Protein kinase C induced calcium influx and sustained enhancement of ciliary beating by extracellular ATP

Cell Calcium (1997) 21(2), 103-113 0 Pearson Professional Ltd 1997

Research

Protein

kinase

C induced calcium and enhancement of ---_ _- sustained _ _- _ beating by extracellular ATP

influx-----

ciiiary

Rachel Levin, Alex Braiman, Zvi Priel Department

of Chemistry,

Ben-Gurion

University,

Beer-Sheva,

Israel

Summary The major purpose of this work was to determine protein kinase C (PKC) influence on ciliary beat frequency (CBF) and to assess participation of PKC in purinergic ciliary stimulation. The experiments were performed by simultaneous measurement of [Ca*+], and CBF on tissue culture of frog esophagus epithelium. The PKC activators TPA and DiC, produced significant elevation of [Ca’+], and strong frequency enhancement. The calcium elevation was inhibited by lowering the extracellular calcium level, or by La 3+,but was unaffected by verapamil and the phospholipase C inhibitor U-73122, suggesting that Ca*+ influx was via non-voltage-operated calcium channels. The inhibition of [Ca*+], elevation resulted in corresponding inhibition of CBF enhancement. The effect of TPA was blocked by the selective PKC inhibitors chelerythrine, calphostin C, and GF109203X, and by the enzyme downregulation. The downregulation of PKC, or the enzyme inhibitors did not affect the immediate response to extracellular ATP but caused rapid decay of initially stimulated [Ca*+], and CBF to the basal level. These results suggest that PKC produces CBF enhancement via activation of calcium influx through non-voltage-operated calcium channels. This calcium influx seems to be responsible for the duration of ciliary stimulation produced by the extracellular ATP.

INTRODUCTION Cilia are tiny hairlike protrusions enveloped by a membrane contiguous with the cell membrane. They are thin (0.25-0.3 pm), relatively long (6-50 Frn) and densely packed on the cell surface (100-200). Their primary function is transport either of a single cell through water or of a mucus layer over the cell. For this task to be performed efficiently, cilia beat in an approximately periodic, spatial and temporal pattern called a metachronal wave. Cilia exist in a wide range of organisms and tissues from protozoa to the epithelial cells of the digestive, reproductive

Received 13 June 1996 Revised 6 November 1996 Accepted 1.5 November 1996 The first two authors

contributed

equally

and respiratory, systems of vertebrates. These cells are excitable, in the sense that they dramatically change their activity in response to various stimuli. Obviously, ciliary regulation by a broad range of physiological mediators implies the existence of several types of receptors on the same ciliary cell. For example, ciliary cells from frog palate or esophagus can be stimulated by acetylcholine, through a muscarinic receptor [I]; and by extracellular ATP, through a purinergic P, receptor [2]. It has been found that extracellular ATP is one of the most potent ciliary activators, stimulating a variety of ciliary cells, including human nasal polyps 13-51, rabbit trachea [6,7], rabbit oviduct [S], dog trachea [9], frog palate and esophagus [lo]. Given the important role that extracellular ATP may play in stimulating cilia in vivo, we have extensively

to this work.

Correspondence to: Prof. Z. Priel, Department of Chemistry, University, PO Box 653, Beer-Sheva 84105, Israel Tel. +972 7 646 1184: Fax. +972 7 690 0046 E-mail [email protected]

Ben-Gurion

Abbreviations: PKC, protein kinase 0-tetradecanoyl-phorbol-13-acetate; phosphatidylinositol phospholipase diacylglycerol;

C; CBF, ciliary beat frequency; TPA, 12. AC, adenylyl cyclase; PI-PLC, C; IP,, inositol-I ,4,5trisphosphate; DAG,

