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Am J Physiol Gastrointest Liver Physiol 294: G1369-G1375, 2008. First published March 6, 2008; doi:10.1152/ajpgi.00063.2008
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INFLAMMATION/IMMUNITY/MEDIATORS

Constitutive nitric oxide differentially regulates Na-H and Na-glucose cotransport in intestinal epithelial cells

Steven Coon, Ramesh Kekuda, Prosenjit Saha, Jamilur R. Talukder, and Uma Sundaram

Section of Digestive Diseases, Department of Medicine, West Virginia University Medical Center, Morgantown, West Virginia

Submitted 8 February 2008 ; accepted in final form 26 February 2008


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Previous in vivo studies suggest that constitutive nitric oxide (cNO) can regulate Na- glucose cotransport (SGLT1) and Na-H exchange (NHE3) in rabbit intestinal villus cells. Whether these two primary Na absorbing pathways are directly regulated by cNO and the mechanisms of this regulation in the enterocyte is not known. Thus nontransformed rat small intestinal epithelial cells (IEC-18) were treated with NG-nitro-L-arginine methyl ester (L-NAME), which directly decreased cNO in these cells. L-NAME treatment decreased SGLT1 in IEC-18 cells. Kinetic studies demonstrated that the mechanism of inhibition was secondary to a decrease in the affinity of the cotransporter for glucose without a change in the number of cotransporters. In contrast, L-NAME treatment increased NHE3 in IEC-18 cells. Kinetic studies demonstrated that the mechanism of stimulation was by increasing the number of the exchangers without a change in the affinity for Na. Quantitative RT-PCR (RTQ-PCR) and Western blot analysis of SGLT1 demonstrated no change in mRNA and protein, respectively. RTQ-PCR and Western blot analysis of NHE3 indicated that NHE3 was increased by L-NAME treatment by an increase in mRNA and protein, respectively. These results indicate that decreased cNO levels directly mediate the inhibition of SGLT1 and stimulation of NHE3 in intestinal epithelial cells. Thus cNO directly but uniquely regulates the two primary Na-absorptive pathways in the mammalian small intestine.

glucose absorption; regulation of transport; NHE3; SGLT1; Na absorption

IN THE MAMMALIAN SMALL INTESTINE there are two major Na-absorptive pathways, coupled NaCl absorption and Na-glucose cotransport (28, 29). Coupled NaCl absorption occurs via the duel operation of Na-H exchange (NHE3) and Cl-HCO3 exchange. Na-glucose cotransport (SGLT1) is a secondary active transport process requiring a favorable intracellular Na gradient provided by Na-K-ATPase on the basolateral membrane (BLM) of villus cells (7). Both NHE3 and SGLT1 have been demonstrated to be present in the brush border membrane (BBM)-absorptive villus cells, but not in the secretory crypt cells in the rabbit small intestine (4, 7, 11, 26).

Nitric oxide (NO) is an important and highly active molecule that regulates many functions in the gastrointestinal tract. Small amounts of constitutive NO (cNO) is produced by intestinal epithelial cells as well as the nervous system, musculature, and endothelium to regulate normal physiological functions. cNO has been shown to regulate mucosal blood flow, mucus secretion, and intestinal motility (12, 19, 23, 27). In contrast, large quantities of NO produced by inducible nitric oxide (iNO) synthase is thought not to be beneficial. For example, it is thought to contribute to as well as prolong the course of intestinal inflammation in conditions such as inflammatory bowel disease (6, 16, 21).

Whether cNO specifically and directly affects Na absorption by the enterocytes is poorly understood. In fact, the effect of cNO on intestinal absorption vs. secretion is also not well understood. cNO has been suggested to have little to no effect on rat small intestinal electrolyte transport. However, other studies suggest that inhibition of cNO in the rat small intestine may promote secretion (14). Similarly, inhibition of NO production vs. stimulation of NO production has also been demonstrated to produce conflicting results. In the rat jejunum inhibition of NO production as well as stimulation of NO production have been shown to promote secretion (20). Potential explanation for these conflicting observations may be species differences. Furthermore, an inability to produce constitutive vs. inducible NO levels consistently in these studies may explain the discrepancies since these two different levels of NO may very well produce opposite effects.

