From the
Glutamate is believed to be the major excitatory transmitter in
the mammalian central nervous system. Keeping the extracellular
concentration of glutamate low, the glutamate transporters are required
for normal brain function. Arachidonic acid (AA) inhibits glutamate
uptake in relatively intact preparations (cells, tissue slices, and
synaptosomes (Rhoads, D. E., Ockner, R. K., Peterson, N. A., and
Raghupathy, E. (1983) Biochemistry 22, 1965-1970 and
Volterra, A., Trotti, D., Cassutti, P., Tromba, C., Salvaggio, A.,
Melcangi, R. C., and Racagni, G. (1992b) J. Neurochem. 59,
600-606). The present study demonstrates that the effect of AA
occurs also in a reconstituted system, consisting of a purified
glutamate transporter protein incorporated into artificial cell
membranes (liposomes). The characteristics of the AA effect in this
system and in intact cells are similar with regard to specificity,
sensitivity, time course, changes in V
Glutamate is probably the major mediator of excitatory signals
in the mammalian central nervous system (Fonnum, 1984). Not only is
glutamate required for most aspects of normal brain function, but it is
believed to have pathological implications as well, because
glutamatergic overstimulation can damage neurons (Olney, 1990).
Consequently, the extracellular concentration of glutamate has to be
kept low (in the order of 1 µM). As the brain contains
huge amounts of glutamate (about 10 mmol/kg, wet weight), powerful
uptake systems are present in both glial cells and neurons (for review,
see Danbolt (1994)). The driving forces of the uptake process are the
membrane potential and the gradients of Na
Arachidonic acid (a fatty acid consisting of a
20-atom-long carbon chain with 4 double bonds and a carboxylic group at
one end, 20:4) has been reported to inhibit several sodium-coupled
amino acid transporters including the uptake systems for glutamate,
glycine, and GABA
All of the previous studies on arachidonic
acid inhibition of amino acid uptake have been based on intact cells or
complex preparations such as tissue slices and synaptosomes. It could
therefore be argued that the effect of arachidonic acid is not a direct
action on the transporter itself or its lipidic environment but an
indirect effect through other mechanisms. Arachidonic acid influences
several molecules (such as the
Na
In some experiments,
liposomes were formed in the presence of labeled glutamate. 150 µl
of phospholipid/cholate/salt mixture was mixed with 100 µl of
buffer (no protein) and gel filtered on spin columns equilibrated with
sodium phosphate buffer containing labeled glutamate. By collecting
these liposomes on Millipore filters (0.45-µm pores) and comparing
the radioactivity retained with that in the buffer, the internal volume
of the liposomes was found to be in the order of 20 µl/ml of
liposome suspension. The integrity of the liposome membranes after
exposure to arachidonic acid was tested by incubating the liposomes
with arachidonic acid (50, 125, or 300 µM) for up to 120
min and checking if arachidonic acid induced a glutamate leak from the
liposomes ().
When the
liposomes were incubated with low concentrations of glutamate (below
100 nM), the uptake was fairly linear with time for 1 min, but
when high concentrations (above 3 µM) were used, the
transport was linear with time for much shorter periods. In this study,
an incubation time of 2 s has been used for the determinations of
kinetic parameters. This is the shortest time consistent with
reproducible results using available equipment.
The amount of arachidonic acid dissolved in the
liposome membranes was assessed by treating liposomes as above, but
using tritiated arachidonic acid diluted with unlabeled arachidonic
acid to 300 µM final concentration. The liposomes were
diluted as above and centrifuged (Beckman TL-100 microultracentrifuge,
100,000 rpm for 15 min), and the radioactivity in pellet and
supernatant were compared.
Although the lipidic
arachidonic acid that had accumulated in the membranes during the
preincubation, did not affect the transport activity, it dramatically
increased the sensitivity of the transport to aqueous arachidonic acid.
Thus, as shown in Fig. 4, liposomes preincubated with 300
µM arachidonic acid (15 min) and diluted as above were
strongly inhibited by as little as 5 µM arachidonic acid
in the dilution medium. 5 µM arachidonic acid had no
effect on nonpretreated liposomes (compare with Fig. 1).
