Regulation of human airway ciliary beat frequency by intracellular pH

Abstract

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 NH 4 Cl, pH i decreased to 7.24 ± 0.02 and CBF to 5.8 ± 0.1 Hz (P < 0.05). Removal of extracellular CO 2 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 [Ca 2+ ] i changes. CBF of basolaterally permeabilized human tracheobronchial 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 [Ca 2+ ] i changes did not seem to mediate pH i -induced CBF changes, pH i may directly act on the ciliary motile machinery.

Impact Assessment of Repeated Exposure of Organotypic 3D Bronchial and Nasal Tissue Culture Models to Whole Cigarette Smoke

“…For ciliary beating measurement, ensuring that the tissues are handled and treated in a similar manner is critical, for example if medium change is implemented, it should be applied for all samples. Sutto and colleagues reported that pH affected the mammalian ciliary beating frequency 24 . Thus, when comparing beating frequencies between cells treated with different compunds/mixtures, pH adjustment should be considered to minimize the variability of ciliary beating measurement.…”

Acquired Cilia Dysfunction in Chronic Rhinosinusitis

“…An increase in intracellular pH produces an increase in CBF, whereas a decrease in pH produces a decrease in CBF. 35 However, it is not known whether this effect is caused by modulation of kinase activity, even though an acidic pH has been shown to inhibit PKA function, 46 or the direct activation of ODAs of the axoneme. 47 Temperature has also been shown in many investigations to influence CBF, most likely through protein kinase C modulation., 36,37,48 Lower temperature tends to slow CBF.…”

“…Ciliary activity accelerates in response to a variety of mechanical, [27][28][29] chemical, 30,31 hormonal, [32][33][34] pH, 35 and thermal stimuli (Table 1). 36,37 Extracellular nucleotides (adenosine and uridine) are especially potent regulators of epithelial functions stimulating mucociliary clearance through mucus secretion, increasing CBF, and gating ion channels involved in the maintenance of epithelial surface liquid volume.…”

Real-time analysis of cAMP-mediated regulation of ciliary motility in single primary human airway epithelial cells

“…ALI cultures were placed in a customized chamber allowing independent perfusion of the basolateral and apical compartment, and mounted at room temperature onto the stage of the Nikon Eclipse microscope. If required, airway epithelial cells were permeabilized as described before (Sutto et al, 2004).…”