Despite the popularity of Na+-binding benzofuran isophthalate (SBFI) to measure intracellular free Na+ concentrations ([Na+](i)), the in situ calibration techniques described to date do not favor the straightforward determination of all of the constants required by the standard equation (Grynkiewicz G, Poenie M, and Tsien RY. J Biol Chem 260: 3440-3450, 1985) to convert the ratiometric signal into [Na+]. We describe a simple method in which SBFI ratio values obtained during a "full" in situ calibration are fit by a three-parameter hyperbolic equation; the apparent dissociation constant (K(d)) of SBFI for Na+ can then be resolved by means of a three-parameter hyperbolic decay equation. We also developed and tested a "one-point" technique for calibrating SBFI ratios in which the ratio value obtained in a neuron at the end of an experiment during exposure to gramicidin D and 10 mM Na+ is used as a normalization factor for ratios obtained during the experiment; each normalized ratio is converted to [Na+](i) using a modification of the standard equation and parameters obtained from a full calibration. Finally, we extended the characterization of the pH dependence of SBFI in situ. Although the K(d) of SBFI for Na+ was relatively insensitive to changes in pH in the range 6.8-7.8, acidification resulted in an apparent decrease, and alkalinization in an apparent increase, in [Na+](i) values. The magnitudes of the apparent changes in [Na+](i) varied with absolute [Na+](i), and a method was developed for correcting [Na+](i) values measured with SBFI for changes in intracellular pH.

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Mechanisms that contribute to Na ϩ influx during and immediately after 5 min anoxia were investigated in cultured rat hippocampal neurons loaded with the Na ϩ -sensitive fluorophore sodium-binding benzofuran isophthalate. -dependent mechanism(s). The results provide insight into the intrinsic mechanisms that contribute to disturbed internal Na ϩ homeostasis during and immediately after anoxia in rat hippocampal neurons and, in this way, may play a role in the pathogenesis of anoxic or ischemic cell injury.

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Patch-clamp and Fura-2 experiments were performed in order to investigate the calcium oscillations due to H1 receptor stimulation in HeLa cells. The cytosolic calcium fluctuations occurring directly at the plasma membrane inner face were detected by measuring the activity of calcium-dependent potassium channels. This method also allowed measurement of changes in intracellular potential using as indicator the amplitude of the channel current jump. The average internal calcium concentration was obtained from Fura-2 experiments carried out at either the single-cell level or from a small population of cells in monolayer. The results indicate that the internal calcium oscillations in HeLa cells arise from a biphasic process with an initial phase independent of the presence of external calcium. External calcium was found, however, to become essential once the regular oscillatory process has been established. Removing external calcium after this initial phase produced a rapid decay in the burst frequency and eventually a complete abolition of the oscillations. In addition, the calcium oscillations occurring during the external-calcium-dependent phase could be blocked by calcium entry blockers such as Co2+ or La3+, or abolished by perfusing the external medium with a high-K+ solution. Experiments were also performed in which the cell internal pH (pHi) was changed by removing the external bicarbonate or by adding NH4Cl to the bathing solution. The results obtained under these conditions indicate that an increase in internal pH abolishes selectively the appearance of calcium spikes without increasing the basal calcium level, while a cellular acidification maintains or stimulates the calcium oscillatory process. It was also observed that the inhibitory effect of alkaline pH was independent of external calcium, and that calcium oscillations could always be seen at alkaline pH during the initial phase of histamine stimulation. On the basis of these results, it is proposed that the internal calcium oscillations in HeLa cells depend on the release of calcium from internal pools, which are reloaded via a pH-dependent mechanism. Part of the calcium sequestration occurring during the oscillatory process would be carried out, however, by pH-insensitive calcium compartments.