Interestingly, a similar increase in sensitivity to arachidonic acid
was observed after preincubation with arachidonic acid-ethyl ester
(Fig. 4), which in itself is completely inactive on glutamate
transport (Figs. 3-5). Moreover, when arachidonic acid and its
ethyl ester were added together (100 µM of each) in a
competition experiment using liposomes that had not been pretreated,
the inactive ester increased the effect of arachidonic acid rather than
reducing it (Fig. 5).
The finding that arachidonic acid inhibits the uptake of
glutamate at concentrations compatible with intact membranes,
demonstrates that the inhibitory effect is a direct action on the
transporter protein itself or its lipidic environment. The effect of
arachidonic acid on the purified and reconstituted transporter shows
characteristics very similar to those observed in cultured astrocytes
and Müller cells. The time course of the inhibition closely
resembles that previously reported in astrocytes (see Fig. 3 A in Volterra et al. (1992b)) with a characteristic fast
onset (0-1 min), a slower rise (1-15 min in liposomes
versus 1-10 min in astrocytes), and a plateau phase
(after 15 min), indicating saturation of the effect with time. The
relative potencies of different fatty acids and the changes in
V
The
prevailing view, as expressed in the cited literature (Rhoads et
al., 1983; Yu et al., 1986; Barbour et al.,
1989; Zafra et al., 1990; Volterra et al., 1992a,
1992b), is that arachidonic acid probably affects the lipidic
environment of the transporters. In contrast to the prevailing view,
the data presented in this study suggest that the major component of
the arachidonic acid effect is a direct action on the protein or the
protein-lipid boundary caused by the arachidonic acid present in the
water phase. The main support for this idea are the data presented in
; the inhibition disappears after dilution of the
liposomes, although about 95% of the arachidonic acid is still present
in the membranes. Thus, dilution of the water phase does not
appreciably affect the lipid phase, but it removes the inhibition. An
unspecific effect on the membrane fluidity cannot be the predominant
mode of action. This interpretation is supported by the finding
(Fig. 3) that esterification of the carboxylic ``head''
of the fatty acid destroys the inhibitory activity in spite of the fact
that the esterification increases the lipophilicity and thereby the
tendency of the molecules to associate with the membranes. Furthermore,
as is shown in Fig. 4, it is possible to increase the sensitivity
of the glutamate transporters to freshly added aqueous arachidonic acid
by pretreating the liposomes with either arachidonic acid or the
inactive ethyl ester. Finally, Fig. 5shows that the ethyl ester
strongly potentiates the effect of arachidonic acid. The easiest way to
explain these data is to assume that there are two different types of
binding sites for arachidonic acid on the liposomes. The first type is
sites directly involved in the inhibition of glutamate transport. These
sites require aqueous arachidonic acid and have to be on the
transporter protein or protein-lipid boundary. The second type is sites
in the lipids. These sites act like a sink and remove the fatty acid
from the water phase, making it unavailable for interaction with the
transporter protein. The ethyl ester probably substitutes for the free
acid only at these sites. When these lipidic sites are saturated by the
ethyl ester, added arachidonic acid will be more potent because more of
it will be available for interaction with the transporter protein.
The substrate affinity of the glutamate transporters has been a
matter of discussion (for review, see Danbolt (1994)). We show here
that the uptake in proteoliposomes is not linear with time for more
than 2 s at highest glutamate concentrations. Because of this, the true
K
In
conclusion, our data suggest that inhibition of glutamate uptake by
arachidonic acid derives from the reversible binding of free aqueous
arachidonic acid to either the transporter protein or the protein-lipid
boundary. The binding, which requires both a hydrophobic
cis-polyunsaturated carbon chain and a hydrophilic carboxyl
group, is probably of low affinity, since it is easily reversed by
simple dilution. Binding of arachidonic acid to the phospholipid
membranes does not seem to affect the function of the glutamate
transporter. However, the lipids seem to bind arachidonic acid tightly,
thereby extracting it from the water phase, i.e. from the
active pool. This would imply that the active arachidonic acid
concentration is significantly lower than that nominally applied. Since
the GABA transporter(s) were inhibited in a similar manner although
they belong to a different protein family with no significant primary
structure identity with the glutamate transporters (for review, see
Danbolt (1994)), the effect of arachidonic acid on membrane proteins in
general could be due to the aqueous rather than the lipid bound fatty
acid. To our knowledge, this is the first report where it has been
possible to conclude whether the fatty acid effect is directly on the
target protein or secondary to changes in the membrane fluidity. Direct
modulation by arachidonic acid of integral membrane proteins has been
reported ( e.g. Ordway et al. (1991)), but these
studies were not able to distinguish the two possibilities.
