Regulation of human airway ciliary beat frequency by intracellular pH

pH_i affects a number of cellular functions, but the influence of pH_i on mammalian ciliary beat frequency (CBF) is not known. CBF and pH_i of single human tracheobronchial epithelial cells in submerged culture were measured simultaneously using video microscopy (for CBF) and epifluorescence microscopy with the pH-sensitive dye BCECF. Baseline CBF and pH_i values in bicarbonate-free medium were 7.2 ± 0.2 Hz and 7.49 ± 0.02, respectively (n = 63). Alkalization by ammonium pre-pulse to pH_i 7.78 ± 0.02 resulted in a 2.2 ± 0.1 Hz CBF increase (P < 0.05). Following removal of NH₄Cl, pH_i decreased to 7.24 ± 0.02 and CBF to 5.8 ± 0.1 Hz (P < 0.05). Removal of extracellular CO₂ to change pH_i resulted in similar CBF changes. Pre-activation of cAMP-dependent protein kinase (10 μM forskolin), broad inhibition of protein kinases (100 μM H-7), inhibition of PKA (10 μM H-89), nor inhibition of phosphatases (10 μM cyclosporin + 1.5 μM okadaic acid) changed pH_i-mediated changes in CBF, nor were they due to [Ca²⁺]_i changes. CBF of basolaterally permeabilized human trachcobronchial cells, re-differentiated at the air-liquid interface, was 3.9 ± 0.3, 5.7 ± 0.4, 7.0 ± 0.3 and 7.3 ± 0.3 Hz at basolateral i.e., intracellular pH of 6.8, 7.2, 7.6 and 8.0, respectively (n = 18). Thus, intracellular alkalization stimulates, while intracellular acidification attenuates human airway CBF. Since phosphorylation and [Ca²⁺]_i changes did not seem to mediate pH_i-induced CBF changes, pH_i may directly act on the ciliary motile machinery.