Hu B et al / Acta Pharmacol Sin 2004 Jun; 25 (6): 714-720

NMDA and AMPA receptors mediate intracellular calcium increase in rat cortical astrocytes¹

Bo HU, Sheng-gang SUN², E-tang TONG

Neurology Department, Union Hospital, Tongji College, Huazhong University of Science and Technology, Wuhan 430022, China

¹ Project supported by the National Natural Science Foundation of China (No 30040037).

² Correspondence to Prof Sheng-gang SUN.

Received 2003-04-28 Accepted 2004-01-12

KEY WORDS glutamate; astrocytes; NMDA receptors; AMPA receptors; calcium

ABSTRACT

AIM: To study the effect of glutamate on the intracellular calcium signal of pure cultured rat astrocytes and the role of N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) receptors in the procedure. METHODS: The fluorescence of calcium was measured by Fura-2/AM (F₃₄₅/F₃₈₀). RESULTS: L-Glutamate induced [Ca²⁺]_i increase in most of the cells in concentration- and time-dependent manner. NMDA 50 mmol/L induced the fluorescence increase by almost three to four times, while the effect of AMPA 50 mmol/L was just half of that of D-(-)-2-amino-5-phosphonopentanoic acid (D-AP-5; a selective antagonist of the NMDA receptor). 6-Cyano-7-nitroquinoxaline-2,3-dione (CNQX, a selective antagonist of the AMPA receptor) abolished the effects of NMDA and AMPA, respectively. D-AP-5 and CNQX simultaneously or respectively attenuated the effect of L-glutamate at different degrees, but could not abolish it entirely. CONCLUSION: Glutamate modulated intracellular Ca²⁺ of pure cultured rat astrocytes through different pathways. The activation of NMDA and AMPA receptors took part in the complex mechanisms.

INTRODUCTION

Almost 50 % of cells in central nervous system are astrocytes. They play an important role in normal physiological activity and have intimate relationship with neurons. There is a close bidirectional communication existing between neurons and astrocytes^[1]. Glutamate, as the most important excitatory transmitter in central nervous system, is proved to be a crucial bridge between astrocytes and neurons. Astrocytes responded to glutamate released from neurons by intracellular Ca²⁺ increase under physiological conditions^[2]. On the other hand, recent Ca²⁺ imaging studies in cell culture and in situ showed that Ca²⁺ elevations in astrocytes induced glutamate release in a calcium-dependent manner^[3,4]. Therefore, the modulation of Ca²⁺ elevations in astrocyte by glutamate should aid in understanding the interaction between astrocytes and neurons^[5].

The mechanisms of calcium mobilization in astrocytes include: (1) Calcium stores. There are two types of calcium stores within the cultured cortex astrocytes of rats: one is IP₃-sensitive, the other is IP₃-insensitive, and mitochondria could serve as an intracellular calcium buffering system in astrocytes^[6,7]. (2) Na⁺/Ca²⁺ exchange. It regulated [Ca²⁺]_i when cytosolic Ca²⁺ increased ten times higher than the resting level. Immunohistochemistry revealed that exchanger molecules distributed in a reticular pattern over the astrocyte surface^[8]. (3) Voltage-operated calcium channel and receptor-operated calcium channels. Glutamate receptor(GluR) was one of the most important receptors which took part in the astrocytic Ca²⁺ mobilization^[9].

There are two classes of glutamate receptors on astrocytes: one is ionotropic receptor linked directly to an ion channel, and the other is metabotropic receptor which induces internal mobilization of Ca²⁺ via inositol phospholipid hydrolysis^[10,11]. Glutamate receptor- mediated responses were detected by imaging Ca²⁺ in astrocytes. N-Methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) receptors were found to be expressed in astrocytes through immunocytochemistry methods^[12,13]. Intracellular astrocyte calcium played an important role in astrocyte-to-neuron signaling^[14,15]. But astrocytes from different animals, different developing periods, even different positions expressed different glutamate receptors. By Northern blot analysis, GluR1 mRNA was the highest in astrocyte cultures from cerebellum and hippocampus and moderate in astrocyte cultures from neocortex and striatum. GluR3 mRNA was detectable in astrocyte cultures from cerebellum and neocortex. GluR2 (the subunit that limits the Ca²⁺ permeability of AMPA receptor) and NR1 mRNA expression were not detected in astrocytes cultured from any brain region examined. In situ hybridization studies showed wide expression of GluR1 mRNA in cultured astrocytes, but GluR2 and GluR3 mRNAs were near background levels^[16]. The AMPA type-glutamate receptor channels without the GluR2 (GluR-B) subunit were characterized by high Ca²⁺ permeability^[17].

