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Journal of Neurochemistry, Volume 68(2) February 1997 pp 786-794
Stimulation of [3H]GABA and -[3H]Alanine Release
from Rat Brain Slices by cis-4-Aminocrotonic Acid
Mary Chebib and Graham A. R.Johnston,
Department of Pharmacology, University of Sydney, Sydney, New South Wales,
Australia
Received June 27, 1996; revised manuscript received September 17, 1996; accepted September 17,
1996.
Address correspondence and reprint requests to Dr. M. Chebib at Department of Pharmacology,
University of Sydney, Sydney, New South Wales, Australia.
Abstract: cis-4-Aminocrotonic acid (CACA; 100
µM), an analogue of GABA in a folded
conformation, stimulated the passive release of
3
[ H]GABA from slices of rat cerebellum, cerebral
cortex, retina, and spinal cord and of β3
[ H]alanine from slices of cerebellum and spinal
cord without influencing potassium-evoked
release. In contrast, CACA (100 µM) did not
3
stimulate the passive release of [ H]taurine from
slices of cerebellum and spinal cord or of D3
[ H]]aspartate from slices of cerebellum and did
not influence potassium-evoked release of
3
[ H]taurine from the cerebellum and spinal cord
3
and D-[ H]aspartate from the cerebellum. These
results suggest that the effects of CACA on
GABA and β-alanine release are due to CACA
acting as a substrate for a β-alanine-sensitive
GABA transport system, consistent with CACA
3
inhibiting the uptake of β-[ H]alanine into slices
of rat cerebellum and cerebral cortex. The
3
observed Ki for CACA against β-[ H]alanine
uptake in the cerebellum was 750 ± 60 µM.
CACA appears to be 10-fold weaker as a
substrate for the transporter system than as an
agonist for the GABAC receptor. The effects of
CACA on GABA and β-alanine release provide
indirect evidence for a GABA transporter in
cerebellum, cerebral cortex, retina, and spinal
cord that transports GABA, β-alanine, CACA,
and nipecotic acid that has a similar
pharmacological profile to that of the GABA
transporter, GAT-3, cloned from rat CNS. The
structural similarities of GABA, β-alanine, CACA,
and nipecotic acid are demonstrated by
computer-aided molecular modeling, providing
information on the possible conformations of
these substances being transported by a
common carrier protein. Key Words: GABA
transporters; cis-4-Aminocrotonic acid; βAlanine; Nipecotic acid; CNS; Release.
J, Neurochem. 68, 786-794
GABA
is
the
main
inhibitory
neurotransmitter in the CNS, activating two
major classes of GABA receptors, the GABAA
and GABAB receptors. The GABAA receptors
are heterooligomeric receptors that gate chloride
channels. These were found to be inhibited by
the alkaloid bicuculline (Johnston, 1986). The
GABAB receptors are transmembrane receptors
coupled to second messenger systems and
Ca2+ and K+ channels via G proteins. These
receptors are not blocked by bicuculline but are
activated by (-)-baclofen (Hill and Bowery,
1981). Both GABAA and GABAB receptors play
a significant role in regulating neurotransmitter
release.
There is increasing evidence and
interest in a third class of GABA receptors, the
GABAC (sometimes called GABANANB and ρ
receptors). These are bicuculline-insensitive,
baclofen-insensitive GABA receptors and have
been described in retina, cerebellum, cerebral
cortex, optic tectum, spinal cord, insects, and
bacteria (Johnston, 1994). This receptor was
first described when a series of conformationally
restricted GABA analogues that had bicucullineinsensitive depression actions on neuronal
3
activity and no activity with [ H]baclofen binding
3
were studied against [ H]GABA binding. The
most interesting compound from the study was
cis-4-aminocrotonic acid (CACA) (Drew et al.,
1984; Johnston, 1994). Subsequently, two ρ
subunits have been cloned, ρ1 and ρ2, from
retina cDNA and when the mRNAs are
expressed
in
Xenopus
oocytes
form
homooligomeric
receptors.
Like
GABAA
receptors, these ρ receptors were found to also
gate chloride channels (Cutting et al., 1991,
1992; Polenzani et al., 1991; Shimada et al.,
1992; Kusama et al., 1993a,b; Wang et al.,
1994).
GABAC
receptors
with
similar
pharmacology have been found in neurons in rat
and fish retinas (Feigenspan et al., 1993; Qian
and Dowling, 1993). Although a physiological
role for GABAC receptors has not been
identified, there is electrophysiological evidence
that GABAC receptors, like the GABAA and
GABAB receptors, play a role in regulating
neurotransmitter release (Lukasiewicz and
Werblin, 1994).
spread of synaptically released GABA to
neighboring synapses, and providing a reusable
pool of neuronal GABA for subsequent release.
