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 microM 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+]0, suggesting that changes in [Ca2+]i alone can modulate CBF. In a second independent manipulation, cells were treated with 1 microM thapsigargin, an irreversible inhibitor of the endoplasmic reticulum Ca(2+)-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 microM 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 peak-to-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.(ABSTRACT TRUNCATED AT 400 WORDS)

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