In this study, we investigated effects of NMDA and AMPA receptors on type-1 astrocytes from cortex of neonatal rats on intracellular Ca²⁺.

MATERIALS AND METHODS

Chemicals and drugs Fetal bovine serum, DMEM/F12 were purchased from Gibco Company. HEPES, NMDA, AMPA, D-(-)-2-Amino-5-phosphono-pentanoic acid (D-AP-5; a selective antagonist of NMDA receptor), 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, a selective antagonist of AMPA receptor), L-glutamate, poly-lysine, trypsin, egtazic acid, and thapsigargin were purchased from Sigma Company. Fura-2/AM was purchased from Molecular Probe Company. Other ordinary drugs were from local chemical drug companies.

Cell culture Sprague-Dawley rats (1-3 d post-natal) were obtained from the Experimental Animal Center of Tongji Medical School, Huazhong University of Science and Technology. All experiments were carried out under the National Animal Protection Protocol. The rats were decapitated under ether anaesthesia. Cortex slice of 500-800 µm thick was cut. The slices were treated with 0.25 % trypsin and incubated in culture medium saturated with 95 % O₂ and 5 % CO₂ at room temperature (21-23 ºC) for 30 min. Culture medium consisted of 78 % DMEM/F12, 10 % fetal calf serum, 1 % benzylpenicillin, and streptomycin 100 mg/L. Cells were maintained in an incubator of 10 % CO₂ at 37 ºC. Seven to nine days later, the culture bottles were put in 37 ºC vibratory device for 15 to 18 h, then passaged every week, and plated on poly-lysine-coated coverslip at cell density of 5×10⁸ L^-1. Coverslips incubated for 3-7 d were used. The cytoimmunochemical results showed that 98 % of the cells were glial fibrilary acidic protein-positive glial cells. Most of the cells were type-1 astrocytes with multiple dendrites. So we chosed type-1 astrocytes as research samples.

Fura-2 loading The cell-bearing coverslips were rinsed three times with buffer solusion without calcium before loading and then incubated with Fura-2/AM (2-5 µmol/L) at 37 ºC for 30 min. The buffer solution contains (mmol/L): NaCl 140, KCl 5.0, MgSO₄ 1, D-glucose 10, 1,4-(2-hydroxyethyl)-1-piperazineethane-sulfonic acid (HEPES) 10, egtazic acid 100 µmol/L (pH=7.3).

Intracellular Ca²⁺ measurement The fluorescent microscopy is constructed around an Axiovert 100 (Zeiss, Germany). Excitation wave length was set at 345 nm or 380 nm by monochromator system (TILL, Photonics, Germany) controlled by computer. The fluorescence signals were collected by a photomultipler (Hammamashu R928) and converted to voltage values. X-CHART (Heka, Germany) was used to calculate and monitor the intracellular [Ca²⁺]_i . The fluorescence ratio of F₃₄₅/F₃₈₀ was used to reflect the fluctuation of [Ca²⁺]_i^[18].

Solutions The bath solution in most experiments was (in mmol/L): NaCl 140, HEPES 10, KCl 2, D-glucose 10, MgCl₂ 2, CaCl₂ 2.5, pH=7.3, with Osmolarity=320±5 mOsm/L. The solution without calcium was the same as above by replacing CaCl₂ with MgCl₂.

RESULTS

Glutamate induced astrocytic [Ca²⁺]_i increase The response of astrocytic [Ca²⁺]_i to glutamate was variable, about 83 % of cells had obvious response to the application of L-glutamate (n=120), while others had mild response, and some cells exhibited calcium oscillation (data not shown).

Glutamate elevated [Ca²⁺]_i in a time- and concentration-dependent manner The magnitude of fluorescence increase induced by glutamate was related to simulation time. When glutamate 100 µmol/L was applied to stimulate the same cell at intervals at least 5 min to avoid receptor desensitization, the longer the stimulation time was, the higher the fluorescence was. As the response time became longer, the decline of fluorescence became slower (n=7, Fig 1A).

Fig 1. Effect of L-glutamate (Glu) on [Ca²⁺]_i of astrocytes in normal extracellular solution. A) Glu 100 µmol/L was applied for 5 s, 10 s, 20 s, and 30 s on the same cell at intervals at least 5 min to avoid receptor desensitization; B) Glu 1 mmol/L-5 mmol/L was applied on the same cell for 10 s at intervals at least 5 min to avoid receptor desensitization.

