Relative contribution of chloride channels and transporters to regulatory volume decrease in human glioma cells.

Habela CW, Ernest NJ, Swindall AF, Sontheimer H. Chloride accumulation drives volume dynamics underlying cell proliferation and migration. J Neurophysiol 101: 750 -757, 2009. First published November 26, 2008 doi:10.1152/jn.90840.2008. During brain development, progenitor cells migrate over long distances through narrow and tortuous extracellular spaces posing significant demands on the cell's ability to alter cell volume. This phenotype is recapitulated in primary brain tumors. We demonstrate here that volume changes occurring spontaneously in these cells are mediated by the flux of Cl Ϫ along with obligated water across the cell membrane. To do so, glioma cells accumulate Cl Ϫ to ϳ100 mM, a concentration threefold greater than predicted by the Nernst equation. Shunting this gradient through the sustained opening of exogenously expressed GABA-gated Cl Ϫ channels caused a 33% decrease in cell volume and impaired the ability of cells to migrate in a spatially constrained environment. Further, dividing cells condense their cytoplasm prior to mitosis, a phenomenon which is associated with the release of intracellular Cl Ϫ as indicated by a 40-mM decrease in [Cl Ϫ ] i . These findings provide a new framework for considering the role of intracellular Cl Ϫ in glioma cells. Here, Cl Ϫ serves as an important osmotically active regulator of cell volume being the energetic driving force for volume changes required by immature cells in cell migration and proliferation. This mechanism that was studied in CNS malignancies may be shared with other immature cells in the brain as well.

Programmed cell death (apoptosis) is important in tissue maintenance. Hallmarks of apoptosis include caspase activation, DNA fragmentation and an overall reduction in cell volume. Whether this apoptotic volume decrease (AVD) is a mere response to initiators of apoptosis or whether it is functionally significant is not clear. In this study, we sought to answer this question using human malignant glioma cells as a model system. In vivo, high grade gliomas demonstrate an increased percentage of apoptotic cells as well as upregulation of death ligand receptors. By dynamically monitoring cell volume, we show that the induction of apoptosis, via activation of either the intrinsic or extrinsic pathways with staurosporine or TRAIL, respectively, resulted in a rapid AVD in D54-MG human glioma cells. This decrease in cell volume could be prevented by inhibiting the efflux of Cl– through channels. Such suppression of AVD also reduced the activation of caspases 3, 8 and 9 and suppressed DNA fragmentation. Importantly, experimental manipulations that reduce the cell volume to 70% of the original volume for periods of at least 3 hours were sufficient to initiate apoptosis even in the absence of death ligands. Hence, this data suggests that cell condensation is both necessary and sufficient for the induction of apoptosis.

Mammalian cells regulate their volume to prevent unintentional changes in intracellular signaling, cell metabolism, and DNA integrity. Intentional cell volume changes occur as cells undergo proliferation, apoptosis, or cell migration. To regulate cell volume, cells use channels and transport systems to flux osmolytes across the plasma membrane followed by the obligatory movement of water. While essentially all cells are capable of regulatory volume decrease (RVD), regulatory volume increase (RVI) mechanisms have only been reported in some cell types. In this investigation, we used human glioma cells as a model system to determine conditions necessary for RVI. When exposed to hyperosmotic conditions through the addition of 30 mosM NaCl or sucrose, D54-MG and U251 glioma cell lines and glioma cells from acute patient biopsies shrunk transiently but were able to fully recover their original cell volume within 40-70 min. This ability was highly temperature sensitive and absolutely required the presence of low millimolar concentrations of l-glutamine in the extracellular solution. Other known substrates of glutamine transporters such as methyl-amino isobutyric acid (MeAIB), alanine, and threonine were unable to support RVI. The ability of cells to undergo RVI also required the presence of Na+, K+, and Cl- and was inhibited by the NKCC inhibitor, bumetanide, consistent with the involvement of a Na+/K+/2Cl- cotransporter (NKCC). Moreover, the expression of NKCC1 was demonstrated by Western blot. We concluded that regulatory volume increase in human glioma cells occurs through the uptake of Na+, K+, and Cl- by NKCC1 and is modulated by the presence of glutamine.