title: In situ fluorescence imaging of glutamate-evoked mitochondrial Na+ responses in astrocytes.
abstract: Astrocytes can experience large intracellular Na+ changes following the activation of the Na+-coupled glutamate transport. The present study investigated whether cytosolic Na+ changes are transmitted to mitochondria, which could therefore influence their function and contribute to the overall intracellular Na+ regulation. Mitochondrial Na+ (Na+(mit)) changes were monitored using the Na+-sensitive fluorescent probe CoroNa Red (CR) in intact primary cortical astrocytes, as opposed to the classical isolated mitochondria preparation. The mitochondrial localization and Na+ sensitivity of the dye were first verified and indicated that it can be safely used as a selective Na+(mit) indicator. We found by simultaneously monitoring cytosolic and mitochondrial Na+ using sodium-binding benzofuran isophthalate and CR, respectively, that glutamate-evoked cytosolic Na+ elevations are transmitted to mitochondria. The resting Na+(mit) concentration was estimated at 19.0 +/- 0.8 mM, reaching 30.1 +/- 1.2 mM during 200 microM glutamate application. Blockers of conductances potentially mediating Na+ entry (calcium uniporter, monovalent cation conductances, K+(ATP) channels) were not able to prevent the Na+(mit) response to glutamate. However, Ca2+ and its exchange with Na+ appear to play an important role in mediating mitochondrial Na+ entry as chelating intracellular Ca2+ with BAPTA or inhibiting Na+/Ca2+ exchanger with CGP-37157 diminished the Na+(mit) response. Moreover, intracellular Ca2+ increase achieved by photoactivation of caged Ca2+ also induced a Na+(mit) elevation. Inhibition of mitochondrial Na/H antiporter using ethylisopropyl-amiloride caused a steady increase in Na+(mit) without increasing cytosolic Na+, indicating that Na+ extrusion from mitochondria is mediated by these exchangers. Thus, mitochondria in intact astrocytes are equipped to efficiently sense cellular Na+ signals and to dynamically regulate their Na+ content.
authors.affiliation: Department of Physiology, University of Lausanne, Switzerland.
authors.forename: Yann
authors.initials: Y
authors.lastname: Bernardinelli
authors.forename: Guillaume
authors.initials: G
authors.lastname: Azarias
authors.forename: Jean-Yves
authors.initials: JY
authors.lastname: Chatton
mesh_terms: D027322:Amino Acid Transport System X-AG / Q000187:drug effects / Q000378:metabolism; D000818:Animals; D001253:Astrocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D001921:Brain / Q000166:cytology / Q000378:metabolism*; D020013:Calcium Signaling / Q000187:drug effects / Q000502:physiology; D002540:Cerebral Cortex / Q000166:cytology / Q000378:metabolism; D002614:Chelating Agents / Q000494:pharmacology; D003600:Cytosol / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005456:Fluorescent Dyes; D018698:Glutamic Acid / Q000378:metabolism* / Q000494:pharmacology; D007424:Intracellular Fluid / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D008856:Microscopy, Fluorescence; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D012964:Sodium / Q000378:metabolism*; D026941:Sodium Channel Blockers / Q000494:pharmacology; D019831:Sodium-Calcium Exchanger / Q000037:antagonists & inhibitors / Q000378:metabolism; D017923:Sodium-Hydrogen Exchangers / Q000037:antagonists & inhibitors / Q000378:metabolism
chemical_list: D027322:Amino Acid Transport System X-AG; D002614:Chelating Agents; D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D026941:Sodium Channel Blockers; D019831:Sodium-Calcium Exchanger; D017923:Sodium-Hydrogen Exchangers; D018698:Glutamic Acid; D012964:Sodium