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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-5-1 21
methyl-4-isoxazole propionic acid (AMPA) class of glutamate receptors, and Na / Ca exchangers. Each of these pathways is thought to
21 21
be capable of mediating a significant increase in Ca concentration ([Ca ] ); however, the relative importance of each and theiri
21
interdependence in the regulation astrocyte [Ca ] is not known. We demonstrate here that 100i mM AMPA in the presence of 100mM
21 21
cyclothiazide (CTZ) causes an increase in [Ca ] in cultured cerebral astrocytes that requires transmembrane Cai influx. This increase
21 1 21
of [Ca ] is blocked by 100i mM benzamil or 0.5mM U-73122, which inhibit reverse-mode operation of the Na / Ca exchanger by
21 21
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
21 21 21
intracellular Ca stores, although depletion of these Ca stores does not decrease the peak [Ca ] response to AMPA. We propose thati
21 1 21
activation of AMPARs in astrocytes can cause [Ca ] to increase through the reverse mode operation of the Na / Cai exchanger with
21 21
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
21
these pathways would be capable of mediating a significant The regulation of intracellular Ca concentration
21
21
increase in [Ca ] ; however, the relative importance of ([Ca ] ) plays a central role in many astrocyte functions,i i
21
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each in the regulation of astrocyte [Ca ] and whether
and modulation of [Ca ] by transmembrane Cai flux i
they are functionally linked in astrocytes is not known has been implicated in the control of astrocyte-neuron
21
[13,18]. For example, AMPAR permeability to Ca can signaling [9,32,49], astrocyte proliferation and
differentia-1
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
21
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
1 21
tion of the Na / Ca exchanger has been shown to be AMPARs (a-amino-3-hydroxy-5-methyl-4-isoxazole
propi-21
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- 21
simultaneously examined all three transmembrane Ca 8082.
influx pathways in cultured cerebral astrocytes using E-mail address: [email protected] (L.D. Partridge).
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
21
While [Ca ] responses have been identified in variousi were performed on astrocytes that had been maintained in 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.
21
substantial elevation of [Ca ] , which is dependent on thei
presence of extracellular calcium. We provide evidence
2.2. Immunocytochemistry
21
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
21
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
21
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
1
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
21
crease in [Ca ]i that we observed. We also provide stain. Alternatively, biotinylated anti-GFAP secondary
21
evidence that AMPAR-dependent transmembrane Ca
antibodies were visualized using the ABC-peroxidase
1 21
influx via reverse mode operation of the Na / Ca
method (Vecor Elite kit, Vector Laboratories Inc.,
Burling-21
exchanger, is associated with increased [Ca ] , throughi ame, CA). Cells were mounted on glass slides and 21
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
1 21
Na / Ca exchanger activity may be functionally linked
astrocytes.
21
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
Total RNA was isolated and purified from cultured
2.1. Cell culture astrocytes by a modification of the method of
Chomczynsky [8], and quantified by spectrophotometry Cerebral astrocytes were grown from the cerebral cortex using a Beckman DU-64 spectrophotometer. All samples and hippocampus of newborn Sprague–Dawley rats using produced absorbance ratios between 1.5 and 2.0 (l5260 / a modification of the method of McCarthy and de Vellis 280 nm). Samples were immediately reverse transcribed [29]. Briefly, on the day of birth (postnatal day 0), rat pups into cDNA using the Superscript preamplification system were decapitated, meninges were removed, and cerebral according to the manufacturers recommended protocol cortices or hippocampi were dissected. Tissues were (Gibco / BRL). RNA templates were digested with Es-mechanically dissociated and plated in Corning T75 or T25 cherichia coli RNase H (2 units /ml, GibcoBRL). cDNAs
6 2
PCR Thermocycler System 9600 (Perkin–Elmer, Branch- (40mg protein per lane) for electrophoresis. Gel running
