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Am J Physiol Cell Physiol 280: C100-C109, 2001;
0363-6143/01 $5.00
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Vol. 280, Issue 1, C100-C109, January 2001

Role of calcium and calmodulin in ciliary stimulation induced by acetylcholine

Orna Zagoory, Alex Braiman, Larisa Gheber, and Zvi Priel

Department of Chemistry, Ben-Gurion University of the Negev, Beer-Sheva 84105, Israel


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The goal of this work was to elucidate the molecular events underlying stimulation of ciliary beat frequency (CBF) induced by acetylcholine (ACh) in frog esophagus epithelium. ACh induces a profound increase in CBF and in intracellular Ca2+ concentration ([Ca2+]i) through M1 and M3 muscarinic receptors. The [Ca2+]i slowly decays to the basal level, while CBF stabilizes at an elevated level. These results suggest that ACh triggers Ca2+-correlated and -uncorrelated modes of ciliary stimulation. ACh response is abolished by the phospholipase C (PLC) inhibitor U-73122 and by depletion of intracellular Ca2+ stores but is unaffected by reduction of extracellular Ca2+ concentration and by blockers of Ca2+ influx. Therefore, ACh activates PLC and mobilizes Ca2+ solely from intracellular stores. The calmodulin inhibitors W-7 and calmidazolium attenuate the ACh-induced increase in [Ca2+]i but completely abolish the elevation in CBF. Therefore, elevation of [Ca2+]i is necessary for CBF enhancement but does not lead directly to it. The combined effect of Ca2+ elevation and of additional factors, presumably mobilized by Ca2+-calmodulin, results in a robust CBF enhancement.

cilia; mucociliary tissue; thapsigargin; cholinergic receptors; atropine


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

CILIA ARE SMALL ORGANELLES protruding from the cell surface that beat in a cooperative pseudoperiodic, spatial, and temporal pattern called the metachronal wave. They exist in a variety of organisms ranging from unicellular organisms and mollusks to epithelial cells in digestive, reproductive, and respiratory systems of vertebrates. Highly cooperative beating of cilia at high frequencies enables the mucociliary system to carry relatively large objects at remarkable velocities. Furthermore, a high frequency of ciliary beating results in increased energy expenditure. Therefore, under "normal" conditions, cilia either beat with low frequency or may even be at rest. However, they can dramatically change their activity in response to a variety of receptor-mediated stimuli. For example, ciliary cells from frog palate or esophagus possess purinergic P1 and P2 (13, 33), cholinergic (1, 12, 30), and adrenergic (21) receptors. Activation of these receptors triggers a strong and prolonged enhancement of ciliary beat frequency (CBF), which is an essential feature for effective mucociliary transport.

Acetylcholine (ACh) is known as a stimulator of ciliary cells from mammalian and nonmammalian organisms (29). ACh has been shown to accelerate the rate of particle transport (30) and to stimulate the CBF (1, 12) in frog palate and esophagus. Similar observations were recorded in other systems, i.e., mammalian trachea (20, 27) and human respiratory epithelium (29, 34). In these systems, the cholinergic agonists stimulate ciliary beating via the muscarinic receptors. ACh stimulation is of special interest in the cilia of frog palate and esophagus because it has been shown that cholinergic nervous stimulation of the ciliary activity takes place in these systems (5, 30). However, the molecular mechanism of muscarinic stimulation in mucociliary epithelia has not as yet been elucidated.

The five subclasses of muscarinic receptors, M1-M5, trigger a variety of intracellular signaling pathways. Generally, activation of the M2 and M4 receptors leads to an inhibition of adenylate cyclase and regulation of membrane channels. M1, M3, and M5 subtypes have been linked to multiple signaling events, including stimulation of phospholipases (PLA2, PLC, and PLD), cAMP accumulation, elevation of intracellular Ca2+ level, and induction of cation influx. The most common activity is PLC stimulation, which results in an increase in intracellular Ca2+ concentration ([Ca2+]i) (4, 10).

Calcium ions are ubiquitous second messengers that take part in regulation of virtually all cellular processes. They exert their function by binding and/or regulating the activity of a wide range of cellular proteins, such as calmodulin (CaM), protein kinases and phosphatases, adenylate cyclase, phosphodiesterases, cytoskeleton elements, membrane channels, ATPases, and others (25). In ciliary cells, calcium ions play a key role in regulating ciliary activity. The direction of swimming in Paramecium is regulated according to the cytosolic Ca2+ level (9). A rise in [Ca2+]i in mucociliary tissue results in CBF enhancement (17, 32). Moreover, the coupling between [Ca2+]i and CBF has been reported during cholinergic stimulation in sheep tracheal epithelium (26, 28). However, the mechanism by which Ca2+ regulates ciliary stimulation remains unclear. Moreover, the available data on the involvement of CaM in this regulation is contradictory. For example, application of a CaM inhibitor blocks the increase in CBF normally produced by ionomycin in human respiratory epithelium (6). On the other hand, application of a CaM inhibitor is ineffective in blocking the CBF rise induced by ACh in cultured ovine trachea. (27).

