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.

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 Ca 2+ 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 Ca 2+ ions, and the response of intracellular Ca 2+ to SP was monitored.SP ( 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 Ca 2+ into cells. The small magnitude of the rise in CBF makes its physiological role uncertain at present.