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
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ABSTRACT |
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
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INTRODUCTION |
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.
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MATERIALS AND METHODS |
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 (
[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.
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RESULTS |
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.
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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 ( ), pirenzepine
(M1 ACh receptor; ),
N,N-dimethyl-4-piperidinyl diphenylacetate
(4-DAMP) (M3 ACh receptor; ), 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.
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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.
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).
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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 ( [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.
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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.
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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 |
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.
 |
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