(D Macmillan Press Ltd, 1991
Br. J. Pharmacol. (1991), 102, 113-118
Effects of glibenclamide on cytosolic calcium concentrations and on contraction of the rabbit aorta Kiyonobu Yoshitake, Katsuya Hirano & 'Hideo Kanaide Division of Molecular Cardiology, Research Institute of Angiocardiology, Faculty of Medicine, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka, 812, Japan 1 Using fluorometry of fura-2 and rabbit aortic strips, we studied the effects of glibenclamide (GLB), a sulphonylurea anti-diabetic drug and an inhibitor of opening of K+ channels, on cytosolic calcium concentrations ([Ca2"]j) and on force development. 2 Both high K+-depolarization and noradrenaline (NA) increased [Ca2+]i and force, in a concentrationdependent manner, in the presence of extracellular Ca2+ (1.25mM). However, force development in relation to [Ca21]i ([Ca2+]J-force relationship) observed with NA was much greater than that observed with K+-depolarization. 3 Pretreatment with GLB (10-6-10-4M) for 10min partially inhibited, in a concentration-dependent manner, both [Ca2 +]i elevation and the force development induced by 118 mm K + -depolarization or NA 1O- 5M in the presence of extracellular Ca2 . The [Ca2+ ]i-force relationship induced by both 118mM K+ physiological salt solutions and by NA 10- M in the GLB-treated strips overlapped that obtained in the non-treated strips, thereby suggesting that GLB has no effect on the Ca2 +-sensitivity of the intracellular contractile apparatus. Only high concentrations (10-4M) of GLB decreased [Ca2+]i and the force, when applied after the force induced by 118 mM K+ PSS or NA 10- s M reached the maximum level. 4 In the absence of extracellular Ca2+, NA induced a transient increase in [Ca2+ ], and in the force and these increases were inhibited when the vascular strips were pretreated with GLB for 10min. The [Ca2+]J-force relationship obtained in the GLB-treated strips overlapped that in the non-treated ones. 5 An ATP-sensitive K+ channel opener, cromakalim (10-5M) reduced the increased [Ca2 + ]i and force induced by 25mm K+-depolarization and NA 10-SM. Subsequent application of GLB concentrationdependently reversed this relaxant effect of cromakalim on the NA-induced contraction (IC50 = 2 10 7 M). Complete reversal of the effect was observed with 10IsM GLB. 6 We suggest that GLB inhibits both high K+-depolarization- and NA-induced contraction of the rabbit aorta, by decreasing [Ca2+]i and with no effect on the [Ca2+]i-force relationship. However, when NA-induced contractions were inhibited by a K+-channel opener, GLB reversed this inhibitory effect by inhibiting K+-channel opening and increasing [Ca2 +]. x
Introduction K + channels inhibited by intracellular adenosine 5'triphosphate (ATP) have been identified in vascular smooth muscle (Standen et al., 1989), and these channels are also present in pancreatic fl-cells (Ashcroft et al., 1984), cardiac cells (Noma, 1983), skeletal muscle cells (Spruce et al., 1985) and cortical neurones (Ashford et al., 1988). The most potent and selective inhibitor of this channel known at present is the sulphonylurea hypoglycaemic agent, glibenclamide (GLB) (Schmidt-Antomarchi et al., 1987). High-affinity sulphonylurea receptors have been noted on pancreatic cells (Gaines et al., 1988) but no evidence of these receptors on vascular smooth muscle has been documented. In pancreatic # cell membranes, the sulphonylurea receptor may be closely linked to or be part of an ATP-sensitive K+ channel (Gerich, 1989), and inhibition of the efflux of K+ by GLB may lead to depolarization; as a consequence, voltage-dependent Ca2+ channels on the membrane would then open and permit entry of Ca2+ (Boyd, 1988). An increase in cytosolic Ca2+ concentration ([Ca2"]j) and the resultant activation of myosin light chain kinase play a critical role in initiating contractions of smooth muscles. In the present study, we examined the effect of GLB on [Ca2+] and the force of rabbit aortic smooth muscle strips, using fluorometry of fura-2.
the level of the renal arteries were immediately excised. Fat and adventitia were removed by dissection, under a binocular microscope. The endothelial cells were removed by rubbing the luminal surface with a cotton swab (Furchgott & Zawadzki, 1980). Medial preparations were cut into 1 x 3 mm circular strips 0.2mm thick. The wet weight of the strips was 0.6 + 0.1 mg (n = 12). Tissue density of the strips was assumed to be 1.05 gcm3 (Murphy et al., 1974) and the cross sectional area of each strip was calculated from the following equation: Cross-sectional area (m2) = wet weight (mg)/length (mm)/1.05 (gcm-3) x 10-6. The mean value of cross-sectional area was 2.14 + 0.19 x 10- 7 m2 (n = 12).
Fura-2 loading
Methods
The vascular strips thus obtained were loaded with the [Ca2+]i indicator dye, fura-2 by incubation in medium containing 50pM fura-2/AM (an acetoxymethyl ester form of fura-2) and 1.25% foetal bovine serum for 6-8 h at 37°C. Subsequently, the strips were washed in physiological salt solution (PSS) containing 1.25 mm Ca2+ at 37°C to remove the dye from the extracellular space and were then incubated with PSS for about 1 h before initiation of the measurements. Strips thus treated showed the specific peak of the fluorescence emission spectrum for fura-2 (500nm) and the specific peak and the valley of the fluorescence excitation spectrum for fura-2, 340nm and 380nm, respectively, determined with a fluores-
Tissue preparation
cence spectrophotometer (model
Japanese white rabbits (male, 16-20 weeks old, body weight 2.5-3.0 kg) were killed with sodium pentobarbitone (lOOmgkg-1, intravenously) and the abdominal aortae below '
Author for correspondence.
650-40, Hitachi, Tokyo, Japan). Loading the vascular strips with fura-2 did not alter the time course or the maximum levels of force development during 118 mm K + depolarization, thereby suggesting that no tissue damage occurred by possible acidification of the cells due to formaldehyde released on AM-ester hydrolysis (Tsien et al., 1982; Rink & Pozzan, 1985).
114
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Measurement of tension
depolarization (100%) were 121 + 36 and 779 + 125 nm, respectively.
Strips were mounted vertically in a quartz organ bath, (strain gauge TB-612T, Nihon Koden, Tokyo, Japan). During a 1 h equilibration period, the strips were stimulated with 118mm K+ depolarization every 15min, and the resting tension was increased in a stepwise manner. After equilibration, the resting tension was adjusted to 200mg. The force development of the steady state was expressed as a % assuming the values in PSS (5.9mM K+) and 118mM K+ PSS to be 0% and 100%, respectively.
