Nitric oxide regulates energy metabolism and Bcl-2 expression
in intestinal epithelial cells
Manabu
Nishikawa1,2,
Kenta
Takeda1,
Eisuke F.
Sato1,
Tetso
Kuroki2, and
Masayasu
Inoue1
Departments of 1 Biochemistry
and 2 Internal Medicine, Osaka
City University Medical School, Osaka 545, Japan
 |
ABSTRACT |
Nitric oxide (NO) inhibits the respiration of
mitochondria and enteric bacteria, particularly under low
O2 concentration, and induces
apoptosis of various types of cells. To gain insight into
the molecular role of NO in the intestine, we examined its effects on
the respiration, Ca2+ status, and
expression of Bcl-2 in cultured intestinal epithelial cells (IEC-6). NO
reversibly inhibited the respiration of IEC-6 cells, especially under
physiologically low O2
concentration. Although NO elevated cytosolic Ca2+
as determined by the fura 2 method, the cells were
fairly resistant to NO. Kinetic analysis revealed that prolonged
exposure to NO elevated the levels of Bcl-2 and suppressed the
NO-induced changes in Ca2+ status
of the cells. Because Bcl-2 possesses antiapoptotic function, toxic NO
effects might appear minimally in enterocytes enriched with Bcl-2. Thus
NO might effectively exhibit its antibacterial action in anaerobic
intestinal lumen without inducing apoptosis of Bcl-2-enriched mucosal
cells.
mitochondria; respiration; apoptosis
 |
INTRODUCTION |
NITRIC OXIDE (NO) plays important roles in various
biological processes, including relaxation of smooth muscle,
neurotransmission, and host defense mechanisms (5, 28, 30). Activated
macrophages and neutrophils produce substantial amounts of NO that
exhibit cytotoxic activity to mammalian cells and microorganisms (28). Recent studies (6, 11, 17, 32, 40, 43) have revealed that NO reversibly
interacts with cytochrome-c oxidase
and other components in mitochondria and inhibits respiration of normal cells as well as of cancer cells.
Under physiological conditions, substantial amounts of NO occur in the
lumen of stomach and intestine (7, 13, 16, 46). Generation of NO in
intestine is markedly enhanced by bacterial lipopolysaccharide (LPS)
and inflammatory cytokines (12, 44). The lifetime of NO has been
postulated to be extremely short, presumably because of its rapid
reaction with O2 (37). However, NO
is fairly stable under low O2
concentration (42). Because O2
concentration in the intestinal lumen is extremely low, the metabolic
effects of NO might be significantly strong in this compartment. Hence
the effects of NO on intestinal bacteria would be stronger than on
intestinal cells. We previously reported that the respiration and
growth of Escherichia coli and other
enteric bacteria were inhibited by NO, particularly under low
O2 concentration (49). However,
the pathophysiological significance of NO in the metabolism of
intestinal cells remains to be elucidated. The present study
demonstrates the effects of NO on the energy metabolism, Ca2+ homeostasis, and expression
of Bcl-2 in intestinal epithelial cells.
 |
MATERIALS AND METHODS |
Chemicals.
NO and argon gas were obtained from Kinkisanki (Osaka, Japan). Rotenone
and antimycin A were obtained from Nakalai Tesque (Kyoto, Japan) and
Sigma Chemical (St. Louis, MO), respectively. Fura 2-AM and
2,2'-(hydroxynitrosohydrazono)bis-ethanamine (NOC18) were
obtained from Dojindo Chemical (Kumamoto, Japan). All other reagents
used were of analytical grade.
Preparation of NO solution.
NO solution was prepared by bubbling NO gas through 50 mM HEPES-NaOH
buffer, pH 7.4, as described previously (10). Briefly, two small tubes
were fitted with an air-tight septum with glass tubes inserted for
delivery and escape of gases, with a first tube containing 5 M KOH and
a second tube containing the HEPES-NaOH buffer. Argon was delivered
into two tubes at a flow rate of 100 ml/min. After 15 min, argon was
replaced with NO at a flow rate of 100 ml/min. After another 15 min,
the saturated NO solution (1.9 mM) was kept on ice and used for
experiments within 3 h; the concentration of NO in the stock solution
remained unchanged during the experiments. NO concentration was
determined by using electron spin resonance (ESR) and the NO trapping
agent 2-(4-carboxyphenyl)-4,4,5,5-tetramethyl-imidazoline-1-oxyl 3-oxide (1, 3).
