Effects of azelastine on membrane currents in tracheal smooth muscle cells isolated from the guinea-pig

ELSEVIER

ejp European Journal of Pharmacology 259 (1994) 143 150

Effects of azelastine on membrane currents in tracheal smooth muscle cells isolated from the guinea-pig Hisanori H a z a m a ", Toshiaki Nakajima ..a, Tetuya Hisada ~', Eiji H a m a d a a, Masao O m a t a a, Yoshihisa Kurachi b " Second Department of Internal Medicine, Faculty of Medicine, University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113, Japan ~'Division of Cardiovascular Diseases, Department of Internal Medicine, and Department of PharmacologLv, Mayo Clinic, Mayo Foundation, Roehesto; MN 55905, USA Received 9 December 1993; revised MS received 30 March 1994; accepted 31 March 1994

Abstract

Azelastine [4-(p-chlorobenzyl)-2-(hexahydro- 1-methyl - 1H-azepin-4-yl)- 1-(2 H)-phthalazinone hydrochloride], an anti-allergic agent, inhibited the high K+-induced contraction in tracheal smooth muscle cells isolated from the guinea-pig. In order to investigate the ionic mechanisms, we examined the effects of azelastine on membrane currents, using the tight-seal whole cell voltage clamp technique. Azelastine (1-100 # M) caused an inhibition of the Ba 2 + inward current (IBm) through the voltage-dependent L-type C a 2+ channel in a concentration-dependent manner. The inhibitory effect of azelastine on IBa was fully reversible. The ICs0 value for azelastine-induccd inhibition of IB~ was approximately 8 /~M, and 100 #M azelastine completely suppressed IBm. Azclastine exerted mainly a tonic block of ln~ but did not show use dependence. Azelastine (10 p.M) shifted the quasi-steady-state inactivation curve of 1B~ to more negative membrane potentials by approximately - 20 mV, suggesting that the inhibitory effect of azelastine on IB~, was voltage-dependent. In addition, azelastine produced inhibitory actions on other membrane currents (i.e. the voltage-dependent transient outward K + current and the Ca2+-activated oscillatory K + current) at doses higher than 10 /zM. These results suggest that azelastine inhibits the voltage-dependent L-type Ca ~+ current in single tracheal smooth muscle cells, which may contribute to the anti-allergic actions of azelastine in airways. Key words: Smooth muscle cell; Single; Tracheal; Azelastine; Voltage-dependent Ca :+ current; K + current

1. Introduction

Azelastine, 4-(p-chlorobenzyl)-2-(hexahydro-1methyl- 1H-azepin-4-yl)- 1-(2 H ) - p h t h a l a z i n o n e hydrochloride, is an anti-allergic and anti-asthmatic drug with a wide spectrum of pharmacological activities (Tasaka and Akagi, 1979; Zechel et al., 1981; Motojima et al., 1985; Oilier et al., 1986; G o u l d et al., 1988; McTavish and Sorkin, 1989). It has b e e n r e p o r t e d that azelastine inhibits the release of histamine and leukotrienes from mast cells ( K a t a y a m a et al., 1981; C h a n d et al., 1983; K a t a y a m a et al., 1987), and also antagonizes the effects of various chemical mediators in airways ( C h a n d et al., 1986; Magnussen, 1987; Richards et al., 1990). Senn et al. (1991) showed that azelastine

* Corresponding author. Fax + 81-3-3814-0021. 0014-2999/94/$07.00 ~C 1994 Elsevier Science B.V. All rights reserved SSDI 0 0 1 4 - 2 9 9 9 ( 9 4 ) 0 0 2 1 1 - 0

