Originally published In Press as doi:10.1074/jbc.M904498199 on April 28, 2000
J. Biol. Chem., Vol. 275, Issue 28, 21241-21246, July 14, 2000
Inhibitory Effects of Nitric Oxide and Nitrosative Stress on
Dopamine-
-Hydroxylase*
Xiaoling
Zhou
,
Michael G.
Espey§,
James X.
Chen,
Lorne J.
Hofseth,
Katrina M.
Miranda§,
S. Perwez
Hussain,
David A.
Wink§, and
Curtis C.
Harris¶
From the Laboratory of Human Carcinogenesis,
§ Radiation Biology Branch, NCI, National Institutes of
Health, Bethesda, Maryland 20892
Received for publication, June 8, 1999, and in revised form, April 25, 2000
 |
ABSTRACT |
Dopamine-
-hydroxylase (DH) is a
copper-containing enzyme that uses molecular oxygen and ascorbate to
catalyze the addition of a hydroxyl group on the -carbon of dopamine
to form norepinephrine. While norepinephrine causes vasoconstriction
following reflex sympathetic stimulation, nitric oxide (NO) formation
results in vasodilatation via a guanylyl cyclase-dependent
mechanism. In this report, we investigated the relationship between NO
and DH enzymatic activity. In the initial in vitro
experiments, the activity of purified DH was inhibited by the NO
donor, diethylamine/NO (DEA/NO), with an IC50 of 1 mM. The inclusion of either azide or GSH partially restored
DH activity, suggesting the involvement of the reactive nitrogen
oxide species, N2O3. Treatment of human neuroblastoma cells (SK-N-MC) with diethylamine/NO decreased cellular DH activity without affecting their growth rate and was augmented by
the depletion of intracellular GSH. Co-culture of the SK-N-MC cells
with interferon-
and lipopolysaccharide-activated macrophages, which
release NO, also reduced the DH activity in the neuroblastoma cells.
Our results are consistent with the hypothesis that nitrosative stress,
mediated by N2O3, can result in the inhibition
of norepinephrine biosynthesis and may contribute to the regulation of
neurotransmission and vasodilatation.
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INTRODUCTION |
Norepinephrine and epinephrine are critical determinants of
transient neuronal regulation of local vascular tone and arterial pressure. DH,1 the third
enzyme in the biosynthetic pathway of norepinephrine, can hydroxylate
the -carbon of a variety of phenylethylamine derivatives. DH is a
tetrameric copper-containing oxidoreductase with conserved homology
among mammalian species (1) that is localized in both noradrenergic and
adrenergic neurons of the central nervous system, sympathetic ganglia,
and adrenal medulla (1, 2). The physiological regulation of DH
activity includes axonal transport rate, transcriptional factors such
as nerve growth factor, and cyclic AMP (3, 4). Germ line mutations in
DH result in systemic hypotension mediated by increased dopamine levels in cerebrospinal fluid and blood (5, 6). Nitric oxide (nitrogen
monoxide, NO) is a prominent cellular messenger involved in several
important biological processes including the regulation of
neurotransmission and cardiovascular function (7). The physiological effects of NO occur with relatively low concentrations and primarily involve direct reactions with metal-containing proteins and radicals (7). Nitric oxide fluxes, generated from nitric-oxide synthase (NOS)
isoenzymes, are present in endothelia, and selected neurons mediate
these direct effects (8). In contrast, indirect effects of NO are
likely to occur during pathological states. Indirect effects require
relatively higher levels of NO and involve the formation of reactive
nitrogen oxide species (RNOS) prior to target modification. In general,
this chemistry is a consequence of the NO fluxes generated by the
isoenzyme, inducible nitric-oxide synthase (iNOS) (7). Nitric oxide
regulates the release and uptake of dopamine and the activity of a
number of catecholamines in the striatum and other dopaminergic cells
(9-13). L-N-monomethylarginine, a competitive
inhibitor of NOS, enhances nerve-induced contraction in the guinea pig
ileum and pulmonary artery and in the dog mesenteric artery segments
(14, 15). In anococcygeus muscle preparations, the contractile response
to noradrenergic stimulation is enhanced in the presence of
L-N-monomethylarginine as well (16, 17). These
studies show that an interrelationship exists between catecholamines and NO in the neuronal-endothelial control of blood flow. In this study, we sought to determine if there was an effect of NO on DH and
under what conditions these might exist in vivo.