burg, NJ). buffer consisted of 25 mM Tris, 192 mM glycine, and
0.1% SDS. Proteins were electrophoretically transferred to
2.3.1. GluR subunits immobilon-P membranes (Millipore Corp.) at 48C.
GluR subunit cDNA sequences were detected using the Membranes were blocked for 1.5 h at room temperature following primers specific for the conserved sequences in blocking buffer consisting of 5.0% nonfat dry milk, 10 shared by GluR1, 2, 3 and 4 cDNAs as described by mM Tris (pH 7.4), 150 mM NaCl, and 0.05% Tween-20. Lambolez [25]: sense 59-CCTTTGGCCTATGAGATCTG- Membranes were probed with primary rabbit polyclonal GATGTG-39and antisense 59-TCGTACCACCATTTGTT- antibodies at room temperature for 1.5 h and then rinsed. TTTCA-39. cDNA was amplified by 35 cycles of 30 s at Primary antibodies were used as follows (all from 948C, 30 s at 498C and 30 s at 728C, which yielded an Chemicon International): 2.5 mg / ml anti-GluR1 amplified product of expected 750 base pairs. To provide (AB1504), 2.0 mg / ml anti-GluR2 (AB1768), 1 mg / ml enough material for restriction analysis, the GluR PCR anti-GluR2 / 3 (AB1506), and 3 mg / ml anti-GluR4 product was reamplified using a 1:7000 dilution of initial (AB1508). Membranes were incubated in blocking buffer PCR product for 35 cycles of 30 s at 968C, 30 s at 558C containing anti-rabbit Ig, horseradish peroxidase-linked and 30 s at 728C. To distinguish between the different (HRP) secondary antibody (NIF824; Amersham, Arlington GluR subunit sequences in this reaction mixture, four Heights, IL) for 90 min at room temperature and then equal aliquots were each digested with one of the follow- rinsed. Secondary antibodies were diluted 1:4000. Western ing restriction enzymes: BgII, Bsp1286I, EcoR47II, or blots were detected using the ECL Western Blotting EcoRI, each of which recognizes a restriction site unique Analysis System (Amersham), and developed with Hy-to the GluR1, 2, 3, or 4 rtPCR products, respectively, perfilm-ECL (Amersham). Controls for non-specific sec-yielding two fragments of 300 and 449 bp for GluR1 ondary antibody binding were completed in parallel re-(BgII), 478 and 271 bp for GluR2 (Bsp1286I), 359 and 396 actions with the omission of the primary antibody solu-bp for GluR3 (EcoR47II), and 411 and 338 solu-bp for GluR4 tions.
(EcoRI) [25,33]. Digestions were carried out to completion
at 378C for 2 h in the appropriate restriction buffers. 2.5. Whole-cell patch clamp
2.3.2. a1 subunit of the L-type calcium channel Membrane currents were measured by the whole-cell The following primers were used to amplify sequences patch clamp technique using an EPC-7 amplifier (Medical that are conserved among the a1 subunits of all L-type Systems, Darmstadt, Germany) and pCLAMP6 software calcium channels: sense 59-CGAAGCTTCTTCATGATG- (Axon Instruments Inc., Foster City, CA). Recordings were AACATCTT-39, antisense 59-GCGGATCCATGTAGAA- made in extracellular recording solution containing (in GCTGATGAA-39. Amplification reactions were carried mM): 140 NaCl, 5 KCl, 2 CaCl , 1 MgCl , 10 HEPES, 252 2 21
out at 35 cycles of 30 s at 948C, 30 s at 508C, and 60 s at dextrose, pH 7.4, |320 mosmol. For detection of Ba 728C, which yielded an expected 974 bp product [36]. currents, cells were rapidly superfused with barium and
tetraethylammonium (Ba-TEA) solution containing (in
2.3.3. b-Actin mM): 10 TEA–Cl, 140 NaCl, 5 KCl, 2 or 12 BaCl , 12
The following primer pair was used to amplifyb-actin MgCl , 10 HEPES, 25 dextrose, pH 7.4,2 |320 mosmol. cDNA sequences as a positive control: sense 59- The pipette solution contained (in mM): 140 KCl, 4 TCCTTCCTGGGTATGGAATC-39, antisense 39-ACTCA- MgCl , 0.1 EGTA, 2 ATP, 10 HEPES, pH 7.4,2 |290 TCGTACTCCTGCTTG-59. Amplification reactions were mosmol. Cells were stepped from a holding potential of carried out as described above for each reaction in which 260 mV to the indicated test potentials for 25 ms. Data this primer pair was used. were low pass filtered at 30 kHz and corrected for leak currents by graphic linear leak subtraction. Since we were 2.4. Western blot analysis interested in the amplitude of slow AMPA and L-type calcium currents and we did not attempt to measure Confluent cultures were harvested by scraping and then reversal potentials at large holding potentials, series resist-collected in ice cold HEPES buffer (0.5 mM) containing 1 ance was not an important consideration and was not mg / ml aprotinin and 10mg / ml leupeptin. After centrifu- compensated. Experiments were carried out at room tem-gation at 14 0003g at 48C, samples were resuspended, perature (22–278C).
positioned within 1.5 cell diameters of an astrocyte and were continuously perfused during bath exchange, and used for rapid application of AMPA. Dose–response AMPA or TGN were added directly to the bath to curves were generated from peak AMPA-induced inward transiently produce the desired bath concentration. Cells current amplitudes using a least squares regression iterated were pretreated with benzamil and U73122 for no more by the Marquardt–Levenburg method (Prostat V 1.02 than 15 min during which time fluorescence ratios reached software, Poly Software International, Salt Lake City, UT). a stable baseline, cells showed no changes in morphology, Because A7r5 cells are well characterized for their con- and ionomycin responses remained robust indicating that
21
stitutive expression of L-type Ca channels [28], these there were no toxic effects of these drugs at the con-cells were used as a positive control for VGCC-mediated centrations used.
currents in our patch clamp experiments.
