Intracellular sodium modulates mitochondrial calcium signaling in vascular endothelial cells

We have investigated the role of extramitochondrial Na+ for the regulation of mitochondrial Ca2+ concentration ([Ca2+](m)) in permeabilized single vascular endothelial cells. [Ca2+], was measured by loading the cells with the membrane-permeant Ca2+ indicator fluo-3/AM: and subsequent removal of cytoplasmic fluo-3 by surface membrane permeabilization with digitonin. An elevation of extramitochondrial Ca2+ resulted in a dose-dependent increase in the rate of Ca2+ accumulation into mitochondria (k(0.5) = 3 muM) via the mitochondrial Ca2+ uniporter. In the presence of 10 mM extramitochondrial Na+ ([Na+](em)), repetitive application of brief pulses of high Ca2' (2-10 muM) to simulate cytoplasmic [Ca2+] oscillations caused transient increases of [Ca2+], characterized by a fast rising phase that was followed by a slow decay. Removal of extramitochondrial Naf or inhibition of mitochondrial Na+/Ca2+ exchange with clonazepam blocked mitochondrial Ca2+ efflux and resulted in a net accumulation of Ca2+ by the mitochondria. Half-maximal activation of mitochondrial Na+/Ca2+ exchange occurred at [Na+](em) = 4.4 mM, which is well within the physiological range of cytoplasmic [Na+]. This study provides evidence that Ca2+ efflux from the mitochondria in vascular endothelial cells occurs solely via Na+/Ca2+ exchange and emphasizes the important role of intracellular Nat for mitochondrial Ca2+ regulation.