It is widely accepted that changes in intracellular levels of either Ca2+ or cAMP or cGMP lead to CBF enhancement. Indeed, it has been shown that, in tissue cultures from frog esophagus, the increase in cAMP concentration [achieved by forskolin or dibutyryl cAMP (DBcAMP)] induces CBF enhancement, even when a rise in [Ca2+]i is deterred (3). Similar results have been obtained in tissue cultures from rabbit trachea with the use of DBcAMP (31) and from human nasal polyps (11) with 8-bromo-cGMP. On the other hand, a clear decoupling between a rise of [Ca2+]i and CBF enhancement was recently revealed in tissue cultures from rabbit trachea (31). It was shown that inhibition of protein kinase G (PKG) almost completely abolished CBF enhancement in the presence of strongly elevated [Ca2+]i . These findings indicate that, at least in rabbit ciliary epithelia, a rise of [Ca2+]i alone is not sufficient to induce CBF enhancement. In addition to the rise in [Ca2+]i, PKG, which presumably phosphorylates axonemal protein(s), is essential for achieving an appreciable stimulation of CBF (31). Because these findings were based on a single tissue type and one stimulant, their generality is yet to be established.

In the present study, we examined the molecular events underlying ciliary activity stimulation in frog esophagus by extracellular ACh. The goal was twofold. First, we intended to examine the role of a rise in [Ca2+]i in CBF stimulation and to reveal a possible cross talk between [Ca2+]i and CaM-dependent enzymatic pathways. Second, we intended to examine the generality of our findings in rabbit trachea (31) that elevation of [Ca2+]i alone is necessary but not sufficient for enhancement of CBF.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Tissue culture preparation for frequency measurement. Experiments were performed on monolayer tissue culture grown from frog esophagus and palate of locally supplied frogs (Rana ridibunda) according to the procedure described previously (7). Briefly, the esophagus or palate was removed from frogs and washed three times in sterile medium. The epithelia were minced in culture medium (15% fetal calf serum, 64% L-15 Leibovitz medium, and 20% sterile distilled water, supplemented with 20 U/ml penicillin, 2.5 U/ml nystatin, and 20 µg/ml streptomycin). Two to four tissue pieces were placed on plastic petri dishes (35 mm; Nunc) and overlaid with 0.7 ml of culture medium. The culture medium was changed every 2 days, and 5- to 21-day-old tissue cultures were used for measurements. According to Chu and Kennedy (5), the muscarinic receptors on the membrane of ciliated cells apparently are not lost during culture and can be maintained throughout the 3-wk culture period.

Chemicals and solutions. The frequency measurement experiments were performed in Ringer solution containing (in mM) 120 NaCl, 2.3 KCl, 1.8 CaCl2, 1.8 MgCl2, 0.85 Na2HPO4, and 0.85 NaH2PO4, pH 7.2. Ringer solution for Ca2+ measurement experiments contained 120 mM NaCl, 2.5 mM KCl, 1.8 mM CaCl2, 1.8 MgCl2, 5 mM HEPES, and 0.5 mM probenecid.

Solutions with low Ca2+ concentration were obtained by adding 0.5 mM EGTA, 1.8 Mg2+, and an excess of Ca2+ to achieve the required concentration. The needed total Ca2+, Mg2+, and EGTA concentrations were calculated according to known equilibrium constants. The external calibration solution for fura 2 was composed of 115 mM KCl, 20 mM NaCl, 5 mM MgCl2, 5 mM D-glucose, 5 mM HEPES, 10 mM EGTA, and 1 µM K5-fura 2.

U-73122, U-73343, and ionomycin were dissolved in ethanol as concentrated stock solutions and diluted into Ringer solution just before use. The final concentration of ethanol in the assay solution did not exceed 0.5%. Thapsigargin was dissolved in DMSO as a concentrated stock solution. The final concentration of DMSO in the assay solution did not exceed 0.05%.

Atropine, verapamil, pirenzepine HCl, HEPES, DMSO, 12-O-tetradecanoylphorbol 13-acetate (TPA), EGTA, thapsigargin, and W-7 were obtained from Sigma. Acetylcholine chloride, KCl, NaCl, NiCl2, CaCl2, and La(NO3)3 were obtained from Merck (Darmstadt, Germany). Pertussis toxin was obtained from Biomol; cholera toxin and ionomycin from CalBiochem; and N,N-dimethyl-4-piperidinyl diphenylacetate (4-DAMP) methiodide and methoctramine HCl from RBI (Natick, MA). Fura 2-AM was from either Molecular Probes (Eugene, OR) or Teflabs (Austin, TX). K5-fura 2 and Pluoronic F-127 were from Molecular Probes. All tissue culture media and supplements were supplied by Biological Industries (Bet-Haemek, Israel).

All reagents and drugs were dissolved in Ringer solution. All solutions were adjusted to pH 7.2-7.4 before use.

