Section of Digestive and Liver Diseases, Department of Medicine, University of Illinois at Chicago and West Side Veterans Affairs Medical Center, Chicago, Illinois 60612
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ABSTRACT |
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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 103 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
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INTRODUCTION |
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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.
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MATERIALS AND METHODS |
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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
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.
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RESULTS |
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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
103 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).
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NO
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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 103 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.
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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 (103 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.
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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 103 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).
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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
protein1 · 5 min
1 for control,
SNAP, and SNAP + 200 nM calphostin C, respectively).
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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
(103 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.
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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.
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DISCUSSION |
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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
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
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
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.
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ACKNOWLEDGEMENTS |
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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).
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FOOTNOTES |
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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.
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