Liposomes (without
protein) loaded with labeled glutamate and suspended in glutamate free
medium were exposed to arachidonic acid (50, 125, or 300
µM) for 15, 30, or 120 min and collected on Millipore
filters as described under ``Experimental Procedures.'' The
results represent the mean ± S.E. of three experiments expressed
as percent of control (values obtained without arachidonic acid).
Control values for the experiments with exposure time of 15, 30, and
120 min were 4511 ± 221, 4667 ± 15, and 5371 ± 653
cpm. Addition of Triton X-100 (0.5 µl/ml) for 30 s reduced the
radioactive content to less than 1%.
Liposomes with purified glutamate transporters and internal
potassium were suspended in potassium-free PBSG and preincubated (15
min) with 300 µM arachidonic acid (AA).
, and
affinity. AA-ethyl ester is inactive, suggesting that the free
carboxylic group is required for inhibitory activity. When incubated
with proteoliposomes, AA (300 µM, 15 min) mostly
partitions to the lipid phase (lipid/water about 95:5). However, uptake
inhibition is abolished by rapid dilution (6.5-fold) of the incubation
medium (water phase), a procedure that does not modify the amount of AA
associated with lipids. On the contrary, inhibition remains sustained
if the same dilution volume contains as little as 5 µM AA,
a concentration inactive before saturation of liposome lipids with 300
µM AA. The same degree of inhibition (60%) is obtained by
5 µM AA following preincubation with the inactive AA-ethyl
ester (300 µM) instead of AA. The lipids apparently
inactivate AA by extracting it from the water phase. The results
suggest that AA acts on the transporter from the water phase rather
than via the membrane. This could be true for other proteins as well
since
-aminobutyric acid uptake is similarly affected by AA.
,
K
, and OH
(Kanner and Sharon, 1978;
Bouvier et al., 1992). A change in the function of these
transporters may have significant effects on synaptic transmission
(Mennerick and Zorumsky, 1994; Barbour et al., 1994). Despite
their obvious importance, little is known about their regulation
(Danbolt, 1994). Impaired uptake may play a role in epilepsy (During
and Spencer, 1993) and in neurodegenerative diseases (Rothstein et
al., 1993).
(
)
(Rhoads et al.,
1983; Chan et al., 1983; Yu et al., 1986; Barbour
et al., 1989; Zafra et al., 1990; Volterra et
al., 1992b; Lynch et al., 1994; Volterra et al.,
1994). Arachidonic acid is released from both neurons (Dumuis et
al., 1988, 1990, 1994; Lazarewicz et al., 1990; Sanfeliu
et al., 1990) and glial cells (Stella et al., 1994)
upon activation of glutamate receptors. Free fatty acids, including
arachidonic acid, are known to accumulate in the brain under
pathological conditions such as ischemia and seizures (Siesjö
et al., 1989)
/K
-ATPase (Okun et al.,
1992; Caspers et al., 1993) and protein kinase C (Hardy et
al., 1994)) that are known to affect glutamate uptake.
Furthermore, arachidonic acid in high concentrations has detergent-like
physicochemical properties and might damage cell membranes and
subsequently impair the ion gradients driving the uptake process. The
successful purification of a glutamate transporter (Danbolt et
al., 1990) has made it possible to study the function of this
protein in the absence of other proteins. The present report, which is
based on highly or partly purified glutamate transporters reconstituted
into artificial membranes (liposomes), demonstrates that the effect of
arachidonic acid is similar to that reported in intact preparations and
therefore does not require the presence of other proteins. We provide
evidence that the glutamate transporter protein itself is sensitive to
low micromolar concentrations of aqueous arachidonic acid.
