Intracellular free sodium concentrations in GH4C1 cells.

Abstract

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.