2.6. Characterization of AMPARs
3. Results
A series of electrophysiological and pharmacological 21
3.1. AMPAR stimulation causes a [Ca ] -dependento experiments were initially performed to determine whether 21
increase in astrocyte[Ca ]i astrocytes, under our culture conditions, fulfilled
previous-ly established criteria for the expression of functional 21
To characterize their [Ca ] response to AMPA, thei
AMPA receptors. As described by Seeburg [40], we
astrocytes were loaded with Fura-2-AM and stimulated confirmed that 100 mM AMPA produced a desensitizing
with 100 mM AMPA, in the presence of 100 mM inward current with an average amplitude of255 pA, and
cyclothiazide (AMPA / CTZ), to block desensitization. Ap-that cyclothiazide (CTZ), which has a specific action on
proximately 80% of the astrocytes responded with signifi-AMPARs [35,42], eliminated the desensitization and in- 21
cant increases in [Ca ] that peaked at over 360% of thei
creased the average inward current amplitude to295 pA. 21
resting [Ca ] (ni 5202 cells, seven experiments). One We also found that the EC50 for the AMPA response was hundredmM AMPA alone, or 100mM cyclothiazide alone,
consistent with that for recombinant AMPARs [44,51] and 21
had no significant effect on [Ca ] (Fig. 1A). To de-i
that the AMPA response was blocked by the highly
termine whether AMPA / CTZ-dependent increases in
specific AMPAR blocker NBQX at 1 mM [56]. These 21 21
[Ca ]i required extracellular Ca , we repeated this results clearly indicate that AMPA / CTZ activates an 21
experiment in Ca -free medium containing 1 mM EGTA. essentially pure population of AMPARs in these astrocytes. 21
These cells were exposed to Ca -free medium for less than 1 min prior to recording to minimize the possibility of
2.7. Fura-2 calcium imaging 21
depleting intracellular Ca stores. As shown in Fig. 1B, astrocytes did not respond to AMPA / CTZ in the absence
Semi-confluent astrocytes (about 50%), grown on glass 21 21
of [Ca ] , suggesting that the increased [Cao ] was mosti
coverslips, were loaded with 4mM Fura-2 AM (Sigma) in 21
likely due to influx of extracellular Ca across the plasma 0.05% pluronic acid for 1 h at 378C in growth medium and
membrane (see also David et al. [10]). rinsed in recording solution for 15 min prior to imaging at
room temperature (22–278C). Images were acquired with
an inverted microscope (Nikon Diaphot 300, Melville, 3.2. Astrocyte AMPARs contain the GluR2 subunit NY), a cooled CCD video imaging system (SenSys 1400,
Photometrics, Tucson, AZ), and the Metafluor image AMPARs are heteromers composed of one or more of processing and analysis system version 2.76 (Universal GluR1, 2, 3, and 4 subunits (for review see Bettler and Imaging Corp., West Chester, PA). Cells were alternately Mulle [3]). The presence of the GluR2 subunit
substantial-21
excited at 340 and 380 nm for 100 ms and the emitted ly reduces AMPAR Ca permeability such that cells with fluorescence was measured at 510 nm. Background-sub- increasing numbers of GluR2 subunits have AMPARs with
21
tracted ratio images (340 / 380 nm) were then stored for very low Ca permeability [12]. As shown in Fig. 2A, all off-line analysis using Prostat software (Poly Software). four GluR subunit mRNAs were expressed in our cultures,
21
Approximate [Ca ] was determined using the method ofi as assessed by non-quantitative rtPCR followed by size Grynkiewicz [16]. At the end of each experiment, 1 mM analysis of restriction enzyme digests. The restriction assay ionomycin was added to the bath as a positive control. that we used to identify each subunit results in two
21
Ionomycin caused [Ca ] to increase in each experimenti restriction fragments that uniquely identify each subunit
21
(mean 613%) in which there was Ca in the bathing [25]. Importantly, the GluR2 subunit mRNA was readily
21 21
solution. With 0 Ca in the bathing solution, [Ca ]i detectable in these astrocyte cell cultures.
decreased by 50% in the presence of ionomycin. Western blot analysis also indicated expression of During measurements, the bath solution was constantly abundant GluR2 subunit protein (Fig. 2B). Immuno-exchanged at the rate of 2 ml / min through the 1-ml bath cytochemical analysis of the cultures demonstrated a producing a 95% exchange every 3 min. CTZ, Bay K8644, uniform distribution of immunostaining, such that the vast
1
mine is known to inhibit only AMPAR-mediated currents in cells that lack functional edited GluR2 subunits [53]. Taken together, these electrophysiological, molecular, and immunocytochemical data strongly suggest that the as-trocyte AMPARs contained functional GluR2 subunits and, therefore, would be expected to have less than maximal
21
Ca permeability. It is important to note that the maximal permeability of AMPARs that completely lack GluR2 subunits is itself thought to be extremely low [12].