Materials
L-[H]Glutamic
acid (50 Ci/mmol) and [
H]GABA were purchased from
Amersham Corp. [5,6,8,11,12,14,15-
H]arachidonic
acid (210 Ci/mmol) was from DuPont NEN (Boston, MA). Sephadex G-50 fine
was from Pharmacia Biotech Inc. Affi-Gel ( N-hydroxysuccinimide
esters of cross-linked agarose) and hydroxylapatite were from Bio-Rad.
Diethylaminoethyl (DEAE)-cellulose (DE52) was from Whatman (Maidstone,
Kent, United Kingdom). CHAPS, fatty acids, wheat germ agglutinin as
free lectin were from Sigma. Stock solutions of the fatty acids were
made in dimethyl sulfoxide (Me
SO) and aliquoted and stored
at
20 °C. Wheat germ agglutinin was immobilized to agarose
as described previously (Danbolt et al., 1992). All other
reagents were of analytical grade or better.
Rapid Gel Filtration on Spin Columns
Spin columns
have been used in this study both for reconstitution of purified
glutamate and GABA transporters and for replacing the external medium
of the liposomes with another medium. They have also been used to
remove substances that interfere with protein measurement. Sephadex
G-50 fine is swollen in buffer overnight and packed in plastic syringes
(1 ml) from which the pistons have been removed and the outlets closed
by cotton fiber. The columns are then centrifuged (1200 rpm, 240
g; 2 min; Heraeus-Christ Minifuge-T) to remove the
void volume. After applying the sample (0.2 ml) and allowing it to sink
into the gel, the columns are centrifuged again (2 min, 1740 rpm, 500
g). The conditions are adjusted using phenol red and
blue dextran 2000 as low and high molecular mass markers, respectively.
The efficiency of the spin columns is illustrated by the addition of
[
H]glutamate to the sample. The columns reduce
the concentration about 5000-fold.
Purification and Reconstitution of Glutamate and GABA
Transporters into Liposomes
This has been done as described
previously (Danbolt et al., 1990, 1992). Briefly, crude rat
brain plasma membranes were solubilized with a zwitterionic detergent
(CHAPS) and centrifuged. The supernatant (CHAPS extract) was passed
through a wheat germ agglutinin-agarose column, and the glycoproteins
were eluted with N-acetylglucosamine. The glycoproteins
(``partially purified transporter'') in this fraction were
either reconstituted directly or further purified by chromatography on
hydroxylapatite and DEAE-cellulose before reconstitution (``highly
purified''). The first purification step results in about a
10-fold purification of both glutamate and GABA transporters with
respect to protein, but removes virtually all low molecular mass
species like ATP and endogenous lipids. After the second and third
purification steps, one predominant band is seen on SDS-polyacrylamide
gel electrophoresis, and the specific glutamate transport activity is
increased 30-fold in spite of inactivation (Danbolt et al.,
1990). This preparation contains the glutamate transporter GLT-1 (Pines
et al., 1992; Levy et al., 1993), but neither the
glutamate transporter GLAST (Lehre et al., 1995) nor any GABA
transporters (Danbolt et al., 1990). For reconstitution, the
fractions (containing 20 mM CHAPS and 0.1-0.2 mg of
protein/ml) were mixed with 1.5 volumes of a phospholipid/cholate/salt
mixture (Danbolt et al., 1990), incubated on ice, and gel
filtered to remove detergent and sodium ions on spin columns (see
above) equilibrated with a medium containing potassium phosphate. The
liposomes form spontaneously during this gel filtration, and the buffer
these columns are equilibrated with becomes the internal medium of the
liposomes. The liposome suspension contained 40-80 µg of
protein and 30 mg of phospholipids/ml.
