The present studies were undertaken to determine the direct effects of nitric oxide (NO) released from an exogenous donor,S-nitroso-N-acetyl pencillamine (SNAP) on Cl−/OH− exchange activity in human Caco-2 cells. Our results demonstrate that NO inhibits Cl−/OH− exchange activity in Caco-2 cells via cGMP-dependent protein kinases G (PKG) and C (PKC) signal-transduction pathways. Our data in support of this conclusion can be outlined as follows: 1) incubation of Caco-2 cells with SNAP (500 μM) for 30 min resulted in ∼50% inhibition of DIDS-sensitive36Cl uptake; 2) soluble guanylate cyclase inhibitors Ly-83583 and (1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one significantly blocked the inhibition of Cl−/OH− exchange activity by SNAP; 3) addition of 8-bromo-cGMP (8-BrcGMP) mimicked the effects of SNAP; 4) specific PKG inhibitor KT-5823 significantly inhibited the decrease in Cl−/OH− exchange activity in response to either SNAP or 8-BrcGMP; 5) Cl−/OH− exchange activity in Caco-2 cells in response to SNAP was not altered in the presence of protein kinase A (PKA) inhibitor (Rp-cAMPS), demonstrating that the PKA pathway was not involved; 6) the effect of NO on Cl−/OH− exchange activity was mediated by PKC, because each of the two PKC inhibitors chelerythrine chloride and calphostin C blocked the SNAP-mediated inhibition of Cl−/OH− exchange activity; 7) SO /OH− exchange in Caco-2 cells was unaffected by SNAP. Our results suggest that NO-induced inhibition of Cl−/OH− exchange may play an important role in the pathophysiology of diarrhea associated with inflammatory bowel diseases.
- chloride absorption
- human intestine
- guanylate cyclase
- protein kinase C regulation
nitric oxide (no) is considered to be an important biological mediator in a number of cellular functions (26). NO is generated froml-arginine by NO synthase (NOS), which exists as a constitutive or inducible enzyme in many tissue types (25). NO has also been implicated in regulating an increasing number of physiological and pathophysiological pathways in the gastrointestinal tract (44). For example, NO has been shown to regulate gastrointestinal motility (6, 15, 28), mucosal permeability (19) and intestinal ion transport (23, 46, 54). In pathological conditions, elevated levels of NO have been found in patients with ulcerative colitis and gastroenteritis (9) and in an experimental model of hypoxia-induced colonic dysfunction (4).
Most of the previous studies concerning the effects of NO on ion transport were mainly based on electrophysiological recordings of short-circuit currents and transmural potential difference without direct measurements of unidirectional ion fluxes (36, 43,46). These studies showed that NO-donating compounds stimulated electrogenic Cl− secretion and inhibited Na+and Cl− absorption in the intestines of guinea pig (23) and rat (54). Previous findings of Plato et al. (35) have also indicated the role of both exogenous and endogenous NO in the inhibition of Cl− absorption in the rat thick ascending loop of Henle (TALH). However, the effects of NO on epithelial cell absorption and secretion appear contradictory, supporting both the proabsorptive or prosecretory functions. For example, under pathophysiological conditions, NO has been suggested to provoke net secretion, whereas under physiological conditions, it has been shown to be a proabsorptive molecule (17). The observed increase in NO-induced diarrhea associated with inflammatory bowel disease (IBD) suggests an important role of NO in either stimulating Cl− secretion or decreasing Na and Cl absorption. However, to date, the effects of NO on the intestinal apical membrane Cl−/OH− exchange activity have not been investigated, and the signal-transduction pathways involved in NO-mediated modulation of electrolyte transport have not been delineated.
In this regard, most of the effects of NO on the electrolyte transport have been shown to be mediated through different signal-transduction pathways. The major mechanism of action of NO is considered to be the activation of soluble guanylate cyclase (sGC) with the formation of cGMP (second messenger) (39), which, in turn, can exert its physiological effects by interacting with various downstream effectors including cGMP-gated channels, cGMP-regulated phosphodiesterases, cGMP-dependent protein kinases (PKGs) and cAMP-dependent protein kinases (PKAs) (21, 51). Alternatively, NO can also mediate its effects via activation of protein kinase C (PKC) (30, 41) or through ADP-associated ribosylation (5).
The current studies were, therefore, undertaken to examine1) the possible regulation of Cl−/OH− exchange activity in Caco-2 cells (a well-established model for the human intestinal transport studies) by exogenous NO donor SNAP and 2) the signal-transduction pathways involved in this process. Our current studies demonstrate that NO inhibits apical Cl−/OH− exchange activity in Caco-2 cells via a combination of cGMP/PKG- and PKC-mediated pathways.
