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