Regulation of NHE3 by nitric oxide in Caco-2 cells

Ravinder K. Gill, Seema Saksena, Irfan Ali Syed, Sangeeta Tyagi, Waddah A. Alrefai, Jaleh Malakooti, Krishnamurthy Ramaswamy, and Pradeep K. Dudeja

Section of Digestive and Liver Diseases, Department of Medicine, University of Illinois at Chicago and West Side Veterans Affairs Medical Center, Chicago, Illinois 60612


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The effect of nitric oxide (NO) on Na+/H+ exchange (NHE) activity was investigated utilizing Caco-2 cells as an experimental model. Incubation of Caco-2 cells with 10-3 M S-nitroso-N-acetylpenicillamine (SNAP), a conventional donor of NO, for 20 min resulted in a ~45% dose-dependent decrease in NHE activity, as determined by assay of ethylisopropylamiloride-sensitive 22Na uptake. A similar decrease in NHE activity was observed utilizing another NO-specific donor, sodium nitroprusside. SNAP-mediated inhibition of NHE activity was not secondary to a loss of cell viability. NHE3 activity was significantly reduced by SNAP (P < 0.05), whereas NHE2 activity was essentially unaltered. The effects of SNAP were mediated by the cGMP-dependent signal transduction pathway as follows: 1) LY-83583 and 1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one (ODQ), specific inhibitors of soluble guanylate cyclase, blocked the inhibitory effect of SNAP on NHE; 2) 8-bromo-cGMP mimicked the effects of SNAP on NHE activity; 3) the SNAP-induced decrease in NHE activity was counteracted by a specific protein kinase G inhibitor, KT-5823 (1 µM); 4) chelerythrine chloride (2 µM) or calphostin C (200 nM), specific protein kinase C inhibitors, did not affect inhibition of NHE activity by SNAP; 5) there was no cross activation by the protein kinase A-dependent pathway, as the inhibitory effects of SNAP were not blocked by Rp-cAMPS (25 µM), a specific protein kinase A inhibitor. These data provide novel evidence that NO inhibits NHE3 activity via activation of soluble guanylate cyclase, resulting in an increase in intracellular cGMP levels and activation of protein kinase G.

S-nitroso-N-acetylpenicillamine; cGMP; sodium/hydrogen exchanger


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

NITRIC OXIDE (NO) is a free radical diatomic molecule synthesized by mammalian cells from the guanidino nitrogen of L-arginine by a five-electron oxidation reaction catalyzed by a family of NO synthase (NOS) enzymes (34). A significant role for NO has been demonstrated in the physiology and pathophysiology of the gastrointestinal tract by a series of in vivo and in vitro studies (55). NO has been shown to be involved in regulation of intestinal motility (8), maintenance of blood flow within the layers of the wall of the gut (41), and protection of the mucosal barrier (34). However, besides its important biological functions under normal conditions and in acute inflammation, there is strong evidence that overproduction of NO plays an important role in the pathogenesis of experimental and clinical inflammatory bowel disease (IBD) (14). Potential sources of NO production in the intestinal epithelium include vascular endothelium (15), mesenteric neurons (37), enterocytes (51), macrophages and mast cells of the lamina propria (36). The effects of NO on intestinal epithelial functions have been suggested to occur through different mechanisms. Evidence suggests that NO can exert its effects through stimulation of soluble guanylate cyclase, resulting in the production of second messenger cGMP (53). Also, NO can act through activation of protein kinase C (PKC) in a number of cell types (53) or through ADP-associated ribosylation (53).

Previous functional studies indicate that NO directly affects Na+ and Cl- absorption and osmotic water permeability (17). NO has also been found to inhibit Na+ transport in the rabbit proximal tubule (45) and in the thick ascending limb (18). Plato et al. (42) demonstrated that NO inhibits Cl- transport in the thick ascending limb. Electrophysiological studies utilizing NO donors indicated a decrease in NaCl absorption in rat colon (58) and guinea pig intestine (29). In contrast, the NO precursor L-arginine at low concentrations increased Na+ absorption in rat intestine (56) and mouse cecum (21). In this regard, however, the effects of NO on Na+ absorption in the human intestine have not been investigated.

The effects of NO on electrolyte transport are influenced by whether the conditions under study are physiological or pathophysiological. Under physiological conditions, NO is considered to be a proabsorptive molecule, whereas it causes net secretion or inhibits absorption under pathophysiological conditions (53). The decreased NaCl absorption can be a result of a decrease in Na+ transport mediated by membrane proteins known as Na+/H+ exchangers (NHEs), which mediate the electroneutral exchange of extracellular Na+ with intracellular H+. Molecular cloning studies have demonstrated the existence of the NHE gene family, of which at least seven members, designated NHE1-NHE7, have been described with different tissue distributions and functional properties. Previous studies from our laboratory have shown that NHE1-NHE3 are expressed in the human intestine (13). NHE1 is localized to the basolateral membrane (53) and is suggested to be involved in housekeeping functions, whereas NHE2 and NHE3 are localized to the apical membrane and are suggested to be involved in vectorial Na+ transport (22).

The present study was undertaken to study the direct effects of NO on possible regulation of NHE activity and mechanism of regulation utilizing Caco-2 cells as an experimental model. The Caco-2 cell line is a human colonic carcinoma cell line that, on differentiation, manifests many anatomic and functional similarities to absorptive small intestinal enterocytes and has been used to study regulation of electrolyte uptake by various hormones and growth factors (3, 35, 44). Our present data demonstrate for the first time that NO decreased Caco-2 cell NHE activity by differential inhibition of the activity of NHE3 but not NHE2. This occurs through the activation of soluble guanylate cyclase, resulting in increased production of intracellular cGMP and activation of protein kinase G (PKG). There was no involvement of PKC- or protein kinase A (PKA)-mediated pathways in this process.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Materials. S-nitroso-N-acetylpenicillamine (SNAP) and Rp-cAMPS were obtained from Sigma Chemical (St. Louis, MO), 22Na from NEN Life Science Products (Boston, MA), Caco-2 cells from the American Type Culture Collection (Manassas, VA), and chelerythrine chloride, calphostin C, 1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one (ODQ), 6-anilinoquinoline-5,8-quinonine (LY-83583), 8-bromo-cGMP (8-BrcGMP), and KT-5823 from Biomol (Plymouth Meeting, PA). HOE-694 was a generous gift from Dr. Hans J. Lang (Aventis, Pharma Deutschland Chemical Research, Frankfurt/Main, Germany). All other chemicals were of at least reagent grade and were obtained from Sigma Chemical or Fisher Scientific.

Cell culture. Caco-2 cells were grown routinely in 75-cm2 plastic flasks at 37°C in a 5% CO2-95% air environment. The culture medium consisted of high-glucose DMEM, 20% FBS, 100 IU/ml penicillin, and 100 µg/ml streptomycin. Cells reached confluence after 5-7 days in culture. They were used for these studies between passages 25 and 55 and were plated in 24-well plates at a density of 2 × 104 cells/ml. Cells were used for experiments 10 days after plating and were fed fresh incubation medium on alternate days.

