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

Abstract

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 approximately 20 min, followed by a decrease to 118+/-9 nM in the next approximately 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 (deltapsim) was lost in 20 min of MI, excluding mitochondrial Ca2+ uptake via the deltapsim-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.

Excitation-contraction coupling and mitochondrial energetics

“…While elevated [Na + ] i reduced steady-state [Ca 2+ ] m , TCA cycle activity and rate of oxidative phosphorylation, inhibiting the mNCE with CGP-37157 improved mitochondrial Ca 2+ -accumulation and energetics in isolated mitochondria and cardiac myocytes [47,114,117]. Furthermore, during ischemia and reperfusion with cytosolic Ca 2+ overload and a collapse of Δψ m , the mNCE in reverse mode has been shown to contribute to mitochondrial Ca 2+ overload [180], which eventually may lead to PTP opening and cell death [29]. In this setting, CGP-37157 inhibited mitochondrial Ca 2+ overload [180].…”

“…Furthermore, during ischemia and reperfusion with cytosolic Ca 2+ overload and a collapse of Δψ m , the mNCE in reverse mode has been shown to contribute to mitochondrial Ca 2+ overload [180], which eventually may lead to PTP opening and cell death [29]. In this setting, CGP-37157 inhibited mitochondrial Ca 2+ overload [180]. Thus, inhibiting the mNCE may improve energetics and potentially protect from apoptosis in various pathologic situations.…”

Regulation of the Human Cardiac Mitochondrial Ca 2+ Uptake by 2 Different Voltage-Gated Ca 2+ Channels

“…Although the remaining Ca 2ϩ uptake in the presence of nanomolar concentrations of Ru360 might be due to alternative Ca 2ϩ pathways via the mitochondrial Na ϩ -Ca 2ϩ exchanger 12,27 or mitochondrial ryanodine receptor, 28 addition of CGP-37157 (10 mol/L), cyclosporin A (10 mol/L), dantrolene (10 mol/L), S(-)-BayK 8644 (10 mol/L), or xestospongin C (10 mol/L) to the bath or pipette solution did not affect I mCa1 or I mCa2 (supplemental Table IV), indicating that these 2 Ca 2ϩ currents were not mediated by the mitochondrial Na ϩ -Ca 2ϩ exchanger, 29 mitochondrial permeability transition pore, 30 mitochondrial ryanodine receptor, 28 mitochondrial dihydropyridine site, 31 or an IP 3 receptor, 32 respectively.…”

PKD1 Haploinsufficiency Causes a Syndrome of Inappropriate Antidiuresis in Mice

“…The tubule segments were seeded onto glass coverslips and examined using an inverted Nikon TMD35 epifluorescence microscope (Analis, Namur, Belgium) in a thermostated chamber at 37°C. The intracellular Ca 2ϩ concentration ([Ca 2ϩ ] i ) was measured as described previously (30). Briefly, after measurement of the background signal, isolated tubules were loaded with Fura-2 by incubation with the membrane-permeant acetoxymethyl (AM) ester form of the dye (10 M) for 1 h at 37°C.…”