Received for publication December 2, 2005.
Revised May 30, 2006.
Accepted for publication June 6, 2006.

Purine Release from Spinal Cord Microglia Following Elevation of Calcium by Glutamate

Abstract

The propagation of Ca²⁺ waves in a network of microglial cells, following its initiation by glutamate, is mediated by purinergic transmission. Here we investigate the mechanisms by which glutamate releases ATP from cultured spinal cord microglia. The four-fold increase in ATP release from microglia in response to glutamate (0.5 mM) is blocked by alpha-amino-hydroxy-5-methyl-isoxazole-4-proprionate (AMPA)/kainate receptor antagonist 6-cyano-7-nitroguinoxaline-2,3-dione (CNQX) 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 (NMDA) or metabotropic glutamate receptor antagonists. Glutamate acting on AMPA receptors evokes an ATP release which is blocked by antagonizing the rise in intracellular Ca²⁺ due to its release from internal stores as well as by antagonizing protein kinase C with chelerythrine. Glutamate-stimulated ATP release is 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 (SPQ) fluorescence assay revealed functional CFTR in microglia. These observations suggest that glutamate acts 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.

Key words: Purinergic, Glutamate, Purinergic, Gi family, Gq/11 family, Protein Kinase C, Ion transporters (SERCA, Na/K ATPase, CFTR), Ca imaging, Fluorescence techniques, Immunocytochemistry