Apoptotic inducers activate the release of D-aspartate through a hypotonic stimulus-triggered mechanism in PC12 cells

We have characterized release of D-aspartate (D-Asp), a regulator of hormone synthesis and secretion, via a volume-sensitive organic anion channel (VSOC) in PC12 cells by studying its response to apoptotic stimuli. PC12 cells have been demonstrated to endogenously synthesize D-Asp. Apoptotic inducers, including staurosporin (STS), tumor necrosis factor (TNF)-alpha, H2O2, and C2-ceramide, activate the release Of D-Asp through a hypotonic stimulus-triggered mechanism. Putative blockers of the anion channel, 5-nitro-2(3-phenylpropylamino)benzoic acid (NPPB) and 4,4'-diisothiocyanostilbene-2,2'-sulphonic acid (DIDS), significantly inhibited stress-induced D-Asp release under hypotonic conditions following the application of apoptotic inducers. Hypotonic conditions are essential for activation by apoptotic inducers. Phorbol 12-mirystate 13-acetate and the Ca2+ ionophore A23187 increased D-Asp efflux via the VSOC, implying the involvement of intracellular Ca2+ in the activation of the D-Asp efflux. However, hypotonic stress and STS had no effect on the concentration of intracellular Ca2+ in PC12 cells. Furthermore, an unknown EGTA-sensitive factor(s), other than Ca2+, and peroxynitrite may play pivotal roles in STS-enhanced D-Asp release. (C) 2009 Elsevier Inc. All rights reserved.