21
The low AMPAR Ca permeability predicted by these experiments was further supported by our experiments with benzamil and U-73122. As described below, these
21
agents completely eliminated AMPAR-mediated Ca responses, but they are not known to affect AMPARs
21
directly. Thus it is highly questionable whether Ca influx through AMPAR channels accounted for the entirety of the
21
large AMPA / CTZ-dependent [Ca ] responses that wei
observed in our cultures.
21
3.3. AMPAR-dependent increases in [Ca ] were noti mediated by voltage-gated calcium channels(VGCCs)
One possible mechanism to explain the AMPA /
CTZ-21 21
dependent increase in [Ca ] , without substantial Cai
influx through AMPARs, is via activation of VGCCs following AMPAR-mediated depolarization. Since the
21
Fig. 1. (A) Fura-2 Ca imaging experiment showing the mean change in
expression of VGCCs in cultured astrocytes may vary [1],
21
[Ca ]i6S.E.M. for a representative example of 33 cells. Similar results
it was important to determine whether VGCCs were were observed in six additional experiments involving a total of 202 cells.
expressed under our culture conditions. We were unable to In control bath solution, addition of 100mM AMPA caused no change in
21
[Ca ] . With the addition of 100i mM CTZ to the bath, the same dose of detect VGCC expression using a number of different
21 21
AMPA elicited a large increase in [Ca ] that fell to baseline over ai detection methods. First, inward Ba currents were not 21
period of about 5 min. (B) Fura-2 Ca imaging experiment showing
detectable in whole-cell patch clamped astrocytes (Fig.
21
mean change in [Ca ]i6S.E.M. for 15 cells. In a bath solution
21 3A), nor were they induced by a 1-h pretreatment with the
containing 100 mM CTZ, and zero [Ca ] , plus 1 mM EGTA, theo
adenylate-cyclase activator, forskolin (10mM), which has addition of 100mM AMPA caused no response.
been previously reported to enhance VGCC-mediated
21
currents in astrocytes [1]. Likewise, using Fura-2 Ca subunit (data not shown). Taken together, these data imaging, we did not detect depolarization-dependent
in-21 1
strongly suggest abundant GluR2 subunit expression in creases in [Ca ] during stimulation with 55 mM K ,i 21
most if not all astrocytes within the cultures, suggesting even in the presence of the L-type Ca channel agonist, that calcium permeability of the astrocyte AMPARs would Bay K 8644 (1 mM). These negative results are most be very low [12]. likely due to the absence of VGCC expression, rather than Evidence for the function of GluR2 subunits in astrocyte to the presence of silent channels, since we did not detect AMPARs was derived from electrophysiological whole- mRNA encoding the pore-forming a1 subunit of the
L-21
cell patch recordings. Previous studies of recombinantly type Ca channel in astrocytes using rtPCR. a1 subunit expressed AMPARs in mammalian cells have shown that mRNA was, however, readily detectable in smooth muscle the current–voltage (I –V ) curves of AMPARs lacking A7r5 cells, which displayed inward currents (Fig. 3C). functional edited GluR2 subunits are doubly rectifying Taken together, these data strongly suggest that few, if with reversal potentials near 230 mV, whereas the I –V any, VGCCs were expressed in the astrocyte cultures thus relationship for GluR2 subunit-containing AMPARs are making it highly unlikely that VGCCs made any
signifi-21
linear with reversal potentials near 0 mV [50]. We observed cant contribution to AMPAR-mediated Ca responses. a linear I –V relationship for AMPAR-induced currents in
21
our astrocytes (linear regression R50.9955), that inter- 3.4. AMPAR-dependent increases in [Ca ] require thei
1 21
cepted the voltage axis at 0.53 mV (data not shown). In reverse-mode operation of the Na /Ca exchanger addition, administration of 500 mM HPP-spermine at a
21 21
Fig. 2. (A) rtPCR results for astrocytes. GluR1, 2, 3, and 4 mRNA were detected in these cerebral astrocytes as indicated by the presence of restriction products that appear below 600 bp in each lane that is labeled GluR. Unrestricted products are shown for comparison. Also included are 100 bp size standards that show 100 bp increments around 600 bp, which appears as the brightest band. (B) ECL Western blot SDS–PAGE results. As shown by both the GluR2 and the GluR2 / 3 antibodies, GluR2 subunits were expressed in relatively high proportions in cultured cerebral astrocytes. Brain tissue containing neurons and astrocytes was used as a positive control. COS-7 cells, which do not express GluRs, were used as a negative control. GluRs migrate near 110 kDa during SDS–PAGE, therefore the region around 110 kDa is shown for each experiment.
