Elsevier

Neuropharmacology

Volume 37, Issue 2, February 1998, Pages 169-178
Neuropharmacology

Pharmacological characterisation of metabotropic glutamatergic and purinergic receptors linked to Ca2+ signalling in hippocampal astrocytes

https://doi.org/10.1016/S0028-3908(98)00012-4Get rights and content

Abstract

Intracellular Ca2+ ([Ca2+]i) signals induced by metabotropic glutamate receptor (mGluR) agonists and by purinergic agonists in cultured hippocampal astrocytes were investigated using [Ca2+]-sensitive fluorophores. The mGluR agonists (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (ACPD) and (R,S)-3,5-dihydroxyphenylglycine (DHPG) induced [Ca2+]i responses in 76 and 93% of the cells, respectively. The broad-spectrum mGluR antagonist (+)-α-methyl-4-carboxyphenylglycine (MCPG) and the mGluR1 antagonists (S)-4-carboxy-3-hydroxyphenylglycine (4C3HPG) and (S)-4-carboxyphenylglycine (4CPG) suppressed the agonist-evoked [Ca2+]i response in about 25% of the cells completely and in about 60% partially, depending on the agonist concentration employed. Together with immunohistochemical receptor localisations these results suggest the presence of at least two subpopulations of class I mGluRs recruited from the truncated splice variants of mGluR1 (mGluR 1b, 1c, 1d) and/or hitherto unknown glial-specific class I mGluRs. Of the hippocampal astrocytes 88, 92 or 83% of the cells responded with a [Ca2+]i elevation (mostly oscillations) to application of ATP, ADP, or 2-methylthio-ATP (2-MeS-ATP), respectively, whereas only 14 and 5% responded to AMP and adenosine, respectively, indicating the predominance of P2 receptors. The ATP-induced [Ca2+]i signal was suppressed by suramin. Release of Ca2+ from intracellular stores was involved in the response to ATP because the cells also exhibited [Ca2+]i elevations in Ca2+-free medium. Cells did not respond to 10 μM UTP. We conclude that the P2Y subtype represents the main [Ca2+]i-linked purinoceptor in hippocampal astrocytes. Sequential application of ATP and DHPG in Ca-free medium showed that metabotropic glutamate and purinergic receptors initiate release of Ca2+ from subsets of cyclopiazonic acid-sensitive Ca2+ stores which are partly independent.

Introduction

Metabotropic receptors (MRs) are G protein-coupled receptors which affect the electrical and metabolic properties of neuronal and glial cells not by direct activation of plasma membrane ion channels but by modulating the concentration of intracellular messengers such as Ca2+ and cAMP. MRs are involved in the complex mechanisms occurring in hippocampal pyramidal cells during long-term potentiation or in neuronal degeneration (Riedel and Reymann, 1996, Ribeiro et al., 1996). Until now it has not been clear whether hippocampal glial cells and their MRs may participate in these processes. Astrocytes have intimate contacts with synaptic structures and express a variety of neurotransmitter and hormonal receptors suggesting that they can modulate synaptic signal transmission (Peters et al., 1991, Keyser and Pellmar, 1994). Moreover, glial cells were demonstrated to play a critical role in the maintenance of synaptic transmission (Keyser and Pellmar, 1994).

It is generally accepted that the level and dynamics of intracellular Ca2+ ([Ca2+]i) plays a central role in crucial brain processes such as synaptic plasticity and neurodegeneration (Malenka et al., 1992, Mattson et al., 1993, Frenguelli et al., 1996, Kristian and Siesjö, 1996). Similar to neurons, astrocytes exhibit [Ca2+]i elevations following stimulation with various neurotransmitters (McCarthy and Salm, 1991, Finkbeiner, 1993, Kim et al., 1994, Salter and Hicks, 1994). For hippocampal astrocytes, there are also indications of the presence of MRs coupled to release of Ca2+ from intracellular Ca2+ stores (ICSs). [Ca2+]i responses in Ca2+-free medium were observed upon activation of metabotropic glutamate receptors (mGluRs) by quisqualate (Cornell-Bell et al., 1990, Glaum et al., 1990) or (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (ACPD) (Cornell-Bell and Finkbeiner, 1991). In hippocampal slice preparations, ACPD elicited [Ca2+]i elevations in glial fibrillary acidic protein (GFAP)-positive cells (Porter and McCarthy, 1995a). Stimulation of the Schaffer collaterals caused [Ca2+]i elevations in CA1 GFAP-positive cells which were blocked by the specific mGluR antagonist (+)-α-methyl-4-carboxyphenylglycine (MCPG) (Porter and McCarthy, 1996). In addition to mGluRs, purinergic MRs were also found to be linked to Ca2+ signalling in hippocampal glial cells. GFAP-positive cells in acute hippocampal slices showed [Ca2+]i elevation following application of purinergic P1 agonists but not of specific P2 agonists (Porter and McCarthy, 1995b). It was recently reported that neuronal Ca2+ signalling can be modulated by astrocytic [Ca2+]i elevations in mixed astrocytic–neuronal cultures (Nedergaard, 1994, Hassinger et al., 1995). Therefore, Ca2+ signalling in hippocampal astrocytes could be involved in the modulation of synaptic signal transmission.