Cell culture.
Well-characterized rat intestinal epithelial crypt cells (IEC-6) were
obtained from Riken Cell Bank (Tsukuba, Japan). IEC-6 cells were grown
to confluence in DMEM (GIBCO, Gaithersburg, MD) containing 5% FCS at
37°C and 5% CO2 and 20% or
5% O2.
Analysis of cell respiration.
O2 consumption by IEC-6 cells was
determined polarographically using a Clark-type
O2 electrode fitted to a 2-ml
water-jacketed closed chamber at 37°C (34). Cellular respiration
was analyzed in Krebs-Ringer phosphate buffer (KRP; pH 7.4) consisting
of 50 mM HEPES, 100 mM NaCl, 5 mM KCl, and 1 mM each of
MgCl2,
NaH2PO4, and CaCl2. The reaction was
started by adding 107 cells to the
assay mixture. During the experiments, aliquots of NO-saturated
solution were added to the reaction mixture.
Analysis of DNA fragmentation.
Cells (2 × 106) were
incubated for 10 min in 100 µl ice-cold lysis buffer (10 mM
Tris · HCl, pH 7.4, 10 mM EDTA, 0.5% Triton X-100)
at 4°C. After incubation for 20 min, the mixture was centrifuged at
10,000 g for 20 min. The supernatant
fraction was incubated with 40 µg RNase A at 37°C for 1 h, then
with 40 µg proteinase K for 1 h. DNA was precipitated by incubating
with 1 volume of isopropanol and 0.2 volume of 5 M NaCl at
20°C for 12 h and centrifuged at 10,000 g for 20 min. Pellets were air dried
and dissolved in 10 µl of buffer composed of 10 mM
Tris · HCl, pH 8.0, and 1 mM EDTA. The DNA samples
thus obtained were subjected to 1.7% agarose gel electrophoresis at
100 V using 40 mM Tris-acetate, pH 8.0, and 1 mM EDTA as a running
buffer. The gel was stained with 0.1 µg/ml of ethidium bromide and
visualized under ultraviolet light.
Western blot analysis of Bcl-2.
Proteins (10 µg) in IEC-6 cell lysates were separated in 15%
SDS-PAGE and blotted onto nitrocellulose sheets using a Pharmacia semidry blot system (2 mA/cm2 for
1 h in 192 mM Tris-glycine buffer). The sheets were incubated in TBS
solution (140 mM NaCl, 50 mM Tris · HCl, pH 7.2)
containing 0.1% Tween 20 and 5% low-fat milk powder for 12 h at
4°C. Then the sheets were incubated with rabbit anti-rat Bcl-2
antibody (1:1,000 in TBS solution with 0.5% low-fat milk powder) for
12 h at 4°C. The incubated sheets were washed five times to
eliminate nonspecific binding of the antibody. After
incubation with horseradish peroxidase-conjugated goat anti-rabbit
antibody (1:1,000 in TBS with 0.5% low-fat milk powder) at 25°C
for 1 h, immunoreactive spots were analyzed by enhanced
chemiluminescence (Amersham, Buckinghamshire, UK).
Analysis of mRNA.
Total RNA was extracted from IEC-6 cells by the acid
guanidium-phenol-chloroform method, as described previously (9). Levels of mRNA for
2-microglobulin and
bcl-2 were analyzed by RT-PCR method
(15). RT was carried out at 42°C for 15 min. PCR was carried out by 1 min denaturation at 95°C, 1 min hybridization at
55°C, and 1.5 min extension at 72°C. The primers used were 5'-TCAGATCTGTCCTTCAGCAA-3' and
5'-CATGTCTCGGTCCCAGGTGA-3' for
2-microglobulin and
5'-TAACCGGGAGATCGTG-3' and
5'-ACATCTCTGCAAAGTCGCGA-3' for
bcl-2. PCR for
2-microglobulin and
bcl-2 mRNA was performed for 35 cycles. PCR products were subjected to 1.7% agarose gel electrophoresis. The gel was stained with 0.1 µg/ml of ethidium bromide and visualized under ultraviolet light.
Measurement of intracellular
Ca2+ level.
