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Na+-Ca2+ exchange has been shown to contribute to reperfusion- and reoxygenation-induced cellular Ca2+ loading and damage in the heart. Despite the fact that both [Na+]i and [Ca2+]i have been documented to rise during ischemia and hypoxia, it remains unclear whether the rise in [Ca2+]i occurring during hypoxia is linked to the rise in [Na+]i via Na+-Ca2+ exchange before reoxygenation and how this relates to cellular injury. Single electrically stimulated (0.2 Hz) adult rat cardiac myocytes loaded with Na+-sensitive benzofuran isophthalate (SBFI), the new fluorescent probe, were exposed to glucose-free hypoxia (Po2<0.02 mm Hg), and SBFI fluorescence was monitored to index changes in [Na+]i. Parallel experiments were performed with indo-1-loaded cells to index [Ca2+]i. The SBFI fluorescence ratio (excitation, 350/380 nm) rose significantly during hypoxia after the onset of ATP-depletion contracture, consistent with a rise in [Na+]i. At reoxygenation, the ratio fell rapidly toward baseline levels. The indo-1 fluorescence ratio (emission, 410/490 nm) also rose only after the onset of rigor contracture and then often showed a secondary rise early after reoxygenation at a time when [Na+]i fell. The increase in both [Na+]i and [Ca2+]i, seen during hypoxia, could be markedly reduced by performing experiments in Na+-free buffer. These experiments suggested that hypoxic Ca2+ loading is linked to a rise in Na+i via Na+-Ca2+ exchange. To show that Na+-Ca2+ exchange activity was not fully inhibited by profound intracellular ATP depletion, cells were exposed to cyanide, and then buffer Na+ was abruptly removed after contracture occurred. The sudden removal of buffer Na+ would be expected to stimulate cell Ca2+ entry via Na+-Ca2+ exchange. A large rapid rise in the indo-1 fluorescence ratio ensued, which was consistent with abrupt cell Ca2+ loading via the exchanger. The effect of reducing hypoxic buffer [Na+] on cell morphology after reoxygenation was examined. Ninety-five percent of cells studied in a normal Na+-containing buffer (144 mM NaCl, n=38) and reoxygenated 30 minutes after the onset of hypoxic rigor underwent hypercontracture. Only 12% of cells studied in Na+-free buffer (144 mM choline chloride, n=17) hypercontracted at reoxygenation (p<0.05). Myocytes were also exposed to hypoxia in the presence of R 56865, a compound that blocks noninactivating components of the Na+ current. R 56865 blunted the rise in [Na+]i typically seen after the onset of rigor, suggesting that Na+ entry may occur, in part, through voltage-gated Na+ channels. These experiments provide evidence that [Na+]i rises during hypoxia and leads to cellular Ca2+ loading and cell destruction via Na+-Ca2+ exchange. Prevention of the rise in [Na+]i during hypoxia reduces cellular injury in this model. Further studies are required to fully elucidate the mechanisms underlying the rise in [Na+]i that occurs during hypoxia and ischemia.