In three separate in vivo studies in rabbits cNO production was inhibited and electrolyte transport was studied in the small intestinal enterocytes (3, 4, 4a). Interestingly, these studies demonstrated that in vivo inhibition of cNO has opposite effects on the two primary Na-absorptive pathways in the mammalian small intestine. Specifically, administration of NG-nitro-L-arginine methyl ester hydrochloride (L-NAME) inhibited SGLT1 in the BBM of villus cells by diminishing its affinity for glucose without a change in the number of cotransporters (3). In contrast, NHE3, also on the BBM of villus cells, was stimulated secondary to an increase in transporter numbers without a change in the affinity of the transporter for Na (4). Whether the observed effects of in vivo inhibition of cNO imply compensatory regulation and whether this is secondary to altered blood flow, enteric nervous system activity and/or a direct effect of reduced cNO in villus cells could not be deciphered in these studies.

To more directly determine the effect of cNO on NHE3 and SGLT1, the effect of L-NAME on nontransformed rat small intestinal epithelial cells (IEC-18), an excellent in vitro model of intestinal absorptive villus cells, was studied. Therefore, the aims of the study were to determine whether L-NAME directly reduces cNO in IEC-18 cells, what effect this direct reduction in cellular cNO has on NHE3 and SGLT1 in these cells, and the mechanism of regulation of NHE3 and SGLT1 by cNO in IEC-18 cells.


   METHODS
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 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
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Tissue culture. Rat IEC-18 were grown in high glucose Dulbecco's modified Eagle's medium supplemented with 2 U/ml of insulin, 0.5 mM β-hydroxybutyrate, and 10% fetal calf serum and incubated at 37°C with 10% CO2. Cells were grown on permeable supports and were used 10 days postconfluent.

Drug treatment. Cells were treated with agents once 24 h prior to the experiment. These drugs include L-NAME (1 mM), D-NG-nitroarginine methyl ester hydrochloride (D-NAME; 1 mM), or vehicle alone.

Na-K-ATPase assay. Na-K-ATPase was measured from cell homogenates as previously described (3, 8). Specific activity was expressed as nanomoles Pi released per milligram protein per minute.

Na-glucose cotransport in IEC-18 cells. At 10 days postconfluent IEC-18 cells were washed and incubated with Leibowitz-15 medium supplemented with 10% fetal bovine serum and incubated for 1 h. The cells were then washed and incubated with Na-free medium containing 130 mM trimethyl ammonium chloride (TMACl), 4.7 mM KCl, 1.2 mM KH2PO4, 1 mM MgSO4, 1.25 mM CaCl2, 20 mM HEPES and gassed with 100% O2 (pH 7.4 at 37°C). Uptakes were performed at desired time intervals in reaction medium containing either 130 mM NaCl or 130 mM TMACl in HEPES medium (as described above) with 10 µCi of 3H-O-methyl glucose (OMG) and 100 µM OMG. Cells were then washed with cold Na-free medium and then incubated with 1 N NaOH for 20 min at 70°C before addition of 4 ml of scintillation fluid (Ecoscint; National Diagnostics). Radioactivity was determined in a Beckman 6500 Beta scintillation counter.

Na-H exchange in IEC-18 cells. IEC-18 cells at 10 days post confluence were washed with Leibowitz-15 medium. The cells were then incubated with Na free medium for 10 min. Uptakes were performed at desired time intervals in reaction medium containing: 130 mM TMACl HEPES medium, 10 µCi of 22Na, 1 mM NaCl with and without 50 µM EIPA to determine Na-H activity. Cells were then washed with cold Na-free medium and processed as described above.