Determination of nitrite levels. The levels of NO in the culture medium can be measured by estimating nitrite (NO<UP><SUB>2</SUB><SUP>−</SUP></UP>), a stable metabolite of NO, utilizing the Greiss reaction (20). For these studies, the medium from confluent cells grown in 24-well plates was aspirated, and cells were incubated for 20 min in the presence or absence of the exogenous donor of NO (SNAP). NO<UP><SUB>2</SUB><SUP>−</SUP></UP> levels were measured in aliquots of the incubation medium by using a commercially available NO colorimetric assay kit (Biomol).

Assay of cGMP. For measuring cGMP levels in Caco-2 cells, confluent monolayers were preincubated for 15 min at room temperature in fresh medium supplemented with 0.5 mM IBMX. SNAP was then added for a further 20 min in the acid load buffer, and the reaction was stopped by addition of 10% (vol/vol, final concentration) trichloroacetic acid. Cell-associated cGMP content was measured in aliquots of the buffer extracted four times with four volumes of water-saturated ether, brought to pH 7.0 with Tris, and assayed for cGMP by radioimmunoassay using a commercially available kit (cGMP kit, Amersham). Results are expressed as picomoles of cGMP per milligram of protein.

Measurement of intracellular pH. Intracellular pH (pHi) was measured in Caco-2 cells grown on coverslips using the pH-sensitive fluorescent dye 2',7'-bis(2-carboxyethyl)-5(6-carboxyfluorescein) (BCECF; Sigma Chemical), as previously described (28, 40). Briefly, the cells were washed with buffer containing (in mM) 140 NaCl, 5 KCl, 1 CaCl2, 1 MgCl2, 5 glucose, and 6 HEPES-KOH, pH 7.4. Cells were then incubated at 37°C for 30 min with the same buffer containing 10 µM BCECF-AM. The Caco-2 cells were washed twice and loaded with acid load medium, in the absence or presence of 1 mM SNAP, for 30 min. The ratio of fluorescence of the intracellularly trapped BCECF dye was determined with excitation at wavelengths of 490 and 440 nm and emission at 530 nm utilizing a luminescence spectrometer (model LS50, Perkin-Elmer, Beaconsfield, UK). To estimate pHi, the BCECF excitation fluorescence ratios were calibrated utilizing the K+/nigericin methods, as previously described (28). The calibration curve demonstrated that the fluorescence ratios were linear as a function of pHi 6.0-8.0, as previously reported (28).

Assay of NHE activity. The NHE activity was determined in acid-loaded Caco-2 cells as ethylisopropylamiloride (EIPA)-sensitive 22Na uptake, as described (3). The activity of NHE isoforms (NHE2 and NHE3) was measured in the presence of HOE-694 (NHE2-specific inhibitor at 50 µM). NHE2 activity was calculated as NHE activity sensitive to 50 µM HOE-694. NHE3 activity was calculated by subtracting 50 µM HOE-694-sensitive NHE activity from total NHE activity (50 µM EIPA-sensitive NHE activity).

For these studies, the cells were placed at room temperature for 15-20 min to allow for equilibration before determination of 22Na uptake. Briefly, confluent cell monolayers were preincubated for 20 min at room temperature in acid load solution containing (in mM) 50 NH4Cl, 70 choline chloride, 5 KCl, 1 MgCl2, 2 CaCl2, 5 glucose, and 15 MOPS (pH 7.0). Cells were then incubated further for 20 min with or without SNAP, a conventional donor of NO, in the acid load solution. In a separate set of experiments, cells were also coincubated with 6-anilinoquinoline-5,8-quinonine (LY-83583) and ODQ (inhibitors of soluble guanylate cyclase), calphostin and chelerythrine chloride (inhibitors of PKC), and KT-5823 (an inhibitor of PKG). The cells were then washed with a solution containing (in mM) 120 choline chloride and 15 Tris-HEPES, pH 7.5. The solution was then aspirated, and the cells were incubated in uptake buffer containing (in mM) 10 NaCl, 110 choline chloride, 1 MgCl2, 2 CaCl2, and 20 HEPES (pH 7.4) and 1 mCi/ml of 22Na, with or without 50 µM EIPA or 50 µM HOE-694. After 5 min, the 22Na-containing uptake solution was aspirated and the cells were washed twice with ice-cold PBS. The cells were then solubilized by incubation with 0.5 N NaOH, and incorporated radioactivity was determined. The protein content of cell uptakes was estimated by the method of Bradford (4a). 22Na uptake was normally measured at 5 min (inasmuch as this was in the linear range of the time course) and expressed as nanomoles per milligram of protein per 5 min.

Apical membrane unidirectional 22Na influx utilizing Transwell inserts. For the experiments using Transwell inserts, Caco-2 cells were plated at a density of 4 × 104 cells/ml for 14 days before experimentation. Unidirectional apical membrane 22Na uptake was measured from the apical side, as described previously (44). Briefly, Caco-2 cells were exposed to SNAP from the apical and basolateral sides for 20 min in the acidifying solution, in the absence or presence of 0.5 mM ouabain applied basolaterally. Data are presented as EIPA (50 µM)-inhibitable component per Transwell insert and are expressed as picomoles per Transwell insert per 5 min.

Statistical analysis. Values are means ± SE. Student's t-test was utilized in statistical analysis. P < 0.05 was considered statistically significant.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Effects of NO on NHE activity. To determine the effects of NO on NHE activity, Caco-2 cells were incubated with 0-1,000 µM SNAP, a conventional donor of NO, for 20 min, and NHE activity was measured as EIPA-sensitive 22Na uptake after acidification of the cells by NH4Cl prepulse. As shown in Fig. 1, incubation with 500 µM and 10-3 M SNAP resulted in a dose-dependent decrease in NHE activity. There was up to ~45% inhibition with 10-3 M SNAP (P < 0.05). Therefore, 10-3 M SNAP was utilized for all subsequent experiments. To exclude the possibility that the effects of SNAP on NHE activity were due to cytotoxic effects of NO, trypan blue exclusion studies were carried out with different concentrations of SNAP. No alteration in cell viability was observed in Caco-2 cells incubated with SNAP compared with controls (15 ± 3% nonviable cells in control vs. 16 ± 1% at 10-3 M SNAP).


View larger version (11K):
[in this window]
[in a new window]
 
Fig. 1.   Concentration-dependent inhibition of apical Na+/H+ exchange (NHE) activity by S-nitroso-N-acetylpenicillamine (SNAP) in Caco-2 cells. Caco-2 cells were preincubated with Na+-free acid load solution for 20 min and then exposed to 0-10-3 M SNAP or vehicle (DMSO) in the acid load solution for 20 min. NHE activity was determined as ethylisopropylamiloride (EIPA)-sensitive (50 µM) 22Na uptake at 5 min. Values are means ± SE of 4-6 separate experiments performed in triplicate. * P < 0.05 and ** P < 0.005 compared with control.

