Reversed Na+/Ca2+ exchange contributes to Ca2+ influx and respiratory burst in microglia.
Phagocytosis and the ensuing NADPH-mediated respiratory burst are important aspects of microglial activation that require calcium ion (Ca(2+)) influx. However, the specific Ca(2+) entry pathway(s) that regulates this mechanism remains unclear, with the best candidates being surface membrane Ca(2+)-permeable ion channels or Na(+)/Ca(2+) exchangers. In order to address this issue, we used quantitative real-time RT-PCR to assess mRNA expression of the Na(+)/Ca(2+) exchangers, Slc8a1-3/NCX1-3, before and after phagocytosis by rat microglia. All three Na(+)/Ca(2+) exchangers were expressed, with mRNA levels of NCX1 > NCX3 > NCX2, and were unaltered during the one hour phagocytosis period. We then carried out a biophysical characterization of Na(+)/Ca(2+) exchanger activity in these cells. To investigate conditions under which Na(+)/Ca(2+) exchange was functional, we used a combination of perforated patch-clamp analysis, fluorescence imaging of a Ca(2+) indicator (Fura-2) and a Na(+) indicator (SBFI), and manipulations of membrane potential and intracellular and extracellular ions. Then, we used a pharmacological toolbox to compare the contribution of Na(+)/Ca(2+) exchange with candidate Ca(2+)-permeable channels, to the NADPH-mediated respiratory burst that was triggered by phagocytosis. We find that inhibiting the reversed mode of the Na(+)/Ca(2+) exchanger with KB-R7943, dose dependently reduced the phagocytosis-stimulated respiratory burst; whereas, blockers of store-operated Ca(2+) channels or L-type voltage-gated Ca(2+) channels had no effect. These results provide evidence that Na(+)/Ca(2+) exchangers are potential therapeutic targets for reducing the bystander damage that often results from microglia activation in the damaged CNS.
Newell
Evan W
EW
Toronto Western Research Institute, University of Toronto, Toronto, Ontario, Canada.
Stanley
Elise F
EF
Schlichter
Lyanne C
LC
D000818:Animals; D001921:Brain / Q000378:metabolism; D002118:Calcium / Q000737:chemistry / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000494:pharmacology; D008297:Male; D008564:Membrane Potentials; D017628:Microglia / Q000378:metabolism*; D009249:NADP / Q000737:chemistry; D010587:Phagocytosis; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D016897:Respiratory Burst; D012964:Sodium / Q000737:chemistry; D019831:Sodium-Calcium Exchanger / Q000378:metabolism*
D005456:Fluorescent Dyes; D012333:RNA, Messenger; D019831:Sodium-Calcium Exchanger; D009249:NADP; D012964:Sodium; D002118:Calcium