Pore size of swelling-activated channels for organic osmolytes in Jurkat lymphocytes, probed by differential polymer exclusion.

The present study explores the impact of the molecular size on the permeation of low-molecular-weight polyethylene glycols (PEG200-1500) through the plasma membrane of Jurkat cells under iso- and hypotonic conditions. To this end, we analyzed the cell volume responses to PEG-substituted solutions of different osmolalities (100-300 mOsm) using video microscopy. In parallel experiments, the osmotically induced changes in the membrane capacitance and cytosolic conductivity were measured by electrorotation (ROT). Upon moderate swelling in slightly hypotonic solutions (200 mOsm), the lymphocyte membrane remained impermeable to PEG300-1500, which allowed the cells to accomplish regulatory volume decrease (RVD). During RVD, lymphocytes released intracellular electrolytes through the swelling-activated pathways, as proved by a decrease of the cytosolic conductivity measured by electrorotation. RVD also occurred in strongly hypotonic solutions (100 mOsm) of PEG600-1500, whereas 100 mOsm solutions of PEG300-400 inhibited RVD in Jurkat cells. These findings suggest that extensive hypotonic swelling rendered the cell membrane highly permeable to PEG300-400, but not to PEG600-1500. The swelling-activated channels conducting PEG300-400 were inserted into the plasma membrane from cytosolic vesicles via swelling-mediated exocytosis, as suggested by an increase of the whole cell capacitance. Using the hydrodynamic radii R(h) of PEGs (determined by viscosimetry), the observed size-selectivity of membrane permeation yielded an estimate of approximately 0.74 nm for the cut-off radius of the swelling-activated channel for organic osmolytes. Unlike PEG300-1500, the smallest PEG (PEG200, R(h)=0.5 nm) permeated the lymphocyte membrane under isotonic conditions thus leading to a continuous isotonic swelling. The results are of interest for biotechnology and biomedicine, where PEGs are widely used for cryopreservation of cells and tissues.

Intracellular delivery of 2-deoxy-D-glucose into tumor cells by long-term cultivation and through swelling-activated pathways: implications for radiation treatment.

2-Deoxy-D-glucose (2DG), a well-known inhibitor of anaerobic glycolysis, is expected to exert cytotoxic and radiosensitizing effects. In order to test this hypothesis, the response of four tumor cell lines (U87-MG, GaMG, A549 and HT1080) to 2DG was analyzed for cell proliferation, changes in cell volume and nucleus size, as well as for radiation-induced DNA fragmentation, measured by the alkaline Comet assay. Two methods were used for loading cells with 2DG. The long-term method included cell cultivation in the presence of 5 mM 2DG for 24 h, while rapid intracellular delivery of 2DG was achieved by exposing the cells for 20 min to a hypotonic solution containing 100 mM 2DG. Irrespective of the loading method, 2DG inhibited the growth of HT1080 and A549 cells. In contrast, two glioblastoma lines (U87 and GaMG) were resistant to 2DG. In three of the four cell lines (all except HT1080), long-term treatment with 2DG reduced radiation-induced DNA fragmentation in conjunction with 2DG-mediated nucleus shrinkage (probably via chromatin condensation) in non-irradiated cells. Complementary volumetric experiments revealed the avid hypotonic uptake of 2DG by all tumor lines. Nonetheless, only HT1080 cells exhibited a significant increase in radiation-induced DNA fragmentation upon hypotonic loading with 2DG, associated with marked nucleus expansion in non-irradiated samples. Our data suggest that, dependant on cell type as well as on medium composition and tonicity, sugar treatment can induce the compaction or expansion of chromatin, thus decreasing or increasing radiation-induced DNA fragmentation. These results raise interesting questions for further studies on the mechanistic links between the sugar-modulated cell volume changes, chromatin structure and radiosensitivity of tumor and normal cells.