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 Ca ²⁺ 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 ∼20 min, followed by a decrease to 118 ± 9 nM in the next ∼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 (δΨ_m) was lost in 20 min of MI, excluding mitochondrial Ca²⁺ uptake via the δΨ _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.