Regulatory volume decrease and intracellular Ca2+ in murine neuroblastoma cells studied with fluorescent probes.
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 approximately 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 approximately -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 approximately 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.
Departamento de Neurobiología, Instituto Mexicano de Psiquiatría, México 14370, D.F. México.
J
J
Altamirano
M S
MS
Brodwick
F J
FJ
Alvarez-Leefmans
D000818:Animals; D002118:Calcium / Q000378:metabolism* / Q000494:pharmacology; D048429:Cell Size / Q000502:physiology; D002614:Chelating Agents / Q000494:pharmacology; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D005452:Fluoresceins / Q000493:pharmacokinetics; D005456:Fluorescent Dyes / Q000493:pharmacokinetics; D016257:Fura-2 / Q000493:pharmacokinetics; D005910:Glioma; D006822:Hybrid Cells / Q000187:drug effects / Q000378:metabolism; D007038:Hypotonic Solutions / Q000494:pharmacology; D051379:Mice; D009447:Neuroblastoma; D009474:Neurons / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism*; D009994:Osmolar Concentration; D051381:Rats; D014407:Tumor Cells, Cultured / Q000187:drug effects / Q000378:metabolism
D002614:Chelating Agents; D005452:Fluoresceins; D005456:Fluorescent Dyes; D007038:Hypotonic Solutions; D004533:Egtazic Acid; C025603:1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; D002118:Calcium; D016257:Fura-2; C007740:fluorexon