DiC,, dioctanoyl-sn-glycerol

103

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Z Priel

investigated the mechanism of action of extracellular ATP on frog palate and esophagus epithelial cells. We found that micromolar concentrations of extracelktlar ATP enhanced ciliary beat frequency 2-3-fold [lo] and induced pronounced changes in the metachronal wave parameters [ 111. These effects enhanced ciliary transport ability by at least 5-fold. In addition, appreciable membrane fluidization was observed [12]. We further demonstrated that an increase in cytosolic calcium concentration (principally from internal stores), coupled to membrane hyperpolarization, were necessary to activate all these cellular effects [ 131. Finally, we revealed the existence of two proteins which contain extracellular ATP binding site(s) and which may be P, receptors, one of them probably a PZY subtype [ 141. It is well established that P, and P,” receptors activate the phospholipase C (PLC) pathway cascade which leads to mobilization of Caz+from intracellular store(s). Indeed, it has been shown that extracellular ATP causes a rapid and strong increase (4-5-fold) in [Ca2+liin a variety of ciliary cell [4,8,12,13]. This effect is transient, decaying after 3-4 min, while ciliary frequency enhancement by extracellular ATP is a rapid and prolonged effect, continuing for 20-30 min. Careful examination of these phenomena, using a technique which enables simultaneous measurement of [Caz+], and ciliary frequency from the same cell [7,15], revealed that [Caz+],decayed to a sustained, higher than basal, plateau. Manipulations which lowered this sustained plateau of [Caz+li caused the ciliary frequency to decay rapidly to its normal value. Moreover, it was shown that the elevated plateau was caused by calcium in&x which occurred after mobilization of calcium from internal stores. Contrary to expectations, voltage-activated calcium channels were not involved in the process of calcium influx. Although the work described above was performed on ciliary tissue culture from rabbit tracheal epithelium, the phenomenon of sustained plateau of [Caz+],is even more pronounced in tissue culture from frog palate or esophagus [ 12,131. Since single cell organisms, such as Paramecium, have been shown to possess voltage dependent calcium channels [ 16,171, such channels have been postulated to exist in mucociliary systems. To the best of our knowledge, there is no direct evidence for the presence of such channels in ciliary epithelial cells. Consequently, the mechanism of calcium influx into ciliary cells from vertebrates, in general, and with extracellular ATP, in particular, is still not clear. Stimulation of PLC by extracellular ATP inevitably results in production of diacylglycerol (DAG), the natural activator of protein kinase C (PKC). PKC is a family of serine/threonine protein kinases that have been proven to be important mediators of diverse cellular functions. There are a number of proteins that can be phosphorylated by PKC, including receptors, enzymes, and various membrane Cell Calcium

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and cytoskeleton proteins [ 18,191. Much effort is presently being concentrated on elucidation of the PKC role as a desensitizer in the purinergic signal transduction pathway It has also been shown that PKC is able to modulate ion channel activity 1201.Therefore, we considered the possibility that modification of Caz+ channel activity by PKC might be a plausible mechanism for controlling calcium entry in ciliary cells. Unfortunately, the available data on the effect of PKC on Caz+influx are ambiguous. For example, the contractile response of some vascular smooth muscle, induced by PKC activators, has been inconsistently reported to be both [Caz+], dependent and independent [2 11.Direct measurement of the effect of PKC activators on intracellular Caz+concentration also produced conflicting results. In cardiac cells, PKC has been shown to exert both inhibitory and stimulatory effects on dihydropyridine-sensitive Caz+ channels [22]. These examples clearly demonstrate the wide variation in experimental observation regarding the role of PKC on Caz+-induced mechanisms. Very little information exists concerning the role of PKC in ciliary cells of vertebrates. Addition of PKC activators to ciliated rabbit or sheep trachea results in a slight reduction of CBF [23,24]. No mechanism for this observed effect has been suggested. Therefore, the present study has been undertaken to investigate the effect of PKC activation, using either phorbol ester (TPA) or cell permeable diacylglycerol (dioctanoyl-sn-glycerol, DiC,), on ciliary activity in mucociliary cells from frog esophagus or palate. In addition, we examined the involvement of PKC in CBF enhancement induced by extracellular ATP. MATERIALS

AND

METHODS

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 1251. Chemicals and solutions

4a- 12-0-Tetradecanoyl-phorbol-13-acetate (4a-TPA) and U-73 122 were purchased from RBI (Natick, MA, USA). Nickel chloride was from Merck (Darmstadt, Germany). Calphostin C and bisindolylmaleimide (GF 109203X) were from Calbiochem (La Jolla, CA, USA). H-89 and SQ22536 were from Biomol (Plymouth Meeting, PA, USA). Fura-2/AM was from either Molecular Probes (Eugene, OR, USA) or from Teflabs (Austin, TX, USA). K,Fura-2 and Pluronic F-127 were from Molecular Probes. Fetal calf serum, L- 15 Leibovitz medium, and antibiotics were from Biological Industries (Bet-Haemek, Israel). All other chem0 Pearson

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Measurement of ciliary beat frequency

icals were obtained from Sigma Chemical Co. (St Louis, MO, USA). Ringer’s solution contained (mM) 1.20 NaCl, 2.5 KCl, 1.8 CaCI,, 1.1 Na,HPO, and 0.85 NaH,PO,. Ringer’s solution for calcium measurement experiments contained (mM) 120 NaCl, 2.5 KCI, 1.8 CaCl,, 5 HEPES. Solutions with a low concentration 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 1261to reach the desired free ion concentration. The external calibration solution for Fura- was composed of (mM) 115 KCl, 20 NaCl, 5 MgCl,, 5 D-glucose, 5 HEPES, 10 EGTA, and 1 PM K,Fura-2. TPA, DiC,, calphostin C and GF 109203X were dissolved in dimethyl sulfoxide (DMSO) and chelerythrine chloride in DMSO/H,O (1: 1) as stock solutions and diluted into the Ringer’s solution just prior to use. The final concentration of DMSO was not more than 0.1%. All the solutions were adjusted to pH Z2-74 before use. Table

1 The

averaged

results

127,281.

