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Vol. 60, Issue 4, 838-846, October 2001
Laboratoire de Neurobiologie Cellulaire et Moléculaire (L.G., Y.M.G.-T.), Institut de Chimie des Substances Naturelles (C.D.), Centre National de la Recherche Scientifique, Gif-sur-Yvette, France
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Abstract |
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 Top
 Abstract
 Introduction
Experimental Procedures
Results
Discussion
References
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Evidence of an overload of reactive oxygen species and peroxynitrite, a derivative of nitric oxide, in sporadic amyotrophic lateral sclerosis suggests that peroxynitrite could impair cholinergic functions. Because of the impossibility of obtaining synaptosomes from vertebrate neuromuscular junctions, we used cholinergic synaptosomes purified from Torpedo marmorata electroneurons to characterize the defects triggered by peroxynitrite in more detail. Addition of peroxynitrite or its donor 3-morpholinosydnonimine abolished high-affinity choline uptake and synthesis of acetylcholine from acetate. T. marmorata choline acetyltransferase (ChAT) was impaired to the same extent as bovine brain ChAT. A hallmark of peroxynitrite action is the nitration of tyrosine residues in proteins. Peroxynitrite induced a concentration-dependent appearance of nitrotyrosines in several neuronal proteins from synaptosomes and, more readily, from synaptic vesicles. Peroxynitrite also triggered tyrosine nitrations in purified ChAT. Peroxynitrite-dependent nitrations were impaired when synaptosomes were pretreated with thioreductants (glutathione, N-acetyl cysteine, dithiothreitol) or antioxidants (uric acid, melatonin, bovine serum albumin, desferrioxamine). Deleterious effects of peroxynitrite on choline transport and ChAT activity were prevented by the thioreductants but only partially by the antioxidants, suggesting a mechanism other than tyrosine nitration, which may involve cysteine oxidation. Further development of protective agents acting on choline transport and on ChAT activity may offer interesting therapeutic possibilities with respect to cholinergic dysfunction occurring in neurodegenerative diseases.
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Introduction |
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Top
Abstract
Introduction
Experimental Procedures
Results
Discussion
References
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Nitric
oxide (NO·) modulates neurotransmission by
cholinergic neurons (Mothet et al., 1996
). At the level of the
neuromuscular junction, NO· is produced
endogenously, mainly in the skeletal muscle by type-1 NO· synthase (Nakane et al., 1993), where it
can interfere with the acetylcholine (ACh) release mechanism after
diffusion and participates in the control of muscular force.
NO· donors are administered therapeutically to
treat various diseases characterized by a deficiency in
NO· synthesis. For example, molsidomine, a drug
used for the last 30 years, is hydrolyzed to 3-morpholinosydnonimine
(SIN-1), which has been shown to release consecutively
O2
; Feelisch et al.,
1989). In other pathological conditions or in response to stressful
situations, NO· is produced at high
concentrations and can spread from cellular compartments.
NO· can then combine with
O2; Pryor and Squadrito,
1995) such as mitochondria, or through enzyme activity (e.g., monoamine
oxidase, xanthine oxidase), to form peroxynitrite
(ONOO
). Recent studies have revealed that
ONOO is a highly reactive molecule that is able
to induce many changes in proteins by oxidizing the sulfhydryl groups
of cysteine and methionine as well as tryptophan residues and
selectively nitrating tyrosine residues (Pryor and Squadrito, 1995;
Radi et al., 2001).
Recently, the detection of nitrotyrosine residues in the brains of
patients with Parkinson's disease (Good et al., 1998) and in model
animals (Ara et al., 1998) led to a search to determine the molecular
targets of ONOO. Clinical studies showed that
tyrosine hydroxylase, the first and rate-limiting enzyme in
catecholamine biosynthesis that is selectively affected in Parkinson's
disease, is nitrated, suggesting that the cause of the pathology may be
an ONOO overload (Ara et al., 1998). In
cholinergic pathologies, brain areas of patients with Alzheimer's
disease showed an increase of ONOO-mediated
nitration of proteins (Smith et al., 1997), and the cerebrospinal fluid
of patients with sporadic amyotrophic lateral sclerosis showed an
increase in 3-nitrotyrosine (Beal et al., 1997). Despite the detection
of tyrosine nitration in synaptic proteins (Di Stasi et al., 1999;
Koppal et al., 1999), no information on peripheral cholinergic proteins
is available. Faced with the impossibility of purifying nerve endings
of the neuromuscular junction, we investigated the effects of
NO· and ONOO on
synaptosomes of a well-characterized model: the electromotoneurons of
Torpedo marmorata. We demonstrated previously the presence of type-1 NO· synthase in the cell bodies and
nerve endings of T. marmorata electroneurons and showed that
NO· increased ACh release, whereas
ONOO inhibited ACh synthesis (Morot
Gaudry-Talarmain et al., 1997).
