Department of Chemistry, Ben-Gurion University, Beer-Sheva 84105, Israel
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ABSTRACT |
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The intent of this work was to evaluate the role of cAMP in regulation of ciliary activity in frog mucociliary epithelium and to examine the possibility of cross talk between the cAMP- and Ca2+-dependent pathways in that regulation. Forskolin and dibutyryl cAMP induced strong transient intracellular Ca2+ concentration ([Ca2+]i) elevation and strong ciliary beat frequency enhancement with prolonged stabilization at an elevated plateau. The response was not affected by reduction of extracellular Ca2+ concentration. The elevation in [Ca2+]i was canceled by pretreatment with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-AM, thapsigargin, and a phospholipase C inhibitor, U-73122. Under those experimental conditions, forskolin raised the beat frequency to a moderately elevated plateau, whereas the initial strong rise in frequency was completely abolished. All effects were canceled by H-89, a selective protein kinase A (PKA) inhibitor. The results suggest a dual role for PKA in ciliary regulation. PKA releases Ca2+ from intracellular stores, strongly activating ciliary beating, and, concurrently, produces moderate prolonged enhancement of the beat frequency by a Ca2+-independent mechanism.
adenosine 3',5'-cyclic monophosphate; cilia; intracellular calcium pools; adenylyl cyclase; phospholipase C; protein kinase A
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INTRODUCTION |
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THE PRIMARY FUNCTION OF cilia is to transport a single cell through water or to propel a mucous layer over the cell surface. To perform this task efficiently, ciliary cells dramatically change their activity in response to various stimuli. It has been shown that intracellular Ca2+ concentration ([Ca2+]i) plays a crucial role in the regulation of ciliary activity. In single cell organisms, increase in [Ca2+]i reverses ciliary beat direction (2). In mucociliary tissue of higher organisms, [Ca2+]i is essential for ciliary beating (42), with a rise in [Ca2+]i correlating with a rise in ciliary beat frequency (CBF) (2, 9, 18, 24, 27, 33, 36, 41, 44).
It is widely accepted that cAMP is also an important modulator of
ciliary beating in a variety of ciliary systems. -Adrenergic agonists known to elevate cAMP levels stimulate CBF of respiratory cilia (43). cAMP-dependent modulation of CBF has been reported in human
and rabbit ciliary cells (10, 40, 45). The cAMP-dependent protein
kinase (PKA) has been shown to phosphorylate specific axonemal targets
that increase the forward swimming speed (determined by CBF) in
Paramecium (20, 21). Similar axonemal targets for PKA phosphorylation
have been identified in mammalian respiratory cilia (34).
Cross talk between Ca2+ and cAMP pathways has been reported in many cells. Several types of adenylyl cyclases and cAMP phosphodiesterases (the enzymes responsible for cAMP synthesis and degradation, respectively) have been shown to be modulated by either Ca2+ or Ca2+-dependent phosphorylation (3, 30). On the other hand, an increase in cAMP has been reported to enhance (6, 32) or inhibit (39) inositol 1,4,5-trisphosphate (IP3)-induced or Ca2+-induced Ca2+ release from intracellular stores. Phosphorylation by PKA may be responsible for these effects. cAMP itself has been reported to induce Ca2+ release from the internal stores by a PKA-independent mechanism (7). In addition, both PKA-dependent and -independent modulation of Ca2+ flux across the plasma membrane by cAMP have been reported (5, 31).
Although Ca2+ and cAMP are well known and widely investigated modulators of ciliary activity, little is known about cross talk between their pathways in ciliary cells. It was suggested that there is dual control of ciliary activity, with Ca2+ and cAMP acting through independent pathways that interact or, possibly, converge at an axonemal or a subaxonemal level (26, 36, 37).
