glutamate transport in the brain: its importance
TRANSCRIPT
Glutamate transport in the brain: Its importance,
regulation, alterations underlying the development of
synaptopathy, and possible peripheral markers
Borisova Tatiana Ph.D. in biochemistry,
Doctor Biol. Sci.
Dept. Neurochemistry
Palladin Institute of Biochemistry NAS of Ukraine
Lithuania, 2012
2007–2013 m. Žmogiškųjų išteklių plėtros veiksmų programos 3 prioriteto „Tyrėjų gebėjimų stiprinimas“
VP1-3.1-ŠMM-05-K priemonės „MTTP tematinių tinklų, asociacijų veiklos stiprinimas“
projektas „Lietuvos Biochemikų draugijos potencialo kurti žinių visuomenę didinimas“
(Nr. VP1-3.1-ŠMM-05-K-01-022)
Lecture Roadmap
Part1 (data of literature)
• CNS disorders and problems
• Ambient glutamate
• Spontaneous exocytosis
• Glutamate receptors and signaling
Part 2 (results)
• Model of cholesterol deficiency
• Glutamate transporters and glutamate uptake
• Extracellular level of glutamate in synaptosomes
• Transporter-mediated glutamate release
• Regulated exocytosis
• Synaptopathy
Part 3 (results)
• Brain-blood glutamate balance
• Blood platelets as potential peripheral models of presynapse
Lithuania, 2012
• Glutamate is the major excitatory neurotransmitter in
the CNS, which is implicated in many aspects of normal
brain functioning, i.e. learning, memory, cognition.
• Abnormal glutamate homeostasis is involved in the
pathogenesis of major neurological and
neurodegenerative disorders and contributes to
neuronal dysfunction in epilepsy, schizophrenia,
amyotrophic lateral sclerosis, Alzheimer’s disease, as
well as hepatic encephalopathy, ischemia, hypoxia,
traumatic brain injury, heavy metals poisoning, brain
cancer, etc.
• Prevention of these disorders and their successful
treatment is restrained by lack of fundamental and
comprehensive knowledge about key processes of
glutamatergic neurotransmission in the CNS.
The human brain
http://ccnmtl.columbia.edu/projects/neuroethics/module4
Lithuania, 2012
Part 1
Discovery timeline highlights major
advances and trends in understanding of
glutamate importance
Bowie, 2008
Lithuania, 2012
Therefore, although L-Glu was originally identified in 1866 by H. Ritthausen, it
took almost another 100 years before its role in the CNS was considered
Bowie 2008
Multiple factors predispose the CNS to disease
Lithuania, 2012
With the advent of reliable
diagnostic criteria, it is now
clear that CNS disorders can
affect any member of any
society at any point in life.
Amongst the many notable
historical personages afflicted
with variousnervous ailments,
both Aristotle and Julius Caesar
were said to have suffered from
epilepsy whereas Mozart may
have been a manic depressive.
Behaviour of the 15 century
French heroine, Joan of Arc was
consistent with schizophrenia
and/or epilepsy.
Although most CNS disorders have unknown aetiology, dysfunction is commonly
attributed to not one but a combination of defects that often include excitatory or
inhibitory neurotransmission, its modulation by other neurotransmitter pathways (e.g.
dopaminergic, cholinergic, serotonergic), environmental or epigenetic factors as well as
genetics.
CNS disorders are treated by targeting
the symptoms
Bowie, 2008
A list of the major CNS
disorders that afflict society and
the drugs commonly used to
treat them
Despite advances in
understanding of the CNS, most
conditions are treated by
attempting
to alleviate symptoms rather
than the roots caused the
disease
Lithuania, 2012
The NMDAR channel blocker, memantine, is currently
used in Europe, the US and Canada in the treatment
of moderate-to-severe Alzheimer’s disease
representing the only therapeutic compound targeted
to iGluRs currently in clinical use.