RTQ-PCR. Total RNA was isolated from IEC-18 cells using RNeasy total RNA purification kit (Qiagen). First strand cDNA was synthesized by using oligo(dT) primer, random hexamers, and SuperScript III Reverse Transcriptase (Invitrogen). The cDNAs synthesized were used as templates for quantitative RT-PCR (RTQ-PCR) by using TaqMan universal PCR master mix (Applied Biosystems) according to the manufacturer's protocol. β-Actin RTQ-PCR was run along with SGLT1 and NHE3 RTQ-PCR to normalize their expression between samples. RTQ-PCR was performed for 45 cycles at 95°C for 15 s and 60°C for 1 min using an ABI 7300 RTQ-PCR system. Experiments using different dilutions of the SGLT1, NHE3, and β-actin cDNAs were also performed to ensure proper PCR efficiency. RTQ-PCR analyses were performed in triplicate with RNA isolated from at least three sets of IEC-18 cells.

Western blot. Western blot analysis of BBM were performed as described earlier (1, 15). BBM solubilized in RIPA buffer (50 mM Tris·HCl pH 7.4, 1% Igepal, 150 mM NaCl, 1 mM EDTA, 1 mM PMSF, 1 mM Na3VO4, 1 mM NaF) containing protease inhibitor cocktail (SAFC Biosciences) was mixed with sample buffer (100 mM Tris, 25% glycerol, 2% SDS, 0.01% bromophenol blue, 10% 2-ME, pH 6.8) and separated on a 4–20% gradient Ready Gel (Bio-Rad). The separated proteins were transferred to Hybond-C extra (Amersham) and probed with SGLT1 and NHE3 antibodies raised in chicken. Chicken anti-rat coupled to horseradish peroxidase was used to detect the binding of SGLT1 and NHE3 antibodies. The resulting chemiluminescence was measured by autoradiography. SGLT1 and NHE3 abundance was quantitated via a densitometric scanner (Molecular Dynamics).

NO measurement. NO concentration in IEC-18 cells was measured by quantitating the amount of nitrite and nitrate, which are end products of NO metabolism. Measurements are made using a NO (NO2–/NO3–) assay kit from Assay Designs. The assay was performed according to kit instructions. Briefly, IEC-18 cells were grown into confluent monolayers on Transwell plates until 10 days postconfluent. Cells were treated with L-NAME for 24 h prior to performance of the assay. Cells were then washed with PBS, scraped from the Transwell membranes, and then sonicated in PBS. The cell lysates were then spun and the supernatant was used for the assay. Nitrite and nitrate concentrations are then measured (all nitrates are enzyme reduced to nitrites) with a nitrite standard curve by a colorimetric enzyme assay. Samples were read at 540 nm. Protein was then estimated as described below.

Protein assay. Proteins were assayed with the RC DC protein assay kit according to manufacturer's protocols (Bio-Rad).

Statistics. Results presented represent means ± SE of experiments performed and calculated by the GraphPad InStat program. All uptakes were done in triplicate. Student's t-test was performed for statistical analysis.


   RESULTS
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 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Effect of L-NAME on Na-glucose cotransport. First it was essential to establish that IEC-18 cells possessed Na-glucose cotransport. Thus uptake studies of 3-OMG were done in the presence and absence of extracellular Na. As demonstrated in Fig. 1A, Na stimulates 3-OMG uptake in these cells, which is consistent with the presence of Na-glucose cotransport. Then, the effect of inhibition of cellular cNO production on Na-glucose cotransport was determined. As shown in Fig. 1B, Na-glucose cotransport is significantly inhibited by L-NAME treatment in IEC-18 cells. In contrast, an inactive analog of L-NAME, D-NAME, had no effect on Na-glucose cotransport in these cells. These data indicates that Na-glucose cotransport is present in IEC-18 cells and inhibition of intracellular cNO production significantly inhibits Na-glucose cotransport in these cells.


