Introduction 1 3 3 5 + + 3 5 + + 6 9 − − 1 2 10 12 The question is thus raised of how hypertonic stress might interfere with AVD. Would osmotic shrinkage facilitate the process? Or would the activation of HICCs or RVI oppose the induction of apoptosis? Materials and methods Cell culture 2 Determination of cell volume 13 2 2 3 2 Patch-clamp experiments Membrane currents were recorded in the fast whole-cell mode of the patch-clamp technique using 2 MΩ borosilicate pipettes. Currents were recorded with an Axopatch 200B amplifier (Molecular Devices, Union City, CA), filtered at 5 kHz with a four-pole Bessel filter and digitized at 20 kHz. pClamp 9.02 software (Molecular Devices) was used for control of the pulse protocol, as well as for data acquisition and analysis. Series resistance was <5 MΩ and was compensated (by 70–80%) to minimize voltage errors. In the experiments, voltage ramps from −80 to +20 mV and of 1 s duration were applied every 10 s; holding voltage was −30 mV. 2 2 d 2 2 2 2 2 + − 13 Monitoring apoptotic cell death Cell viability and caspase-3/7 activity were determined by use of a calorimetric MTT assay (Cell Counting Kit-8; Dojindo, Kumamoto, Japan) and a fluorometric probe (Apo-ONE Homogeneous Caspase-3/7 Assay; Promega, Madison, WI), respectively. PLUS g Testing for necrosis and late apoptosis Necrosis and late apoptosis of cells were quantified fluorometrically on the basis of a propidium iodide staining of nuclei (excitation/emission at 530 nm/620 nm) performed with reference to an overall staining of cells by Hoechst 33342 (excitation/emission at 350 nm/460 nm). Chemicals All reagents were purchased from Sigma–Aldrich (Tokyo, Japan). Statistical analysis n t P Results and discussion 2 2 n 1 3 14 y 1 1 Fig. 1 a 2 2 2 2 b n  6 15 6 2 2 n 2 2 cf. 1 2 minus 2 16 Fig. 2 a 2 1 b minus minus n  2 3 t t t t 3 minus minus minus 2 2 3 3 n P 2 P 3 3 Fig. 3 a 2 b a minus minus n  4 n Fig. 4 a b c a  3 5 + + + + − 5 13 5 2 4 5 13 plus minus n P 5 5 plus P 5 17 5 P 5 Fig. 5 a 2 plus  t b minus a n P #,## P P  − 2 18 − 19 t n n n 2 6 6 6 2 2 2 15 Fig. 6 a 2 n b 2 n c 2 n  2 2 2 2 2 n 6 2 2 P n 7 2 7 3 4 Fig. 7 2 a n b n c  n  + + 20 22 + + 23 + + − + + − 24 25 2 2 26 29 4 30 32 Conclusions Taken together, our results indicate that hypertonic stress caused a significant reduction in the staurosporine (STS)-induced apoptosis of HeLa cells. When analysing the functional interplay between apoptotic volume decrease (AVD) and regulatory volume increase (RVI) through the use of different activation protocols and effective blockers, the activation of hypertonicity-induced cation channels (HICCs) could be identified as the molecular mechanism by which HeLa cells are actually rescued from STS-induced apoptosis in hypertonic conditions. The precise mechanism by which HICC activation inhibits AVD and apoptosis remains to be elucidated. 33 4 5