IEC-6 cells (108 cells/ml) were
incubated in KRP containing 1 µM of fura 2-AM at 37°C for 15 min.
After washing twice with KRP, cells (5 × 104/ml) were incubated in the same
buffer at an O2 concentration of
25 µM. Fura 2 fluorescence was measured in a Hitachi F-2000 fluorescence spectrophotometer with excitation and emission wavelengths of 340/380 and 510 nm, respectively. Cytosolic levels of
Ca2+ were determined from the
ratio of fura 2 fluorescence intensities at excitation of 340 and 380 nm (18). The data were processed with a computer fitted to a Hitachi
F-2000 fluorescence spectrophotometer, and estimated intracellular
Ca2+ concentration was recorded
continuously.
 |
RESULTS |
Effect of NO on respiration of IEC-6 cells.
IEC-6 cells revealed a marked O2
consumption without adding respiratory substrates (Fig.
1). Because
O2 consumption by IEC-6 cells was
enhanced by dinitrophenol and completely inhibited by potassium cyanide
(data not shown), it fully reflected the respiration of their
mitochondria. The respiration was transiently inhibited by a fairly low
concentration of NO and recovered after certain periods of incubation.
The inhibitory effect of NO increased with a concomitant decrease in
O2 concentration.

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Fig. 1.
Effect of nitric oxide (NO) on O2
consumption. Intestinal epithelial cells (IEC-6 cells) (5 × 106 cells/ml) were incubated in a
closed chamber containing 2 ml of Krebs-Ringer phosphate buffer at
37°C. At the indicated times (arrows), NO was added to give a final
concentration of 2 µM. During incubation,
O2 concentration in the medium was
monitored as described in text. Experiments were repeated 5 times with
similar results.
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DNA fragmentation.
Some compounds that inhibit mitochondrial electron transfer have been
shown to induce apoptosis of cells (47). Fragmentation of
internucleosomal DNA has been used as a marker of apoptosis of various
cell types. To test the possible occurrence of apoptosis of NO-treated
IEC-6 cells, their DNA samples were isolated and analyzed by agarose
gel electrophoresis. When cells were cultured for 24 h under 20%
O2 in the presence of ~100 µM
NOC18, no appreciable fragmentation of DNA was found to occur (Fig.
2). When
O2 concentration was decreased to
5%, NOC18 slightly enhanced the fragmentation of cellular DNA. The
presence of extremely high concentrations of NO (1 mM) strongly induced
DNA fragmentation; the fragmentation was more apparent at an
O2 concentration of 5% than at
20%. Under identical conditions, both rotenone (1 µM) and antimycin
A (0.1 µM), specific inhibitors of mitochondrial electron transport, failed to induce DNA fragmentation and to affect the viability of IEC-6
cells (data not shown). The concentrations of the inhibitors used in
the experiments effectively inhibited the respiration of IEC-6 cells.
Thus, unlike with other types of cells, inhibition of cellular
respiration did not induce apoptosis of IEC-6 cells.

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Fig. 2.
Effect of NO on DNA fragmentation. IEC-6 cells were cultured without
(lanes 1 and
5) or with NOC18 (10 µM,
lanes 2 and
6; 100 µM,
lanes 3 and
7; 1 mM,
lanes 4 and
8) under
O2 concentration of 20%
(lanes 1-4)
or 5% (lanes 5-8)
for 24 h. C, control (cells before treatment). DNA was extracted and
subjected to agarose gel electrophoresis as described in text.
Experiments were repeated 3 times with similar results.
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Effect of NO on Bcl-2 expression.
Bcl-2 exhibits antiapoptotic activity by stabilizing membrane potential
of mitochondria (39). To elucidate the mechanism by which IEC-6 cells
showed strong resistance to NO, the effect of NOC18 on the expression
of bcl-2 in IEC-6 cells was studied. At 20% O2, NOC18 enhanced the
expression of bcl-2 gene only slightly (Fig. 3). However, the expression of
bcl-2 gene was strongly enhanced under
low O2 concentration (5%),
particularly at 24 h of incubation. NO did not affect the expression of
Bcl-2 protein under 20% O2 but
increased it under 5% O2 (Fig.
