Abstract
We analysed the kinetics of coupling between cytoplasmic calcium ([Ca
2+
]
i
) and ciliary beat frequency (CBF) using simultaneous single cilium recording and single cell [Ca
2+
]
i
measurements from cultured ovine tracheal epithelial cells.
CBF and [Ca
2+
]
i
(indicated by fura-2) were measured at rest and in response to activation of the G-protein coupled M3 muscarinic receptor by 10 μM acetylcholine (ACh).
Fourier transform analysis of 3 s data segments of light intensity from phase-contrast microscopy showed no significant delay between changes in [Ca
2+
]
i
and CBF during a 2 min exposure to ACh and subsequent washout.
CBF time resolution was improved by computing instantaneous beat frequency. This revealed that CBF lagged the rapid increase in [Ca
2+
]
i
in response to ACh with a delay of less than 1 beat cycle (143 ms at 7 Hz). When CBF was estimated by an improved Fourier method, this delay was observed to be 70 ± 30 ms (mean ±
s.e.m
.;
n
= 20 cilia). During the slower return to baseline, a lag of 8 ± 3.2 s was observed, indicative of hysteresis.
While calmodulin inhibitors (calmidazolium and W-7; each
n
= 5) decreased baseline CBF by an average of 1.1 ± 0.1 Hz, they did not alter the kinetic relationship between [Ca
2+
]
i
and CBF. Similarly, phosphatase inhibitors (okadaic acid and cyclosporin A; each
n
= 5), changed neither baseline CBF nor the kinetic coupling between [Ca
2+
]
i
and CBF.
These data suggest that the timing of Ca
2+
action on CBF in ovine airway epithelial cells, is unlikely to be determined by phosphorylation reactions involving calmodulin or kinase/phosphatase reactions.
A simple model for Ca
2+
stimulation of CBF is presented. Fits of the model to the data suggest four or more Ca
2+
ions bind cooperatively to speed up CBF.