These
transporters
have
been
pharmacologically described in neuronal and
glial cultures (Balcar et al., 1979; Larsson et al.,
1980,
1983;
Borden
et
al.,
1995a),
synaptosomes (Iversen and Johnston, 1971),
and brain slices (Iversen and Neal, 1968;
Iversen and Johnston, 1971; Johnston and
Stephanson, 1976).
The most potent GABAC agonists are
muscimol, trans-4-aminocrotonic acid, and
GABA. Although these are potent for the GABAC
receptors, they also have activity for other GABA
receptors and are substrates for GABA
transporters. CACA is a conformationally
restricted analogue of GABA in a folded
conformation. It has an inhibitory action on
central neurons that does not appear to be
related to either GABAA or GABAB receptors,
and it appears to be a weak but selective
agonist for the GABAC receptors (Johnston,
1994).
Chemicals
3
[ H]GABA
(71-110
Ci/mmol),
β3
3
[ H]alanine (51 Ci/mmol), D-[ H]aspartic acid
3
(10-25 Ci/mmol), and [ H]taurine (24.1 Ci/mmol)
were purchased from NEN DuPont (Boston, MA,
U.S.A.). Tetrodotoxin (TTX), β-alanine, EGTA,
nipecotic acid, (aminooxy)acetic acid, and L-2,4diamino-n-butyric
acid
(L-DABA)
were
purchased from Sigma Chemical Co. (St. Louis,
MO, U.S.A.). Bicuculline methochloride was
obtained from Tocris Cookson Ltd. (Bristol,
U.K.). Ultima Gold and Emulsifier Safe were
purchased from Packard Instrument, B.V.
Chemical
Operations
(Groningen,
The
Netherlands). CACA was prepared as previously
described (Johnston et al., 1975). 4,4-Diphenyl-
GABA transporters in neurons and glia
play important roles in maintaining low
extracellular levels of GABA, preventing the
Nipecotic acid is a potent inhibitor of
GABA uptake, and, conversely, GABA can
inhibit the uptake of nipecotic acid. GABA can
release preloaded nipecotic acid from brain
slices, and nipecotic acid can release preloaded
GABA, indicating that GABA and nipecotic acid
can be countertransported using the same
mobile carrier (Johnston et al., 1976b). This
study investigates the release of radiolabeled
neurotransmitters from rat CNS slices by CACA
on potassium-stimulated release and for
countertransport. The effects of CACA were
studied on the release of preloaded radioactive
GABA,
β-alanine,
D-aspartate
(a
nonmetabolizable marker for glutamate release),
and taurine from slices of rat cerebellum,
cerebral cortex, retina, and spinal cord.
MATERIALS AND METHODS
3-butenylnipecotic acid (SKF 89976A) was a gift
from Smith Kline & Beecham.
Release experiments
The potassium-stimulated release of
3
3
[ H]GABA, D-[ H]aspartate (a nonmetabolizable
substrate for high-affinity excitatory amino acid
3
transport
systems),
β-[ H]alanine,
and
3
[ H]taurine from slices of rat CNS tissues was
studied using the methodology developed by
Davies and Johnston (1976). Male SpragueDawley rats (weighing 300-350 g) were killed by
first stunning followed by decapitation. The
brain, retinas, and spinal cord were rapidly
removed into iceCold Krebs buffer. Krebs buffer
consisting of 125 mM NaCl, 3 mM KCl, 1 mM
NaH2PO4, 1.2 mM MgSO4, 2.4 mM CaCl2, 22
mM NaHCO3, and 10 mM glucose was
oxygenated with 95% O2 and 5% CO2. The
contents of the Krebs buffer used in the
depolarizing medium were almost identical to
those in the standard medium except for the
concentrations of KCl, which were increased to
3
15 mM in [ H]GABA and 35 mM in D3
3
3
[ H]aspartate, β-[ H]alanine, and [ H]taurine
experiments. KCl replaced an equal amount of
NaCl to maintain osmolarity concentrations. In
3
[ H]GABA experiments, the standard and
depolarizing media also contained 50 µM
(aminooxy)acetic acid, which was used to inhibit
GABA:2-oxoglutarate aminotransferase. The
uptake blockers SKF 89976A (30 µM) (Yunger
et al., 1984) and nipecotic acid (100 µM) were
used in the standard and depolarizing media in
3
some [ H]GABA experiments. L-DABA (200 µM)
3
was used in some [ H]GABA and some β3
[ H]alanine experiments in the spinal cord.
