Calcium and the regulation of mammalian ciliary beating

This report summarizes our recent work on the role of intracellular Ca2+ ([ Ca2+](i)) in regulating mammalian ciliary beat frequency (CBF). CBF from a single ovine cilium and [Ca2+](i) from the same cell were measured by digit al video phase contrast microscopy and fura-2 ratiometric imaging video mic roscopy, respectively. Cells were stimulated with two exposures to 10 mu M acetylcholine (ACh). CBF was recorded during the first and [Ca2+](i) during the second stimulation. ACh increased [Ca2+](i) and CBF transiently with i ndistinguishable kinetics and, early in culture, even induced [Ca2+](i) osc illations and ciliary frequency modulations with the same peak-to-peak time interval. Cells treated with 1 mu M thapsigargin, an inhibitor of the endo plasmic-reticulum Ca2+-ATPase, showed transient [Ca2+](i) and CBF increases , again with similar kinetics, which often remained at an elevated plateau. Application of ACh to cells pretreated with thapsigargin produced decrease s in both [Ca2+](i) and CBF. Finally, changing extracellular Ca2+-concentra tions induced corresponding changes in [Ca2+](i) that were associated with kinetically similar CBF changes. These data strongly suggested that [Ca2+]( i) is a critical signal to regulate CBF in mammalian tracheal epithelial ce lls. In an initial effort to provide constraints on the number and type of reactions that link changes in [Ca2+](i) to changes in CBF, simultaneous re cordings of both signals from a single cell were analyzed. Such recordings provided higher resolution of the kinetic responses of CBF and [Ca2+](i) to ACh as well as they allowed direct assessment of the coupling between [Ca2 +](i) and CBF. Simultaneous measurements revealed that [Ca2+](i) and CBF we re perfectly correlated within the CBF measurement time resolution, except for the period of the fastest changes in both signals during the initial AC h exposure. There, changes in CBF lagged the changes in [Ca2+](i) by 1-3 ci liary beat cycles (ca. 150-450 ms).