Simultaneous measurement and ciliary beating

TPA 80 nM DiC, 20 ug/ml 4a-TPA 80 nM [Ca2+], = 1 0-6 M [Ca2+], = 10” M [Ca*+], = 10d M [Ca2+], = lO-3 M

8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26

Verapamil 10 ).LM La3+ 500 PM Ni*+ 1 mM Ni2+ 5 mM U-731 22 10 uM Chelerythrine 10 uM GFI 09203X 10 uM Calphostin C 500 nM Downregulation K+ 60 mM K+ 120 mM Quinidine 1 mM SQ-22536 500 uM H-89 1 uM H-89 5 FM ATP 10kM Chelerythrine 10 uM GFlO9203X 10 PM Calphostin C 500 nM

27

Downregulation

89 f 7 (35) 9Okl9(5) 7 f 7 (5) 13&10(5) 125f9(5) 61 k 7 (7) 85fll (7) 30 f 6 (7) 92flO(5) 66+12(7) 80fl9(5) 12f7(8) 11 &7(10) 5 k 8 (7) 12 f 10 (5) 65 + 8 (10) 65 + IO (8) 80fl6(5) 98&15(6) 104f22(7) 390 f80 370 f 84 372 !I 78 355 !I 91 366 + 95

AICa*+li nM 4 min

(11) (7) (8) (6) (5)

2.5 2.5 1.1 1.3

f 0.1 + 0.2 20.1 20.2

(42) (5) (5) (15)

1.2 k 0.2 (15) 2.2 f 0.2 (18) 2.5 f. 0.3(7) 1.9 * 0.2 1.4 + 0.2 2.6 f 0.3 2.1 kO.1 2.8 f. 0.3 1.2 IL 0.2 1.2 kO.2 1.1 kO.2 1.2 rf 0.2 1.6?0.1 1.7 f 0.2 1.5 z!c 0.2 2.6 + 0.3 2.9 5~ 0.3 2.6 + 0.3 3.1 20.1 3.0 AZ0.2 3.1 k 0.2 2.8 f. 0.3 2.6 f 0.3

(17) (7) (5) (25) (5) (8) (10) (8) (5) (18) (16) (8) (5) (6) (7) (24) (7) (8) (6) (5)

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f/f0 4min

73+15(32) 72 + 24 (5) 3 + 8 (5) 3 + 5 (5) 2 zk 4 (5) 51 + 15 (7) -

2.4 2.4 1.1 1.1 1 .l 2.0 -

f 0.2 zk 0.2 fO.l kO.1 f 0.2 f. 0.3

(39) (5) (5) (5) (5) (7)

78*15(7) 8+10(7) 75 + 15 (5) 61 +12(7)

1.8 1 .I 2.5 1.9 2.5 1 .l 1 .l 1 .l 1.1 1.4 1.4

f 0.3 + 0.2 + 0.3 f 0.2 It 0.3 + 0.2 dc 0.2 f 0.2 kO.2 + 0.2 k 0.2

(7) (7) (5) (7) (5) (8) (10) (8) (5) (IO) (8)

1.1 2.4 2.4 2.5 2.5 1 .l 1.2 1 .I 1 .o

to.1 f 0.3 + 0.3 t 0.2 i 0.3 + 0.2 i 0.2 k 0.2 k 0.1

(8) (5) (6) (7) (11) (7) (8) (6) (5)

71 +16(5) 2 + 4 (8) 3 +5 (10) 2 f. 4 (7) 3 + 4 (5) 55&12(10) 58kll (8) 72&15(5) 70+10(6) 74 k 14 (7) 83?25(11) 4 + 6 (7) 3 k 6 (8) 2 f 4 (6) 2 t 4 (5)

number represents average f SE (number of experiments): max columns - maximal response - response 4 min after reaching the maximum. l-3, 23 - results obtained by addition of the indicated substances to cells in regular conditions, 4-22 - results obtained by addition of TPA (80 nM) to cells treated as indicated in the table. 24-27 - results obtained by addition of ATP (10 FM) to cells treated as indicated in the table.