1 1
produced the observed AMPAR-mediated increases in ing [Na ] directly affects both the function of the Na /o
21 21
[Ca ] , we examined the contribution of the reverse-modei Ca exchanger as well as of AMPARs. Therefore, to test
1 21 1 21
operation of the Na / Ca exchanger [5], apparently the involvement of the Na / Ca exchanger, we exposed
1
driven by Na influx through the AMPAR. Indeed, Gold- the cells to AMPA / CTZ after a 10-min pretreatment in 100
1 21
man et al. [14] have previously demonstrated the reverse- mM benzamil, a well known inhibitor of the Na / Ca mode operation of this transporter following increased exchanger [22,24,26]. As shown in Fig. 4A, benzamil
1
[Na ]i in astrocytes. Additionally, David et al. [10] completely eliminated the AMPA / CTZ-dependent increase
1 21
showed that removal of [Na ] completely blocks AMPA /o in [Ca ] in 94% of the astrocytes. Benzamil pretreatmenti 21
CTZ-dependent increases in protoplasmic astrocyte caused an increase of resting [Ca ] to 147% of that ini
21 1 21
[Ca ] , suggesting the possibility that AMPAR activityi untreated cells, consistent with inhibition of the Na / Ca
1 21 21
and the Na / Ca exchanger are functionally linked. It is, exchanger; however, this [Ca ] level was still well belowi
1 21
however, not possible to alter [Na ] in order to test theo the average AMPAR-mediated increase in [Ca ] . Thesei
1 21 1 21
possible functional relationship between the Na / Ca results strongly suggest that the Na / Ca exchanger was
21
exchanger and AMPARs, with respect to understanding the involved in the [Ca ] response to AMPA / CTZ.i
21 1 21
Fig. 3. (A) Whole-cell patch clamp results from cerebral astrocytes grown in FCS. Recordings were made in 10 cells and no inward currents were observed
21
within the sensitivity of our measurements. Error bars represent S.E.M.s. The extracellular recording solution contained Ba -TEA, and the results were
21 21
adjusted by linear leak subtraction (see Section 2). (B) Fura-2 Ca imaging results in astrocytes showing no increase in [Ca ] in depolarizing solutioni
1 21 21
(55 mM K ), or in depolarizing solution with the L-type Ca channel agonist Bay K 8466 (1mM). Average [Ca ]i6S.E.M. is shown from data pooled
21
from nine groups of 10–30 cells. (C) rtPCR results for astrocytes grown in FCS and for A7r5 cells. A7r5 cells (lane 1) express L-type Ca channela1
subunit mRNA (932 bp). L-Type calcium channela1subunit mRNA was not detected in astrocytes (lane 2).b-Actin sequences (300 bp) were amplified from astrocyte cDNA in parallel reactions as a positive control to confirm the integrity of the rtPCR reactions.
1 21
is known to require phospholipase-C (PLC)-activation and reverse-mode operation of the Na / Ca exchanger
lead-21
hydrolysis of phosphoinositol-bis-phosphate (PIP )2 ing to elevated [Ca ] in astrocytes exposed to AMPA /i
1 21
CTZ. [2,7,17]. Therefore, as a second measure of the Na / Ca
21
exchanger involvement in AMPAR-mediated [Ca ] re-i
3.5. AMPA /CTZ depletion of thapsigargin (TGN)-sponses, we pretreated astrocytes in 0.5 mM U-73122, an
21
sensitive Ca stores inhibitor of PLC in astrocytes, and subsequently exposed
the cells to AMPA / CTZ [47,49]. As shown in Fig. 4B,
Because of the many instances of interactions between U-73122 completely eliminated the AMPAR-dependent
21 21
21
transmembrane Ca flux and the release of Ca from increase in astrocyte [Ca ] . In this experiment, restingi
21
intracellular stores, we investigated the possibility of a role [Ca ] was again elevated, consistent with an inhibitioni
1 21 21
for thapsigargin-sensitive stores in the AMPAR-dependent of the Na / Ca exchanger, but again this [Ca ] leveli
21
increase in [Ca ] . Thus we treated astrocytes with was well below the average AMPAR-mediated increase in i
21
21
thapsigargin, to deplete intracellular TGN-sensitive Ca [Ca ] .i
stores both prior to and after AMPA / CTZ treatment (see One possible explanation for the block of
AMPAR-21
Wictome et al. [55]). As shown in Fig. 5A, AMPAR-mediated Ca responses following PLC inhibition is
21
21
dependent increases in [Ca ] were not attenuated by through the inhibition of Ca release from internal stores. i
pre-treatment with TGN. However, as shown in Fig. 5B, This is, however, unlikely because benzamil had a similar
21
pre-treatment with AMPA / CTZ severely decreased the blocking effect to U-73122 and since release of Ca from
21
TGN-dependent [Ca ] response. internal stores has not been linked previously to AMPAR i
Fig. 5C summarizes the results of 17 experiments stimulation. Thus, the simplest explanation of our data is
1
21
regulating internal Ca stores and where it may also help
21
to regulate other Ca phenomena [14,15,46]. Despite its potential importance in astrocytes, very little is known
1
about physiological mechanisms that can stimulate Na /
21
Ca exchange and its role in astrocyte-brain physiology.
