Intracellular ATP depletion inhibits swelling-induced D-[3H]aspartate release from primary astrocyte cultures.

Volume expansion-sensing outward rectifier (VSOR) anion channel, also referred to as volume-sensitive organic osmolyte-anion channel (VSOAC), appears to be responsible for cell swelling-induced amino acid release in a variety of cells. One prominent feature of the VSOR/VSOAC is that non-hydrolyzed intracellular ATP binding to the channel or an accessory protein is required for its activation. In this study, the effect of intracellular ATP depletion on the swelling-induced release of D-[3H]aspartate from rat primary astrocyte cultures due to exposure to either high K(+) or hypotonic media was studied. When the cells were pretreated for 10 min with a combination of the metabolic inhibitors 2-deoxyglucose and rotenone, 100 mM K(+) media- or hypotonic media-induced D-[3H]aspartate release was completely suppressed. Added separately, each inhibitor showed only partial or no inhibition of D-[3H]aspartate release, which correlated with its relative effectiveness in decreasing intracellular ATP levels. These data are consistent with the view that during high [K(+)](o) or hypotonic media-induced swelling of primary astrocyte cultures an ATP-dependent swelling-activated VSOAC channel is responsible for D-[3H]aspartate release and close to normal ATP is required for full channel activation.

Pharmacological characterization of swelling-induced D-[3H]aspartate release from primary astrocyte cultures.

During stroke or head trauma, extracellular K+ concentration increases, which can cause astrocytes to swell. In vitro, such swelling causes astrocytes to release excitatory amino acids, which may contribute to excitotoxicity in vivo. Several putative swelling-activated channels have been identified through which such anionic organic cellular osmolytes can be released. In the present study, we sought to identify the swelling-activated channel(s) responsible for D-[3H]aspartate release from primary cultured astrocytes exposed to either KCl or hypotonic medium. KCl-induced D-[3H]aspartate release was inhibited by the anion channel inhibitors 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB), dideoxyforskolin, L-644711, ATP, ITP, 3'-azido-3'-deoxythymidine, DIDS, and tamoxifen but not by cAMP. The cell swelling caused by raised KCl was not inhibited by extracellular ATP or tamoxifen as measured by an electrical impedance method, which suggests that these anion channel inhibitors directly blocked the channel responsible for efflux. Extracellular nucleotides and DIDS, however, had no or only partial effects on D-[3H]aspartate release from cells swollen by hypotonic medium, but such release was inhibited by NPPB, dideoxyforskolin, and tamoxifen. Of the swelling-activated channels so far identified, our data suggest that a volume-sensitive outwardly rectifying channel is responsible for D-[3H]aspartate release from primary cultured astrocytes during raised extracellular K+ and possibly during hypotonic medium-induced release.

Release of [3H]-D-aspartate from primary astrocyte cultures in response to raised external potassium.

There are significant Ca2+-independent increases in extracellular glutamate and aspartate during various CNS insults such as ischemia and anoxia. However, the cellular sources of such presumed nonvesicular excitatory amino acid (EAA) release have not been established. To further explore potential mechanisms and sites for EAA release, we studied the release of preloaded [3H]-D-aspartate from primary cultured astrocytes prepared from the cerebral cortices of rat pups. Two phases of release were seen in response to raised KCl. The first phase was small and transient, and the second phase was slower and increased progressively. The initial phase of [3H]-D-aspartate release was greatly enhanced by ouabain pretreatment and was inhibited when astrocytes were preexposed to the EAA transport inhibitor threo-hydroxy beta-aspartic acid (THBA). Neither of these manipulations affected the second release component. The second phase of release was inhibited by an anion channel blocker, L-644,711, which is known to inhibit hypotonic swelling-induced release of EAA. Ouabain also resulted in the first phase of release occurring at lower [K+]o. Omission of Ca2+ had no effect on either phase of [3H]-D-aspartate release. These results support the hypothesis that the first component of release in cultured astrocytes is a reversal of the glutamate transporter, and the second component is a result of high KCl-induced swelling. Because marked increases in [K+]o are well established in CNS pathologies such as ischemia, such release may represent a significant source for the increased extracellular EAAs seen in such conditions.