Glutamatergic neurotransmission
glutamate
transporters
glutamate Na+
synaptic
vesicles
presynaptic
nerve terminal
Presynaptic
nerve terminal
glutamate
receptors
http://www.wormbook.org/chapters Danbolt, 2000 Lithuania, 2012
Tzingounis, 2007
Lithuania, 2012
Ambient extracellular glutamate is the most
important characteristic of glutamatergic
neurotransmission
• Plasma glutamate is 30 -100 µM
• Cerebrospinal fluid glutamate is 3-10 µM
• Synaptic vesicle glutamate transporters in glutamatergic neurons produce a 10,000-fold gradient.
• The concentration of glutamate in the cytoplasm of glutamatergic neurons is ~10,000 µM and synaptic vesicles within these neurons have glutamate concentrations near 100,000 µM
• Excitotoxicity: with swelling and apoptosis predominating at <20 µM glutamate and fast necrosis at >100 µM glutamate
Lithuania, 2012
Ambient extracellular glutamate and
synaptic transmission
In norm:
• Extracellular glutamate controls several important neurological
processes, including neuronal and glial cell differentiation and migration during development.
• Ambient extracellular glutamate also likely plays an important nonpathological role in control of glutamatergic synapse strength. Synapse strength essentially determines information flow in the brain, and activity-dependent changes in glutamatergic synapse strength are now recognized as the basis of learning and memory.
• Ambient extracellular glutamate varies spatially and temporally in healthy brains.
• Ambient extracellular glutamate may be an important unrecognized determinant of neuronal circuit function and plasticity.
Lithuania, 2012
Spatial variation in ambient extracellular
glutamate concentration could differentially
modulate neuronal circuit function
A hypothetical point source (asterisk) for ambient extracellular glutamate is present near the left center of this image. As a
result, ambient extracellular glutamate concentration (green shading) is highest there but drops with distance. Four
hypothetical glutamatergic neural circuits, each possibly leading to a different behavioral output, are represented by
arrows and numbered 1 to 4. All else being equal, circuits 3 and 4 will be weaker than circuits 1 and 2 because of
suppression of synapse strength by ambient extracellular glutamate. This will decrease the probability of behaviors
triggered by circuits 3 and 4, compared to 1 and 2. Temporal variations in glutamate uptake or ambient extracellular
glutamate secretion could alter the relative strength of the circuits at different times. In this way, ambient extracellular
glutamate could serve as a potent modulator of spatially “connected” circuits and regulate behavior.
Featherstone, 2008 Lithuania, 2012
Ambient extracellular glutamate and
synaptic transmission
Under pathological conditions:
• Ambient extracellular glutamate rises substantially during acute
pathological conditions such as seizure, ischemia, or fever, amyotrophic lateral sclerosis (ALS).
• Circadian changes in ambient extracellular glutamate may be caused by circadian rhythms in glial glutamate uptake that are themselves regulated by melatonin.
• Changes in ambient extracellular glutamate and glial activity may also contribute to mood.
• The possibility that ingestion of monosodium glutamate, a common food additive, can alter extracellular glutamate levels in certain regions of the brain has always been controversial and is now generally discounted.
Lithuania, 2012
Regulation of ambient extracellular
glutamate
• Ambient extracellular glutamate is the steady-state balance between glutamate secretion (which will increase ambient extracellular glutamate concentration) and glutamate uptake (which will decrease ambient extracellular glutamate)
• Vesicular release: spontaneous exocytosis
• Nonvesicular release:
• swelling-activated anion channels,
• gap junction hemi-channels,
• purinergic (P2X) receptors,
• cystine-glutamate exchangers,
• diffusion
Lithuania, 2012
Spontaneous exocytosis
Wasser, 2009 Lithuania, 2012
Regulation of synaptic transmission by
ambient extracellular glutamate
• The easiest way to imagine ambient extracellular glutamate affecting any biological process, including synaptic transmission, is via glutamate receptors. A typical initial thought is that ambient extracellular might perpetually activate glutamate receptors.
• Another possibility is that ambient extracellular glutamate might lead to constitutive desensitization of receptors, and therefore, suppression of glutamatergic signaling.
• Which actually occurs depends on the glutamate sensitivity of receptor activation and desensitization relative to the concentration of ambient extracellular glutamate. The exact glutamate sensitivity of activation and desensitization depends on glutamate receptor type.
