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 less than 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 less than 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.(ABSTRACT TRUNCATED AT 400 WORDS)