Calcium and the regulation of mammalian ciliary beating

Summary 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 M 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 M 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).