monensin and sodium-binding-benzofuran-isophthalate

In the present investigation, intracellular sodium ([Na+]i) levels were determined in GH4C1 cells using the fluorescent probe SBFI. Fluorescence was determined by excitation at 340 nm and 385 nm, and emission was measured at 500 nm. Intracellular free sodium ([Na+]i) was determined by comparing the ratio 340/385 to a calibration curve. The ratio was linear between 10 and 60 mM Na+. Resting [Na+]i in GH4C1 cells was 26 +/- 6.2 mM (mean +/- SD). In cells incubated in Na(+)-free buffer [Na+]i decreased to 3 +/- 3.6 mM. If Na+/K+ ATPase was inhibited by incubating the cells with 1 mM ouabain, [Na+]i increased to 47 +/- 12.8 mM in 15 min. Stimulating the cells with TRH, phorbol myristyl acetate, or thapsigargin had no effect on [Na+]i. Incubating the cells in Ca(2+)-free buffer rapidly increased [Na+]i. The increase was not inhibited by tetrodotoxin. Addition of extracellular Ca2+, nimodipine, or Ni2+ to these cells immediately decreased [Na+]i, whereas Bay K 8644 enhanced the influx of Na+. In cells where [Na+]i was increased the TRH-induced increase in intracellular free calcium ([Ca2+]i) was decreased compared with control cells. Our results suggest that Na+ enters the cells via Ca2+ channels, and [Na+]i may attenuate TRH-induced changes in [Ca2+]i in GH4C1 cells.

Topics: Animals; Benzofurans; Calcium; Calcium Channels; Cell Line; Clone Cells; Ethers, Cyclic; Fluorescent Dyes; Membrane Potentials; Monensin; Ouabain; Pituitary Gland; Rats; Sodium; Sodium Channels; Terpenes; Tetradecanoylphorbol Acetate; Thapsigargin; Thyrotropin-Releasing Hormone

The new fluorescent Na+ indicator sodium-binding benzofuran isophthalate (SBFI) was used for determination of the cytosolic free Na+ concentration, [Na+]i, in human platelets. The dye could be loaded into platelets in the form of its acetoxymethyl ester (SBFI-AM). Calibration of the fluorescence in terms of [Na+]i was done by measuring the 345/385 nm excitation ratio (emission 490 nm) at various extracellular Na+ concentrations, [Na+]o, in the presence of gramicidin D. The 345/385 intensity ratio increased almost linearly when [Na+]i was stepwise raised from 20 to 60 mM. The basal value for [Na+]i was found to be 26.0 +/- 4.5 mM (n = 15). Incubation of platelets in Na(+)-free buffer decreased [Na+]i, whereas inhibition of the (Na+ + K+)-ATPase by 0.5 mM ouabain increased [Na+]i to 56 +/- 4 mM (n = 4) within 60 min. Activation of Na+/H+ exchange by exposing platelets to propionic acid also raised [Na+]i, and a comparable effect was produced by the Na+/H+ ionophore monensin. Activation of platelets with thrombin (0.1-0.5 unit/ml) also increased the 345/385 nm intensity ratio, an effect that was not seen in Na(+)-free buffer or after raising intracellular cAMP by treatment of platelets with prostaglandin E1. On the average, [Na+]i was raised to 59.5 +/- 5.3 mM (n = 15) at 10 min after addition of thrombin without a significant decrease for further 10 min. An increase in [Na+]i was also seen when platelets were challenged with the Ca2+ ionophore ionomycin, an effect that did not occur in the absence of Na+o. Our findings confirm earlier reports which demonstrated a rise in [Na+]i in stimulated platelets and show that SBFI is a useful tool for determination of [Na+]i in resting and stimulated platelets.

Topics: Alprostadil; Benzofurans; Blood Platelets; Carrier Proteins; Cyclic AMP; Cytosol; Ethers, Cyclic; Fluorescent Dyes; Humans; Ionomycin; Monensin; Ouabain; Platelet Activation; Propionates; Sodium; Sodium-Hydrogen Exchangers; Sodium-Potassium-Exchanging ATPase; Spectrometry, Fluorescence; Thrombin

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