The ciliary cells from frog esophagus and palate can be stimulated by at least two agents that act through different specific receptors, namely, ACh, through a muscarinic receptor (1), and extracellular ATP (29), through a purinergic receptor (17). Extracellular ATP has been shown to produce a variety of effects in frog ciliary cells through mechanisms involving an intricate combination of Ca2+ release from the internal stores and Ca2+ influx from the extracellular medium (27). In addition to CBF enhancement, extracellular ATP induces membrane hyperpolarization (41) and fluidization (2), creating dramatic changes in several parameters of the metachronal wave and ciliary beating (16). There is substantial evidence suggesting the important role of [Ca2+]i in the generation of these effects.
The goal of this work was to evaluate the significance of the cAMP pathway in the regulation of ciliary activity in frog mucociliary epithelium and to assess the possibility of cross talk between cAMP and Ca2+ pathways in this system.
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MATERIALS AND METHODS |
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Tissue preparation. Experiments were carried out on monolayer tissue cultures grown from frog esophagus of locally supplied frogs (Rana ridibunda) using the procedure described previously (12).
Chemicals and solutions. Forskolin was purchased from Research Biochemicals International (Natick, MA). H-89 was from Biomol (Plymouth Meeting, PA). Fura 2-AM was from either Molecular Probes (Eugene, OR) or Teflabs (Austin, TX). K5fura 2 and Pluronic F-127 were from Molecular Probes. FCS, L-15 Leibovitz medium, and antibiotics were from Biological Industries (Bet-Haemek, Israel). All other chemicals were obtained from Sigma Chemical (St. Louis, MO). Ringer solution contained (in mM) 120 NaCl, 2.5 KCl, 1.8 CaCl2, 1.1 Na2HPO4, and 0.85 NaH2PO4. Ringer solution for Ca2+ measurement experiments contained (in mM) 120 NaCl, 2.5 KCl, 1.8 CaCl2, 5 HEPES, and 0.5 probenecid. Solutions with low concentrations of Ca2+ were prepared by adding to Ca2+-free Ringer solution 0.5 mM EGTA, 1.8 mM Mg2+, and Ca2+ in a concentration calculated by a computer program (23) to reach the desired free Ca2+ concentration. 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 K5fura 2. Forskolin and thapsigargin were dissolved in DMSO as concentrated stock solutions and diluted into the Ringer solution just before use. The final concentration of DMSO did not exceed 0.1%. All the solutions were adjusted to pH 7.2-7.4 before use.
Measurement of CBF. CBF was determined by simultaneous measurement of scattered light from two points on the monolayer ciliary epithelium as described in detail previously (13, 15).
Simultaneous measurement of intracellular Ca2+ and ciliary beating. Simultaneous measurement of intracellular Ca2+ and ciliary beating was carried out as previously described (25). 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). The 340/380 nm fluorescence ratio, averaged over a period of 1 s, was stored in a computer (586 IBM compatible). CBF was measured by transilluminating the same ciliary area with light at 600 nm (so as not to interfere with the 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 (19). The Ca2+ concentration was calculated directly from the calibration curve by interpolation using a table look-up algorithm.Procedure. Before any treatment, the 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 the 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% Pluronic F-127, and 500 µM probenecid, for 60 min at 37°C in a rotating water bath, followed by washing in the Ringer solution for 30 min. Pluronic 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 (fo) and [Ca2+]i level were measured for 2-5 min in 900-950 µl of the appropriate solution. These measurements were taken as reference values. Then 50-100 µl of solution containing the test substance were added to reach the desired final concentration. 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 (fo), that is, f/fo = frequency enhancement. Intracellular Ca2+ elevation was represented by the difference between the observed Ca2+ level and the reference level ( ![]() |
RESULTS |
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Effect of cAMP-elevating agents on [Ca2+]i and CBF. To determine the effect of cAMP elevation on CBF and intracellular Ca2+ levels, we treated the cells with forskolin, an activator of adenylyl cyclase.
Forskolin did not produce any effect at 0.1 µM. It showed apparently unvarying potency to stimulate ciliary beating in the concentration range of 1-50 µM. The average responses to 25 µM forskolin at the maximal increases of [Ca2+]i and CBF were
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Source of
Ca2+ for the
[Ca2+]i
elevation.
