gramicidin-a and sodium-binding-benzofuran-isophthalate

Palytoxin was found to equilibrate sodium ions (Na+) across the cell membrane much faster than dose gramicidin, which has been frequently used to calibrate the intracellular Na+ concentration ([Na+]in), in cells treated with a Na+-sensitive fluorescent dye, sodium-binding benzofuran isophthalate (SBFI). Palytoxin was capable of equilibrating Na+ in cells treated with SBFI-acetoxymethyl ester (SBFI-AM) and in voltage-clamped cells loaded with SBFI through a patch pipette. Nicotine caused a dose-dependent increase in ([Na+]in) in porcine adrenal chromaffin cells treated with SBFI-AM and caused a simultaneous increase in [Na+]in and inward current in the voltage-clamped cells loaded with SBFI. Palytoxin has an advantage of calibrating ([Na+]in) in a shorter time than dose gramicidin because of its powerful ionophoretic activity.

Topics: Acrylamides; Animals; Benzofurans; Calibration; Cells, Cultured; Chromaffin Cells; Cnidarian Venoms; Dialysis; Drug Evaluation, Preclinical; Ethers, Cyclic; Fluorescent Dyes; Gramicidin; Ionophores; Membrane Potentials; Nicotine; Patch-Clamp Techniques; Sodium; Swine

1. Mag-fura-2 fluorescence microscopy and whole-cell patch-clamp recordings were used to measure glutamate-induced changes in the intracellular free Mg2+ concentration ([Mg2+]i) and Mg2+ currents, respectively, in cultured forebrain neurones from fetal rats in the absence of extracellular Na+ (Nao+) and Ca2+ (Cao2+). 2. Increasing the extracellular Mg2+ concentration ([Mg2+]o) from 9 to 70 mM significantly enhanced the maximum [Mg2+]i induced by a 5 min 100 microM glutamate plus 1 microM glycine stimulation ([Mg2+]i,5 min) from 2.04 +/- 0.07 to 2.98 +/- 0.20 mM. Increasing [Mg2+]o from 9 to 70 mM also significantly enhanced the initial rate of rise in [Mg2+]i upon glutamate stimulation from 0.41 +/- 0.02 to 0.81 +/- 0.08 mM min-1. 3. The glutamate-stimulated increase in [Mg2+]i was not altered by prior depletion of intracellular free Na+ (Nai+). For paired stimulations in single neurones, the mean [Mg2+]i,5 min was 1.95 +/- 0.17 mM under Na(+)-depleted conditions and 1.94 +/- 0.16 mM under control conditions. 4. The glutamate-stimulated increase in [Mg2+]i was significantly reduced when NMDA channel-permeant Cs+ or K+ ions were used as the Na+ substitute instead of the presumably NMDA channel-impermeant ions N-methyl-D-glucamine (NMDG), Tris or sucrose. The mean [Mg2+]i,5 min was 0.56 +/- 0.06 and 0.74 +/- 0.08 mM in the presence of Cs+ or K+, respectively, compared with 2.13 +/- 0.10, 1.93 +/- 0.11 and 2.07 +/- 0.22 mM in the presence of NMDG, Tris or sucrose, respectively. 5. In whole-cell recordings performed with Cs+ as the primary intracellular cation, application of 100 microM NMDA plus 10 microM glycine induced inward currents that reversed around -55 mV in an extracellular solution containing 70 mM Mg2+ and 31 mM NMDG as the only cations. The currents were reversibly inhibited by DL-2-amino-5-phosphonovaleric acid (APV). In an extracellular solution containing 2 mM Mg2+ and 140 mM NMDG, NMDA plus glycine activated outward currents at potentials more depolarized than -90 mV. 6. In whole-cell recordings made with NMDG as the principal cation in the patch pipette, application of NMDA plus glycine in the 70 mM Mg2+ extracellular solution induced inward currents at voltages more negative than +15 mV. The ratio of the current measured under these conditions to the current measured in an extracellular solution containing Na+ as the principal cation (0.073:1) was nearly constant from cell to cell. 7. Following a 5 min glutamate stimulation in the presence

Topics: Animals; Antiporters; Benzofurans; Brain; Cells, Cultured; Electrophysiology; Ethers, Cyclic; Fluorescent Dyes; Fura-2; Glutamic Acid; Gramicidin; Magnesium; Membrane Potentials; N-Methylaspartate; Neurons; Potassium; Rats; Rats, Sprague-Dawley; Receptors, N-Methyl-D-Aspartate; Sodium

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.

Topics: Amphotericin B; Animals; Benzofurans; Cell Line; Cell Nucleus; Chlorides; Cytosol; Ethers, Cyclic; Fibroblasts; Fluorescent Dyes; Gramicidin; Humans; Kinetics; Lymphocytes; Mathematics; Monensin; Nigericin; Rats; Rats, Inbred F344; Sodium; Spectrometry, Fluorescence