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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.