The elevation in
[Ca2+]i
produced by either forskolin or DBcAMP could be a result of
Ca2+ release from intracellular
stores or, alternatively, a result of
Ca2+ influx from the extracellular
medium. To discriminate between those two possibilities, either
forskolin or DBcAMP was added to cells bathed in
low-Ca2+ medium
(106 M external
Ca2+). The addition of 25 µM
forskolin produced similar responses to those obtained in the normal
medium (Fig. 3). The maximal elevations in
[Ca2+]i
and CBF were
[Ca2+]i = 208 ± 80 nM (n = 26) and
f/fo = 2.3 ± 0.3 (n = 35) with
prolonged stabilization of CBF at
f/fo = 1.7 ± 0.2 (n = 35). The response to DBcAMP also was essentially unaffected by reducing the extracellular Ca2+ concentration, with
[Ca2+]i = 266 ± 127 nM (n = 8),
f/fo = 2.4 ± 0.4 (n = 8) and prolonged stabilization of CBF at
f/fo = 1.6 ± 0.3 (n = 8) (data not
shown). These results suggest that
Ca2+ influx does not play a
significant role in the effects of either forskolin or DBcAMP.
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Dependence of CBF enhancement on Ca2+ elevation. To evaluate the dependence of CBF enhancement on [Ca2+]i elevation, we used two different methods: 1) loading the cells with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), a Ca2+ chelator that binds free cytosolic Ca2+, thus preventing the elevation of its concentration; and 2) depletion of the intracellular Ca2+ stores by thapsigargin, an endoplasmic Ca2+ pump inhibitor.
Loading the cells with 5 µM BAPTA-AM canceled the rise in [Ca2+]i in response to forskolin. However, the CBF enhancement induced by forskolin was not abolished, although the magnitude and the pattern of the CBF elevation were altered dramatically (Fig. 4).
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Role of PKA in [Ca2+]i elevation and CBF enhancement. To determine whether the effects produced by forskolin and DBcAMP are due to PKA activity or are induced by cAMP through a PKA-independent mechanism, the cells were treated for 3-15 min before the addition of the stimulant with H-89, a selective PKA inhibitor [IC50 = 48 nM for PKA, IC50 = 480 nM for protein kinase G (PKG), IC50 > 28 µM for other kinases (obtained from the supplier)]. Forskolin (25 µM) did not induce detectable elevation in [Ca2+]i or CBF in six of eight experiments in cells treated with 5 µM H-89 (Fig. 1, A and B). Similarly, 200 µM DBcAMP did not elevate [Ca2+]i or CBF in five of six experiments in cells treated with 0.5 µM H-89 (Fig. 2).
These results suggest that Ca2+ release from intracellular stores and CBF enhancement produced by forskolin or DBcAMP are due to PKA activity.Characterization of the PKA-releasable stores. Several additional experiments were carried out to assess a mechanism of Ca2+ release induced by PKA and to characterize the PKA-releasable stores.
To evaluate the possibility that PKA utilizes the IP3 pathway to mobilize Ca2+ from intracellular stores, we applied a phospholipase C (PLC) inhibitor U-73122 (2 µM) 3-6 min before the addition of forskolin. As can be seen in Fig. 5, the PLC inhibitor abolished the rise in [Ca2+]i, normally induced by forskolin. The frequency behavior was essentially identical to the response obtained when BAPTA and thapsigargin were used to prevent Ca2+ elevation (described above). The CBF enhancement induced by forskolin after pretreatment with 2 µM U-73122 was f/fo = 1.7 ± 0.1 (n = 9).
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DISCUSSION |
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It has been widely accepted that cAMP is an important stimulator of ciliary beating in a variety of ciliary systems. The mechanism underlying this cAMP-dependent ciliary stimulation and its interplay with other ciliary activity modulators are the subject of intensive investigation.
