Calcium and the regulation of mammalian ciliary beating

Abstract

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 digital video phase contrast microscopy and fura-2 ratiometric imaging video microscopy, respectively. Cells were stimulated with two exposures to 10 μM acetylcholine (ACh). CBF was recorded during the first and [Ca2+]i during the second stimulation. ACh increased [Ca2+]i and CBF transiently with indistinguishable kinetics and, early in culture, even induced [Ca2+]i oscillations and ciliary frequency modulations with the same peak-to-peak time interval. Cells treated with 1 μM thapsigargin, an inhibitor of the endoplasmic-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 decreases in both [Ca2+]i and CBF. Finally, changing extracellular Ca2+-concentrations 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 cells. 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 recordings 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 were perfectly correlated within the CBF measurement time resolution, except for the period of the fastest changes in both signals during the initial ACh exposure. There, changes in CBF lagged the changes in [Ca2+]i by 1–3 ciliary beat cycles (ca. 150–450 ms).