Lithuania, 2012
Ionotropic glutamate receptors
• There are two main types of glutamate receptor in the nervous system: ionotropic (pore-forming) glutamate receptors and metabotropic (G-protein coupled) glutamate receptors.
• Mammalian ionotropic glutamate receptors are functionally and molecularly differentiated into three subgroups based on agonist pharmacology and subunit composition:
• 1) N-methyl-D-aspartate (NMDA) receptors,
• 2) amino-3-hydroxy-5-methylisoxazole-4-propionic acid
• (AMPA) receptors, and
• 3) kainate receptors.
• As their names imply, NMDA receptors display particular sensitivity to NMDA,AMPA receptors display particular sensitivity to AMPA, and kainate receptors display particular sensitivity to kainate.
• Differences between the three subtypes are attributed to the fact that, although all ionotropic glutamate receptors are thought to be tetrameric, each receptor subtype is assembled from a different set of subunit proteins.
• NMDA receptors are assembled from NR1,NR2,or NR3 subunits,
• AMPA receptors are composed of various combinations of GluR1, GluR2, GluR3, and GluR4 subunits,
• and kainate receptors are composed of GluR5, GluR6,
• GluR7,KA1,and KA2 subunits Lithuania, 2012
Glutamate dependence of activation for various
glutamate receptor subtypes, compared to the
probable concentration of ambient extracellular
glutamate
NMDA receptors (NMDAR) and metabotropic receptors (mGluR) are activated by relatively
low concentrations of glutamate (1 to 20 µM) compared to AMPA receptors (AMPAR) and
kainate receptors (KAR), which are activated only by glutamate concentrations of 100 to
2000 µM. If ambient extracellular glutamate is ~2 µM, then about 40% of NMDARs and 10% of
mGluRs could be constitutively activated in vivo. Featherstone, 2008
Glutamate dependence of desensitization for various
glutamate receptor subtypes, compared to the
probable conсentration of ambient extracellular
glutamate
Compared to activation, steady-state desensitization of ionotropic glutamate receptor occurs
at much lower glutamate concentrations (0.1 to 10 µM). If ambient extracellular glutamate is ~2
µM, then one-half to three-quarters of glutamate receptors might be constitutively
desensitized, and thus functionally silent, in vivo. However, slight changes in ambient
extracellular glutamate concentration or dose-dependence of steady-state desensitization
could have dramatic effects on glutamate receptor availability and synaptic strength. Featherstone, 2008
Structure and domain organization of
glutamate receptors
A, linear representation of the subunit polypeptide chain and schematic illustration of the subunit
topology. Glutamate receptor subunits have a modular structure composed of two large extracellular
domains [the ATD (green) and the LBD (blue)]; a TMD (orange) that forms part of the ion channel pore; and
an intracellular CTD. The LBD is defined by two segments of amino acids termed S1 and S2. The TMD
contains three membrane-spanning helices (M1, M3, and M4) and a membrane re-entrant loop (M2). B,
crystal structure at 3.6 A of the membrane-spanning tetrameric GluA2 AMPA receptor
Traynelis 2010 Lithuania, 2012
Binding sites for the agonists, antagonists,
and modulators shown for the glutamate
receptors
Traynelis, 2010
AMPA and kainate indicates that the ligand selectively targets GluA or GluK receptor
subunits, respectively. The ATDs,LBDs,TMDs,and linkers are shown in purple, orange, green,
andgray, respectively Lithuania, 2012
Conformational changes in the
functioning AMPA receptor
Traynelis, 2010
Lithuania, 2012
Ionotropic glutamate receptors fulfill
distinct roles in the CNS
Table identifying each iGluR subfamily (left column) with the individual subunits that assemble as mature receptors (middle column) and their respective roles within the CNS (right column)
Bowie, 2008 Lithuania, 2012
AMPA receptors and CNS disorders
Bowie, 2008 Lithuania, 2012
Table listing a number of disease states (left column) where defective signaling
through AMPARs has been established. The middle column summarizes key
characteristics associated with each disorder and the right column refers to the
drug classes whose actions may have therapeutic value.
