Ca2+ uptake in mitochondria occurs via the reverse action of the Na+/Ca2+ exchanger in metabolically inhibited MDCK cells

In ischemic or hypoxic tissues, elevated Ca2+ levels have emerged as one of the main damaging agents among other Ca2+-independent mechanisms of cellular injury. Because mitochondria, besides the endoplasmic reticulum, play a key role in the maintainance of cellular Ca2+ homeostasis, alterations in the mitochondrial Ca2+ content ([Ca2+](m)) were monitored in addition to changes in cytosolic Ca2+ concentration ([Ca2+](i)) during metabolic inhibition (MI) in renal epithelial Madin-Darby canine kidney (MDCK) cells. [Ca2+](i) and [Ca2+](m) were monitored via, respectively, fura 2 and rhod 2 measurements. MI induced an increase in [Ca2+](i) reaching 631 +/- 78 nM in similar to20 min, followed by a decrease to 118 +/- 9 nM in the next similar to 25 min. A pronounced drop in cellular ATP levels and a rapid increase in intracellular Na+ concentrations in the first 20 min of MI excluded Ca2+ efflux in the second phase via plasma membrane ATPases or Na+/Ca2+ exchangers (NCE). Mitochondrial rhod 2 intensities increased to 434 +/- 46% of the control value during MI, indicating that mitochondria sequester Ca2+ during MI. The mitochondrial potential (DeltaPsi(m)) was lost in 20 min of MI, excluding mitochondrial Ca2+ uptake via the DeltaPsi(m)-dependent mitochondrial Ca2+ uniporter after 20 min of MI. Under Na+-free conditions, or when CGP-37157, a specific inhibitor of the mitochondrial NCE, was used, no drop in [Ca2+](i) was seen during MI, whereas the MI-induced increase in mitochondrial rhod 2 fluorescence was strongly reduced. To our knowledge, this study is the first to report that in metabolically inhibited renal epithelial cells mitochondria take up Ca2+ via the NCE acting in the reverse mode.