ACh in neuromuscular junctions is synthesized from two extracellular
precursors. The first is acetate, which is converted into acetyl-CoA by
acetyl-CoA synthetase. The second is choline, which is internalized by
a sodium-dependent and hemicholinium-sensitive transporter (Okuda et
al., 2000). Synthesis of ACh from choline and acetyl-CoA is performed
by choline acetyltransferase (ChAT), a cytosolic enzyme (Wu and Hersh,
1994). In a subsequent step, newly synthesized ACh is transported into
synaptic vesicles by the energy-dependent vesicular ACh transporter.
This article presents the inhibitory effects of
ONOO and SIN-1, a donor of
ONOO, on high-affinity choline uptake, ACh
synthesis from radiolabeled acetate, and ChAT activity.
ONOO-dependent changes in several presynaptic
proteins (ChAT, synaptophysin, VAMP/synaptobrevin, actin, tubulin)
present in T. marmorata synaptosomes and synaptic vesicles
were examined and showed examples of nitration of tyrosines and
covalent oligomerization. To counteract ONOO
toxicity, several endogenous and exogenous compounds were tested. Antioxidants and thioreductants prevented nitration, but only thioreductants fully protected high-affinity choline uptake and ChAT activity.
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Experimental Procedures |
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Top
Abstract
Introduction
Experimental Procedures
Results
Discussion
References
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Animals
T. marmorata were purchased from the marine station in Roscoff, France, and kept in oxygenated artificial sea water tanks.
Materials
Stock Solutions of ChAT. Bovine brain ChAT (E.C. 2.3.1.6, C-3388 batches 36F9625 and 100H9500) was obtained from Sigma (St. Louis, MO). For each experiment, the lyophilized powder of partially purified ChAT was solubilized in 50 mM sodium-phosphate buffer, pH 7.3 (10 mg/ml or as specified), and used for analysis by Western blotting and measurement of ChAT activity.
Stock Solutions of SIN-1.
SIN-1 was obtained from BIOMOL
(Plymouth Meeting, PA). Aqueous solutions of SIN-1 (100 mM) were
stored at 
20°C before use. Monoclonal antibodies against
nitrotyrosine, ChAT, and actin (JLA20) were obtained from Upstate
Biotechnology, Inc. (Lake Placid, NY), Chemicon International
(Temecula, CA), and DSHB (University of Iowa, Iowa City, IA),
respectively. Polyclonal antibody against T. marmorata
synaptophysin and monoclonal antibodies against VAMP/synaptobrevin and
tubulin were developed and characterized in the laboratory by Dr.
Nicolas Morel (Laboratoire de Neurobiologie Cellulaire et Moleculaire,
CNRS, Gif-sur-Yvette, France). Polyclonal antibody against
muscle lactate dehydrogenase was obtained from Chemicon. Other reagents
were obtained from commercial sources, and the highest purity available
was obtained.
Chemical ONOO Synthesis
Alkaline ONOO was synthesized at room
temperature using the procedure described by Uppu and Pryor (Uppu and
Pryor, 1996) in the two-phase system using isoamyl nitrite and hydrogen
peroxide. After a freezing step (20°C, overnight) of the obtained
solution, the upper yellow phase containing concentrated
ONOO (between 1.3 and 2.4 M) was collected.
Residual hydrogen peroxide was not removed, and
diethylenetriaminepentaacetic acid was added to minimize inadvertent
trace-metal contamination. The final stock concentration of this
solution was quantified spectrophotometrically after dilution in 0.1 N
NaOH at 302 nm (
M = 1670 M1 · cm1) immediately
before each experiment, and appropriate dilutions of
ONOO were performed in 0.1 N NaOH (100 µM to
500 mM ONOO).
Methods
Preparation of Synaptosomes and Synaptic Vesicles.
T.
marmorata electric organ was used to purify cholinergic nerve
endings and synaptic vesicles. Synaptosomes were isolated on isoosmotic
sucrose-saline Krebs' gradients and were collected at the interface of
0.3 to 0.5 M sucrose according to the method described by Morel et al.
(1977). The synaptosomal fraction (40-50 ml) derived from 25 g of
electric organ was collected from a gradient made of discontinuous
isoosmotic saline-sucrose Krebs' solution devoid of
Ca2+ and equilibrated to pH 7.4 with 5 mM
NaHCO3. Aliquots were used immediately for
functional studies. For Western blot analysis of proteins, after
treatment with drugs, the synaptosomal suspension was diluted in
Krebs' buffer and centrifuged at 12,000g for 20 min.
and collected at the interface of a 0.38 to
0.5 M sucrose-saline isoosmotic gradient, then further concentrated by
centrifugation in 10 mM Tris, 350 mM KCl, and 0.1 M sucrose buffer, pH
7.2.
