SODIUM-CALCIUM EXCHANGE IN RAT CORTICAL ASTROCYTES

Regulation of the cytosolic free Ca2+ concentration ([Ca2+](cyt)) by a n Na/Ca exchanger was studied in primary cultured rat cortical astrocy tes. [Ca2+](cyt) was measured by digital imaging in cells loaded with fura-2. The resting [Ca2+](cyt), approximate to 150 nM, was only sligh tly increased by reducing the extracellular Na+ concentration ([Na+](o )) to 6.2 mM, or by treating the cells with ouabain for 15 min (to rai se cytosolic Na+). Following treatment with ouabain, however, lowering [Na+](o) caused [Ca2+](cyt) to rise rapidly to approximate to 1300 nM . When Ca2+ sequestration in intracellular stores was blocked by thaps igargin, lowering [Na+](o) increased [Ca2+](cyt) to approximate to 150 0 nM in the absence of ouabain. The low-[Na+](o)-stimulated rise in [C a2+](cyt) was abolished by removal of external Ca2+, but was not block ed by the Ca2+ channel blocker verapamil, or by caffeine or ryanodine, which deplete an intracellular Ca2+ store responsible for Ca2+-induce d Ca2+ release. These data suggest that Na+ gradient reduction promote s 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 sequestrat ion with thapsigargin, or overwhelmed by enhancing net Ca2+ gain by pr etreating the cells with ouabain. The presence of Na/Ca exchanger prot ein and mRNA in the astrocytes was confirmed by Western and Northern b lot analyses, respectively. Immunohistochemistry revealed that exchang er molecules are distributed in a reticular pattern over the astrocyte surface. We suggest that the Na/Ca exchanger plays a role in regulati ng both [Ca2+](cyt) and the intracellular stores of Ca2+ in astrocytes , and may thus contribute to the control of astrocyte responsiveness t o neurotransmitters and neurotoxins.