Determination of Glutamate and GABA Transport
Activity
This was done essentially as described (Danbolt et
al., 1990). Briefly, 20 µl of proteoliposomes, containing and
suspended in 0.12 M potassium phosphate buffer, pH 7.4, were
diluted into 500 µl of PBSG (140 mM NaCl, 10 mM
sodium phosphate buffer, 1% (v/v) glycerol, pH 7.4). MeSO
with or without fatty acids was included in the incubation medium. The
final Me
SO concentration was kept constant at 1% (v/v). The
mixture was preincubated (unless stated otherwise, 15 min at room
temperature) before starting the uptake reaction by adding 1.4 µCi
of labeled amino acid and 3 µM valinomycin (a selective
potassium ionophore to ensure a negative membrane potential). The
liposomes were incubated (room temperature for 70 s with glutamate or 7
min with GABA, unless stated otherwise). The reaction was terminated by
dilution in 2 ml of ice-cold PBSG and filtration through Millipore HAWP
filters (0.45-µm pores). The filters were rinsed 3 times and
dissolved in Filter Count (Packard) for liquid scintillation counting.
Addition of nigericin (a nonselective potassium-sodium ionophore
abolishing ion gradients) to the incubation medium resulted in a
complete loss of transport activity (equivalent to omitting the
liposomes from the mixture) and served as negative control.
Dilution of Liposomes Preincubated with Arachidonic
Acid
The liposomes obtained after reconstitution were loaded and
suspended in the same potassium-containing buffer. By filtering the
liposome suspension through another set of spin columns (as described
above) equilibrated with PBSG, the free external potassium was removed
from the proteoliposomes. The washed liposomes (still with potassium on
the inside) were diluted 1 + 3 with PBSG containing arachidonic
acid or arachidonic acid ethyl ester (0 or 300 µM, final
concentrations) and incubated (15 min at room temperature). Aliquots of
80 µl of diluted preincubated liposomes were added to different
volumes of uptake medium (0, 80, 160, 240, or 440 µl of PBSG with
1.4 µCi of labeled amino acid, valinomycin, and 1%
MeSO) with arachidonic acid (0, 5, 15, or 300
µM). The reaction was terminated by dilution and
filtration as above.
Protein Determination
Protein content was
determined by the method of Lowry et al. (1951). Interfering
substances (EDTA, CHAPS, and N-acetyl-D-glucosamine),
when present, were removed from the samples by gel filtration on
Sephadex equilibrated with 0.4% SDS as described previously (Danbolt
et al., 1990). Bovine serum albumin was used as standard.
Inhibition of Glutamate Uptake by Arachidonic
Acid
The effect of arachidonic acid was studied in preparations
of glutamate transporters purified and reconstituted as described
previously (Danbolt et al., 1990). The inhibition of glutamate
uptake by arachidonic acid in these preparations is dose-dependent
(Fig. 1) with more than 65% inhibition at 300 µM (15
min preincubation). The effect of 10 µM arachidonic acid
was not significant. No differences were found between the partially
and highly purified glutamate transporter preparations (Fig. 1).
At 125 µM arachidonic acid, 14.2 ± 1.8, 23.8
± 3.5, 45.7 ± 6.6, 57.6 ± 4.4, and 56.6 ±
5.2% inhibition was obtained after 1, 5, 10, 15, and 30 min,
respectively. In the absence of arachidonic acid, the
Vand K
was 10.6
± 0.9 nmol/min/mg of protein and 8.3 ± 0.45
µM, respectively (Fig. 2). Arachidonic acid (125
µM) reduced the V
by 50 ± 5%
( p < 0.01) and increased the affinity slightly, by 19
± 4% ( p < 0.05).
Figure 1:
Concentration-dependent inhibition of
partially and highly purified glutamate transporters by arachidonic
acid. Glutamate uptake activity was determined as described under
``Experimental Procedures'' with a 15-min preincubation time.
The results are expressed as percent inhibition + S.E. of control
uptake. Bars represent the average of three separate
determinations done in triplicate. The inhibition observed was
significantly different from control ( p < 0.01) for all
arachidonic acid concentrations tested except 10 µM. No
differences were noted between the partially and highly purified
preparations.