Exposure of Synaptosomes, Synaptic Vesicles, and Bovine Brain
ChAT to ONOO or SIN-1.
Preincubation of synaptosomes
(in Krebs' buffer), synaptic vesicles (in Tris buffer), or purified
bovine brain ChAT (in 50 mM sodium-phosphate buffer, pH 7.3) with
ONOO or SIN-1 was carried out at room
temperature for 1 to 2 h before freezing. For
ONOO and SIN-1 incubations, various increasing
concentrations of drugs were added in one bolus directly on the
neuronal fractions and stirred immediately, respectively, by a vortex
and magnetic agitation in oxygenated T. marmorata medium.
Usually, ONOO concentration of stock solutions
was 1 to 2 M. ONOO was then diluted in 0.1 N
NaOH and used for physiological studies at final concentrations of less
than 4 mM, leading to a minimal dilution (v/v) of 1/250. According to
Uppu and Pryor (1996), we estimated that the residual
H2O2 concentration in the
peroxynitrite stock solution may reach 0.2 to 0.5 M, leading to a
maximal final concentration of 1 to 2 mM in treated samples.
did not induce a significant lysis of
synaptosomes. Synaptosomes were treated by increasing concentrations of
ONOO and concentrated by centrifugation.
Released protein content after ONOO treatment
was measured in the supernatants: 1) by the Lowry method for total
protein; 2) by immunodetection of muscle lactate dehydrogenase after
Western blotting; and 3) by the measurement of the volume variations of
the synaptosomes. The three different sorts of analysis gave comparable
results and confirmed that for up to 500 µM
ONOO, there is no lysis of synaptosomes. A 25 to 30% leakage of proteins started to be observed at 1 mM
ONOO (data not shown).
ChAT activity was measured using aliquots of the same synaptosomal
samples, and a protein analysis was done on the concentrated preparations. Protein content was determined by the Lowry method of assay.
Synthesis and Compartmentalization of ACh.
Synthesis of ACh
by 400 µl of synaptosomes was measured using
[14C]acetate (100 µM) and choline (100 µM)
as ACh precursors. At the end of the synthesis time (usually 60 min),
aliquots (20 µl) were saved and kept frozen for further determination
of ChAT activity. Formation of newly synthesized radioactive ACh in
synaptosomes (180 µl) was stopped by the addition of 5%
trichloroacetic acid (TCA), whereas in the other aliquot (180 µl),
radioactive ACh accumulation in the vesicular pool was determined after
one freezing and thawing cycle of the synaptosomes (Dolezal et al.,
1993), followed by 5% TCA addition. After extraction on ice, TCA was eliminated from all the samples by three ether washes, and radioactive ACh was extracted by an allylcyanide-tetraphenylboron organic extraction procedure (Fonnum, 1975). The organic lipophilic phase containing radioactive ACh was collected, and radioactivity was counted
in Lipoluma scintillation liquid (Lumac, Landgraaf, the Netherlands).
ChAT Assay.
ChAT activity was determined using 10-µl
aliquots. The treated enzyme and synaptosomes were collected at the end
of the incubation and frozen until the assay. Samples were warmed to
room temperature and mixed with detergent [0.02% Triton X-100 (w/v)]
for 1 min to disrupt membranes. The reaction was started by the
addition of the substrates [1-14C]acetyl-CoA
(14 µM) and choline (2.5 mM) in the presence of 250 µM eserine, 10 µM bovine serum albumin (BSA), and 75 mM NaCl and stopped after
1 h by dilution with cold sodium-phosphate buffer. Radioactive ACh
was extracted and determined according to the method used by Fonnum
(1975). Typical ChAT activity of control samples was 207.7 ± 24.1 nmol · h1 · g1, in
accordance with the findings of Morel et al. (1977).
High-Affinity Choline Uptake Assay.
Choline accumulation in
nerve endings was measured by use of the following method: after 15 min
of rewarming at room temperature, freshly prepared synaptosomes (300 µl) were pretreated with ONOO, SIN-1, or
vehicle (0.1 N NaOH) for 45 min. High-affinity choline uptake was
initiated by the addition of [14C]choline (5 µM) and terminated by filtration on a 1.2-µm filter (Millipore
Corporation, Bedford, MA). After subsequent washing of the filter with
10 ml of cold physiological medium, pH 7.4, the incorporated
[14C]choline was determined by liquid
scintillation counting.
