Naunyn-Schmiedeberg's Arch Pharmacol (1992) 346:462-468
Naunyn-Schmiedeberg's
Archivesof Pharmacology © Springer-Verlag 1992
The relaxant effects of cromakalim (BRL 34915) on human isolated airway smooth muscle J. Cortijo 1, B. Sarrifi 1, C. Pedr6s 1, M. Perpififi 1, F. Paris 2 and E. Moreillo t 1 Department of Pharmacology, Faculty of Medicine, University of Valencia, Av. Blasco Ibafiez 15, E-46010 Valencia, Spain 2 Department of ThoraCic Surgery, C. S. "La Fe", Valencia, Spain Received February 26, 1992/Accepted June 16, 1992
Summary. Cromakalim (BRL 34915) is a potassium channel opener with therapeutic potential as a bronchodilator in asthma. Cromakalim (0.1-30~tmol/1) inhibited the spontaneous tone of human isolated bronchi in a concentration-related manner being nearly as effective as isoprenaline or theophylline. The order of relaxant potencies (expressed as -logxo IC5o mol/1; mean + SEM) was isoprenaline (7.29+0.27; n = 8) > cromakalim (5.89+0.12; n = 7) > theophylline (4.07_+ 0.13; n = 10). In human bronchi where tone had been raised by addition of histamine (0.1 mmol/l), acetylcholine (0.1 mmol/1) or leukotriene D4 (LTD4, 0.1 gmol/1), the relaxant effect of cromakalim was substantially reduced. Cromakalim suppressed the contraction produced by KC1 (25 mmol/1) but not that produced by KC1 (120 mmol/1). Tetraethylammonium (8 mmol/1) was without effect against the relaxant action of cromakalim but procaine ( 0 . 5 - 5 mmol/1) and glibenclamide (0.3 gmol/1) antagonised it. Cromakalim (10 gmol/1) produced an upward displacement of concentration-effect curves for KC1 (1 - 100 mmol/1), acetylcholine (1 nmol/l-1 mmol/1) and histamine (1 nmol/1-1 mmol/l) but it did not alter the concentration-effect curve for LTD4 (0.1 nmol/1-0.1 lamol/1). When tissues were challenged in the presence of cromakalim (10 lamol/1) with KC1 (100 mmol/1), acetylcholine (1 mmol/1) or histamine (1 mmol/1), an enhanced contraction was observed compared to control tissues. This enhancement by cromakalim was absent when tissues were challenged with acetylcholine or histamine in either a Ca2+-free medium (plus EGTA 0.1 mmol/1) or in the presence of verapamil (10 gmol/1). It is concluded that cromakalim is an effective relaxant of human airway smooth muscle in vitro and this activity may depend on the opening of K + channels in the plasma membrane of smooth muscle cells but other actions cannot be ruled out.
Correspondence to." J. Cortijo at the above address
Key words: Cromakalim muscle
Human airway smooth
Introduction Potassium channel activators represent a new class of smooth muscle relaxant agents (Hamilton and Weston 1989). One of these substances, cromakalim (BRL 34915; Hamilton et al. 1986) is currently being evaluated for the treatment of nocturnal asthma (Williams et al. 1990). Cromakalim relaxes guinea-pig (Allen et al. 1986) and human (Taylor et al. 1988; Black et al. 1990) isolated airways and prevents bronchospasm in vivo both in the guinea-pig (Arch et al. 1988) and in man (Baird et al. 1988). Pharmacomechanical studies with selective antagonists (Murray et al. 1989, 1991; Jones et al. 1990) and results from electrophysiological (Green et al. 1991) and radioisotopic (Longmore et al. 1991) experiments have confirmed the presence of K + channels in the plasmalemma of airway smooth muscle cells although their characterisation has not been completed. The effects of cromakalim on airway smooth muscle appear to be mainly the consequence, as in vascular smooth muscle, of the opening of a single or different populations of K + channels (Longmore et al. 1991). The subsequent membrane hyperpolarization is considered crucial for relaxation (Allen et al. 1986; Jones et al. 1990). However, the precise links between this action and Ca 2+ movements in the airway smooth muscle cells is mostly unknown and other direct (Bray et al. 1991) and indirect (McCaig and De Jonckheere 1989) mechanisms may contribute to the final relaxation. Cromakalim has been extensively studied in the guinea-pig trachea (Allen et al. 1986; Arch et al. 1988; McCaig and De Jonckheere 1989; Murray et al. 1989; Jones et al. 1990) but there are fewer studies in human isolated airways (Taylor et al. 1988; Black et al. 1990).
