Determination of the intravesicular ionized sodium concentration in a cell-free brain membrane vesicle preparation using the fluorescent indicator, SBFI.

The intravesicular ionized Na concentration (Nai) was measured using the fluorescent Na indicator, SBFI, in microsacs, a cell-free brain vesicle preparation. SBFI fluorescence was monitored by a dual excitation-wavelength method at the same wavelengths commonly employed for Fura-2 determination of intracellular ionized calcium concentrations (Cai). Calibration of SBFI fluorescence was reliably performed in brain microsacs in situ. Resting Nai was dependent on the extravesicular Na concentration (Nao) and was about 36 mM in the presence of 120 mM extracellular Nao. In the presence of ouabain, an inhibitor of the plasma membrane Na/K-ATPase, Nai increased by 27 mM over 60 s. Nai was also increased by resuspension of microsacs in buffers of low free Ca concentrations (0 to 0.8 mM), indicating that the extravesicular Ca concentration (Cao) is an important regulator of Nai. Alkaloids active at voltage-sensitive Na channels, veratridine and aconitine, also increased Nai. These results demonstrate the presence of homeostatic mechanisms for neuronal Nai regulation and show that Nai can be measured in a cell-free brain vesicle preparation using SBFI.