Regulation of intracellular pH in LLC-PK1 cells by Na+/H+ exchange

Abstract

Summary Suspensions of LLC-PK₁ cells (a continuous epitheliod cell line with renal characteristics) are examined for mechanisms of intracellular pH regulation using the fluorescent probe BCECF. Initial experiments determine suitable calibration procedures for use of the BCECF fluorescent signal. They also determine that the cell suspension contains cells which (after 4 hr in suspension) have Na⁺ and K⁺ gradients comparable to those of cells in monolayer culture. The steady-state intracellular pH (7.05±0.01,n=5) of cells which have recovered in (pH 7.4) Na⁺-containing medium is not affected over several minutes by addition of 100 μM amiloride or removal of extracellular Na⁺ (Na ⁺_o /H ⁺_i and Na ⁺_i /H ⁺_o exchange reactions are functionally inactive (compared to cellular buffering capacity). In contrast, Na ⁺_o /H ⁺_i exchange is activated by an increased cellular acid load. This activation may be observed directly either as a stimulation of net H⁺ efflux or net Na⁺ influx with decreasing intracellular pH. The extrapolation of this latter data suggests a ‘set point” of Na⁺/H⁺ exchange of approximately pH 7.0, consistent with the observed resting intracellular pH of approximately 7.05.