MATERIALS AND METHODS
DIDS, S-nitroso-N-acetyl penicillamine (SNAP), and Rp-cAMPs were obtained from Sigma (St. Louis, MO). Radionuclide[35S]sulphuric acid and [36Cl]hydrochloric acid were obtained from NEN Life Science Products (Boston, MA). Caco-2 cells were obtained from American Type Culture Collection (Manassas, VA). 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 were obtained from Biomol (Plymouth Meeting, PA). All other chemicals were of at least reagent grade and were obtained from Sigma or Fisher Scientific (Pittsburgh, PA).
Caco-2 cells were grown in DMEM supplemented with 4.5 g/l glucose, 2 mM glutamine, 50 U/ml penicillin, 50 μg/ml streptomycin, 10 mM HEPES, 1% essential and nonessential amino acids, and 20% fetal bovine serum, pH 7.4, in 5% CO2-95% O2 at 37°C. For the uptake experiments, cells from passages between 20 and 25 were plated in 24-well plates at a density of 2 × 104cells/cm2. Confluent monolayers were then used for experiments at day 10 after plating and were fed with fresh medium every alternate day.
Determination of nitrite (NO ) levels. The levels of NO in the culture medium can be estimated by measuring NO , a stable metabolite of NO utilizing Greiss reaction (13). For these studies, the medium from confluent cells grown in 24 well plates was aspirated and cells were incubated for 30 min in the presence or absence of exogenous donor of NO (SNAP). Nitrite levels were measured in the aliquots of the incubation medium by using a commercially available NO colorimetric assay kit (Biomol, PA, USA). Incubation of cells with SNAP (500 μM) resulted in a significant increase (49.76 ± 6.25 μM in SNAP-treated vs. 2.30 ± 0.05 μM in control) in NO production.
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 30-min period, and the reaction was stopped by the addition of 10% trichloroacetic acid [TCA (vol/vol), final concentration]. Cell-associated cGMP content was measured in aliquots of the medium 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 were expressed as picomoles cGMP per milligram protein.
36Cl− and35SO uptake.
Chloride and sulphate uptake experiments were performed to assess Cl−/OH− and SO /OH− exchange activities by the method of Olsnes et al. (32) with some modification, as previously described by us (2). Caco-2 cells were incubated with DMEM base medium containing 20 mM HEPES/KOH, pH 8.5, with or without SNAP, an exogenous NO donor, for 30 min at room temperature. In some experiments, cells were also incubated with inhibitors of soluble guanylate cyclase, ODQ, and Ly-83583, specific inhibitors of PKC, chelerythrine chloride, and calphostin C, Rp-cAMPs (specific PKA inhibitor), and KT-5823 (PKG inhibitor). The medium was removed, and the cells were rapidly washed with 1 ml tracer-free uptake mannitol buffer containing 260 mM mannitol and 20 mM Tris/MES, pH 7.0. The cells were then incubated with the uptake buffer for a 5-min time period. This time period was chosen because this falls within the linear range of Cl− uptake in this system (2). For 35SO uptake studies, the uptake buffer was the mannitol buffer containing 2.4 μCi/ml of 35SO of sulphuric acid (specific activity = 1325.0 Ci/mmol) and 50 μM unlabeled K2SO4. For 36Cl−uptake studies, the uptake buffer was the mannitol buffer containing 1.3 μCi/ml 36Cl− (2.7 mM) of hydrochloric acid (specific activity = 17.12 mCi/g). For the uptake studies, the acid forms of the radionucleotides were neutralized with equimolar concentrations of KOH. The uptake was terminated by removing the buffer and washing the cells rapidly two times with 1 ml of ice-cold PBS, pH 7.2. Finally, the cells were solubilized by incubation with 0.5 N NaOH for 4 h. The protein concentration was measured by the method of Bradford (3), and the radioactivity was counted by Packard Liquid Scintillation Analyzer, TRI-CARB 1600-TR (Packard Instruments, Downers Grove, IL). The uptake values were expressed as nanomoles per milligram per 5 min.
Measurement of intracellular pH.
The intracellular pH (pHi) was measured in Caco-2 cells grown on coverslips using the pH-sensitive florescent dye 2′,7′-bis(2-carboxyethyl)-5(6-carboxyfluorescein) (BCECF-AM; Sigma) as previously described (22, 33). 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 DMEM-KOH base medium, pH 8.5, in the absence or presence of SNAP (500 μM) 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 using a luminescence spectrometer (model LS50, Perkin-Elmer, Beaconsfield, UK). To estimate pHi, the BCECF excitation fluorescence ratios were calibrated using the K+/nigericin methods, as previously described (22). The calibration curve demonstrated that the fluoresence ratios were linear as a function of pHi in the range of 6.0–8.0, as previously reported (22).
