Section of Digestive and Liver Diseases, Department of Medicine, University of Illinois at Chicago and Chicago Veteran's Affairs System: West Side Division, Chicago, Illinois 60612
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ABSTRACT |
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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-sensitive
36Cl 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
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
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INTRODUCTION |
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NITRIC OXIDE (NO) IS CONSIDERED to be an important biological mediator in a number of cellular functions (26). NO is generated from L-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 examine
1) 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.
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MATERIALS AND METHODS |
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Materials
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).Methods
Cell culture. 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 × 104 cells/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 (NOAssay 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 and
35SO
/OH
and
SO
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 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. Cell viability was assessed by trypan blue exclusion (47), and the acridine orange/ethidium bromide methods as described previously (34).
Statistical analysis. 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.
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RESULTS |
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Effects of NO Donor on
Cl/OH
and
SO
Exchange Activities
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Role of cGMP in SNAP-Mediated Inhibition of
Cl/OH
Exchange 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 (21, 52). To assess the role of PKG in the regulation of Cl
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Effect of Specific Inhibitor of PKA on SNAP-Mediated Inhibition of
Cl/OH
Activity
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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
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DISCUSSION |
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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
exchange activity
(2) in Caco-2 cells. The results showed that the
SO
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
exchange processes are mediated
via two distinct transporters, thereby indicating that DRA might
primarily be an SO
exchanger but
capable of transporting chloride as well. Thus our current data of
modulation of only Cl
/OH
but not of
SO
exchange process by SNAP
further supports the notion that the Cl
/OH
and SO
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
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).
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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.
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ACKNOWLEDGEMENTS |
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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).
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FOOTNOTES |
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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: 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.00395.2001
Received 8 September 2001; accepted in final form 19 April 2002.
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