Induction of 86Rb Fluxes by Ca2+ and volume changes in thymocytes and their isolated membranes

Abstract

Cell swelling and elevated intracellular Ca²⁺ increase K⁺ permeability in lymphocytes. Experiments were performed to test whether these effects can also be elicited in isolated plasma membrane vesicles. Rabbit thymocytes, used as a source of membrane vesicles, were found to regain their volume after swelling in hypotonic, low‐K⁺ media. This regulatory volume decrease (RVD) was inhibited by quinine and trifluoperazine, but not affected by ouabain. Both efflux and uptake of K⁺ (⁸⁶Rb) were stimulated by hypotonicity. Addition of A23187 plus Ca²⁺ also increased ⁸⁶Rb fluxes. Ca²⁺ ‐ and volume‐induced ⁸⁶Rb fluxes were also studied in isolated membranes. A plasma membrane‐rich vesicle fraction, enriched over 11‐fold in 5′‐nucleotidase, was isolated from thymocytes. The vesicles were about 35% inside‐out and trapped ⁸⁶Rb in an osmotically active compartment of ∼1.3 μl/mg protein. Equilibrium exchange fluxes of ⁸⁶Rb in the vesicles were unaffected by Ca²⁺ with or without A23187. Calmodulin had no effect on ⁸⁶Rb permeability but stimulated ATP‐dependent Ca²⁺ accumulation. Hypotonic swelling increased both uptake and efflux of ⁸⁶Rb from vesicles. However, this increase was not blocked by either quinine or trifluoperazine, was not specific for K⁺ (⁸⁶Rb), and is probably unrelated to RVD. It is concluded that components essential for the volume‐ and Ca²⁺‐induced changes in K⁺ permeability are lost or inactivated during membrane isolation. An intact cytoarchitecture may be required for RVD.