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EXPERIMENTAL PROCEDURES |
NO Inhibition of DH Activity in Vitro--
A modified
spectrophotometric assay (18) was used to detect DH activity
in vitro. This assay was based on the conversion of
substrate tyramine to octopamine, and the octopamine was converted to
p-hydroxybenzaldehyde by sodium periodate treatment. The
p-hydroxybenzaldehyde is measured spectrophotometrically.
Bovine adrenal DH (40 units/liter; Sigma) in PBS was mixed with
DEA/NO (a generous gift from Dr. Joseph Saavedra, SCAI, Frederick, MD)
and incubated at 37 °C for 30 min. Then the DH with or without
exposure to DEA/NO was added to a substrate mixture (600 µl). The
substrate mixture contained N-ethylmaleimide (40 mM), sodium acetate buffer (200 mM, pH 5.0), tyramine (20 mM), sodium fumarate (10 mM),
pargyline (1 mM), catalase (0.1 mg), fresh ascorbic acid
(10 mM), and PBS (400 µl). The reaction mixtures were
incubated at 37 °C for 30 min with shaking. The reaction was
terminated by adding 0.2 ml of 3 M cold trichloroacetic acid. The mixture was immediately centrifuged at 2500 rpm for 10 min
(MX/TX-160, TOMY Technology), and the supernatant was transferred to a
microbio-spin column (400-µl bed volume) of GA-50W-X4 (H+, 200-400
mesh; Bio-Rad). After washing with 2 ml of distilled water, the
absorbed reaction product octopamine was eluted with 1 ml of 4 M ammonia. Octopamine in the eluate was converted to
p-hydroxybenzaldehyde by adding 0.2 ml of 2% sodium
periodate, and the excess sodium periodate was reduced by adding 0.2 ml
of 10% sodium metabisulfite. The absorbance was measured by a dual
wavelength spectrophotometer at 330 nm. The blank value was obtained by
substituting water for the enzyme. One hundred mM DEA/NO in
NaOH and NaNO2 in water were used as stock to detect the NO
inhibition effect. Preventing the inhibition of DH activity by
DEA/NO was preformed by directly adding GSH (Sigma) and sodium azide
(Sigma) into the reaction before incubation. To eliminate the
possibility that DEA/NO might interact with octopamine directly, the
absorbance was measured after the co-incubation of 5 mM
octopamine (Sigma) with 5 mM DEA/NO.
Determination of DH Activity and NO Inhibition of DH
Activity in a Neuroblastoma Cell Line--
The neuroblastoma cell line
SK-N-MC (ATCC, Rockville, MD) was grown in Eagle's minimum essential
medium supplemented with 10% fetal bovine serum at 37 °C in a
humidified atmosphere containing 5% CO2. When the culture
was 85-90% confluent, cells were harvested, suspended at a density of
107 cells/ml in PBS, and stored at 
70 °C. To
investigate the dose response and time course of inhibition of DH
activity, various amounts of DEA/NO were added to the cell cultures
with different incubation times according to the experimental design.
The cells were trypsinized, harvested, counted, and lysed by freezing
and thawing three times and then centrifuged for 30 min at 14,000 rpm
at 4 °C. The supernatant was transferred to a new tube, and the
DH activity was measured as described above. To eliminate the
possibility that the reduction of DH activity was caused by a
decreased number of viable cells, the cell growth rate was examined by
a nonradioactive cell proliferation assay (MTS assay; Promega). The
SK-N-MC cells, with and without DEA/NO treatment, were washed with the
culture medium and subcultured into a 96-well plate. The number of
viable cells was measured by the MTS assay in the first, second, third,
and fourth day after 24 h of subculture.
Irreversible Inhibition of DH Activity by DEA/NO in Vitro and
in Vivo--
Purified bovine adrenal DH (40 units/liter) was
exposed to 5 mM fresh and decomposed DEA/NO in PBS for
2 h at room temperature before the enzyme reaction. The decomposed
DEA/NO was made by incubating DEA/NO at 37 °C for 1 h in PBS
with shaking. The enzyme activities were measured at 15-min intervals
throughout the total 2-h incubation time. The activity of the purified
DH without DEA/NO was measured as a positive control. The cultured
neuroblastoma cells (SK-N-MC) were incubated with 0 and 5 mM DEA/NO for 1 h. The cells were washed with PBS four
times and continually cultured with fresh medium. The cells were
collected for DH activity measurement at 0, 1, 2, 3, 4, and 5 h
after DEA/NO exposure.