Kainate receptors and CNS disorders
Summary table identifying which KAR subunits are implicated in distinct disorders of
the CNS. As work progress, it is likely that this information will be more complete. (For example, it is commonly assumed that native KARs are heteromers assembled from GluR5–7 subunits with KA1
and/or KA2. In view of this, in disease states where GluR5–7 have been implicated, it is possible that future work will
also implicate KA1 and/or KA2 subunits.) Lithuania, 2012 Bowie, 2008
Metabotropic glutamate receptors
• Mammalian metabotropic glutamate receptors are also divided into three subfamilies based on molecular and pharmacological differences.
• Group I metabotropic glutamate receptors are formed from mGluR1 or mGluR5 subunits and often activate phospholipase C pathways.
• Group II and Group III metabotropic receptors are composed of mGluR2/mGluR3 or mGluR4/mGluR6/mGluR7/mGluR8 subunits, respectively, and generally suppress adenylate cyclase activity.
Lithuania, 2012
Schematic diagram of the mGluR dimer
in different activity states
mGluR dimers contain two large extracellular domains called the Venus flytrap domains (VFDs),
which bind glutamate and other orthosteric ligands. The cysteine-rich domain links the VFDs to
seven transmembrane-spanning domains; the C-terminus faces intracellularly and is often subject
to alternative splicing to generate different C-terminal protein tails. The open-open state (left) is the
inactive state and can be stabilized by antagonists. Either one or two VFDs can then bind
glutamate, resulting in active receptor conformations
Traynelis, 2010 Lithuania, 2012
Glutamatergic neurotransmission
glutamate
transporters
glutamate Na+
synaptic
vesicles
presynaptic
nerve terminal
glutamate
receptors
Danbolt, 2001 universe-review.ca/R10-16-ANS.htm Lithuania, 2012
Part 2
Glutamate transporters
Tzingounis, 2007
Lithuania, 2012
EAAT1
and EAAT2 are
predominantly glial,
whereas EAAT3,
EAAT4 and EAAT5 are
expressed by neurons
throughout
the brain. Notably,
EAAT4 and EAAT5 are
specifically
located in Purkinje
cells (PCs) in the
cerebellum and the
retina, respectively.
DL-threo-b-benzyloxyaspartate L-threo-b-hydroxyaspartate Dihydrokainate
(DL-TBOA) (DL-ТНА) (DHK)
Synaptic vesicles and exocytosis
(Sudhoff, 2004) Lithuania, 2012
Confocal imaging of synaptosomes
labeled with the fluorescent dye R18
Preparations used in the research
Synaptic vesicle
Lithuania, 2012
synaptosomes
www.coloradocollege.edu
Electron microscopy of synaptosomes
brain homogenate crude synaptosomal
fraction
synaptosomes purified
according to Cotman, 1970
Lithuania, 2012
Nervous system cholesterol
homeostasis failure
?
Impairment of
neurotransmission, synaptic
function and plasticity
Impairment of learning and
memory,
neuronal loss
Cholesterol is an
essential component of
mammalian cell
membranes where it is
required to establish
proper membrane
permeability and
fluidity
The central nervous
system, which is equal
to two percent of body
mass, keeps a special
place among other
systems of organism,
since it contains
approximately a
quarter of total
unesterified
cholesterol
Cholesterol
homeostasis breaks are
associated with the
pathogenesis of certain
neurological disorders:
•Niemann-Pick disease C is
due to mutations in either
the NPC1 or NPC2 genes,
resulting in defective
cholesterol transport
•Defective synthesis of
brain cholesterol is the
cause of Smith-Lemli-Optiz
Syndrome.
