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Received May 14, 2004
Revised June 25, 2004
Accepted after revision August 6, 2004
1 University of Miami School of Medicine
* To whom correspondence should be addressed. E-mail: msalathe{at}miami.edu.
pHi affects a number of cellular functions, but the influence of pHi on mammalian ciliary beat frequency (CBF) is not known. CBF and pHi of single human tracheobronchial epithelial cells in submerged culture were measured simultaneously using videomicroscopy (for CBF) and epifluorescence microscopy with the pH-sensitive dye BCECF. Baseline CBF and pHi in bicarbonate free medium were 7.2 ± 0.2 Hz and 7.49 ± 0.02, respectively (n = 63). Alkalisation by ammonium prepulse to pHi 7.78 ± 0.02 resulted in a 2.2 ± 0.1 Hz CBF increase (p < 0.05). Following removal of NH4Cl, pHi decreased to 7.24 ± 0.02 and CBF to 5.8 ± 0.1 Hz (p < 0.05). Removal of extracellular CO2 to change pHi resulted in similar CBF changes. Neither 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 cyclosporine + 1.5 µM okadaic acid) changed the coupling of pHi and CBF. pHi-induced CBF changes were not due to [Ca2+]i changes. CBF of basolaterally permeabilized human tracheobronchial cells re-differentiated at the air-liquid interface was 3.9 ± 0.3 Hz, 5.7 ± 0.4 Hz, 7.0 ± 0.3 Hz, and 7.3 ± 0.3 Hz at basolateral or intracellular pH of 6.8, 7.2, 7.6, and 8.0, respectively (n = 18). Thus, intracellular alkalisation stimulates, while intracellular acidification attenuates human airway CBF. Since phosphorylation and [Ca2+]i changes did not seem to mediate pHi-induced CBF changes, pHi may directly act on the ciliary motile machinery.
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