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Am J Physiol Cell Physiol 290: C524-C538, 2006. First published August 31, 2005; doi:10.1152/ajpcell.00290.2005
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METHODS IN CELL PHYSIOLOGY

Spectral imaging microscopy demonstrates cytoplasmic pH oscillations in glial cells

Sergio Sánchez-Armáss,1,* Souad R. Sennoune,2,* Debasish Maiti,2 Filiberta Ortega,1 and Raul Martínez-Zaguilán2,3

1Departamento de Fisiología y Farmacología, Facultad de Medicina, Universidad Autónoma de San Luis Potosí, San Luis Potosí, Mexico; and 2Department of Physiology and 3Southwest Cancer Center, Texas Tech University Health Sciences Center, Lubbock, Texas

Submitted 15 June 2005 ; accepted in final form 24 August 2005

Glial cells exhibit distinct cellular domains, somata, and filopodia. Thus the cytoplasmic pH (pHcyt) and/or the behavior of the fluorescent ion indicator might be different in these cellular domains because of distinct microenvironments. To address these issues, we loaded C6 glial cells with carboxyseminaphthorhodafluor (SNARF)-1 and evaluated pHcyt using spectral imaging microscopy. This approach allowed us to study pHcyt in discrete cellular domains with high temporal, spatial, and spectral resolution. Because there are differences in the cell microenvironment that may affect the behavior of SNARF-1, we performed in situ titrations in discrete cellular regions of single cells encompassing the somata and filopodia. The in situ titration parameters apparent acid-base dissociation constant (pK'a), maximum ratio (Rmax), and minimum ratio (Rmin) had a mean coefficient of variation approximately six times greater than those measured in vitro. Therefore, the individual in situ titration parameters obtained from specific cellular domains were used to estimate the pHcyt of each region. These studies indicated that glial cells exhibit pHcyt heterogeneities and pHcyt oscillations in both the absence and presence of physiological HCO3–. The amplitude and frequency of the pHcyt oscillations were affected by alkalosis, by acidosis, and by inhibitors of the ubiquitous Na+/H+ exchanger- and HCO3–-based H+-transporting mechanisms. Optical imaging approaches used in conjunction with BCECF as a pH probe corroborated the existence of pHcyt oscillations in glial cells.

proton gradients; proton waves; carboxyseminaphthorhodafluor-1; 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein


Address for reprint requests and other correspondence: R. Martínez-Zaguilán, Dept. of Physiology, Texas Tech Univ. Health Sciences Center, 3601 4th St., STOP 6551, Lubbock, TX 79430-6551 (e-mail: Raul.MartinezZaguilan{at}ttuhsc.edu)






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