Co-culture of the Neuroblastoma Cells with
Microglia--
Immortalized C3 murine microglial cells (kindly
provided by Dr. C. A. Colton, Georgetown University, Washington,
D. C.) were seeded (2 × 106 cells) into
100-mm2 Petri dishes and were stimulated for 12 h with
both IFN- (20 ng/ml) and LPS (20 ng/ml). The nitrosative capacity of
C3 cells under these conditions was monitored by the release of nitrite and the ability to form triazole from the target compound,
2,3-diaminonaphthalene, present in the medium. Nitric oxide was
measured using fluorescence spectroscopy (19). SK-N-MC cells were
trypsinized, washed with the culture medium, and seeded into the C3
cultures at an effector-to-target ratio of 1:3. The iNOS inhibitor
L-N-methylarginine (NMA) was added into the
co-culture either at the beginning of co-culture or 4 h before
harvest to investigate whether the inhibition can be prevented or
restored. Following 12 h of co-incubation, the cells were
suspended, washed, and lysed as described previously. The activity of
DH was detected with a dual wavelength spectrophotometer. To rule
out the possibility that the overnight co-culture may affect the
SK-N-MC cells' growth and viability, the number of viable SK-N-MC
cells after co-culture was counted by bright line hemocytometer (Sigma)
with trypan blue staining.
Effects of GSH on NO-induced DH Inhibition--
The
concentration of GSH was determined in both DEA/NO-treated (5 mM, 1 h) and nontreated SK-N-MC cells. Intracellular
GSH was determined as described previously (20). The cells were washed
with PBS four times, harvested, and then suspended in 2 ml of 0.69%
sulfosalicylic acid and stored at 70 °C. Spectra were performed on
a UV-visible spectrophotometer, model U-3000/U-3300 (Hitachi). To
investigate whether a selective inhibitor of GSH synthesis, buthionine
suloximine (BSO; Sigma), affected DH inhibition by NO, SK-N-MC cells
were treated with 5 mM fresh BSO for 12 h before
DEA/NO was added into the culture. After DEA/NO treatment, the cells
were washed with PBS, harvested, and lysed as described above.
In each experiment, experimental and control cells were matched for
seeding density, number of passages, and the percentage of confluence.
The experiments were repeated at least three times. Student's paired
t tests were used to determine the significance of
differences between the means of experiments and controls.
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RESULTS |
To verify the reliability of the spectrophotometric assay for the
detection of DH activity, purified bovine adrenal DH activity was
measured. In this assay, the reaction containing both enzyme and
substrate was incubated, and the absorbance was measured. The enzyme
activities (absorbance) showed a linear relation with the amount of
DH added from a range of 0-80 units/liter (Fig. 1A). To determine the effect
of NO on DH activity, the protein was exposed to the reaction buffer
with different amounts of the NO donor DEA/NO. Exposure of DH to
increasing concentrations of DEA/NO in vitro resulted in a
marked decrease in the conversion of tyramine to octopamine (Fig.
1B). The absolute absorbance of octopamine remained the same
with and without DEA/NO co-incubation (data not shown). The
concentration of DEA/NO required to reduce DH activity by 50% was 1 mM.
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Fig. 1.
DH activity and
inhibition of DH activity by DEA/NO and
NaNO2 in vitro. A, the indicated
amounts (units/liter) of purified bovine adrenal DH were added into
the spectrophotometric assay reaction. The activities of DH
(absorbance) were measured by a dual wavelength spectrophotometer at
330 nm. B, the purified enzymes were mixed with different
amounts (mM) of DEA/NO ( ) and NaNO2 ( )
and were incubated for 30 min at 37 °C. The y
axis numbers represent relative activities. The
results are representative of three repeated experiments. S.D. is
indicated.
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Nitrite is a by-product of the decomposition of DEA/NO, which can
produce both NO and RNOS under acidic conditions. Because the assay for
DH was performed under acidic conditions (21), we determined the
effect of nitrite on DH. The effect of increasing nitrite on DH
caused a decrease in DH activity (Fig. 1B). This result
suggests that NO/RNOS, produced in the presence of nitrite under acidic
conditions, could be responsible for this inhibition (Fig.