0 Pearson

of intracellular calcium

Simultaneous measurement of intracellular calcium and ciliary beating was carried out as previously described [7]. Briefly, [Caz+], was measured with the fluorescent indicator Fura-2. The dye-loaded cells were epi-illuminated with light from a 150 W xenon lamp (Oriel Corp. Stamford, CT, USA) filtered by 340 and 380 nm interference filters (Oriel) mounted on a four position rotating filter wheel. The fluorescence, emitted at 5 10 nm, was

f/f0 max

max

Every columns Rows Rows Rows

We used the method of simultaneous measurement of scattered light from two points on the monolayer ciliary epithelium that was extensively described previously

of all experiments

AICaz+li nM

1 2 3 4 5 6 7

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in calcium

and frequency;

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detected by a photon-counting photomultiplier (H346053, Hamamatsu, Japan). The 340/380 fluorescence ratio, averaged over a period of 1 s, was stored in a computer (IBM 486). CBF was measured by trans-illuminating the same cells with light at 600 rm-r (so as not to interfere with the Fura- fluorescence at 510 nm). The light scattering from the beating cilia created amplitude modulations of the 600 nm light that were detected by a photomultiplier (R2014, Hamamatsu, Japan). A calibration curve of the calcium concentration was created by titration of an external calibration solution with a solution of the same composition but containing 10 n-&l CaCl, 1291.The calcium concentration was calculated directly from the calibration curve by interpolation using a table look-up algorithm. Procedure

Prior to any treatment, the Ringer solution over the tissue culture was changed 3 times. The tissue was then preincubated in a fourth change of Ringer’s solution (containing

the test substances if required by experimental procedure) for 15-30 min prior to the experiment, to prevent any transient effects on the ciliary motility. In experiments where [Caz+li was measured, the cells were preloaded with Fura- by incubation of the tissue in the growth medium containing 8.5 w Fura-2/AM and 0.03 % Pluronic F-127 for 60 min at 37°C in a rotating water bath. We reported previously that loading of Furainto the frog esophagus epithelium culture is extremely difficult 1131. The low permeability of the membrane of these cells in intact culture seems to be the probable reason. Disruption of culture integrity profoundly increases the amount of the dye that enters the cells. Unfortunately, such treatment impairs the cell responsiveness and viability. Since our last publication [ 121, the procedure has been markedly improved, enabling us to reduce the rate of loading failure significantly without interference with cell function. The basal ciliary beat frequency (fo) and [Caz+li level were measured for 2-5 min in 900 ~1 of the appropriate solution. These were taken as reference values. Then, 100 C

1001 0

5

TPA

250

TPA

10

M,,,.,,..,,,,,.,,,,~ 0 2

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6

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25 20 15 10 5 0

.

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time, min

10

15

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..I.

*

2

.,m

m

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6 time, min

I

m,.

*.,

8

10

Fig. 1 The effect of TPA on [Ca2+]i and CBF. Two representative experiments of [Ca2+li (A, C) and CBF (B, D) rise induced by TPA (80 nM). [Ca2+li and CBF were measured srmultaneously from one cell that showed slow gradual elevation of the parameters (A, B) and a second cell that showed strong irregular oscillations of the parameters (C, D). Strong correlation between [Ca’+], and CBF is evident.

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~1 of solution containing the test substance was added to reach the desired final concentration. The frequency (f) and [Caz+li were monitored on the same ciliary cell for 15-40 min. Beat frequency enhancement was represented by normalization of the observed frequency to the reference frequency (fo); f/f0 = frequency enhancement. The intracellular calcium elevation is represented by the difference AICaz+li between the observed calcium level and the reference level. Every experiment was performed using 5-42 tissue cultures taken from 2-3 animals. RESULTS

The original goal of this work was to determine the role of PKC in regulation of ciliary activity. Hence, only CBF measurements were performed, using equipment designed for this purpose [27]. When the results strongly suggested involvement of calcium in the effects produced by PKC activators, a major effort was made to overcome the technical difficulties of Fura- loading and most of the experiments were redone on apparatus that permitted simultaneous measurement of both intracellular calcium and CBF. Since the results of ciliary beat frequency measurements performed by both techniques were essentially the same, they were merged and are presented in this paper as combined data. Effect of PKC activators on cytosolic calcium concentration and ciliary beat frequency To determine whether PKC activators affect ciliary activity, 80 nM TPA or 20 pg/ml DiC, were added and then the CBF and intracellular calcium were measured. The two activators significantly enhanced the CBF (2-3-fold) and caused considerable elevation of [Ca2+li (Table 1, rows 12). In contrast, 4a-TPA, a phorbol ester that does not stimulate PKC, had no effect on CBF and [Caz+], (Table 1, row 3). The pattern of cellular response to TPA varied in different experiments from slow gradual elevation of [Caz+ll and CBF to strong irregular oscillations in both parameters. The two extreme cases are shown in Figure 1. The intensity of the fluctuations in the rest of the experiments distributed uniformly over the range between these two cases. In all experiments, strong correlation between the changes in calcium and the changes in frequency was evident. This correlation is most clearly illustrated by Figure 1 CD. Every change of calcium level corresponds to a simultaneous change of CBF in the same direction. CBF and [Caz+li responses reached their maximum within 160 f 28 s after TPA addition. Maximum enhancement of the CBF was already reached with 1 nM TPA, though a significant percentage of the cells in the culture did not respond at all at that concentration. The elevation of TPA concentration 0 Pearson Professional Ltd 1997