463 T h e a i m o f the p r e s e n t s t u d y was to investigate the effects o f c r o m a k a l i m o n s p o n t a n e o u s t o n e a n d a g a i n s t v a r i o u s s p a s m o g e n s i n h u m a n isolated airway s m o o t h muscle, c o m p a r i n g its activity with t h a t o f other r e l a x a n t agents.
Methods Tissue preparation. Human lung tissue was obtained from patients who, in the majority of cases, were undergoing surgery for bronchial carcinoma. Within a few minutes after lung resection, part of the bronchus was dissected free from macroscopically abnormal lung tissue and placed in Krebs solution (composition, mmol/l: NaC1 118.4, KC1 4.7, CaCI22.5, MgSO4 1.2, KHzPO4 1.2, NaHCO3 25.0, glucose 11.1). After rapid transport to the laboratory, bronchi of 2 to 3 mm internal diameter were dissected from the specimen. The tissues were either immediately set up (fresh tissue) or were first stored in Krebs solution, equilibrated with 5% COz in Oz, at 4° C for 1 2 - 2 4 h (stored tissue). Previous experiments have shown that tissue reactivity is not altered by storage for this period of time. Several bronchial rings, 3 to 4 mm in length, were prepared and suspended on tissue hooks in 10 ml double-jacketed organ baths containing Krebs solution, gassed with 5% CO/: 95% 02, at 37°C (pH = 7.35). Each ring was connected to a force-displacement transducer (Grass FT03) and isometric tension changes recorded on a Grass polygraph (model 7P). The preparations were equilibrated for 1 - 2 h and the medium changed every 20 min. The extensive washing of the tissue will remove anaesthetics. An optimal load of 2 g was maintained throughout the equilibration period. A steady resting level of tone (between 1.8 and 2.0 g) was present at the end of the equilibrationperiod and before any drug addition to the bath. Effects of drugs on spontaneous and induced bronchial tone. The relaxant effects of cromakalim (0.1 to 30 txmol/l), theophylline (10 nmol/1 to 1 mmol/l) and isoprenaline (1 nmol/1 to 1 I.tmol/1)on the spontaneous tone were studied by constructing cumulative concentration-effect curves for each relaxant agent. When testing the effects of K+-channel inhibitors an initial concentration-effect curve for cromakalim (0.1-10 ~tmol/1) was constructed. After washing, baseline tone was re-established and tissues were allocated randomly to test or time matched control groups. Test tissues were exposed to Krebs solution containing tetraethylammonium (TEA, 8 mmol/1), procaine (0.25, 0.5, 1 or 5 retool/l) or glibenclamide (0.3 gmol/1) for 20 min before and during the generation of a second concentration-effect curve to cromakalim. Control tissues were treated similarly but not exposed to the inhibitors. Concentration-effect curves for isoprenaline (1 nmol/1-1 gmol/l) were also generated in the absence or presence of glibenclamide (0.3 gmol/1). In other experiments increased tone was induced by addition of a spasmogen (KC1 25 mmol/1, acetylcholine 0.1 mmol/1, histamine 0.1 retool/1 or leukotriene D4 0.1 gmol/1) and then cromakalim was cumulatively added (0.1 to 30 gmol/1). Cromakalim was also tested against KCI (120mmol/1)-induced spasm. Cumulative concentration-effect curves for theophylline (10 nmol/1 to 1 mmol/1) and isoprenaline (1 nmol/1 to 1 gmol/1) were also generated in preparations contracted with histamine (0.1 mmol/1). Only one concentration-effect curve was constructed in each ring. Experiments were terminated by the addition of theophylline (10 retool/l), the effect of which was taken to represent the maximum relaxation. The molar concentration required to produce 50% of maximum relaxation (ICso) was calculated graphically from plots of individual log concentration-effect curves and expressed as lOglo ICso. Cumulative concentration-effect curves were constructed for KC1 (i -- i00 mmol/1), acetylcholine (1 nmol/1-- 1 mmol/1), histamine (1 nmol/1-1 mmol/1) or leukotriene D4 (0.1 nmol/1-0.1 ~tmol/1). Following the construction of an initial concentration-
