Naunyn-Schmiedeberg's Arch Pharmacol (1992) 346:691- 700

Naunyn-Schmiedeberg's

Archivesof Pharmacology © Springer-Verlag1992

K + channel openers, cromakalim and Ki4032, inhibit agonist-induced Ca 2 + release in canine coronary artery Toshio Yamagishi, Teruyuki Yanagisawa, and Norio Taira Department of Pharmacology, Tohoku University School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai 980, Japan Received May 25, 1992/Accepted August 14, 1992

Summary.

The effects of K + channel openers, cromakalim and an acetoxyl derivative of KRN2391 (Ki4032), were studied on force of contraction, increases in intracellular calcium concentration ([Ca2+]i) measured by fura-2 and inositol 1,4,5-trisphosphate (IP3) production induced by the thromboxane A 2 analogue, U46619, in canine c o r o n a r y arteries. U p o n single dose applications of U46619 at 300 nmol/1, phasic and tonic increases in [Ca2+]i and force were seen, which were almost abolished by cromakalim (10 ~mol/1) and Ki4032 (100~tmol/1). In the absence of extracellular Ca 2+, U46619 induced a transient increase in [Ca2+]i with a contraction. Cromakalim ( 0 . 0 1 - 1 0 ~tmol/l) and Ki4032 (0.1 - 100 Ixmol/1) concentration-dependently inhibited the increases in [Ca2+] i and contraction. The inhibitory effects of cromakalim and Ki4032 were blocked by the K + channel blocker tetrabutylammonium (TBA) and counteracted by 20 mmol/1 KCl-induced depolarization. Cromakalim and Ki4032 did not affect caffeine-induced Ca 2+ release. Cromakalim reduced U46619-induced IP 3 production significantly and TBA blocked this inhibitory effect. These results suggest that the hyperpolarization of the plasma membrane by K + channel openers inhibits the production of IP 3 and Ca 2+ release from intracellular stores related to stimulation of the thromboxane A 2 receptor.

Key words: K + channel openers - Tetrabutylammonium (TBA) Thromboxane A 2 analogue 1,4,5-trisphosphate - Ca 2+ release

-

Inositol

smooth muscle (Nelson et al. 1990). Nevertheless, contractions induced by agonists are effectively inhibited by the K + channel opener cromakalim (Hamilton et al. 1986; Cook 1988). At present the relaxant action of cromakalim is generally understood to be a consequence o f its ability to hold the membrane potential of vascular smooth muscle cells close to their K + equilibrium potential (Hamilton et al. 1986) and thus deactivate voltage dependent Ca 2+ channels. However, the finding that the dihydropyridine Ca 2+ channel blocker isradipine was less potent than cromakalim in inhibiting angiotensin II-induced contractions in rabbit aorta suggested that cromakalim could exert effects additional to the indirect closure of L-type Ca 2+ channels (Cook et al. 1988). Cromakalim may also exert a direct inhibitory action on Ca 2+ uptake into and release from Ca 2+ stores and additionally inhibit the pathway through which Ca 2+ passes from the extracellular space to intracellular Ca 2+ stores (Bray et al. 1991). To clarify the relaxant mechanism of K + channel openers, we examined contractions induced by an agonist, U46619 (a thromboxane A2 analogue) and relaxations produced by K + channel openers using simultaneous measurements of force and [Ca z+ ]i with a fura-2 microscopic fluorimetric method. As K + channel openers are structurally different and therefore likely to possess additional actions unrelated to K + channel opening action (Edwards and Weston 1990; Yanagisawa et al. 1990), we used cromakalim (Yanagisawa et al. 1990) and an acetoxy derivative of KRN2391, Ki4032 (Okada et al. 1991), as selective K + channel openers in canine coronary arteries.

Introduction

Methods

Mechanisms involved in contractions following receptor stimulation by agonists are far more complex than those following simple membrane depolarization in vascular

Preparation of arterial rings. Hearts were obtained from mongrel dogs of either sex, weighing 6 to 15 kg, anaesthetized with sodium pentobarbital (30 mg/kg, i.v.). Coronary arterial rings (0.6-1.2 mm in diameter,

Correspondence to T. Yanagisawa at the above address

about 1 mm in width) were dissected and connective tissues were carefully removed in a dissecting chamber under a binocular microscope. The endothelium was removed by gentle rubbing and the luminal side was turned to face outwards.