Different concentrations of glutamate were applied to stimulate the same cell for 10 s at least for 5 min to avoid receptor desensitization. Glutamate induced Ca²⁺ increase in a concentration-dependent manner. When the concentration was 50 mmol/L, the elevated [Ca²⁺]_i reached a plateau and did not decline to baseline, which was followed by cell death. The EC₅₀ was about 5 mmol/L in normal calcium extracellular solution (Fig 1B).

Effect of NMDA receptor on astrocytes response to glutamate Of all the cells respond to glutamate, the response of 87 % (n=66) cells induced by glutamate 1 mmol/L were diminished by D-AP-5 100 µmol/L, but could not be abolished entirely (Fig 2). It demonstrated that effect of glutamate on astrocytic calcium signal was partially through activation of NMDA receptors.

Fig 2. Effect of D-AP-5 on the increase of astrocytic [Ca²⁺]_i induced by glutamate.

About 72 % of all the cells responded to NMDA with [Ca²⁺]_i increase (n=50). The response induced by NMDA 50 µmol/L could be almost abolished by D-AP-5 100 µmol/L, and it recovered after a long enough interval (Fig 3), which indicated that the activation of NMDA-receptor took part in astrocytic calcium signal modulation.

Fig 3. Effect of NMDA on astrocytic [Ca²⁺]_i in normal extracellular solution.

Effect of non-NMDA receptor on response of astrocytes to glutamate Of all the cells respond to glutamate, the response of 83 % (n=70) cells induced by glutamate 1 mmol/L was diminished by CNQX 45 µmol/L, but could not be abolished entirely (Fig 4).

Fig 4. Effect of CNQX on increase of astrocytic [Ca²⁺]_i induced by glutamate.

About 81 % of all the cells responded to AMPA with [Ca²⁺]_i increase (n=56). The response induced by AMPA 50 µmol/L was markedly diminished by CNQX 45 µmol/L, and it recovered after a long enough interval (Fig 5). It demonstrated that the activation of AMPA-receptor took part in astrocytic calcium signal modulation.

Fig 5. Effect of AMPA on increase of astrocytic [Ca²⁺]_i in normal extracellular solution.

When CNQX and D-AP-5 was applied simultaneously on the same cell (n=23), the increased [Ca²⁺]_i was inhibited significantly, but could not be abolished completely (Fig 6, 7). It suggested that perhaps there were other mechanism such as metabotropic receptor might take part in the reaction induced by glutamate.

Fig 6. Effect of CNQX and D-AP-5 on increase of astrocytic [Ca²⁺]_i induced by glutamate in normal extracellular solution.

Fig 7. Comparison of the responses of astrocytes to Glu 1 mmol/L, AMPA 50 µmol/L, NMDA 50 µmol/L, Glu 1 mmol/L+CNQX 45 µmol/L, Glu 1 mmol/L+D-AP-5 100 µmol/L, and Glu 1 mmol/L+CNQX 45 µmol/L+D-AP-5 100 µmol/L. n=23. Mean±SD. ^cP<0.01 vs Glu+CNQX group.

Effect of extracellular Ca²⁺ in the response of astrocyte to glutamate In extracellular solution without calcium or after Ca²⁺-chelator egtazic acid 2 mmol/L was added in extracellular solution, the response of astrocytes to glutamate became weaker (Fig 8, n=22). In normal extracellular solution, the elevation of astrocytic [Ca²⁺]_i induced by glutamate could be inhibited entirely after stimulation with thapsgargin (Fig 9). It shows that response of glutamate is extracellular-Ca²⁺ independent, and there is other mechanism except Na⁺/Ca²⁺ exchange.

Fig 8. Effect of L-glutamate on [Ca²⁺]_i of astrocytes after addition of egtazic acid or in extracellular solution without calcium.

Fig 9. Effect of thapsgargin 1 µmol/L on astrocytic [Ca²⁺]_i increase induced by glutamate 1 mmol/L in extracellular solution without calcium.

DISCUSSION

When cerebral ischemia or damage occurr, gluta-mate receptors are activated excessively and extracellular glutamate is elevated rapidly^[19-21]. Increase in the concentration of intercellular glutamate protected astrocytes during this process. Concentration and diffusion of glutamate in the extracellular space are associated with the degree of astrocytic coverage to neu- rons^[22,23]. Previous work discovered that glutamate reuptake by astrocytes existed not only under pathological states but also under physiological states^[2,24-26].

