title: 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 +/- 1.8%, P < 0.05). 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-isoquinolinyl-sulphonyl)-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.
authors.affiliation: Department of Child Health (Centre for Research into Human Development), Ninewells Hospital and Medical School, King's Cross Hospital, Dundee, UK.
authors.forename: R P
authors.initials: RP
authors.lastname: Smith
authors.forename: R
authors.initials: R
authors.lastname: Shellard
authors.forename: D P
authors.initials: DP
authors.lastname: Dhillon
authors.forename: J
authors.initials: J
authors.lastname: Winter
authors.forename: A
authors.initials: A
authors.lastname: Mehta
mesh_terms: D000293:Adolescent; D000328:Adult; D003994:Bucladesine / Q000494:pharmacology; D000001:Calcimycin / Q000031:analogs & derivatives / Q000494:pharmacology; D002147:Calmodulin / Q000378:metabolism*; D002648:Child; D002923:Cilia / Q000502:physiology*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D007476:Ionophores / Q000494:pharmacology; D007700:Kinetics; D018715:Microscopy, Video; D008875:Middle Aged; D009297:Nasal Mucosa / Q000187:drug effects / Q000502:physiology*; D010766:Phosphorylation; D015203:Reproducibility of Results; D013997:Time Factors; D014420:Turbinates
chemical_list: D002147:Calmodulin; D007476:Ionophores; D000001:Calcimycin; D003994:Bucladesine; C033170:4-bromo-A-23187; D017868:Cyclic AMP-Dependent Protein Kinases