692

Fluorescence measurements. We performed fluorescence measurements as previously described (Yanagisawa et al. 1989). In brief, coronary arterial rings were exposed to 10 ~mol/1 fura-2 acetoxymethyl ester (fura-2 AM) for about 2 h at 37 °C. The noncytotoxic detergent pluronic F-127 (0.1% W/V) was premixed in the loading physiological salt solution (PSS) to help dissolve the fura-2 AM in PSS. After the fura-2 loading, muscles were rinsed with normal PSS for more than 1 h and then used for experiments. Fluorescence was measured in a fluorimeter equipped with a dual wavelength excitation device (CAM-200 or CAM-220, Japan Spectroscopic, Tokyo, Japan) connected to an inverted microscope (TMD-8, Nikon, Tokyo, Japan). The fluorescence image was obtained by focussing on the smooth muscle cells in the medial layer with a Nikon CF UV (Fluor) 10× objective lens. The muscle ring was placed horizontally in a temperature-controlled 0.4 ml tissue bath which was mounted on the inverted microscope and perfused with PSS at a rate of 4 ml/min. The muscle ring was stretched to a resting tension of about 5 mN between two tungsten needles, one of which was glued to a transducer element (AE801, AME, Horten, Norway or U-10230, Shinko, Tokyo, Japan). The photosignals and the mechanical activity were measured simultaneously and both were recorded on a chart recorder (Rectihoriz-8K, NEC-San-ei, Tokyo, Japan). They were also digitized by A / D converters and fed into a microcomputer (PC-9801, NEC, Tokyo, Japan) for further calculation and graphic analysis (Himpens and Somlyo 1988). Every experiment commenced with perfusion with 90retool/1 KC1-PSS for 10 min. At the end of each experiment, the cell membrane was lysed with the detergent Triton X-100 (1%0), and autofluorescence signals were determined by quenching fura-2 signals with MnC12. We subtracted the F340 and F380 values due to autofiuorescence from the corresponding values of F340 and F380 obtained under conditions of fura-2 loading to derive a recalculated ratioro. Since it is difficult to calculate the absolute concentration of [Ca~+] i (Konishi et al. 1988; Karaki 1989), we used the relative F340/F380 ratio as an indicator of [Ca2+] i (Sato et al. 1988; Yanagisawa et al. 1989). Changes in the ratiOrc (changes in [Ca2+]i) and those in force were expressed as percentages of the differences between basal values and those obtained with a 10 min perfusion with 90 mmol/1 KC1-PSS. None of the drugs and chemicals used in the present study, with the exception of fura-2 and MnC12, affected fluorescence signals at the concentrations used.

Protocol Effects of cromakalim and Ki4032 on Ca2+ release and influx induced by U46619. After perfusion with 90 nmol/1 KC1-PSS for 10 rain followed by the perfusion with 5 mmol/1 KC1-PSS for 10 rain, the arterial rings were exposed to U46619 (300 mmol/1) for 5 min. After washout with drug-free 5 mmol/1 KC1-PSS for 20 rain, the rings were perfused with 90 retool/1 KC1-PSS for 10 rain. This was washed out by the drugfree 5 retool/1 KC1-PSS for 5 rain and the preparation was perfused with 5mmol/1 KC1-PSS containing cromakalim (10~tmol/1) or Ki4032 (100 ~tmol/l) for 5 rain. The effects of U46619 were examined in the presence of cromakaiim or Ki4032. After a 20-min period of washout with drug-free PSS, 10-min perfusion with 90mmol/1 KC1-PSS and 10-min perfusion with 5 mmol/1MKC1-PSS, the effects of U46619 were examined in the absence of drugs to check the reversibility of the responses of the preparation.

