Effects of chloride and potassium channel blockers on apoptotic cell shrinkage and apoptosis in cortical neurons

K⁺ and Cl^– homeostasis have been implicated in cell volume regulation and apoptosis. We addressed the hypothesis that K⁺ and Cl^– efflux may contribute to apoptotic cell shrinkage and apoptotic death in cultured cortical neurons. CLC-2 and CLC-3 chloride channels were detected in cultured cortical neurons. The Cl^– channel blockers 4,4-diisothiocyanatostilbene-2,2-disulfonic acid (DIDS), 4-acetamido-4-isothiocyanatostilbene-2,2-disulfonic acid (SITS) and 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB) inhibited the outwardly rectifying Cl^– current, prevented apoptotic cell shrinkage, and mildly attenuated cell death induced by staurosporine, C₂-ceramide, or serum deprivation. Cl^– channel blockers, however, at concentrations that prevented cell shrinkage had no significant effects on caspase activation and/or DNA fragmentation. Cell death in the presence of a Cl^– channel blocker was still sensitive to blockade by the caspase inhibitor z-Val-Ala-Asp(OMe)-fluoromethyl ketone (z-VAD-fmk). Electron microscopy revealed that, although DIDS prevented apoptotic cell shrinkage, certain apoptotic ultrastructural alterations still took place in injured neurons. On the other hand, the K⁺ channel blocker tetraethylammonium (TEA), clofilium, or the caspase inhibitor z-VAD-fmk prevented cell shrinkage as well as caspase activation and/or DNA damage, and showed stronger neuroprotection against apoptotic alterations and cell death. The results indicate that neurons may undergo apoptotic process without cell shrinkage and imply distinct roles for Cl^– and K⁺ homeostasis in regulating different apoptotic events.