The phosphorylation profile of ciliary proteins under basal conditions and after stimulation by extracellular ATP was investigated in intact tissue and in isolated cilia from porcine airway epithelium using anti-phosphoserine and anti-phosphothreonine specific antibodies. In intact tissue, several polypeptides were serine phosphorylated in the absence of any treatment (control conditions). After stimulation by extracellular ATP, changes in the phosphorylation pattern were detected on seven ciliary polypeptides. Serine phosphorylation was enhanced for three polypeptides (27, 37, and 44 kD), while serine phosphorylation was reduced for four polypeptides (35, 69, 100, and 130 kD). Raising intracellular Ca2+ with ionomycin induced identical changes in the protein phosphorylation profile. Inhibition of the NO pathway by inhibiting either NO syntase (NOS), guanylyl cyclase (GC), or cGMP-dependent protein kinase (PKG) abolished the changes in phosphorylation induced by ATP. The presence of PKG within the axoneme was demonstrated using a specific antibody. In addition, in isolated permeabilized cilia, submicromolar concentrations of cGMP induced protein phosphorylation. Taken together, these results suggest that the axoneme is an integral part of the intracellular NO pathway. The surprising observation that ciliary activation is accompanied by sustained dephosphorylation of ciliary proteins via NO pathway was not detected in isolated cilia, suggesting that the protein phosphatases were either lost or deactivated during the isolation procedure. This work reveals that any pharmacological manipulation that abolished phosphorylation and dephosphorylation also abolished the enhancement of ciliary beating. Thus, part or all of the phosphorylated polypeptides are likely directly involved in axonemal regulation of ciliary beating.

Using confocal microscopy we demonstrate that ciliary cells from airway epithelium maintain two qualitatively distinct cytosolic regions in terms of pH regulation. While the bulk of the cytosol is stringently buffered and is virtually insensitive to changes in extracellular pH (pHo), the values of cytosolic pH in the vicinity of the ciliary membrane is largely determined by pHo. Variation of pHo from 6.2 up to 8.5 failed to affect ciliary beat frequency (CBF). Application of NH(4)Cl induced profound localized alkalization near cilia, which did not depress ciliary activity, but resulted in strong and prolonged enhancement of CBF. Calmodulin and protein kinase A (PKA) functionality was essential for the alkalization-induced CBF enhancement. We suggest that the ability of airway epithelium to sustain unusually strong but localized cytosolic alkalization near cilia facilitates CBF enhancement through altering the binding constants of Ca2+ to calmodulin and promotion of Ca2+-calmodulin complex formation. The NH4Cl-induced elevations in cytosolic pH and Ca2+ concentration act synergistically to activate calmodulin-dependent processes, cAMP pathway, and, thereby, stimulate CBF.