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increased the percentage of the responding cells with no influence on maximal effect. At 10 nM concentration, 65% of the ciliary cells responded to TPA. At 80 r&I, 90% responded. This behavior may be due to the low permeability of the cell membrane mentioned above. The cells that are less resistant to TPA penetration already respond at 1 r&I. The cells more resistant to TPA penetration require much higher concentrations of TPA in the extracellular medium before they respond. Since each measurement was performed on a single, randomly chosen cell, the experiments were carried out at a concentration of 80 nM TPA (at which concentration 90% of the ciliary cells responded to TPA, as noted above). Effect of PKC inhibitors and PKC downregulation on intracellular calcium elevation and ciliary stimulation induced by TPA The effects of some reported PKC selective inhibitors on the enhancement of CBF by TPA were tested. All three inhibitors interfere with the enzyme at different sites. Chelerythrine (IC,, = 660 nM) competes with the protein substrate [30]. GF 109203X (IC,, = 5-70 r&I) which resembles staurosporine structurally, but is highly selective for the inhibition of PKC [3 l] competes with ATP. Calphostin C (IC,, = 50 r&I) competes with DAG and TPA [32]. All three inhibitors effectively antagonized the TPA induced [Ca2+J and CBF elevation. Chelerytrine and GF 109203X, at 10 PM concentration, and calphostin C, at 500 nM concentration, almost completely eliminated the calcium and frequency response to TPA (Table 1, rows 13-15). It has been reported that prolonged activation of PKC results in the enzyme’s degradation (PKC downregulation) [ 181. To produce this effect, the cells were incubated for 22-24 h in growth medium containing 160 nM TPA. Then, TPA was rinsed away, the cells were loaded with Furaand the response to TPA was measured as described above. After the treatment, TPA failed to produce any significant elevation of [Caz+], or CBF enhancement (Table 1, row 16). Effect of extracellular Ca2+and Ca2+channel blockers on TPA stimulation and the source of calcium mobilization To assess whether extracellular Ca2+influences the elevation of intracellular calcium and the enhancement of the ciliary activity in response to TPA, two separate examinations were made: (i) ciliary stimulation and [Caz+], produced by TPA were measured over a wide range of free Ca2+ concentrations in the external solution; and (ii) the cellular response was measured after TPA addition to cells pretreated with the Ca2+channel blockers verapamil, Ni2+ and La3+. Cell Calcium (1997) 21(2), 103-l 13

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approximately equally (Fig. 3). Ni2+, which blocks voltagegated calcium channels and sodium-calcium exchange at submillimolar concentration failed to inhibit the TPA effect at 1 mM and only slightly attenuated it at 5 mM (Fig. 3). To evaluate the possibility of phospholipase C activation and stimulation of calcium release from the internal stores, we tested the potent PLC inhibitor U-73 122. Incubation of the cells with 10 ).tM U-73 122 for lo-30 min did not affect their response to TPA (Table 1, row 12). As can be clearly seen in both Figure 3 and Table 1, with the exception of verapamil, for each substance tested the magnitude of the calcium response correlates strongly with the magnitude of the frequency response.

A 100 80 i

20

/

0 10-7

10-6

10-5

10-4

10-3

, 10-z

fCa2+10,M

100 2.5 80 2-

3

0

“,

8 1.5 -

10-7

10‘6

IO.5

lo-

4

10-3

lo-

2

TPA 80 nM (control)

fCa2+10,M Fig. 2 [Ca’+], and CBF elevation produced by TPA versus extracellular Ca*+ concentration. Maximal values of A[Ca2+li and f/f0 from each simultaneous experiment at a given [Ca*+], were averaged over 5-l 8 experiments (error bar represents SE deviation). The concentration of TPA was 80 nM.

verapamil 1OpM

La3+ 0.5mM

Ni2+ 1 mM

Ni2+ 5&

[Ca2+] o

verapamil 10pM

Q+ 0.51x&f

Ni2+ 1mM

Ni2+ 5rnM

D2+l,

1O‘5 M

B

2.6 -

o

2.2 -

s

Figure 2 shows that ciliary stimulation by TPA is dependent on free extracellular Ca2+ concentration ([Ca2+],) and correlates with the similar dependence of the intracellular calcium elevation. Maximal effect of TPA is observed at 1O-3 M free Ca2+ concentration, whereas the TPA effect is completely blocked already in 1O-5M free Ca2+ concentration. Verapamil (10 p&l), which is known to block voltage gated calcium channels in the 1O”- 1O-5M concentration range, produced moderate inhibition of CBF enhancement, but had no effect on the calcium response (Fig. 3). La3+ (500 PM), a potent non-selective calcium channel blocker which has been shown to be an effective inhibitor of non-voltage dependent calcium influx [33,34], significantly reduced both calcium and frequency responses Cell Calcium

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1.8 -

TPA 80 nM (control)

1O-5 M

Fig. 3 Effect of calcium channel blockers on [Ca*+], and CBF elevation caused by TPA. Maximal values of A[Ca2+], and f/f0 from each simultaneous experiment were averaged. The effect of low extracellular Caz+ concentration is also given to emphasize what appears to be a strong dependence of the TPA effect on calcium influx. Each column represents mean + SE averaged over 5-25 experiments.

