Regulation of cytosol–nucleus pH gradients by K+/H+ exchange mechanism in the nuclear envelope of neonatal rat astrocytes

Abstract In order to study the subcellular heterogeneity of intracellular H + concentration in reactive astrocytes, the pH in the nucleus and cytosol of cultured astrocytes was measured using a confocal laser scanning microscope (CLSM) and pH indicator dye, 5′(and 6′)-carboxyseminaphthofluorescein (carboxy SNAFL-1). The change in intracellular pH was indexed by the fluorescence ratio ( F 535 / F 610 ) at an excitation wavelength of 514.5 nm. The in vitro fluorescence ratio increased as pH decreased. This ratio in the nucleus was significantly lower than that in the cytosol of astrocytes when perfused by HEPES-buffered Hanks' balanced salt solution (HHBSS) at pH 7.4. Acid stimulations of cells (pH 5.0) raised the fluorescence ratio in both nucleus and cytosol. However, the increase in the fluorescence ratio of the nucleus was less than that of cytosol. Treatment with a K + /H + ionophore, nigericin (20 μM), reversibly nullified this cytosol–nucleus pH gradient. These findings suggest that a buffering mechanism(s) for maintaining of intracellular pH exists between the nucleus and cytosol, and a K + /H + exchanger may act on the nuclear envelope to eventuate intranuclear pH maintenance in the living cells.