SDS-PAGE Analysis.
ONOO-treated
samples (synaptosomes or purified enzymes) were analyzed by
SDS-polyacrylamide gel electrophoresis (SDS-PAGE) in reducing
conditions (
-mercaptoethanol 10% in lysis buffer) followed by
Western blotting onto nitrocellulose. Western blots were probed with
primary antibodies for 3 to 12 h at room temperature. Probed
proteins were detected by secondary antibodies linked to horseradish
peroxidase and visualized by the enhanced chemiluminescence kit (ECL;
Amersham Pharmacia Biotech AB, Uppsala, Sweden).
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Results |
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Top
Abstract
Introduction
Experimental Procedures
Results
Discussion
References
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Exogenous ONOO or SIN-1 Inhibits High-Affinity
Choline Uptake and [14C]ACh Synthesis from
[14C]Acetate.
As shown in Fig.
1, increasing concentrations of
ONOO (Fig. 1A) or SIN-1 (Fig. 1B) in the
preincubation medium of synaptosomes resulted in a dose-dependent
inhibition of high-affinity choline uptake. An inhibition of 50% was
obtained at approximately 500 µM for ONOO and
800 µM for SIN-1. After pretreatment with 1 mM
ONOO, choline uptake was nearly totally
abolished. H2O2 was without effect (Fig. 1C), even in the millimolar range, showing that
ONOO effects were not related to residual
traces of H2O2 present in the preparation of ONOO (Uppu and Pryor, 1996).
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dose-dependently
inhibited [14C]ACh synthesis
(IC50 = 300 µM; Fig. 2A) but not its
incorporation into vesicles (Fig. 2B). Continuous infusion with SIN-1
(Fig. 2A', 2B') led to similar results, with a lesser potency
(IC50 = 900 µM).
H2O2 (200 µM) did not
induce any significant changes in the synthesis and incorporation of
ACh (data not shown).
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ONOO Inhibits ChAT Activity.
Having shown that
ACh synthesis from acetate was defective in
ONOO-treated synaptosomes, we wanted to
establish whether the enzymatic activity of ChAT, the enzyme
responsible for ACh synthesis from acetyl-CoA and choline, was altered,
or if this inhibition was subsequent to the inactivation of choline
uptake, which provides the other ACh precursor, choline. Therefore,
synaptosomes pretreated with ONOO or SIN-1 were
lysed by Triton X-100 (0.02%) and assessed for the activity of
intracellular ChAT at the end of the measurement of the ACh synthesis.
The enzyme activity was inhibited by ONOO
(IC50 = 350 µM) and by SIN-1
(IC50 = 40 µM) (Fig. 2, C and C'). SIN-1 was
surprisingly a more potent inhibitor of ChAT activity of synaptosomes
than ONOO. ChAT activity was determined after
solubilization of the synaptosomal membrane with Triton X-100 and
dilution of the cytosol, which leads to better oxygen accessibility
during SIN-1 decomposition. Moreover, SIN-1 decomposition in tissues
can lead to the continuous formation of oxidized toxic substances such
as peroxidized fatty acids and proteins (Koppal et al., 1999; Tien et
al., 1999), which might interfere with the measurement of enzyme
activity (Currier and Mautner, 1976).
on
ChAT depended on its subcellular distribution in T. marmorata motoneurons, we compared the activity of synaptosomal
ChAT with that contained in the cell bodies concentrated in the
electric lobe (data not shown). ChAT activity was inhibited by
ONOO in both structures with comparable
IC50 values (400 µM, n = 8; and
300 µM, n = 3, respectively). We showed that 1 mM
H2O2 was without effect on
the ChAT activity of synaptosomes (100.96 ± 1.55% of untreated
control, n = 3).
As in the previous experiments performed with T. marmorata
nerve endings showing an inhibition of the activity of ChAT by ONOO and SIN-1, we investigated the effect of
ONOO and SIN-1 on partially purified bovine
brain ChAT in the presence of Triton X-100 (0.02%) in sodium-phosphate
buffer, pH 7.3. ChAT activity was inhibited by
ONOO and, to a lesser extent, by SIN-1,
possibly because of a lack of oxygen in the medium (Fig.
3). An inhibition of 50% was obtained with 300 µM ONOO and was total at
concentrations of nearly 1 mM ONOO. This result
shows that ONOO probably directly modifies the
ChAT protein itself.
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Nitrotyrosine Detection in Purified ChAT after ONOO
Treatment.