4). After 24 h of incubation under 5%
O2, Bcl-2 levels increased by
about fivefold. Under the standard culture conditions (20%
O2-5%
CO2), the enhancing effect of
NOC18 also depended on its dose; 100 µM of NOC18 increased Bcl-2
levels by ~50% (Fig. 5). However, doses
of NOC18 >1 mM strongly inhibited the expression of Bcl-2,
especially under low O2
concentration.

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Fig. 3.
Regulation of bcl-2 gene expression by
NO. IEC-6 cells were cultured in the presence of 100 µM NOC18 under
O2 concentration of 20% or 5%
for 4 and 24 h. mRNAs for bcl-2 and
2-microglobulin
( 2-MG) were analyzed with these cells by RT-PCR. PCR for
bcl-2 and
2-microglobulin was performed
for 35 cycles each. Experiments were repeated 3 times with similar
results.
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Fig. 4.
Regulation of Bcl-2 protein expression by NO. IEC-6 cells were cultured
in the presence of 100 µM NOC18 under
O2 concentration of 20% or 5%
for 4, 8, 12, and 24 h. Western blot of 10 µg protein of cell lysate
developed with specific antibody demonstrates Bcl-2 protein.
Experiments were repeated 3 times with similar results.
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Fig. 5.
Dose-dependent effect of NO on Bcl-2 protein expression. IEC-6 cells
were cultured without (lanes 1 and
5) or with NOC18 (10 µM,
lanes 2 and
6; 100 µM,
lanes 3 and
7; 1 mM,
lanes 4 and
8) under
O2 concentration of 20%
(lanes 1-4)
or 5% (lanes 5-8)
for 24 h. Western blot of 10 µg protein of cell lysate developed with
specific antibody demonstrates Bcl-2 protein. Experiments were repeated
3 times with similar results.
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Effect of specific inhibitors of mitochondrial electron transport on
Bcl-2 expression.
Western blot analysis revealed that both rotenone and antimycin A
markedly enhanced cellular levels of Bcl-2 in IEC-6 cells (Fig.
6).

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Fig. 6.
Effect of mitochondrial electron transport inhibitors on Bcl-2 protein
expression. IEC-6 cells were cultured in the presence of 1 µM
rotenone or 0.1 µM antimycin A for 24 h. Western blot of 10 µg
protein of cell lysate developed with monoclonal antibody demonstrates
Bcl-2 protein. Control, cells before treatment. Experiments were
repeated 3 times with similar results.
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Effect of NO on cellular
Ca2+ status.
Mitochondria have been known to regulate cellular
Ca2+ homeostasis by an
ATP-dependent mechanism (19). Because NO inhibited the mitochondrial
respiration of IEC-6 cells, its effect on the cytosolic levels of
Ca2+ was determined. Cytosolic
levels of Ca2+ were markedly
elevated by NO in a concentration-dependent manner (Fig.
7). The elevating effect of NO was stronger
in control cells expressing low levels of Bcl-2 than in
NOC18-pretreated cells with enhanced expression of this protein.

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Fig. 7.
Effect of NO on cytosolic Ca2+
levels. Changes in intracellular
Ca2+ concentration
([Ca2+]i)
in IEC-6 cells were determined with control
(A) or Bcl-2-overexpressed cells,
which were pretreated with 100 µM NOC18 under 5%
O2 for 24 h
(B), by the fura 2-AM method at an
O2 concentration of 25 µM. At
the indicated times (arrows), NO solution was added to reaction mixture
to make final concentrations of 10 (2), 20 (3) and 50 µM
(4).
1, Experiments performed in the
absence of NO. Experiments were repeated 5 times with similar
results.
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 |
DISCUSSION |
The present study demonstrates that NO reversibly inhibits the
respiration of intestinal epithelial cells and enhances the expression of Bcl-2 in dose- and
O2 concentration-dependent
manners. Because NO reacts with molecular
O2
(k = 6 × 106
M
2s
1),
it is fairly stable under low O2
concentration. NO reversibly binds to cytochrome
c-oxidase and increases its apparent
Michaelis constant value for O2
(6). Hence the effects of NO on the metabolism of
enterocytes facing the anaerobic lumen might be stronger than those
expected from in vitro experiments performed under air
atmospheric conditions.