CBF measurements. CBF measurements were performed by using the dual-photoelectric method, as described previously (8). Briefly, the method is based on measurement of scattered light from a small ciliary area. The light is collected by an optical fiber placed in the focal plan of the ocular. The CBF is determined by the fast Fourier transform of the photoelectric signals.

Simultaneous measurement of intracellular Ca2+ and ciliary beating. Simultaneous measurement of intracellular Ca2+ and ciliary beating was carried out as previously described (17). Briefly, [Ca2+]i was measured with the fluorescent indicator fura 2. The dye-loaded cells were epi-illuminated with light from a 75-W xenon lamp (Oriel, Stamford, CT) filtered through 340- and 380-nm interference filters (Oriel) mounted on a four-position rotating filter wheel. The fluorescence, emitted at 510 nm, was detected by a photon-counting photomultiplier (H3460-53, Hamamatsu, Hamamatsu, Japan). The 340/380 fluorescence ratio, averaged over a period of 1 s, was stored in a computer (Pentium). CBF was measured by transilluminating the same ciliary area with light at 600 nm (so as not to interfere with fura 2 fluorescence at 510 nm). Scattering of the 600-nm light from the beating cilia created amplitude modulations that were detected by a photomultiplier (R2014, Hamamatsu).

A calibration curve of the Ca2+ concentration was created by titrating an external calibration solution with a solution of the same composition containing 10 mM CaCl2 (14, 17). To account for the difference between the fura 2 fluorescence signal in the intracellular medium and in the calibration solution, the maximal and minimal values of the 340/380 fluorescence ratio were measured from the cells. The maximal value was obtained by addition of 5 µM ionomycin to the cells, which resulted in flooding of the cells with Ca2+. The minimal value was obtained by addition of ionomycin to the cells in a 0-Ca2+ medium. The calibration curve was corrected according to the obtained results. The nonspecific signal was estimated by addition of ionomycin to the cells in the presence of 1 mM Mn2+, which leads to quenching of fura 2 fluorescence (14). The Ca2+ concentration was calculated directly from the corrected calibration curve by interpolation using a table look-up algorithm.

Procedure. Before any treatment commenced, Ringer solution over the tissue culture was changed twice. The tissue was then preincubated in a third change of the Ringer solution for 15-30 min before the experiment to prevent any transient effects on ciliary motility.

In experiments in which [Ca2+]i was measured, the cells were preloaded with fura 2 by incubating the tissue in serum-free growth medium containing 5 µM fura 2-AM, 0.03% Pluoronic F-127, and 500 µM probenecid for 60 min at 37°C in a rotating water bath, followed by washing in Ringer solution for 30 min. Pluoronic F-127 is a nonpolar polymeric detergent that increases solubility of hydrophobic fura 2-AM in aqueous media. Probenecid, an inhibitor of membrane organic anion transporters, is used widely to prevent extrusion of loaded fura 2 from the cells.

The basal CBF (F0) and [Ca2+]i levels were measured for 2-5 min in 900-950 µl of the appropriate solution. These measurements were taken as reference values. Next, 50-100 µl of solution containing the test substance were added to reach the desired final concentration. Alternatively, the solution was rapidly changed to the one containing the test substance with the use of a constant flow perfusion system. Our previous experience indicates that the pattern and magnitude of the response in either CBF or [Ca2+]i do not depend on the manner of substance addition (16). The frequency (F) and [Ca2+]i were monitored on the same ciliary cell for 10-40 min. Beat frequency enhancement was represented as the observed frequency normalized to the reference frequency, that is, F/F0 = frequency enhancement. The intracellular Ca2+ elevation was represented by the difference between the observed Ca2+ level and the reference level (Delta [Ca2+]i). The results are presented as means ± SE with n equal to the number of experiments. Every experiment was performed with 5-97 tissue cultures taken from at least 2 animals. Each tissue culture was used only once.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

ACh enhances CBF via muscarinic receptors. The effect of ACh (10 µM) on CBF is demonstrated in Fig. 1A. After ACh was added, CBF increased 2.74 ± 0.07-fold within ~10 s, from a basal value of 10.80 ± 0.88 Hz to 29.59 ± 0.85 Hz (n = 97 tissues from 30 frogs). The frequency then declined slowly over >10 min to a moderately elevated level, remaining stable for more than an additional 10 min. In 15% of the tested tissue cultures, the response to 10 µM ACh lasted only a few seconds. These abnormal lines of tissue preparation were not further used or explored.


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  		Fig. 1.   The effect of acetylcholine (ACh) on ciliary beat frequency (CBF). A: typical time course of CBF stimulation by extracellular ACh. Each point represents the average over 5 consequent seconds. The basal CBF (F0) was measured for 3 min before the addition of 0.1 ml ACh to 0.9 ml of solution, to give a final concentration of 10 µM ACh. The frequency was measured for an additional 10 min. The normalized effect is expressed as the obtained frequency (F) divided by F0 (F/F0). B: dose-response curve of CBF stimulation by extracellular ACh. Each point represents the mean frequency of the maximal enhancement (Fmax/Fo) that is achieved in a given ACh concentration (, results obtained by bolus addition of ACh; , results obtained using perfusion; see MATERIALS AND METHODS). Data represent means ± SE of 5-30 experiments.

