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+ depe ndence of RVD was investigated. Using a method that measures extracell ular electrical resistance to measure cell volume changes in substratu m-attached cells, we found that when astrocytes were exposed to hypoto nic media without calcium, RVD was abolished. The addition of CaCl2 to astrocytes swollen in hypotonic media without calcium caused an almos t immediate initiation of volume regulation, with an EC50 of approxima tely 0.1 mM CaCl2. Swelling of astrocytes in hypotonic medium caused a n increased influx of Ca-45(2+), which was partially blocked (60%) by 1 muM nimodipine, suggesting that voltage-gated L-type calcium channel s were being opened. Previous work had shown that hypotonic media-indu ced swelling of astrocytes caused membrane potential depolarizations s ufficient to open such channels (Kimelberg and O'Connor, 1988). By mea suring intracellular free calcium with fura-2, we found that astrocyte s swollen in hypotonic medium showed a rapid increase in [Ca2+]i, reac hing a peak of approximately 600 nM, followed by a decrease to a susta ined plateau (approximately 250 nM) mirroring the time course of volum e 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 s uggest 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 o f 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 o f 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 n ot affect H-3-D-aspartate efflux. We suggest that the swelling of astr ocytes in hypotonic media which leads to a rapid membrane depolarizati on first opens voltage-gated calcium channels. Extracellular Ca2+ then enters the cell, leading to a sustained increase in intracellular fre e calcium ([Ca2+]i), triggering activation of Ca2+-dependent ion chann els and the release of K+ and Cl followed by osmotically obligated wat er, thus leading to RVD. Although intimately associated with this proc ess, swelling-induced release of amino acids, because of its independe nce of extracellular Ca2+, does not seem to be involved in RVD.