21
It has been previously suggested that Ca influx through
1 21
reverse mode function of the Na / Ca exchanger might result from ionotropic glutamate receptor (iGluR) activa-tion in astrocytes [10,21,22]. Demonstraactiva-tion of this cou-pling would greatly improve our understanding of the
1 21
Na / Ca exchanger in astrocytes. In contrast, modulation
21
of [Ca ] by ionotropic glutamate receptor stimulation ofi
1 21
reverse mode operation of the Na / Ca exchanger has been demonstrated in Bergmann glia, oligodendrocyte progenitors, and rat forebrain neurons, clearly
demon-21
strating that these two transmembrane Ca influx path-ways can be functionally related [19,23,26]. In this report we provide several lines of evidence to suggest that
1 21
AMPAR activation and reverse-mode Na / Ca exchange are indeed coupled in cultured astrocytes leading to
21 1 21
AMPAR-dependent Ca influx through the Na / Ca
21
exchanger and an associated release of Ca from intracel-lular stores.
21
21 4.1. AMPAR stimulation causes a [Ca ] -dependent
Fig. 4. (A) Fura-2 Ca imaging experiment showing the mean change in o
21
21
increase in astrocyte[Ca ] [Ca ]i6S.E.M. for 64 cells from three separate experiments. In a bath i
solution containing 100mM CTZ, and 100mM benzamil, the addition of
21
100mM AMPA caused no response. (B) Fura-2 Ca imaging experiment It is well established that application of glutamate,
21
showing the mean change in [Ca ]i6S.E.M. for 55 cells from two
quisqualate, kainate, and / or AMPA to astrocytes can lead separate experiments. In a bath solution containing 100mM CTZ, and 0.5 21 21
to the [Ca ] -dependent elevation of [Ca ]
mM U-73122, the addition of 100 mM AMPA caused no change in o i
21
[10,13,18,21,22]. Fewer studies have specifically focused [Ca ] .i
on the role that the AMPA-preferring subtype of glutamate receptor might play in these responses. Our studies re-produce the results of David et al. [10] who showed the
21 21 21
average peak [Ca ] response evoked by AMPA / CTZi [Ca ] -dependent elevation of [Cao ] in astrocytes fol-i
alone, or AMPA / CTZ after a pretreatment with TGN, were lowing AMPAR stimulation (Fig. 1B). This result raises
21
not significantly different from one another. Treatment of the question: how does AMPAR-stimulation lead to Ca the cells with TGN after pretreatment with AMPA / CTZ, influx across the plasma membrane? Since some AMPARs
21
however, resulted in the significant attenuation of the TGN are known to have a limited permeability to Ca , the
21
response to less that 66% of its original magnitude. The simplest conclusion is that the Ca enters directly through
21
results of Fig. 5 indicate that TGN-sensitive Ca stores the AMPAR; however, our results bring this interpretation are partially depleted by AMPA / CTZ, but that release of into question in our cells.
21
Ca from these stores does not significantly add to the
21
AMPA / CTZ-stimulated increase in [Ca ] . Thus AMPARi
stimulation appears to be linked to the reverse mode 4.2. Astrocyte AMPARs contain functional GluR2
1 21
operation of the Na / Ca exchanger and to an associated subunits
21
release of Ca from TGN sensitive stores, to achieve a
21
much more complex intracellular [Ca ] response thani The molecular biological data shown in Fig. 2 clearly
21
would be expected by the function of either of these Ca indicate the presence of the GluR2 subunit mRNA and
effectors alone. protein in our astrocytes, moreover, by determining that
the I –V relationship for AMPA / CTZ induced currents was linear, reversed very close to zero mV, and was
unrespon-4. Discussion sive to HPP-spermine, we were able to confirm the
presence of functional GluR2 subunits in our astrocyte
1 21
The Na / Ca exchanger is expressed by astrocytes AMPARs. The presence of these subunits strongly suggests
21
21 21
Fig. 5. (A) Top: Fura-2 Ca imaging experiment showing the mean change in [Ca ]i6S.E.M. for 33 cells. In a bath solution containing 100mM CTZ,
21 21
application of 50 nM TGN caused a large increase in [Ca ] . Subsequent treatment with 100i mM AMPA caused a similar large increase in [Ca ] .i
21 21
Ionomycin was used as a positive control (not shown). Bottom: Fura-2 Ca imaging experiment showing the mean change in [Ca ]i6S.E.M. for 40 cells.
21
In a bath solution containing 100mM CTZ, pretreatment with 100mM AMPA diminished the increase in [Ca ] caused by 50 nM TGN. (B) Summary ofi
21 21
data from Fura-2 Ca imaging experiments. Average [Ca ]i6S.E.M. is shown under basal conditions (11 experiments, 314 cells; baseline). After treatment with 100mM AMPA (seven experiments, 202 cells; AMPA), and after treatment with 50 nM TGN (four experiments, 107 cells; TGN), after pretreatment with 100 mM AMPA (three experiments, 92 cells; TGN(AMPA)), and after pretreatment with 50 nM TGN (three experiments, 78 cells;
21 21
AMPA(TGN).6S.E.M. (C) Summary of data from Fura-2 Ca imaging experiments. Average [Ca ]i6S.E.M. is shown under basal conditions, 11 experiments, 314 cells, baseline. After treatment with 100mM AMPA, seven experiments, 202 cells, AMPA. After treatment with 50 nM TGN, four experiments, 107 cells, TGN. After pretreatment with 100mM AMPA, three experiments, 92 cells, TGN(AMPA). After pretreatment with 50 nM TGN, three experiments, 78 cells, AMPA(TGN).