The magnitude of CBF enhancement and its duration depends only moderately on the concentration of ACh (Fig. 1B). A strong and sustained enhancement of CBF is already achieved at 10 nM of ACh (Fmax/F0 = 1.87 ± 0.21, n = 12 tissues from 4 frogs), and a further increase of ACh concentration to 0.1 mM (4 orders of magnitude) induced a relatively similar response (Fmax/F0 = 2.64 ± 0.41, n = 17 tissues from 9 frogs). To exclude a possible error that might result from bolus injection of ACh to the bath, several experiments at three different concentrations of ACh (8-10 experiments at each concentration) were performed by perfusing the tissue with a solution containing a final concentration of ACh (Fig. 1B). As clearly shown, no significant difference in the results produced by the two different methods was obtained. A similar dose-response pattern was obtained by Aiello et al. (1), yet the magnitude of the response that we obtained (Fig. 1B) at each concentration was considerably higher. Because of the weak dependence of the effect on ACh concentration, most of the experiments in the present study were performed in the range of 0.5-10 µM ACh. The ability of ACh to induce considerable enhancement of CBF at a 1 nM concentration indicates a very low ACh esterase activity in our preparations (1, 24).

Characterization of the receptor subtypes in the cultured frog esophagus was performed with the use of antagonists that are specific for particular subtypes of muscarinic receptors (4). Atropine, a potent antagonist of all muscarinic receptor subtypes, completely antagonized ACh stimulation with an IC50 of 18 nM (Fig. 2). Similar to atropine, 4-DAMP, which is selective for M3 receptors but has low affinity for M1 and M5 as well, completely antagonized the ACh response, but at a higher concentration (IC50 = 0.1 µM). Pirenzepine, which has the highest affinity for the M1 subtype, had only a partial inhibitory effect. Even when used at high concentrations, 10 or 100 µM, only one-half of the response was blocked. Thus, with regard to its maximal inhibition, the IC50 of pirenzepine was 0.1 µM. Metoctramine, which has the highest affinity for M2 and partial selectivity for M4, affected the ACh response only when applied at a very high concentration (100 µM). At this concentration its activity is no longer specific. On the basis of these results, the relative inhibitory effect for muscarinic antagonists is of the following potency rank: atropine > 4-DAMP > pirenzepine > methoctramine. This potency profile indicates that M3 and M1 muscarinic receptor subtypes mediate the ACh effect in cultured frog esophagus. Interestingly, muscarinic receptors with a similar subtype classification were found in ciliated tissue cultures from sheep trachea (28).


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  		Fig. 2.   Inhibition of ACh enhancement of CBF by muscarinic antagonists: normalized CBF enhancement induced by 10 µM ACh after treatment with muscarinic receptor antagonists. The following antagonists were used: atropine (open circle ), pirenzepine (M1 ACh receptor; ), N,N-dimethyl-4-piperidinyl diphenylacetate (4-DAMP) (M3 ACh receptor; black-triangle), and methoctramine (M2 ACh receptor; ). The tissues were incubated for 15-40 min with each antagonist before ACh was added. Data represent the frequency (means ± SE) of the maximal enhancement achieved at a given muscarinic inhibitor concentration. Those measurements were performed during different seasons. The muscarinic antagonists showed the following potency rank: atropine > 4-DAMP > pirenzepine > methoctramine.

Muscarinic receptors belong to the seven transmembrane receptors, which transduce their signals by coupling to G-binding proteins. The common toxins pertusis toxin (PTX) and cholera toxin (CTX) are known to uncouple the receptor from its effector. Treatment of the cells with 1 µg/ml PTX or 1 µg/ml CTX for 3-4 h did not affect stimulation of the ciliary beating induced by ACh (Table 1). Even prolonged preincubation with PTX for 24 h failed to inhibit ACh-induced stimulation (Table 1). Therefore, we suggest that M1 and M3 muscarinic receptors mediate the signal in frog esophagus via PTX- and CTX-insensitive G-binding proteins.