Figure 1
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  		Fig. 1. SGLT1 in IEC-18 cells. Na-dependent 3H-O-methyl glucose (3H-OMG) uptake is present in IEC-18 cells (A). Na-dependent 3H-OMG uptake (e.g., Na-glucose cotransport) is markedly diminished when IEC-18 cells are treated with NG-nitro-L-arginine methyl ester hydrochloride (L-NAME) to reduce intracellular constitutive nitric oxide (cNO) levels (B). An inactive analog of L-NAME, D-NG-nitroarginine methyl ester hydrochloride (D-NAME), had no effect on Na-dependent glucose uptake in IEC-18 cells. Therefore, Na glucose cotransport is present in IEC-18 cells and it is inhibited by L-NAME.

 
Effect of cNO on Na-H exchange. First the presence of Na-H exchange in IEC-18 cells was demonstrated. Na-H exchange is defined as intracellular proton-stimulated uptake of Na that is inhibitable by cationic exchange inhibitor EIPA. As seen on Fig. 2A, an intracellular proton load stimulates 22Na uptake in IEC-18 cells. This proton stimulated Na uptake is inhibited by EIPA. These observations are consistent with the presence of Na-H exchange in IEC-18 cells. Next we looked at the effect of inhibition of cNO production with L-NAME on Na-H exchange in IEC-18 cells. L-NAME treatment significantly increased Na-H exchange in these cells. The inactive analog of L-NAME, D-NAME, had no effect on Na-H exchange. These data indicate that Na-H exchange is present in IEC-18 cells and that Na-H exchange is markedly stimulated when intracellular cNO production is inhibited by L-NAME.


Figure 2
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  		Fig. 2. Na-H exchange in IEC-18 cells. A proton gradient-driven EIPA-sensitive Na uptake is present in IEC-18 cells (A). Proton gradient-dependent, EIPA-sensitive Na uptake (e.g., Na-H exchange) is markedly increased when cNO levels are reduced by L-NAME treatment in IEC-18 cells (B). D-NAME, the inactive analog of L-NAME, had no effect on Na-H exchange in these cells. Thus Na-H exchange is present in IEC-18 cells and when cNO levels are directly inhibited in these cells; unlike SGLT1, NHE3 is markedly stimulated.

 
Effect of L-NAME on intracellular NO levels. L-NAME is a competitive inhibitor of cNO synthase. Thus we measured NO in IEC-18 cells. L-NAME treatment significantly decreased cellular NO production as shown in Fig. 3. These data along with the previous uptake studies indicate that L-NAME indeed mediates its effect on Na-glucose cotransport and Na-H exchange by decreasing intracellular cNO in IEC-18 cells.


Figure 3
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  		Fig. 3. Effect of L-NAME treatment on intracellular cNO levels in IEC-18 cells. Treatment of IEC-18 cells with L-NAME significantly diminishes cNO levels measured as cellular nitrites. The inactive analog D-NAME had no effect on cNO levels in IEC-18 cells. Thus L-NAME directly inhibits cNO in IEC-18 cells.

 
Effect of L-NAME on Na-K-ATPase. Intracellular Na homeostasis is important for the functioning of BBM Na-dependent transport processes. For example, it is well accepted that Na-glucose cotransport is dependent on the favorable Na gradient across the BBM that is maintained by Na-K-ATPase on the BLM. Therefore, the effect of L-NAME on Na-K-ATPase was determined in IEC-18 cells. As shown in Fig. 4, L-NAME stimulates Na-K-ATPase in IEC-18 cells. Thus the inhibition of Na-glucose cotransport in IEC-18 cells treated with L-NAME is not secondary to diminished Na gradient across the BBM.