Drugs and solutions Physiological salt solution (PSS) was of the following composition (in mM): NaCl 123, KCl 4.7, NaHCO3 15.5, KH2PO4 1.2, MgCl2 1.2, CaCl2 1.25, and D-glucose 11.5. High potassium PSS was identical, except for an equimolar substitution of KCl for NaCl. Ca 2+-free solution was identical except for a substitution of 2mm ethylene glycol bis (f-aminoethyl ether) N,N,N',N'-tetraacetic acid (EGTA) for 1.25 mm CaCl2. PSS was gassed with a mixture of 95% 02-5% CO2 (pH adjusted to 7.4 at 370C). Glibenclamide (GLB, 5-chloro-N-[2-
Fluorometry
[4- [[[(cyclohexylamino)carbonyl]amino]sulphonyl]phenyl]
Changes in the fluorescence intensity of the fura-2-Ca2 + complex were simultaneously monitored during measurement of the force. The fluorescence intensity was measured by use of a fluorometer specially designed for fura-2 fluorometry (CAMOF-1), in collaboration with Japan Spectroscopic Co, Tokyo, Japan (Hirano et al., 1989). In brief, the strips were illuminated by guiding the alternating (400 Hz), 340nm and 380nm, excitation light from a Xenon light source through quartz optic fibres arranged in a concentric inner circle (diameter = 3 mm). Surface fluorescence of the strips was collected by glass optic fibres arranged in an outer circle (diameter = 7 mm) and introduced through a 500 nm band-pass filter (full width at half maximum transmission 10 nm) into a photon-counting photomultiplier. Special care was taken to keep the distance between a strip and the end of the optic fibres short and constant during each measurement. The ratio of the 500nm fluorescence at 340nm excitation to that at 380nm excitation was expressed as a %, assuming the values in PSS (5.9 mm K+) and 118 mm K+ PSS to be 0% and 100%, respectively. The absolute value of [Ca2+]i was estimated as described by Grynkiewicz et al. (1985). The mean value of 7 different measurements of [Ca2+]i at rest (0%) and during 118mm K+-
Statistical analysis Values are expressed as means + s.e.mean (n = 3). Student's t test was used to determine the statistical significance.
Results
Characteristics of changes in [Ca2j]i andforce induced by high external K + -depolarization and noradrenaline Representative recordings of changes in [Ca2+]i and force
induced by 118 mm K+-depolarization followed by NA 10 -M are shown in Figure 1. When the vascular strip was exposed to high external K+ (118 mM) solution containing 1.25 mm [Ca 2 1] (nM)
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ethyl]-2-methoxybenzamide) and cromakalim (3,4-dihydro3 - hydroxy - 2,2 - dimethyl - trans - 4 - (2 - oxo - 1 - pyrrolidinyl) 2H-1-benzopyran-6-carbonitrile) were generous gifts from Hoechst Japan and Chugai (Japan), respectively. GLB was disolved in dimethylsulphoxide (DMSO) and diluted in the organ bath (final concentration of DMSO was 1%). Fura-2/ AM was purchased from Molecular Probes (Eugene, OR, U.S.A.).
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-log [NaJ (M) [K+1 (mM) Figure 1 (a) Representative recordings of changes in fluorescence ratio (upper panel) and force development (lower panel) induced by high external K+ (118mM) physiological salt solution (PSS) followed by noradrenaline (NA) 10-5M in the presence of 1.25mM extracellular Ca2l. (b) Dose-response curve for the maximum force development (0) and the fluorescence ratio (0) induced by high external K + -depolarization in the presence of 1.25 mm Ca2 +. The abscissa scale is the concentration of K + in isosmotic high K + PSS. Measurements were done 5 min after application of high K+. Ordinates indicate percentage fluorescence ratio and force. Vertical lines indicate s.e.mean (n = 3). Note that the maximum [Ca2+]i and force induced by 40mM K+ are larger than those induced by 118mM K+. (c) Dose-response curve for fluorescence ratio (0) and the maximum force (0) induced by NA. Measurements were done at 6min after NA application. The abscissa scale is -log[NA]. Each value is expressed as percentage fluorescence ratio or force, assuming the values obtained during 118mM K+ application to be 100%.
GLIBENCLAMIDE EFFECTS ON SMOOTH MUSCLE CONTRACTION
Ca2", [Ca2 +]i rose abruptly by 111% in the first 20s, and
then, decreased slightly to reach a steady level at 6min; this level was assumed to be 100%. The force also rose rapidly and reached about 80% of the maximum level in the first 20s, the maximum level (100%) at about 5min, and remained at this level for at least 30 min. When 118 mm K+ PSS was changed to normal PSS, both [Ca2]i and the force rapidly reverted to the normal, pre-depolarization level, 0%. Subsequent application of 10-5M NA rapidly elevated [Ca2 ], with a first peak (67 + 18%, n = 3), reached in the first i5s. After a dip (to 38 + 7%, n = 3) at about 30s, [Ca2+]i gradually reached a second peak (57 + 9%, n = 3) at 3 min, and was then sustained at a slightly decreased level (42 + 11%, n = 3) for at least 20min. The force induced by NA 10- 5 M also rose rapidly to reach a level of 115% in the first 15s, the maximum level (178 + 12%, n = 3) at 6 min, followed by the steady state level (167 + 9%, n = 3), which was sustained for at least 20min. Figure lb and c show that the elevations of [Ca2+]i and force at the steady state induced by both external high K+ PSS and NA application were dose-dependent. It should be noted that in the rabbit aorta, the dose-response curves for [Ca2+]i and force induced by external high K+ PSS showed somewhat bell-shaped curves; the maximum [Ca2+]i and force were obtained with 40mm K+ PSS, and concentrations of K+ over 40 mm caused less steady state [Ca 2+ ]i and force development. In addition, force development in relationship to the increase in [Ca21]i ([Ca 2+]i-force relationship) observed with NA (Figure ic) was much greater than observed with K+-depolarization.
Effects ofglibenclamide on increases in [Ca2j]i andforce induced by K +-depolarization and noradrenaline in the presence ofextracellular Ca2 + When vascular strips were incubated with 10- GLB for 10min in PSS (5.9mM K+, 1.25mM Ca2"), there was no change in either [Ca2+]i or force. To examine the effect of GLB on increases in the [Ca2+]i and force induced by K+depolarization and NA, GLB was administered for 10min before and during application of these stimulations. Figures 2 and 3 show levels of [Ca2"]i and force induced by K+ 118 mm and NA 10-SM, respectively, in the presence of various concentrations of GLB. Treatment of the strips with GLB inhibited increases both in [Ca2+]i and in force development at all time points of contractions induced by both 118mm K+ PSS and 10- 5M NA, in a concentration-dependent manner within the range between 1- 6M and 104 M (Figure 2a,b and Figure 3a,b). We did not examine the effect of GLB in concentrations over 10-4M, because it gave an optical artifact and interfered with the fura-2 fluorometry. The [Ca2+]J-force relationships obtained at the steady state of contraction are shown in Figure 2c and 3c. The [Ca2j]i-force relationships of contractions induced by 118 mm K+-depolarization (Figure 2c) and NA (Figure 3c) observed in the presence of GLB did not dissociate from those of the counterparts observed in the absence of GLB. These findings indicate that there was no change in Ca2 +-sensitivity of the intracellular contractile apparatus during the vasorelaxation induced by GLB. When GLB was applied at the steady state of contraction induced by 118mm K+-depolarization and 10-SM NA, only higher concentrations of GLB (10-4M) induced reduction of [Ca2+]i and force ([Ca24]i, from 42% to 35%, and force from 167% to 150%). Thus, differing from the results of pretreatment with GLB, application of GLB during K+depolarization- or NA-induced contractions had little effect on [Ca2 +]i and force (data not shown).
