Rebuttal from Sergei N. Orlov, Michael M. Model and Ryszard Grygorczyk

Abstract

We agree with the title of the counter-argument article by Florian Lang and Else Hoffmann, Cell volume changes are essential step in the cell death machinery (Lang & Hoffmann, 2013), albeit with one important proviso: this statement is true for selected rather than for all cell types and cell death stimuli. Indeed, as shown in the major section of our paper as well as in our recent review (Orlov et al. 2013), cell volume perturbations do not contribute to the triggering or progression of the cell death machinery in several types of cells, including serum-deprived vascular smooth muscle cells (VSMC) or ouabain-treated renal epithelial cells, possessing biochemical markers of apoptosis and necrosis, respectively (Orlov et al. 1999a; Platonova et al. 2011). The proapoptotic action of ouabain documented in Fas-ligand treated Jurkat cells (Panayiotidis et al. 2010) contradicts its antiapoptotic action in rat VSMC (Orlov et al. 1999b). We also would like to point out methodological limitations of several studies cited in the counter argument article. Thus, in demonstrating that pro-caspase-3 activity (Bortner et al. 1997) and DNA degradation (Ajiro et al. 2008) is inhibited by high-K+ medium, the authors did not employ high-Na+ medium as a negative control. This comment seems to be important because in vascular smooth muscle cells, the pro-caspase-3 activity is equally inhibited by elevation of [KCl] and [NaCl], thus showing that augmented ionic strength rather than elevated [Na+]i/[K+]i ratio occurring in dying cells is responsible for the effect. The death of U937 and HeLa cells documented in Cl−-depleted medium (Maeno et al. 2006) might be caused by modulation of the activity of diverse [Cl−]i-sensitive enzymes (Orlov & Hamet, 2006) rather than by inhibition of inwardly directed Na+–K+–2Cl− cotransporter NKCC1 and cell shrinkage. Indeed, to the best of our knowledge, there are no reports of cell death triggered by sustained exposure to bumetanide or other potent NKCC1 inhibitors.