Figure 4
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  		Fig. 4. Effect of L-NAME on Na-K-ATPase activity in IEC-18 cells. Na-K-ATPase activity measured as an organic phosphate released is stimulated in IEC-18 cells upon treatment with L-NAME to reduce cNO levels. Thus inhibition of cNO does not reduce the favorable Na gradient in IEC-18 cells for the functioning of SGLT1.

 
Na-glucose cotransport kinetic studies. To determine the mechanism of inhibition of Na-glucose cotransport, kinetic studies were performed. Figure 5 shows Na-dependent glucose uptake as a function of increasing concentrations of extracellular glucose in IEC-18 cells. As the concentration of extracellular glucose was increased, the uptake of Na-dependent glucose was stimulated and subsequently became saturated in all conditions (Fig. 5). The maximal rate of uptake (Vmax) of glucose was not altered by L-NAME treatment (Vmax for glucose uptake in control was 4.9 ± 0.2 nM/mg protein at 30 s and 5.6 ± 0.6 in L-NAME-treated cells, n = 4). However, the affinity (1/Km) for glucose uptake was significantly diminished in IEC-18 cells treated with L-NAME compared with control (Km for glucose uptake in control IEC-18 cells was 5.7 ± 0.5 mM and 61.0 ± 1.6 in L-NAME-treated cells, n = 4, P < 0.05). These results indicate that the mechanism of Na glucose cotransport inhibition by L-NAME in IEC-18 cells was secondary to a decrease in the affinity of the cotransporter for glucose rather than a decrease in the number of cotransporters.


Figure 5
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  		Fig. 5. Kinetics of SGLT1 in IEC-18 cells. Na-dependent glucose uptake is shown as a function of varying concentrations of extravesicular glucose. Isosmolarity was maintained by adjusting the concentration of mannitol. Uptake for all concentrations was determined at 30 s. As the concentration of extravesicular glucose was increased, uptake of glucose was stimulated and subsequently became saturated in IEC-18 cells in all conditions. The affinity (1/Km) for glucose uptake was significantly reduced by L-NAME treatment. However, the maximal rate of uptake of glucose (Vmax) was unaffected in the L-NAME-treated IEC-18 cells. The data shown are an average of 3 experiments, and each uptake was done in triplicate.

 
Na-H exchange kinetic studies. To determine the mechanism of stimulation of Na-H exchange by L-NAME kinetic studies were performed. Figure 6 shows that as the concentration of extracellular Na was increased the uptake of proton dependent EIPA sensitive Na was stimulated and subsequently became saturated in all conditions (Fig. 6). The affinity for Na was not altered by L-NAME treatment (Km for Na uptake in IEC-18 cells was 4.7 ± 1.5 mM in control and 4.1 ± 0.5 in L-NAME, n = 4). However, the Vmax or maximal rate of uptake of Na was significantly increased in cells treated with L-NAME compared with control (Vmax for Na uptake in control was 8.7 ± 0.8 nM/mg protein at 30 s and 12.7 ± 1.0 in L-NAME-treated cells, n = 3, P < 0.01). These studies indicate that the mechanism of Na-H exchange stimulation by L-NAME in IEC-18 cells was secondary to an increase in the number of transporters rather than an increase in the affinity of the exchangers for Na.


Figure 6
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  		Fig. 6. Kinetics of Na-H exchange in IEC-18 cells. Na uptake is shown as a function of varying concentrations of extravesicular Na. Isosmolarity was maintained by adjusting the concentration of mannitol. Uptake for all concentrations was determined at 30 s. As the concentration of extravesicular Na was increased, uptake of Na was stimulated and subsequently became saturated in IEC-18 cells in all conditions. The maximal rate of uptake of Na (Vmax) was significantly stimulated by L-NAME treatment. However, affinity (1/Km) for Na uptake was unaffected in IEC-18 cells. The data shown are an average of 3 experiments, and each uptake was done in triplicate.