Effect of glibenclamide on the increases in [Ca2j]i and force induced by noradrenaline (10 5M) in the absence of extracellular Ca2 + Typical recordings of increases in [Ca2+]i and force induced by NA 10- 5M in the absence of extracellular Ca2+ are shown
115
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p[Ca2411 Figure 2 Effects of glibenclamide (GLB) on (a) fluorescence ratio and (b) force development induced by 118mm K+. Vascular strips were incubated with GLB for 10min before and during 118mM K+application. GLB 16M (-), 10-5M (@), 10-4M (A), and without GLB (control, 118mM K4 (0)). (c) Effects of GLB on [Ca2l],-force relationship induced by high K+. GLB 10-6M (U), 1O-5M (0), 10-4M (A), and without GLB (control, 5.9mM K+ (0), 20mM K+ (A), 30 mM K4 (El), 40mM K+ (V), 60mM K4 (0), 118mM K+ (+)). [Ca2 4]i-force relationship in the absence of GLB was obtained from the data in Figure lb.
in Figure 4a. After recording the response to 118 mM K+ PSS, the vascular strip was exposed for 10min to Ca2 + -free PSS containing 2 mM EGTA. In the absence of extracellular Ca2+, [Ca2 +]i gradually decreased to a new steady level (-17 + 2%, n = 3) within 10min, however, no change in force was observed. Subsequent application of 10-I M NA induced a rapid and transient elevation of [Ca2 +]i and force: [Ca2+]i rapidly rose to reach the maximum level (30 + 11%, n = 3) in 12s, then declined to the pre-stimulation level within 8min (Figures 4a and 5a), and force developed rapidly to reach a maximum (97 + 7%, n = 3), gradually declining to the prestimulation levels after about 30min (Figures 4b and 5b). Changes in [Ca24+]i and force induced by NA were concentration-dependent (Figure 4b,c). When GLB was applied, in the absence of extracellular Ca2 , for Omin before and during the application of 10- 5M NA, transient increases both in [Ca2+]1 and force induced by NA were concentrationdependently reduced. In the absence of extracellular Ca2 +, the [Ca22+]-force relationship of the contraction induced by
K. YOSHITAKE et al.
116
Cromakalim 10-'M had no effect on [Ca2+]i increase and force development during 118mm K+-depolarization. However, it decreased [Ca2+]i and force during 25mM K+depolarization; these changes were not reversed by GLB (data not shown).
[Ca2+l (nm)
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p[Ca2+1] Figure 3 Effects of glibenclamide (GLB) on (a) fluorescence ratio and (b) force development induced by noradrenaline (NA). Vascular strips were incubated with GLB for 10min before and during application of NA. GLB 10-6M (), 10- 5M (0), 10-4M (A), without GLB (control, NA 10--M (0)). (c) Effects of GLB on [Ca2l],-force relationship induced by NA. GLB 10-6M (0), 10-5M (0), 10-4M (A), and without GLB (control, NA 3 x 10-9M (0), 1O-8M (*), 3 x 10-8M (V), 10-7M (V), 3 x
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(x)). Control [Ca2+]j-force relationship in the absence of GLB obtained from the data in Figure Ic.
was
10- 5M NA with various concentrations of GLB did not differ from that induced by various concentrations of NA without GLB (Figure Sc).
Effects of glibenclamide on [Ca2]i andforce in the of cromakalim
presence
Cromakalim, an ATP-sensitive K+ channel opener, decreased the increased levels of [Ca2']i and force, when applied at the steady state of contractions induced by 10-SM NA (Figure 6a). Subsequent application of GLB, 10- 5M, completely reversed the effect of 10- IM cromakalim on [Ca2" j and force in the NA-induced contraction (Figure 6a). There was no statistical difference (P > 0.05) between [Ca2+]i and force before the relaxation induced by cromakalim and after reversal by GLB, 10- M. The reversal effect of GLB against cromakalim was concentration-dependent (Figure 6b) (IC50 of GLB = 2 x 10-7M). However, 10-5M cromakalim had no effect on the reduction in [Ca2+]i and force induced by 10-'M GLB applied at the steady state of contraction induced by 10- M NA (data not shown).
While using fura-2 fluorometry and rabbit aortic strips we found that both high K+-depolarization and NA increased [Ca2+]i and force, in a concentration-dependent manner, and that the force development in relation to the [Ca2+]i observed with NA was much greater than that observed with K+depolarization. Also we found that GLB, a sulphonylurea antidiabetic drug known to be an ATP-sensitive K+ channel blocker, inhibited the [Ca2]i increase and contraction induced both by K+-depolarization and by NA, without altering the [Ca2 ]i-force relationship. Thus, this inhibitory effect of GLB on contraction is not due to an effect on the Ca2+ sensitivity of the intracellular contractile apparatus. However, we also found that when GLB was applied during the relaxation induced by a K+ channel opener, GLB caused contraction with increasing [Ca2+]i, and thus, reversed the effect of cromakalim. This seems to be the first observation that the contraction of vascular strips induced by GLB is accompanied by an increase in [Ca2+]i. However, vasoconstriction with an increase in [Ca2 ] is consistent with the report that GLB inhibited ATP-sensitive K+ channel activity, activated by cromakalim and contracted rabbit mesenteric arterial strips (Standen et al., 1989). It was suggested that a blockade of the K+ channel depolarizes the membrane, and consequently activates voltage-dependent Ca2+ channels, followed by influx of extracellular Ca2" and elevation of [Ca2+]i (Peterson & Findlay, 1987). In pancreatic 11 cells, even in the absence of K+ channel openers, GLB actively inhibited K+ channel activity (Peterson & Findlay, 1987), depolarized the membrane and elevated [Ca2+]i, resulting in a secretion of insulin (Sturgess et al., 1985, Schmid-Antomarchi et al., 1987, Boyd, 1988). Conversely, in the present study, GLB decreased [Ca2+]i and inhibited the force development of the rabbit aorta in the absence of cromakalim. When the K+ channel was activated by cromakalim, GLB increased [Ca2+]i and induced contraction. It was observed that the basal activity of the ATP-sensitive K+ channel is high in pancreatic I) cells (Findlay et al., 1985) but low in vascular smooth muscle (Standen et al., 1989). This may explain the difference in the response to GLB in the absence of K+ channel openers between pancreatic P6 cells and smooth muscle cells, that is, the inhibitory effects of GLB on the K+ channel and the resultant increase in [Ca2+]i and contraction become apparent, when K+ channel activity is kept high with cromakalim. In the present study, however, the reduction of 25mm K+-induced contraction by cromakalim was not reversed by GLB. This observation is consistent with the finding that cromakalim (>3yM) also inhibits Ca2 + flux through voltage-operated Ca2 + channels, independently of K+ channel opening (Okabe et al., 1990). It was shown that cromakalim caused hyperpolarization of the membrane potential at rest or during NAinduced depolarization, in the rat aorta (Doggrell et al., 1989). In the present study, since GLB completely reversed the cromakalim-induced reduction of the NA-induced contraction, a mechanism independent of the K+ channel opening might not be functioning in the cromakalim-induced reduction of the NA-induced contraction. The vasorelaxation induced by GLB was accompanied by a decrease in [Ca2+]i in the present study but the mechanism was not determined. Inhibition of contraction of rat uterine smooth muscle (Villar et al., 1986) and reduction in myocardial contractility induced by GLB (Pogasta & Dubecz, 1977) has been reported. Since the [Ca2+]i-force relationship in GLB-induced inhibition of contraction was not affected, the inhibitory effect may be related to initial steps involved in
GLIBENCLAMIDE EFFECTS ON SMOOTH MUSCLE CONTRACTION [Ca 2+1; (nm)
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Figure 4 Changes in [Ca2+]i and force induced by noradrenaline (NA) 10-5M in the absence of extracellular Ca2+. (a) Representative recordings of changes in fluorescence ratio and force development. After recording the responses of [Ca2 ] and force to 118mm K+application in the presence of 1.25mm extracellular Ca2", the strips were incubated with Ca2"-free PSS containing 2mM EGTA for 10min, and then NA was applied. (b) Dose-dependent responses of fluorescence ratio and (c) force development to NA. Data are means with s.e. shown by vertical lines (n = 3).
