Asymmetric interactions between phosphorylation pathways regulating ciliary beat frequency in human nasal respiratory epithelium in vitro.

Abstract

1. The effects of the sequential stimulation of ciliary beat frequency (CBF) via two different phosphorylation cascades (dependent on protein kinase A (PKA) and calmodulin, respectively) were determined using video microscopy applied to a perfused preparation of human nasal respiratory epithelium in vitro. Dibutyryl cyclic AMP (db-cAMP) (10-3 M) was used to stimulate PKA and the calcium ionophore 4-Br-A23187 (10-5 M) was used to stimulate calmodulin-dependent phosphorylation.2. Perfusion with db-cAMP (10-3 M) alone showed an early rise in CBF (15-0 + 4%, mean + S.E.M., P < 0 05) by 10 min which remained elevated for 35 min; in contrast, the highest CBF response to 4-Br-A23187 (10-5 M) alone was not achieved until 35 min (16-1 +1P8%, P< 005).3. When a db-cAMP stimulus was applied to cells which had been pre-incubated with 4-Br-A23187 for 30 min, a further rise in CBF (maximal at 20 min, 14 3 + 2%, P< 0 05) was observed. Reversing the sequence of perfusions, cells pre-incubated with db-cAMP showed no further rise in response to stimulation with 4-Br-A23187. 4. We hypothesized that PKA inhibited the response to the 4-Br-A23187. This notion was supported by the restoration of the CBF response (22-8 + 4%, P< 0 05) to 4-Br-A23187 when the cells were pre-incubated with the protein kinase inhibitor 1-(5-isoquinolinylsulphonyl)-2-methylpiperazine (10-3 M), before the sequential perfusions with db-cAMP and 4-Br-A23187. We conclude that the A23187-dependent pathway, which regulates intrinsic CBF, is inhibited by db-cAMP but not vice versa.

Effects of terbutaline in combination with mannitol on mucociliary clearance

“…In general, the effects of terbutaline in combination with mannitol on MCC in the present study are in agreement with those observed in in vitro studies of the effects of cAMP and terbutaline combined with a calcium agonist on the CBF in vitro [26,27]. In the present study, the only different finding was that the inhibition observed with terbutaline inhaled before mannitol was transient, lasting only for the duration of the intervention (y20 min).…”

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

“…al., 2002; Morse et al, 2001;Smith et al, 1996;Tamaoki et al, 1989b;Wyatt et al, 2005;Wyatt et al, 1998). Adrenergic drugs, known to increase CBF (Sanderson and Dirksen, 1989;Wyatt et al, 1998;Yang et al, 1996), also increase intracellular cAMP levels.…”

Nitric Oxide Dependent Ethanol Stimulation of Ciliary Motility Is Linked to cAMP‐Dependent Protein Kinase (PKA) Activation in Bovine Bronchial Epithelium

“…Because PKA activation has been tightly linked to stimulation of ciliary motility in many airway cell types (Braiman et al, 1998;Smith et al, 1996;Wyatt et al, 1998a), we next determined if any linkage was present between ethanol activation of PKA activity and ethanol stimulation of CBF. Similar to PKA activation, ethanol, at a concentration threshold of 20 mM, stimulated CBF with maximal stimulation observed at 100 mM (Fig.…”