 
SGLT1 molecular studies. Na-glucose cotransport is mediated by SGLT1 in intestinal epithelial cells. To determine the molecular mechanism of inhibition of SGLT1 by L-NAME, mRNA levels of SGLT1 was determined by RTQ-PCR. L-NAME had no effect on SGLT1 mRNA in IEC-18 cells (Fig. 7 ). These data indicate that L-NAME mediated inhibition of SGLT1 is not at the level of the number of cotransporters. Since mRNA levels may not necessarily correlate with functional protein on the BBM, Western blot analysis was performed. Immunoreactive SGLT1 levels were measured in IEC-18 cells as shown in Fig. 8. L-NAME treatment did not affect immunoreactive protein levels of SGLT1. Densitometric quantitation shown in Fig. 8C confirmed these findings. These data in conjunction with the kinetic data above indicate that the mechanism of inhibition of SGLT1 by L-NAME in IEC-18 cells is secondary to alteration in the affinity of the cotransporter for glucose rather than an alteration in the number of cotransporters.


Figure 7
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  		Fig. 7. Real-time PCR analysis demonstrated that the message for SGLT1 is unaffected in IEC-18 cells treated with L-NAME. This data indicates that L-NAME does not alter SGLT1 mRNA in IEC-18 cells.

 

Figure 8
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  		Fig. 8. Western blot analysis demonstrated that the brush border membrane (BBM) protein levels of SGLT1 was not altered in IEC-18 cells treated with L-NAME. A: a representative blot of 4 separate experiments is shown. B: a representative blot of gel loading control using ezrin. C: densitometry quantitation confirms that treatment with L-NAME does not alter SGLT1 in IEC-18 cells.

 
NHE3 molecular studies. Na-H exchange is mediated by NHE3 in the BBM of intestinal epithelial cells. To determine the molecular mechanism of stimulation of NHE3 by L-NAME, mRNA levels of NHE3 were determined by RTQ-PCR. L-NAME treatment markedly increased NHE3 mRNA levels in IEC-18 cells (Fig. 9). These data indicate that the mechanism of stimulation of NHE3 by L-NAME is at the level of the number of transporters. Since mRNA levels do not necessarily correlate with functional protein levels in the BBM, Western blot studies were also performed. As shown in Fig. 10 L-NAME markedly increased NHE3 levels in the BBM of IEC-18 cells. Densitometric quantitation shown in Fig. 10B confirmed these findings. These data indicate that L-NAME stimulation of Na-H exchange is via increasing NHE3 transporter numbers in the BBM of IEC-18 cells rather than an alteration in the affinity of the exchanger for Na.


Figure 9
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  		Fig. 9. Real-time PCR analysis demonstrated that the message for NHE3 is significantly increased in IEC-18 cells when treated with L-NAME. These data indicate that L-NAME increases NHE3 mRNA in IEC-18 cells. Error bars are inclusive when not seen in the figure.

 

Figure 10
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  		Fig. 10. Western blot analysis demonstrated that the BBM protein levels of NHE3 were significantly increased in IEC-18 cells treated with L-NAME. A: representative blot of 4 separate experiments is shown. B: densitometric quantitation confirms that treatment with L-NAME significantly increases BBM NHE3 in IEC-18 cells. The same ezrin gel loading condition as in Fig. 8B was used here as well.

 

   DISCUSSION
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
This study for the first time demonstrates that epithelial cell cNO directly regulates the brush border Na-absorptive pathways in intestinal epithelial cells. A novel observation of this study is that intracellular NO compensatorily regulates the two primary Na-absorptive pathways in the BBM of intestinal epithelial cells to presumably maintain cellular Na homeostasis. This observation indicates that there is functional linkage between the BBM NHE3 exchange and SGLT1 that is mediated by cNO in intestinal epithelial cells.

