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Research Papers

Reversed Na⁺/Ca²⁺ Exchange Contributes to Ca²⁺ Influx and Respiratory Burst in Microglia

Evan W. Newell, Elise F. Stanley and Lyanne C. Schlichter

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Phagocytosis and the ensuing NADPH-mediated respiratory burst are important aspects of microglial activation that require calcium ion (Ca²⁺) influx. However, the specific Ca²⁺ entry pathway(s) that regulates this mechanism remains unclear, with the best candidates being surface membrane Ca²⁺-permeable ion channels or Na⁺/Ca²⁺ exchangers. In order to address this issue, we used quantitative real-time RT-PCR to assess mRNA expression of the Na+/Ca2+ exchangers, Slc8a1-3/NCX1-3, before and after phagocytosis by rat microglia. All three Na⁺/Ca²⁺ 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⁺/Ca²⁺ exchanger activity in these cells. To investigate conditions under which Na⁺/Ca²⁺ exchange was functional, we used a combination of perforated patch-clamp analysis, fluorescence imaging of a Ca²⁺ 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⁺/Ca²⁺ exchange with candidate Ca²⁺-permeable channels, to the NADPH-mediated respiratory burst that was triggered by phagocytosis. We find that inhibiting the reversed mode of the Na⁺/Ca²⁺ exchanger with KB-R7943, dose dependently reduced the phagocytosis-stimulated respiratory burst; whereas, blockers of store-operated Ca²⁺ channels or L-type voltage-gated Ca²⁺ channels had no effect. These results provide evidence that Na⁺/Ca²⁺ exchangers are potential therapeutic targets for reducing the bystander damage that often results from microglia activation in the damaged CNS.

Authors

Evan W. Newell

Stanford University

Elise F. Stanley

Toronto Western Research Institute

Lyanne C. Schlichter

Toronto Western Research Institute

This is an open-access article

 Download PDF

If the document does not open, please right-click on the link (control-click on a Macintosh) and select the option to save the file to disk.