EFFECT OF ATP-INDUCED PERMEABILIZATION ON LOADING OF THE NA-CELLS( PROBE SBFI INTO ENDOTHELIAL)

The fluoroprobe sodium-binding benzofuran isophthalate (SBFI) is used to measure intracellular cytosolic sodium concentration ([Na](i)). A p roblem with the use of this probe is the difficulty in loading it into cells. ATP reversibly increases membrane permeability of some cells v ia activation of receptors of the tetrabasic form of ATP (ATP(4-)). We investigated the effect of ATP-induced membrane permeabilization on l oading of the acetoxymethyl ester (AM) form of SBFI (SBFI-AM) into bov ine pulmonary arterial endothelial cells. Monolayers were incubated in a series of solutions that reversibly opened pores, loaded the fluoro probe, and finally sealed the pores. ATP (1-5 mM) or 3'-O-(4-benzoyl)b enzoyl-ATP (0.1-1 mM), an analogue 30-100x more specific for ATP(4-) r eceptors, was utilized to permeabilize the cell membrane. The signal-t o-background ratio of the intracellular SBFI fluorescent signal was us ed as an indicator of the effectiveness of dye loading. ATP and 3'-O-( 4-benzoyl)benzoyl-ATP significantly increased the signal-to-background ratio compared with the values obtained with the standard dye-loading procedure without ATP, indicating that permeabilization increased SBF I-AM entry into the cells. The permeabilization procedure produced a s mall decrease in cell viability, as determined with a fluorescent viab ility assay (ethidium dimer uptake), compared with the standard method of loading SBFI-AM. We used the procedure to measure baseline [Na](i) and changes in [Na]i after the administration of ouabain (10(-4) M) a nd monensin (10(-5) M). Baseline [Na](i) with this procedure (19.7 +/- 2.7 mM; n = 15 monolayers) was similar to measurements made in other cell types with the standard method of loading the probe. We conclude that i) the ATP-induced permeabilization technique is an improved dye- loading method for SBFI-AM in endothelial cell monolayers that facilit ates measurement of [Na](i) and 2) these data suggest the presence of an ATP(4-) pore-forming mechanism in this cell type.