caused an inhibition of Ca 2+ mobilization in tracheal smooth muscle. In addition, it has been shown that azelastine directly inhibits tracheal smooth muscle contractions evoked by high K + or t e t r a e t h y l a m m o n i u m (Lee et al., 1990) as well as histamine or acetylcholine (Lee and Sperelakis, 1989). These findings suggest that azelastine may inhibit the v o l t a g e - d e p e n d e n t Ca 2+ channels in tracheal smooth muscle ceils. Recently, M a s u o et al. (1992) showed that azelastine inhibits the v o l t a g e - d e p e n d e n t L-type Ca 2+ current in single ileal smooth muscle ceils from the guinea-pig. However, little is known about the ionic mechanisms of azelastine's action on tracheal smooth muscle cells. In order to investigate the ionic mechanisms of the relaxing action of azelastine in tracheal smooth muscle cells, we applied the whole-cell voltage-clamp technique to single smooth muscle cells from the guinea-pig trachea. Here, we report that azelastine inhibits the

ft. Hazama el al. / Et~ropean Journal q/ Pharmacology 259 (1994) 143-150

144

high K+-induced contraction in single tracheal smooth muscle cells, probably due to the direct inhibition of the voltage-dependent L-type Ca e* current. In addition, the actions of azelastine on the K ~ currents in single tracheal smooth muscle cells were also investigated.

The tissue was transferred to the enzyme-free dissociation medium and kept at 4°C for later experiments. The tracheal smooth muscle cells were obtained by gentle mechanical agitation. This procedure yielded an acceptable number of viable single smooth muscle cells. All cxperiments were done at 3,~ o C.•

2.2. Solution amt drugs 2. Materials and m e t h o d s

2.1. Cell preparation Single smooth muscle cells were isolated from the guinea-pig by enzymatic dispersion using a technique similar to that previously described by Hisada et al. (1990) and Iguchi et al. (1992). Briefly, after the animals were killed, the surrounding connective tissue of the trachea was removed, and the cartilaginous portion of the trachea was cut open longitudinally. Then, the membranous portion of the trachea was cut into small pieces and incubated in the dissociation medium containing 0.5 m g / m l papain (Sigma Chemical Co., St. Louis, MO) and 0.(/5% bovine serum albumin at 4°C for approximately 15 h. Thereafter, 0.15 mM dithiothreitol (Sigma) was added to the enzyme solution, and the tissue was incubated with 3 m g / m l collagenase (Worthington CLS II, Freehold, N J) and 0.5 m g / m l trypsin inhibitor (Sigma type l-S) at 37°C fl)r 45 rain.

The control bathing solution was as follows (in mM): NaCI 136.5, KC1 5.4, CaCI, 1.8, MgCI 2 0.53, glucose 5.5, Hepes-NaOH buffer 5 (pH 7.4). The high K + solution contained (in mM): KCI 75, NaC[ 70, CaCI~ 1.8, MgC1 e 0.53, glucose 5.5, Hepes-KOH buffer 5 (pl~ 7.4). The patch pipette solution contained (in mM): KCI 130, E G T A 0.1, MgCI: 2, N a z A T P 3, guanosine5'-triphosphate (sodium salt, Sigma) 0.1 and HepesKOH buffer 5 (pH 7.2). In the experiments on the voltage-dependent Ca 2+ current, KCI in the pipette solution was replaced by equimolar CsC1, and the pH was adjusted to 7.2 with CsOH. The Ba>+-containing bathing solution was the same as the control bathing solution, with the exception that CaCI. was changed to BaCI, (5 mM). The dissociation medium contained (in raM): NaCI 110, NaHCO_~ 10, KCI 5, MgC1 e 0.5, N a H 2 P O ~ 0.5, CaCI 2 0.16, EDTA 0.49, taurine 10, phenol red 0.02, Hepes-NaOH buffer 10 and glucose 11 (pH 8.0). Azelastine was a gift from Eisai Pharmaceuticals, Japan.

Azelastine High K +

control

ttigh K +

o

3

<%

o

o

0

0

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Fig. 1. Azelastine inhibits the high K+-induced contraction in single tracheal smooth muscle cells from the guinea-pig. A cell pretreated with azelastine (50 g,M) for approximately 1 min was superfused with the high K + solution (middle panel). Note that azelastine inhibits the high K ~-evoked contraction. After the washout of azelastine, the cell contracted during the perfusion of high K + solution (right panel).