Determination of cell viability.
Results are expressed as means ± SE. Each independent set represents means ± SE of data from at least nine wells analyzed on at least three different days. Student's t-test was used for statistical analysis. P < 0.05 was considered statistically significant.
Effects of NO Donor on Cl−/OH−and SO /OH−Exchange Activities
To determine the effects of NO on Cl−/OH− and SO /OH− exchange activities, Caco-2 cells were incubated with 500 μM concentration of SNAP (a conventional donor of NO) for 30 min and DIDS-sensitive Cl−/OH− and SO /OH− exchange activities were assessed as described above. As shown in Fig.1, SNAP (500 μM) resulted in ∼50% inhibition (P < 0.005) of DIDS-sensitive36Cl uptake. However, DIDS-sensitive SO /OH− exchange activity in Caco-2 cells was unaffected by SNAP treatment (1.76 ± 0.09 nmol · mg protein−1 · 5 min−1in SNAP-treated vs. 1.69 ± 0.11 nmol · mg protein−1 · 5 min−1 in control). To ascertain appropriate base loading as well as to rule out possible changes in pH of the cells in response to SNAP treatment, the pHi was determined in Caco2 cells grown on coverslips loaded with the DMEM-KOH base medium, pH 8.5, for 30 min using the pH-sensitive BCECF dye method (22, 33). The results indicated a significant increase in the intracellular pH on base loading the cells (pH 7.79 ± 0.015) compared with the baseline pH (pH 7.15 ± 0.03, n = 3), which appears to be sufficient to establish an outwardly directed OH− gradient at the time of uptake. Moreover, our results also demonstrated that there was no significant change in the baseline pHi of these cells in response to SNAP treatment compared with controls (7.1 ± 0.02 and 6.9 ± 0.04, respectively, n= 3). To exclude the possibility that the effects of SNAP on Cl−/OH− exchange activity may be due to cytotoxic effects of NO, trypan blue exclusion studies were carried out. No alteration in cell viability was observed in Caco-2 cells incubated with SNAP compared with control [10 ± 3% nonviable cells in control vs. 12 ± 1% in SNAP (500 μM)-treated cells]. Similar results were obtained using the acridine orange/ethidium bromide method to measure cell viability [6.5 ± 0.78% nonviable cells in control vs. 6.25 ± 0.88% nonviable cells in response to SNAP (500 μM) treatment].
Role of cGMP in SNAP-Mediated Inhibition of Cl−/OH−Exchange Activity
We next examined the signal-transduction pathways mediating the effects of NO on Cl−/OH− exchange activity in Caco-2 cells. NO stimulates cGMP production in many cell types via activation of soluble guanylate cyclase (39). Experiments were thus performed to measure cGMP levels in response to SNAP treatment of Caco-2 cells. SNAP caused an approximately fourfold increase in the cGMP levels compared with control (data not shown). However, in the presence of the soluble guanylate cyclase inhibitor Ly-83583, the levels of cGMP almost reverted back to the control levels. To investigate further the role of cGMP in regulation of Cl−/OH− exchange, cells were incubated with SNAP (500 μM) alone or in the presence of the inhibitors of soluble guanylate cyclase, ODQ (1 μM) or Ly-83583 (10 μM), and DIDS-sensitive Cl−/OH− exchange activity was assessed. As shown in Fig. 2 A, 500 μM SNAP inhibited the Cl−/OH− exchange activity by ∼50%, but in the presence of ODQ, the inhibitory effect of SNAP was significantly reduced. Similarly, Ly-83583 (10 μM), another specific inhibitor of soluble guanylate cyclase that acts as an NO scavenger by specifically inhibiting soluble guanylate cyclase, also blocked the inhibitory effect of SNAP (Fig. 2 B). These results suggested that production of cGMP through activation of soluble guanylate cyclase mediates NO-induced inhibition of Cl−/OH− exchange activity. To further investigate the role of cGMP, we also studied the effect of the membrane-permeant analog of cGMP, 8-BrcGMP, on apical Cl−/OH− exchange. As shown in Fig.3, preincubation of cells for 30 min with 8-BrcGMP (500 μM) resulted in ∼50% decrease in Cl−/OH− exchange activity. These observations suggest that 8-BrcGMP mimicked the effects of NO in inhibiting Cl−/OH− exchange activity.