Nitration of tyrosine residues in proteins is an
important post-translational modification triggered by
ONOO. Therefore, we sought the presence of
nitrotyrosine in untreated bovine brain ChAT and in
ONOO-treated samples by Western blot analysis.
treatment, a decrease of the
Coomassie blue staining was noticed after treatment of ChAT at the
highest ONOO concentrations (Fig. 4A),
suggesting possible degradation of the protein. Likewise, the
immunoreactivity of the 65- and 67-kDa bands decreases at
concentrations of ONOO greater than 200 µM
(Fig. 4C).
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(10 µM). At
100 µM ONOO, the 63-kDa band is labeled, as
well as another band at 61 kDa that was not detected otherwise. At
concentrations greater than 100 µM ONOO, we
showed a concentration-dependent increase of the chemiluminescent signal that became saturated at 1 mM ONOO. At
high concentrations of ONOO, other bands have
nitrotyrosine immunoreactivity at higher and lower molecular masses.
This suggests that a possible oligomerization of ChAT (by di-tyrosine
cross-linking, for example) and lysis may occur. We were unable to
detect nitration by SIN-1, and no significant change in the
immunoreactivity for ChAT was noticed after SIN-1 treatment.
Nitrotyrosine Appearance in Presynaptic Proteins after
ONOO Treatment Is Prevented by Thioreductants and
Antioxidants.
Analysis of the possible changes triggered by an
ONOO attack on neuronal proteins was further
pursued by tracking down nitrotyrosines or other modifications on
synaptic proteins after treatment with ONOO.
Therefore, we treated a synaptic vesicles
preparation (Fig. 5) or intact, functional synaptosomes
(Fig. 6) with increasing ONOO concentrations.
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than the synaptosomal fraction. In synaptic vesicles, the nitrotyrosine
immunoreactivity was observed at low concentrations of
ONOO (100 µM) and, even at higher
ONOO concentrations, was specific to certain
proteins because VAMP/synaptobrevin, for example, was not nitrated
(Fig. 5, A and B). Among the nitrated proteins, two proteins migrating
at molecular masses of 43 kDa and 52 kDa seemed to be nitrated early.
They comigrate with actin (Fig. 5C) and tubulin (Fig. 5D),
respectively. Immunoreactivity against tubulin was revealed by an
antibody developed against T. marmorata tubulin, which
recognizes tubulin at 50 kDa and another uncharacterized 
40-kDa band
specific for T. marmorata nerve terminals (Sbia et al.,
1991). When blots were stripped and reprobed with antiactin and
antitubulin antibodies (Fig. 5, C and D, respectively), we observed the
coincident disappearance of their immunoreactivity with the appearance
of nitrotyrosines.
Synaptophysin, an integral vesicular 38-kDa glycoprotein, was detected
in the controls (Fig. 5E) as a doublet using an antibody directed
against the C-terminal epitope
265GYQPNYGQ277Q (Cowan et
al., 1990; N. Morel, personal communication). We found that its
immunoreactivity was lost upon treatment with
ONOO.
VAMP/synaptobrevin was not nitrated after ONOO
action, and it did not lose its immunoreactivity after peroxynitrite
action. Furthermore, we observed that the immunoreactivity of a 30- to 32-kDa VAMP-containing complex resistant to SDS, -mercaptoethanol, and boiling increased after ONOO treatment of
synaptic vesicles (Fig. 5A). In contrast, the B-regulatory subunit of
calcineurin, which is present in the synaptic vesicles preparation, is
not nitrated by peroxynitrite and nevertheless loses its
immunoreactivity after peroxynitrite action (not shown).
By comparison with isolated synaptic vesicles, only a few proteins, yet
uncharacterized, were sensitive to 1 mM ONOO
when functional synaptosomes were treated with increasing
concentrations of ONOO (Fig. 6). Among these
proteins, the faint 63-kDa nitrated protein may be ChAT. As in synaptic
vesicles, no nitration occurs for VAMP/synaptobrevin, but
oligomerization was observed (data not shown).
When synaptosomes were treated with increasing concentrations of SIN-1
(100-4000 µM) for 1 h under continuous agitation and ambient
aerobic conditions, no immunoreactivity for nitrotyrosine was detected,
and only faint bands appeared at 4 mM SIN-1 (data not shown).
As shown for tryptophan hydroxylase (Kuhn and Geddes, 1999), protection
against ONOO-induced tyrosine nitrations may
prevent the loss of enzymatic activity. To assess further the
interrelationship between the nitration of synaptosomal proteins
induced by ONOO and the inhibition of
cholinergic functions, we tried to prevent the damage or to intercept
the oxidizing and nitrating reactive species, as suggested previously
(Arteel et al., 1999; Blanchard et al., 2000). Synaptosomes were
pretreated with the following thioreductants: 5 mM glutathione (GSH),
1.25 mM N-acetylcysteine (NAC), and 1 mM dithiothreitol
(DTT); and with the following antioxidants: 1 mM uric acid, 1 mM
melatonin, 0.2 mM desferrioxamine, and 1 mM BSA, for 30 min before the
addition of 1 mM ONOO. In Fig. 6, it can be
seen that all these compounds were able to prevent effectively the
appearance of nitrotyrosine in synaptosomes.
