Mode of Ca²⁺ action on ciliary beat frequency in single ovine airway epithelial cells

Abstract

We analysed the kinetics of coupling between cytoplasmic calcium ([Ca2+]i) and ciliary beat frequency (CBF) using simultaneous single cilium recording and single cell [Ca2+]i measurements from cultured ovine tracheal epithelial cells. CBF and [Ca2+]i (indicated by fura‐2) were measured at rest and in response to activation of the G‐protein coupled M3 muscarinic receptor by 10 μM acetylcholine (ACh). Fourier transform analysis of 3 s data segments of light intensity from phase‐contrast microscopy showed no significant delay between changes in [Ca2+]i and CBF during a 2 min exposure to ACh and subsequent washout. CBF time resolution was improved by computing instantaneous beat frequency. This revealed that CBF lagged the rapid increase in [Ca2+]i in response to ACh with a delay of less than 1 beat cycle (143 ms at 7 Hz). When CBF was estimated by an improved Fourier method, this delay was observed to be 70 ± 30 ms (mean ± s.e.m.; n= 20 cilia). During the slower return to baseline, a lag of 8 ± 3.2 s was observed, indicative of hysteresis. While calmodulin inhibitors (calmidazolium and W‐7; each n= 5) decreased baseline CBF by an average of 1.1 ± 0.1 Hz, they did not alter the kinetic relationship between [Ca2+]i and CBF. Similarly, phosphatase inhibitors (okadaic acid and cyclosporin A; each n= 5), changed neither baseline CBF nor the kinetic coupling between [Ca2+]i and CBF. These data suggest that the timing of Ca2+ action on CBF in ovine airway epithelial cells, is unlikely to be determined by phosphorylation reactions involving calmodulin or kinase/phosphatase reactions. A simple model for Ca2+ stimulation of CBF is presented. Fits of the model to the data suggest four or more Ca2+ ions bind cooperatively to speed up CBF.

Nervous control of ciliary beating by Cl-, Ca2+ and calmodulin inTritonia diomedea

“…Could there be different pathways controlling coordination and CBF increase? We also provide evidence that IP 3 may not be a significant player in CBF excitation, but that does not rule out a PLC dependent pathway using PKC, as found in Helisoma embryos (Christopher et al, 1999;Doran et al, 2004), nor the adenylate cyclase/cAMP/ PKA pathway found to be necessary in Mytilus (Stommel and Stephens, 1985;Aiello, 1990) (preliminary findings suggest that cAMP plays no role in CPE cells; O. M. Woodwood, unpublished observation), nor other non-calmodulin dependent Ca 2+ actions (Salathe and Bookman, 1999). Electrically induced excitation of CPE cells in Tritonia diomedea is consistent with their role as primary locomotory effectors, and like muscle, their RyR-gated stores and voltage-gated Ca 2+ channels may be an adaptation permitting parallel CNS control.…”

“…Calmodulin has been implicated in many of the described CBF control pathways (Stommel and Stephens, 1985;Zagoory et al, 2001;Zagoory et al, 2002). Some researchers attribute W-7 and calmidazolium reduction of CBF excitation to nonspecific chelating effects, instead believing that Ca 2+ directly binds to ciliary beating mechanism (Salathe and Bookman, 1999). Others support the idea that Ca 2+ -calmodulin dependent kinases and phosphodiesterases influence dynein behavior (Zagoory et al, 2002).…”

“…There is no consensus as to the relationship between second messenger Ca 2+ and the increases in dynein motor activity and subsequent global increases in CBF. It was proposed that Ca 2+ might act directly on the axoneme in epithelial cells (Salathe and Bookman, 1999); however, numerous other intermediaries remain possibilities. Calmodulin is one option.…”

“…Ciliary beating may be under hormonal control (Verdugo, 1980;Korngreen and Priel, 1996;Korngreen et al, 1998;Lieb et al, 2002;Barrera et al, 2004) or nervous control via dopamine and neuropeptides (Willows et al, 1997;Woodward and Willows, 2006), serotonin (Goldberg et al, 1994;Christopher et al, 1996;Willows et al, 1997;Nguyen et al, 2001; Doran et al, 2004), acetylcholine (ACh) (Salathe and Bookman, 1999;Zagoory et al, 2001;Zagoory et al, 2002) or depolarization (Aiello and Guideri, 1964;Mackie et al, 1969;Mackie et al, 1976;Murakami and Takahashi, 1975). Work permitting any distinction between these control mechanisms is sparse and this may be due, in part, to technical challenges presented by vertebrate models.…”

“…Studies of CBF have yielded different possible mechanisms for increasing internal free Ca 2+ , including the phospholipase C (PLC) pathway (Christopher et al, 1999;Zagoory et al, 2001), both protein kinase C (PKC) (Gertsberg et al, 1997;Christopher et al, 1999;Barrera et al, 2004) and protein kinase A (PKA) (Braiman et al, 1998;Zagoory et al, 2002;Lieb et al, 2002), inositol 1,4,5-triphosphate (Barrera et al, 2004), nitrous oxide (Uzlander and Priel, 1999;Runer and Lindberg, 1999;Doran et al, 2003) and cAMP (Stommel and Stephens, 1985;Aiello, 1990). The next step in signal transduction after an increase in internal calcium may be direct binding of Ca 2+ to the dynein motor (Salathe and Bookman, 1999) or Ca 2+ -calmodulin activation of kinases or phosphodiesterases (Zagoory et al, 2001), or possibly both. Unfortunately, little is known of possible electrical control of these pathways, though most early results were consistent with such a hypothesis (Aiello and Guideri, 1964;Mackie et al, 1969;Mackie et al, 1976;Murakami and Takahashi, 1975;Saimi et al, 1983a;Saimi et al, 1983b).…”