H. Hazama et al. / European Journal of Pharmacolo~,~' 259 (l 994) 143-150

m V were a p p l i e d from a holding p o t e n t i a l of - 8 0 mV. A t 10 ms after the e n d of each c o n d i t i o n i n g pulse, a test pulse to + 10 m V (200 ms in d u r a t i o n ) was applied to evoke In, ,. T h e ratio of the a m p l i t u d e s of IB~, with a n d w i t h o u t the c o n d i t i o n i n g pulse was plotted for the m e m b r a n e p o t e n t i a l of each c o n d i t i o n i n g pulse (inactivation curve). T h e interval b e t w e e n the sets of d o u b l e pulses was 60 s. Statistical data are expressed as m e a n s _+ S.D.

2.3. Current measurement and data analysis T h e m e m b r a n e c u r r e n t s were r e c o r d e d by using the tight-seal whole cell voltage c l a m p t e c h n i q u e (Hamill et al., 1981; K u r a c h i et al., 1986). T h e h e a t - p o l i s h e d patch p i p e t t e with the i n t e r n a l solution had a tip resistance of 3 - 5 MI2. T h e series resistance was comp e n s a t e d . T h e m e m b r a n e c u r r e n t s were c o n t i n u o u s l y m o n i t o r e d with a high-gain storage oscilloscope (COS 5020-ST, Kikusui Electronic, Tokyo, Japan). T h e data were stored o n line on v i d e o t a p e by using the P C M c o n v e r t i n g system (RP-880, N F electronic i n s t r u m e n t , Tokyo, Japan). T h e data were r e p r o d u c e d , low-passed filtered at 1 kHz ( - 3 dB) with a Bessel filter (FV-665, NF, 48 d B / o c t a v e slope a t t e n u a t i o n ) , s a m p l e d at 5 kHz a n d analyzed off-line on a p e r s o n a l c o m p u t e r (PC-386LS, Epson, Tokyo, Japan). T h e quasi-steadystate inactivation p a r a m e t e r s (f~) of the Ba 2+ inward c u r r e n t t h r o u g h the v o l t a g e - d e p e n d e n t Ca 2+ c h a n n e l (IBm,) at various m e m b r a n e p o t e n t i a l s were e s t i m a t e d with the use of d o u b l e - p u l s e protocol (Iguchi et al., 1992). T h e c o n d i t i o n i n g voltage pulses (3 s in d u r a t i o n ) to various m e m b r a n e p o t e n t i a l s b e t w e e n - 70 a n d + 30

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3. Results

3.1. Azelastine inhibits contraction o f single tracheal smooth muscle cells induced by high K + T h e effects of azelastine on high K+-evoked contraction were e x a m i n e d in single tracheal smooth muscle cells from the guinea-pig. Fig. 1 shows the m o r p h o logical characteristics of a single tracheal smooth muscle cell a n d its response to the high K + - b a t h i n g solution with or without the application of azelastine (50 /zM). T h e high c o n c e n t r a t i o n of K + ions (75 m M KCI) 1

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Fig. 2. Inhibition of the voltage-dependent L-type Ca2+ channel current by azelastine. (A) The cell was held at -40 mV, and command voltage pulses to + 0 mV were applied at 0.2 Hz. The patch pipette contained the CsCI-internal solution, and the bath was superfused with 5 mM Ba2+-bathing solution. The traces (a-d) were recorded at the times indicated by the letters, a: control, b: azelastine 10/zM, c: after washout, d: azelastine 100 p.M. The zero current is indicated by an arrow. In (B), the time courses of the changes in the amplitude of In~, are illustrated. (C) Concentration-dependent inhibition of ll~, by azelastine. The relations between the percent inhibition of In,, and the concentration of azelastine are illustrated. The cell was held at -40 mV, and the command voltage pulses to + 10 mV were applied at 0.2 Hz. Various concentrations of azelastine (1-100 ~M) were examined. The amplitude of the peak IBa during the application of azelastine was compared with the control value. The percent inhibition by azelastine of In,, is illustrated. The mean + S.D. value is shown, and the data were obtained from five different cells.