                              
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  		Table 1.   Maximal normalized CBF enhancements induced by 10 µM ACh

Ca2+ mobilization from internal stores is a necessary condition for initiation of CBF enhancement by ACh. Muscarinic receptors of the M1, M3, and M5 subtypes have been found to be functionally linked to PLC activation (4). Activated PLC generates inositol 1,4,5-trisphosphate (IP3), which releases Ca2+ from internal stores. To evaluate the role of intracellular Ca2+ in the ciliary stimulation produced by ACh, simultaneous measurements of intracellular Ca2+ concentrations and CBF from the same cell were performed (17). Figure 3, A and B, demonstrates the typical response to 10 µM ACh obtained in these simultaneous measurements. Addition of ACh produced rapid elevation of [Ca2+]i, followed by slow decline of [Ca2+]i to its basal level. On the average, the maximal increase in [Ca2+]i in response to 10 µM ACh was 349 ± 18 nM (n = 41 tissues from 9 frogs). To test the possibility that the rise in [Ca2+]i is mediated by PLC, the cells were pretreated with U-73122 (a potent blocker of PLC), and then ACh was applied. U-73122 (8 µM) abolished completely the rise of both [Ca2+]i and CBF induced by 10 µM ACh (Fig. 3, A and B). The nonactive structural analog of this PLC inhibitor, U-73433, did not affect the response to ACh (Table 1). These results confirm previous findings for different organs or animals that muscarinic subtype receptors of the kind found in our system mobilize [Ca2+]i via the PLC pathway.


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  		Fig. 3.   Effect of ACh on intracellular Ca2+ concentration ([Ca2+]i) and CBF. Experiments represent the [Ca2+]i (A and C) and CBF (B and D) responses to 10 µM ACh. [Ca2+]i and CBF were measured simultaneously from 1 cell. A and B: response obtained in the normal medium; C and D: response obtained in medium containing 0.5 µM Ca2+. The dotted line (A) and open circles (B) represent the response obtained from another cell treated with U-73122 (8 µM).

The products of PLC activity are IP3 and diacylglycerol. The latter is an endogenous second messenger that activates protein kinase C (PKC). A recent study in our laboratory demonstrated that PKC played an important role in the purinergic stimulation of ciliary activity in frog esophagus (19). Therefore, the possible involvement of PKC was examined. The tissue cultures were treated with 150 nM TPA for 20 h, to downregulate PKC isozymes, or with the PKC inhibitor chelerythrin (28 µM) for 90-120 min. Surprisingly, none of these treatments affected CBF enhancement induced by ACh (Table 1). Although PKC takes an important part in the ciliary stimulation produced by a different PLC activator (19), the cellular events induced by ACh seem to be independent of PKC activity.

Because ACh may activate Ca2+ channels through intracellular second messengers or G proteins (4) and, thereby, induce Ca2+ influx, we tested several potent blockers of Ca2+ channels at relatively high concentrations. Verapamil, Ni2+, La3+, and Cd2+ were tested for their ability to inhibit ACh-induced stimulation of CBF. Ni2+ and La3+ were also tested for their ability to inhibit ACh-induced elevation in [Ca2+]i. All the Ca2+ blockers failed to inhibit the stimulatory effect of ACh on CBF and [Ca2+]i (Table 1 and Figs. 4 and 5). Moreover, we found that ACh-induced rises in CBF and in [Ca2+]i did not depend on free extracellular Ca2+ concentrations in the range from 0.1 µM to 1.8 mM. Figure 3, C and D, and Fig. 4 show that, in the medium containing 0.5 µM Ca2+, the responses in both [Ca2+]i and CBF produced by 10 µM ACh are similar to those obtained in the regular medium. However, pretreatment of the cells with 50 nM thapsigargin or 2 µM ionomycin in the low-Ca2+ medium, which brought about depletion of the intracellular Ca2+ stores, completely abolished ACh (10 µM)-induced enhancement of CBF (Table 1).


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  		Fig. 4.   Time course of the response in [Ca2+]i and CBF to ACh under various conditions. A: difference between maximal and basal Ca2+ levels (Delta [Ca2+]i); B: normalized CBF (F/Fo) at 3 time points after stimulation by 10 µM ACh. These time points were the time of maximal CBF enhancement (maximum) and 5 and 10 min after the maximum. Each group of bars represents responses to 10 µM ACh obtained under the following conditions: control (open bars), in the presence of 5 mM Ni2+ (solid bars), in the presence of 0.1 mM La3+ (hatched bars), and in the medium containing 0.5 µM Ca2+ (crosshatched bars). [Ca2+]i and CBF were measured simultaneously from the same cell. Note that, at 10 min after maximum, [Ca2+]i returns to its basal level, while CBF remains substantially elevated. Data represent means ± SE of 10-41 experiments.



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  		Fig. 5.   Effect of ACh on [Ca2+]i and CBF in the presence of 5 mM Ni2+. Results are representative of [Ca2+]i (A) and CBF (B) responses to 10 µM ACh in the presence of 5 mM Ni2+. [Ca2+]i and CBF were measured simultaneously from 1 cell. Note that the results of [Ca2+]i measurements obtained in the presence of 5 mM Ni2+ do not differ from the results obtained in its absence.

It is worth mentioning that Ni2+ is an effective quencher of fura 2 fluorescence. Thus, in the presence of 5 mM Ni2+ in the external solution, any signal from the dye, which may have leaked from the cell, should be eliminated. The fact that the results of [Ca2+]i measurements obtained in the presence of 5 mM Ni2+ do not differ from the results obtained in its absence (Fig. 5) indicates that in our preparations there is no significant contribution of the "leaked" fura 2 to the signal.