To evaluate whether the observed decrease in NHE activity by SNAP is due to the release of NO, and not a nonspecific effect of the NO donor, we determined the response to another NO donor, sodium nitroprusside (SNP), which is structurally unrelated to SNAP. The results showed that, similar to SNAP, SNP (0-500 µM) also decreased NHE activity in a dose-dependent manner (Fig. 2).


View larger version (10K):
[in this window]
[in a new window]
 
Fig. 2.   Effects of sodium nitroprusside (SNP) on apical NHE activity in Caco-2 cells. Caco-2 cells were preincubated with Na+-free acid load solution for 20 min and then exposed to 0-500 µM SNP in the acid load solution for 20 min. NHE activity was determined as EIPA-sensitive (50 µM) 22Na uptake at 5 min. Values are means ± SE of 3 separate experiments performed in triplicate. * P < 0.05 or less compared with control.

To further rule out the possibility that the effects of SNAP on NHE activity are mediated by changes in the buffering capacity of the cells after SNAP treatment, pHi was measured in Caco-2 cells grown on coverslips and incubated in the absence or presence of 1 mM SNAP in the acidifying solution for 20 min using the pH-sensitive dye BCECF-AM. The results demonstrated that, after incubation of Caco-2 cells in the acid load, there was a significant decrease in pHi (7.20 ± 0.02 and 6.50 ± 0.01 for control and acid load, respectively, n = 3). This drop in pHi was essentially unaltered in the presence of SNAP (6.48 ± 0.03 and 6.50 ± 0.01 in SNAP-treated and control cells, respectively). These data indicate almost no change in the buffering capacity of the cells in response to SNAP, and the observed effects of SNAP on NHE activity were indeed due to the effects of NO generated.

NO<UP><SUB>2</SUB><SUP><UP>−</UP></SUP></UP> levels. To estimate the concentration of NO in solution, the levels of NO<UP><SUB>2</SUB><SUP>−</SUP></UP>, a stable metabolite of NO (30), were measured after exposure of cells to SNAP for 20 min. As shown in Table 1, incubation of cells with SNAP resulted in a concentration-dependent increase in NO<UP><SUB>2</SUB><SUP>−</SUP></UP> production.

                              
View this table:
[in this window]
[in a new window]
 
Table 1.   NO<UP><SUB>2</SUB><SUP>−</SUP></UP> in control and SNAP-treated Caco-2 cells

Effect of NO on NHE3 vs. NHE2. The amiloride analogs EIPA and HOE-694 were used to measure the effect of SNAP on apical NHE isoforms NHE2 and NHE3. As described in MATERIALS AND METHODS, NHE2 activity was calculated as NHE activity sensitive to 50 µM HOE-694 and determined as total NHE activity minus NHE activity in the presence of 50 µM HOE-694. NHE3 activity was calculated as NHE activity (50 µM EIPA sensitive) remaining in the presence of 50 µM HOE-694. Under control conditions, 42 ± 6% of total NHE activity was contributed by NHE2, and 58 ± 8% was contributed by NHE3 (Fig. 3A). Figure 3B shows the differential effect of SNAP at two different concentrations on NHE3 activity. NHE3 activity was reduced to 58% and 41% in the presence of 500 µM and 10-3 M SNAP, respectively, compared with the control values. In contrast, however, NHE2 activity essentially remained unaltered in the presence of SNAP (47.5 ± 2.85 and 45.3 ± 6.8% at 500 µM and 10-3 M SNAP, respectively). These observations indicate specific effects of SNAP on NHE3 activity.


View larger version (9K):
[in this window]
[in a new window]
 
Fig. 3.   A: contribution of NHE2 and NHE3 to total NHE activity. B: NHE3 activity after treatment with SNAP in Caco-2 cells. NHE activity was determined in the presence of 50 µM EIPA or 50 µM HOE-694. Values are means ± SE of 4-7 separate experiments performed in triplicate. SNAP decreased NHE3 activity. * P < 0.05 or less compared with control.

Role of cGMP in NHE inhibition by NO. We next examined the signal transduction pathways mediating the effects of NO on NHE activity in Caco-2 cells. NO stimulates cGMP production in many cell types via activation of soluble guanylate cyclase (45). Experiments were thus performed to measure cGMP levels stimulated by NO. As shown in Table 2, SNAP caused a concentration-dependent increase in cGMP production. There was ~4.0-fold increase in cGMP levels at 1 mM SNAP compared with control. However, in the presence of the soluble guanylate cyclase inhibitor LY-83583 (10 µM), the cGMP levels were equivalent to that of control. To investigate further the role of cGMP in regulation of NHE activity, cells were incubated with the inhibitor of guanylate cyclase, ODQ (1 µM), in the presence of SNAP (10-3 M SNAP), and NHE activity was assayed. As shown in Fig. 4, 10-3 M SNAP inhibited NHE activity by ~50%, as expected, but, in the presence of ODQ and LY-83583, specific inhibitors of soluble guanylate cyclase, which act as NO scavengers by specifically inhibiting soluble guanylate cyclase, also blocked the inhibitory effect of SNAP. These results suggested that production of cGMP through activation of guanylate cyclase mediates NO-induced inhibition of NHE activity. cGMP has been shown to inhibit NHE activity in different cell types (9, 47). We also studied the effect of the membrane-permeable analog of cGMP, 8-BrcGMP, on apical NHE activity. As shown in Fig. 5, preincubation of cells for 20 min with 8-BrcGMP resulted in a dose-dependent decrease in NHE activity (P < 0.05). These observations suggest that 8-BrcGMP mimicked the effects of NO in inhibiting NHE activity.

                              
View this table:
[in this window]
[in a new window]
 
Table 2.   Effect of SNAP on cGMP production in Caco-2 cells



View larger version (10K):
[in this window]
[in a new window]
 
Fig. 4.   Role of cGMP in SNAP-induced inhibition of NHE activity in Caco-2 cells. Caco-2 cells were preincubated with Na+-free acid load solution for 20 min and then exposed to 10-3 M SNAP or vehicle alone (DMSO) in the acid load solution for 20 min in the absence or presence of 1 µM 1H-(1,2,4)-oxadiazolo(4,3-a)quinoxalin-1-one (ODQ) or 10 µM LY-83583, and NHE activity was measured as EIPA-sensitive (50 µM) 22Na uptake at 5 min. Values are means ± SE of 4-6 separate experiments performed in triplicate. Absolute value for EIPA-sensitive 22Na uptake for control was 1.138 ± 0.080 nmol · mg protein-1 · 5 min-1. * P < 0.05 or less compared with control.



View larger version (9K):
[in this window]
[in a new window]
 
Fig. 5.   Effect of 8-bromo-cGMP (8-BrcGMP) on NHE activity in Caco-2 cells. Caco-2 cells were preincubated with acid load solution for 20 min and then exposed to 0-10-3 M 8-BrcGMP in the acid load solution for 20 min. NHE activity was determined as EIPA-sensitive (50 µM) 22Na uptake at 5 min. Values are means ± SE of 3 separate observations. * P < 0.05 or less compared with control.