146

H. Hazama et al. / European .lournal of Pharmacology 259 (1904) 143-150

in the bathing solution caused a significant shortening of muscle length (Fig. 1, right panel), indicating that single tracheal smooth muscle cells contract in the high K+-bathing solution. However, in cells pretreated with azelastine (50/xM), the cells did not contract on application of the high K + solution, as illustrated in Fig. 1 (middle panel). Similar results were obtained from three different cells. These results indicate that azelastine inhibits the contraction of single tracheal smooth muscle cells induced by high K ~, which is comparable with previous results obtained with tracheal smooth muscle tissues (Sanagi et al., 1992). 3.2. Azelastine inhibits the L-type Ca-' + current in tracheal smooth muscle cells To clarify the underlying mechanism of the inhibitory effects of azelastine on the high K+-induced contraction, we examined the effects of azelastine on the lu, through the voltage-dependent L-type Ca 2~ channels in single tracheal smooth muscle cells by using the whole-cell voltage-clamp technique. In guinea-pig tracheal smooth muscle cells, we have already reported that only voltage-dependent L-type Ca 2+ currents exist (Hisada et al., 1990; Iguchi et al.. 1992). As shown in Fig. 2(A,B), the cell was held at - 4 0 mV, and the command pulses (300 ms in duration) to + 0 mV were applied at 0.2 Hz. The addition of azelastine (Ill /zM) caused a decrease in I m from 320 to 180 pA, and moreover, azelastine (100 /xM) completely abolished ll~,. The inhibitory effects of azelastine on I m were completely reversible. Fig. 3 shows the effects of azelastine (10/zM) on the current-voltage relationships of 1B:,. The cell was held at - 4 0 mV, and the command voltage pulses to various membrane potentials were applied, Azelastine decreased the amplitude of I m at any command pulse, as illustrated in Fig. 3. The configuration of the currentvoltage relationships was not altered by azelastine significantly. Also, the apparent reversal potential of ll~. failed to be changed in the absence or presence of azelastine (10/xM). Similar results were obtained from three different cells. Fig. 2C shows the relationship between the concentrations of azelastine and the percent inhibition of / m. The data were obtained from five different cells tested. Azelastine (1-100 /xM) inhibited IB~~ in a concentration-dependent manner. The half-maximal concentration required for the inhibitory effect of azelastine was approximately 8 /xM, and 100 /zM azelastine almost completely suppressed I m. These results clearly indicate that azelastine has a significant Ca-antagonistic effect in single tracheal smooth muscle cells. This effect may underlie the ionic mechanisms of the inhibition of azelastine on the high K+-induced contraction, as shown in Fig. 1.

3.3. Voltage and use dependence o1" azelastine-induced inhibition of" Ca: + current To characterize the inhibitory effects of azelastine on I~,,, we further examined the voltage dependence of the effect of azelastine on lB,. Fig. 4A shows the quasi-steady-state inactivation (.ft,) curve of 1~,, with the addition of azelastine (10 #M). The double pulse protocol (sec under Materials and mcthods) was used. A 3-s conditioning pulse to various membrane potentials preceded the test pulse (200 ms in duration) to + 10 mV from a holding potential of 80 mY. The relationships between the membrane potentials and the /'~_ value with or without the application of azelas-

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100 Fig. 3. l n h i b i t o ~ effects of azelastine on the IAypc C a " currcnt (lu~0 in single tracheal smooth muscle cells from the guinea-pig. The cell was held at - 4 0 mV, and c o m m a n d voltage pulses to various m e m b r a n e potentials (300 ms in duration) were applied at 0.2 Hz. 111 (A), the original current traces are shown in control (left panel), after the application of azelastine (1[) /.tM) (middle panel), and after washout of the drug (right panel). The current-voltage relationships of the peak llS. in control (open circle), after the application of azelastine (closed circle) and after washout of the drug (open square) are illustrated in (B).

