Sodium/calcium exchange in rat cortical astrocytes

Abstract

Regulation of the cytosolic free Ca2+ concentration ([Ca2+](cyt)) by an Na/Ca exchanger was studied in primary cultured rat cortical astrocytes. [Ca2+](cyt) was measured by digital imaging in cells loaded with fura-2. The resting [Ca2+](cyt) ≃150 nM, was only slightly increased by reducing the extracellular Na+ concentration ([Na+](o)) to 6.2 mM, or by treating the cells with ouabain for 15 min (to raise cytosolic Na+). Following treatment with ouabain, however, lowering [Na+](o) caused [Ca2+](cyt) to rise rapidly to ≃1300 nm. When Ca2+ sequestration in intracellular stores was blocked by thapsigargin, lowering [Na+](o) increased [Ca2+](cyt) ≃1500 nM in the absence of ouabain. The low-[Na+](o)-stimulated rise in [Ca2+](cyt) was abolished by removal of external Ca2+, but was not blocked by the Ca2+ channel blocker verapamil, or by caffeine or ryanodine, which deplete an intracellular Ca2+ store responsible for Ca2+-induced Ca2+ release. These data suggest that Na+ gradient reduction promotes net Ca2+ gain via Na/Ca exchange. Normally, however, a large rise in [Ca2+](cyt) is prevented by sequestration of the entering Ca2+; this buffering of cytosolic Ca2+ can be circumvented by blocking sequestration with thapsigargin, or overwhelmed by enhancing net Ca2+ gain by pretreating the cells with ouabain. The presence of Na/Ca exchanger protein and mRNA in the astrocytes was confirmed by Western and Northern blot analyses, respectively. Immunohistochemistry revealed that exchanger molecules are distributed in a reticular pattern over the astrocyte surface. We suggest that the Na/Ca exchanger plays a role in regulating both [Ca2+](cyt) and the intracellular stores of Ca2+ in astrocytes, and may thus contribute to the control of astrocyte responsiveness to neurotransmitters and neurotoxins.