1 KC1-PSS. Following a 5 min pre-incubation in various solutions containing cromakalim and/or TBA in the presence and in the absence of extracellular Ca 2+ ([Ca2+ ]o), the rings were stimulated with 300 nmol/1 U46619 for a predetermined time (10-300 s) and immediately frozen in liquid nitrogen. After measuring the wet weight of each ring, it was homogenized with 1 ml 10% trichloroacetic acid. The homogenate was centrifuged, the supernatant was extracted three times with diethylether and then neutralized by KOH. The IP 3 content was assayed by a sensitive radioimmunoassay kit from Amersham International plc. The results were expressed as pmol/mg wet weight of tissue.

Drugs and solutions. The composition (mmol/1) of normal or 5 mmol/ 1KC1PSS was as follows: NaCI 140, KC15, CaCI 2 2.5, MgC12 2.5, glucose 11.1, HEPES 3 (pH 7.4). The solution was equilibrated with 100%0 0 2 at 37°C. High KC1-PSS was made by substituting NaC1 with equimolar KC1. Ca 2+-free PSS was made by removing CaC12 from normal PSS and by adding 1 mmol/l EGTA to the solution. Drugs and chemicals were obtained from the following sources: cromakaiim (Beecham, Harlow, UK), Ki4032 (Kirin Brewery, Tokyo, Japan), U46619 (9,11-dideoxy-lla,9a-epoxymethano-prostaglandin F2a; Sigma, St Louis, Mo., USA), EGTA (ethyleneglycol-bis-(B-amiuoethylether)N,N,N,N'-tetra-acetate), HEPES (N-2-hydroxyethyl-piperazine-N'-2-ethanesulphonic acid), fura-2 AM and pluronic F-127 (Dojin, Kumamoto, Japan), glibenclamide (Yamanouchi Pharmaceutical, Tokyo, Japan), gliclazide (Dainippon Pharmaceutical, Osaka, Japan), TBA (tetrabutylammonium chloride), caffeine, DMSO (dimethylsulphoxide), Triton X-100, trichloroacetic acid and diethylether (Wako, Tokyo, Japan). U46619 was dissolved in ethanol to give a concentration of I retool/1. Cromakaiim was dissolved in 70%0 ethanol to give a concentration of 10 mmol/1. Ki4032 was dissolved with PSS immediately before using and the pH of the solution was adjusted to 7.4. Caffeine was dissolved in PSS at a concentration of 25 mmol/1. Fura-2 AM was dissolved in DMSO at a concentration of 1 mmol/1. Pluronic F-127 was dissolved in 25%0W/V in DMSO. Triton X-100 was dissolved in 1%o W/V in PSS. Other drugs or chemicals were dissolved in distilled water and diluted to the desired concentrations by PSS.

Analysis of concentration-response curves and statistical analysis. Experimental values are given as mean+SEM. The concentration-response curves for cromakalim and Ki4032 on changes in [Ca2+] i and force induced by U46619 in Ca 2+-free PSS were computer fitted to the following equation:

E = 100- IO0×AP/(A p +K p) where E is the effect normalized by the differences between the values just before the application of U46619 and the peak values of [CaZ+] i and force expressed by percentage (control= 100%), A is the cromakaiim or Ki4032 concentration, K is ECs0 value of the drug and p is the slope parameter. ECs0 values were presented as pD 2 (pD 2 = - l o g ECs0). Statistical significance of results was evaluated using a one-way analysis of variance followed by Dunnett multiple-comparison test or Kruskal-Wailis test. A P-value smaller than 0.05 was considered to be significant.

Effects of eromakalim and Ki4032 on Ca2+ release induced by U46619 or caffeine. To eliminate the influx of Ca 2+ , Ca2+-free PSS

Results

was perfused 5 rain before the application of U46619 (300 nmol/1) or caffeine (25 mmol/1). The concentration-response curves for cromakalim (0.01 - 10 ~tmol/1) and Ki 4032 (0.1 - 100 ~mol/1) to inhibit the increases in [Ca2+]i and force induced by U46619 were obtained in Ca2+-free PSS. To examine the modification of the effects of cromakalim and Ki4032 on U46619-induced responses by membrane depolarization or TBA, 20 retool/1 KC1-PSS was perfused 5 min before, and during the application of U46619 or TBA (3 mmol/1) was administered 20 min before, and during the application of U 46619.