H. Hazama et al. /European Journal of Pharmacology 259 (1994) 143-150

f~.ma× and b = the slope factor. In control, J~'max = 1, a = - 3 2 . 4 mV, b = 10.1 mV, whereas in the presence of azelastine (10 /xM), f~c.max=0.6, a = - - 4 8 . 0 mV, b = 11.7 mV. Thus, azelastine not only inhibited the maximal conductance of IB~~, but also shifted the f~ curve to the hyperpolarized potential by - 2 0 mV (n = 3). These results indicate that azelastine inhibits

tine (10 p.M) were fitted to the Boltzman equation, using least-square methods: f ~ ( V ) = f~.max/{1 + e x p [ ( V -

147

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where f~.m~ = the maximal value of f~ (in the control condition, the value of f~., .... = 1), V = the m e m b r a n e potential in mV, a = the m e m b r a n e potential at 1 / 2

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Fig. 4. (A) Quasi-steady-state inactivation curve of IBa with and without the addition of azelastine. By the use of the double-pulse protocol, the quasi-steady-state inactivation p a r a m e t e r (f=) of IBa was obtained in the control and with the application of azelastine (10 /xM) (see under Materials and methods). T h e relative amplitude of /Ba in response to the test pulse was determined at each m e m b r a n e potential of the conditioning pulses. The relationships between the m e m b r a n e potential and f~ in the control and with the addition of azelastine (10 ~,M) were fitted to the Boltzman equation (see text for details). Control: closed circle. Azelastine (10/xM): open circle. (B) Tonic and use-dependent block of IBa by azelastine. The cell was held at - 4 0 mV, and c o m m a n d voltage pulses of + 10 mV (200 ms in duration) were applied at 0.2 Hz. The protocol for superfusing azelastine is denoted by the bar indicated in the lower part of the panel. The current traces (a-f) were obtained at the times shown in the lower part. In (B), the alteration of the relative amplitude of 1Ba during the addition of various concentrations of azelastine (10 and 100/xM) is plotted. The amplitude of IBa in the control was taken as 1.0. After the voltage steps were stopped, various concentrations of azelastine were added to the bath. After approximately 90 s in the control BaZ+-bathing solution or azelastine (10 and 100 /zM)-containing solution, repeated depolarizing pulses to + 10 mV at 0.2 Hz were applied again.

H. Hazama et al. / European Journal O#Pharmacology 259 (1994) 143 150

148

the voltage-dependent L-type Ca 2+ current in a voltage-dependent manner in single tracheal smooth muscle cells. Furthermore, the use-dependent property of the inhibition of IB~, by azelastine was characterized. As shown in Fig. 4B, the command voltage pulses from - 4 0 to + 10 mV (200 ms in duration) were applied at 0.2 Hz. The amplitude of IB, , remained constant in the control Ba 2+ bathing solution (Fig. 4B, a and d). The depolarizing pulses were then stopped and various concentrations (10 and 100 /,M) of azelastine were applied to the cells. After 90 s of cessation, repeated depolarizing pulses to + 10 mV at 0.2 Hz were applied to the cells again. The relative amplitude of 1~,, with respect to that before the addition of azelastine was plotted in the lower part of Fig. 4B. After application of 10 # M azelastine, the amplitude of 1B,, evoked by the first voltage step after the 90-s pause was already suppressed by approximately 40% (tonic inhibition, Fig. 4B, e). During the repeated pulse stimuli, the

amplitude of IB~, remained constant (Fig. 4B, f). When the concentration of azelastine was increased to 100 /zM, the amplitude of I~, at the first depohtrizing pulse was fully suppressed (Fig. 4B, b). These results indicate that azelastine produces a tonic bh)ck of the voltage-dependent L-type Ca 2+ current in single tracheal smooth muscle cells. 3.4. £ f f e c t s q/"azelastine on the K ' currents in single tracheal s m o o t h muscle cells

