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, approximately 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 approximately 1300 nM. When Ca2+ sequestration in intracellular stores was blocked by thapsigargin, lowering [Na+]o increased [Ca2+]cyt to approximately 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 Ca(2+)-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.
