VOLUME CHANGES IN SINGLE N1E-115 NEUROBLASTOMA-CELLS MEASURED WITH A FLUORESCENT-PROBE

A non-invasive microspectrofluorimetric technique was used to investigate experimentally induced changes in cell water volume in single N1E-115 murine neuroblastoma cells, using calcein, a derivative of fluorescein, as a marker of the intracellular water compartment. The osmotic behavior of N1E-115 cells exposed to media of various osmolalities was studied. Exposure to hyperosmotic (up to + 28%) or hyposmotic (up to -17%) solutions produced reversible decreases and increases in cell water volume, respectively, which agreed with near-osmometric behavior. Increases in [Ca2+](i) produced by exposing the cells to the ionophore ionomycin (1 mu M) in isosmotic medium, resulted in a gradual decrease in cell water volume. Cells shrank to 40+/-7% (n=7) below their initial water volume at an initial rate of -1.2+/-0.2%/min. It is concluded that N1E-115 cells are endowed with Ca2+-sensitive mechanisms for volume control, which can produce cell shrinkage when activated under isosmotic conditions. Because the technique used for measuring cell water volume changes is new, we describe it in detail. It is based on the principle that relative cell water volume in single cells can be measured by introducing an impermeant probe into cells and measuring its changes in concentration. If the intracellular content of the probe is constant, changes in its concentration reflect changes in cell water volume. Calcein was used as the probe because its fluorescence intensity is directly proportional to its concentration and independent of changes in the concentration of native intracellular ions within the physiological range. Because calcein is two to three times more fluorescent than other fluorophores such as 2',7,-bis-[2-carboxyethyl]-5-[and-6]-carboxyfluorescein or Fura-2, and it is used at its peak excitation and emission wavelengths, it has a better signal to noise ratio and baseline stability than the other dyes. Calcein can also be esterified allowing for cell loading and because of the possibility of reducing the intensity of the excitation light, measurements can be performed producing minimal photodynamic damage. The technique allows for measurements of cell water volume changes of < 5% and it can be applied to single cells which can be grown or affixed to a rigid substratum, e.g., a coverslip.