Astrocyte mGlu 2/3-mediated cAMP potentiation is calcium sensitive: studies in murine neuronal and astrocyte cultures

Abstract

Signal transduction mechanisms of group II metabotropic glutamate receptors (mGlu 2/3) remains a matter of some controversy, therefore we sought to gain new insights into its regulation by studying cAMP production in cultured neurons and astrocytes, and by examining inter-relationships of mGlu 2/3-induced signalling with cellular calcium and various signalling cascades. mGlu 2/3 agonists 2 R,4 R-4-aminopyrrolidine-2,4-dicarboxylic acid (2 R,4 R-APDC) and (–)-2-oxa-4-aminobicyclo[3.1.0]hexane-4,6-dicarboxylic acid (LY379268) inhibited 10 μM forskolin-stimulated production of cAMP in murine cortical neurons, striatal neurons and forebrain astrocytes in the absence of extracellular Ca 2+. These agonists potentiated cAMP production in the presence of 1.8 mM Ca 2+ in astrocytes only. This potentiation was dependent on the extracellular Ca 2+ concentration (0.001–10 mM) and inhibited by the mGlu 2/3 antagonist LY341495 (1 μM), adenosine deaminase (1 U/ml) and the adenosine A 2A receptor antagonist ZM241385 (1 μM). Pre-incubation with the phospholipase C (PLC) inhibitor U73122 (10 μM), L-type Ca 2+-channel blockers nifedipine (1 μM) and nimodipine (1 μM), the calmodulin kinase II (CaMKII) inhibitor KN-62 (10 μM) or pertussis toxin (100 ng/ml) inhibited this potentiation. In the absence of 1.8 mM Ca 2+, thapsigargin (1 μM) facilitated the potentiation of cAMP production. Measurement of the Ca 2+-binding dye Fluo-3/AM showed that, compared to Ca 2+-free conditions, thapsigargin and 1.8 mM Ca 2+ elevated [Ca 2+] i in astrocytes; the latter effect being prevented by L-type Ca 2+-channel blockers. Potentiation of cAMP production was also demonstrated when astrocytes were stimulated with the β-adrenoceptor agonist isoprenaline (10 μM) in the presence of 1.8 mM Ca 2+, but not with the adenosine agonist NECA (10 μM) or the group I mGlu receptor agonist DHPG (100 μM). BaCl 2 (1.8 mM) in place of Ca 2+ did not facilitate forskolin-stimulated mGlu 2/3-potentiation of cAMP. In short, this study in astrocytes demonstrates that under physiological Ca 2+ and adenylate cyclase stimulation an elevation of cAMP production is achieved that is mediated by PLC/IP 3- and CaMKII-dependent pathways and results in the release of endogenous adenosine which acts at G s protein-coupled A 2A receptors. These findings provide new insights into mGlu 2/3 signalling in astrocytes versus neurons, and which could determine the functional phenotypy of astrocytes under physiological and pathological conditions.