ROLE OF CALCIUM IN ASTROCYTE VOLUME REGULATION AND IN THE RELEASE OF IONS AND AMINO-ACIDS

Primary astrocyte cultures exposed to hypotonic media undergo a rapid initial swelling followed by a regulatory volume decrease (RVD), which is associated with the release of ions and amino acids. The Ca2+ dependence of RVD was investigated. Using a method that measures extracellular electrical resistance to measure cell volume changes in substratum-attached cells, we found that when astrocytes were exposed to hypotonic media without calcium, RVD was abolished. The addition of CaCl2 to astrocytes swollen in hypotonic media without calcium caused an almost immediate initiation of volume regulation, with an EC50 of approximately 0.1 mM CaCl2. Swelling of astrocytes in hypotonic medium caused an increased influx of Ca-45(2+), which was partially blocked (60%) by 1 muM nimodipine, suggesting that voltage-gated L-type calcium channels were being opened. Previous work had shown that hypotonic media-induced swelling of astrocytes caused membrane potential depolarizations sufficient to open such channels (Kimelberg and O'Connor, 1988). By measuring intracellular free calcium with fura-2, we found that astrocytes swollen in hypotonic medium showed a rapid increase in [Ca2+]i, reaching a peak of approximately 600 nM, followed by a decrease to a sustained plateau (approximately 250 nM) mirroring the time course of volume regulation. The removal of extracellular calcium totally abolished, and the addition of 1 muM nimodipine partially abolished the elevated plateau, while neither affected the initial [Ca2+]i peak. These data suggest that the initial peak of the hypotonic-induced rise in [Ca2+]i is caused by release from intracellular stores and that the sustained elevated plateau is due to extracellular calcium influx. The removal of extracellular calcium also abolished swelling-induced K+(Rb-86) and Cl-36- efflux, but did not affect the swelling-induced release of H-3-D-aspartate, or H-3-taurine (data not shown). These data indicate that hypotonic-induced aspartate and taurine release is not necessary for RVD in astrocytes swollen by exposure to hypotonic media, since RVD is completely inhibited by the omission of external Ca2+. The addition of 1 mM quinine HCl, which is known to block Ca2+-activated K+ channels, also abolished both volume regulation and Rb-86+ efflux in hypotonic media-swollen astrocytes in the presence of medium calcium, but did not affect H-3-D-aspartate efflux. We suggest that the swelling of astrocytes in hypotonic media which leads to a rapid membrane depolarization first opens voltage-gated calcium channels. Extracellular Ca2+ then enters the cell, leading to a sustained increase in intracellular free calcium ([Ca2+]i), triggering activation of Ca2+-dependent ion channels and the release of K+ and Cl followed by osmotically obligated water, thus leading to RVD. Although intimately associated with this process, swelling-induced release of amino acids, because of its independence of extracellular Ca2+, does not seem to be involved in RVD.