Role of PKG. NO stimulates soluble guanylate cyclase, resulting in production of cGMP, and increases in cGMP lead to PKG activation (54). To assess the role of PKG in regulation of NHE activity, cells were treated with 10-3 M SNAP in the presence of the PKG inhibitor KT-5823. In the presence of 1 µM KT-5823, the SNAP-mediated inhibition of NHE activity was completely reversed, indicating that PKG activation was involved in inhibition of NHE activity (Fig. 6).


View larger version (10K):
[in this window]
[in a new window]
 
Fig. 6.   Regulation of NHE activity by cGMP/protein kinase G (PKG)-dependent pathway. Caco-2 cells were preincubated with Na+-free acid load solution for 20 min and then exposed to 10-3 M SNAP in the acid load solution for 20 min in the absence or presence of 1 µM KT-5823 (KT), and NHE activity was measured as EIPA-sensitive (50 µM) 22Na uptake at 5 min. KT-5823 blocked the inhibitory effect of SNAP. Values are means ± SE of 3 separate observations. * P < 0.05 or less compared with control.

Effect of PKC inhibitors on SNAP-mediated inhibition of NHE activity. To exclude the possibility that PKC might be involved in SNAP-mediated inhibition of NHE activity, experiments were also performed utilizing the specific PKC inhibitors chelerythrine chloride and calphostin C. Caco-2 cells were preincubated with the PKC inhibitors for 60 min before addition of SNAP, and NHE activity was assayed. Results indicated that, in the presence of 2 µM chelerythrine chloride (Fig. 7), there was essentially no effect on SNAP-mediated inhibition of NHE activity. Similar results were obtained utilizing another inhibitor of PKC, calphostin C (2.32 ± 0.48, 1.44 ± 0.14, and 1.19 ± 0.07 nmol · mg protein-1 · 5 min-1 for control, SNAP, and SNAP + 200 nM calphostin C, respectively).


View larger version (10K):
[in this window]
[in a new window]
 
Fig. 7.   Effect of protein kinase C antagonist on SNAP-induced inhibition of NHE activity in Caco-2 cells. Caco-2 cells were preincubated for 60 min in the presence of 2 µM chelerythrine chloride (Chel Cl) in the cell culture medium and then exposed to 10-3 M SNAP or vehicle alone (DMSO) in the acid load solution for 20 min in the absence or presence of chelerythrine chloride, and NHE activity was measured as EIPA-sensitive (50 µM) 22Na uptake. Values are means ± SE of 3-4 observations. * P < 0.05 or less compared with control.

Role of possible cross activation of PKA by cGMP. Previous evidence suggests that cAMP inhibits apical Na+/H+ exchange (7), and high concentrations of cGMP can cross activate PKA (16). To determine whether a PKA-dependent mechanism was involved in NO-mediated inhibition of NHE activity, NHE activity in response to SNAP was measured in the presence of the specific PKA inhibitor Rp-cAMPS (25 µM). As shown in Fig. 8, SNAP (10-3 M)-mediated NHE activity remained unaltered in the presence of Rp-cAMPS, thereby indicating that the effects of NO are specifically mediated by cGMP and activation of PKG, rather than PKA.


View larger version (9K):
[in this window]
[in a new window]
 
Fig. 8.   SNAP does not cross activate protein kinase A in Caco-2 cells. Caco-2 cells were preincubated with Na+-free acid load solution for 20 min and then exposed to 10-3 M SNAP or vehicle alone (DMSO) in the acid load solution for 20 min in the absence or presence of 25 µM Rpc-AMPS, and NHE activity was measured as EIPA-sensitive (50 µM) 22Na uptake. Values are means ± SE of 3 observations. * P < 0.05 or less compared with control.

Possible role of Na+-K+-ATPase in SNAP-mediated effects on NHE activity. Previous studies on the effects of NO showed a decrease in the activity of the Na+-K+-ATPase in kidney (31) and T84 cells (49). Therefore, additional experiments were performed to determine whether inhibition of this enzyme by NO would account for the observed decrease in NHE activity by SNAP. For this purpose, Caco-2 cells were grown in Transwell inserts treated with SNAP (from apical and basolateral sides) in the absence or presence of basolaterally applied ouabain (0.5 mM). As shown in Fig. 9, the observed decrease in apical 22Na uptake in response to SNAP was essentially unaltered in the presence of ouabain. These results rule out the possible involvement of the basolaterally expressed Na+-K+-ATPase in mediating the effects of acute SNAP treatment on NHE activity.


View larger version (7K):
[in this window]
[in a new window]
 
Fig. 9.   Role of Na+-K+-ATPase in SNAP-mediated decrease in NHE activity in Caco-2 cells grown on Transwell inserts. Caco-2 cells grown on Transwell inserts were treated with 1 mM SNAP in the acid load solution for 20 min from the apical and basolateral side in the absence or presence of basolaterally applied ouabain (0.5 mM). EIPA (50 µM)-sensitive 22Na uptake was measured from the apical side. Values are means ± SE of 3 observations. * P < 0.05 or less compared with control.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The present study demonstrated that high concentrations of NO inhibited Na+ uptake mediated by NHE3 in the Caco-2 cells. High levels of NO have been implicated in the pathogenesis of IBD. Increased NO production has been associated with high inducible NOS immunoreactivity and increased nitrate/NO<UP><SUB>2</SUB><SUP>−</SUP></UP> levels in various animal models of colitis (33, 43, 59). Similar results have also been obtained from clinical findings in patients with ulcerative colitis (2). The fact that NO levels are elevated in diarrhea, associated with IBD, suggests an important role of NO in modulating electrolyte and fluid transport in the intestine. In this regard, previous studies in the literature regarding the effect of NO on electrolyte transport appear contradictory and depend on whether the conditions under study are physiological or pathophysiological. For example, in physiological conditions, endogenous NO is a proabsorptive molecule and may decrease fluid secretion stimulated by various agonists (24). In contrast, under pathophysiological conditions, high levels of NO can provoke net secretion and decrease absorption (24).

Previous electrophysiological studies showed that NO stimulates Cl- and bicarbonate secretion and inhibits Na+ and Cl- absorption in rat colon (58). Recent studies by Liang and Knox (27) and Roczniak and Burns (45) suggested a decrease in NHE activity in the proximal tubule cells in response to exogenous donors of NO. In contrast, Marletta et al. (30) reported an increase in Na+ absorption in response to L-arginine (precursor of NO) in mouse cecum (21).

Our studies in Caco-2 cells using SNAP as an exogenous donor of NO demonstrated a dose-dependent decrease in apical NHE activity, with ~50% inhibition at 1 mM. Estimation of NO concentration by measuring NO<UP><SUB>2</SUB><SUP>−</SUP></UP> levels in the culture medium demonstrated a dose-dependent increase in NO<UP><SUB>2</SUB><SUP>−</SUP></UP> levels with increasing concentrations of SNAP. The concentrations of SNAP used in this study produced NO<UP><SUB>2</SUB><SUP>−</SUP></UP> levels within the range of values considered to be obtainable in vivo (19, 48). Furthermore, patients with colorectal carcinoma and IBD show elevated plasma nitrate/NO<UP><SUB>2</SUB><SUP>−</SUP></UP> levels (60-70 µM) (26, 50), which tend to be similar to the values achieved in our studies.