In the present work, we have shown that the CBF can be strongly stimulated through a cAMP-dependent pathway in frog mucociliary epithelium. The adenylyl cyclase activator forskolin and the membrane-permeable cAMP analog DBcAMP produce significant [Ca2+]i elevation and strong prolonged CBF enhancement. These results clearly indicate the existence of cross talk between the cAMP and Ca2+ pathways.
We have shown that a PKA inhibitor, H-89 (IC50 = 48 nM), at a concentration of 0.5 µM, abolished [Ca2+]i elevation and CBF enhancement produced by 200 µM DBcAMP (Fig. 2). At this concentration, H-89 shows high selectivity for PKA. A higher concentration (5 µM) was required to completely inhibit the effect of forskolin (Fig. 1, A and B). At a concentration of 5 µM, H-89 is less specific and is known to inhibit another kinase, PKG (IC50 = 480 nM). The difference in the concentration of H-89 required for full inhibition of the effects produced by forskolin and DBcAMP may be attributed to the fact that forskolin induces powerful and persistent activation of adenylyl cyclase, thus leading to accumulation of large amounts of cAMP that may far exceed levels achieved by addition of DBcAMP. It should also be noted that the actual cytosolic concentrations of the applied materials are unknown and could be less, to various extents (depending on the material), than their concentrations in the extracellular medium. From our experience, the frog esophagus cells in an intact culture are highly resistant to penetration by foreign materials (27). Because the responses produced by forskolin and DBcAMP were otherwise essentially identical and forskolin is not known to activate guanylyl cyclase, it is reasonable to conclude that the effects produced by both stimulants are due to PKA activation.
The elevation in [Ca2+]i induced by PKA gives rise to the fast and strong CBF enhancement. However, three independent sets of experiments [depletion of intracellular stores with thapsigargin, loading of the cells with BAPTA (Fig. 4), and inhibition of PLC (Fig. 5)] clearly demonstrate that the elevated plateau in CBF generated by forskolin can be produced without any detectable elevation in Ca2+. These results suggest that PKA can also stimulate CBF through a Ca2+-independent mechanism, although this stimulation is significantly weaker and slower than the Ca2+-induced enhancement.
These results suggest two distinct modes by which PKA can effect the process of ciliary regulation. One mode is through a Ca2+-independent mechanism, producing moderate but prolonged CBF enhancement. This mechanism likely involves direct phophorylation of axonemal proteins (20, 21, 34). The other mode is through induction of Ca2+ release from the intracellular stores, producing a relatively short but powerful stimulation of CBF.
The mechanism of Ca2+ release induced by PKA deserves separate discussion. PKA has been shown to potentiate Ca2+ release induced by other agents. Thus PKA-induced phosphorylation caused a fourfold shift to the left in the concentration dependence of IP3-induced Ca2+ release in permeabilized hepatocytes (6) so that much lower concentrations of IP3 were needed to induce Ca2+ release. However, in contrast to our results, PKA activation alone, without addition of IP3, could not induce the release of Ca2+. We have shown that application of U-73122, a PLC inhibitor, caused significant reduction of the basal [Ca2+]i, subsequently preventing the PKA-induced Ca2+ release (Fig. 5). Therefore, it is tempting to suggest that PLC is slightly activated in the nonstimulated frog ciliary cells, leading to slightly elevated concentrations of IP3 and [Ca2+]i under basal conditions. The IP3 level is too low to induce massive Ca2+ release under normal circumstances but is high enough to initiate Ca2+ release after the affinity to IP3 is shifted by PKA. The reduction in [Ca2+]i induced by U-73122 can be interpreted as a result of inhibition of the basal activity of PLC and the consequent reduction in the basal concentration of IP3 to levels at which the PKA activation alone is unable to induce Ca2+ release. We cannot rule out, however, the possibility that PKA induces activation of PLC, but such a possibility seems to be unlikely. To the best of our knowledge, such an action of PKA has not yet been reported.
