(Received for publication, August 3, 1995; and in revised form, December 11, 1995)
From the
The reuptake of glutamate in neurons and astrocytes terminates
excitatory signals and prevents the persistence of excitotoxic levels
of glutamate in the synaptic cleft. This process is inhibited by oxygen
radicals and hydrogen peroxide (HO
). Here we
show that another biological oxidant, peroxynitrite
(ONOO
), formed by combination of superoxide (
O
) and nitric oxide
(NO), potently inhibits glutamate uptake by purified or recombinant
high affinity glutamate transporters reconstituted in liposomes.
ONOO
reduces selectively the V
of transport; its action is fast (reaching
90% within 20 s),
dose-dependent (50% inhibition at 50 µM), persistent upon
ONOO
(or by product) removal, and insensitive to the
presence of the lipid antioxidant vitamin E in the liposomal membranes.
Therefore, it likely depends on direct interaction of ONOO
with the glutamate transporters. Three distinct recombinant
glutamate transporters from the rat brain, GLT1, GLAST, and EAAC1,
exhibit identical sensitivity to ONOO
.
H
O
also inhibits reconstituted transport, and
its action matches that of ONOO
on all respects;
however, this is observed only with 5-10 mM H
O
and after prolonged exposure (10 min)
in highly oxygenated buffer. NO, released from NO donors (up to 10
mM), does not modify reconstituted glutamate uptake, although
in parallel conditions it promotes cGMP formation in synaptosomal
cytosolic fraction. Overall, our results suggest that the glutamate
transporters contain conserved sites in their structures conferring
vulnerability to ONOO
and other oxidants.
Glutamate uptake in neurons and astrocytes is essential to
maintain resting extracellular glutamate concentration below levels
inducing significant activation of excitatory amino acid (EAA) ()receptors(1) . Thereby it provides a high signal
to noise ratio for excitatory neurotransmission and prevents harmful
receptor overstimulation. Altered transport function has been
associated with neuronal damage in ischemia/reperfusion injury (2) and amyotrophic lateral sclerosis
(ALS)(3, 4) . The uptake process is mediated by
glycoproteins located in the plasma membrane of neuronal and glial
cells. At least four different transporters are now cloned, i.e. GLAST(5) , GLT1(6) , EAAC1(7) , and
EAAT4(8) , constituting a gene family with specialized brain
distributions (9, 10, 11) . There is
increasing evidence that glutamate transport is regulated, e.g. via protein kinase C-mediated phosphorylation (12) and
arachidonic acid(13, 14) . Oxygen radicals and
H
O
induce persistent inhibition of glial
glutamate uptake, probably via direct interaction with the transport
process(15) . Sodium nitroprusside, a NO generator, decreases
uptake into synaptosomes(16) . When generated simultaneously,
O
and NO react together
at a diffusion-limited rate to form the strong oxidant ONOO
(17) . Several biological or toxic effects originally
attributed to either NO or
O
are now thought to be mediated by
ONOO
(18, 19, 20, 21, 22) .
In the present study, we address the possibility that ONOO
affects glutamate uptake by direct interaction with the glutamate
transporters.
The possible direct interaction of ONOO with glutamate uptake was first studied on a preparation of
partially purified brain glutamate transporters reconstituted in
liposomes. Liposomes were exposed to ONOO
or to
decayed ONOO
as control (see legend to Fig. 1). The agents were then removed by gel filtration and the
uptake assay run. ONOO
(but not decayed
ONOO
) inhibited uptake dose dependently (Fig. 1A). A 70-s exposure to 50 µM ONOO
reduced uptake by 50 ± 6%. Threshold
inhibition (-10 ± 6%) was seen with 5 µM ONOO
, while nearly complete inhibition
(-85 ± 5.1%) required 250 µM ONOO
. The ONOO
effect
developed almost immediately, reaching
90% within 20 s and then
increasing slightly in the next 2-3 min (Fig. 1B). Conversely, 50 µM ONOO
preincubated for 20 s at pH 7.4 before
addition to liposomes was totally devoid of effect. Characterization of
uptake kinetics without ONOO
gave a K
value of 11 ± 0.8 µM and a V
of 5.7 ± 1 nmol/min/mg of protein. ONOO
(50 µM, 70 s) reduced V
by
50% without affecting K
(Fig. 1C).
Different from ONOO
, 3 fast NO donors, failed to
significantly modify reconstituted glutamate uptake. In a few cases,
MAHMA/NO (1 mM) or SNAP (1 mM) were used, while in
most experiments we utilized DEA/NO (0.1-10 mM). Uptake
assay was run after preincubation (2 min) and removal of DEA/NO or in
its presence. In either case, the compound produced weak inhibition
(
10%) that was similar at 0.1, 1, and 10 mM (Fig. 2A). However, in the same condition, NO
release from DEA/NO, observed spectrophotometrically (see
``Experimental Procedures''), was found to proceed dose
dependently, with a t
of 2 min.