Generation of high levels of NO has been reported to lead to induction of cell death in a number of cell types (38, 39). However, measurement of cell viability by trypan blue exclusion in present studies ruled out the possibility that effects of NO on apical NHE activity are a mere manifestation of cytotoxicity. Consistent with our observations, Salzman et al. (47) found no evidence of cytotoxicity in Caco-2/bb2 cells incubated with 1.25 mM sodium nitroprusside (SNP) for 24 h as seen by confocal and ultrastructural microscopy as well as lactate dehydrogenase release.

Studies from our laboratory (3) and others (25, 35, 44) have provided strong evidence for the suitability of Caco-2 cells for investigating the functions of NHE isoforms. NHE3 and NHE2 are suggested to be involved in vectorial Na+ transport across the apical membranes in human small intestine and colon. NHE1 is localized basolaterally (53) and is considered to be important for housekeeping functions such as regulation of cell volume and pH. Several previous studies have demonstrated the differential regulation of the apical NHE isoforms NHE2 and NHE3, and even in some cases these isoforms are regulated reciprocally in intestinal epithelial cells. In C2 intestinal epithelial cells, for example, both isoforms are downregulated by cAMP; however, NHE2 is upregulated by serum and unaffected by cGMP or PKC, whereas NHE3 is downregulated by cGMP, PKC, and serum (4, 32). Our present studies indicate the regulation of NHE3 by NO in Caco-2 cells. There was no effect on NHE2 transport activity as estimated by 22Na uptake studies in the presence of 50 µM HOE-694 to block NHE2 but not NHE3. These observations suggest that NO regulates Na+ absorption in Caco-2 cells by exerting specific effects on NHE3.

It could be argued that elevated levels of NO secondary to 1 mM SNAP treatment may also affect permeability in Caco-2 cell monolayers. In this regard, Salzman et al. (47) showed that NO depletes ATP levels and reversibly dilates the tight junctions in cultured Caco-2/bbe (C2) cells. However, these effects were observed only with long-term (6-24 h) treatment in the presence of high concentrations of NO donor, e.g., 5 mM SNAP. In the present studies, a lower concentration of SNAP (1 mM) for a shorter duration (20 min) was used. Therefore, it is very unlikely that the observed changes in NHE3 activity could be due to alterations in the permeability of the Caco-2 monolayers. On the other hand, considering the fact that even if these shorter incubations, i.e., treatment with 1 mM SNAP for 20 min, possibly caused minor permeability changes, our results showing a decrease in NHE3 activity would remain valid, as NHE3 activity only represents the HOE-694-insensitive component of the total 22Na uptake.

NO has been shown to act via a variety of second messenger cascades (6, 45, 52), although most of its effects are mediated by activation of soluble guanylate cyclase, leading to an increase in intracellular levels of cGMP. The guanylate cyclase enzyme occurs in soluble (cytosolic) and membrane-bound (particulate) forms (24). The soluble guanylate cyclase contains heme moiety with bound iron, which can serve as an intracellular receptor for NO, leading to its activation. Although the intestinal epithelium contains 95% of the membrane-associated form of guanylate cyclase, NO-donating compounds have been shown to activate the soluble guanylate cyclase in colonic mucosa (57) and production of high levels of cGMP in ileal mucosal scrapings (29). Roczniak and Burns (45) found that cGMP partially mediated the observed effects of NO on NHE activity in rabbit proximal tubule cells. Our findings from the present study suggest that apical NHE activity in Caco-2 cells is inhibited by NO through a cGMP/PKG-dependent pathway. The data with measurements of cGMP levels in Caco-2 cells confirmed that SNAP stimulation of Caco-2 cells resulted in a rapid (~4.0-fold) increase in cGMP. This increase was blocked by the specific inhibitor of soluble guanylate cyclase LY-83583. To provide more direct evidence for the role of the cGMP pathway, additional studies were performed; for example, the direct effect of cGMP on NHE activity was examined. Incubation of Caco-2 cells with 8-BrcGMP significantly inhibited NHE activity (Fig. 5). Thus we could mimic the effect of SNAP by a biologically active analog of cGMP. Second, LY-83583 and ODQ (Fig. 4), specific inhibitors of soluble guanylate cyclase, completely abrogated the effect of SNAP on NHE activity. Third, KT-5823, a specific inhibitor of cGMP-dependent PKG, abolished the effect of SNAP. Therefore, the above findings convincingly indicate that inhibition of NHE activity by NO occurs via stimulation of soluble guanylate cyclase with a resultant increase in cGMP and activation of cGMP-dependent PKG in Caco-2 cells. Our studies are also supported by the previous findings of McSwine et al. (32) showing inhibition of NHE3, but not NHE2, by cGMP in the Caco-2/bbe cell line. More recently, Cha et al. (10) demonstrated the role of cGMP kinase II in the regulation of NHE3 by cGMP in the PS120 cell line.

The interactions of PKG with NHE3, whether direct or involving some accessory proteins, however, remain a point of further investigation. In this regard, studies have shown that although NHE3 is a phosphoprotein under basal conditions, changes in phosphorylation of NHE3 are not involved in the regulation by growth factors and protein kinases (60). Further studies are therefore needed to elucidate the downstream effectors of SNAP-mediated inhibition of NHE activity via a cGMP/PKG-mediated pathway.

NO-mediated inhibition of NHE activity in Caco-2 cells could also occur via 1) a PKC-mediated pathway or 2) cross activation of a PKA-mediated pathway by cGMP. PKC isozymes have been involved in mediating the effects of NO in some cell types (52). Our data with specific PKC inhibitors (chelerythrine chloride or calphostin C) exclude a role of this signal transduction cascade as a mediator of NO-induced inhibition of apical NHE activity, as these inhibitors failed to show an effect on decrease in NHE activity by SNAP.

High concentrations of cGMP have been shown to cross activate cAMP-dependent protein kinase in smooth muscle cells (11) and in intestine (16). It was thus considered of interest to determine whether the SNAP-induced decrease in NHE activity was secondary to cross talk and/or activation of cAMP. Our results with the specific PKA inhibitor Rp-cAMPS showing no effect on SNAP-mediated inhibition of NHE activity rule out the possible involvement of PKA in the cGMP/PKG-dependent pathway for inhibition of NHE activity in Caco-2 cells.