Together, these results suggest that [Ca2+]i elevation and CBF enhancement triggered by ACh are independent of extracellular Ca2+ and that Ca2+ is mobilized solely from internal stores. Moreover, a rise in [Ca2+]i is a necessary condition for initiation of the CBF response.

The simultaneous measurements of [Ca2+]i and CBF from the same cell demonstrate that the strong correlation between [Ca2+]i and CBF, observed during the initial phase of ciliary stimulation, is gradually being lost in the course of the response (Figs. 3-6). While [Ca2+]i decays to its initial level, CBF is stabilized at a high excited state, which is maintained for a long time after [Ca2+]i has reached its basal value. To evaluate the dependence of this CBF plateau on the continuous receptor stimulation, we added 5 µM atropine during the sustained phase of the response (Fig. 6). Addition of atropine resulted in a rapid decay of CBF to its basal value, while [Ca2+]i was not affected. Together, these results demonstrate that the sustained excited state of CBF induced by ACh is under tight receptor control. Furthermore, while elevation in [Ca2+]i is apparently required for the creation of the CBF plateau, high [Ca2+]i is not needed for its sustenance.


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  		Fig. 6.   Dependence of the sustained CBF plateau on receptor stimulation. Results are representative of [Ca2+]i (A) and CBF (B) behavior induced by 10 µM ACh followed by addition of 5 µM atropine at the sustained phase of the response. [Ca2+]i and CBF were measured simultaneously from 1 cell.

Inhibition of CaM decouples the rise of [Ca2+]i and the enhancement of CBF induced by ACh. Our results indicate that a rise in [Ca2+]i is a necessary condition for initiation of CBF enhancement by ACh (see Ca2+ mobilization from internal stores is a necessary condition for initiation of CBF enhancement by ACh). To assess whether Ca2+ acts directly on the ciliary axoneme or through the Ca2+-CaM pathway, the CaM inhibitors trifluoperazine (TFP), W-7, and calmidazolium were tested. TFP appeared to be toxic to our preparation. It produced a rapid, profound elevation of [Ca2+]i, followed by degradation of the tissue culture. TFP was previously tried and found toxic for ciliary cells in cultured ovine trachea as well (27). Therefore, it was not used for further investigation. The addition of both W-7 (50 µM) and calmidazolium (2 µM) induced a rise in [Ca2+]i (Fig. 7, A and C). For W-7, this rise was transient, followed by stabilization of [Ca2+]i on a lower basal level. For calmidazolium, the elevation in [Ca2+]i was stronger and was not transient. It is well known that CaM may be involved in the delicately integrated dynamic system of [Ca2+]i homeostasis (15). Therefore, the rise in [Ca2+]i produced CaM inhibitors (Fig. 7, A and B) may be an outcome of this involvement. Although the underlying mechanism of this phenomenon is beyond the scope of this work, this Ca2+ rise was used as an internal control for W-7 and calmidazolium potency.


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  		Fig. 7.   Effect of calmodulin inhibition on the [Ca2+]i and CBF rise induced by ACh. Results are time courses of [Ca2+]i (A and C) and CBF (B and D) during sequential addition of the calmodulin inhibitors W-7 (A and B) or calmidazolium (C and D) and 10 µM ACh. While the rise in [Ca2+]i after ACh was added in the cells pretreated with calmodulin inhibitors is evident, the rise in CBF is completely inhibited.

The initial rise in [Ca2+]i, caused by W-7 and calmidazolium, induced transient rise in CBF, which returned to the basal level in both cases (Fig. 7, B and D). In calmidazolium-treated cells, CBF decayed to its basal value despite the high levels of [Ca2+]i. To test the effect of ACh after the CaM inhibition, we added 10 µM ACh after CBF decayed to its initial value. Our results clearly show that, after CaM inhibition, ACh failed to induce any CBF enhancement, despite the evident rise in [Ca2+]i [in 9 of 11 experiments (3 animals) for W-7 and in 5 of 5 experiments (2 animals) for calmidazolium]. Because ACh was applied to cells with high [Ca2+]i after the preincubation with calmidazolium, the results obtained using this inhibitor may be somewhat ambiguous. Nevertheless, results with both inhibitors demonstrate the same pattern: shortly after application of CaM inhibitors, CBF declines to its basal value and becomes insensitive to elevated [Ca2+]i. These data indicate that activation of the Ca2+-CaM complex (Ca-CaM) is necessary for stimulation of CBF induced by high [Ca2+]i in cholinergic stimulation of CBF.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

ACh induces a wide range of intracellular responses through a number of signaling pathways in various tissues. Our results indicate that, in cultured frog esophagus, ACh activates M1 and M3 muscarinic receptors and produces a strong and sustained CBF enhancement (Figs. 1 and 2). The sensitivity to ACh is quite high, given that a pronounced effect is already achieved at nanomolar concentrations. As expected by the activation of M1 and M3 receptors, this effect is mediated through the PLC pathway. Inhibition of PLC completely abolished the rise in [Ca2+]i and CBF enhancement (Figs. 3, A and B). Moreover, depletion of intracellular Ca2+ stores by thapsigargin or ionomycin abolished CBF enhancement induced by ACh.

