Steady-State pH_i, Buffering Power, and Effect of CO₂ in a Smooth Muscle-Like Cell Line

Intracellular pH (pH _i ) was studied in the smooth muscle- like cell line, BC3H-1, using the pH-sensitive fluorescent dye 2', 7' -bis(2-carboxyethyl)-5,6-carboxyfluorescein (BCECF). The initial pH _i measured in 20 mM Na N -2-hydroxyethylpiperazine- N' -2 ethanesulfonic acid-buffered medium [NHB; external pH (pH ₀ ). 7.4, 37.C] was 6.89 ± 0.01 ( n = 178). pH; was affected by changes in external pH ₀ , pH _i changing by ~70% of the change in pH ₀ . The intrinsic buffering power ( ß _int ) of these cells, measured either with NH ₄ Cl or Na propionate pulses, is low for muscle cells, averaging ~10 mM/pH unit. Steady-state pH _i of BC3H -1 cells in NHB acidified reversibly on exposure to 0.5 mM 4,4' -diisothiocyanostilbene-2,2' -disulfonic acid (DIDS; 1.4 ± 0.3 x 10 ^-4 pH/s), 1 mM amiloride (2.0 ± 0.7 X 10 ^-4 pH/s), or Na-free solution (8.3 ± 2.4 x 10 ^-4 pH/s) and alkalinized upon exposure to Cl-free solutions (9.7 ± 2.2 x 10 ^-4 pH/s). Exposure of BC3H-1 cells to C0 ₂ -HCO ₃ -buffered solutions resulted in a transient acidification followed by an alkalinization of 0.3-0.4 pH unit to a new steady-state pH _i of 7.27 ± 0.01 ( n = 65). This new steady-state pH _i acidified very slowly upon exposure to 1 mM amiloride (0.3 ± 0.1 x 10 ^-4 pH/s), acidified more rapidly upon exposure to 0.5 mM DIDS (5.9 ± 0.6 x 10 ^-4 pH/s) or Na-free solutions (9.8 ± 1.0 x 10 ^-4 pH/s), and alkalinized on exposure to Cl-free solutions (24.5 ± 1.3 x 10 ^-4 pH _i s). The alkalinization seen upon the removal of Cl was completely inhibited by 0.5 mM DIDS but only 25% inhibited by Na-free solutions. These data suggest that the regulation of pH _i in smooth muscle cells is mediated by several membrane transport systems. Under physiological conditions, it is the HC0 ₃ -dependent transport systems that will be predominantly responsible for the maintenance of steady-state pH _i .