NO can interact with superoxide anions in cells, leading to the formation of oxidants such as peroxynitrite, with subsequent liberation of hydroxyl radicals (12). Peroxynitrite has been shown in previous studies (5, 23) to mediate its effect on vascular relaxation through metabolic generation of NO. The present studies suggest that NO directly modulates the activity of NHE3 and that this activity is not due to the possible generation of other reactive intermediates for the following reasons: 1) preliminary studies in our laboratory demonstrated that oxidants and free radicals had no significant effects on NHE activity in Caco-2 cells (unpublished observations); 2) use of LY-83583, which acts as an NO inhibitor by blocking the NO-induced activation of soluble guanylate cyclase, strongly indicated that the observed effects are indeed due to NO and not to reactive intermediates; and 3) we could mimic the effects of SNAP, with a structurally dissimilar NO donor, SNP, suggesting that the effects of SNAP were related to generation of NO and were not due to any direct nonspecific effect of the NO donor. These results also emphasize that the observed effects obtained with 1 mM SNAP were not pharmacological in nature.

In the present studies, the observed effects of NO on NHE3 activity appear to be primary in nature and not secondary to changes in the activity of other transporters. NO has previously been shown to inhibit the activity of basolateral Na+-K+-ATPase. Sugi et al. (49) observed a marked decrease in Na+-K+-ATPase activity in T84 cells grown on Transwell inserts in response to long-term treatment with NO. Similarly, a decrease was observed in response to chronic NO induction in the kidney (31). Our data utilizing Transwell inserts showed that SNAP treatment (20 min) in the presence or absence of ouabain, a well-known inhibitor of Na+-K+-ATPase activity, exhibits no significant effects on the observed inhibition of apical NHE activity by SNAP. These results, although indirect, rule out the possible involvement of Na+-K+-ATPase in SNAP-mediated effects on NHE3 activity in Caco-2 cells. The contribution of NHE1 also seems unlikely, since, in the present studies, basal pHi remained unchanged after acute SNAP treatment. Moreover, NO decreased the activity of only NHE3, and not NHE2, so it appears that NO-induced changes are not secondary to alterations in the Na+ concentration or pHi changes caused by NHE1. Interestingly, previous studies by Aizman et al. (1) showing the effects of NO on K+ transport in the rat distal colon suggested that although NO, being a lipophilic molecule, can traverse the plasma membrane, its effects were observed near its site of production because of its rapid inactivation under aerobic conditions. For example, these studies indicated that NO produced in the lumen affected only apical K+ transporters, whereas NO produced at the serosal side affected only basolateral K+ transporters. Additionally, studies in our laboratory have shown that NO decreased the activity of the apical Cl-/OH- exchanger via a mechanism involving PKG and PKC, different from the mechanism suggested in the present studies (46). Under exactly the same conditions, there was no alteration in the activity of the SO<UP><SUB>4</SUB><SUP>2−</SUP></UP>/OH- exchanger in the Caco-2 cells (46). These results further emphasize that the effects of NO on NHE3 are specific and are not secondary to changes in other transporter activities, pHi, or Na+ concentration.

In summary, the present study, for the first time, demonstrates that NO regulates the apical membrane NHE activity in Caco-2 cells by inhibiting the activity of NHE3 but not NHE2. These studies also present the novel findings of NO-induced inhibition of NHE3 via a PKG-mediated pathway without any involvement of PKC or PKA.

On the basis of our results, we propose a model (Fig. 10) demonstrating the mechanism of inhibition of NHE3 by NO. The observed inhibition appears to be mediated by cGMP/PKG-dependent mechanism(s), whereas there is no involvement of the PKC- or PKA-mediated pathway in this process. Because increased levels of NO and inducible NOS are associated with ulcerative colitis, gastroenteritis, and hypoxia-induced dysfunction, our present data would suggest a crucial role of NO in the regulation of human intestinal electrolyte transport in normal and pathological conditions.


View larger version (18K):
[in this window]
[in a new window]
 
Fig. 10.   Proposed model of mechanism of inhibition of NHE3 by nitric oxide (NO). PKA, PKC, and PKG, protein kinase A, C, and G, respectively; sGC, soluble guanylate cyclase.


    ACKNOWLEDGEMENTS

These studies were supported by the Department of Veterans Affairs and National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-54016 (P. K. Dudeja), DK-33349 (K. Ramaswamy), and DK-09930 (W. A. Alrefai).


    FOOTNOTES

Address for reprint requests and other correspondence: P. K. Dudeja, University of Illinois at Chicago, Medical Research Service (600/151), Chicago VA: West Side Division, 820 South Damen Ave., Chicago, IL 60612 (E-mail: pkdudeja{at}uic.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.

10.1152/ajpgi.00294.2001

Received 11 July 2001; accepted in final form 16 April 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Aizman, R, Brismar H, and Celsi G. Nitric oxide inhibits potassium transport in the rat distal colon. Am J Physiol Gastrointest Liver Physiol 276: G146-G154, 1999[Abstract/Free Full Text].

2.   Alican, I, and Kubes P. A critical role for nitric oxide in intestinal barrier function and dysfunction. Am J Physiol Gastrointest Liver Physiol 270: G225-G237, 1996[Abstract/Free Full Text].

3.   Alrefai, WA, Scaglione-Sewell B, Tyagi S, Wartman L, Brasitus TA, Ramaswamy K, and Dudeja PK. Differential regulation of the expression of Na+/H+ exchanger isoform NHE3 by PKC-alpha in Caco-2 cells. Am J Physiol Cell Physiol 281: C1551-C1558, 2001[Abstract/Free Full Text].

4.   Bookstein, C, Musch MW, Xie Y, Rao MC, and Chang EB. Regulation of intestinal epithelial brush border Na+/H+ exchanger isoforms, NHE2 and NHE3, in C2bbe cells. J Membr Biol 171: 87-95, 1999[ISI][Medline].

4a.   Bradford, M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248-254, 1976[ISI][Medline].

5.   Brown, AS, Moro MA, Masse JM, Cramer EM, Radomski M, and Darley-Usmar V. Nitric oxide-dependent and -independent effects on human platelets treated with peroxynitrite. Cardiovasc Res 40: 380-388, 1998[ISI][Medline].

6.   Brune, B, and Lapetina EG. Activation of a cytosolic ADP-ribosyltransferase by nitric oxide-generating agents. J Biol Chem 264: 8455-8458, 1989[Abstract/Free Full Text].

7.   Burns, KD, Inagami T, and Harris RC. Cloning of a rabbit kidney cortex AT1 angiotensin II receptor that is present in proximal tubule epithelium. Am J Physiol Renal Fluid Electrolyte Physiol 264: F645-F654, 1993[Abstract/Free Full Text].

8.   Calignano, A, Whittle BJ, Di Rosa M, and Moncada S. Involvement of endogenous nitric oxide in the regulation of rat intestinal motility in vivo. Eur J Pharmacol 229: 273-276, 1992[ISI][Medline].

9.   Caramelo, C, Lopez-Farre A, Riesco A, Olivera A, Okada K, Cragoe EJ, Tsai P, Briner VA, and Schrier RW. Atrial natriuretic peptide and cGMP inhibit Na+/H+ antiporter in vascular smooth muscle cells in culture. Kidney Int 45: 66-75, 1994[ISI][Medline].