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80-

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To test the possible involvement of K+ chamrels in the enhancement of ciliary activity by PKC activators, the effect of TPA on CBF was measured in tissues treated with: (i) a K+ channel blocker; and (ii) Ringer’s solutions with K+ concentrations ranging from 2.5-120 mM. In this way, the transmembrane K+ gradient was decreased and K+ efflux through the cell membrane was prevented. The osmolarity was kept constant by replacing Na+ with K+. Quinidine (1 n-&I), a potent inhibitor of Ca2+-activated K+ channels at millimolar concentrations, caused an inhibition of TPA induced enhancement of ciliary activity (Table 1, row 19). Unfortunately, because of the strong fluorescent properties of quinidine, calcium measurements in its presence at 1 mM concentration were not possible. The effect of TPA on CBF was inhibited by raising the extracellular K+ concentration in a dose-dependent manner. It was strongly attenuated at a concentration of 60 mM K+ (Fig. 4). In contrast, the calcium response to TPA was only slightly diminished at either 60 mM or 120 mM K+ (Fig. 4). Involvement of adenylyl cyclase and PKA in intracellular calcium elevation and ciliary stimulation induced by TPA

control (TF’A 80 nM)

K+ 6Omh4

K+ 12omM

Fig. 4 TPA induced [Ca’+], and CBF rise at high extracellular K+ concentration. Maximal values of A[Ca2+], and f/fo were averaged over 8-I 8 experiments, the bars represent SE deviation. The concentration of TPA was 80 nM.

It has been reported that K+ flux can stimulate adenylyl cyclase (AC) production of CAMP, a well known activator of CBF 1351. Furthermore, several isoenzymes of AC can be directly activated by calcium and PKC. In order to determine whether TPA enhanced CBF through a CAMPdependent pathway, we tested the AC selective inhibitor Q-22536 (500 PM) and the PKA selective inhibitor H-89 (1 FM and 5 PM). Both inhibitors failed to produce any attenuation of CBF or calcium response to TPA (Table 1, rows 20-22). Involvement of PKC in purinergic ciliary stimulation

These results suggest that [Caz+li elevation induced by TPA is due to calcium influx, probably via non-voltagegated calcium channels, and blocking of this influx impairs CBF enhancement. The moderate inhibition of CBF (but not [Ca2+]J elevation by verapamil seems to be unrelated to its channel blocking activity and should probably be considered a side effect. Involvement of K+ channels in intracellular calcium elevation and ciliary stimulation induced by TPA It has previously been shown that potassium flux through the cellular membrane, possibly via Caz+-activated K+ channels, is essential for the duration of ATPinduced CBF enhancement [ 10,131. 0 Pearson

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Extracellular ATP produces a powerful and rapid rise in [Ca2+li and CBF. After the initial elevation, the calcium level decays to a sustained, higher than basal, plateau, whereas CBF, decaying slowly, remains at a high level of activation up to 20-30 mm (Fig. 5; Table 1, row 23). We examined the possible involvement of PKC in purinergic stimulation of ciliary cells by extracellular ATP by adding ATP to cells in which PKC activity was attenuated, either by preincubation with PKC inhibitors, or by long term exposure to TPA leading to the downregulation of the enzyme. Preincubation of the cells for 15-30 min with the PKC inhibitors chelerythrine (10 @‘VI), GF 109203X (10 PM) or calphostin C (500 nM) as well as downregulation of PKC did not affect the initial calcium and CBF rise, but resulted in disappearance of the elevated Cell Calcium

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100: 0

. , . , . , . , . , . , . , . , . , . , 2 4 6 8 10

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4

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3 30 25 N 3

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Fig 5 Typical time courses of [Ca’+], and CBF enhancement induced by extracellular ATP with and without involvement of PKC. Pretreatment of ciliary cells with PKC inhibitor GF109203X (10 PM) for 15 min eliminated the elevated sustained plateau of [CaP+], (A) and prolonged CBF enhancement (B) produced by extracellular ATP (10 PM). Similar results were obtained by downregulation of PKC (C, D). The simple line plots correspond to untreated cells, the circles correspond to the treated cells. Note that the initial rise of [Ca’+], and CBF induced by ATP was not altered by pretreatment of the cells.