1B). Despite the effect of nitrite, complete inhibition required 50 mM nitrite, a dose 10 times higher than DEA/NO
to inhibit the enzyme activity to the same extent. These results suggest that the vast majority of the inhibition by DEA/NO resulted from the liberation of nitric oxide and not nitrite; thus, increasing nitric oxide concentrations inhibit DH.
The inhibition of DH activity by DEA/NO could be mediated by either
NO or an RNOS, such as N2O3. A significant
amount of N2O3 is formed at 5 mM
concentrations of DEA/NO, which has been shown to inhibit other enzymes
(22). To determine the nature of the chemical intermediate responsible
for the decomposition, the same experiments were performed in the
presence of azide, an efficient scavenger of
N2O3 (21, 23). Exposure of DEA/NO to DH in
the presence of sodium azide showed abrogation of the inhibitory effect
of DEA/NO (Fig. 2A). The same
concentration of azide had no effect on the activity of the enzyme.
Furthermore, when GSH was substituted with the azide as a scavenger of
N2O3, abrogation of DEA/NO-mediated inhibition
also was observed (Fig. 2B). It should be noted that 10 mM GSH inhibited the assay, which is not surprising,
because previous reports have shown that thiol-containing substances
such as cystamine inhibited DH (24). However, the pretreatment of
the isolated protein in the presence of GSH with excess DEA/NO results
in nearly a 100% conversion to S-nitrosoglutathione (22).
These results suggest that the S-nitrosothiol,
S-nitrosoglutathione, does not inhibit DH. Hence,
scavenging of N2O3 by GSH resulted in the
protection of the enzyme. Because NO does not directly react with
either azide or GSH, these data clearly indicate that N2O3, not NO, was responsible for the
inhibition of DH activity in this in vitro experimental
model.
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Fig. 2.
Attenuation of DEA/NO inhibition of
DH activity by sodium azide and GSH. Two
and 5 mM of either sodium azide (A) or GSH
(B) was added into the enzyme reaction before incubation.
The activities of DH are relative. The results are expressed as
mean ± S.E., n = 3. *, p < 0.05;
**, p < 0.01 versus control. S.D. is
indicated.
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As shown above, the purified enzyme can readily be inhibited by
N2O3. To extend these findings, we selected a
human neuroblastoma cell line as a model to detect and measure the
possible alteration of DH activity by NO. Human neuroblastoma
(SK-N-MC) cells have been shown to contain endogenous DH (25).
Although we did not detect any DH activity in the cell culture
medium (data not shown), we were able to measure the activity of DH
in the SK-N-MC cell lysate. The activity of DH was positively
correlated with the number of cells used for lysis (Fig.
3A). The different negative controls including boiled cell lysate, buffer only, 100 µM specific DH inhibitor, fusaric acid (Sigma) (26),
and zero incubation time showed either very low or no DH activity
(Fig. 3B). The DH activity in the cell lysates was
partially decreased when DEA/NO was added to the culture before
cellular lysing of the cells (Fig. 3C). Unlike the results
with the isolated protein (Fig. 1B), 5 mM DEA/NO
reduced cellular DH activity to a lesser extent, i.e.
50%. An increase of DEA/NO concentrations had no further effect on the
reduction of DH activity. A major difference between the
experiments, exposing DEA/NO directly to the isolated protein or to the
DH-containing cell lysate, is the presence of cellular defenses
against NO/RNOS such as GSH. It has been shown that GSH protects cells
against N2O3 toxicity (22); therefore, GSH may
be expected to attenuate the inhibitory effect of DEA/NO. SK-N-MC cells
contain 20 µg of GSH/mg of protein, which is considerably more than
most other mammalian cells. Exposure of 10 mM DEA/NO only
reduced GSH levels modestly (data not shown). To determine if GSH
affected the DEA/NO-mediated inhibition of DH, cells were pretreated
with BSO to deplete intracellular GSH. In controlled experiments, the
BSO treatment had no effect on the DH activity of the cells.
However, BSO increased the DEA/NO-mediated inhibition from 50 to 90%
(Fig. 3D). These results indicate that the presence of
intracellular GSH plays a role in partially protecting DH from
NO-mediated inhibition. The cells treated with DEA/NO did not show any
significant alteration of cell growth rate in comparison with the
non-DEA/NO-treated control. The doubling times of the cells, with and
without DEA/NO, were 64 and 60 h, respectively.