To investigate further the effects of azcIastine on the other currents, we used the K+-pipette solution. The cell was held at 70 mV and command voltagc pulses to various membrane potentials were applied. In the control, as illustrated in Fig. 5A (left panel), three components of the outward currents were elicited as previously described (Hisada et at., 1990; lguchi et al., 1992): (1) a voltage-dependent transient outward K ~ current, which was actiwtted by depolarizing pulses and

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Fig. 5. Effects of azelastine on K + currents in single tracheal smooth muscle cells from the guinea-pig. In (A), the patch pipetle contained the K+-internal solution. The bath was perfused with the Ca2+-containing normal Tyrode solution. The cell was held at 70 mV, and command voltage pulses to various m e m b r a n e potentials were applied at 0.2 Hz. The original current traces are illustrated in control (left panel), after the application of azelastine (10/,tM) (middle panel) and after washout of the drug (right panel). The zero current level is indicated by an arrow. In (B), the current-voltage relationships are illustrated in control (open circle), azelastine (10 /zM) (closed circle) and after washout of the drug (open triangle). The initial current-voltage (I-V) relationships are indicated in (a), and the I-V relationships measured at the end of the pulse are indicated in (b). In (C), effects of azelastine on the Ca2*-activated oscillatory K ~ current (ST()(?) are indicated. The patch pipette contained the K+-internal solution. The cell was held at - 20 mV, and the continuous chart recording of the holding current is indicated in the tipper part o! the panel. In the lower part, the current traces ( a - d ) are illustrated at the times indicated in the tipper part. The drug sequences arc shown in the upper part of the panel, and the zero current is indicated by a triangle.

H. Hazama et al. /European Journal of Pharmacology 259 (1994) 143-150

declined during the command voltage steps (IT); (2) a Ca2+-activated oscillatory K + current during depolarizing pulses, which is closely related to the release of Ca 2+ from intracellular stores (spontaneous transient outward current, IsToc; Benham and Bolton, 1986); and (3) a steady K + current (Is). After application of azelastine (10 txM, middle part of panel A), the I T and I s components were inhibited. The effects of azelastine on K + currents were fully reversible, as shown in the right part of panel A. The current-voltage relationships of the peak and the current at the end of the pulse are illustrated in panel B, a and b in control, during the application of azelastine (10 mM) and after washout. Fig. 5C illustrates the effects of azelastine on the Ca2+-activated oscillatory K + current (IsToc) at a holding potential of - 2 0 inV. Azelastine (10 /zM) did not significantly inhibit ISTOO but markedly inhibited ISTOC at doses of 100 /zM. The inhibitory effect of azelastine on ISTOC was also reversible.

4. Discussion

In the present study, we showed that azelastine (1-100 /zM) inhibits IBa through the voltage-dependent L-type Ca 2+ channel in a concentration-dependent manner in single tracheal smooth muscle cells from the guinea-pig, which may result in inhibition of contractions evoked by high K + solution. In addition, azelastine has depressant effects on K + currents (i.e. the voltage-dependent transient outward K + current and the Ca2+-activated oscillatory K + current) at doses higher than 10 /~M. These results suggest that the inhibition of the voltage-dependent L-type Ca 2+ channel is one of the ionic mechanisms underlying azelastine-induced relaxation in tracheal smooth muscle cells. In smooth muscle, including trachea, it is known that high K + evokes contraction by depolarizing the m e m b r a n e potential, thereby opening voltage-dependent Ca 2+ channels. Our result illustrates that single tracheal smooth muscle cells contracted during superfusion of high K+-bathing solution, as shown in Fig. 1. The inhibition of the high K+-induced contraction by azelastine suggests that azelastine may act as a Ca 2+ channel antagonist. Our results provide direct evidence that azelastine inhibits the voltage-dependent L-type Ca 2+ current in tracheal smooth muscle cells. Because of these inhibitory effects on the Ca 2+ current, it is likely that azelastine inhibits action potentials and contraction evoked by tetraethylammonium in guinea-pig trachea (Lee et al., 1990). The ICs0 value of azelastine required for the inhibition of the Ca 2+ current was approximately 8 p.M in the present study. In guinea-pig or canine tracheal muscle, it has been reported that azelastine inhibits the "Qm~,x of tetraethylammoniuminduced action potentials with an ICs0 value of 5 /zM,

