Purine release from spinal cord microglia after elevation of calcium by glutamate.
The propagation of Ca2+ waves in a network of microglial cells, after its initiation by glutamate, is mediated by purinergic transmission. In this study, we investigated the mechanisms by which glutamate releases ATP from cultured spinal cord microglia. The 4-fold increase in ATP release from microglia in response to glutamate (0.5 mM) was blocked by alpha-aminohydroxy-5-methyl-isoxazole-4-proprionate (AMPA)/kainate receptor antagonist 6-cyano-7-nitroguinoxaline-2,3-dione and specific AMPA receptor antagonist 1-(4-aminophenyl)-4-methyl-7,8-methylenedioxy-5H-2,3-benzodiazepine hydrochloride (GYKI 52466) but not by N-methyl-d-aspartic acid or metabotropic glutamate receptor antagonists. Glutamate acting on AMPA receptors evoked an ATP release that was blocked by antagonizing the rise in intracellular Ca2+ as a result of its release from internal stores as well as by antagonizing protein kinase C with chelerythrine. Glutamate-stimulated ATP release was significantly antagonized by the cystic fibrosis transmembrane conductance regulator (CFTR) blockers flufenamic acid and glibenclamide. A role for the CFTR was further confirmed using microglia from CFTR knockout mice, which released significantly less ATP than microglia from control wild-type mice in response to glutamate. Use of 6-methoxy-1-(3-sulfopropyl)quinolinium fluorescence assay revealed functional CFTR in microglia. These observations suggest that glutamate acted on microglial AMPA receptors to stimulate release of Ca2+ from intracellular stores as well as a Ca2+-dependent isoform of protein kinase C, which then acts to trigger release of ATP with the CFTR acting as a regulator of the ATP release process, perhaps through another channel or transporter.
Liu
Guo Jun
GJ
Neurobiology Laboratory, Discipline of Physiology, School of Medical Sciences, Institute for Biomedical Research, University of Sydney, NSW 2006, Australia.
Kalous
Adrianna
A
Werry
Eryn L
EL
Bennett
Max R
MR
D018528:ATP-Binding Cassette Transporters / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D020013:Calcium Signaling / Q000187:drug effects*; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism; D005971:Glutamates / Q000494:pharmacology*; D051379:Mice; D017628:Microglia / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism*; D037342:Pertussis Toxin; D011493:Protein Kinase C / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D018091:Receptors, AMPA / Q000378:metabolism; D013116:Spinal Cord / Q000166:cytology / Q000187:drug effects*
D018528:ATP-Binding Cassette Transporters; D005971:Glutamates; D018091:Receptors, AMPA; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D000255:Adenosine Triphosphate; D037342:Pertussis Toxin; D011493:Protein Kinase C; D019204:GTP-Binding Proteins