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Fig. 3.
The cellular DH
activity was affected by DEA/NO. A, DH activity in
the human neuroblastoma cell line SK-N-MC. Indicated amounts of human
neuroblastoma cell lysates (in µl; 10,000 cells/µl) were used and
were incubated with the reaction buffer for 30 min at 37 °C. The
activity of DH is an absolute value. B, negative controls
of DH activity in SK-N-MC cell lines. A boiled cell lysate, the cell
lysate with specific DH inhibitor fusaric acid (100 µM), the cell lysate with 0 min of incubation, and lysate
buffer only were used to detect the DH activity. C,
inhibition of DH activity by DEA/NO in vivo. Various
amounts of the NO donor, DEA/NO, were added into SK-N-MC cell cultures
for 1 h before collecting the cells. The cells were lysed and
incubated with the reaction buffer. The DH activities were measured
and were calculated as a relative value. D, GSH depletion
enhanced the inhibition by DEA/NO in vivo. A GSH depletion
agent, BSO (5 mM), was applied to the cell culture for
12 h before treating the cell with DEA/NO. The cells treated with
5 mM DEA/NO only for 1 h or 5 mM both BSO
and DEA/NO (BSO + DEA/NO) were harvested and lysed. The
cells without any treatment (Control) or treated with BSO
alone (BSO only) were used as controls. All of the
experiments were repeated at least three times, **, p < 0.01. S.D. is indicated.
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We investigated whether the inhibition of DH activity by NO is
reversible or irreversible in our experimental models. The activity of
the purified DH was inhibited completely by 5 mM of the
fresh DEA/NO, and even when the incubation time was prolonged to 2 h, there was no recovery of the enzyme activity (Fig.
4A). Because the half-life of
DEA/NO is about 4 min at 37 °C (27), DEA/NO cannot generate any NO
in this system when the preincubation time is extended to 2 h at
room temperature. The composite spectra for the decomposition of DEA/NO
in our experimental conditions show that the concentration of DEA/NO is
almost down to zero after a 2-h period (data not shown). The activity
of the DH treated with decomposed DEA/NO showed a slight reduction
because of the DEA/NO by-product nitrite (Fig. 4A), which is
consistent with the data showing DH activity after exposure to
NaNO2 only (Fig. 1B). After removing 5 mM DEA/NO exposed for 1 h, no recovery of DH
activity was detected in the continuing culture of SK-N-MC cells, even
up to 5 h (Fig. 4B).
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Fig. 4.
Irreversible inhibition of
DH activity by DEA/NO in vitro
and in vivo. A, purified bovine
adrenal DH (40 units/liter) were exposed to 5 mM of
fresh DEA/NO ( ), decomposed DEA/NO ( ), or solvent ( ) for
2 h at room temperature before the enzyme reaction. The enzyme
activities were measured at 15-min intervals throughout the total 2-h
incubation time. B, the cultured neuroblastoma cells
(SK-N-MC) were incubated with 0 () and 5 mM DEA/NO ()
for 1 h. The cells were washed with PBS four times and continually
cultured with fresh medium. The cells were collected for DH activity
measurement at 0, 1, 2, 3, 4, and 5 h after DEA/NO exposure. The
results are representative of three independent experiments. S.D. is
indicated.
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Although 5 mM of DEA/NO inhibited both the purified and the
cellular DH, it could be argued that these conditions are not relevant in biological systems. Therefore, we sought to determine if
NO, derived from activated microglial cells, could inhibit DH
activity in co-cultured SK-N-MC cells. SK-N-MC cells were plated in
Petri dishes containing murine macrophage C3 cells untreated or
activated by IFN- and LPS. The C3 cells showed no detectable DH
activity (data not shown). A marked inhibition of DH was observed in
the SK-N-MC cells co-cultured with activated C3 cells (Fig.
5). This inhibition was abated in the
presence of the NOS inhibitor NMA during the co-culture (Fig. 5).
However, the activity of DH was not restored by the addition of NMA
for 4 h after 8 h of co-culture (Fig. 5). The number and the
viability of the SK-N-MC cells after co-culture were found to be
similar to those of the companion cultures without C3 cells (>95%).