The cerebellum and cerebral cortex
were dissected free on ice, and minislices (250 x
250 µm) were cut (first in a sagittal direction and
then in a transverse direction) using a McIlwain
tissue chopper. Experiments using spinal cord
(lumbar enlargement where only 250-µm slices
were used) followed similar procedures. Retinal
slices were cut using a sharp scalpel blade and
cut into quarters. The slices were incubated in
oxygenated Krebs buffer (10 ml) for 30 min at
o
37 C followed by a further 30-min incubation
3
3
with [ H]GABA (20 µl, 0.02 µM), β-[ H]alanine
3
(15 µl, 0.03 µM), D-[ H]aspartate (20 µl, 0.11
3
µM), or [ H]taurine (40 µl, 0.04 µM). In some
3
[ H]GABA experiments, β-alanine (100 µM) was
used in the incubation, standard, and
depolarizing media. The slices were loaded onto
presoaked glass fiber filters (type AP15;
borosilicate microfiber glass, with acrylic resin
binder; Millipore, Bedford, MA, U.S.A.; 0.5 ml of
Krebs buffer), layered under moderate vacuum
filtration, and then covered with another
presoaked glass fiber filter before being loaded
onto a set of parallel superfusion chambers
maintained at 37oC. Superfusion was started
with the standard medium, and the slices were
washed for 38 min before collection (t = 0). Ten
fractions were collected at 2-min intervals,
during which time, at t = 4, the system was
depolarized for 2 min with 15 mM KCl (for
3
[ H]GABA experiments) or 35 mM KCl (for β3
3
3
[ H]alanine, D-[ H]aspartate, and [ H]taurine
experiments). The system was washed for 8 min
before being superfused with the standard
medium containing 100 µM CACA. Following
equilibration for 12 min (t = 40), an additional 10
fractions were collected at 2-min intervals, and
at t = 44, the system was again depolarized with
3
15 (for [ H]GABA experiments) or 35 mM KCl
3
3
(for
β-[ H]alanine,
D-[ H]aspartate,
and
3
[ H]taurine experiments) containing 100 µM
CACA for 2 min. In experiments containing
bicuculline methochloride, both bicuculline
methochloride (100 µM) and CACA (100 µM)
were added 12 min before t = 40 and continued
throughout the experiment. In experiments
containing L-DABA (200 µM), L-DABA was
added 20 min before t = 40, whereas CACA was
added 10 min before t = 40. Scintillant (Packard
Ultima Gold; 3 ml) was added to the fractions,
and the slices were collected, placed in
scintillation vials containing 1 ml of water, and
left for 24 h before adding scintillant. The
samples were counted for radioactivity on a
Packard Instrument Co. (Downers Grove, IL,
U.S.A.) model 1500 TriCarb liquid scintillation
analyzer with a counting efficiency of tritium of
57%. The release was determined as a
percentage of the total amount of tritium in the
slices at t = 0.
For experiments where calcium was
omitted, the slices were first exposed to Krebs
buffer containing of 0.1 mM CaCl2, and 8 min
before t = 0, the Krebs buffer (0.1 mM CaCl2)
was changed to Krebs buffer containing no
calcium and 0.5 mM EGTA. The slices were
3
stimulated with 15 mM KCl in [ H]GABA
3
experiments and 35 mM KCl in β-[ H]alanine
experiments.
Uptake experiments
3
The uptake of β-[ H]alanine was studied
in cerebellar minislices and essentially following
a procedure similar to that described by Iversen
and Neal (1968) using radiolabeled GABA. In
brief, the brain was removed and placed into
iceCold Krebs-Ringer-HEPES buffer (pH 7.4)
consisting of 118 mM NaCl, 4.75 mM KCl, 1 mM
NaH2PO4, 1.18 mM MgSO4, 2.5 mM CaCl2, 22
mM HEPES (buffer to pH 7.4 with 1 M NaOH),
and 5.8 mM glucose and oxygenated with 95%
O2 and 5% CO2. The cerebellum was dissected
free and cut (first in a sagittal direction and then
in a transverse direction) using a McIlwain tissue
chopper (three cerebella in 33 ml of KrebsRinger-HEPES buffer, pH 7.4; 0.1 x 0.1 µm).
Smaller slices were used for the uptake
experiments than for the release experiments.
The smaller slices increased the reproducibility
within replicates by allowing greater control of
the quantity of tissue in each replicate. This is
not an issue in release experiments because a
percent release is obtained from the amount of
tissue in each chamber that acts as an inbuilt
control. Tissue (100 µl) was used for uptake in a
total volume of 0.5 ml. Various concentrations of
β-alanine (1-10,000 µM) and CACA (1-50,000
µM) were tested to obtain a dose-response
3
curve. β-[ H]Alanine (8 nM) was used to initiate
uptake, and the mixtures were incubated for 30
min at 37 oC. Uptake was terminated by
harvesting (Brandel, Gaithersburg, MD, U.S.A.)
onto Whatman GF/B filters and washing three
times with iceCold saline (0.9% NaCl). The
slices were extracted with 1 ml of water for 1 h.
