Coupling of [ Ca 2 + ] i and ciliary beating in cultured tracheal epithelial cells

The molecular mechanisms responsible for the regulation of ciliary beating frequency (CBF) are only partially characterized. To determine whether elevation of intracellular Ca2+ ([Ca2+]i) can cause an increase in CBF, we measured CBF and Ca2+ in single cells. Ovine tracheal epithelial cells, obtained by dissociation with protease, were grown in primary culture for 1 to 28 days in a mucus-free system. CBF of a single cilium was measured by digital video phase-contrast microscopy and on-line Fourier-transform analysis. Changes in [Ca2+]i from single cells were determined with fura-2, using ratio imaging video microscopy. Activation of a muscarinic pathway with 10 μM ACh (acetylcholine) increased [Ca2+]i from 53±9 nM (mean ± s.e.m.) to 146±12 nM or to 264±51% above initial baseline. In the same cells, ACh increased CBF from a baseline of 7±0.5 Hz to 9±0.2 Hz or to 31±2.8% above baseline (n=14). The elevations of both [Ca2+]i and CBF were transient and relaxed back to an elevated plateau (10/14 cells) as long as ACh was present. To elevate [Ca2+]i by mechanisms independent of a G-protein-coupled receptor, we measured [Ca2+]i and CBF of the same cells in extracellular solutions with either 0 Ca2+ (+ 1 mM EGTA) or 10 mM Ca2+. Both signals rose and fell with similar kinetics in response to changing [Ca2+]o, suggesting that changes in [Ca2+]i alone can modulate CBF. In a second independent manipulation, cells were treated with 1 μM thapsigargin, an irreversible inhibitor of the endoplasmic reticulum Ca2+-ATPase. Upon thapsigargin addition, 37 of 42 cells showed a transient [Ca2+]i increase and, as measured in different experiments, 8 of 9 cells showed a transient increase in CBF. Interestingly, application of ACh after cells were treated with thapsigargin produced decreases in both [Ca2+]i and CBF in 8/8 cells. Lastly, after 1-3 days in culture, addition of 10 μM ACh often produced [Ca2+]i oscillations rather than transients in [Ca2+]i. Measurements of CBF in these cells showed frequency modulation of CBF with the same peakto-peak time interval as the Ca2+ oscillation. These results show that: (1) CBF can be measured from a single cilium and monitored on-line to track changes; (2) CBF and [Ca2+]i can be measured in the same single cell; (3) transient changes in [Ca2+]i (induced by 4 different manipulations) are associated with kinetically similar changes in CBF; and (4) [Ca2+]i oscillations are coupled to frequency modulation of ciliary beating. Taken together, these results provide strong evidence that [Ca2+]i is a critical intracellular messenger in the regulation of CBF in mammalian tracheal epithelial cells.