Response of [Ca2+]i to ACh. The time course of [Ca2+]i response induced by ACh deserves a separate discussion. After ACh was applied, [Ca2+]i rapidly rose to its highest level, followed by a slow decay (typically taking place between 8-10 min) to its basal values (Fig. 3A). As shown, the rise of [Ca2+]i induced by ACh was via the PLC pathway (Fig. 3A) and was virtually independent of extracellular Ca2+ concentration (Figs. 3C and 4A). The described response of [Ca2+]i is conspicuously different from the time course of [Ca2+]i induced by extracellular ATP for the same type of cells, although both agonists are considered to activate the PLC-dependent pathway. Activation of a P2 purinergic receptor by ATP produces a rapid and transient [Ca2+]i elevation that ceases within 2-4 min (19). Simultaneously, endogenous second messengers activate non-voltage Ca2+ channels producing Ca2+ influx from the extracellular space. As a result, a sustained elevated plateau of [Ca2+]i is formed (19). A similar pattern of [Ca2+]i behavior induced by extracellular ATP was observed in ciliary tissue cultures from rabbit trachea (16, 31). Moreover, this biphasic pattern is not limited to ciliary tissues but is viewed as a classic response of electrically nonexcitable cells to Ca2+ mobilizing agents (23).

The elevated Ca2+ plateau induced by purinergic stimulation of frog ciliary tissue is a result of PKC activation. Activated PKC induces Ca2+ influx from the extracellular fluid (19). Surprisingly, the current work demonstrates that ACh, in contrast to ATP, does not activate PKC (Table 1), which could explain the absence of Ca2+ influx and the absence of an elevated Ca2+ plateau. Such a marked difference between the two PLC pathways, one induced by ATP and the other by ACh, in the same cells is surprising and may derive from the different location of the ATP and ACh receptors. Indeed, it was recently shown that the ciliary membrane is polarized, and even the same receptor (purinoceptor) induces different [Ca2+]i responses when it is activated on either the basal or lateral membrane (22).

The slow decay of [Ca2+]i from its maximum to its basal value (Fig. 3, A and C) might be explained by the involvement of protein kinase A (PKA) in the Ca2+ release process. It has been recently shown that activated PKA induces Ca2+ release from intracellular stores in cultured frog esophagus, even at the basal level of PLC activity (3), presumably by shifting the affinity of the Ca2+ channel to IP3. Therefore, it is tempting to suggest that ACh induces PKA activation prolonging the Ca2+ release in a PKA-dependent manner.

ACh enhances CBF in a Ca2+-correlated and -uncorrelated manner. The time course of CBF enhancement induced by ACh exhibits two distinct phases. The first phase consists of a strong rise followed by a partial decay of the CBF, both of which correlate well with the rise and the partial decay of [Ca2+]i. In the second phase, the CBF is stabilized at an activated state (between 50 and 100% above the basal value), while [Ca2+]i decreases to its original basal value (Figs. 3-6). The initial [Ca2+]i elevation is essential for development of the second phase, but it is not needed for the maintenance of the CBF plateau. Yet, the continuation of the second phase is crucially dependent on continuous receptor stimulation (Fig. 6).

It is important to emphasize that the sustained stimulated state of CBF continues for quite a long time (~20 min) after [Ca2+]i decays to the basal value. A response having a similar pattern and magnitude was observed previously in the same tissue after stimulation by DBcAMP, a cell-permeable analog of cAMP, or forskolin, which induced a powerful and persistent activation of adenylate cyclase, leading to accumulation of large amounts of cAMP (3). While the first stage of CBF enhancement was shown to be Ca2+ dependent, the second stage was Ca2+ independent and was driven by PKA activity. Given all these facts, it is tempting to suggest that ACh, probably via Ca-CaM, activates the cyclic nucleotides pathway. This activation prolongs [Ca2+]i elevation without need of the Ca2+ influx and, after the eventual decay of [Ca2+]i to its basal level, ensures sustained activation of CBF through a Ca2+-independent mechanism. However, further work is needed to verify this hypothesis and to assess the role of PKA in ACh-induced ciliary stimulation.

As demonstrated in cultured frog esophagus, ACh evokes prolonged activation of CBF with the correlation between CBF and [Ca2+]i being gradually lost in the course of the response. In contrast to these results, a different behavior was observed in cultured ovine trachea (26, 28). Despite the similarity in the signaling pathway (receptor subtypes, PLC activation, and dependence on intracellular Ca2+ stores), the response in Ca2+ and CBF decayed within 2 min, and a strict correlation between [Ca2+]i and CBF was preserved. Considering this discrepancy, it is important to emphasize that this strict correlation between [Ca2+]i and CBF is maintained during the first minute of the response in frog esophagus tissue cultures as well. However, the second phase of the response, characterized in frog esophagus by loss of the correlation, was not manifested in ovine trachea tissue. Despite the similar initiation of the response, the enzymatic cascade responsible for the second phase of the ciliary stimulation by ACh was presumably not activated in cultured ovine trachea. A possible reason for this phenomenon could be the thermal conditions of the experiments, which were relatively low for the ovine tissue. Alternatively, the different responses observed in frog esophagus and ovine trachea can be explained by certain functional differences between those two types of tissue cultures.