M o d i f i c a t i o n s by cromakalim and Ki4032 o f changes in [ C J + ] i and f o r c e induced by U46619 in the presence o f [ C ~ + l o

Measurement of inositol 1,4,5-trisphosphate (IP3). Endothelium-denuded arterial rings were equilibrated for 2 h at 37°C in 5 m m o l /

Figure I a and b shows typical examples of changes in [ Ca2+ ]i a n d f o r c e i n d u c e d b y a p p l i c a t i o n o f a s i n g l e c o n centration of U46619 (300nmol/1) in the presence of [Ca2+]o. T h e s u m m a r i z e d d a t a o b t a i n e d f r o m 6 p r e p a r a t i o n s e a c h a r e p r e s e n t e d i n Fig. l c a n d d. U 4 6 6 1 9 p r o d u c e d a n i n c r e a s e i n [CaZ+] i b y a b o u t 40°70 a t t h e p e a k and with a gradual reduction, whereas the force of con-

693 a 90 mmol/I KCI

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U46619(300 nmol/I)

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traction induced by U46619 tended to be maintained. In the presence of cromakalim (101xmol/1) or Ki4032 (100 gmol/1), [Ca2+]i was reduced below the basal level, and cromakalim greatly inhibited the increases in [Ca 2÷ ]i and force in both phases induced by U46619. After washout of cromakalim or Ki4032, the effects of U46619 were almost the same as those induced by the first application (data not shown).

Modifications by cromakalim and Ki4032 of the effects of U46619 in the absence of [Ca2+]o In the absence of [Ca 2+ ]o, U 46619 produced a transient increase in [Ca ]i and force of contraction, the latter was followed by sustained contraction on a reduced level. After washout with 5mmol/1KCI-PSS, [Ca2+]i increased to a level higher than the basal one and a larger force was generated (Fig. 2a). Peak increases in [Ca2+]i and force occurred within 1 rain after exposure to U46619, the peak of [Ca2+] i slightly preceded that of force. Since there was no Ca 2+ to influx, the transient increases in [Ca2+]i was due to the Ca 2+ release from intracellular stores. When the preparation was perfused with Ca2+-free PSS containing cromakalim (10 ~tmol/1), no further reduction in [Ca 2+ ]i was seen. In the presence of cromakalim (10 lxmol/1), transient increases in [Ca2+]i and force induced by U46619 were almost abolished,

0

5

Time (rain)

25

Fig. 1 a - d. Modification by cromakalim and Ki4032 of changes in [Ca2+]i and force of contraction induced by U46619 (300 nmol/1) in the presence of [Ca2+]o. a Typical traces of [Ca2+]i (ratio) and force with and without cromakaiim (CRK; 10 Ixmol/1). After the application of U46619 for 5 min, all preparations were washed with 5 mmol/1 KC1-PSS without drugs, b Typical traces of [Ca2+]i and force with and without Ki 4032 (100 gmol/1), e The summarized data obtained from 6 preparations in the absence (©) and presence ( e ) of 10 gmol/1 cromakalim. d The summarized data obtained from 6 preparations in the absence (©) and presence ( e ) of 100 gmol/l Ki4032

whereas a diminished tonic force of contraction still remained. Ki4032 had effects similar to cromakalim (Fig. 2b). The summarized data obtained from 6 preparations are shown in Fig. 2c, d and Table 1. The concentration-response curves for cromakalim and Ki4032 to inhibit increases in [Ca2+] i and force induced by U46619 are shown in Fig. 3. Cromakalim and Ki4032 inhibited the increases in [Ca2+] i and force in a concentration-dependent manner. At the highest concentration of cromakalim (10 gmol/1) and Ki4032 (100 gmol/1), the increase in [Ca2+]i and force were abolished. The pD2 values of cromakalim to inhibit increases in [CaZ+]i and force are 6.88___0.38 and 6.85 + 0.46, respectively. Those values of Ki4032 are 5.67+0.56 and 4.96+0.14, respectively (n = 4 - 6).