Within the intestine NO is produced at multiple levels. The epithelial cells, endothelial cells, neurons, and leukocytes including mast cells, macrophages, and neutrophils have been shown to produce NO. Thus any in vivo inhibition of cNO production and the observed resultant effect on transporter functions in epithelial cells is likely to be a combination of multiple effects. Additionally, in vivo inhibition of cNO production undoubtedly alters intestinal blood flow and/or motility, which may in turn have further effects on epithelial absorptive properties (12, 19, 27). Thus, to determine whether cNO has a direct effect on intestinal epithelial cell transporter function, it is important to eliminate these confounding factors. Furthermore, to fully understand the physiological effects of cNO on epithelial cell transporters it is necessary to study nontransformed epithelial cells to avoid the influences of oncogenesis as may occur in cancer cell lines.

In this study a nontransformed rat small intestinal epithelial cell line, IEC-18, which as a postconfluent monolayer resembles intestinal absorptive villus cells, was utilized. Similar to the mammalian small intestinal villus cells, these cells have NHE3 and SGLT1 on the BBM. L-NAME, but not the inactive analog D-NAME, significantly inhibits intracellular cNO in IEC-18. The inhibition of cNO inhibits SGLT1 activity in these cells. This inhibition of SGLT1 is not secondary to reduced Na gradient in these cells since L-NAME treatment, if anything, increased BLM Na-K-ATPase activity.

Inhibition of cNO production, in contrast to SGLT1 activity, stimulates NHE3 activity in IEC-18 cells. This compensatory regulation of BBM NHE3 and SGLT1 has not been reported previously in the literature. In fact, in the literature the effects of cNO on NaCl absorption in general is quite confusing. It has been suggested that inhibition of cNO in the rat small intestine promotes secretion (20), although other studies in the rat small intestine suggest that inhibition of cNO has no effect on electrolyte transport. Furthermore, increasing NO via NO donating compounds also appears to produce a host of responses in different species. NO-donating compounds have been shown to be proabsorptive in the rabbit ileum (2), in dog (18), and in mouse (22). Interestingly, in rat (24) and guinea pig ileum (17) and human colon cancer cells (8a), compounds that increase NO appears to inhibit absorption or be prosecretory. It is conceivable that these differences are species related.

The information in the literature is equally unclear as to the effect of inhibition of NO production vs. stimulation of NO production and the subsequent effect on electrolyte transport. In the rat jejunum inhibition of NO production as well as the simulation of NO production both have been shown to promote secretion (20). Other than the species differences, a potential explanation as to why the effect of NO on electrolyte transport in the intestine seems to produce different results in different studies may be secondary to differing amounts of NO produced in these studies. Thus physiological concentrations of NO may have one effect whereas pathophysiological concentrations may have quite a different effect on electrolyte transport. Although there is confounding information on the effects of NO on Na absorption via Na-H exchange, how cNO may affect the other primary Na-absorptive pathway, specifically Na glucose cotransport, has not been considered. Therefore, whether there is in fact a compensatory linkage between these two BBM transport pathways has not been considered before this study. In vivo inhibition of cNO production in rabbits has been demonstrated to inhibit SGLT1 in villus cells (3). In another study stimulation of villus cell NHE3 was demonstrated on in vivo inhibition of cNO in rabbits (4). However, as previously discussed, in vivo inhibition of cNO production undoubtedly has multiple effects on intestinal epithelial cells. Finally, in the chronically inflamed rabbit intestine SGLT1 is diminished secondary to a decrease in cotransporter numbers whereas NHE3 is unaffected (26). However, in the chronically inflamed intestine pathophysiological levels of NO are produced by inducible NO synthase, which is orders of magnitude higher than the NO produced by cNO synthase. In addition during chronic ileal inflammation peroxynitrite is also produced exclusively. Thus both of these in vivo in the inflamed intestine may regulate SGLT1 and NHE3 differently than NO in vitro in intestinal epithelial cells from another species.

