Role of mitochondrial Na+ concentration, measured by CoroNa red, in the protection of metabolically inhibited MDCK cells.
In ischemic or hypoxic tissues, elevated cytosolic calcium levels can induce lethal processes. Mitochondria, besides the endoplasmic reticulum, play a key role in clearing excessive cytosolic Ca2+. In a previous study, it was suggested that the clearance of cytosolic Ca2+, after approximately 18 min of metabolic inhibition (MI) in renal epithelial cells, occurs via the reverse action of the mitochondrial Na+/Ca2+ exchanger (NCX). For further investigating the underlying mechanism, changes in the mitochondrial Na+ concentration ([Na+](m)) were monitored in metabolically inhibited MDCK cells. CoroNa Red, a sodium-sensitive fluorescence probe, was used to monitor [Na+]m. In the first 15 min of MI, a twofold increase of [Na+]m was observed reaching 113 +/- 7 mM, whereas the cytosolic Na+ concentration ([Na+]c) elevated threefold, to a level of 65 +/- 6 mM. In the next 45 min of MI, [Na+]m dropped to 91 +/- 7 mM, whereas [Na+]c further increased to 91 +/- 4 mM. The striking rise in [Na+]m is likely sufficient to sustain the driving force for mitochondrial Ca2+ uptake via the NCX. Furthermore, when CGP-37157, a specific inhibitor of the mitochondrial NCX, was applied during MI, the second-phase drop of [Na+]m was completely abolished. The obtained results support the hypothesis that the mitochondrial NCX reverses after approximately 15 min of MI. Moreover, because the cellular homeostasis can recover after MI, the mitochondria likely protect MDCK cells from injury during MI by the reversal of the mitochondrial NCX. This study is the first to report [Na+]m measurements in nonpermeabilized living cells.
Baron
Szilvia
S
Laboratory of Cell Physiology, University Hasselt and transnationale Universiteit Limburg, Biomedisch Onderzoeksinstituut, Diepenbeek, Belgium.
Caplanusi
Adrian
A
van de Ven
Martin
M
Radu
Mihai
M
Despa
Sanda
S
Lambrichts
Ivo
I
Ameloot
Marcel
M
Steels
Paul
P
Smets
Ilse
I
D002478:Cells, Cultured; D003600:Cytosol / Q000378:metabolism; D004847:Epithelial Cells / Q000166:cytology / Q000378:metabolism; D005456:Fluorescent Dyes; D006801:Humans; D007684:Kidney Tubules / Q000166:cytology / Q000378:metabolism*; D018613:Microscopy, Confocal; D008928:Mitochondria / Q000378:metabolism*; D012430:Ruthenium Red; D012680:Sensitivity and Specificity; D012964:Sodium / Q000378:metabolism*; D019831:Sodium-Calcium Exchanger / Q000378:metabolism
D005456:Fluorescent Dyes; D019831:Sodium-Calcium Exchanger; D012430:Ruthenium Red; D012964:Sodium