•A specific down-regulation
of seladin-1, a protein
involved in cholesterol
synthesis, was shown in
Alzheimer's disease
•Low membrane cholesterol
was observed in
hippocampal membranes of
ApoE4 related case of
Alzheimer's disease
•Cholesterol is the
precursor for neurosteroids
which may be modulators of
the pathophysiology of
schizophrenia and bipolar
disorder Lithuania, 2012
1
Confocal imaging of synaptosomes. Dynamics of
cholesterol depletion by MCD (4s). Fluorescent
dye filipin
5мMcontrol 15мM 30мM 60мM 15мM
MCD+
Chol
0
20
40
60
80
100
120
MCD
ch
ole
ste
rol,
% *
*
*
*
Decrease in the level of cholesterol
Two methodological protocols:
-In the presence of the acceptor in the incubation
media (protocol М1).
-After washing of the acceptor (protocol М2).
Cholesterol deficient brain nerve terminals
C М1 М2
Relevance:
MCD as drug
deliverer and as a
component of
nanoparticles
cholesterol
Decreased level of
cholesterol in
neurological disorders
and as a result of
statin treatment
synaptosomes
Lithuania, 2012
Cholesterol acceptor –
methyl-β-cyclodextrin (MCD)
High-affinity Na+ - dependent glutamate
uptake by nerve terminals
L-[14C]glutamate
Na+
K+
EAAT
EAAT3
MCh
Synaptic
vesicles
Presynaptic nerve
terminal
Glutamate uptake activity
depends from:
-Na/K electrochemical gradient
of the plasma membrane;
-cell surface expression of
glutamate transporters
(protein kinase C –dependent
mechanism of regulation);
-the level of membrane
cholesterol;
-acidification of synaptic
vesicles.
Lithuania, 2012
() Changes in
the level
of membrane
cholesterol
()Glutamate
uptake in the
presence of
MCD (М1)
High-affinity Na+-dependent uptake of glutamate under
conditions of cholesterol deficiency
() Synaptic vesicle
acidification
() Glutamate
uptake
(М2)
Tarasenko A., Krisanova N., Sivko R., Himmelreich N., Borisova T. (2010) J. Mol. Neuroscience
Protein
kinase С -
dependent
regulation
• The effects of MCD on synaptic
vesicle acidification. (1) - control
synaptosomes; (2) – M1; (3) -
synaptosomes in the presence of 15
mM MCD, which was added 35 min
before the addition of acridine
orange; (4)-M2.
*
control 5 mM MCD 15mM 15mM MCD+
cholesterol
0
0.5
1
1.5
2
2.5
3
3.5
*
L-[
14C
]glu
tam
ate
,
nm
ol *
min
-1*
mg
of pro
tein
-1
Decreased L-[14C]glutamate uptake under
cholesterol deficiency
М1
0 200 400 600 800 1000 1200 1400 1600
0,6
0,8
1,0
Ft/F
0
Time, s
MCD
1
2
3
М2
control
15 mM MCD
0
1
2
3
4
5
6
0 100 200 300 400 500 600 700
time, s
glu
tam
te, n
mo
l/m
g p
rote
in The reduction of
glutamate uptake
during the removal
of cholesterol (M1)
Lithuania, 2012
The effect of MCD on glutamate uptake by
isolated synaptic vesicles
The initial velocity of L-[14C]glutamate (50mM) uptake by isolated synaptic
vesicles during the application of 15 mM MCD and 15mM MCD complexed
with cholesterol (2.3 mM).
*
**
0
50
100
150
200
250
300
350
400
450
500
control M CD M CD+cholesterol
glu
tam
ate
, p
mo
l/m
in/m
g p
rote
in
Lithuania, 2012
The ambient extracellular glutamate level
Glutamate receptors NMDA
R, AMPA R, mGlu R
Glu
Cys
Transmembrane diffusion
Glu
Glu
Glu
Glu Anion channels
СВ
ЕААТ3
Presynaptic nerve
terminal
Possynaptic membrane
[glu]extracellular=[glu]tonic release–[glu]uptake
Lithuania, 2012
The changes in ambient extracellular
glutamate followed M1 and M2
Confocal imaging of synaptosomes labeled with
a pH sensitive fluorescent dye acridine orange
following the application of MCD. The
measurements were performed by using the
confocal laser scanning microscope LSM 510
META, Carl Zeiss.