Figure 2:
Effect of arachidonic acid on the kinetics
of glutamate uptake. Liposomes inlaid with partly purified glutamate
transporters were preincubated (15 min) and incubated (2 s) with
different concentrations of glutamate in the absence () and in the
presence (
) of 125 µM arachidonic acid (see
``Experimental Procedures'' for details). The results are
expressed as mean ± S.E. of four experiments performed in
triplicate. The data were analyzed with the direct linear plot method
(Cornish-Bowden, 1974).
Structure-Activity Study of Transporter Inhibition by
Fatty Acids
The effect of arachidonic acid was compared with
that of other long chain saturated and unsaturated fatty acids as well
as arachidonic acid esters (Fig. 3). Neither the
trans-unsaturated fatty acid elaidic acid (18:1) nor the
saturated fatty acids stearic acid (16:0) and arachidic acid (20:0)
inhibited glutamate uptake. However, all of the
cis-unsaturated fatty acids tested (including oleic acid,
which is a cis-unsaturated analogue of elaidic acid) did
inhibit glutamate uptake. The order of potency was as follows:
eicosapentaenoic acid (20:5) > arachidonic acid (20:4) >
docosahexaenoic acid (22:6) > linolenic acid (18:3) > oleic acid
(18:1). The differences in potency between the various
cis-configured fatty acids seem to correlate with the degree
of unsaturation present in the molecule and thereby with the degree of
folding. ( cis-unsaturated fatty acids are bent, while
trans-configured acids are linear.)
Figure 3:
Inhibition of glutamate uptake by
different fatty acids and arachidonate esters expressed as percent of
the inhibition by arachidonic acid. All agents (100 µM)
were incubated (15 min) with proteoliposomes. Then, glutamate was added
and the mixture incubated for 70 s. Arachidonic acid inhibited
glutamate transport by 32 ± 4.5% at 100 µM (100% in
graph). SA, stearic acid; ARA, arachidic acid;
EA, elaidic acid; OA, oleic acid; LA,
linolenic acid; EPA, eicosapentaenoic acid; DHA,
docosahexaenoic acid, AA-ME, methyl arachidonate;
AA-EE, ethyl arachidonate; AA-CE, cholesteryl
arachidonate. Results are mean + S.E. of three experiments
performed in triplicate.
Upon modification of
the carboxyl-group, arachidonic acid lost its inhibitory activity.
Thus, arachidonic acid-methyl ester showed very low activity, while
arachidonic acid-ethyl ester and cholesteryl-arachidonate were
inactive.
Reversal of Inhibition of Glutamate Transporter
Activity
Liposomes were loaded with potassium phosphate,
suspended in PBSG, and preincubated (15 min) with 300 µM
arachidonic acid. Then, glutamate uptake was determined by mixing the
liposome suspension with 0, 1, 2, 3, or 5.5 volumes of
[H]glutamate-containing uptake medium. 70 s
later, the uptake reaction was stopped. Inhibition by about 60% was
observed when the uptake medium contained arachidonic acid
(). However, when the liposomes were diluted into uptake
medium without arachidonic acid, the inhibitory effect of arachidonic
acid was lost proportionally to the volume of the uptake medium.
Surprisingly, dilution with only 5.5 volumes was sufficient to restore
the transport activity. This 1 + 5.5 dilution would reduce the
average arachidonic acid concentration to 46 µM, which is
expected to give almost 20% inhibition (see Fig. 1). These
experiments show the following three things. 1) The arachidonic acid
effect is reversible. 2) The phospholipid membranes have not been
damaged by a detergent-like effect of arachidonic acid, because, if
they had, there would not have been any ion gradients to drive the
uptake. The integrity of the liposomes is also demonstrated in
. 3) The degree of inhibition cannot be predicted from the
average arachidonic acid concentration (see below). This finding
suggested that the partition of arachidonic acid between the lipid and
aqueous phases might be important.