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p[Ca2+ 1i Figure 5 Effects of glibenclamide (GLB) on changes in (a) fluorescence ratio and (b) force development induced by noradrenaline (NA) 10- IM in the absence of extracellular Ca2+. GLB 10-6M (0), 10-5M (@), and without GLB (control, NA 10-5M (0)). (c) Effects of GLB on [Ca2`]i-force relationship induced by NA in the absence of extracellular Ca2+. GLB 10 7M (0), 3 x 10 7M (0), 10 6M (A), 3 x 106-m (V), 10-5M (*), and without GLB (control, NA 10-7M (A), 10- 6 M (0), 10- M (0)). Control [Ca2+]i-force relationship with various concentrations of NA in the absence of GLB were obtained from the data in Figure 4b,c.
[Ca2 ] (nM) 01)o
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Figure 6 Effects of glibenclamide (GLB) and cromakalim on changes in [Ca2+]1 and force induced by noradrenaline (NA) 105-M (a) Representative recordings of changes in fluorescence ratio and force. Cromakalim was applied when the maximum force development was obtained in NA iO- IM. Subsequently, GLB 1O- M was applied when [Ca2 ] and force were reduced to a steady level by cromakalim, 10-5Im. (b) Effects of GLB on [Ca2 ] and force during cromakalim-induced relaxation and during NA-induced contraction. Fluorescence ratio (+) and force (x) of the steady level of NA-induced contraction (1), and following cromakalim-induced relaxation (2). The reversal effects of GLB against cromakalim were dose-dependent (3). GLB 10-9 M (0. 0), 10-8 M (El. *), 10- 7 M (V, V), 10- 6 M (O>, *), 1 0 5 M (A, A). Open and closed symbols are force and [Ca2 +];, respectively.
118
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signal transduction. The reversal of cromakalim-induced relaxation was observed with 10- 8M to 10- 5M GLB (the concentration for the half maximum reversal effect was 2 x 10 7M), while the inhibition of contraction was observed with 10-6M to 1O-4 M GLB. Because of the difference in the concentration-range between the two effects of GLB, the site of action of GLB for the inhibitory effect does not seem to be related to the K' channel which may be closely linked to receptors for sulphonylurea. In conclusion, GLB inhibited contractions induced by K+depolarization and by NA in the rabbit aorta, by decreasing [Ca2+]i and with no effect on the [Ca2"],-force relationship. When contractions induced by NA were inhibited by a K+-
channel opener, GLB reversed this inhibitory effect by inhibiting K+-channel opening and increasing [Ca2+]i. We thank M. Ohara for helpful comments. The present study was supported in part by a Grant-in-Aid for Scientific Research on Priority Areas (No. 02257207, 02223107 and 01641532) and for General Scientific Research (No. 01480250) from the Ministry of Education, Science and Culture, Japan and grants from the 'Research Program on Cell Calcium Signals in the Cardiovascular System', from Suzuken Memorial Foundation, from Tokyo Biochemical Research Foundation, from Kanehara Ichiro Memorial Foundation, from Casio Science Promotion Foundation, from CIBA-GEIGY Foundation (Japan) for the Promotion of Science, and from Uehara Memorial Foundation.
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OKABE, K., KAJIOKA, S., NAKO, K., KITAMURA, K., KURIYAMA, H. &
Glucose induces closure of single potassium channels in isolated rat pancreatic fl-cells. Nature, 312, 446-447.
WESTON, A.H. (1990). Actions of cromakalim on ionic currents recorded from single smooth muscle cells of the rat portal vein. J. Pharmacol. Exp. Ther., 252, 832-839. PETERSON, O.H. & FINDLAY, I. (1987). Electrophysiology of the pancreas. Physiol. Rev., 67, 1054-1116. POGASTA, G. & DUBECZ, E. (1977). The direct effect of hypoglycemic sulfonylureas on myocardial contractile force and arterial blood pressure. Diabetologia, 13, 515-519. RINK, T.J. & POZZAN, T. (1985). Using quin2 in cell suspensions. Cell Calcium, 6, 133-144.
ASHFORD, M.L.J., STURGESS, N.L., TROUT, N.J., GARDNER, N.L. &
HALES, C.N. (1988). Adenosine-5'-triphosphate-sensitive ion channels in neonatal rat cultured central neurons. Pflugers Arch., 412, 297-304. BOYD, A.E. III. (1988). Sulfonylurea receptors, ion channels fruit flies. Diabetes, 37, 847-850. DOGGRELL, S.A., SMITH, J.W., DOWING, O.A. & WILSOM, K.A. (1989). Hyperpolarizing action of cromakalim on the rat aorta. Eur. J. Pharmacol., 174, 131-133. FINDLAY, I., DUNNE, M.J. & PETERSON, O.H. (1985). ATP-sensitive inward rectifier and voltage and calcium-activated K+ channels in cultured pancreatic islet cells. J. Membr. Biol., 88, 165-172. FURCHGOTT, R.F. & ZAWADZKI, J.V. (1980). The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetylcholine. Nature, 288, 373-376. GAINES, K.L., HAMILTON, S. & BOYD, A.E. III (1988). Characterization of the sulfonylurea receptor on beta cell membranes. J. Biol. Chem., 263, 2589-2592. GERICH, J.E. (1989). Oral hypoglycemic agents. N. Engl. J. Med., 321, 1231-1245. GRYNKIEWICZ, G., POENIE, M. & TSIEN, R.Y. (1985). A new generation of Ca2 + indicators with greatly improved fluorescence properties. J. Biol. Chem., 26, 3440-3450. HIRANO, K., KANAIDE, H. & NAKAMURA, M. (1989). Effects of okadaic acid on cytosolic calcium concentration and on contractions of the porcine coronary artery. Br. J. Pharmacol., 98, 12611266. MURPHY, R.A., HERLIHY, J.T. & MEGERMAN, J. (1974). Forcegenerating capacity and contractile protein of arterial smooth muscle. J. Gen. Physiol., 64, 691-705. NOMA, A. (1983). ATP-regulated K' channels in cardiac muscle, Nature, 305, 147-148.