3
4
2
1
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0 10 20 30 40
time, min
L[1
4C
]glu
tam
ate
, n
mo
l/m
g p
rote
in
The extracellular level of L-[14C]glutamate in
synaptosomes after the addition of 0, 5, 15,
30 mМ MCD – curves 1; 2; 3; 4, respectively
M1
Lithuania, 2012
0
0,05
0,1
0,15
0,2
0,25
0,3
0,35
L-{
14C
]glu
tam
ate
, n
mo
l/m
g o
f p
rote
in
M2
The extracellular L-[14C]glutamate level
of control (empty bar) and cholesterol-
depleted synaptosomes (shaded bar)
(Р≤0.05, n=8). *, Р≤0.05 as compared to
control.
*
The extracellular level of endogenous
glutamate and glutamine in control
(empty bar) and cholesterol-depleted
(shaded bar) synaptosomes measured
chromatographycaly (Р≤0.05, n=4).
*, Р≤0.05 as compared to control.
*
*
0
2
4
6
8
10
12
14
16
18
20
endogenous glutamate glutamine
nm
ol/m
g p
rote
in
Lithuania, 2012
The possible interrelation between uptake (black arrows), tonic release
(open arrows), transporter-mediated release (grey arrow) and the
intra/extra - cellular concentration of glutamate (indicated by black dots).
Circles inside of synaptosomes showed filled with the neurotransmitter
(black circles) or empty (open circles) synaptic vesicles
In the presence
of MCD (M1)
Cholesterol-deficient
(M2)
Lithuania, 2012
Stimulated by depolarisation of the plasma membrane Са2+-
dependent (exocytosis) and Са2+- independent (transporter-
mediated) glutamate release from nerve terminals
Transporter- mediated release of
glutamate is the main mechanism of
glutamate release during hypoxia,
stoke, insult, seizures, brain trauma exocytosis
EAAT
Glu
SV
МCH
1K+
1 Glu-
n Na+
m H+
in
out
Plasma membrane
http://www.gs.washington.edu Lithuania, 2012
The alterations in exocytotic release of
glutamate followed M1 and M2
Ca2+-dependent (exocytotic) release of glutamate stimulated by high KCl:
(1) - control synaptosomes;
(2) - synaptosomes in the presence of 15 mM MCD (M1), which was added at zero time point just before application of 35 mM KCl;
(3) - synaptosomes in the presence of 15 mM MCD, which was added 35 min before the application of 35 mM KCl;
(4) - synaptosomes were preliminary treated with 15 mM MCD for 35 min, then were washed with 10 volumes of the buffer (M2), then were loaded with L-[14C]glutamate and depolarized with 35 mM KCl
1 2 3 4
0
1
2
3
4
5
6
7
8
9
10
glu
tam
ate
rele
ase, %
*
*** *
Lithuania, 2012
Borisova T., et al. ( 2010) Cell Moll Neurobiol
The opposite effects of M1 and M2 on
transporter-mediated glutamate release
1 2 3 4
0
2
4
6
8
10
12
14
glu
tam
ate
rele
ase, %
**
*
*
Ca2+-independent (transporter-mediated)
release of L-[14C]glutamate
stimulated by high- KCl:
(1) - control synaptosomes;
(2) - synaptosomes in the presence of 15
mM MCD (M1), which was added at
zero time point just before
application of 35 mM KCl;
(3) - synaptosomes in the presence of 15
mM MCD, which was added 35 min
before application of 35 mM KCl;
(4) - synaptosomes were preliminary
treated with 15 mM MCD for 35 min,
then were washed with 10 volumes of
buffer (M2), and then were loaded
with L-[14C]glutamate and depolarized
with 35 mM KCl
Lithuania, 2012
Borisova T., et al. ( 2010) Cell Moll Neurobiol
The possible mechanisms of the action
of cadmium and lead
0 100 200 300 400 500 600 700 800
0,5
0,6
0,7
0,8
0,9
1,0
F/F
0
Time (s)
cadmium
lead
control