The Relative Importance of Arachidonic Acid in the Water
and Lipid Phases
In order to determine the amount of arachidonic
acid bound to the lipids after the above dilution of preincubated
liposomes (), liposomes were preincubated (15 min) with
tritiated arachidonic acid (mixed with unlabeled arachidonic acid to a
total arachidonic acid concentration of 300 µM), diluted
as above, and centrifuged (15 min) to separate the liposomes from the
water phase. Surprisingly, 95% of the labeled arachidonic acid remained
in the lipids after dilution and centrifugation. Because dilution
removed transport inhibition but had little effect on the amount of
arachidonic acid incorporated into the liposome membranes, the
lipid-bound arachidonic acid must be less important than the
arachidonic acid in the water phase.
Figure 4:
Preexposure increases arachidonic acid
potency. Liposomes loaded with potassium phosphate and inlaid with
purified glutamate transporters were ( 1step)
suspended in potassium-free PBSG (see ``Experimental
Procedures'' for details) and incubated (15 min) with 300
µM arachidonic acid ( AA300), 300 µM
arachidonic acid ethyl ester ( AA- EE300), or 0
µM arachidonic acid ( CTL). Then ( 2 step), the liposome mixtures were diluted (1 + 5.5) with PBSG
containing labeled glutamate and either arachidonic acid (0
µM, CTL; 300 µM, AA300; 15
µM, AA15; 5 µM, AA5) or
arachidonate ethyl ester ( AA- EE300) and incubated for
70 s. The results are expressed as percent of control
( CTL- CTL); mean + S.E. of four experiments
performed in triplicate. One-way analysis of variance analysis shows
that (*) AA300-AA300 is not significantly different from
AA300- AA15, AA300- AA5, or
AAEE300- AA5, which are all significantly different
from CTL ( p < 0.01).
Figure 5:
The inhibitory action of arachidonic acid
on glutamate uptake is potentiated by arachidonic acid ethyl ester.
Liposomes were preincubated (15 min) with either 100 µM
arachidonic acid ( AA), 100 µM arachidonic acid
ethyl ester ( AAEE), or the two in combination. Glutamate
uptake activity was assessed as described under ``Experimental
Procedures.'' The overall inhibition by AA +
AAEE was reversed by >90% following dilution of the
liposome suspension, demonstrating that the potentiation was not due to
disruption of the liposome membranes (data not shown). The results
represent mean + S.E. of three separate experiments each performed
in quadruplicate.
Inhibition of GABA Uptake by Arachidonic Acid
The
sensitivity of the partly purified and reconstituted GABA transporter
was comparable with that of glutamate uptake. At 25, 50, and 100
µM arachidonic acid (15-min preincubation, uptake reaction
terminated after 7 min), 10 ± 5, 26 ± 5, and 55 ±
7% inhibition was observed. Like glutamate uptake, the GABA uptake was
not sensitive to 100 µM arachidonic acid ethyl ester, and
the arachidonic acid induced-inhibition was completely reversed upon
dilution (1 + 5.5), suggesting that the mechanism of inhibition
may be the same for the two types of transporters.
and K
also fit
with previous findings (Volterra et al., 1992a, 1992b; Barbour
et al., 1989). Thus, the simple reconstituted system with a
single protein residing in an artificial membrane, retains all the
characteristics of arachidonic acid sensitivity. Therefore it is
possible to exclude indirect actions of arachidonic acid such as
changes in the ion gradients (inhibition of
Na
/K
-ATPase, modification of ion
channels, etc.), protein kinase C activation, formation of downstream
mediators (enzymatic derivatives of arachidonic acid: eicosanoids), or
changes in membrane lipid composition due to active esterification of
arachidonic acid in phospholipids by acetyltransferase enzymes.
values are at least 10-fold higher than
those previously reported for proteoliposomes (Danbolt et al.,
1990; Pines et al., 1992; Pines and Kanner, 1990), but they
are in agreement with those determined electrophysiologically.
Table:
Integrity of liposomes loaded with labelled
glutamate after exposure to arachidonic acid
Table:
Reversibility of arachidonic acid-induced
inhibition in liposomes reconstituted with glutamate transporter
protein
-aminobutyric acid; CHAPS,
3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.