Sodium Is Not Required for Chloride Efflux via Chloride/Bicarbonate Exchanger from Rat Thymic Lymphocytes

摘要

Sodium-dependent Cl~- /HCO_3~- exchanger acts as a chloride (Cl~-) efflux in lymphocytes. Its functional characterization had been described when Cl~- efflux was measured upon substituting extracellular sodium (Na~+) by N-methyl-D-glucamine (NMDG). For Na~+ and Cl~- substitution, we have used D-mannitol or NMDG. Thymocytes of male Wistar rats aged 7-9 weeks were used and intracellular Cl~- was measured by spectrofluorimetry using MQAE dye in bicarbonate buffers. Chloride efflux was measured in a Cl~--free buffer (Cl~- substituted with isethionate acid) and in Na~+ and Cl~--free buffer with D-mannitol or with NMDG. The data have shown that Cl~- efflux is mediated in the absence of Na~+ in a solution containing D-mannitol and is inhibited by H_2DIDS. Mathematical modelling has shown that Cl~- efflux mathematical model parameters (relative membrane permeability, relative rate of exchanger transition, and exchanger efficacy) were the same in control and in the medium in which Na~+ had been substituted by D-mannitol. The net Cl~- efflux was completely blocked in the NMDG buffer. The same blockage of Cl~- efflux was caused by H_2DIDS. The study results allow concluding that Na~+ is not required for Cl~- efflux via Cl~-/HCO_3~- exchanger. NMDG in buffers cannot be used for substituting Na~+ because NMDG inhibits the exchanger.