NO exhibits cytotoxic effects on target cells and induces DNA
fragmentation (14, 23-25, 41). Fairly high concentrations of NO
(~100 µM NOC18) failed to induce DNA fragmentation and apoptosis of
IEC-6 cells even under low O2
concentration. At an NOC18 concentration of 100 µM, ~0.6 µM/min
of NO would be released under the present experimental conditions by an
O2-independent mechanism. A
protooncogene product, Bcl-2 localizes in biological membranes, such as
endoplasmic reticulum, plasma membranes, mitochondrial inner membranes,
and nuclear membranes, and prevents various cells from apoptotic death (8, 20). Although protective effects of Bcl-2 against hazardous stimuli
leading to apoptosis have been studied extensively (2, 24-27, 45),
the molecular mechanism of its regulation remains to be elucidated.
Based on its preferential association with mitochondrial membranes, the
antiapoptotic effect of Bcl-2 has been postulated to be enclosed in
mitochondrial functions (29, 39, 50). The present study finds that NO
inhibits mitochondrial respiration and enhances expression of Bcl-2
gene and protein, which prevents the fragmentation of cellular DNA.
Because both rotenone (1 µM) and antimycin A (0.1 µM) markedly
increased cellular levels of Bcl-2 by some
O2-independent mechanism, the
expression of Bcl-2 might be regulated principally by mitochondrial
electron flow.
Mitochondria play a critical role in the regulation of
Ca2+ homeostasis (36, 38). NO
deenergized mitochondria and elevated cytosolic levels of
Ca2+ in IEC-6 cells, particularly
under physiologically low O2
concentration. Sustained increase in cellular
Ca2+ levels by a variety of agents
often enhances the process of apoptotic cell death (31).
Ca2+ has been found to activate
endonuclease in nuclei (21) and to stimulate apoptosis of thymocytes
(48). Thus elevation of cytosolic
Ca2+ by NO might induce DNA
fragmentation in intestinal epithelial cells, especially under
physiologically low O2
concentration. It should be noted that NO enhanced the expression of
Bcl-2 and suppressed the NO-induced changes in
Ca2+ status. Bcl-2 enhances
mitochondrial Ca2+ uptake and
exhibits antiapoptotic activity by stabilizing membrane potential of
mitochondria (22, 29). Thus Bcl-2 might stabilize membrane potential of
mitochondria and Ca2+ status in
IEC-6 cells and prevent DNA fragmentation. When mitochondrial respiration was inhibited by various inhibitors, including NO, expression of Bcl-2 was enhanced in IEC-6 cells. Therefore Bcl-2 and
mitochondrial energy metabolism are closely linked. The molecular mechanism by which Bcl-2 affects the energy metabolism in intestinal epithelial cells should be studied further.
Activated neutrophils and macrophages synthesize substantial amounts of
NO by inducible NO synthase (iNOS). Increased production of NO might be
important for host defense mechanisms. When stimulated by LPS,
intestinal epithelial cells also express iNOS and produce NO (12, 44).
In fact, patients with inflammatory bowel diseases, such as ulcerative
colitis and Crohn's disease, have high iNOS activity in the large
intestine and high nitrite levels in their plasma (4, 35). Because
vascular and mucosal permeability increase in inflammation of the
intestine, inhibition of bacterial translocation across intestinal
walls is critically important. We previously reported (49) that the
respiration and growth of E. coli and
other enteric bacteria were inhibited by NO, particularly under low
O2 concentration. However, when
excess amounts of NO were produced locally, NO and/or its
reactive metabolites became toxic to both enteric bacteria and host
cells (33). Thus, to get selective toxicity to bacteria by
NO, intestinal mucosal cells should be more resistant to this gaseous
radical than enteric bacteria. In this context, enhanced expression of
Bcl-2 in intestinal mucosal cells by NO might be essential in selective
inhibition of bacterial growth without injuring intestinal
cells.
 |
ACKNOWLEDGEMENTS |
This work was supported by grants from the Ministry for Education,
Science, and Culture of Japan and from Osaka City University Research
Foundation.
 |
FOOTNOTES |
Address for reprint requests: M. Nishikawa, Dept. of Biochemistry,
Osaka City Univ. Medical School, 1-4-54 Asahimachi, Abeno-ku, Osaka
545, Japan.
Received 3 November 1997; accepted in final form 12 January 1998.
 |
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