Counteraction by KCl-induced depolarization of the effects of cromakalim and Ki4032 Figure 4a and b shows the influence of the perfusion of CaZ+-free 20 mmol/1 KC1-PSS on the inhibitory effects o f cromakalim and Ki4032 on changes in [Ca2+]i and force induced by U46619. [Ca2+]i in the presence of cromakalim (10 vmol/1) or Ki4032 (100 ~mol/1) during the perfusion with Ca2+-free 20 mmol/1 KC1-PSS was not different from that in their absence during the perfu-

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Table1. Effects of cromakalim and Ki4032 on changes in

'

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[Ca2+]i and

force induced by U46619 in the absence of [Ca2+] o n

Peak [Ca2+]i (%)

Peak force (%)

Control + C r o m a k a l i m 10ixmol/1 +Ki4032 1001xmo]/1

11 5 6

100+ 0 9 + 7* 0_+ 0*

100+ 0 10+_ 6* 7 + 5*

2 0 m m o l / l KC1 + Cromakalim 10 ~tmol/1 + Ki4032 100 ~tmol/]

5 6 6

136_+31 77+11 a 81 +_ 18 a

92+ 7 58+11 a 79 _+29 a

3 mmol/1 T B A + Cromakalim 10 pmol/1 +Ki4032 100 Ixmol/1

6 6 6

69 -+ 11 85+_38 a 52+ 8 a

87 _+23 60-+23 a 47-+ 11 a

* P < 0 . 0 1 , compared with control a P < 0 . 0 5 , compared with cromakalim or Ki4032 Peak [Ca2+] i, peak force: differences between the values just before the application of U46619 and the peak values of [Ca 2+ ]i and force expressed as a percentage (control = 100%). The m e a n values of the increases in [Ca 2 + ]i and force in the control are 18 _+2 and 32 + 4 (%; expressed as % of the increases following 10 rain exposure to 90 mmol/1 KC1-PSS; n = 45), respectively

]1

'

25

Fig. 2 a - d . Modification by cromakalim and Ki4032 of changes in [Ca2+] i and force of contraction induced by U46619 (300 nmol/1) in the absence of [Ca2+] o. a Typical traces of [Ca2+] i and force with and without cromakalim (CRK; 10 Ixmol/1). After the application of U46619 for 5 min, all preparations were washed with 5 mmol/1 KC1-PSS without drugs, b Typical traces of [Ca2--]i and the force with and without Ki4032 (100 pmol/1), e The summarized data obtained from 6 preparations in the absence ( o ) and presence ( e ) of 10 pmol/1 cromakalim, d The summarized data obtained from 6 preparations in the absence ( O ) and presence ( • ) of 100 gmol/1 Ki4032

sion with Ca 2+-free PSS (5 mmol/1 KC1). The inhibitory effects of cromakalim and Ki4032 were abolished in Ca 2+-free 20 mmol/1 KC1-PSS (Table 1). It is known that an increase in KC1 concentration does not block opening of K + channels by cromakalim and Ki4032 but rather increases the conductance of K + channels. Thus, the inhibitory effects of cromakalim and Ki4032 on the U46619-induced Ca 2+ release might be exclusively due to their hyperpolarizing action.

Blockade by TBA of the effects of cromakalim and Ki4032 Figure 4c and d shows the influence of TBA (3 mmol/1), o n e of non-selective K + channel blockers, on the effects of cromakalim (10 ~tmol/1) or Ki4032 (100 ~tmol/1) on changes in [Ca2+]i and force induced by U46619 in Ca2+-free PSS. Because sulfonylureas, such as glibenclamide (10 ~tmol/l) and gliclazide (100 Ixmol/1), inhibited the effects of U46619 in our experiments (data not shown) and it has been reported that glibenclamide is an

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Fig. 3. Concentration-response curves for the inhibition by cromakalim (a) and Ki4032 (b) of increases in [Ca2+] i and force of contraction induced by U46619 in the absence of [Ca2+] o. The experimental protocol

20 mmoI/I K, 0Ca I U46619 (300 nmol/I)

