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J. Gen. Physiol.,
Volume 112, Number 2, August 1, 1998 145-160
§
From the * Departamento de Neurobiología, Instituto Mexicano de Psiquiatría, México 14370, D.F. México; the The possible role of Ca2+ 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
[Ca2+]i in single cells loaded with fura-2. [Ca2+]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 [Ca2+]i, whose onset preceded RVD. For hyposmotic solutions (up to ~ Department of Physiology and Biophysics, University of Texas Medical Branch, Galveston, Texas 77555; and the § Departamento de Farmacología y Toxicología, Centro de Investigación y de Estudios Avanzados del IPN, México 07000, D.F. México
40%), [Ca2+]i
increased steeply with the reciprocal of the external osmotic pressure and with the cell volume. Chelation of external and internal Ca2+, with EGTA and 1,2-bis-(o -aminophenoxy) ethane-N,N,N ',N '-tetraacetic acid (BAPTA), respectively, attenuated but did not prevent RVD. This Ca2+-independent RVD proceeded even when there was a
concomitant decrease in [Ca2+]i below resting levels. Similar results were obtained in cells loaded with calcein.
For cells not treated with BAPTA, restoration of external Ca2+ during the relaxation of RVD elicited by Ca2+-free
hyposmotic solutions produced an increase in [Ca2+]i without affecting the rate or extent of the responses. RVD and the increase in [Ca2+]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 Ca2+-free solutions. Hence, our simultaneous measurements of intracellular Ca2+ and volume in single neuroblastoma cells directly demonstrate that an increase in
intracellular Ca2+ is not necessary for triggering RVD or its inactivation. The attenuation of RVD after Ca2+ chelation could occur through secondary effects or could indicate that Ca2+ is required for optimal RVD responses.
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