Therefore, we have shown here that activation of PKA can induce massive release of Ca2+ from the internal stores, producing rapid and strong changes in cellular function. So far it was assumed as almost self-evident that receptors initiating Ca2+ release from intracellular stores in electrically nonexcitable cells were coupled to PLC. Our results strongly suggest that an alternative mechanism should be considered: a Ca2+-mobilizing receptor coupled to adenylyl cyclase, producing Ca2+ release through PKA activation.
The intracellular Ca2+ oscillations and waves have been widely reported and extensively described. The rationalizations suggested for these effects (4, 8, 14, 22) can be used to explain the oscillations observed in our experiments in response to PKA activation.
The PKA activation shifts the affinity of the channel to IP3, thus initiating Ca2+ release. The primary increase in [Ca2+]i accelerates further release by binding of Ca2+ to an activation site on the IP3 channel. However, at high [Ca2+]i, the opening probability of the channel decreases due to binding of Ca2+ to a second site. The cytosolic Ca2+ concentration is then lowered because of Ca2+ pump activity, thus permitting another cycle of Ca2+ release. Note that such a process can take place only as long as the IP3 level remains within a certain range. If it is too low, the initial release will not occur, but, if it is too high, it will overcome the restraining effect of high [Ca2+]i. Because the released Ca2+ is constantly extruded from the cell by Ca2+ pumps, it is reasonable to suggest that the oscillations eventually cease due to depletion of the stores. The [Ca2+]i elevations obtained in response to forskolin and DBcAMP did not show a constant pattern. The type of response is probably determined by an interplay of several factors, like the condition of the stores, the efficiency of pumping, the level of the IP3 channel activation, and so forth.
We have shown that PKA releases most of the Ca2+ from the intracellular thapsigargin-sensitive stores. However, PKA is incapable of mobilizing all the Ca2+ releasable by thapsigargin. The remaining Ca2+ can be discharged by an application of extracellular ATP. The magnitude of the Ca2+ rise induced by ATP after prolonged treatment with forskolin in the low-Ca2+ medium was somewhat higher than the magnitude of the Ca2+ rise induced by thapsigargin in similar conditions. It can be explained by the fact that the kinetics of the Ca2+ release induced by ATP is more rapid. It is also possible that ATP induces Ca2+ release from additional, thapsigargin-insensitive stores (24).
The inability of the elevation in [Ca2+]i produced by either thapsigargin or ATP in the forskolin-treated cells to increase CBF is an interesting phenomenon. It seems that prolonged cAMP elevation not only enhances ciliary beating but also reduces the sensitivity of the cilia to [Ca2+]i. A similar effect has previously been described. It was shown that prolonged perfusion of human ciliary epithelium cells with DBcAMP abolished the enhancement in CBF normally induced by a Ca2+ ionophore (38). The mechanism of this effect is unclear. However, because the effect has already been observed in two different types of mucociliary tissue, namely frogs and humans, it seems to be more than a curiosity and definitely requires intensive investigation.
In conclusion, we have shown that, in ciliary cells from the frog esophagus, PKA can activate ciliary beating by two mechanisms. First, PKA can induce a rapid and massive release of Ca2+ from the intracellular stores, producing strong CBF enhancement. This is the first unequivocal indication that cross talk between Ca2+ and cAMP pathways exists in the mucociliary systems at early stages of the cellular response, namely, at the level of second messenger production. Second, PKA can induce moderate enhancement of beat frequency with a prolonged stabilization at an elevated plateau through a Ca2+-independent mechanism. The ciliary beating, directly enhanced by PKA, becomes virtually insensitive to changes in [Ca2+]i. The involvement of both mechanisms in the physiological stimulation of ciliary activity requires further investigation.
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ACKNOWLEDGEMENTS |
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This work was supported by a grant from the Israeli Science Foundation. A. Braiman gratefully acknowledges the support of the Kreitman School of Advanced Graduate Studies.
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FOOTNOTES |
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Address for reprint requests: Z. Priel, Dept. of Chemistry, Ben-Gurion University, PO Box 653, Beer-Sheva 84105, Israel.
Received 27 October 1997; accepted in final form 8 June 1998.
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