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Fig. 4 a - d. Influences of 20 mmol/1 KCl-induced depolarization or TBA on the inhibitory effects of cromakalim and Ki4032. a The summarized data obtained from 6 preparations in the absence (©) and presence ( e ) of i0 pmol/1 cromakalim (CRK) with Ca2+-free 20 mmol/1 KC1-PSS. b The summarized data obtained from 6 preparations in the absence ( # ) and presence ( e ) of 100 pro©l/1 Ki4032 with Ca2+-free 20 rnmol/1 KC1-PSS. c The summarized data obtained from 6 preparations in the absence (©) and presence ( e ) of 10 pro©l/1 cromakalim and 3 ram©l/1 TBA. d The summarized data obtained from 6 preparations in the absence (©) and presence ( e ) of 100 ~tmol/1 Ki4032 and 3 ram©l/1 TBA

696

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antagonist of the thromboxane A 2 receptor (Cock et al. 1990), we chose TBA to block K ÷ channels including the ATP-sensitive one. Since it takes more than 20 rain to obtain the effective K + channel blocking action of TBA (Yanagisawa et al. 1990), the application of TBA was started 20 rain before the exposure to U46619. TBA by itself increased [Ca2+] i in 5mmol/1KC1-PSS, however, after the perfusion with Ca2+-free PSS, [Ca2+]i was almost at the same level as in the absence of TBA (Fig. 4c and d). The inhibitory effects of cromakalim and Ki4032 on Ca z+ release induced by U46619 were blocked by TBA (Table 1).

No modification by KCl-induced depolarization and TBA of the effects of U46619 in the absence of [Ca?+]o Figure 5 and Table 1 show the summarized results of the influences of 20 mmol/l KCl-induced depolarization and TBA (3 mmol/1) on the increases in [Ca 2+ ]i and force induced by U 46619 in the absence of [Ca 2+ ]o. The increases in [Ca2+]i and force induced by U46619 were not changed in 20 mmol/1 KC1-PSS or by TBA.

No modification by cromakalim and Ki4032 on the effects of caffeine in the absence of [Ca?+]o Figure 6a and b shows typical traces of caffeine-induced increases in [Ca2+]i and force. Cromakalim (10 gmol/1) or Ki4032 gmol/1) did not affect the caffeine-induced Ca z(100 release and force of contraction. Furthermore, in the absence of [Ca2+] o, the application of caffeine (25mmol/1) subsequent to the application of U46619 produced Ca 2+ release but suppressed the force of contraction (Fig. 7). Differences between the values just before the application of U46619 and caffeine and the peak values of [Ca2+] i were 11+2% and 10_+2%, respectively (n = 4). The changes in force were 17_+4% and - 3 +_1%, respectively. In the presence of cromakalim (10 gmol/1), differences in [Ca 2+ ]i induc ed b y U 4 66 19 and caffeine were 1 _+3 % (P < 0.01) and 24_+ 11%, respec-

,

25

Fig. 5a, b. Influences of 20mmol/1 KCl-induced depolarization or TBA on changes in [Ca2+]i and force of contraction induced by U46619 in the absence of [Ca2+] o. a The summarized data obtained from 5 preparations in the absence (©) and presence (A) of 20 mmol/1 KC1-PSS. b The summarized data obtained from 6 preparations in the absence (©) and presence (A) of TBA (3 retool/l)

tively (n = 4). The changes in force were 7_+ 3 % (P < 0.05) and 0+_0%, respectively (n = 4). Thus, in the experiment with the sequential application of U46619 and caffeine, the Ca 2+ release induced by U46619 was inhibited by cromakalim, whereas that by caffeine was not inhibited but rather increased. The decrease in force by caffeine seems to be due to its inhibitory action on phosphodiesterase (Sato et al. 1988).