10.   Cha, BY, Kim JH, Hut H, Kwon WL, Nadarajah J, Tse M, Cavet M, Yun C, Dejonge H, and Donowitz M. E3KARP (NHE3 kinase A anchoring protein) is necessary for cGMP regulation of NHE3: demonstration of a plasma membrane signaling complex containing NHE3, E3KARP, and cGMP kinase II (Abstract). Gastroenterology 87: A85, 2001.

11.   Cornwell, TL, Arnold E, Boerth NJ, and Lincoln TM. Inhibition of smooth muscle cell growth by nitric oxide and activation of cAMP-dependent protein kinase by cGMP. Am J Physiol Cell Physiol 267: C1405-C1413, 1994[Abstract/Free Full Text].

12.   Dijkstra, G, Moshage H, van Dullemen HM, de Jager-Krikken A, Tiebosch AT, Kleibeuker JH, Jansen PL, and van Goor H. Expression of nitric oxide synthases and formation of nitrotyrosine and reactive oxygen species in inflammatory bowel disease. J Pathol 186: 416-421, 1998[ISI][Medline].

13.   Dudeja, PK, Rao DD, Syed I, Joshi V, Dahdal RY, Gardner C, Risk MC, Schmidt L, Bavishi D, Kim KE, Harig JM, Goldstein JL, Layden TJ, and Ramaswamy K. Intestinal distribution of human Na+/H+ exchanger isoforms NHE-1, NHE-2, and NHE-3 mRNA. Am J Physiol Gastrointest Liver Physiol 271: G483-G493, 1996[Abstract/Free Full Text].

14.   Dykhuizen, RS, Masson J, McKnight G, Mowat AN, Smith CC, Smith LM, and Benjamin N. Plasma nitrate concentration in infective gastroenteritis and inflammatory bowel disease. Gut 39: 393-395, 1996[Abstract].

15.   Falcone, JC, and Bohlen HG. EDRF from rat intestine and skeletal muscle venules causes dilation of arterioles. Am J Physiol Heart Circ Physiol 258: H1515-H1523, 1990[Abstract/Free Full Text].

16.   Forte, LR, Thorne PK, Eber SL, Krause WJ, Freeman RH, Francis SH, and Corbin JD. Stimulation of intestinal Cl- transport by heat-stable enterotoxin: activation of cAMP-dependent protein kinase by cGMP. Am J Physiol Cell Physiol 263: C607-C615, 1992[Abstract/Free Full Text].

17.   Garcia, NH, Pomposiello SI, and Garvin JL. Nitric oxide inhibits ADH-stimulated osmotic water permeability in cortical collecting ducts. Am J Physiol Renal Fluid Electrolyte Physiol 270: F206-F210, 1996[Abstract/Free Full Text].

18.   Garvin, JL, and Hong NJ. Nitric oxide inhibits sodium/hydrogen exchange activity in the thick ascending limb. Am J Physiol Renal Physiol 277: F377-F382, 1999[Abstract/Free Full Text].

19.   Geng, Y, Hansson GK, and Holme E. Interferon-gamma and tumor necrosis factor synergize to induce nitric oxide production and inhibit mitochondrial respiration in vascular smooth muscle cells. Circ Res 71: 1268-1276, 1992[Abstract].

20.   Green, LC, Wagner DA, Glogowski J, Skipper PL, Wishnok JS, and Tannenbaum SR. Analysis of nitrate, nitrite, and [15N]nitrate in biological fluids. Anal Biochem 126: 131-138, 1982[ISI][Medline].

21.   Homaidan, FR, Martello LA, Melson SJ, and Burakoff R. Regulation of electrolyte transport by nitric oxide in the mouse cecum. Eur J Pharmacol 350: 93-99, 1998[ISI][Medline].

22.   Hoogerwerf, S, Tsao SC, Devuyst O, Levine SA, Yun CHC, Yip JW, Cohen ME, Wilson PD, Lazenby AJ, Tse CM, and Donowitz M. NHE-2 and NHE-3 are human and rabbit intestinal brush-border proteins. Am J Physiol Gastrointest Liver Physiol 270: G29-G41, 1996[Abstract/Free Full Text].

23.   Iesaki, T, Gupte SA, Kaminski PM, and Wolin MS. Inhibition of guanylate cyclase stimulation by NO and bovine arterial relaxation to peroxynitrite and H2O2. Am J Physiol Heart Circ Physiol 277: H978-H985, 1999[Abstract/Free Full Text].

24.   Izzo, AA, Mascolo N, and Capasso F. Nitric oxide as a modulator of intestinal water and electrolyte transport. Dig Dis Sci 43: 1605-1620, 1998[ISI][Medline].

25.   Janecki, AJ, Montrose MH, Zimniak P, Zweibaum A, Tse CM, Khurana S, and Donowitz M. Subcellular redistribution is involved in acute regulation of the brush border Na+/H+ exchanger isoform 3 in human colon adenocarcinoma cell line Caco-2. Protein kinase C-mediated inhibition of the exchanger. J Biol Chem 273: 8790-8798, 1998[Abstract/Free Full Text].

26.   Levine, JJ, Pettei MJ, Valderrama E, Gold DM, Kessler BH, and Trachtman H. Nitric oxide and inflammatory bowel disease: evidence for local intestinal production in children with active colonic disease. J Pediatr Gastroenterol Nutr 26: 34-38, 1998[ISI][Medline].

27.   Liang, M, and Knox FG. Production and functional roles of nitric oxide in the proximal tubule. Am J Physiol Regul Integr Comp Physiol 278: R1117-R1124, 2000[Abstract/Free Full Text].

28.   Lubman, RL, and Crandall ED. Polarized distribution of Na+-H+ antiport activity in rat alveolar epithelial cells. Am J Physiol Lung Cell Mol Physiol 266: L138-L147, 1994[Abstract/Free Full Text].

29.   MacNaughton, WK. Nitric oxide-donating compounds stimulate electrolyte transport in the guinea pig intestine in vitro. Life Sci 53: 585-593, 1993[ISI][Medline].

30.   Marletta, MA, Yoon PS, Iyengar R, Leaf CD, and Wishnok JS. Macrophage oxidation of L-arginine to nitrite and nitrate: nitric oxide is an intermediate. Biochemistry 27: 8706-8711, 1988[ISI][Medline].

31.   McKee, M, Scavone C, and Nathanson JA. Nitric oxide, cGMP, and hormone regulation of active sodium transport. Proc Natl Acad Sci USA 91: 12056-12060, 1994[Abstract/Free Full Text].

32.   McSwine, RL, Musch MW, Bookstein C, Xie Y, Rao M, and Chang EB. Regulation of apical membrane Na+/H+ exchangers NHE2 and NHE3 in intestinal epithelial cell line C2/bbe. Am J Physiol Cell Physiol 275: C693-C701, 1998[Abstract].

33.   Miller, MJ, Sadowska-Krowicka H, Chotinaruemol S, Kakkis JL, and Clark DA. Amelioration of chronic ileitis by nitric oxide synthase inhibition. J Pharmacol Exp Ther 264: 11-16, 1993[Abstract].

