R Shellard | PubMed Health

PubMed1996

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.

Adolescent↗

PubMed1996

Interaction between calcium, neutral endopeptidase and the substance P mediated ciliary response in human respiratory epithelium.

Following irritation of the airway, the ciliostimulatory effects of the tachykinin, substance P (SP), are thought to be secondary to mucus release. We hypothesized that SP also induces small increases in ciliary beat frequency (CBF) via a calcium-mediated process. Brushed ciliated cells from the nasal epithelium of healthy human subjects were suspended in tissue culture fluid and the acute effects of SP upon these cells were studied in a mucus-free environment. In some preparations, changes in CBF in response to SP were measured with a video-based system. The effect of an SP antagonist, of Ca2+ channel block with verapamil, and of the calcium analogue lanthanum on the SP response were also tested. In other preparations, the ciliated cells were preloaded with Fura-2, a dye which fluoresces with Ca2+ ions, and the response of intracellular Ca2+ to SP was monitored. SP (10(-9)-10(-6) M) transiently increased CBF in a dose-dependent manner, with the maximal response occurring at 10 min. The response was small, with a maximum increase of 8.9%. The SP receptor antagonist (D-pro2,D-trp7,9)-SP (10(-5) M) abolished this effect. Verapamil (10(-5) M) attenuated the response to SP (10(-7) M), whilst lanthanum chloride (250 microM) abolished it. Inhibition of SP destruction by phosphoramidon (10(-6) M) also eliminated the transient rise in CBF. However, compared to SP alone, the combination of SP and phosphoramidon induced a novel delayed lanthanum-sensitive rise in CBF. In other experiments, SP (10(-7) M) induced a transient increase in free intracellular Ca2+ concentration (maximal rise 73%), which returned to baseline before the expected onset of the CBF response. We conclude that substance P induces either a transient or sustained increase in CBF dependent on the rate of destruction of this peptide around tachykinin receptors. These receptors are likely to be linked to lanthanum- and verapamil-sensitive pathways for the entry of Ca2+ into cells. The small magnitude of the rise in CBF makes its physiological role uncertain at present.

Adolescent↗