Non-NMDA receptors contributed to partial elevation of [Ca²⁺]_i. CNQX , a potent competitive antagonist of the AMPA/Kainate (non-NMDA) receptor, decreased the elevation of [Ca²⁺]_i induced by glutamate. Consisted with our results, some evidence showed that astrocytes expressed Ca²⁺-permeable AMPA receptors^[2,11,27]. Aactivation of AMPA receptors in astrocytes caused increase in [Ca²⁺]_i through the reverse mode operation of the Na⁺/Ca²⁺ exchanger with an associated release of Ca²⁺ from intracellular stores^[27,28]. Other conflicting results demonstrated that activation of AMPA and NMDA receptor did not affect intracellular calcium stores^[29].

Smith et al proposed that astrocytes exhibited three transmembrane Ca²⁺ influx pathways: voltage-gated Ca²⁺ channels (VGCCs), AMPA class of glutamate receptors, and Na⁺/Ca²⁺ exchangers^[28]. But our results was not the same as it completely. Immunocytochemistry and physiology proved that astrocytes expressed NMDA receptors^[13,30]. When D-AP-5, a selective NMDA receptor antagonist, was applied, response of glutamate was abolished. It is an indirect proof of existence of NMDA receptor and its effects on [Ca²⁺]_i in astrocytes. The mechanism of NMDA receptor in astrocytic calcium mobilization is not clear yet. Our results conflicted with some previous reports, so we speculated that the difference was mainly caused by the cultured cells from different positions or at different developmental periods.

Glutamate can induce cytosolic Ca²⁺ increase in primary cultured astrocytes in time-dependent and concentration-dependent manner. It not only activated non-NMDA and NMDA receptors, but also induced intracellular calcium mobilization. It enhanced the generation of IP₃ ^[31,32], the later could activate IP₃-insensitive calcium stores. When NMDA and AMPA receptor are blocked simultaneously by their antagonists L-AP-5 and CNQX, respectively, glutamate can still stimulate astrocytic [Ca²⁺]_i increase slightly. There were conflicting views about whether activation of NMDA and AMPA receptors could affect astrocytic calcium stores, and whether they are extracellular Ca²⁺-dependent. When extracellular Ca²⁺ was chelated or absent, the response of glutamate still existed though much weaker than that in normal extracellular solution. But the response could be inhibited by thapsgargin. So we deduced that glutamate could induce the release of Ca²⁺ from calcium stores.

Above all, glutamate triggered a complex response in some astrocytes consisting of Ca²⁺ mobilization from intracellular stores and also Ca²⁺ influx by activation of NMDA and non-NMDA receptors .