[Ca2+li plateau and relatively rapid decay basal level (Fig. 5; Table 1, rows 24-27). suggest that PKC induced calcium influx for the elevated [Ca2+], plateau and the activation produced by ATP.

of CBF to the These results is responsible sustained CBF

DISCUSSION

We have recently shown that the mechanism of CBF enhancement by extracellular ATP in frog esophagus involves calcium mobilisation from intracellular stores through P,-purinergic receptors [ 13,141. The calcium mobilizing P, receptors appear to activate phospholipase C (PLC) which produces IP, and DAG. The former interacts with the calcium stores to initiate the release of calcium and the latter activates PKC. While the involvement of [Ca’+], elevation in CBF enhancement is well established, Cell Calcium

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the role of PKC activation remains a mystery. In addition, the time course of the response to ATP is inconsistent with a simple ‘calcium release-CBF enhancement’ one-to-one model. After the strong initial elevation of both [Ca2+],and CBF resulting from the calcium stores’ discharge, the calcium concentration rapidly decreases to a slightly elevated (over basal value) plateau, whereas the CBF remains highly activated for a much longer time. This behavior suggests that additional factors, besides the calcium mobilization from the intracellular stores, must be switched on and participate in purinergic ciliary stimulation. All these facts dictate the necessity to investigate PKC influence on ciliary activity and its possible role in purinergic ciliary stimulation. In the present work we showed that two distinct PKC activators, TPA and DiC,, at specific concentrations, produced similar effects of strong CBF enhancement and 0 Pearson

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significant [Ca2+], elevation. 4o-TPA failed to produce any response. Three distinct, selective inhibitors of PKC and PKC downregulation completely abolished the effect of TPA. These facts enable us to affirm that activation of PKC in frog esophagus ciliary epithelium leads to intracellular calcium elevation and strong CBF enhancement. In contrast to our results, it has been reported previously that addition of PKC activators to ciliated rabbit or sheep trachea results in slight inhibition of CBF [23,24]. The discrepancy can be explained by tissue specific differences between rabbit or sheep trachea and frog esophagus (such as different PKC isoenzyme expression). In addition, in the trachea experiments, the concentrations of the PKC activators used were relatively high. It could produce unwanted side effect on ciliary activity. The elevation of [Ca2+li was dependent on extracellular calcium concentration, was inhibited by the calcium channel blocker La3+, and was unaffected by the PLC inhibitor U-73 122, suggesting that the elevation was due to Caz+ influx through the plasma membrane from the extracellular fluid. The strong coupling between calcium and CBF enhancement, produced by PKC activation, was observed in two ways. First, any attenuation of the calcium elevation resulted in attenuation of CBF enhancement to the same extent. Second, in every single experiment, any change of [Caz+li, even a tiny one, caused an immediate change of CBF in the same direction. This coupling indicates that the calcium influx is a prime mover of the CBF enhancement produced by the PKC activation. The nature of the PKC induced calcium influx and the mechanism of its activation are not completely understood. According to our results, the influx is not blocked by verapamil or NiZ+at concentrations appropriate for voltage-dependent calcium channel blockage. Moreover, we have recently shown that extracellular ATP, which should activate PKC and, consequently, the PKC induced calcium influx, produces significant membrane hyperpolarization and, therefore, can not open voltage-dependent channels [ 131. These facts suggest that PKC activates calcium influx via non-voltage-operated calcium channels. We assume that these channels resemble the widely discussed capacitative entry calcium channels (very low conductive channels that are activated by an unknown mechanism in response to intracellular calcium store depletion) [33,34]. Because of low conductivity, these channels need sufficient driving force for calcium entry in order to produce significant calcium elevation. Indeed, as we have shown, a decrease in extracellular calcium concentration strongly inhibits the PKC-induced Ca2+ influx and CBF enhancement. The membrane depolarization achieved by high extracellular K+ concentration, which also reduces the driving force for calcium entry, inhibits the influx as well. It should be mentioned that the inhibition of CBF 0 Pearson