Role of PKG
NO stimulates soluble guanylate cyclase resulting in production of cGMP, and increases in cGMP lead to PKG activation (21,52). To assess the role of PKG in the regulation of Cl−/OH− exchange activity, cells were treated with either SNAP (500 μM) alone or in the presence of PKG inhibitor KT-5823. In the presence of KT-5823 (1 μM), the SNAP-mediated inhibition of Cl−/OH− exchange activity was significantly blocked, indicating that PKG activation was involved in SNAP-mediated inhibition of Cl−/OH−exchange activity (Fig. 4).
Effect of Specific Inhibitor of PKA on SNAP-Mediated Inhibition of Cl−/OH−Activity
To exclude the possibility that PKA is involved (via possible cross-activation by cGMP) in the SNAP-mediated inhibition of Cl−/OH− exchange activity, specific PKA inhibitor RpcAMP (25 μM) was used. Results indicated that in the presence of the inhibitor, there was essentially no effect on the SNAP-mediated inhibition of Cl−/OH− exchange activity (Fig. 5).
Role of Possible Cross-Activation of PKC by cGMP
Because previous evidence suggests that NO can activate PKC activity in a number of cell types (30, 41), it was considered of interest to examine the involvement of a PKC-mediated pathway on the inhibitory effect of SNAP on Cl−/OH− exchange activity. Caco-2 cells were preincubated with PKC inhibitors chelerythrine chloride and calphostin C for 60 min before the addition of SNAP and Cl−/OH− exchange activity was assessed. In the presence of chelerythrine chloride (2 μM; Fig.6 A) or calphostin C (200 nM; Fig. 6 B), SNAP-mediated inhibition of Cl−/OH− exchange activity was completely abolished, thereby indicating that the effects of NO were mediated by cGMP/PKG- and PKC-mediated pathways.
The results of our current study demonstrate that the NO donor SNAP, at a concentration of 500 μM, resulted in a significant decrease in Cl−/OH− exchange activity in Caco-2 cells. To determine whether the effects of NO were specific to the Cl−/OH− exchanger, parallel studies were also carried out to examine the effect of SNAP on the recently demonstrated SO /OH− exchange activity (2) in Caco-2 cells. The results showed that the SO /OH− exchange activity remained unaltered by SNAP treatment. Previous studies have suggested that the protein product of downregulated in adenoma (DRA) gene might represent the intestinal apical membrane Cl−/HCO3 − (OH−) exchanger (24, 27). However, the cDNA sequence of DRA has been shown to exhibit high homology with the sulfate transporters but not with any member of the anion exchanger (AE) gene family (42). Additionally, DRA was initially shown to be capable of transporting sulfate and oxalate (42). Moreover, recent studies from our laboratory have demonstrated that in the human colon (48) and Caco-2 cells (2), the Cl−/OH− and SO /OH− exchange processes are mediated via two distinct transporters, thereby indicating that DRA might primarily be an SO /OH− exchanger but capable of transporting chloride as well. Thus our current data of modulation of only Cl−/OH− but not of SO /OH− exchange process by SNAP further supports the notion that the Cl−/OH−and SO /OH− exchange processes may involve two distinct transporters.
Previous studies have shown that NO can cause cell death (apoptosis) in macrophages (1) and cytotoxicity in other cell types (16). Accordingly, we considered the possibility that the SNAP-mediated inhibition of Cl−/OH− exchange activity might be secondary to the cytotoxic effects of NO. However, this mechanism seems unlikely, because our data suggest that the cell viability, as assessed by trypan blue exclusion and acridine orange/ethidium bromide methods, remained unaffected by SNAP treatment. These findings are consistent with the previous studies showing that exposure of Caco-2BBe cells to up to 1.25 mM sodium nitroprusside for 24 h did not reveal any evidence of cell death based on confocal microscopy and lactate dehydrogenase release studies (38). NO is a very labile molecule in biological medium; therefore, the concentration of NO in solution was estimated by determination of nitrite levels, a stable metabolite of NO. In the present study, the NO levels were found to be more likely in the range observed in pathophysiological conditions (20, 45).
