Regulatory Volume Decrease and Intracellular Ca(2+) in Murine Neuroblastoma Cells Studied with Fluorescent Probes

The possible role of Ca(2+) as a second messenger mediating regulatory volume decrease (RVD) in osmotically swollen cells was investigated in murine neural cell lines (N1E-115 and NG108-15) by means of novel microspectrofluorimetric techniques that allow simultaneous measurement of changes in cell water volume and [Ca(2+)](i) in single cells loaded with fura-2. [Ca(2+)](i) was measured ratiometrically, whereas the volume change was determined at the intracellular isosbestic wavelength (358 nm). Independent volume measurements were done using calcein, a fluorescent probe insensitive to intracellular ions. When challenged with ∼40% hyposmotic solutions, the cells expanded osmometrically and then underwent RVD. Concomitant with the volume response, there was a transient increase in [Ca(2+)](i), whose onset preceded RVD. For hyposmotic solutions (up to ∼−40%), [Ca(2+)](i) increased steeply with the reciprocal of the external osmotic pressure and with the cell volume. Chelation of external and internal Ca(2+), with EGTA and 1,2-bis-(o -aminophenoxy) ethane-N,N,N ′,N ′-tetraacetic acid (BAPTA), respectively, attenuated but did not prevent RVD. This Ca(2+)-independent RVD proceeded even when there was a concomitant decrease in [Ca(2+)](i) below resting levels. Similar results were obtained in cells loaded with calcein. For cells not treated with BAPTA, restoration of external Ca(2+) during the relaxation of RVD elicited by Ca(2+)-free hyposmotic solutions produced an increase in [Ca(2+)](i) without affecting the rate or extent of the responses. RVD and the increase in [Ca(2+)](i) were blocked or attenuated upon the second of two ∼40% hyposmotic challenges applied at an interval of 30–60 min. The inactivation persisted in Ca(2+)-free solutions. Hence, our simultaneous measurements of intracellular Ca(2+) and volume in single neuroblastoma cells directly demonstrate that an increase in intracellular Ca(2+) is not necessary for triggering RVD or its inactivation. The attenuation of RVD after Ca(2+) chelation could occur through secondary effects or could indicate that Ca(2+) is required for optimal RVD responses.