SCHMID-ANTOMARCHI, H., DE WEILLE, J.R., FOSSET, M. & LAZ-
DUNSKI, M. (1987). The antidiabetic sulfonylurea glibenclamide is a potent blocker of the ATP-modulated K+ channel in insulinsecreting cells. Biochem. Biophys. Res. Commun., 146, 21-25. SPRUCE, A.E., STANDEN, N.B. & STANFIELD, P.R. (1985). Voltagedependent ATP-sensitive potassium channels of skeletal muscle membrane. Nature, 316, 736-738. STANDEN, N.B., QUAYLE, J.M., DAVIES, N.W., BRAYDEN, J.E.,
HUANG, Y. & NELSON, M.T. (1989). Hyperpolarizing vasodilators activate ATP-sensitive K+ channels in arterial smooth muscle. Science, 245, 177-180. STURGESS, N., ASHFORD, M.L.J., COOK, D.L. & HALES, C.N. (1985).
The sulfonylurea receptor may be an ATP-sensitive potassium channel. Lancet, ii, 474-475. TSIEN, R.Y., POZZAN, T. & RINK, T.J. (1982). Calcium homeostasis in intact lymphocytes: Cytoplasmic free calcium monitored with a new, intracellularly trapped fluorescent indicator. J. Cell. Biol., 94, 325-334. VILLAR, A., D'OCON, M.P. & ANSELMI, E. (1986). Relaxant effects of sulfonylureas on induced contractions of rat uterine smooth muscle: Role of intracellular calcium. Arch. Int. Pharmacodyn., 279,248-257.
(Received June 29, 1990 Revised August 17, 1990 Accepted August 21, 1990)
Br. J. Pharmacol. (1991), 102, 113-118
Effects of glibenclamide on cytosolic calcium concentrations and on contraction of the rabbit aorta Kiyonobu Yoshitake, Katsuya Hirano & 'Hideo Kanaide Division of Molecular Cardiology, Research Institute of Angiocardiology, Faculty of Medicine, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka, 812, Japan 1 Using fluorometry of fura-2 and rabbit aortic strips, we studied the effects of glibenclamide (GLB), a sulphonylurea anti-diabetic drug and an inhibitor of opening of K+ channels, on cytosolic calcium concentrations ([Ca2"]j) and on force development. 2 Both high K+-depolarization and noradrenaline (NA) increased [Ca2+]i and force, in a concentrationdependent manner, in the presence of extracellular Ca2+ (1.25mM). However, force development in relation to [Ca21]i ([Ca2+]J-force relationship) observed with NA was much greater than that observed with K+-depolarization. 3 Pretreatment with GLB (10-6-10-4M) for 10min partially inhibited, in a concentration-dependent manner, both [Ca2 +]i elevation and the force development induced by 118 mm K + -depolarization or NA 1O- 5M in the presence of extracellular Ca2 . The [Ca2+ ]i-force relationship induced by both 118mM K+ physiological salt solutions and by NA 10- M in the GLB-treated strips overlapped that obtained in the non-treated strips, thereby suggesting that GLB has no effect on the Ca2 +-sensitivity of the intracellular contractile apparatus. Only high concentrations (10-4M) of GLB decreased [Ca2+]i and the force, when applied after the force induced by 118 mM K+ PSS or NA 10- s M reached the maximum level. 4 In the absence of extracellular Ca2+, NA induced a transient increase in [Ca2+ ], and in the force and these increases were inhibited when the vascular strips were pretreated with GLB for 10min. The [Ca2+]J-force relationship obtained in the GLB-treated strips overlapped that in the non-treated ones. 5 An ATP-sensitive K+ channel opener, cromakalim (10-5M) reduced the increased [Ca2 + ]i and force induced by 25mm K+-depolarization and NA 10-SM. Subsequent application of GLB concentrationdependently reversed this relaxant effect of cromakalim on the NA-induced contraction (IC50 = 2 10 7 M). Complete reversal of the effect was observed with 10IsM GLB. 6 We suggest that GLB inhibits both high K+-depolarization- and NA-induced contraction of the rabbit aorta, by decreasing [Ca2+]i and with no effect on the [Ca2+]i-force relationship. However, when NA-induced contractions were inhibited by a K+-channel opener, GLB reversed this inhibitory effect by inhibiting K+-channel opening and increasing [Ca2 +]. x
Introduction K + channels inhibited by intracellular adenosine 5'triphosphate (ATP) have been identified in vascular smooth muscle (Standen et al., 1989), and these channels are also present in pancreatic fl-cells (Ashcroft et al., 1984), cardiac cells (Noma, 1983), skeletal muscle cells (Spruce et al., 1985) and cortical neurones (Ashford et al., 1988). The most potent and selective inhibitor of this channel known at present is the sulphonylurea hypoglycaemic agent, glibenclamide (GLB) (Schmidt-Antomarchi et al., 1987). High-affinity sulphonylurea receptors have been noted on pancreatic cells (Gaines et al., 1988) but no evidence of these receptors on vascular smooth muscle has been documented. In pancreatic # cell membranes, the sulphonylurea receptor may be closely linked to or be part of an ATP-sensitive K+ channel (Gerich, 1989), and inhibition of the efflux of K+ by GLB may lead to depolarization; as a consequence, voltage-dependent Ca2+ channels on the membrane would then open and permit entry of Ca2+ (Boyd, 1988). An increase in cytosolic Ca2+ concentration ([Ca2"]j) and the resultant activation of myosin light chain kinase play a critical role in initiating contractions of smooth muscles. In the present study, we examined the effect of GLB on [Ca2+] and the force of rabbit aortic smooth muscle strips, using fluorometry of fura-2.
the level of the renal arteries were immediately excised. Fat and adventitia were removed by dissection, under a binocular microscope. The endothelial cells were removed by rubbing the luminal surface with a cotton swab (Furchgott & Zawadzki, 1980). Medial preparations were cut into 1 x 3 mm circular strips 0.2mm thick. The wet weight of the strips was 0.6 + 0.1 mg (n = 12). Tissue density of the strips was assumed to be 1.05 gcm3 (Murphy et al., 1974) and the cross sectional area of each strip was calculated from the following equation: Cross-sectional area (m2) = wet weight (mg)/length (mm)/1.05 (gcm-3) x 10-6. The mean value of cross-sectional area was 2.14 + 0.19 x 10- 7 m2 (n = 12).