149

which is not significantly different from our results (Lee and Sperelakis, 1989; Lee et al., 1990). Also, recently, in guinea-pig ileal smooth muscle cells, Masuo et al. (1992) reported that azelastine inhibited the voltage-dependent L-type Ca 2+ channel (ICs0 = about 13/zM). This ICs0 value is quite similar to the present value obtained with the guinea-pig tracheal smooth muscle cells. Thus, it is likely that there is not a significant difference in azelastine sensitivity between tissues and species, including human tissue. The inhibitory effect of azelastine on the Ca 2+ current was voltage-dependent, and it shifted the steady-state inactivation curve to the left along the voltage axis (Fig. 4A), which suggests that the inhibitory action of azelastine on the Ca 2+ current seems to be more pronounced as the holding potential is more depolarized. Use-dependent inhibition of the Ca 2+ current was not observed in the case of azelastine. Inhibition of the Ca 2+ current was observed at the first depolarizing pulse after the application of azelastine and further inhibition was not observed during the repeated pulses. Thus, the lack of use-dependent inhibition of azelastine on the Ca 2+ current may relate to the smaller effect of azelastine in the heart compared to that of other Ca 2+ antagonists, such as verapamil (Lee and Tsien, 1983). In addition to the Ca 2+ antagonistic effect, azelastine had inhibitory effects on other m e m b r a n e currents in single tracheal smooth muscle ceils, as illustrated in Fig. 5. Azelastine at doses higher than 10/zM inhibited the voltage-dependent K + currents (Ix, Is) , and Ca 2*activated oscillatory K + c u r r e n t (/STOC) of tracheal smooth muscle cells. The inhibitory effect o n I f and ISTOC may be partly due to the inhibition of Ca 2+ current by azelastine, since these currents are dependent on the submembrane Ca 2+ concentration, which can be affected by Ca 2+ influx through Ca 2+ channels (Hisada et al., 1990). In addition, azelastine inhibits Ca 2+ release from intracellular storage sites (Lee et al., 1990; Senn et al., 1991), which may also contribute to the inhibitory effects of azelastine on these K + currents. However, as Lee and Sperelakis (1989) reported that azelastine failed to affect the resting membrane potential, the inhibitory effect of azelastine on K + currents appears to be relatively mild, in comparison to its Ca2+-antagonistic effect. Actually, in single tracheal smooth muscle cells from the guinea-pig, azelastine (10 # M ) failed to affect the m e m b r a n e potential significantly (not shown). Katayama et al. (1987) reported that the action of azelastine was related to an increase in intracellular cyclic A M P level. However, our previous studies showed that agents which increase intracellular cyclic A M P levels, such as isoproterenol, theophylline or dibutyric cyclic AMP, did not affect the L-type Ca :+ current in single tracheal smooth muscle cells (Iguchi

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et al., 1992). Therefore, it is unlikely that an increase in cyclic AMP is involved in the inhibitory effect of azelastine on the Ca 2+ current. Also, the inhibitory effect of azelastine on K + currents does not appear to be mediated by cyclic AMP, because in tracheal smooth muscle cells, isoproterenol, which increases cyclic AMP, activates Ca2+-dependent K + currents (Kume et al., 1989). Recent papers showed that azelastine also inhibits agonist-induced Ca 2+ release and agonist-induced Ca 2* sensitization of contractile elements in guinea-pig ileal and tracheal smooth muscle cells (Masuo et al., 1992; Sanagi et al., 19921. In the present study, we could not determine the mechanism by which azelastine produces bronchodilator activity. However, the potent effect of azelastine to inhibit Ic~ , as well as the above-mentioned actions makes it a very promising drug to relax airway smooth muscle.

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