Brain Research 887 2000 98–109 www.elsevier.com locate bres
Research report
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Coupling of AMPA receptors with the Na Ca exchanger in
cultured rat astrocytes
Jeffrey P. Smith, Lee Anna Cunningham, L. Donald Partridge
Department of Neurosciences , University of New Mexico, School of Medicine, Albuquerque, NM 87131, USA
Accepted 19 September 2000
Abstract
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Astrocytes exhibit three transmembrane Ca influx pathways: voltage-gated Ca
channels VGCCs, the a-amino-3-hydroxy-5-
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methyl-4-isoxazole propionic acid AMPA class of glutamate receptors, and Na Ca exchangers. Each of these pathways is thought to
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be capable of mediating a significant increase in Ca concentration [Ca
] ; however, the relative importance of each and their
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interdependence in the regulation astrocyte [Ca ] is not known. We demonstrate here that 100 mM AMPA in the presence of 100 mM
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cyclothiazide CTZ causes an increase in [Ca ] in cultured cerebral astrocytes that requires transmembrane Ca
influx. This increase
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of [Ca ] is blocked by 100 mM benzamil or 0.5 mM U-73122, which inhibit reverse-mode operation of the Na Ca
exchanger by
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independent mechanisms. This response does not require Ca influx through VGCCs, nor does it depend upon a significant Ca
influx through AMPA receptors AMPARs. Additionally, AMPA in the presence of CTZ causes a depletion of thapsigargin-sensitive
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intracellular Ca stores, although depletion of these Ca
stores does not decrease the peak [Ca ] response to AMPA. We propose that
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activation of AMPARs in astrocytes can cause [Ca ] to increase through the reverse mode operation of the Na Ca
exchanger with
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an associated release of Ca from intracellular stores. This proposed mechanism requires neither Ca
-permeant AMPARs nor the activation of VGCCs to be effective.
2000 Elsevier Science B.V. All rights reserved.
Theme : Neurotransmitters, modulators, transporters and receptors
Topic : Excitatory amino acid receptors: physiology, pharmacology and modulation
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Keywords : AMPA receptor; Na Ca
exchanger; Ca channel; Astrocyte
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Ca channels VGCCs, and influx via reverse-mode
1. Introduction
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operation of the Na Ca exchanger. In theory, each of
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these pathways would be capable of mediating a significant The regulation of intracellular Ca
concentration
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increase in [Ca ] ; however, the relative importance of
[Ca ] plays a central role in many astrocyte functions,
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each in the regulation of astrocyte [Ca ] and whether
and modulation of [Ca ] by transmembrane Ca
flux
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they are functionally linked in astrocytes is not known has been implicated in the control of astrocyte-neuron
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[13,18]. For example, AMPAR permeability to Ca can
signaling [9,32,49], astrocyte proliferation and differentia-
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be negligible, depending on which of the four receptor tion [11], astrocyte K
channel activity [37], and astrocyte subunits are assembled to form the functional multimeric
release of the excitatory neurotransmitter, glutamate receptor channel [12,39]. In addition, astrocytes do not
[20,34]. Three major pathways for entry of extracellular
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express VGCCs under all physiological or experimental Ca
across the plasma membrane have been described in conditions [6]. Finally, although the reverse-mode opera-
protoplasmic astrocytes; direct influx through activated
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tion of the Na Ca exchanger has been shown to be
AMPARs a-amino-3-hydroxy-5-methyl-4-isoxazole propi-
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capable of mediating substantial Ca influx in astrocytes
onic acid class of glutamate receptors or voltage-gated [14,46], the physiological conditions under which this
could occur have not been clearly established. We have
Corresponding author. Tel.: 11-505-272-8815; fax: 11-505-272-
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simultaneously examined all three transmembrane Ca
8082.
influx pathways in cultured cerebral astrocytes using
E-mail address : dpartridgesalud.unm.edu L.D. Partridge.
0006-8993 00 – see front matter
2000 Elsevier Science B.V. All rights reserved. P I I : S 0 0 0 6 - 8 9 9 3 0 0 0 2 9 7 3 - 5
J .P. Smith et al. Brain Research 887 2000 98 –109
99
physiological and molecular techniques to better under- positive, protoplasmic astrocytes, as assessed using im-
stand their individual contributions and interactions. munofluorescent staining see below. All experiments
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While [Ca ] responses have been identified in various
were performed on astrocytes that had been maintained in
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types of glia in response to ionotropic glutamate receptor culture for 17–30 days. Data from cortical and hippocam-
agonists [13,18,30], the mechanisms responsible for these pal astrocytes were always analyzed separately, but were
responses remain to be clearly established in protoplasmic never found to be significantly different, and therefore
astrocytes. Our results demonstrate that AMPAR activa- were combined and presented simply as data from cerebral
tion, under non-desensitizing conditions, results in a astrocytes.
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substantial elevation of [Ca ] , which is dependent on the
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presence of extracellular calcium. We provide evidence 2.2. Immunocytochemistry
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that the majority of this Ca influx is unlikely to be
carried directly by the AMPAR channel, given the rectifi- Astrocyte cultures were fixed with 4 paraformal-
cation properties of these AMPARs, the abundant expres- dehyde, blocked in 4 normal donkey serum Chemicon
sion of the GluR2 AMPAR subunit by these astrocytes, International Inc., Temecula, CA, 1 bovine serum
and the inhibition of the entire influx by agents that are not albumin BSA, and 0.4 Triton X-100 Sigma. Cells
known to interact with AMPARs. While it is possible that were incubated overnight at 48C in phosphate-buffered
AMPAR-mediated depolarization could activate VGCCs to saline PBS plus 0.4 Triton X, and 1.0 bovine serum
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allow transmembrane Ca entry, we found no evidence
albumin BSA containing a 1:200 dilution of guinea pig for the expression of VGCCs in these cultured astrocytes,
anti-GFAP antibody 031223; Advanced Immunochemical, as assessed using both electrophysiological and molecular
Long Beach, CA or a 1:1500 dilution of rabbit anti-GluR2
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methods. However, Ca entry following AMPAR stimula-
polyclonal antibody AB1768; Chemicon International. tion was found to be completely blocked by benzamil and
Cells were rinsed and incubated under subdued light for 1
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U-73122, two pharmacological inhibitors of Na Ca h at room temperature in PBS containing a 1:150 dilution
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exchanger activity, suggesting that Na influx through the
of fluorescein-conjugated, affinity-purified, donkey anti- AMPAR may drive reverse-mode operation of this ex-
guinea pig IgG Jackson Immuno Research Laboratories changer, thereby mediating the rapid and significant in-
Inc., West Grove, PA and 1.0 mg ml Hoescht nuclear
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crease in [Ca ]
that we observed. We also provide
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stain. Alternatively, biotinylated anti-GFAP secondary
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evidence that AMPAR-dependent transmembrane Ca antibodies were visualized using the ABC-peroxidase
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influx via reverse mode operation of the Na Ca method Vecor Elite kit, Vector Laboratories Inc., Burling-
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exchanger, is associated with increased [Ca ] , through
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ame, CA. Cells were mounted on glass slides and
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the release of Ca from thapsigargin-sensitive intracellu-
photographed. Cells were .95 GFAP positive and lar stores. We conclude that the AMPAR and reverse mode
exhibited a flattened morphology typical of protoplasmic
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Na Ca exchanger activity may be functionally linked
astrocytes.
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in the co-regulation of intracellular Ca homeostasis in
astrocytes under certain conditions. 2.3. Reverse transcription–polymerase chain reaction
rtPCR
2. Materials and methods
