibition of epithelial chloride secretion by butyrate: role of reduced adenylyl cyclase expression and activity. Am J Physiol Cell Physiol 281: C1837–C1849, 2001.[Abstract/Free Full Text]
  27. Ruppin H, Bar-Meir S, Soergel KH, Wood CM, Schmitt MG Jr. Absorption of short-chain fatty acids by the colon. Gastroenterology 78: 1500–1507, 1980.[Web of Science][Medline]
  28. Schmitt MG Jr, Soergel KH, Wood CM, Steff JJ. Absorption of short-chain fatty acids from the human ileum. Am J Dig Dis 22: 340–347, 1977.[CrossRef][Web of Science][Medline]
  29. Sellin JH. SCFAs: the enigma of weak electrolyte transport in the colon. News Physiol Sci 14: 58–64, 1999.[Abstract/Free Full Text]
  30. Stieger B, Marxer A, Hauri HP. Isolation of brush-border membranes from rat and rabbit colonocytes: is alkaline phosphatase a marker enzyme? J Membr Biol 91: 19–31, 1986.[CrossRef][Web of Science][Medline]
  31. Vidyasagar S, Barmeyer C, Geibel J, Binder HJ, Rajendran VM. Role of short-chain fatty acids in colonic HCO3 secretion. Am J Physiol Gastrointest Liver Physiol 288: G1217–G1226, 2005.[Abstract/Free Full Text]
  32. Vidyasagar S, Ramakrishna BS. Effects of butyrate on active sodium and chloride transport in rat and rabbit distal colon. J Physiol 539: 163–173, 2002.[Abstract/Free Full Text]
  33. Wormmeester L, Sanchez de Medina F, Kokke F, Tse CM, Khurana S, Bowser J, Cohen ME, Donowitz M. Quantitative contribution of NHE2 and NHE3 to rabbit ileal brush-border Na+/H+ exchange. Am J Physiol Cell Physiol 274: C1261–C1272, 1998.[Abstract/Free Full Text]
  34. Yun CH, Tse CM, Nath SK, Levine SA, Brant SR, Donowitz M. Mammalian Na+/H+ exchanger gene family: structure and function studies. Am J Physiol Gastrointest Liver Physiol 269: G1–G11, 1995.[Abstract/Free Full Text]
  35. Zachos NC, Tse M, Donowitz M. Molecular physiology of intestinal Na+/H+ exchange. Annu Rev Physiol 67: 411–443, 2005.[CrossRef][Web of Science][Medline]
  36. Zaharia V, Varzescu M, Djavadi I, Newman E, Egnor RW, Alexander-Chacko J, Charney AN. Effects of short chain fatty acids on colonic Na+ absorption and enzyme activity. Comp Biochem Physiol A Mol Integr Physiol 128: 335–347, 2001.[CrossRef][Medline]



This article has been cited by other articles:


Home page
 J. Pharmacol. Exp. Ther.Home page
M. W. Musch, D. L. Arvans, H. Paris, and E. B. Chang
{alpha}2-Adrenergic Receptors Attenuate Secretagogue-Induced Endocytosis and Promote Exocytosis of Intestinal NHE2 and NHE3
J. Pharmacol. Exp. Ther., September 1, 2009; 330(3): 818 - 825.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
293/4/G857    most recent
00462.2006v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Subramanya, S. B.
Right arrow Articles by Binder, H. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Subramanya, S. B.
Right arrow Articles by Binder, H. J.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2007 by the American Physiological Society.