Measurements of intracellular pH in single LLC-PK1 cells: recovery from an acid load via basolateral Na+/H+ exchange

Abstract

LLC-PK1 cells (a continuous epithelioid cell line with renal characteristics) are examined by microspectrofluorometry as single cells, in order to determine the mechanism of intracellular pH (pHi) recovery from an acid load imposed by ammonium preincubation and removal (NH4 prepulse). Initial experiments evaluate the intracellular K+ levels through a null point analysis of total cellular K+ with flame photometry. The response of BCECF (a pH-sensitive fluorescent dye) is then calibrated, using saturating concentrations of nigericin to cause defined changes in pH. For experiments with the microspectrofluorometer, LLC-PK1 cells were grown on either glass coverslips or filters (the latter attached to plastic coverslips with a hole under the filter). The cells on glass coverslips demonstrate a Na+-dependent recovery from an (NH4 prepulse) acid load which is sensitive to 1 microM ethylisopropylamiloride. They also demonstrate a 'set point' of activation of Na+/H+ exchange. When examined for changes in pH, due to changes in membrane potential, plasma membrane proton conductance could not be detected at resting pHi. Cells grown on filters also demonstrate a pHi recovery from an acid load which is Na+ dependent and ethylisopropylamiloride sensitive, but in this configuration, the majority of cells (22/23 preparations) require Na+ at the basolateral membrane for rapid pHi recovery. The morphology and polarity of the cells grown on permeable supports appears normal at the electron-microscopic level. The results are not affected by changes in cell seeding density or collagen treatment of the filters.