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

Abstract

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.