Fura-2 loading
Methods
The vascular strips thus obtained were loaded with the [Ca2+]i indicator dye, fura-2 by incubation in medium containing 50pM fura-2/AM (an acetoxymethyl ester form of fura-2) and 1.25% foetal bovine serum for 6-8 h at 37°C. Subsequently, the strips were washed in physiological salt solution (PSS) containing 1.25 mm Ca2+ at 37°C to remove the dye from the extracellular space and were then incubated with PSS for about 1 h before initiation of the measurements. Strips thus treated showed the specific peak of the fluorescence emission spectrum for fura-2 (500nm) and the specific peak and the valley of the fluorescence excitation spectrum for fura-2, 340nm and 380nm, respectively, determined with a fluores-
Tissue preparation
cence spectrophotometer (model
Japanese white rabbits (male, 16-20 weeks old, body weight 2.5-3.0 kg) were killed with sodium pentobarbitone (lOOmgkg-1, intravenously) and the abdominal aortae below '
Author for correspondence.
650-40, Hitachi, Tokyo, Japan). Loading the vascular strips with fura-2 did not alter the time course or the maximum levels of force development during 118 mm K + depolarization, thereby suggesting that no tissue damage occurred by possible acidification of the cells due to formaldehyde released on AM-ester hydrolysis (Tsien et al., 1982; Rink & Pozzan, 1985).
114
K. YOSHITAKE et al.
Measurement of tension
depolarization (100%) were 121 + 36 and 779 + 125 nm, respectively.
Strips were mounted vertically in a quartz organ bath, (strain gauge TB-612T, Nihon Koden, Tokyo, Japan). During a 1 h equilibration period, the strips were stimulated with 118mm K+ depolarization every 15min, and the resting tension was increased in a stepwise manner. After equilibration, the resting tension was adjusted to 200mg. The force development of the steady state was expressed as a % assuming the values in PSS (5.9mM K+) and 118mM K+ PSS to be 0% and 100%, respectively.
Drugs and solutions Physiological salt solution (PSS) was of the following composition (in mM): NaCl 123, KCl 4.7, NaHCO3 15.5, KH2PO4 1.2, MgCl2 1.2, CaCl2 1.25, and D-glucose 11.5. High potassium PSS was identical, except for an equimolar substitution of KCl for NaCl. Ca 2+-free solution was identical except for a substitution of 2mm ethylene glycol bis (f-aminoethyl ether) N,N,N',N'-tetraacetic acid (EGTA) for 1.25 mm CaCl2. PSS was gassed with a mixture of 95% 02-5% CO2 (pH adjusted to 7.4 at 370C). Glibenclamide (GLB, 5-chloro-N-[2-
Fluorometry
[4- [[[(cyclohexylamino)carbonyl]amino]sulphonyl]phenyl]
Changes in the fluorescence intensity of the fura-2-Ca2 + complex were simultaneously monitored during measurement of the force. The fluorescence intensity was measured by use of a fluorometer specially designed for fura-2 fluorometry (CAMOF-1), in collaboration with Japan Spectroscopic Co, Tokyo, Japan (Hirano et al., 1989). In brief, the strips were illuminated by guiding the alternating (400 Hz), 340nm and 380nm, excitation light from a Xenon light source through quartz optic fibres arranged in a concentric inner circle (diameter = 3 mm). Surface fluorescence of the strips was collected by glass optic fibres arranged in an outer circle (diameter = 7 mm) and introduced through a 500 nm band-pass filter (full width at half maximum transmission 10 nm) into a photon-counting photomultiplier. Special care was taken to keep the distance between a strip and the end of the optic fibres short and constant during each measurement. The ratio of the 500nm fluorescence at 340nm excitation to that at 380nm excitation was expressed as a %, assuming the values in PSS (5.9 mm K+) and 118 mm K+ PSS to be 0% and 100%, respectively. The absolute value of [Ca2+]i was estimated as described by Grynkiewicz et al. (1985). The mean value of 7 different measurements of [Ca2+]i at rest (0%) and during 118mm K+-
Statistical analysis Values are expressed as means + s.e.mean (n = 3). Student's t test was used to determine the statistical significance.
Results
Characteristics of changes in [Ca2j]i andforce induced by high external K + -depolarization and noradrenaline Representative recordings of changes in [Ca2+]i and force
induced by 118 mm K+-depolarization followed by NA 10 -M are shown in Figure 1. When the vascular strip was exposed to high external K+ (118 mM) solution containing 1.25 mm [Ca 2 1] (nM)
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ethyl]-2-methoxybenzamide) and cromakalim (3,4-dihydro3 - hydroxy - 2,2 - dimethyl - trans - 4 - (2 - oxo - 1 - pyrrolidinyl) 2H-1-benzopyran-6-carbonitrile) were generous gifts from Hoechst Japan and Chugai (Japan), respectively. GLB was disolved in dimethylsulphoxide (DMSO) and diluted in the organ bath (final concentration of DMSO was 1%). Fura-2/ AM was purchased from Molecular Probes (Eugene, OR, U.S.A.).
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-log [NaJ (M) [K+1 (mM) Figure 1 (a) Representative recordings of changes in fluorescence ratio (upper panel) and force development (lower panel) induced by high external K+ (118mM) physiological salt solution (PSS) followed by noradrenaline (NA) 10-5M in the presence of 1.25mM extracellular Ca2l. (b) Dose-response curve for the maximum force development (0) and the fluorescence ratio (0) induced by high external K + -depolarization in the presence of 1.25 mm Ca2 +. The abscissa scale is the concentration of K + in isosmotic high K + PSS. Measurements were done 5 min after application of high K+. Ordinates indicate percentage fluorescence ratio and force. Vertical lines indicate s.e.mean (n = 3). Note that the maximum [Ca2+]i and force induced by 40mM K+ are larger than those induced by 118mM K+. (c) Dose-response curve for fluorescence ratio (0) and the maximum force (0) induced by NA. Measurements were done at 6min after NA application. The abscissa scale is -log[NA]. Each value is expressed as percentage fluorescence ratio or force, assuming the values obtained during 118mM K+ application to be 100%.
GLIBENCLAMIDE EFFECTS ON SMOOTH MUSCLE CONTRACTION
Ca2", [Ca2 +]i rose abruptly by 111% in the first 20s, and
then, decreased slightly to reach a steady level at 6min; this level was assumed to be 100%. The force also rose rapidly and reached about 80% of the maximum level in the first 20s, the maximum level (100%) at about 5min, and remained at this level for at least 30 min. When 118 mm K+ PSS was changed to normal PSS, both [Ca2]i and the force rapidly reverted to the normal, pre-depolarization level, 0%. Subsequent application of 10-5M NA rapidly elevated [Ca2 ], with a first peak (67 + 18%, n = 3), reached in the first i5s. After a dip (to 38 + 7%, n = 3) at about 30s, [Ca2+]i gradually reached a second peak (57 + 9%, n = 3) at 3 min, and was then sustained at a slightly decreased level (42 + 11%, n = 3) for at least 20min. The force induced by NA 10- 5 M also rose rapidly to reach a level of 115% in the first 15s, the maximum level (178 + 12%, n = 3) at 6 min, followed by the steady state level (167 + 9%, n = 3), which was sustained for at least 20min. Figure lb and c show that the elevations of [Ca2+]i and force at the steady state induced by both external high K+ PSS and NA application were dose-dependent. It should be noted that in the rabbit aorta, the dose-response curves for [Ca2+]i and force induced by external high K+ PSS showed somewhat bell-shaped curves; the maximum [Ca2+]i and force were obtained with 40mm K+ PSS, and concentrations of K+ over 40 mm caused less steady state [Ca 2+ ]i and force development. In addition, force development in relationship to the increase in [Ca21]i ([Ca 2+]i-force relationship) observed with NA (Figure ic) was much greater than observed with K+-depolarization.