21
responsible for the observed [Ca ] responses in our cells,i further support our interpretation that these AMPARs have
21
since it is known that the incorporation of these subunits at most extremely low Ca permeability. Additionally, it
21
into AMPARs severely reduces their Ca permeability is very possible that previous attempts to directly measure
21 21
[12,39,44]. Furthermore, Ca influx accounts for only a the Ca permeability of astrocyte AMPARs may have
21
very small portion of the total current through AMPARs greatly overestimated their Ca permeability due to the
1 21
that completely lack GluR2 subunits, thus it is unlikely possible contribution of the Na / Ca exchanger to
21 21
that direct Ca flux through AMPARs could account for AMPA-dependent [Ca ] responses.i
21 21
the large [Ca ] responses that we observed in thesei Because AMPA only induces an increase in [Ca ]i
GluR2-containing astrocytes. when AMPAR desensitization is blocked by CTZ, the Despite our evidence to the contrary, our data do not question is raised: Under what physiological conditions
21
rule out a small contribution of direct Ca influx through could this pathway be operative? Alternative ‘flip’ and AMPARs. However, if these AMPARs have even a very ‘flop’ splice variants of AMPAR subunit mRNAs exert
21
low permeability to Ca , then benzamil or U-73122 control over AMPAR desensitization [43]. Because as-would not have been expected to completely eliminate the trocyte AMPAR desensitization appears to be
developmen-21
AMPA / CTZ-evoked [Ca ]i responses. Therefore, the tally regulated [41], it is possible that functional
AMPAR-1 21 1 21
data in Fig. 4 not only indicate coupling of the Na / Ca Na / Ca exchanger coupling could be developmentally
21
1 21
also possible that endogenous modulatory agents could effects were most likely via blockade of Na / Ca directly alter AMPAR function and thereby regulate the exchanger activity.
1 21 1 21
effectiveness of AMPAR-Na / Ca exchanger coupling. To further examine the role of the Na / Ca exchanger
21
Evidence for this hypothesis comes from neostriatal in our AMPA / CTZ stimulated [Ca ]i responses, we medium spiny neurons, where AMPAR-mediated excitat- utilized the phospholipase C (PLC) inhibitor U-73122. ory postsynaptic currents are rapidly modulated by dopa- PLC has been shown in a variety of preparations to exert a minergic influences [48]. It is also possible that AMPAR- very powerful modulatory effect on the reverse-mode
1 21 1 21
Na / Ca exchanger coupling causes continual small operation of the Na / Ca exchanger. Initial studies
21 1 21
changes in [Ca ] under normal conditions, which arei showed that the reverse mode operation of the Na / Ca amplified when desensitization is blocked. exchanger is positively regulated by synthesis and
degra-dation of PIP2 through a mechanism that appears to involve the physical association of PIP with the exchanger
21 2
4.3. AMPAR-dependent increases in [Ca ] were noti
[2,17]. Other investigators have described a similar
regula-mediated by VGCCs 1 21
tion of reverse mode operation of the Na / Ca ex-changer by PLC in oligodendroglial precursor cells and It has been hypothesized that AMPAR activation might
cerebral microvessels [7,26,45]. Most recently, it was
21 21
result in an increase of [Ca ] because of Cai currents demonstrated that PLC plays a specific and critical role in
through VGCCs activated as a result of the AMPAR- 1 21
the amplification of reverse-mode action of the Na / Ca dependent depolarization [22]. The AMPAR-dependent
exchanger [52]. Given the specific dependence of the
21
changes in [Ca ] that we observed are unlikely to occuri 1 21
operation of the Na / Ca exchanger on PLC, we were by this mechanism, since we were unable to find any
not surprised to see the dramatic inhibition of AMPA /
evidence for the expression of VGCCs in our cultured 1 21
CTZ-stimulated reverse mode Na / Ca exchanger opera-astrocytes. While the expression of functional VGCCs in
tion with the PLC inhibitor U73122 (Fig. 4B). Because astrocytes depends on many factors, our observations in
U-73122 did not affect AMPAR responses in CA1 neurons cultured astrocytes are consistent with the recent report of
[38] or in oligodendrocyte progenitors [27], it is highly Carmignoto et al. [6] in which VGCCs were not expressed
unlikely that the blockade of the AMPAR / CTZ-stimulated in adult rat brain astrocytes in situ. Perhaps the upregula- 21
rise in [Ca ] by U-73122 was due to a non-specifici
tion of VGCCs by astrocytes under certain pathological
inhibition of the AMPAR channel, particularly at the low conditions [54], could create conditions whereby
gluta-concentrations U73122 used here. Rather, these inhibitory
21
mate-stimulated [Ca ] signaling might be accentuated byi effects were most likely via PLC blockade leading to
a VGCC-dependent mechanism. 1 21
Na / Ca exchanger inhibition.
