Asymmetric interactions between phosphorylation pathways regulating ciliary beat frequency in human nasal respiratory epithelium in vitro.
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.
Smith
R P
RP
Department of Child Health (Centre for Research into Human Development), Ninewells Hospital and Medical School, King's Cross Hospital, Dundee, UK.
Shellard
R
R
Dhillon
D P
DP
Winter
J
J
Mehta
A
A
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
D002147:Calmodulin; D007476:Ionophores; D000001:Calcimycin; D003994:Bucladesine; C033170:4-bromo-A-23187; D017868:Cyclic AMP-Dependent Protein Kinases