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

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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 (pH i ) 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 i . 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 μM ethylisopropylamiloride. They also demonstrate a ‘set point’ of activation of Na+/H+ exchange. When examined for changes in pH i due to changes in membrane potential, plasma membrane proton conductance could not be detected at resting pH i . Cells grown on filters also demonstrate a pH i 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 pH i 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.