Nonspecific uptake was measured at zero-time.
Scintillant (Emulsifier Safe; 4 ml) was added to
the filters and left for 24 h before counting. The
samples were counted for radioactivity on a
Packard model 1500 TriCarb liquid scintillation
analyzer with a counting efficiency of tritium of
57%. Six experiments were performed, and
each
concentration
was
assayed
in
quadruplicate.
Calculations
The amount of radioactivity released
into each fraction was expressed as a
percentage of the total tritium content in the
slices at t = 0. The depolarization-evoked
release (S1) was estimated by adding the
percent release of fractions 6 and 7 followed by
subtracting twice the average percentage of
passive release, which was taken from fractions
1-5 (R1). This was repeated for the second
depolarization-evoked release (S2). The percent
releases of fractions 16 and 17 were added
followed by subtracting twice the average
percentage of passive release, which was taken
from fractions 11-15 (R2).
The effect of the drug was evaluated as
the ratio of the depolarization-evoked release
calculated in the presence of the drug
(S2drug/S1drug) compared with that obtained
under control conditions (S2control/S1control)
and determined as a percentage of the control.
The unpaired two-tailed Student's t test using
StatView
SE+Garaphics
(1988;
Abacus
Concepts, Berkeley, CA, U.S.A.) was used to
evaluate the significance of the results (p <
0.05). The effect of drug on the passive release
was evaluated as the ratio of the passive
release in the presence of drug (R2drug/R1drug)
compared with the passive release obtained
under control conditions (R2control/R1control)
and determined as a percentage of the control.
The unpaired two-tailed Student's t test using
Stat-View SE+Garaphics was used to evaluate
the significance of the results (p < 0.05).
In uptake experiments, the program
Prism (1994; Graph-Pad Software, San Diego,
CA, U.S.A.) was used to determine the IC50 of
CACA against β-alanine, whereas the Ki was
calculated using the Cheng-Prusoff equation
(Cheng and Prusoff, 1973) using the average
3
KD value for β-[ H]alanine, calculated from
EBDA and LIGAND programs (G. A.
McPherson, 1987; Elsevier Biosoft), as 3 mM
and a final ligand concentration of 8 nM.
Molecular modeling
A computer-assisted study was carried
out on CACA, GABA, β-alanine, and R(-)nipecotic acid using the programs Chem-X
(1994; Chemical Design Ltd., Oxford, U.K.) and
Chem 3-D (Cambridge Scientific Computing,
Cambridge, MA, U.S.A.) to determine the
conformation by which transport occurs. The
three-dimensional matrices of the compounds
were optimized using the molecular mechanics
optimization
routines
in
Chem-X.
The
conformers of each compound were then
subjected to conformational search routines
about the bonds at which torsional rotations
were possible. A search was then undertaken to
determine the low-energy conformations of each
compound that fitted both R(-)-nipecotic acid
and β-alanine. It is proposed that these
conformations are the conformations of each
respective compound that is acting at the
transporter site.
RESULTS
Figure 1 illustrates the average
3
[ H]GABA outflow from cerebellar slices.
Potassium chloride (15 mM) stimulated the
3
release of [ H]GABA by twofold. CACA (100
µM) does not affect the stimulated release
(Table 1) but increases the passive release of
3
[ H]GABA by 55 ± 10%. The effect of CACA on
3
the passive release of [ H]GABA was more
marked in the spinal cord (200 ± 33%) than the
cerebral cortex (85 ± 9%), the retina (63 ± 2%),
or the cerebellum (55 ± 10%; Fig. 2). CACA (100
µM) had no significant effect on the potassium3
stimulated release of [ H]GABA in the spinal
cord, cortex, and retina (Table 1). Preincubation
of cerebellar and retinal slices with unlabeled βalanine (100 µM) to block uptake of GABA into
3
"glial" cells and then incubation with [ H]GABA
blocked the increase in the passive release of
3
[ H]GABA (Table 1).
FIG. 1. Effect of CACA on KCl-induced and
3
passive release of [ H]GABA in cerebellar
slices. Superfused cerebellar slices (250 x 250
3
µm) loaded with [ H]GABA (0.01 µM) were
depolarized twice (period indicated by solid
bars) with 15 mM KCl. The second
depolarization stimulus was conducted in the
absence or presence of 100 µM CACA. CACA
was added for the duration of the hatched bar.
