title: Dependence of hypoxic cellular calcium loading on Na(+)-Ca2+ exchange.
abstract: 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)
authors.affiliation: Laboratory of Cardiovascular Science, National Institute on Aging, National Institutes of Health, Baltimore, Md.
authors.forename: M C
authors.initials: MC
authors.lastname: Haigney
authors.forename: H
authors.initials: H
authors.lastname: Miyata
authors.forename: E G
authors.initials: EG
authors.lastname: Lakatta
authors.forename: M D
authors.initials: MD
authors.lastname: Stern
authors.forename: H S
authors.initials: HS
authors.lastname: Silverman
mesh_terms: D000818:Animals; D001572:Benzofurans; D052160:Benzothiazoles; D002118:Calcium / Q000378:metabolism*; D015687:Cell Hypoxia; D004988:Ethers, Cyclic; D006863:Hydrogen-Ion Concentration; D007211:Indoles; D007511:Ischemia / Q000378:metabolism; D009206:Myocardium / Q000166:cytology / Q000378:metabolism*; D010880:Piperidines / Q000494:pharmacology; D051381:Rats; D012964:Sodium / Q000378:metabolism*; D015222:Sodium Channels / Q000187:drug effects / Q000378:metabolism; D000254:Sodium-Potassium-Exchanging ATPase / Q000502:physiology*; D013844:Thiazoles / Q000494:pharmacology
chemical_list: D001572:Benzofurans; D052160:Benzothiazoles; D004988:Ethers, Cyclic; D007211:Indoles; D010880:Piperidines; D015222:Sodium Channels; D013844:Thiazoles; C061665:sodium-binding benzofuran isophthalate; C054551:R 56865; D012964:Sodium; D000254:Sodium-Potassium-Exchanging ATPase; C048960:indo-1; D002118:Calcium