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(Received for publication, December 2, 1994) From the
Arachidonic acid has been proposed to be a messenger molecule
released following synaptic activation of glutamate receptors and
during ischemia. Here we demonstrate that micromolar levels of
arachidonic acid inhibit glutamate uptake mediated by EAAT1, a human
excitatory amino acid transporter widely expressed in brain and
cerebellum, by reducing the maximal transport rate approximately 30%.
In contrast, arachidonic acid increased transport mediated by EAAT2, a
subtype abundantly expressed in forebrain and midbrain, by causing the
apparent affinity for glutamate to increase more than 2-fold. The
results demonstrate that the response of different glutamate
transporter subtypes to arachidonic acid could influence synaptic
transmission and modulate excitotoxicity via positive or negative
feedback according to the transporter(s) present in a particular
region.
Reuptake of L-glutamate in brain is mediated by a
recently isolated family of membrane
proteins(1, 2, 3) . Because of its role as an
excitatory neurotransmitter, regulation of the L-glutamate
concentration in the synaptic space is critical for normal
neurotransmission (for review see (4) ). Another important
aspect of glutamate transport relates to its role in limiting
neurotoxicity, which results from elevated levels of this transmitter
during ischemia(5) . Recently, members of an excitatory amino
acid transporter (EAAT) ( Arachidonic acid is released by
synaptic activation of ionotropic and metabotropic glutamate receptors (8, 9) as well as during
ischemia(10, 11) . Inhibition of glutamate uptake by
arachidonic acid has been proposed to play a role in modulation of
synaptic transmission (12) and
neurotoxicity(10, 11) . However, previous studies of
arachidonic acid actions on glutamate transport have relied upon
experimental systems, which are likely to reflect the activity of
multiple subtypes of transporters such as brain slice and synaptosomal
preparations (13, 14, 15) .The present study
characterizes the actions of arachidonic acid on three cloned human
brain glutamate transporter subtypes (6) expressed in Xenopus oocytes and reveals a heretofore unknown heterogeneity
in the transporter response to arachidonic acid.
Uptake assays were performed in
transfected HEK-293 cells grown to a density of approximately 10
Xenopus oocytes were injected with cRNAs encoding
the human excitatory transporters EAAT1, EAAT2, and EAAT3(6) .
The inward transport current resulting from superfusion of 30
µML-glutamate in voltage-clamped oocytes
expressing EAAT1 was decreased 20-30% by co-application of 100
µM arachidonate (Fig. 1). In contrast, the
transport current mediated by EAAT2 was increased approximately 2-fold
by arachidonate, while the EAAT3 current was increased only slightly (Fig. 1). Both the inhibition and the increase in transport
currents had a rapid onset and reversed slowly upon washout of
arachidonate (Fig. 1). These effects were due to specific
interaction with the transporters, as application of arachidonate alone
or with glutamate did not induce a current in uninjected oocytes (Fig. 1). In addition, arachidonate alone did not induce any
current in oocytes expressing the transporters nor were the glutamate
transport currents altered by co-application of vehicle (0.1% dimethyl
sulfoxide) with glutamate (not shown). In order to determine whether
the arachidonate-induced changes in transport currents mediated by
EAAT1 and EAAT2 resulted from changes in glutamate uptake, measurements
were made of radiolabeled L-glutamate transport mediated by
these subtypes. Uptake of 10 µML-[
Figure 1:
Differential effects of arachidonic
acid on glutamate-induced currents recorded in voltage-clamped
uninjected oocytes (control) and oocytes injected with RNA transcribed
from human EAAT cDNAs. Cells were clamped at -60 mV, and
compounds were superfused for the times indicated by open (30
µML-glutamate) and closed (100
µM arachidonic acid) bars. Identical results were
seen in batches of oocytes from five frogs.
Figure 2:
Arachidonic acid inhibits uptake of L-[
The effects
of arachidonate on transport in oocytes expressing EAAT1 and EAAT2 were
dose-dependent and saturable. The K
Figure 3:
Concentration response for arachidonic
acid effects on glutamate transport currents mediated by EAAT1 (A) and EAAT2 (B). The percentage decrease or
increase was calculated by comparing the current amplitudes induced by
30 µML-glutamate co-applied with varying
concentrations of arachidonic acid to the control transport current
amplitudes in response to 30 µML-glutamate in
the same oocytes. Data points represent mean ± S.E. (n = 3-4).