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enhancement in the latter case is much stronger than the inhibition of [Ca”], rise. This suggests that K+ efflux and/or membrane hyperpolarization produced by such efflux has an additional role in the ciliary stimulation besides providing the driving force for calcium entry. In ciliated tissue cultures from rabbit trachea, verapamil at quite high concentrations produced conflicting results. At 1 mM concentration, the CBF response to mechanical stimulation was abolished 1361. On the other hand, verapamil at 0.2 mM concentration completely prevented calcium influx induced by maitotoxin but did not significantly inhibit CBF increase [37]. While at 10 FM concentration, which is the upper limit of its specificity as a voltage-dependent calcium channel blocker, verapamil did not influence either calcium influx or frequency enhancement induced by extracellular ATP [ 151. In ciliated tissue cultures from frog esophagus, verapamil at high concentration (0.1 mM) abolished almost completely the CBF enhancement, while at 10 cl.n/rthe inhibition was moderate [lo]. In this work, we showed that verapamil at 10 @I already produced significant inhibition of CBF enhancement induced by TPA without any influence on the calcium influx. Therefore, the inhibition of CBF stimulation in the discussed system may be considered a side effect of verapamil and not a result of calcium channel blocking. Naturally this effect is more pronounced at higher concentrations. Hence, verapamil can not be used to prove the involvement of voltagedependent calcium channels in mucociliary systems. In fact, there is no direct evidence of such involvement to the best of our knowledge. The relative magnitude of the [Ca2+], elevation and the CBF enhancement at different stages of ATP and TPA stimulation deserves separate discussion. First, it should be mentioned that the technique used in these experiments enabled us to measure the average calcium concentration in a cell. When the activation of P, receptors by extracellular ATP caused calcium release from the intracellular stores, [Ca2+li rose rapidly throughout the whole cell. This stage is monitored as the initial strong elevation of both calcium and CBF. On the other hand, when TPA activates PKC and induces calcium influx, due to very low conductivity of the channels, [Caz+li rises significantly only near the membrane, in the immediate proximity of the cilia. The local calcium concentration in this region, which may be as high as the concentration produced by ATP throughout the cell, is what determines the CBF. The average calcium concentration in the whole cell appears to be only slightly elevated, but the CBF is almost as high as in response to ATP. After the initial elevation of calcium and CBF produced by ATP, [Ca2+lidrops over most of the cell volume due to re-uptake by the stores and extrusion from the cell. But, because of PKC-induced calcium influx activation, near Cell Calcium

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R Levin, A Braiman, Z Priel

) ATP \

K+,m

Intracellular

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-

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Fig. 6 Schematic diagram of the suggested model for ciliary stimulation by extracellular ATP. The suggested model is based on our previous findings and the PKC involvement revealed in this work.

the membrane [Ca2+li remains high and CBF does not decrease. The measured calcium concentration does not reach the basal value but remains slightly elevated due to the contribution of the near membrane region. If PKC is inhibited or downregulated, ATP fails to activate calcium influx. In this case, CBF starts to decrease with [Caz+li decreasing immediately after the initial elevation, both reaching the basal level a short time after the stimulation. As can be seen from our results, [Caz+li and CBF obtained after the TPA treatment are approximately equal to the calcium and frequency values at the plateau stage of the response to ATP, suggesting that in both cases we are observing essentially the same effect produced by two different methods. Based on the present results, we would like to extend the previously suggested model [lo] of ciliary stimulation by extracellular ATP (Fig. 6). The activation of P,purinergic receptor results in production of IP, and DAG by phospholipase C. IP, produces intracellular calcium stores discharge that results in strong rapid [Caz+], elevation throughout the cell and strong CBF enhancement. Both effects produced by the calcium stores discharge are transient and cease within 3-4 min of onset. Concurrently, DAG activates PKC which induces calcium influx from the extracellular space via non-voltage-operated calcium channels. The calcium released from the intracellular stores may also assist in activation of calcium dependent PKC isoenzymes. The calcium influx produces local elevation of [Ca”], near the membrane in the immediate proximity of the cilia and maintains prolonged CBF enhancement when the initially released cal-

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cium is no longer available and [Caz+], inside the cell drops presumably to the basal level. The localization of the [Ca2+], rise only to the area near the cilia is very important because high [Caz+li is toxic to cells and prolonged calcium elevation all over the cell would be fatal. The influx may also further enhance the activity of Ca2+dependent PKC. [Ca2+li elevation in the membrane region activates Caz+-dependent K+ channels producing K+ efflux and hyperpolarization of the membrane. This effect itself seems to be important for CBF activation though the mechanism is still unknown. But, in addition, the hyperpolarization enlarges the driving force for calcium entry and further intensifies the influx. The role of PKC as a negative feedback factor in purinergic stimulation which serves to restrain and eventually terminate the effect has been discussed in a great number of publications. In this work, we show that PKC can also be an integral part of the purinergic stimulatory system that enforces the effect and is critical for its continuation. In conclusion, we have shown for the first time to the best of our knowledge that PKC can activate calcium influx through non-voltage-operated calcium channels. The mechanism of this activation is not clear and requires further investigation. The activation of PKC results in strong CBF enhancement due to calcium influx induction. So far, only inhibition of ciliary beating by PKC has been reported. The duration of the ciliary stimulation by extracellular ATP is PKC dependent. Prolonged CBF enhancement produced by ATP is maintained by PKC-induced calcium influx. 0 Pearson Professional Ltd 1997

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ACKNOWLEDGEMENT

This work was supported Science Foundation.

by a grant from the Israel

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