Effects ofglibenclamide on increases in [Ca2j]i andforce induced by K +-depolarization and noradrenaline in the presence ofextracellular Ca2 + When vascular strips were incubated with 10- GLB for 10min in PSS (5.9mM K+, 1.25mM Ca2"), there was no change in either [Ca2+]i or force. To examine the effect of GLB on increases in the [Ca2+]i and force induced by K+depolarization and NA, GLB was administered for 10min before and during application of these stimulations. Figures 2 and 3 show levels of [Ca2"]i and force induced by K+ 118 mm and NA 10-SM, respectively, in the presence of various concentrations of GLB. Treatment of the strips with GLB inhibited increases both in [Ca2+]i and in force development at all time points of contractions induced by both 118mm K+ PSS and 10- 5M NA, in a concentration-dependent manner within the range between 1- 6M and 104 M (Figure 2a,b and Figure 3a,b). We did not examine the effect of GLB in concentrations over 10-4M, because it gave an optical artifact and interfered with the fura-2 fluorometry. The [Ca2+]J-force relationships obtained at the steady state of contraction are shown in Figure 2c and 3c. The [Ca2j]i-force relationships of contractions induced by 118 mm K+-depolarization (Figure 2c) and NA (Figure 3c) observed in the presence of GLB did not dissociate from those of the counterparts observed in the absence of GLB. These findings indicate that there was no change in Ca2 +-sensitivity of the intracellular contractile apparatus during the vasorelaxation induced by GLB. When GLB was applied at the steady state of contraction induced by 118mm K+-depolarization and 10-SM NA, only higher concentrations of GLB (10-4M) induced reduction of [Ca2+]i and force ([Ca24]i, from 42% to 35%, and force from 167% to 150%). Thus, differing from the results of pretreatment with GLB, application of GLB during K+depolarization- or NA-induced contractions had little effect on [Ca2 +]i and force (data not shown).
Effect of glibenclamide on the increases in [Ca2j]i and force induced by noradrenaline (10 5M) in the absence of extracellular Ca2 + Typical recordings of increases in [Ca2+]i and force induced by NA 10- 5M in the absence of extracellular Ca2+ are shown
115
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p[Ca2411 Figure 2 Effects of glibenclamide (GLB) on (a) fluorescence ratio and (b) force development induced by 118mm K+. Vascular strips were incubated with GLB for 10min before and during 118mM K+application. GLB 16M (-), 10-5M (@), 10-4M (A), and without GLB (control, 118mM K4 (0)). (c) Effects of GLB on [Ca2l],-force relationship induced by high K+. GLB 10-6M (U), 1O-5M (0), 10-4M (A), and without GLB (control, 5.9mM K+ (0), 20mM K+ (A), 30 mM K4 (El), 40mM K+ (V), 60mM K4 (0), 118mM K+ (+)). [Ca2 4]i-force relationship in the absence of GLB was obtained from the data in Figure lb.
in Figure 4a. After recording the response to 118 mM K+ PSS, the vascular strip was exposed for 10min to Ca2 + -free PSS containing 2 mM EGTA. In the absence of extracellular Ca2+, [Ca2 +]i gradually decreased to a new steady level (-17 + 2%, n = 3) within 10min, however, no change in force was observed. Subsequent application of 10-I M NA induced a rapid and transient elevation of [Ca2 +]i and force: [Ca2+]i rapidly rose to reach the maximum level (30 + 11%, n = 3) in 12s, then declined to the pre-stimulation level within 8min (Figures 4a and 5a), and force developed rapidly to reach a maximum (97 + 7%, n = 3), gradually declining to the prestimulation levels after about 30min (Figures 4b and 5b). Changes in [Ca24+]i and force induced by NA were concentration-dependent (Figure 4b,c). When GLB was applied, in the absence of extracellular Ca2 , for Omin before and during the application of 10- 5M NA, transient increases both in [Ca2+]1 and force induced by NA were concentrationdependently reduced. In the absence of extracellular Ca2 +, the [Ca22+]-force relationship of the contraction induced by
K. YOSHITAKE et al.
116
Cromakalim 10-'M had no effect on [Ca2+]i increase and force development during 118mm K+-depolarization. However, it decreased [Ca2+]i and force during 25mM K+depolarization; these changes were not reversed by GLB (data not shown).
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p[Ca2+1] Figure 3 Effects of glibenclamide (GLB) on (a) fluorescence ratio and (b) force development induced by noradrenaline (NA). Vascular strips were incubated with GLB for 10min before and during application of NA. GLB 10-6M (), 10- 5M (0), 10-4M (A), without GLB (control, NA 10--M (0)). (c) Effects of GLB on [Ca2l],-force relationship induced by NA. GLB 10-6M (0), 10-5M (0), 10-4M (A), and without GLB (control, NA 3 x 10-9M (0), 1O-8M (*), 3 x 10-8M (V), 10-7M (V), 3 x
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(x)). Control [Ca2+]j-force relationship in the absence of GLB obtained from the data in Figure Ic.
was
10- 5M NA with various concentrations of GLB did not differ from that induced by various concentrations of NA without GLB (Figure Sc).
Effects of glibenclamide on [Ca2]i andforce in the of cromakalim
presence
Cromakalim, an ATP-sensitive K+ channel opener, decreased the increased levels of [Ca2']i and force, when applied at the steady state of contractions induced by 10-SM NA (Figure 6a). Subsequent application of GLB, 10- 5M, completely reversed the effect of 10- IM cromakalim on [Ca2" j and force in the NA-induced contraction (Figure 6a). There was no statistical difference (P > 0.05) between [Ca2+]i and force before the relaxation induced by cromakalim and after reversal by GLB, 10- M. The reversal effect of GLB against cromakalim was concentration-dependent (Figure 6b) (IC50 of GLB = 2 x 10-7M). However, 10-5M cromakalim had no effect on the reduction in [Ca2+]i and force induced by 10-'M GLB applied at the steady state of contraction induced by 10- M NA (data not shown).