Figure 4:
Differential effects of arachidonic acid
on transport kinetic parameters for EAAT1 (A) and EAAT2 (B). Data points representing normalized mean ± S.E.
for 5 oocytes were fit to I = I
The pharmacological
mechanism of action of arachidonic acid was investigated by testing
structural analogues and inhibitors of its metabolism. The increase in
EAAT2-mediated transport of 10 µML-glutamate
caused by 100 µM arachidonic acid was compared with equal
concentrations of linolenic, linoleic, or arachidic acid. The order of
efficacy relative to arachidonic acid was linolenic acid (91 ±
21%) > linoleic acid (49 ± 9%) Arachidonic acid has been proposed to be a messenger molecule
that influences synaptic transmission released by synaptic activation
of ionotropic and metabotropic glutamate
receptors(8, 9) . Its release also occurs following
ischemia(10, 11) . Arachidonic acid-mediated
inhibition, but not stimulation, of glutamate transport has been
reported
previously(13, 14, 15, 16) . Similar
to the results in the present study, these inhibitory actions appear to
be mediated by arachidonic acid itself, as are its potentiating actions
on synaptic transmission in hippocampus(12) . Although net L-[ The glutamate receptor-mediated
stimulation of arachidonate synthesis in glial cells (17) and
neurons (8, 9, 18) suggests that arachidonic
acid could modulate uptake in both cell types according to which
transporter subtype is expressed. In one well defined system, the
salamander retinal Mueller cell, arachidonic acid directly inhibits
glutamate transport currents(16) . In accord with this result,
a transcript encoding a glutamate transporter homologous to EAAT1 is
highly abundant in these cells, and expression of this salamander
transporter in Xenopus oocytes confirms that it is negatively
modulated by arachidonate. ( The present results suggest the possibility that arachidonic acid
could differentially influence the rate of clearance of synaptically
released glutamate, which can in turn influence the kinetics of
glutamatergic transmission at some synapses(21, 22) .
Moreover, modulation of interstitial glutamate levels could lead to
changes in synaptic efficacy by tonic activation of receptors (23) or receptor desensitization(24) . The actions of
arachidonic acid on glutamate transport kinetics would allow for
complex regulation of synaptic transmission as well as excitotoxicity
via either positive or negative feedback according to which transporter
subtypes are present in a particular pathway.
Volume 270,
Number 12,
Issue of March 24, 1995 pp. 6433-6435
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
)gene family expressed in human
brain have been molecularly cloned and expressed (6) . These
human transporters as well as corresponding rat homologs are
differentially expressed in various brain
regions(6, 7) .
Expression of EAAT Subtypes in Oocytes and HEK293
Cells
Capped RNA was transcribed from linearized pOTV plasmids
containing the coding region of EAAT1-3 (6) using T7
polymerase (Boehringer Mannheim). 50 ng of RNA was injected into stage
V oocytes, and experiments were performed 2-6 days later. Stably
transformed HEK-293 cells expressing various EAATs were obtained by
transfection with pCEP4
E constructs that confer hygromycin
resistance and utilize the cytomegalovirus promoter to drive
transporter expression. pCEP4E was derived from pCEP4 (Invitrogen)
by restriction digest with EcoRV and StuI and
religation to delete the EBNA-1 gene and OriP. EAAT coding sequences
were isolated as KpnI/BamHI fragments and subcloned
into pCEP4E. HEK-293 cells stably transformed with the
pCEP4E-EAAT plasmids were selected using hygromycin (Sigma) at a
concentration of 250 µg/ml.L-[
Uptake was measured in
control (uninjected) oocytes and in oocytes expressing EAAT1-3
during a 10-min incubation in ND-96 buffer (96 mM NaCl, 2
mM KCl, 1.8 mM CaCl
H]Glutamate Uptake
Measurements in Oocytes and HEK293 Cells
, 1 mM MgCl, 5 mM HEPES pH 7.4) containing 10
µML-[H]glutamate.
Radiolabeled L-glutamate (Amersham) was diluted with an
appropriate concentration of unlabeled L-glutamate to give a
final specific activity of 100 µCi/µmol. Uptake was terminated
by three rapid washes in ice-cold buffer followed by lysis in 0.1% SDS
and scintillation counting.
cells/well by incubation with 10 µML-[H]glutamate in serum-free
Dulbecco's modified Eagle's medium for 10 min followed by
three washes in ice-cold buffer.Electrophysiology
Two-electrode voltage clamp
recordings were made with an Axon Instruments Geneclamp interfaced to a
Macintosh computer with a MacLab 2E analog/digital converter
(ADInstruments, Milford, MA). Oocytes were voltage-clamped at -60
mV and continuously superfused with ND-96 recording solution or
solution containing test compounds at various concentrations. The
normalized mean concentration response of currents induced by L-glutamate was fitted by least squares to the equation, I = I
([Glu]/([Glu]
+ K
). K values are
expressed as mean ± S.E from fits to individual oocytes. The
effects of arachidonic acid on glutamate-induced currents were
determined by co-application as indicated by bars above current traces.
The percentage decrease (EAAT1) or increase (EAAT2) in the transport
current was calculated by comparing the current amplitudes induced by
30 µML-glutamate co-applied with varying
concentrations of arachidonic acid to the control transport current
amplitudes in the same oocytes. Data were fitted to the expression, %
change = % change([arachidonic
acid]
/([arachidonic acid] + K
). K values
are expressed as mean ± S.E. from fits to individual oocytes.
