Evidence for several mechanisms of volume regulation in neuroblastoma×glioma hybrid NG108-15 cells

Abstract

Volumes of neuroblastoma×glioma hybrid NG 108-15 cells were electronically measured in order to characterize the mechanisms involved in volume regulation in isosmotic and anisosmotic conditions. The cells behave as perfect osmometers when tonicity was changed at constant chloride concentration by adding sucrose or replacing NaCl with CaCl 2 or MgCl 2. In contrast, the cell volume was poorly dependent on tonicity when the Cl − concentration was changed by adding NaCl or H 2O. Cell shrinkage was induced by cell stirring or after a hypotonicity-induced swelling. These volume decreases were abolished by caffeine but not by ryanodine or EGTA. Shrinkage was also induced by the Ca 2+ ionophore ionomycin. The ionomycin-induced volume decrease was abolished by EGTA. Cell swelling induced an outwardly rectifying Cl − current which was blocked by 5-nitro-2-(3-phenylpropylamino)benzoic acid, 4,4′-diisothiocyanatostilbene-2,2′-disulphonic acid and dihydroindenyl-oxy-alkanoic acid. When the tonicity was reduced at constant Cl − concentration by replacing NaCl with CaCl 2 or MgCl 2, the volume increased and then slowly decreased towards its control value. This regulatory volume decrease was blocked by 5-nitro-2-(3-phenylpropylamino)benzoic acid, 4,4′-diisothiocyanatostilbene-2,2′-disulphonic acid and dihydroindenyl-oxy-alkanoic acid. Long-term (hours–days) cell shrinkage was induced by a reduction of the culture medium osmolarity. Long-term cell swelling was induced by an increase of the culture medium osmolarity. These volume changes were abolished by the protein translation inhibitor cycloheximide. The results suggest that NG 108-15 cell volume is regulated by at least four interacting mechanisms controlled, respectively, by intracellular Ca 2+, extracellular NaCl, cell volume and intracellular ionic strength. The speculative nature of ionic systems responsible for these volume regulating mechanisms is discussed.