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

Signal transduction mechanisms of group 11 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 2R,4R-4-aminopyrrolidine-2,4-dicarboxylic acid (2R,4R-APDC) and (-)-2-oxa-4-aminobicyclo[3.1.0]hexane-4,6-dicarboxylic acid (LY379268) inhibited 10 muM forskolin-stimulated production of cAMP in murine cortical neurons, striatal neurons and forebrain astrocytes in the absence of extracellular Ca2+. These agonists potentiated cAMP production in the presence of 1.8 mM Ca2+ in astrocytes only. This potentiation was dependent on the extracellular Ca2+ concentration (0.001-10 mM) and inhibited by the mGlu(2/3) antagonist LY341495 (I muM), adenosine deaminase (I U/ml) and the adenosine A(2A) receptor antagonist ZM241385 (I muM). Preincubation with the phospholipase C (PLC) inhibitor U73122 (10 muM), L-type Ca2+-channel blockers nifedipine (I muM) and nimodipine (I muM), the calmodulin kinase 11 (CaMKII) inhibitor KN-62 (10 muM) or pertussis toxin (100 ng/ml) inhibited this potentiation. In the absence of 1.8 mM Ca2+, thapsigargin (I muM) facilitated the potentiation of cAMP production. Measurement of the Ca2+ binding dye Fluo-3/AM showed that, compared to Ca2+-free conditions, thapsigargin and 1.8 mM Ca2+ elevated [Ca2+], in astrocytes; the latter effect being prevented by L-type Ca2+-channel blockers. Potentiation of cAMP production was also demonstrated when astrocytes were stimulated with the beta-adrenoceptor agonist isoprenaline (10 muM) in the presence of 1.8 mM Ca2+, but not with the adenosine agonist NECA (10 muM) or the group I mGlu receptor agonist DHPG (100 pM). BaCl2 (1.8 mM) in place of Ca2+ did not facilitate forskolin-stimulated mGlu(2/3)-potentiation of cAMP. In short, this study in astrocytes demonstrates that under physiological Ca2+ and adenylate cyclase stimulation an elevation of cAMP production is achieved that is mediated by PLC/IP3- 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 phenotype of astrocytes under physiological and pathological conditions. (C) 2002 Elsevier Science Ltd. All rights reserved