34.   Moncada, S, Palmer RM, and Higgs EA. Nitric oxide: physiology, pathophysiology, and pharmacology. Pharmacol Rev 43: 109-142, 1991[ISI][Medline].

35.   Musch, MW, Bookstein C, Xie Y, Sellin JH, and Chang EB. SCFA increase intestinal Na absorption by induction of NHE3 in rat colon and human intestinal C2/bbe cells. Am J Physiol Gastrointest Liver Physiol 280: G687-G693, 2001[Abstract/Free Full Text].

36.   Nathan, C. Nitric oxide as a secretory product of mammalian cells. FASEB J 6: 3051-3064, 1992[Abstract/Free Full Text].

37.   Nichols, K, Staines W, and Krantis A. Nitric oxide synthase distribution in the rat intestine: a histochemical analysis. Gastroenterology 105: 1651-1661, 1993[ISI][Medline].

38.   Nishio, E, and Watanabe Y. Nitric oxide donor-induced apoptosis in smooth muscle cells is modulated by protein kinase C and protein kinase A. Eur J Pharmacol 339: 245-251, 1997[ISI][Medline].

39.   O'Connor, KJ, and Moncada S. Glucocorticoids inhibit the induction of nitric oxide synthase and the related cell damage in adenocarcinoma cells. Biochim Biophys Acta 1097: 227-231, 1991[ISI][Medline].

40.   Paradiso, AM. Identification of Na+-H+ exchange in human normal and cystic fibrosis ciliated airway epithelium. Am J Physiol Lung Cell Mol Physiol 262: L757-L764, 1992[Abstract/Free Full Text].

41.   Pique, JM, Whittle BJ, and Esplugues JV. The vasodilator role of endogenous nitric oxide in the rat gastric microcirculation. Eur J Pharmacol 174: 293-296, 1989[ISI][Medline].

42.   Plato, CF, Stoos BA, Wang D, and Garvin JL. Endogenous nitric oxide inhibits chloride transport in the thick ascending limb. Am J Physiol Renal Physiol 276: F159-F163, 1999[Abstract/Free Full Text].

43.   Ribbons, KA, Zhang XJ, Thompson JH, Greenberg SS, Moore WM, Kornmeier CM, Currie MG, Lerche N, Blanchard J, Clark DA, Potential role of nitric oxide in a model of chronic colitis in rhesus macaques. Gastroenterology 108: 705-711, 1995[ISI][Medline].

44.   Rocha, F, Musch MW, Lishanskiy L, Bookstein C, Sugi K, Xie Y, and Chang EB. IFN-gamma downregulates expression of Na+/H+ exchangers NHE2 and NHE3 in rat intestine and human Caco-2/bbe cells. Am J Physiol Cell Physiol 280: C1224-C1232, 2001[Abstract/Free Full Text].

45.   Roczniak, A, and Burns KD. Nitric oxide stimulates guanylate cyclase and regulates sodium transport in rabbit proximal tubule. Am J Physiol Renal Fluid Electrolyte Physiol 270: F106-F115, 1996[Abstract/Free Full Text].

46.   Saksena, S, Gill RK, Tyagi S, Syed IA, Alrefai WA, Ramaswamy K, and Dudeja PK. Modulation of Cl-/OH- exchange process in Caco2 cells by nitric oxide (Abstract). Gastroenterology 120: A210, 2001.

47.   Salzman, AL, Menconi MJ, Unno N, Ezzell RM, Casey DM, Gonzalez PK, and Fink MP. Nitric oxide dilates tight junctions and depletes ATP in cultured Caco-2BBe intestinal epithelial monolayers. Am J Physiol Gastrointest Liver Physiol 268: G361-G373, 1995[Abstract/Free Full Text].

48.   Stuehr, DJ, and Nathan CF. Nitric oxide. A macrophage product responsible for cytostasis and respiratory inhibition in tumor target cells. J Exp Med 169: 1543-1555, 1989[Abstract].

49.   Sugi, K, Musch MW, Field M, and Chang EB. Inhibition of Na+,K+-ATPase by interferon-gamma down-regulates intestinal epithelial transport and barrier function. Gastroenterology 120: 1393-1403, 2001[ISI][Medline].

50.   Szaleczky, E, Pronai L, Nakazawa H, and Tulassay Z. Evidence of in vivo peroxynitrite formation in patients with colorectal carcinoma, higher plasma nitrate/nitrite levels, and lower protection against oxygen free radicals. J Clin Gastroenterol 30: 47-51, 2000[ISI][Medline].

51.   Tepperman, BL, Brown JF, and Whittle BJ. Nitric oxide synthase induction and intestinal epithelial cell viability in rats. Am J Physiol Gastrointest Liver Physiol 265: G214-G218, 1993[Abstract/Free Full Text].

52.   Tepperman, BL, Chang Q, and Soper BD. The involvement of protein kinase C in nitric oxide-induced damage to rat isolated colonic mucosal cells. Br J Pharmacol 128: 1268-1274, 1999[Abstract/Free Full Text].

53.   Tyagi, S, Joshi V, Alrefai WA, Gill RK, Ramaswamy K, and Dudeja PK. Evidence for a Na+-H+ exchange across human colonic basolateral plasma membranes purified from organ donor colons. Dig Dis Sci 45: 2282-2289, 2000[ISI][Medline].

54.   Vaandrager, AB, and de Jonge HR. Signalling by cGMP-dependent protein kinases. Mol Cell Biochem 157: 23-30, 1996[ISI][Medline].

55.   Wallace, JL, and Miller MJ. Nitric oxide in mucosal defense: a little goes a long way. Gastroenterology 119: 512-520, 2000[ISI][Medline].

56.   Wapnir, RA, Wingertzahn MA, and Teichberg S. L-Arginine in low concentration improves rat intestinal water and sodium absorption from oral rehydration solutions. Gut 40: 602-607, 1997[Abstract].

57.   Wilson, KT, Vaandrager AB, De Vente J, Musch MW, De Jonge HR, and Chang EB. Production and localization of cGMP and PGE2 in nitroprusside-stimulated rat colonic ion transport. Am J Physiol Cell Physiol 270: C832-C840, 1996[Abstract/Free Full Text].

58.   Wilson, KT, Xie Y, Musch MW, and Chang EB. Sodium nitroprusside stimulates anion secretion and inhibits sodium chloride absorption in rat colon. J Pharmacol Exp Ther 266: 224-230, 1993[Abstract].

59.   Yamada, T, Sartor RB, Marshall S, Specian RD, and Grisham MB. Mucosal injury and inflammation in a model of chronic granulomatous colitis in rats. Gastroenterology 104: 759-771, 1993[ISI][Medline].

60.   Yip, JW, Ko WH, Viberti GC, Huganir RL, Donowitz M, and Tse CM. Regulation of epithelial brush border NHE3 expressed in fibroblasts by fibroblast growth factor and phorbol esters is not through changes in phosphorylation of the exchanger. J Biol Chem 272: 18473-18480, 1997[Abstract/Free Full Text].


Am J Physiol Gastrointest Liver Physiol 283(3):G747-G756