These data indicate that NO, derived from iNOS, could produce an
environment to inhibit DH similar to 5 mM DEA/NO
in vivo.
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Fig. 5.
Inhibition of DH
activity induced by cytokine-activated microglia. Immortalized C3
murine microglial cells were seeded (2 × 106 cells)
into 100-mm2 Petri dishes and were stimulated for 12 h
with both IFN- (20 ng/ml) and LPS (20 ng/ml). SK-N-MC cells
(MC) were seeded into the IFN- and LPS-stimulated
(MC + C3IL) or nonstimulated (MC + C3) C3
cultures. The iNOS inhibitor NMA was added into the co-culture either
at the beginning of co-culture (MC + C3IL + NMA) or 4 h
before harvest (MC + C3IL + NMA*). Following 12 h of
co-incubation, the cells were harvested and lysed. The relative DH
activities are representative of three independent experiments. S.D. is
indicated.
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Because either DEA/NO or cytokine-activated C3 cells inhibited DH
activity in the SK-N-MC cells, we determined the relative NO
concentration that was produced by these two methods. A solution of 10 mM DEA/NO was dissolved in an appropriate buffer at
37 °C. Aliquots of samples were taken every 30 s at different
time points and evaluated by a chemiluminescent method. The flux of NO
rose to 50-100 µM and then decreased as described
previously (23). We then examined NO production by the
cytokine-activated C3 cells in which aliquots were obtained hourly. As
seen in Table I, the activated C3 cells
produced 10-15 µM of nitrite during the course of the
experiment where DEA/NO generated 10 mM (data not shown).
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Table I
Nitrosative capacity of activated microglia
Data are derived from triplicate values ± S.E. of the nitrite and
N-nitrosated product of 2,3-diaminonaphthalene, triazole
that accumulated over a period of 3 h. Blank values have been
subtracted.
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DISCUSSION |
We have investigated whether nitrosative stress generated by
activated macrophages can alter the noradrenergic function by regulating the DH enzymatic activity. Our results show that the activity of DH, the enzyme that catalyzes the conversion of dopamine to norepinephrine, is inhibited by NO, derived from either a synthetic donor or macrophage/microglial iNOS.
DEA/NO decomposes to release a high flux of NO at a physiological pH
(27). In an aerobic environment, NO can combine with molecular oxygen
to form RNOS such as N2O3 (28). The RNOS
scavenger compounds, GSH and azide (21), attenuated DEA/NO-mediated
inhibition of DH (Fig. 2, A and B), indicating
that N2O3, rather than NO alone, was the
effector molecule.
N2O3 can transfer an NO+ equivalent
to nucleophilic sites in a reaction known as nitrosation (22, 29).
Enzymes containing cysteine-zinc finger motifs, unprotonated lysyl, or
reduced cystyl residues at key sites are susceptible to
nitrosation-mediated inhibition (29). In addition,
N2O3 can nitrosate tyrosine residues in
proteins (30). Previous studies indicated that multiple residues are
critical for DH activity (31). Incubation of DH with diazonium tetrazole, iodoacetamide, or diethylpyrocarbonate caused complete inhibition of the enzyme activity, indicating that lysine, tyrosine, and histidine residues are critical for catalytic function.
Interestingly, the only thiol sites in DH are oxidized on cystine
bridges that maintain the tetrameric structure of the protein complex.
This precludes their accessibility to N2O3. The
reaction of the DH complex with dithiothreitol reduces it into a
dimeric form without affecting DH activity (31). Similar to other
metalloenzymes, such as P450 hydroxylase and cytochrome c
oxidase in the mitochondrial respiration chain (7), our data showed
that DH activity was irreversibly inhibited by 5 mM of
DEA/NO and microglia-derived NO. The higher local concentration of NO
could form N2O3 and cause indirect effects on
DH. The formation of a benzylic radical intermediate in the
hydroxylation of DH has been proposed (32). Our data show that the
unpaired electron of NO does not contribute to DH inhibition.
However, the benzylic radical mechanism could play a role under
turnover conditions (data not shown). Taken together, these data
suggest that nitrosation-mediated inhibition of DH activity may
occur at either lysine and/or tyrosine residues. Additional studies are
needed to further test this hypothesis.