Data are mean ± SEM (bars) values, displayed
as fractional release observed for each 2-min
fraction collected over two 20-min periods from
four independent experiments performed in
quadruplicate. CACA at 100 µM affects the
3
passive release of [ H]GABA by 55 ± 10% (by
unpaired two-tailed Student's t test, p < 0.05) but
not the potassium-stimulated release.
3
The CACA-stimulated increase in [ H]GABA
release was found not to need calcium, whereas
the potassium-stimulated release required
calcium (2.4 mM) as little release was observed
when calcium was omitted (Table 1). The
3
CACA-stimulated increase in [ H]GABA release
was not blocked by either TTX (1 µM; Table 1)
or bicuculline methochloride (100 µM; Table 1),
suggesting that the effect is not acting by
propagation of an action potential or by GABAA
receptors. This indicated that a transporter
sensitive to GABA may be involved.
3
TABLE 1. Effect of various treatments on passive release of [ H]GABA in brain slices
SKF 89976A (30 µM), a potent GABA uptake
inhibitor, did not block the CACA-stimulated
3
[ H]GABA release in cerebellar slices, whereas
nipecotic acid (100 µM) greatly stimulated
3
[ H]GABA release equally potently in the
presence and absence of CACA (Fig. 3). There
was no observed effect due to CACA (100 µM)
in the presence of nipecotic acid. However, in
contrast, nipecotic acid (100 µM) had no effect
3
on the passive release of [ H]GABA in either the
presence or absence of CACA in the retina
(Table 1). L-DABA (200 µM) had no effect on the
CACA-stimulated
or
potassium-stimulated
3
[ H]GABA release in spinal cord slices (Table 1).
3
[ H]GABA in the presence and absence of SKF
89976A (30 µM). Nipecotic acid (100 µM)
3
increased the passive release of [ H]GABA
([black small square]). CACA (100 µM) had no
significant effect in the presence of nipecotic
acid ([white square]). Data are mean ± SEM
(bars) values, displayed as an average of
baseline fractional release (R2) collected from
two to four independent experiments performed
in quadruplicate. Unpaired two-tailed Student's t
test (p < 0.05) was used for statistical analysis.
FIG. 2. Effect of CACA on the passive release of
3
[ H]GABA in slices of cortex, cerebellum, retina,
and spinal cord. Slices of cortex,
cerebellum, retina, and spinal cord were loaded
3
with [ H]GABA (0.01 µM) and depolarized twice
with 15 mM KCl. CACA (100 µM) ([white
square]) increases significantly the passive
3
release of [ H]GABA under control conditions
([black small square]) from slices of cortex (85 ±
9%), cerebellum (55 ± 10%), retina (63 ± 2%),
and spinal cord (200 ± 33%). Data are mean ±
SEM (bars) values, displayed as an average of
baseline fractional release (R2) collected from
two to four independent experiments performed
in quadruplicate. Unpaired two-tailed Student's t
test (p < 0.05) was used for statistical analysis.
FIG. 3. Effect of CACA on the passive release of
3
[ H]GABA in cerebellar slices in the presence of
the uptake blockers SKF 89976A and nipecotic
acid. Slices of cerebellum were loaded with
3
[ H]GABA (0.01 µM) and depolarized twice with
15 mM KCl. SKF 89976A (30 µM) had no effect
3
on the passive release of [ H]GABA ([black
small square]). CACA (100 µM) ([white square])
increases significantly the passive release of
FIG. 4. Effect of CACA on KCl-induced and
3
passive release of β-[ H]alanine in cerebellar
slices. Superfused cerebellar slices (250 x 250
3
µm) loaded with β-[ H]alanine (0.03 µM) were
depolarized twice (period indicated by solid
bars) with 35 mM KCl. The second
depolarization stimulus was conducted in the
absence or presence of 100 µM CACA. CACA
was added for the duration of the hatched bar.
Data are mean ± SEM (bars) values, displayed
as fractional release observed for each 2-min
fraction collected over two 20-min periods from
four independent experiments performed in
quadruplicate. CACA at 100 µM affects the
3
passive release of β-[ H]alanine by 25 ± 6% (by
unpaired two-tailed Student's t test, p < 0.05) but
not the potassium-stimulated release.