Thioreductants and Uric Acid Protect Choline Uptake.
In
further studies, we changed the synaptosomal oxidation state by
pretreatment with the same thioreductants (GSH, NAC, DTT) and
antioxidants (uric acid, BSA, desferrioxamine, melatonin). Apart from
BSA, which increased choline uptake, these compounds did not affect the
basal rate of choline transport. We show in Fig.
7A that all thioreductants, uric acid,
and BSA significantly protected choline uptake from
ONOO inhibition. Surprisingly, desferrioxamine
and melatonin did not affect choline transport, but they prevented all
the nitration reactions.
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Thioreductants Fully Protect and Partially Restore ChAT
Activity.
Similarly, we tested the protective properties of
thioreductants and antioxidants against the loss of ChAT activity of
synaptosomes caused by ONOO. Although the
thioreductants fully protected ChAT activity (Fig. 7B), we did not
observe protection with any of the other antioxidant compounds tested
(uric acid, BSA, desferrioxamine, melatonin).
; Radi et al., 2001),
formation of disulfides or nitrosothiol products by
ONOO is reversible by thiol-reducing agents.
The reversibility of ONOO action on ChAT
activity was assessed by treating synaptosomes first with
ONOO and then with DTT (5 mM). As shown in Fig.
8, DTT was able to significantly improve
ChAT activity after 200 µM and 1 mM ONOO
action.
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Discussion |
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Top
Abstract
Introduction
Experimental Procedures
Results
Discussion
References
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The pathological production of ONOO at the
presynaptic side of neuromuscular junctions was mimicked in vitro using
either a dose of the chemically synthesized
ONOO or a donor, SIN-1, which released
ONOO by a regular flux associated with
molecular degradation. In physiological buffers containing
NaHCO3, ONOO is
transformed into the species nitroso-peroxicarbonate
(ONOOCO2) (Lymar et al.,
1996). Both ONOO and
ONOOCO2 decompose into mineral
nitrate and nitrite and in the presence of reactive molecules,
they are powerful nitrating and oxidizing agents. However, the rapid
decay of ONOO added extracellularly explains
why high doses of ONOO (up to 1 mM) are
required to affect intracellular targets.
The present results show that under physiological conditions of pH and
bicarbonate concentrations, ONOO is a powerful
inhibitor of two major cholinergic processes: choline transport and ACh
synthesis. Furthermore, the analysis by Western blotting of presynaptic
proteins, which are modified by ONOO (either by
nitration of tyrosines, loss of immunoreactivity, or cross-linking),
support the idea that it may induce severe alterations of the
cholinergic functions.
In cholinergic neurons, the synthesis of ACh by ChAT is dependent on
intracellular pools of choline provided by the high-affinity choline
uptake and of acetyl-CoA replenished by metabolism. Very recently, the
cholinergic-specific high-affinity choline transporter was cloned
(Okuda et al., 2000). High-affinity choline uptake, which is present in
all cholinergic neurons, is Na+- and
Cl-dependent, uses the
Na+/K+-ATPase-maintained
membrane potential as a driving force, and is electrogenic (O'Regan,
1988). In this report, choline transport was found to be inactivated in
a concentration-dependent manner both by ONOO
and SIN-1, but not by millimolar ranges of
H2O2. We did not establish whether this inhibition is direct: it could also be mediated by perturbations of ionic gradients because
Na+/K+-ATPase was
previously shown to be inhibited by NO·-donors,
ONOO, and other oxidants (Muriel and Sandoval,
2000). Previous reports showed that liposome-reconstituted
Na+-dependent high-affinity glutamate transport
was inhibited by similar doses of ONOO, but
also by high doses (more than 1 mM) of
H2O2 (Trotti et al., 1996).
These effects, reversed by disulfide-reducing agents such as DTT, were
attributed to cysteine oxidation. Our results show that selective
modifications of choline transport are triggered by
ONOO and not by the other oxidant
H2O2 and that these
modifications could be prevented by thiols and uric acid.
ONOO is a stronger oxidant than
H2O2 and may induce
cysteine oxidation beyond disulfide (e.g., into sulfenic, sulfinic, or
sulfonic acid) or may oxidize other residues (methionine and
tryptophan). It can also trigger tyrosine nitrations. The fact that
some compounds (e.g., melatonin or desferrioxamine) that were fully
protective for ONOO-induced nitration were
ineffective in protecting choline uptake suggests that tyrosine
nitration is not the mechanism that explains selective
ONOO toxicity toward choline uptake.
