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© The Rockefeller University Press, 0022-1295/1998//145/ $5.00
Journal of General Physiology, Volume 112, Number 2, 1998

research-article

Regulatory Volume Decrease and Intracellular Ca2+ in Murine Neuroblastoma Cells Studied with Fluorescent Probes



J. Altamirano*,{ddagger}, M.S. Brodwick{ddagger}, and F.J. Alvarez-Leefmans*,{ddagger},§

From the * Departamento de Neurobiología, Instituto Mexicano de Psiquiatría, México 14370, D.F. México; the {ddagger} 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


ABSTRACT
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 ~–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.

Key Words: calcium • volume regulation • neuroblastoma • regulatory volume decrease

Address correspondence to Dr. F.J. Alvarez-Leefmans, Departamento de Neurobiología, Instituto Mexicano de Psiquiatría, Av. México-Xochimilco 101, México 22 D.F. C.P. 14370, México. Fax: 525-655-9980; E-mail: falvarea{at}mailer.main.conacyt.mx


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