To determine the mechanism of compensatory regulation of SGLT1 and NHE3 when intracellular cNO is inhibited, kinetic studies were performed. These kinetic studies demonstrated that the mechanism of inhibition of SGLT1 in IEC-18 cells when cNO is inhibited is secondary to a decrease in the affinity of the cotransporter for glucose without a change in the number of cotransporters in the BBM. Subsequent molecular studies to further define the mechanistic changes demonstrated that when cNO is inhibited there is no change in SGLT1 message levels. Since mRNA levels do not necessarily correlate with functional protein levels, Western blot studies that were subsequently undertaken demonstrated that indeed when cNO is inhibited in IEC-18 cells there is no significant alteration in BBM SGLT1 cotransporter numbers. Taken together these studies clearly indicate that inhibition of cNO inhibits SGLT1 at the posttranslational level perhaps via phosphorylation and/or glycosylation of this protein.

In contrast to SGLT1, kinetic studies demonstrated that the mechanism of stimulation of NHE3 when cNO is inhibited in IEC-18 cells is secondary to an increase in the maximal rate of uptake or exchangers numbers in the BBM of these cells. The molecular mechanism of compensatory stimulation of NHE3 when cNO is inhibited is quite different from the mechanism of inhibition of SGLT1 under the same conditions. The stimulation of NHE3 appears to be at the transcriptional level rather than at the posttranslational level as observed with SGLT1. When cNO is inhibited in IEC-18 cells the message for NHE3 is dramatically increased. Again, since mRNA may not necessarily correlate with functional protein on the BBM, Western blot studies were undertaken. These studies demonstrated that when cNO is inhibited in IEC-18 cells the number of NHE3 exchangers are also markedly increased in the BBM of IEC-18 cells. Taken together with the kinetic data, these results clearly indicate that, unlike SGLT1, inhibition of cNO compensatorily stimulates NHE3 by increasing transporter numbers in the BBM of IEC-18 cells.

It is interesting to note that in vivo inhibition of cNO production in a different species, specifically rabbits, produced similar results. Similar to what was observed in vitro in IEC-18 cells, in vivo inhibition of cNO inhibited SGLT1 in rabbit intestinal villus cell BBM while stimulating NHE3 in the same cells. Furthermore, the mechanism of alteration of SGLT1 and NHE3 when cNO is directly inhibited in vitro in IEC-18 cells is similar to that seen during in vivo inhibition of cNO in rabbits (3, 25). Taken together these results suggest that the effects of cNO on Na absorption in the intestine are potentially broadly applicable.

Specifically how cNO mediates this compensatory regulation of BBM NHE3 and SGLT1 remains to be elucidated. NO may mediate this effect directly or via cyclic GMP and its dependent kinases (5, 10). Whether these two primary Na-absorptive pathways are fundamentally physiologically linked to maintain cellular homeostasis is another interesting question. If so, cNO maybe one of many mediators of this physiological linkage. This compensatory regulation of Na absorption by these two absorptive villus cell BBM transporters maybe very important for designing therapy for severe diarrheal conditions such as cholera (9, 13).

In summary, direct inhibition of intestinal epithelial cell cNO production results in the inhibition of SGLT1 and stimulation of NHE3. The mechanism of inhibition of SGLT1 is likely posttranslational whereas the mechanism of stimulation of NHE3 is likely transcriptional. Furthermore, these studies demonstrate that the compensatory mechanism of regulation of SGLT1 and NHE3 by cNO in intestinal epithelial cells appears to be a broad-spectrum phenomena across two species whether the inhibition of cNO occurs in vivo or in vitro.


   GRANTS
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 GRANTS
REFERENCES
 
This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-45062 and DK-58034 to U. Sundaram.


   FOOTNOTES
 

Address for reprint requests and other correspondence: U. Sundaram, Section of Digestive Diseases, West Virginia Univ. School of Medicine, One Medical Center Dr., Morgantown, WV 26506 (e-mail: usundaram{at}hsc.wvu.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
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 REFERENCES
 

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