control
cadmium
lead
-10
-8
-6
-4
-2
0
2
4
6
0 2 4 6 8 10 12
time,min
extr
acellu
lar
glu
tam
ate
,% o
f to
tal
0 100 200 300 400 500 600
0,2
0,4
0,6
0,8
1,0
Ft/F
o
time,s
Decrease in synaptic
vesicle proton gradient
Decrease in
transporter-
mediated
glutamate uptake
Decrease in
exocytotic
release of
glutamate
Blockage of
potential-
dependent calcium
channels
Binding to SH-
groups of
glutamate
transporters
Synaptic
pathology
Cadmium
Lead
Lithuania, 2012
Borisova et al., 2011, Neurochemistry Int
The effects of 200 mM CdCl2 and 200 mM PbCl2
on the accumulation of acridine orange into
the acidic compartments of synaptosomes
The changes in the extracellular L-
[14C]glutamate level in digitonin-
permeabilised synaptosomes in the control
and in the presence of 200 mM CdCl2 and
PbCl2
-50 0 50 100 150 200 250 300 350 400
0,4
0,6
0,8
1,0
Ft/F
o
time,s
The effect of cadmium on acidification of
isolated synaptic vesicles
The effect of manganese
on acidification of isolated
synaptic vesicles
The possible mechanisms of the action
of cadmium and lead
Nerve terminals
Cd 2+ Pb 2+
Lithuania, 2012
Borisova et al., 2011, Neurochemistry Int
Part 3
Glutamate transport in blood platelets
EAAT
Glu
VGLUT
Secretory
granules
Glutamate receptors
NMDA R, AMPA R
Glu
Blood platelets contains:
-glutamate transporters
ЕААТ 1-3;
-glutamate receptors
NMDA, AMPA, mGlu;
-vesicular glutamate
transporters V GLUT;
-neuronal protein
involved in exocytosis
Lithuania, 2012
Confocal imaging of
blood platelets labeled
with the fluorescent
dye filipin
Blood platelets
Brain-blood glutamate balance
Teichberg, 2009 Lithuania, 2012
Time course of L-
[14C]glutamate uptake by
platelets
А
L-[14C]glutamate uptake by platelets
Km Vmax
Platelets 36 8 mМ 5.2 1.3 pmol х min-1 х mg protein-1
Nerve terminals 10.7 2.5 mМ 12.5 3.2 nmol х min-1 х mg protein-1
Na+-dependent uptake by platelets
1/[glutamate], мicroМ -1
1/V
0, (n
mol/m
in/m
g p
rote
in
Час,с Time,min
n
molm
in/m
g o
f pro
tein
inh
ibit
ion
, %
Influence of DL-ТНА on glutamate uptake by platelets
10 100 mM
Lithuania, 2012
Depolarization of the plasma membrane of
platelets labeled with rodamine 6 G
0 200 400 600 800 1000
0,75
0,80
0,85
0,90
0,95
1,00
Ft /
Fo
Time, s
35 mM KCl
20 mM KCl
контроль
Flow cytometry of platelets in the
presence of KCl
Na+- dependent glutamate uptake by platelets during
depolarization of the plasma membrane
Kasatkina L., Borisova T. ( 2010) Neurochemistry International
Time,s
Secretory granule acidification of
platelets labeled with АО
*
0
0.2
0.4
0.6
0.8
1
1.2
1.4
glu
tam
ate
, p
mo
l/ m
in/
mg
pro
tein
A decrease in the initial velocity of
L-[14C]glutamate uptake by platelets
during the depolarization of the
plasma membrane
In vivo, glutamate uptake in platelets could be targeted under
conditions of hyperkalemia or preudohyperkalemia, i.e.
activation and clotting of platelets, hemolysis, leucocytosis,
acute renal failure, hypofunction of adrenal cortex, lack of
aldosterone, stroke, trauma, etc. In this context, malfunction
of glutamate transporters may be one of the causes for the
development of neurological consequences.
Also, lack function of glutamate transporters has to take place
during depolarization of the platelet plasma membrane
associated with their activation by ADP, thrombin, platelet-
activating factor, etc.