3
Figure 4 illustrates the release of β-[ H]alanine
from cerebellar slices. Potassium chloride (35
3
mM) stimulated the release of β-[ H]alanine by
twofold. A higher concentration of potassium
chloride was used with β-alanine to obtain a
similar amount of release as GABA. CACA (100
µM) does not affect the stimulated release but
3
increases passive release of β-[ H]alanine
(Table 2). As with the effects of CACA on
3
[ H]GABA release, CACA was significantly more
3
effective in stimulating β-[ H]alanine release in
the spinal cord (120 ± 20%) than in the
cerebellum (25 ± 6%; Fig. 5 and Table 2). LDABA (200 µM) had no effect on the CACA- or
3
potassium-stimulated β-[ H]alanine release in
spinal cord slices (Table 2). The CACA3
stimulated increase of β-[ H]alanine release was
found not to need calcium, whereas the
potassium-stimulated release required calcium
(2.4 mM) as little release was observed when
calcium was omitted (Table 2). An increase in
the passive release (31 ± 4%) was observed
when calcium was omitted from the system
(Table 2).
Potassium chloride (35 mM) stimulated the
3
3
release of [ H]taurine and D-[ H]aspartate by
twofold. A higher concentration of potassium
chloride was used with taurine and D-aspartate
to obtain a similar amount of release as GABA.
CACA (100 µM) does not affect the stimulated
3
release or the passive release of [ H]taurine in
the cerebellum and spinal cord or D3
[ H]aspartate in the cerebellum (Table 2). This
indicates that the transporter involved in this
study is more likely to be a GABA transporter
sensitive to β-alanine than a taurine transporter
that also transports GABA and β-alanine.
TABLE 2. Effect of various treatments on
3
passive
release
of
β-[ H]alanine,
D3
3
[ H]aspartate, and [ H]taurine in brain slices
FIG. 5. Effect of CACA on the passive release of
3
β-[ H]alanine in slices of cerebellum and spinal
cord. Slices of cerebellum and spinal cord were
3
loaded with β-[ H]alanine (0.03 µM) and
depolarized twice with 35 mM KCl. CACA (100
µM) ([white square]) increases significantly the
3
passive release of β-[ H]alanine under control
conditions ([black small square]) from slices of
cerebellum (25 ± 6%) and spinal cord (120 ±
20%). Data are mean ± SEM (bars) values,
displayed as an average of baseline fractional
release (R2) collected from two to four
independent
experiments
performed
in
quadruplicate. Unpaired two-tailed Student's t
test (p < 0.05) was used for statistical analysis.
3
CACA was found to inhibit β-[ H]alanine uptake
in cerebellar slices. The KD of β-alanine was
found to be 3.0 mM. The Ki of CACA in the
cerebellum was 750 ± 60 µM (n = 6). At 100 µM
CACA, the concentration used in the release
3
experiments, β-[ H]alanine uptake was inhibited
by 25 ± 5% (Fig. 6).
There are two main classes of transporter
systems that carry GABA and β-alanine, the
GABA and taurine transporters. CACA did not
3
affect the passive release of [ H]taurine. This
provides evidence that CACA is not a substrate
of a taurine transporter sensitive to β-alanine
and is most likely being transported by the
GABA transporters. CACA does not affect
3
stimulated release of [ H]GABA in cerebellum,
3
cortex, spinal cord, and retina, [ H]taurine and β3
[ H]alanine in cerebellum and spinal cord, or D3
[ H]aspartate in the cerebellum but is found to
be a substrate for GABA transport in these
tissues.
FIG. 6. Dose-response curve for CACA against
β-alanine uptake in cerebellar slices. A doseresponse curve was determined for CACA (13
50,000 µM) using β-[ H]alanine (8 nM) as the
competitive ligand in cerebellar slices (0.1 x 0.1
µm) and the program Prism (1994; GraphPad
Software, San Diego, CA, U.S.A.). Data are
mean ± SEM (bars) values, expressed as the
average of six independent experiments
performed in quadruplicate. The Ki of CACA
(750 ± 60 µM) was determined using the KD of
β-alanine (3 mM) and the Cheng-Prusoff
equation.
The modeling results suggest that for transport
to occur, the compounds need to attain a
partially extended conformation (Fig. 7). This
was shown with R(-)-nipecotic acid as this
enantiomer is more potent for the transporter
systems than the S(+) enantiomer (Johnston et
al., 1976a). The low-energy chair conformation
with the carboxylate group in the equatorial
position of the ring provides the basis for
analyzing the conformation for which transport
may occur. CACA, GABA, and β-alanine were
able to attain low-energy conformations to fit R()-nipecotic acid in the chair and boat forms. The
fitted root mean square values of CACA, βalanine, GABA, and R(-)-nipecotic acid in the
chair and boat forms were 0.342 and 0.316,
respectively.
