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J. Altamirano^*,, M.S. Brodwick, and F.J. Alvarez-Leefmans^*,,

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

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