While using fura-2 fluorometry and rabbit aortic strips we found that both high K+-depolarization and NA increased [Ca2+]i and force, in a concentration-dependent manner, and that the force development in relation to the [Ca2+]i observed with NA was much greater than that observed with K+depolarization. Also we found that GLB, a sulphonylurea antidiabetic drug known to be an ATP-sensitive K+ channel blocker, inhibited the [Ca2]i increase and contraction induced both by K+-depolarization and by NA, without altering the [Ca2 ]i-force relationship. Thus, this inhibitory effect of GLB on contraction is not due to an effect on the Ca2+ sensitivity of the intracellular contractile apparatus. However, we also found that when GLB was applied during the relaxation induced by a K+ channel opener, GLB caused contraction with increasing [Ca2+]i, and thus, reversed the effect of cromakalim. This seems to be the first observation that the contraction of vascular strips induced by GLB is accompanied by an increase in [Ca2+]i. However, vasoconstriction with an increase in [Ca2 ] is consistent with the report that GLB inhibited ATP-sensitive K+ channel activity, activated by cromakalim and contracted rabbit mesenteric arterial strips (Standen et al., 1989). It was suggested that a blockade of the K+ channel depolarizes the membrane, and consequently activates voltage-dependent Ca2+ channels, followed by influx of extracellular Ca2" and elevation of [Ca2+]i (Peterson & Findlay, 1987). In pancreatic 11 cells, even in the absence of K+ channel openers, GLB actively inhibited K+ channel activity (Peterson & Findlay, 1987), depolarized the membrane and elevated [Ca2+]i, resulting in a secretion of insulin (Sturgess et al., 1985, Schmid-Antomarchi et al., 1987, Boyd, 1988). Conversely, in the present study, GLB decreased [Ca2+]i and inhibited the force development of the rabbit aorta in the absence of cromakalim. When the K+ channel was activated by cromakalim, GLB increased [Ca2+]i and induced contraction. It was observed that the basal activity of the ATP-sensitive K+ channel is high in pancreatic I) cells (Findlay et al., 1985) but low in vascular smooth muscle (Standen et al., 1989). This may explain the difference in the response to GLB in the absence of K+ channel openers between pancreatic P6 cells and smooth muscle cells, that is, the inhibitory effects of GLB on the K+ channel and the resultant increase in [Ca2+]i and contraction become apparent, when K+ channel activity is kept high with cromakalim. In the present study, however, the reduction of 25mm K+-induced contraction by cromakalim was not reversed by GLB. This observation is consistent with the finding that cromakalim (>3yM) also inhibits Ca2 + flux through voltage-operated Ca2 + channels, independently of K+ channel opening (Okabe et al., 1990). It was shown that cromakalim caused hyperpolarization of the membrane potential at rest or during NAinduced depolarization, in the rat aorta (Doggrell et al., 1989). In the present study, since GLB completely reversed the cromakalim-induced reduction of the NA-induced contraction, a mechanism independent of the K+ channel opening might not be functioning in the cromakalim-induced reduction of the NA-induced contraction. The vasorelaxation induced by GLB was accompanied by a decrease in [Ca2+]i in the present study but the mechanism was not determined. Inhibition of contraction of rat uterine smooth muscle (Villar et al., 1986) and reduction in myocardial contractility induced by GLB (Pogasta & Dubecz, 1977) has been reported. Since the [Ca2+]i-force relationship in GLB-induced inhibition of contraction was not affected, the inhibitory effect may be related to initial steps involved in
GLIBENCLAMIDE EFFECTS ON SMOOTH MUSCLE CONTRACTION [Ca 2+1; (nm)
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Figure 4 Changes in [Ca2+]i and force induced by noradrenaline (NA) 10-5M in the absence of extracellular Ca2+. (a) Representative recordings of changes in fluorescence ratio and force development. After recording the responses of [Ca2 ] and force to 118mm K+application in the presence of 1.25mm extracellular Ca2", the strips were incubated with Ca2"-free PSS containing 2mM EGTA for 10min, and then NA was applied. (b) Dose-dependent responses of fluorescence ratio and (c) force development to NA. Data are means with s.e. shown by vertical lines (n = 3).
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p[Ca2+ 1i Figure 5 Effects of glibenclamide (GLB) on changes in (a) fluorescence ratio and (b) force development induced by noradrenaline (NA) 10- IM in the absence of extracellular Ca2+. GLB 10-6M (0), 10-5M (@), and without GLB (control, NA 10-5M (0)). (c) Effects of GLB on [Ca2`]i-force relationship induced by NA in the absence of extracellular Ca2+. GLB 10 7M (0), 3 x 10 7M (0), 10 6M (A), 3 x 106-m (V), 10-5M (*), and without GLB (control, NA 10-7M (A), 10- 6 M (0), 10- M (0)). Control [Ca2+]i-force relationship with various concentrations of NA in the absence of GLB were obtained from the data in Figure 4b,c.
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Figure 6 Effects of glibenclamide (GLB) and cromakalim on changes in [Ca2+]1 and force induced by noradrenaline (NA) 105-M (a) Representative recordings of changes in fluorescence ratio and force. Cromakalim was applied when the maximum force development was obtained in NA iO- IM. Subsequently, GLB 1O- M was applied when [Ca2 ] and force were reduced to a steady level by cromakalim, 10-5Im. (b) Effects of GLB on [Ca2 ] and force during cromakalim-induced relaxation and during NA-induced contraction. Fluorescence ratio (+) and force (x) of the steady level of NA-induced contraction (1), and following cromakalim-induced relaxation (2). The reversal effects of GLB against cromakalim were dose-dependent (3). GLB 10-9 M (0. 0), 10-8 M (El. *), 10- 7 M (V, V), 10- 6 M (O>, *), 1 0 5 M (A, A). Open and closed symbols are force and [Ca2 +];, respectively.
118
K. YOSHITAKE et al.
signal transduction. The reversal of cromakalim-induced relaxation was observed with 10- 8M to 10- 5M GLB (the concentration for the half maximum reversal effect was 2 x 10 7M), while the inhibition of contraction was observed with 10-6M to 1O-4 M GLB. Because of the difference in the concentration-range between the two effects of GLB, the site of action of GLB for the inhibitory effect does not seem to be related to the K' channel which may be closely linked to receptors for sulphonylurea. In conclusion, GLB inhibited contractions induced by K+depolarization and by NA in the rabbit aorta, by decreasing [Ca2+]i and with no effect on the [Ca2"],-force relationship. When contractions induced by NA were inhibited by a K+-
channel opener, GLB reversed this inhibitory effect by inhibiting K+-channel opening and increasing [Ca2+]i. We thank M. Ohara for helpful comments. The present study was supported in part by a Grant-in-Aid for Scientific Research on Priority Areas (No. 02257207, 02223107 and 01641532) and for General Scientific Research (No. 01480250) from the Ministry of Education, Science and Culture, Japan and grants from the 'Research Program on Cell Calcium Signals in the Cardiovascular System', from Suzuken Memorial Foundation, from Tokyo Biochemical Research Foundation, from Kanehara Ichiro Memorial Foundation, from Casio Science Promotion Foundation, from CIBA-GEIGY Foundation (Japan) for the Promotion of Science, and from Uehara Memorial Foundation.
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(Received June 29, 1990 Revised August 17, 1990 Accepted August 21, 1990)