CaM mediates between a rise in [Ca2+]i and CBF enhancement induced by ACh. The necessity of a [Ca2+]i increment seems to be a general feature of CBF stimulation by exogenous ligands. This was also observed in tissue cultures from sheep trachea stimulated by ACh (28), from frog palate or esophagus (19), and from rabbit trachea (16, 18, 31) stimulated by extracellular ATP. The mechanism by which a rise in [Ca2+]i induces CBF enhancement is still unknown. In principle, Ca2+ can directly interact with axonemal proteins. Alternatively, it can regulate the activities of various enzymes and/or induce configurational changes in endogenous proteins. One of the goals of this work was to differentiate between these two alternatives.

CaM is a ubiquitous Ca2+-binding protein. It has been identified in all animal and plant cells and appears to be an intracellular Ca2+ receptor that participates in a majority of Ca2+-regulated processes. Therefore, a possible involvement of CaM in CBF stimulation by ACh was examined. Indeed, in the presence of the CaM blockers, ACh failed to enhance CBF despite a significant increase in [Ca2+]i, i.e., inhibition of CaM led to decoupling between the rise of [Ca2+]i and CBF stimulation induced by ACh (Fig. 7). It is important to emphasize that an extensive involvement of CaM in the cellular regulation presents a difficult challenge to the investigators, because inhibition of CaM tends to disrupt multiple processes and consequently gives rise to unwanted "side effects." For example, CaM is known to participate in the regulation of Ca2+ homeostasis in the cell (15). Interference with this function is the most probable reason for the [Ca2+]i elevation produced by CaM inhibitors. According to the product catalogs, CaM inhibitors exhibit different efficiency at inhibition of different CaM-dependent enzymes. This may explain the difference in the magnitude of the [Ca2+]i elevation produced by the inhibitors. Nevertheless, despite the side effects, our results strongly indicate the important role of Ca-CaM as a mediator between [Ca2+]i and CBF. Moreover, the development of the decoupling between [Ca2+]i and CBF can be watched in the process of preincubation with the blockers, when CBF declines rapidly to its basal level despite the high [Ca2+]i produced by the blockers (Fig. 7). It is important to mention that the CaM blocker W-7 produced a similar inhibition of CBF without affecting the rise in [Ca2+]i in rabbit airway ciliary epithelium stimulated by ATP (2). Moreover, in the rabbit ciliary epithelium, the side effect of [Ca2+]i elevation induced by the inhibitor itself was not observed (2). In addition, it was shown that inhibition of the nitric oxide/cGMP pathway also led to decoupling between a rise in [Ca2+]i and CBF in rabbit airway epithelium (31), and it has been well established that the synthesis of nitric oxide can be induced by Ca-CaM. These facts conform to our findings regarding the role of Ca-CaM in ciliary stimulation. Demonstration of decoupling between CBF and [Ca2+]i responses in two different species, mammalian (31) and amphibian (present study), suggests a general role of Ca-CaM in ciliary stimulation.

The mechanism by which Ca-CaM stimulates CBF is not yet clear. It can activate, for instance, adenylate cyclase and/or guanylate cyclase, thereby increasing cAMP and/or cGMP levels in the cell. Both these cyclic nucleotides are known to stimulate ciliary activity. Alternatively, Ca-CaM can directly interact with axoneme to induce CBF enhancement. Further work is needed to distinguish between these alternatives.

In summary, the elevation of [Ca2+]i alone, without participation of CaM, is insufficient to induce CBF enhancement. The elevation in [Ca2+]i is necessary for initiation of the strong and prolonged ciliary response to ACh, but high [Ca2+]i is not needed for its sustenance. The combined effect of Ca2+ elevation and of additional factors, presumably activated by Ca-CaM, manifests itself in robust and sustained CBF enhancement. Such a strong CBF enhancement with the accompanied changes in metachronal wave characteristics (12) may explain the amazing ability of cilia to transport heavy objects at a high speed.


    ACKNOWLEDGEMENTS

A. Braiman gratefully acknowledges the fellowship support of the Kreitman Foundation. This work was partially supported by the Israeli Science Foundation founded by the Israeli Academy of Sciences and Humanities.


    FOOTNOTES

Address for reprint requests and other correspondence: Z. Priel, Dept. of Chemistry, Ben-Gurion Univ. of the Negev, PO Box 653, Beer-Sheva 84105, Israel (E-mail: alon{at}bgumail.ac.il).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 28 December 1999; accepted in final form 7 August 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Cell Physiol 280(1):C100-C109
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