FIG. 7. A computer-assisted study was carried
out using the program Chem-X (1994; Chemical
Design Ltd., Oxford, U.K.) to obtain low-energy
conformations of R(-)-nipecotic acid in both the
chair (as seen in the diagram) and boat (data
not shown) forms, CACA, GABA (data not
shown), and β-alanine. The fitted root mean
square of CACA, β-alanine, GABA, and R(-)nipecotic acid in the chair and boat forms were
0.342 and 0.316, respectively. The overlay of
R(-)-nipecotic acid in the low-energy chair form
and CACA was drawn by the program Chem-3D
(Cambridge Scientific Computing, Cambridge,
MA, U.S.A.) using solid circles to represent the
nitrogen atoms, open circles to represent the
oxygen atoms, and the hatched circles to
represent the carbon atoms. For simplicity, the
hydrogen atoms are not shown, and GABA is
not shown. CACA, β-alanine, and GABA can
obtain low-energy conformations to fit R(-)nipecotic acid in the chair form.
DISCUSSION
β-Alanine is a selective substrate of GABA
transport into glial cells (Schon and Kelly, 1974,
1975), although there is increasing evidence for
β-alanine transport into neurons (Iversen and
Kelly, 1975; Johnston, 1977; Cummins et al.,
1982; Levi et al., 1983). β-Alanine is a substrate
for a taurine transporter found in cultured
cerebellar granule cells (Saransaari and Oja,
1993), neurons and astrocytes (Larsson et al.,
1986), glial cells (Breckenridge et al., 1981),
spinal glioma cells (Martin and Shain, 1979),
and brain slices (Kaczmarek and Davison,
1972). β-Alanine was shown to be transported
equally by cultured neurons and astrocytes, and
it was proposed by Larsson et al. (1986) that βalanine should not be used as a glial marker. It
was also shown that β-alanine and taurine share
a common transport system and that neither βalanine nor taurine was a competitive inhibitor of
GABA uptake (Larsson et al., 1986). This
contrasts with a study in brain slices that
concluded that different transport systems
mediate taurine and β-alanine versus GABA and
β-alanine (Johnston and Stephanson, 1976).
Subsequently, cloning studies have shown that
a high-affinity taurine transporter, rB16a (Smith
et al., 1992), exists that transports β-alanine and
GABA, and that two GABA transporters, GAT-2
(Borden et al., 1992) and GAT-3 (Borden et al.,
1992; Clark et al., 1992; Clark and Amara, 1994)
exist that also transport β-alanine and GABA.
The mRNAs of these transporters were further
studied in cultured neurons and astrocytes and
revealed that GAT-3 was mainly neuronal and to
a lesser extend glial, whereas GAT-2 was purely
glial, and that different astrocyte types contained
either the taurine transporter or the GABA
transporters (Borden et al., 1995b).
Uptake studies have shown that CACA acts as
an inhibitor of β-alanine uptake in rat cerebral
cortex slices, inhibiting by 36 ± 8% at 100 µM
(Johnston and Stephanson, 1976). The present
study found a Ki value of 750 ± 60 µM in
cerebellar slices, which corresponds to CACA
(100 µM) inhibiting uptake by 25 ± 5%, which is
not significantly different from that in cerebral
cortex slices. CACA is a substrate for a GABA
transporter in isolated Müller cells in guinea pig
retina, with 100 µM CACA uptake inducing a
current that was 26 ± 6% of the current induced
by the same concentration of GABA
(Biedermann et al., 1994). CACA (100 µM) also
3
increased passive release of [ H]GABA in rat
retina (63%; present study). CACA was shown
3
to be both a potent inhibitor of [ H]GABA uptake
mediated by gab permease (GabP) in
Escherichia coli SK5 cells and a counterflow
substrate (Brechtel et al., 1996).
CACA showed no significant effect on the
3
passive release of [ H]taurine in either the
cerebellum or spinal cord; hence, the transporter
system in this study may be related to a GABA
transporter sensitive to β-alanine rather than a
taurine transporter sensitive to GABA and βalanine. Nipecotic acid increased the passive
3
release of [ H]GABA in cerebellar slices, which
supports the study by Johnston et al. (1976b);
however, this did not occur in retinal slices.
Nipecotic acid blocked the CACA-stimulated
GABA release in cerebellar slices.
These studies show that CACA not only acts as
an agonist for the GABAC receptor subclass but
also is a substrate for a subclass of GABA
transporters that also transports β-alanine.
CACA did not influence the stimulated release of
the neurotransmitters GABA, taurine, β-alanine,
and D-aspartate in the cerebellum, GABA,
taurine, and β-alanine in the spinal cord, or
GABA in the retina; hence, the GABAC
receptors described in these regions were not
shown to modulate neurotransmitter release
under our conditions.