NO
accumulated accordingly. Finally, we
confirmed that active NO was indeed released from DEA/NO. Thus, the
compound-induced dose-dependent accumulation of cGMP in synaptosomal
soluble fraction from rat cortex cGMP was 0.1 ± 0.03 nmol/sample
in controls (2 min, PBSG), 0.08 ± 0.03 in 1 mM DEA,
0.17 ± 0.02 in 0.1 mM DEA/NO (+117%), and 0.33
± 0.05 in 1 mM DEA/NO (+312%) (n =
3 in duplicate). Different from NO and similar to
ONOO
, H
O
, another biological
oxidant, inhibited reconstituted glutamate transport. Like
ONOO
, H
O
selectively reduced
uptake V
; however, it induced a comparable level
of inhibition only at mM concentrations (-38.9 ±
8.2% at 10 mM), after long exposure (10 min) and in highly
oxygenated buffer (Fig. 2B).
Figure 1:
Characteristics of the inhibitory
effect of ONOO on reconstituted glutamate transport.
[
H]Glutamate uptake was run on partly purified
glutamate transporters reconstituted in liposomes and exposed to
ONOO
or decayed ONOO
as control
(see ``Experimental Procedures'' for details). Decayed
ONOO
is an ONOO
solution left to
decompose for at least 1 min in PBSG, pH 7.4. Time course of decay
indicates that the species active on glutamate transport disappear
within 20 s from addition of ONOO
to PBSG. A, dose dependence of ONOO
inhibition. Data
are expressed as % inhibition ± S.D. of control uptake (n = 3 in triplicate). ONOO
inhibition was
significant at all tested concentrations (p < 0.01). B, time course of ONOO
inhibition. Liposomes
were exposed for 10, 20, 70, 190, or 310 s to 50 µM ONOO
and uptake assay run in the last 15 s,
except for the 10-s point where uptake was run during ONOO
exposure. Data are expressed as % inhibition ± S.D. of
control (n = 3 in triplicate). C, effect of
ONOO
on the kinetics of glutamate uptake. Assay run
for 2 s on liposomes pre-exposed to control (
) or 50 µM ONOO
(
): (n = 2 in
quadruplicate, see (13) for details). Kinetic analysis was
performed with the Enzpack 3.0 program and the direct linear plot
method(13) . D, left, effect of ONOO
on the
Rb content of liposomes. Radioactivity in
liposomes with KP
+
Rb as internal medium
was measured after exposure to either control (=100%) or
ONOO
(5 or 50 µM). No significant
difference was observed (n = 2 in triplicate). D,
right, effect of ONOO
on glutamate transport
reconstituted in liposomes with different vitamin E content. The effect
of ONOO
(50 µM, 70 s) was compared in
liposomes formed without (=100%) or with vitamin E as a membrane
constituent (1:100 or 1:25 (w/w) with phospholipid). No significant
difference was observed (n = 3 in triplicate). The
effective presence of vitamin E in the membranes was confirmed by
chloroform extraction from liposomes blotted on filters after uptake
assay and fluorescence detection (30) .
Figure 2:
Concentration-dependent inhibition of
reconstituted glutamate transport by HO
but not
by NO. [
H]glutamate uptake was run on partly
purified glutamate transporters reconstituted in liposomes and exposed
to either DEA/NO (hatched bars) or H
O
(hollow bars) (see ``Experimental
Procedures''). Data are expressed as percent inhibition ±
S.D. of control uptake (n = 4 in triplicate). Control
for DEA/NO was DEA (diethylamine hydrochloride). Other controls tested
and without effect on uptake included NaNO
at pH 7.4,
NaNO
, and a decomposed DEA/NO solution (for 24 h).
H
O
effect, but not that of DEA/NO, was
significantly different from control at all the reported concentrations (p < 0.01).
Glutamate uptake
inhibition with ONOO or H
O
could be due to peroxidation of the liposome membranes resulting
either in loss of the ion gradients fueling the uptake process or
changes in the lipidic environment of the transporter proteins. To
exclude the first possibility, liposomes were preloaded with
Rb and exposed to ONOO
(5-50
µM). No release of radioactivity was observed at times
parallel to uptake inhibition (Fig. 1D, left).
To address the second possibility, we prepared liposomes containing the
lipophilic antioxidant vitamin E among the lipid constituents (1:100 or
1:25 w/w). The effect of ONOO
(50 µM, 70
s) was compared in liposomes with and without vitamin E, finding the
same extent of uptake inhibition in all types of liposomes, independent
of their vitamin E content (Fig. 1D, right).
Moreover, by use of a standard assay, we failed to detect any
significant signal of lipid peroxidation in conjunction with uptake
inhibition. H
O
(10 mM, 10 min) behaved
identically to ONOO
in the above experiments. We then
tested the effect of ONOO
and H
O
on uptake by recombinant transporter subtypes. HeLa cells were
transfected with cDNAs encoding the glutamate transporters GLT1, GLAST,
or EAAC1 and their cell membranes reconstituted into liposomes. Uptake
capacity was found about 100- (GLT1), 20- (GLAST), and 50-fold (EAAC1)
higher with respect to mock-transfected cells similarly reconstituted.