ACh synthesis was inhibited by either ONOO or
SIN-1, but neither was inhibited by
H2O2 up to 1 mM (this
study) or by similar concentrations of the NO·
donors S-nitroso-N-acetylpenicillamine and sodium
nitroprusside (Morot Gaudry-Talarmain et al., 1997). To determine
whether ChAT itself was affected or whether ACh synthesis inhibition
was caused by the prevention of choline uptake, we tested the effect of
ONOO on T. marmorata synaptosomal
ChAT or on partially purified bovine brain ChAT. In both preparations,
ONOO totally inhibited ACh synthesis
(IC50 500 µM), confirming that both ChAT
from mammalian central nervous system and from T. marmorata peripheral motoneurons were direct targets of
ONOO. Moreover, we have shown that the
inhibition of ChAT by ONOO coincides with the
appearance of nitrotyrosine and starts at concentrations as low as 50 to 100 µM ONOO. Hersh et al. (1984) showed
previously that several variants of ChAT were present in partially
purified bovine brain ChAT preparations differing in their isoelectric
points, molecular masses, and affinities for specific antibodies, but
not in their enzymatic activities. The pattern of immunoreactivity of
bovine brain ChAT after ONOO treatment is
complex. We showed that the anti-ChAT signal for the 65-kDa and 67-kDa
bands decreased as the antinitrotyrosine immunoreactivity of the 63-kDa
band increased, leading us to question whether there was a preferential
sensitivity of the 63-kDa band of ChAT to nitration of tyrosine. Our
work indicates that variants of ChAT may differ in their sensitivity to
action of ONOO. At concentrations of
ONOO higher than 200 µM, several protein
bands presented nitrotyrosine immunoreactivity with higher and lower
molecular masses. They were not characterized, but we cannot eliminate
the possibility that oligomerization of ChAT, by dityrosine
cross-linking, for example, as described previously for
-synuclein by Souza et al. (2000), or proteolysis may occur,
requiring further studies.
The complex regulation of ChAT activity has been reported previously.
The state of phosphorylation, controlled proteolysis, and subcellular
localization (cytosolic versus membrane-bound isoforms) and the
presence of thiol agents can all modulate the activity of the enzyme
posttranslationally (Oda, 1999; Wu and Hersh, 1994). Tyrosine nitration
could interfere with some of these pathways. As proposed previously
(Beckman and Koppenol, 1996; Di Stasi et al., 1999), there is a
modulation of tyrosine-dependent signaling in motoneurons when
tyrosines are nitrated. ChAT is phosphorylated by several
serine/threonine kinases (protein kinase C, casein kinase II, protein
kinase G, and -calcium/calmodulin-dependent protein kinase II)
(Bruce and Hersh, 1989; Dobransky et al., 2000). There is only one
conserved tyrosine phosphorylation consensus site on human ChAT,
without experimental evidence of its functionality.
ONOO is also a potent oxidant known to react
with cysteine residues (Viner et al., 1999; Radi et al., 2001). Human
ChAT isoforms contain 20 or 24 cysteines, and thiol reagents are known
to affect the activity of the enzyme (Currier and Mautner, 1976). Our
data are in accordance with these observations:
ONOO blocks the activity of the enzyme, and the
deleterious effect of ONOO can be prevented by
agents that maintain a high content of free reducing compounds (NAC,
GSH, DTT). We did not measure the variations of GSH content in synaptic
vesicles and synaptosomes in the presence of
ONOO and SIN-1. Nevertheless, according to many
reports in the literature and data obtained from brain synaptosomes
(Koppal et al., 1999), we suspect that a decrease of reducing
equivalents and the formation of S-S bonds between cysteines may occur
after ONOO treatment of synaptosomes. The fact
that ONOO inhibition can be at least partially
reversed by DTT favors this hypothesis, but the possibility of other
DTT-reversible cysteine modifications such as
S-nitrosylation awaits further investigation. The protective
role of thioreductant agents on ChAT activity can be explained by a
maintaining action on neuronal GSH pools and on critical reduced
cysteines close to the active site of the enzyme. Functional analysis
of conserved histidines in ChAT by site-directed analysis revealed
their essential role in catalysis as an acid/base sensor (Wu and Hersh,
1994). In this article, we suggest that tyrosines and cysteines may be
redox sensors for ChAT. Inactivation by ONOO of
the enzyme activity by sulfhydryl oxidation was recently shown to be
essential for tryptophan hydroxylase, another neurotransmitter synthesis enzyme (Kuhn and Geddes, 1999). Nevertheless, we do not know
the impact of the nitration of tyrosines that we observed.