Effect of cromakalim on IP3 production induced by U46619 As shown in Fig. 8a, 300 nmol/1 U46619 increased the IP3 content in canine coronary artery in 5 mmol/1 KC1PSS containing 2.5 mmol/1 CaC1/. The production of IP 3 peaked around 20 s. The effect of cromakalim on the increase in IP3 was measured a t 20 s after the application of U46619. Cromakalim did not modify basal IP3 level but inhibited the production of IP 3 induced by U46619 significantly ( P < 0.05, Fig. 8b). TBA (3 mmol/1) blocked the inhibitory effect of cromakalim without affecting basal IP 3 level and U46619-induced IP 3 production. In the absence of [Ca2+] o, the IP3 contents were about onetenth of those in the presence of [Ca2+] o. Even in the absence of [Ca2+] o, U46619-induced increase in IP3 content was inhibited by cromakalim but the inhibition by cromakalim was not significant in Ca z+-free 20 retool/1 KC1-PSS (Fig. 8 c). Thus, the inhibitory effect of cromakalim was counteracted by the depolarization caused by 20 mmol/l KCI. Discussion

In the present study, it has become evident that, in canine coronary arteries, cromakalim and Ki4032 selectively inhibited the Ca 2÷ release from intracellular stores induced by a thromboxane A2 analogue U 46619 but not by caffeine. The inhibitory effect of K + channel openers seems to be dependent on membrane hyperpolarization and due to the inhibition of U46619-induced IP 3 production.

697

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Fig. 6a-d. Modification by cromakalim and Ki4032 of changes in [Ca2+]i and force of contraction induced by caffeine (25 mmol/1) in the absence of [Ca2+]o. a Typical traces of [Ca2+]i and force with and without cromakalim (CRK; 10 ~tmol/1). After the application caffeine for 5 min, all preparations were washed with 5 retool/1 KC1-PSS without drugs, b Typical traces of [Ca2+]i and force with and without Ki4032 (100 Ixmol/1). c The summarized data obtained from 4 preparations in the absence (©) and presence ( e ) of 10 ~tmol/1 cromakalim, d The summarized data obtained from 4 preparations in the absence (©) and presence (e) of 100 ~tmol/1 Ki4032

T h r o m b o x a n e A 2 is a potent constrictor o f vascular s m o o t h muscle as well as a strong inducer o f platelet aggregation ( H a m b e r g et al. 1975; Ellis et al. 1976; M o n cada and Vane 1978). U46619 is a stable and full agonist analogue o f t h r o m b o x a n e A 2 (Hanasaki et al. 1988; Tymkewycz et al. 1991). Recently, the h u m a n thromboxane A2 receptor has been cloned, and its amino-acid sequence shows that it is one o f the G-protein coupled receptors (Hirata et al. 1991). The vasoconstriction by t h r o m b o x a n e A 2 analogues occurs t h r o u g h b o t h release o f Ca 2+ f r o m intracellular stores and increase in transmembrane Ca 2+ influx (Toda 1982; K a n m u r a et al. 1987). C r o m a k a l i m and Ki4032 are selective K + channel openers in canine c o r o n a r y arteries (Yanagisawa et al. 1990; O k a d a et al. 1991). They inhibit phasic and tonic increases in [ C a > ] i and force o f contraction induced by U46619 in the presence o f [CaZ+] o. Since the tonic increase in [Ca2+]i induced by U46619 was abolished by removal o f [Ca2+] o, it seems to be due to Ca 2÷ influx stimulated by U46619, similar to that induced by histamine (Mori et al. 1990). Thus, the inhibitory m e c h a n i s m o f K ÷ channel openers on Ca 2+ influx induced by

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Fig. 8. a Time course of IP 3 production stimulated by U46619 (300 nmol/1) and the effect of cromakalim on U46619-induced IP 3 production in the presence (b) and absence (e) of 2.5 mmol/1 [Ca 2+ ]o. Peak IP 3 production occurred around 20 s. The effects of cromakalim on this response were examined at 20 s after the application of U46619. Cromakalim inhibited the IP 3 production. TBA (3 mmol/1) blocked and 20 mmol/1 KC1 counteracted this inhibitory effect. Open columns: not stimulated by U46619; closed columns: stimulated by U46619. # represents a significant difference (P

K+ channel openers, cromakalim and Ki4032, inhibit agonist-induced Ca2+ release in canine coronary artery.

The effects of K+ channel openers, cromakalim and an acetoxyl derivative of KRN2391 (Ki4032), were studied on force of contraction, increases in intra...
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