Neuroblastoma is composed of primitive cells derived from the neural
crest and contains a high level of DH activity roughly equivalent to
adrenal medulla tissue (25). While 5 mM DEA/NO inhibited
the purified enzyme completely, this concentration of DEA/NO reduced
the activity of DH within intact SK-N-MC neurons by only 50% (Fig.
3C). The inclusion of BSO resulted in full (>90%) inhibition, showing that the thiol-containing tripeptide GSH was a
protective agent against nitrosative DH inhibition for only 50% of
the intracellular enzyme population. GSH concentrations of 5 mM were sufficient to protect purified DH (Fig.
2B). SK-N-MC cells contain a reduced GSH pool in the range
of 12-30 mM. These data demonstrated that GSH was unable
to protect approximately half of the intracellular DH against
nitrosative inhibition despite relatively high levels.
DH exists in both cytoplasmic and membrane-bound forms in
approximately equal amounts (5, 33). The reaction between NO and
molecular oxygen to form N2O3 is accelerated
greatly in the hydrophobic phase of lipid bilayers (34). The insertion of DH into membranes may augment nitrosative inhibition by 1) placing it in an environment of relatively higher NO+
equivalents and 2) concealing lysine and/or tyrosine sites from the GSH
protection. The hydroxylation reaction catalyzed by DH requires the
reduction of ascorbate as the electron donor. Sequestration of the
active site of DH into the hydrophobic phase of the membrane may be
necessary to avoid interference from the high cytoplasmic pool of
reduced GSH. By this mechanism, GSH may maintain DH in an inactive
state as it is transported down axons restricting norepinephrine
synthesis to nerve terminals.
The term nitrosative stress has been used to describe biological
components that are nitrosated by species derived from
N2O3 (7). A nitrosative stress microenvironment
can occur under very distinct conditions including high concentrations
of NO for prolonged periods of time. Constitutive NOS are unlikely to
produce these conditions, because they produce NO for only short
periods of time generally, and they also are inhibited by high fluxes of NO (35). Therefore, the iNOS would be the most likely source. Nitrosative stress can occur following the expression of iNOS in
activated leukocytes (36). Co-culture of SK-N-MC cells with macrophages/microglia stimulated to produce
N2O3, resulted in DH inhibition equivalent
to that observed with 5 mM DEA/NO (Fig. 5). The inhibition
of iNOS with L-N-monomethylarginine fully
restored DH activity, ruling out the potential influence of other
inflammatory mediators. These findings suggest that a condition of
nitrosative stress is required in vivo to inhibit DH. The
possibility of DH inhibition in cortex, cerebellum, hypothalamus,
and adrenal medulla may be a significant feature of nitrosative stress,
particularly during infection that localizes the leukocytes to express
iNOS in the proximity of catecholaminergic systems. Under these
circumstances, the resultant increase in both dopamine and NO with a
decrease in norepinephrine would increase vasodilation, regional blood flow, and tissue permeability. Nitrosated DH may then be replaced by
the GSH-protected pool mobilized from the cellular perikarya. Chronic
nitrosation, however, may lead to a more severe dysregulation of the
catecholamine balance. NO, as an important intercellular messenger, may
regulate catecholamine and blood flow by the modulation of DH activity.
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ACKNOWLEDGEMENT |
We thank D. Dudek for editorial assistance.
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FOOTNOTES |
*
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.
¶
To whom correspondence should be addressed: Laboratory of
Human Carcinogenesis, NCI, National Institutes of Health, Bldg. 37, Rm.
2C05, 37 Convent Dr., Bethesda, MD 20892-4255. Tel.: 301-496-2048; Fax:
301-496-0497; E-mail: Curtis_Harris@nih.gov.
Published, JBC Papers in Press, April 28, 2000, DOI 10.1074/jbc.M904498199
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ABBREVIATIONS |
The abbreviations used are:
DH, dopamine--hydroxylase;
NO, nitric oxide;
NOS, nitric-oxide synthase;
iNOS, inducible nitric-oxide synthase;
RNOS, reactive nitrogen oxide
species;
DEA, diethylamine;
BSO, buthionine suloximine;
LPS, lipopolysaccharide;
IFN-, interferon-;
PBS, phosphate-buffered
saline;
NMA, L-N-methylarginine.
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Copyright © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.
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Copyright © 2000 by the American Society for Biochemistry and Molecular Biology.
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