To complete our functional studies, we undertook biochemical
characterization of proteins that were affected by
ONOO through nitration of tyrosines or other
modifications. We observed that after purification, the synaptic
vesicles devoid of the protection afforded by endogenous reducing
compounds present in the cytosol are very sensitive to
ONOO-mediated nitration of tyrosines. We
confirmed in peripheral nerve endings results previously obtained in
studies of mammalian cells or brain extracts (Beckman and Koppenol,
1996; Eiserich et al., 1999) showing nitrotyrosine immunoreactivity at
the molecular masses of tubulin and actin. We showed that after
ONOO attack of the proteins, the monoclonal
antitubulin and antiactin antibodies no longer recognized their
targets. This coincident loss of recognition of the epitopes may be
caused by changes in the protein structure. As proposed by Eiserich and
colleagues, these changes could compromise the function of these
proteins and interfere with their binding properties, e.g., with dynein for tubulin (Eiserich et al., 1999). Another protein that was affected
was synaptophysin/p38. Thus, we have confirmed with peripheral cholinergic synaptic vesicles the results that Di Stasi et al. obtained
using brain synaptosomes (Di Stasi et al., 1999). Moreover, we were
able to define affected residues by taking advantage of a specific
antibody raised against a well-conserved sequence
(265GYQPNYGQ273Q) of the
T. marmorata synaptophysin (Cowan et al., 1990). This epitope is located at the C terminus of the protein (Cowan et al.,
1990) and faces the cytosol of synaptosomes. We showed that ONOO (up to 500 µM) induced the loss of
recognition by this antibody, indicating that the conserved
266Y and 270Y could be
potential targets. We also observed that VAMP/synaptobrevin was not
nitrated on tyrosines but formed at 32 kDa a covalent complex that may
be the dimer previously obtained with the use of cross-linking agents
(Laage and Langosch, 1997).
In summary, we demonstrated that ONOO can
affect numerous proteins at the presynaptic side of a neuromuscular
junction in several subcellular compartments; these include choline
transporter at the plasma membrane, ChAT in the cytosol, and
synaptophysin, tubulin, or actin at the periphery of synaptic vesicles.
ONOO can act by various mechanisms including
cross-link formation, tyrosine nitration, cysteine oxidation or
S-nitrosylation that are differentially protected by various
antioxidants. These results can be of potential interest for
therapeutic research on ONOO-related neuronal
diseases such as amyotrophic lateral sclerosis, Alzheimer's disease,
or Parkinson's disease.
| |
Acknowledgments |
|---|
We thank Dr. Maurice Israël and Dr. Pierre Potier, in
whose laboratories this work was performed, for continuous support; Dr.
Nicolas Morel for help on immunological studies and the kind gift of
antibodies against VAMP/synaptobrevin, synaptophysin, and tubulin; Dr.
François-Marie Meunier for sequence alignments; Dr. Seana
O'Regan for expertise in choline uptake mechanisms and critical
reading; and Dr. Michael Spedding and Dr. Esther Schenker from the
Institut de Recherches Internationales Servier for their valuable
advices. We are grateful to Dr. Peter Lea for reading and helping with
the manuscript, Dr. Jacques Stinnakre for help with software,
Jean-Baptiste Milandri for measurement of synaptosomal volume under
ONOO treatment, and Jean-Paul Bouillot for photography.
| |
Footnotes |
|---|
Received February 12, 2001; Accepted June 29, 2001
This work was supported by the Centre National de la Recherche Scientifique and the Institut de Recherches Internationales Servier.
Dr. Yvette Morot-Gaudry-Talarmain, Laboratoire de Neurobiologie Cellulaire et Moléculaire, Center National de la Recherche Scientifique, 91198 Gif-sur-Yvette-Cedex, France. E-mail: morot{at}nbcm.cnrs-gif.fr.
| |
Abbreviations |
|---|
ONOO, peroxynitrite;
ACh, acetylcholine;
SIN-1, 3-morpholinosydnonimine;
CoA, Coenzyme A;
ChAT, choline acetyltransferase;
VAMP/synaptobrevin, vesicular-associated
membrane protein;
TCA, trichloroacetic acid;
BSA, bovine serum albumin;
PAGE, polyacrylamide gel electrophoresis;
ECL, enhanced
chemiluminescence;
GSH, glutathione;
NAC, N-acetylcysteine;
DTT, dithiothreitol.
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References |
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Top
Abstract
Introduction
Experimental Procedures
Results
Discussion
References
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) or in the absence (
) of DTT, and
expressed as a percentage of control values obtained without
ONOO