REFERENCES

• 1 Carmignoto G. Reciprocal communication systems between astrocytes and neurones. Prog Neurobiol 2000; 62: 561-81.
• 2 Shelton MK, McCarthy KD. Mature hippocampal astrocytes exhibit functional metabotropic and ionotropic glutamate receptors in situ. Glia 1999; 26: 1-11.
• 3 Sanzgiri RP, Araque A, Haydon PG. Prostaglandin E(2) stimulates glutamate receptor-dependent astrocyte neuromodula-tion in cultured hippocampal cells. J Neurobiol 1999; 41: 221-9.
• 4 Parpura V, Haydon PG. Physiological astrocytic calcium levels stimulate glutamate release to modulate adjacent neurons. Proc Natl Acad Sci USA 2000; 97: 8629-34.
• 5 Cormier RJ, Mennerick S, Melbostad H, Zorumski CF. Basal levels of adenosine modulate mGluR5 on rat hippocampal astrocytes. Glia 2001; 33: 24-35.
• 6 Hu B, Sun SG, He LM. Features of endoplasmic reticulum calcium stores in rat astrocytes. Chin J Neurosci 2002; 18: 596-601.
• 7 Hu B, Sun SG, He LM. Property of IP₃-sensitive and non-IP₃-sensitive calcium stores in rat astrocytes and their relationship. Chin J Phys Med Rehabil 2002; 24: 272-6.
• 8 Golovina VA, Bambrick LL, Yarowsky PJ, Krueger BK, Blaustein MP. Modulation of two functionally distinct Ca²⁺ stores in astrocytes: role of the plasmalemmal Na/Ca exchanger. Glia 1996; 16: 296-305.
• 9 Glaum SR, Holzwarth JA, Miller RJ. Glutamate receptors activate Ca²⁺ mobilization and Ca²⁺ influx into astrocytes. Proc Natl Acad Sci USA 1990; 87: 3454-8.
• 10 Jensen AM, Chiu SY. Differential intracellular calcium responses to glutamate in type 1 and type 2 cultured brain astrocytes. J Neurosci 1991; 11: 1674-84.
• 11 Iino M, Goto K, Kakegawa W, Okado H, Sudo M, Ishiuchi S, et al. Glia-synapse interaction through Ca²⁺-permeable AMPA receptors in Bergmann glia. Science 2001; 292: 926-9.
• 12 Kondoh T, Nishizaki T, Aihara H, Tamaki N. NMDA-responsible, APV-insensitive receptor in cultured human astrocytes. Life Sci 2001; 68: 1761-7.
• 13 Schipke CG, Ohlemeyer C, Matyash M, Nolte C, Kettenmann H, Kirchhoff F. Astrocytes of the mouse neocortex express functional N-methyl-D-aspartate receptors. FASEB J 2001;15: 1270-2.
• 14 Fiacco TA, McCarthy KD. Intracellular astrocyte calcium waves in situ increase the frequency of spontaneous AMPA receptor currents in CA1 pyramidal neurons. J Neurosei 2004; 24: 722-32.
• 15 Parpura V, Haydon PG. Physiological astrocytic calcium levels stimulate glutamate release to modulate adjacent neurons. Proc Natl Acad Sci USA 2000; 97: 8629-34.
• 16 Fan D, Grooms SY, Araneda RC, Johnson AB, Dobrenis K, Kessler JA,et al. AMPA receptor protein expression and function in astrocytes cultured from hippocampus. J Neuro-sci Res 1999; 57: 557-71.
• 17 Ishiuchi S, Tsuzuki K, Yamada N,Okado H, Miwa A, Kuromi H, et al. Extension of glial processes by activation of Ca²⁺-permeable AMPA receptor channels. Neuroreport 2001; 12: 745-8.
• 18 Chen LG, Zhou SB, LOU XL, Kang HG. Different stimulatory opioid effects on intracellular Ca²⁺ in SH-SY5Y cells. Brain Res 2000; 882: 256-65.
• 19 Nishizawa. Glutamate release and neuronal damage in ischemia. Life Sci 2001; 69: 369-81.
• 20 Koinig H, Vornik V, Rueda C, Zornow MH. Lubeluzole inhibits accumulation of extracellular glutamate in the hippocampus during transient global cerebral ischemia. Brain Res 2001; 898: 297-302.
• 21 Crossman SD, Rosenberg LJ, Wrathall JR. Relationship of altered glutamate receptor subunit mRNA expression to acute cell loss after spinal cord contusion. Exp Neurol 2001; 168: 283-9.
• 22 Chen Y, Vartiainen NE, Ying W, Chan PH, Koistinaho J, Swanson RA. Astrocytes protect neurons from nitric oxide toxicity by a glutathione-dependent mechanism. J Neurochem 2001; 77: 1601-10.
• 23 Oliet SH, Piet R, Poulain DA. Control of glutamate clearance and synaptic efficacy by glial coverage of neurons. Science 2001; 292: 923-6.
• 24 Rothstein JD, Dykes-Hoberg M, Pardo CA, Bristol LA, Jin L, Kuncl RW, et al. Knockout of glutamate transporters reveals a major role for astroglial transport in excitotoxicity and clearance of glutamate. Neuron 1996;16: 675-86
• 25 Bergles DE, Jahr CE. Contribution to glutamate uptake at Schaffer collateral-commissural synapses in the hippo-campus. J Neurosci 1998; 18: 7709-16.
• 26 Tanaka K, Watase K, Manabe T, Yamada K, Watanabe M, Takahashi K, et al. Epilepsy and exacerbation of brain injury in mice lacking the glutamate transporter GLT-1. Science 1997; 276: 1699-702.
• 27 Seifert G, Zhou M, Steinhauser C. Analysis of AMPA receptor properties during postnatal development of mouse hippocampal astrocytes. J Neurophysiol 1997; 78: 2916-23.
• 28 Smith JP, Cunningham LA, Partridge LD. Coupling of AMPA receptors with the Na(+)/Ca(2+) exchanger in cultured rat astrocytes. Brain Res 2000; 887: 98-109.
• 29 Ahmed Z, Lewis CA, Faber DS. Glutamate stimulates release of Ca²⁺ from internal stores in astroglia. Brain Res 1990; 516: 165-9.
• 30 Nishizaki T, Matsuoka T, Nomura T, Kondoh T, Tamaki N, Okada Y. Store Ca²⁺ depletion enhances NMDA responses in cultured human astrocytes. Biochem Biophys Res Commun 1999; 259: 661-4.
• 31 Kim WT, Rioult MG, Cornell-Bell AH. Glutamate-induced calcium signaling in astrocytes. Glia 1994; 11: 173-84.
• 32 Liu HN, Molina-Holgado E, Almazan G. Glutamate-stimulated production of inositol phosphates is mediated by Ca²⁺ influx in oligodendrocyte progenitors. Eur J Pharmacol 1997;338: 277-87.