КСl C
0 200 400 600 800 1000
0,75
0,80
0,85
0,90
0,95
1,00
Ft /F
o
час, сTime,s
KCl KCl
Lithuania, 2012
The proton gradient of secretory granules and glutamate
transport in blood platelets during cholesterol depletion of
the plasma membrane by methyl--cyclodextrin
Borisova T., Kasatkina L., ( 2011) Neurochemistry International
Confocal imaging: MCD-
evoked depletion of
membrane cholesterol from
platelets labeled with the
fluorescent dye filipin. The
profiles of the fluorescence
intensity of filipin recorded
along red arrows of the
confocal images.
0 60 120 180 240 300 360 420 480
0,4
0,5
0,6
0,7
0,8
0,9
1,0
15 mM MCD-cholesterol complex (1:0.15)
5 mM MCD
Ft/F
0
Time, s
15 mM MCD
Control
Acidification of secretory granules of platelets
after the application of MCD
0 60 120 180 240 300
4
6
8
10
12
14
5 mM MCD
F
Time, s
1 mM L-GluMCD 15 mM MCD
0 30 60 90 120 150 180 210 240
4
6
8
10
12
14
16
F
Time, s
1 mM L-Glu
thrombin
1 NIH U/ml
Glutamate dehydrogenase assay. Analysis of
the effect of MCD on platelets.
Release of endogenous glutamate from
platelets stimulated by thrombin (1 NIH U/ml). Lithuania, 2012
Related publications 2010-2011:
• T. Borisova, N. Krisanova, R. Sivko, L. Kasatkina, A. Borysov, S. Griffin, M. Wireman Presynaptic malfunction: The neurotoxic effects of cadmium and lead on the proton gradient of synaptic vesicles and glutamate transport. Neurochemistry International - 2011 .- V.59 .- Р. 272-279.
• T. Borisova, L. Kasatkina, L. Ostapchenko The proton gradient of secretory granules and glutamate transport in blood platelets during cholesterol depletion of the plasma membrane by methyl-beta-cyclodextrin. Neurochemistry International-2011.- V.59 .- Р. 965-975.
• T.Borisova, R.Sivko, A.Borysov, N.Krisanova Diverse presynaptic mechanisms underlying methyl-beta-cyclodextrin – mediated changes in glutamate transport. Cellular and Molecular Neurobiology - 2010.- V.30, № 7.- Р. 1013-1023.
• T.Borisova, N.Krisanova, R.Sivko, A.Borysov Cholesterol depletion attenuates tonic release but increases the ambient level of glutamate in rat brain synaptosomes. Neurochemistry International- 2010.- V.56.- Р. 466-478.
• A. S.Tarasenko, R. V. Sivko, N. V.Krisanova, N. H.Himmelreich, T. A. Borisova Cholesterol depletion from the plasma membrane impairs proton and glutamate storage in synaptic vesicles of nerve terminals. Journal of Molecular Neuroscience - 2010.- V.41, № 3.- Р. 358-367.
• L.Kasatkina, T.Borisova Impaired Na+- dependent glutamate uptake in platelets during depolarization of their plasma membrane. Neurochemistry International - 2010.- V.56.- Р. 711-719.
• Krisanova, N.V. Synaptopathy under conditions of altered gravity: Changes in synaptic vesicle fusion and glutamate release / N.V.Krisanova, I.O.Trikash, T.A.Borisova // Neurochemistry International.- 2009.- V.55.- Р. 724-731.
Lithuania, 2012
Thank you for the attention!
Acknowledgements:
Dr. Natalia Krisanova
Roman Sivko
Ludmila Kasatkina
Lithuania, 2012
Acknowledgements:
2007–2013 m. Žmogiškųjų išteklių
plėtros veiksmų programos 3
prioriteto „Tyrėjų gebėjimų
stiprinimas“VP1-3.1-ŠMM-05-K
priemonės „MTTP tematinių tinklų,
asociacijų veiklos stiprinimas
“projektas „Lietuvos Biochemikų
draugijos potencialo kurti žinių
visuomenę didinimas“ (Nr. VP1-3.1-
ŠMM-05-K-01-022)