Many of the biological actions of NO are mediated through the activation of soluble guanylate cyclase leading to increased intracellular levels of cGMP (39), and the intestine is highly responsive to such stimulation (17). Guanylate cyclase exists in both soluble (cytosolic) and particulate (membrane associated) forms (49). It contains heme with bound iron, which serves as the receptor for NO. Although the intestinal epithelial cells contain 95% of the particulate guanylate cyclase and 5% of soluble guanylate cyclase, previous studies have demonstrated that the small intestine (23) and colon (53) are highly responsive to NO agonists to produce high levels of cGMP. Our current results using inhibitors of soluble guanylate cyclase in blocking the effects of NO on Cl−/OH−exchange activity as well as the role of cGMP in mimicking the effects of NO indicate that production of cGMP, through the activation of soluble guanylate cyclase, mediates NO-induced inhibition of Cl−/OH− exchange activity. Our findings are in agreement with the previous studies showing that cGMP inhibits Cl− absorption in the TALH (29) and Na+/H+ exchange in rabbit proximal tubule (37), avian intestinal cells (40), and vascular smooth muscle cells (7). Recently, we have also shown that the effects of NO on Na+/H+exchanger (NHE)3 activity in Caco-2 cells are mediated by cGMP (12).
Second-messenger cGMP is well known to exert its physiological effects by interacting with various downstream effectors, especially PKG. It was thus considered of interest to delineate the role of PKG in SNAP-mediated inhibition of Cl−/OH− exchange activity in Caco-2 cells using a specific inhibitor of PKG, KT-5823. It has been previously demonstrated that NO stimulates activation of cGMP-dependent PKG in cortical collecting ducts (11) and could directly phosphorylate the Na-K-2 Cl transporter, Na+-K+ -ATPase, apical K+ channels, or basolateral Cl− channels, which, in turn, directly or indirectly decrease Cl− transport. Recently, PKG was shown to regulate net fluid absorption by a dual action: stimulation of electrogenic Cl secretion via cystic fibrosis transmembrane regulator Cl channel and inhibition of electroneutral Na+ absorption in mouse small intestine and colon (50). Our current data are also in agreement with the above findings, because the inhibitory effect of SNAP on Cl−/OH− exchange activity in Caco-2 cells was completely abolished in the presence of PKG inhibitor, suggesting the role of cGMP-dependent PKG pathway in SNAP-mediated inhibition of Cl−/OH− exchange activity in Caco-2 cells.
High concentrations of cGMP have been shown to cross-activate cAMP-dependent PKA in various systems (8, 10, 18, 31). However, in the present study, the inhibitory effect of SNAP on Cl−/OH− exchange activity remained essentially unaffected in the presence of RpcAMPS, a specific PKA inhibitor. Therefore, our data do not support a role for NO-induced cross-activation of PKA resulting in the inhibition of Cl−/OH− exchange activity.
An increase in PKC activity in response to NO has also been shown in a variety of cell types (30, 41). To examine the possible involvement of PKC in the SNAP-induced decrease in Cl−/OH− exchange activity in Caco-2 cells, we treated the cells with specific PKC inhibitors chelerythrine chloride and calphostin C. Our results showed that the inhibition of Cl−/OH− exchange activity was completely abolished by these inhibitors. These observations clearly indicate the involvement of PKC as well in mediating the inhibitory actions of NO. In another set of parallel studies, we recently showed that NO decreases NHE3 activity in Caco-2 cells via stimulation of sGC and activation of PKG but not PKC (12). As in that study, the specific PKC inhibitors did not affect the SNAP-mediated inhibition of NHE3 activity (12). Because in the current studies, blocking either PKG or PKC pathways resulted in complete reversal of SNAP-mediated inhibition, we speculate that PKC may act as a downstream effector of PKG in mediating the inhibitory effect of SNAP on Cl−/OH− exchange activity. In this regard, the involvement of both PKG and PKC pathways has also been demonstrated in cGMP-mediated long-term depression in cerebellar Purkinje cells (14). On the basis of our data, we have proposed a model for the effect of NO on Cl−/OH− exchange activity in Caco-2 cells (Fig. 7).
In conclusion, our studies, for the first time, demonstrate inhibition of the human intestinal apical membrane Cl−/OH− exchange activity by NO (using Caco-2 cells as an experimental model) and indicate that the mechanism of NO-mediated decrease in apical Cl−/OH−exchange activity involves cGMP/PKG- and PKC-mediated pathways. In light of our recent findings demonstrating the inhibition of the NHE3 activity in Caco-2 cells by NO and the data of our current report, we speculate that a decrease in Na and Cl absorption by NO (at levels obtained under inflammatory conditions) may play an important role in diarrhea associated with IBDs.
These studies were supported by the Department of Veterans Affairs and the National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-54016 (to P. K. Dudeja), DK-33349 (to K. Ramaswamy), and DK-09930 (to W. A. Alrefai).
Address for reprint requests and other correspondence: P. K. Dudeja, Univ. of Illinois at Chicago, Medical Research Service (600/151), Chicago Veterans Affairs: West Side Division, 820 South Damen Ave, Chicago, IL 60612 (E-mail:).
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