Experiments were repeated in batches of oocytes from five different
frogs. Arachidonic acid (Calbiochem) was stored in 100 mM stock solutions in dimethyl sulfoxide and dissolved in recording
solution by sonication immediately prior to use. All other compounds
were from Sigma.
H]glutamate into oocytes
expressing the transporters was linear for at least 20 min and was
increased 10-100-fold over control (uninjected or water-injected)
oocytes. In agreement with voltage clamp measurements, addition of
arachidonic acid (100 µM) resulted in differential effects
on the transporters (Fig. 2). Specific uptake in oocytes
expressing EAAT1 was decreased from 275 ± 9 to 210 ± 10
fmol/oocyte/s (mean ± S.E. n = 6). In contrast,
uptake in oocytes expressing EAAT2 was increased from 75 ± 6 to
143 ± 11 fmol/oocyte/s (n = 6; Fig. 2).
Similar effects were seen in measurements of 10 µML-[H]glutamate into the human
embryonic kidney cell line HEK-293 stably transfected with EAAT1 or
EAAT2. Co-application of 100 µM arachidonate reduced
EAAT1-mediated uptake from 158 ± 8 to 123 ± 9
pmol/10 cells (n = 6), while uptake of L-[H]glutamate mediated by EAAT2 was
increased from 120 ± 6 to 175 ± 6 pmol/10 cells (n = 6). The background in control
(non-transfected) cells was not significantly changed by addition of
arachidonate (10.9 ± 1.0 control versus 9.9 ±
1.0 pmol/10 cells; n = 6).
H]glutamate into oocytes expressing
EAAT1 and stimulates uptake mediated by EAAT2. Uptake in control
(uninjected) oocytes was unaffected by arachidonic
acid.
for the
maximal inhibition of the EAAT1 current was 16 ± 6
µM, while the K for maximal
increase of the EAAT2 current was 59 ± 6 µM (n = 5; Fig. 3, A and B). The kinetic
mechanism underlying the modulation of transport was investigated by
analyzing the effect of arachidonic acid on the glutamate concentration
response. Co-application of 100 µM arachidonic acid with
varying doses of glutamate to oocytes expressing EAAT1 resulted in a 29
± 1% decrease in the maximal current relative to control, while
the apparent affinity for glutamate was not significantly altered (K = 42 ± 3 versus 37
± 1 µM, n = 5; Fig. 4A). In contrast, the EAAT2 transport currents
were enhanced via a decrease in the transport affinity constant for
glutamate from 36 ± 7 to 14 ± 1 µM (n = 5; Fig. 4B).
([Glu]/([Glu] + K
). A, co-application of 100
µM arachidonic acid reduced the maximal EAAT1 current 29
± 1% without significantly changing the apparent affinity for
glutamate. B, glutamate concentration dependence of EAAT2
transport currents was shifted by 100 µM arachidonic acid
from 36 ± 7 to 14 ± 1 µM without
significantly affecting the I.
arachidic acid (1
± 5%; n = 3). The same rank order of efficacy
relative to arachidonic acid was observed for inhibition of EAAT1:
linolenic (109 ± 19%) > linoleic (43 ± 14%)
arachidic (5 ± 5%; n = 3). Neither the
cyclooxygenase inhibitor indomethacin (100 µM) nor the
lipoxygenase inhibitor nordihydroguaritic acid (50 µM)
affected arachidonate inhibition of EAAT1 (n = 3) or
stimulation of EAAT2 (n = 6). These results, together
with the rapid onset of modulation (Fig. 1), suggest that both
effects on glutamate transport are mediated directly by arachidonic
acid itself, rather than via a metabolite.
H]glutamate uptake into rat brain
synaptosomes and slices is reduced by arachidonic
acid(13, 14, 15) , regional heterogeneity in
transporter expression would not be resolved in such an
assay(6, 7) . EAAT1 is relatively abundant in many
human brain regions, particularly in cerebellum, while EAAT2 is highly
abundant in forebrain and midbrain regions including cortex and basal
ganglia(6) . EAAT3 is uniformly expressed in brain and
periphery at lower levels(6) . These distributions are
consistent with the immunohistochemical localization in rat brain of
the analogous glutamate transporters with which they share >90%
sequence identities (7) .)While some studies on cultured
mammalian glial cells have demonstrated arachidonic acid inhibition of
glutamate uptake(15, 19) , a recent study utilizing
cultured astroglial cells demonstrated that inhibition of arachidonic
acid synthesis during ischemia potentiates toxicity, suggesting a
possible neuroprotective action of arachidonic acid(20) .
))
We thank Alan North, Gary Westbrook, Scott Eliasoff,
Jacques Wadiche, and Nancy Zahniser for discussion.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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