Fluorescence ratio imaging of cytosolic free Na+ in individual fibroblasts and lymphocytes.

New fluorescent Na+ indicators, SBFI (short for sodium-binding benzofuran isophthalate) and SBFP (short for sodium-binding benzofuran phthalate) (Minta, A., and Tsien, R. Y. (1989) J. Biol. Chem. 264, 19449-19457), were tested in Jurkat tumor lymphocytes and in REF52 rat embryo fibroblasts. Both dyes could be introduced by direct microinjection. However, when cells were incubated with the tetra(acetoxymethyl) esters of the dyes, only SBFI gave intracellular loading that was reasonably responsive to [Na+]i. Because some compartmentation of the SBFI was visible and because the indicator properties are somewhat affected by cytoplasm, the relationship between intracellular free Na+ [( Na+]i and the 340/385 nm excitation ratio of the indicator was calibrated in situ using poreforming antibiotics to equilibrate cytosolic [Na+] [( Na+]i) with extracellular [Na+]. The excitation ratio was sufficiently sensitive to resolve small changes, less than or equal to 1 mM, in [Na+]i in single cells. Basal [Na+]i values in lymphocytes and serum-starved fibroblasts were 9.4 and 4.2 mM, respectively. As expected, large increases in [Na+]i were elicited by blocking the Na+ pump with ouabain or withdrawal of extracellular K+. Mitogens such as phytohemagglutinin acting on the lymphocytes, or serum or vasopressin in fibroblasts, caused [Na+]i to increase up to 2-fold. In fibroblasts, the rise in [Na+]i was due at least partly to a stimulation of Na+ influx, which was not wholly through the Na+/H+ exchanger. The mitogen-induced increases in [Na+]i and the rate of Na+ influx are consistent with earlier estimates based on measurements of total [Na+] or tracer fluxes. However, the absolute values for free [Na+]i are much lower than previous values for total intracellular Na+, suggesting that much of the latter is bound or sequestered.