During electrical activity of neurons, glutamate and ATP are simultaneously released (Wieraszko et al., 1989, Hamann and Attwell, 1996) resulting in a co-activation of purinergic and glutamatergic MRs. In the present work we used Ca2+-imaging to study the functional expression and interaction of [Ca2+]i-linked purinergic and glutamatergic MRs in cultured hippocampal astrocytes. The receptors were pharmacologically and immunologically characterised and classified by means of specific agonists and antagonists as well as subtype-specific antibodies.

Section snippets

Cell cultures

Cells were prepared from postnatal day 1 to 5 Wistar rat pups using standard procedures. After rapid cervical dislocation, hippocampi were isolated in ice-cold preparation medium containing 10 mM HEPES, 1 mM pyruvate, 10 mM glucose, 6 μg/ml DNAse I type IV, 1 mg/ml bovine serum albumine, 0.25 mM CaCl2, 5.8 mM MgCl2, 2 mM stabilised glutamine and 1% (v/v) antibiotics mixture (Sigma A9099) in phosphate-buffered saline with reduced Cl content (130 mM sodium gluconate, 20 mM NaCl). Hippocampi were

Metabotropic glutamate receptors

Initial experiments showed that the non-NMDA glutamatergic agonist quisqualate (20 μM) in Ca2+-free medium induced a transient increase of [Ca2+]i in five out of eight hippocampal astrocytes, due to Ca2+ release from intracellular stores. In subsequent experiments, we applied ACPD, a mGluR agonist specific for classes I and II, or (R,S)-3,5-dihydroxyphenylglycine (DHPG), a class I mGluR agonist, in Ca2+-free medium. ACPD (200 μM) and DHPG (20 μM; see Fig. 1) elicited a [Ca2+]i rise in 83% (n

Discussion

Our results provide substantial experimental evidence that the majority of hippocampal astrocytes possess mGluRs which are linked to the Ca2+ release from intracellular stores. Of the three mGluR agonists used in this study, quisqualate elicited [Ca2+]i responses in 63%, ACPD in 83%, and DHPG in 90% of the cells. The [Ca2+]i rise upon agonist application persisted in Ca2+-free medium indicating that Ca2+was released from intracellular stores. This is consistent with previous reports showing

Acknowledgements

We thank V. Wilsch and K. Böhm for help in establishing the cell cultures. The investigation was partly funded by grants to G. Reiser from Land Sachsen-Anhalt (1899A/0025) and BMBF (07 NBL 04/01 ZZ 9505) and to K.G. Reymann from Land Sachsen-Anhalt (1877A/0025).

References (43)

  • Y Shao et al.

    Regulation of astroglial responsiveness to neuroligands in primary culture

    Neuroscience

    (1993)
  • A Verkhratsky et al.

    Calcium signalling in glial cells

    Trends Neurosci.

    (1996)
  • A Wieraszko et al.

    Stimulation-dependent release of adenosine triphosphate from hippocampal slices

    Brain Res.

    (1989)
  • W Beindl et al.

    Inhibition of receptor/G protein coupling by suramin analogues

    Mol. Pharmacol.

    (1996)
  • C Centemeri et al.

    Characterization of the Ca2+ responses evoked by ATP and other nucleotides in mammalian brain astrocytes (in process citation)

    Br. J. Pharmacol.

    (1997)
  • D.F Condorelli et al.

    Metabotropic glutamate receptor expression in cultured rat astrocytes and human gliomas

    Neurochem. Res.

    (1997)
  • P.J Conn et al.

    Pharmacology and functions of metabotropic glutamate receptors

    Annu. Rev. Pharmacol. Toxicol.

    (1997)
  • A.H Cornell-Bell et al.

    Glutamate induces calcium waves in cultured astrocytes: long-range glial signaling

    Science

    (1990)
  • S Duffy et al.

    Adrenergic calcium signalling in astrocyte networks within the hippocampal slice

    J. Neurosci.

    (1995)
  • S.M Finkbeiner

    Glial calcium

    Glia

    (1993)
  • S.R Glaum et al.

    Glutamate receptors activate Ca2+ mobilization and Ca2+ influx into astrocytes

    Proc. Natl. Acad. Sci. USA

    (1990)
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