21
4.4. AMPAR-dependent increases in [Ca ] requirei 4.5. AMPA /CTZ-dependent depletion of thapsigargin
1 21 21
reverse mode operation of the Na /Ca exchanger (TGN) sensitive Ca stores
1 21
In their studies using the Na / Ca exchanger-in- Our data indicating that AMPA / CTZ depletes astrocyte
21
hibitors benzamil and amiloride, Kim et al. [22] have TGN-sensitive Ca stores are very interesting, since clearly demonstrated that the reverse mode function of the AMPAR activation is not thought to have the capacity for
1 21 21
Na / Ca exchanger has an important role in glutamate or directly releasing Ca from intracellular stores. However,
21
kainate stimulated [Ca ] responses in cultured astrocytes.i because of the strong effect of PLC inhibition in our Our studies support those results and help to clarify the experiments, and since PLC stimulation has been shown to
1
mechanisms involved since we used the selective AMPAR exert downstream effects through stimulation of the Na /
21
agonist, AMPA / CTZ with benzamil (Fig. 4A), rather than Ca exchanger [7], we were led to hypothesize that
1 21
glutamate, which simultaneously activates iGluRs of the reverse mode operation of the Na / Ca exchanger might
21
kainate and AMPA-preferring subtypes, as well as be associated with the release of TGN-sensitive Ca metabotropic glutamate receptors. Kim et al. [22] also stores in a PLC-dependent mechanism. Our results in Fig.
21
show the complete elimination of kainate induced [Ca ]i 5 are consistent with this interpretation. Our data do not responses by benzamil further supporting our results that rule out an additional involvement of ryanodine-sensitive
21 21
21 [10] J.C. David, K.A. Yamada, M.R. Bagwe, M.P. Goldberg, AMPA
entire AMPA / CTZ-dependent [Ca ] increase. Any addi-i
receptor activation is rapidly toxic to cortical astrocytes when tional contribution to this increase, due to release from
desensitization is blocked, J. Neurosci. 16 (1996) 200–209.
21
Ca stores, appeared only to enhance the forward-mode [11] T. Florio, M. Grimaldi, A. Scorziello, M. Salmona, O. Bugiani, F.
21 21
extrusion of Ca as the exchanger reaches its [Ca ]i Tagliavini, G. Forloni, A. Schettini, Intracellular calcium rise threshold for reversing direction, since pretreatment with through L-type calcium channels, as molecular mechanism for prion
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TGN did not reduce the magnitude of the observed [Ca ]i
Biophys. Res. Commun. 228 (1996) 397–405. increase (Fig. 5A).
[12] J.R. Geiger, T. Melcher, D.S. Koh, B. Sakmann, P.H. Seeburg, P. Taken together, our data clearly indicate that the func- Jonas, H. Monyer, Relative abundance of subunit mRNAs
de-1 21
21
tional coupling of AMPARs and Na / Ca exchangers is termines gating and Ca permeability of AMPA receptors in
21
a potentially important aspect of astrocyte [Ca ] regula-i principal neurons and interneurons in rat CNS, Neuron 15 (1995)
21 193–204.
tion. By simultaneously examining Ca permeable
AM-1 21 [13] S.R. Glaum, J.A. Holzwarth, R.J. Miller, Glutamate receptors
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the functional interdependency of these [Ca ] effectori [14] W.F. Goldman, P.J. Yarowsky, M. Juhaszova, B.K. Krueger, M.P. pathways in astrocytes suggesting that the integration of Blaustein, Sodium / calcium exchange in rat cortical astrocytes, J.
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Wallace, Ph.D., Paul McGuire, Ph.D., Rebecca Lee, Ph.D., [18] J.A. Holzwarth, S.J. Gibbons, J.R. Brorson, L.H. Philipson, R.J. Thomas Resta, Ph.D., and Benjimen Walker, Ph.D. This Miller, Glutamate receptor agonists stimulate diverse calcium re-work was funded by NIH grant NS32562 and HHMI grant sponses indifferent types of cultured rat cortical glial cells, J
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![Fig. 1. (A) Fura-2 Ca1addition of 1002imaging experiment showing the mean change in[Ca21] 6S.E.M](https://thumb-ap.123doks.com/thumbv2/123dok/3137678.1382424/5.612.48.284.63.359/fig-fura-addition-imaging-experiment-showing-mean-change.webp)
![Fig. 5. (A) Top: Fura-2 Ca1experiments, 107 cells, TGN. After pretreatment with 100pretreatment with 1002imaging experiment showing the mean change in [Ca21] 6S.E.M](https://thumb-ap.123doks.com/thumbv2/123dok/3137678.1382424/9.612.86.514.64.388/experiments-cells-pretreatment-pretreatment-imaging-experiment-showing-change.webp)