As shown in Table 1, µM ONOO
inhibited uptake by any transporter subtype dose-dependently and
with equivalent potency. Again, mM H
O
behaved comparably.
ONOO, a biological oxidant and the
combination product of
O
and NO, potently inhibits purified and recombinant glutamate
transporters reconstituted in liposomes. NO alone appears unable to
directly modify glutamate transport. Thus, 3 fast NO releasers,
MAHMA/NO (1 mM), SNAP (1 mM), and DEA/NO (0.1, 1, and
10 mM), failed to inhibit reconstituted transport. To be sure
that active NO indeed was released, we checked that the dose- and
time-dependent NO disappearance from the DEA/NO adduct paralleled
NO
accumulation and that DEA/NO
dose-dependently enhanced cGMP levels in synaptosomal soluble fraction.
We reported previously that oxygen radicals and HO
induce long-lasting decreases of glutamate transport in
astrocytic cultures, probably due to protein oxidation (15) .
In agreement, here we find that H
O
directly
affects reconstituted glutamate transport. Its mode of action is
superimposable to that of ONOO
. Thus, both agents
selectively reduce uptake V
. This effect
involves some persistent modification of proteoliposomes, because it is
observed after removal of the compounds and their by products via gel
filtration. However, it unlikely depends on oxidation of the lipid
component. Thus, (a) ONOO
or
H
O
inhibition is not attenuated in liposomes
containing vitamin E among the membrane constituents (up to
1:10
molar ratio with phospholipids) and (b) a standard lipid
peroxidation assay does not reveal detectable levels of malonaldehyde
or 4-hydroxyalkenals paralleling uptake changes. Generalized membrane
damage is also ruled out because no radioactive leakage is observed
from
Rb-filled liposomes exposed to 5-50 µM ONOO
or to 10 mM H
O
. Therefore, uptake inhibition likely
depends on direct interaction of ONOO
and
H
O
with the glutamate transporter proteins
resulting in covalent modification of their structure. Recombinants of
the 3 major cloned rat brain subtypes, GLT1, GLAST (both glial), and
EAAC1 (neuronal) are all similarly inhibited by ONOO
(or H
O
). Lack of differential sensitivity
suggests that one or more ``oxidant-vulnerable site(s)'' are
present in conserved regions of these proteins.
Although similar in
the mode of action, ONOO is significantly more potent
and rapid than H
O
in inhibiting reconstituted
glutamate transport. Thus, while H
O
effect is
seen at mM concentrations and after several minutes,
ONOO
acts in the µM range and within
seconds (
90% of inhibition at 20 s, paralleled by disappearance of
the active species), in line with its reported half-life and rate of
decomposition at pH 7.4, resulting in the formation of potent oxidant
intermediates with reactivity of hydroxyl radical (
OH)
and other reactive species such as nitronium ion and nitrogen
dioxide(31) . Moreover, ONOO
is effective in
normal air-equilibrated buffer, while H
O
only
in a hyperoxygenated buffer, suggesting that its transformation into
OH
via
O
-driven Fenton reaction
may be required(32) . A common primary target for
ONOO
, H
O
, or downstream
products such as
OH is oxidation of cysteine
sulfhydryl groups. Thiol oxidation by ONOO
proceeds
10
-fold faster than with
H
O
(32) . In addition, ONOO
could induce nitrosylation and/or nitration of aromatic amino
acids. Targeting of transporter SH groups by H
O
and ONOO
would be consistent with our previous
observation that glial uptake, inhibited by oxygen radicals, is
significantly restored with dithiothreitol, a disulfide-reducing
agent(15) .
Inhibition of glutamate uptake by
ONOO may be of pathological significance,
contributing to the build up of excitotoxic extracellular glutamate.
The conditions for local formation of ONOO
at
glutamate synapses exist. Thus, both
O
and NO can be
generated as a result of activation of EAA
receptors(33, 34) . If formed in conjunction,
O
and NO react together
to give ONOO
at a diffusion-limited
rate(17) . Several pathological situations would favor this
process, e.g. because NO levels are enhanced (via activation
of inducible NO synthase(35, 36) ), because
O
catabolism is reduced,
or superoxide dismutase (SOD) activity is altered, as proposed for
mutant SOD1 in familial ALS(37, 38) . Indeed, enhanced
protein tyrosine nitration, a marker for ONOO
formation, was recently reported in mutant SOD1 transgenic mice (39) as well as in other animal models of neurodegenerative
diseases thought to involve excitotoxicity (40, 41) .
The case of ALS is intriguing, as this pathology has been associated
with defect of glutamate transport(2) . µM ONOO
is highly neurotoxic to cultured
neurons(21) . Due to its half-life, ONOO
can
travel quite a distance from the site of production to damage critical
neuronal constituents, such as the neurofilament proteins (37, 38) or the mitochondrial enzyme
aconitase(19, 20) . The present study indicates that
the glutamate transporters could be other important targets of
ONOO
toxicity.