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First published online June 29, 2006
Journal of Experimental Biology 209, 2765-2773 (2006)
Published by The Company of Biologists 2006
doi: 10.1242/jeb.02377
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Nervous control of ciliary beating by Cl-, Ca2+ and calmodulin in Tritonia diomedea

Owen M. Woodward1,2,* and A. O. Dennis Willows1,2

1 Department of Biology, University of Washington, Box 351800, Seattle, WA 98195, USA
2 Friday Harbor Laboratories, University of Washington, 620 University Road, Friday Harbor, WA 98250, USA


Figure 1
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  		Fig. 1. Methods for recording ciliary beating frequency (CBF). (A) Pedal ciliated epithelial tissue is removed from the foot margin of Tritonia diomedea and dissected to small pieces with vigorously beating cilia, accessible for experimentation. (B) Phototransducer measurement of a video projection of beating cilia produces an oscillatory voltage output corresponding to the ciliary beating rate. (C) Using a Fast Fourier transform the dominant frequency from the voltage signal can be measured.

 

Figure 2
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  		Fig. 2. Depolarization in high external K+ causes CBF excitation. (A) 30 mmol l-1 K+ bath (filled circles) significantly increases CBF as compared to ASW controls (N=5, P≤0.0003) in CPE cell explants. After 3 min, 30 mmol l-1 K+ is still excitatory (P≤0.02) but less so. After 6 min, 30 mmol l-1 K+ is no longer excitatory. 30 mmol l-1 K+ seawater was not excitatory in a zero Ca2+ bath (open squares, N=6), and only transiently so (P≤0.02) in a bath containing 50 µmol l-1 of the Ca2+ channel blocker nifedipine (triangles, N=5). (B) 30 mmol l-1 K+ significantly excites CBF in isolated, individual CPE cells (N=5, P≤0.007). *Significant change from mean basal levels.

 

Figure 3
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  		Fig. 3. Ca2+ influx is necessary for increases in CBF. (A) Zero Cl- bath (filled circles) excites CBF (N=5, P≤0.001), and the effect does not diminish over time. The excitatory effect is abolished (N=5, P≤0.10 compared to control) when 50 µmol l-1 nifedipine (Nif; open squares) is included in the bath solution. (B) Excitation induced by 100 µmol l-1 dopamine (DA) and 10 µmol l-1 TPep-NLS (TPep) is also dependent on Ca2+ influx. Zero external Ca2+ (N=5, P≤0.008) and 50 µmol l-1 nifedipine (N=5, P≤0.003) both significantly reduce dopamine excitation. Similarly, zero external Ca2+ (N=5, P≤0.00009) and 50 µmol l-1 nifedipine (N=6, P≤0.01) also significantly reduce TPep-NLS excitation. *Significant change from mean basal levels (A) or significant reduction from stimulated levels (B).

 

Figure 4
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  		Fig. 4. Ca2+ release from stores contributes to the excitation of CBF. (A) Caffeine stimulated CBF. Both 1 mmol l-1 (open circles, N=5, P≤0.001) and 10 mmol l-1 (filled squares, N=6, P≤0.0003) caffeine increases CBF, however, the effects of 1 mmol l-1 caffeine decline over time. (B) Caffeine (Caff) excitation is reduced by the ryanodine receptor blocker dantrolene (Dant). 10 mmol l-1 Caffeine (N=6) induced CBF excitation is significantly reduced in 50 µmol l-1 dantrolene (N=7, P≤0.00004). 100 µmol l-1 dopamine and 10 µmol l-1 TPep-NLS excitation is also reduced by 50 µmol l-1 dantrolene (N=6, P≤0.0002; N=6, P≤0.00009, respectively). *Significant change from mean basal levels.

 

Figure 5
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  		Fig. 5. IP3 gated internal stores do not contribute to CBF excitation pathway. (A) CBF excitation caused by 100 µmol l-1 dopamine (DA) in CBF explants either bathed in FSW (open squares, N=5) or SW including 1 µmol l-1 of the IP3 inhibitor xestospongin C (Xesto; filled circles, N=5). (B) Comparisons of percent change in CBF with and without 1 µmol l-1 xestospongin C show it has no effect on dopamine or TPep-NLS (TPep) induced CBF excitation. 100 µmol l-1 Dopamine (N=5, P≤0.27) and 10 µmol l-1 TPep-NLS (N=5, P≤0.06) induced CBF excitation in a bath containing 1 µmol l-1 xestospongin C was indistinguishable from excitation induced by either alone.

 

Figure 6
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  		Fig. 6. Calmodulin dependent processes necessary for CBF excitation. Calmodulin antagonists W-7 (50 µmol l-1) and calmidazolium (Calmid; 5 µmol l-1) both significantly reduced 10 mmol l-1 caffeine (Caff) excitation (W-7: N=5, P≤0.003; calmidazolium: N=4, P≤0.006) and 100 µmol l-1 dopamine (DA) excitation (W-7: N=5, P≤0.007; calmidazolium: N=5, P≤0.001). W-7 also reduced 10 µmol l-1 TPep-NLS (TPep) excitation (N=5, P≤0.0001), however, calmidazolium did not (N=3, P≤0.17). *Significant reduction from stimulated levels.

 

Figure 7
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  		Fig. 7. Model of transmitter and neuropeptide action on CPE cells. Binding of dopamine (DA) or TPep-NLS to a receptor leads to a reduction in ICl(Ca), carried by Ca2+ dependent Cl- channels (CaCC) and I(Cl-)leak, also carried by CaCC channels. We hypothesize that the blockage of Cl- currents leads to a depolarization of the cell and the activation of voltage-gated Ca2+ channels (CavC) leading to an influx of Ca2+. This initial influx triggers the release of further Ca2+ from ryanodine receptor channel (RyR)-gated endoplasmic reticulum (ER) stores. The sharp rise of [Ca2+]in activates the Ca2+-calmodulin complex, which in turn, upregulates kinases and phosphodiesterases critical for increasing ciliary beating rate. Also present are voltage activated proton channels that play a significant role in pH maintenance and may activate after long depolarizations to aid in the repolarization of the membrane potential. Pluses indicate molecules or actions that increase current amplitude or beating rate, minuses represent inhibitory molecules or actions.

 

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