This study provides evidence for the interaction
of GABA in a folded conformation, accessible to
CACA, with these receptors and transporters.
CACA appears to be more potent in its
interactions with GABAC receptors [EC50 = 75
µM for CACA as an agonist at ρ1 receptors
expressed in oocytes (Kusama et al., 1993b;
Woodward et al., 1993) than with GABA
transporters (Ki = 750 µM; present study). CACA
is 10-fold weaker as a substrate for the
transporter system than as an agonist for the
GABA ρ1 receptor. β-Alanine appears to be a
very weak agonist at GABAC receptors [EC50 =
660 ± 110 µM at ρ1 receptors expressed in
oocytes (Calvo and Miledi, 1995). Modeling
results show that CACA can achieve a lowenergy conformation similar to both β-alanine
and R(-)-nipecotic acid in the chair and boat
forms.
The effects of CACA on GABA and β-alanine
release provide evidence for a GABA transporter
in the cerebellum, cerebral cortex, and spinal
cord that transports GABA, β-alanine, CACA,
and nipecotic acid. These results are similar to
those described in guinea pig retinal Müller cells
(Biedermann et al., 1994). Pharmacological
studies on GABA transporters have described
two major high-affinity transporters that exist in
the CNS, a neuronal transporter that is cis-3aminocyclohexanecarboxylic acid sensitive and
a glial transporter that is β-alanine sensitive.
With increasing evidence suggesting that βalanine can block transport in both neurons and
glial cells, one can no longer use this as a
marker for glial uptake. The cloning of four
GABA transporters (GAT-1, GAT-2, GAT-3, and
BGT-1) from the rat, mouse, and human has
provided some insight into the localization of
these high-affinity GABA transporters. Both
GAT-1 and GAT-3 are neuronal and glial,
whereas GAT-2 and BGT-1 are purely glial
(Borden et al., 1995b).
The rat GAT-1 transporter (Guastella et al.,
1990; Cutting et al., 1991) is a high-affinity
transporter,
sensitive
to
cis-3aminocyclohexanecarboxylic acid but not βalanine. SKF 89976A is a potent and selective
blocker of uptake in the rat GAT-1 transporter
(Borden et al., 1994) but had no significant effect
on blocking the CACA-stimulated release of
3
[ H]GABA in cerebellar slices; hence, CACA
cannot be a substrate for this transporter.
Human and canine BGT-1 (Yamauchi et al.,
1992; Borden et al., 1995a) and the species
homologue, mouse GAT2 (Liu et al., 1993),
have been cloned. The mouse GAT2 is a lowaffinity transporter that is sensitive to betaine
and not sensitive to nipecotic acid or β-alanine
(Lopez-Corcuera et al., 1992); hence, CACA is
not a substrate for the species homologue of
mouse GAT2. Rat GAT-2 (Borden et al., 1992)
and GAT-3 (Borden et al., 1992; Clark et al.,
1992; Clark and Amara, 1994) are sensitive to βalanine and nipecotic acid. Rat GAT-3 is more
abundant in the CNS and is highly expressed in
spinal cord but weakly expressed in the
cerebellum and cerebral cortex (Clark et al.,
1992). From the four cloned GABA transporters,
the pharmacological profile observed in this
study is most closely related to that observed
with the rat GAT-2 and GAT-3 transporters. Rat
GAT-2 and GAT-3 have similar pharmacology
but can be distinguished with L-DABA: GAT-2 is
more sensitive than GAT-3 to L-DABA (Borden
et al., 1992). L-DABA (100 µM) inhibits
3
[ H]GABA uptake at GAT-2 and GAT-1 by 43
and 49%, respectively, whereas at GAT-3, there
was no significant inhibition (Borden et al.,
1992). L-DABA (200 µM) had no effect on the
3
CACA-stimulated release of [ H]GABA in the
spinal cord; hence, we propose that CACA is
more likely a substrate for the rat GAT-3
transporter than the rat GAT-2 transporter. This
is in agreement with Biedermann et al. (1994),
who found CACA to be a substrate for a GABA
transporter in guinea pig retinal Müller cells and
proposed the transporter in these cells to be the
species homologue of the GAT-3 transporter.
3
In conclusion, the effects of CACA on [ H]GABA
3
and β-[ H]alanine release provide indirect
evidence for a GABA transporter in cerebellum,
cerebral cortex, and spinal cord that transports
GABA, β-alanine, CACA, and nipecotic acid.
This transporter may be related to the GAT-3
protein cloned from rat CNS.
Acknowledgment: The authors wish to thank
the National Health and Medical Research
Council of Australia for financial support and
Nina Krogsgaard-Larsen for excellent technical
assistance.
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