effect curve for one of these spasmogens, tissues were allocated randomly to test or time matched control groups. In test tissues, concentration-effect curves for spasmogens were reconstructed following 20 min incubation with and in the presence of cromakalim (10 gmol/1). Time matched control tissues were treated similarly but were not exposed to cromakalim. In other experiments the same protocol was followed except that tissues were treated with papaverine (10 gmol/1) instead of cromakalim (10 gmol/l). Efficacy (estimated as Emax) and potency (assessed as -loglo molar ECso) of the spasmogens was calculated from individualconcentration-effect curves. In an additional set of experiments tissues were challenged two times with acetylcholine (1 mmol/1), histamine (1 mmol/1) or KC1 (100 mmol/1) with a 30 rain interval between challenges. In test tissues, cromakalim (10 gmol/l) was present for 20 min before and throughout the second challenge with the spasmogens. Time matched control tissues were not exposed to cromakalim but were otherwise treated identically. In other experiments the same protocol was followed except that after the first challenge with a spasmogen, the test tissues were exposed to a Ca 2+-free Krebs solution containing EGTA (0.I mmol/1); cromakalim (10 gmol/1) was present for 20 min before and throughout the second challenge. Time matched control tissues were treated similarly but were not exposed to cromakalim. The same protocol was followed in other experiments but after the first challenge with a spasmogen, the test tissues were incubated with Krebs solution containig verapamil (10 ~mol/1) and cromakalim (10 ~tmol/1) for 20 min before and throughout the second challenge whereas time matched control tissues were treated similarly but were not exposed to cromakalim. In all experiments the response to the second challenge was expressed as a percentage of that to the first challenge.
Drugs and solutions. Drugs concentrations are expressed in terms of the molar concentration of the active substances. The following drugs were used: acetylcholine chloride, glibenclamide, histamine phosphate, isoprenaline hydrochloride, papaverine hydrochloride, procaine hydrochloride, theophylline, and tetraethylammonium bromide; all were purchased from Sigma (St. Louis, Mo., USA). Cromakalim (BRL 34915; (+) 6-cyano-3,4-dihydro-2,2-dimethyltrans-4-(2-oxo-l-pyrrolidyl)-2H-benzo[b]pyran-3-ol)was obtained from Smith Kline Beecham Pharmaceuticals (Surrey, UK). KC1 was of analytical grade. Stock solutions of cromakalim and isoprenaline were prepared in 70% v/v ethanol and 0.1 M HC1 respectively, those of glibenclamide in absolute ethanol, and those of other drugs in twice-distilled water. Dilutions of isoprenaline were prepared in distilled water containing 0.57 mmol/1 ascorbic acid as an antioxidant. The final concentrations of vehicles in the bath did not alter either baseline tension or drug-induced responses.
Statistical analysis of results. Results are given as means _ SEM of observations in tissues fron different patients. Occasionally two observations were derived from the same patient. Statistical analysis of the results was performed by analysis of variance (ANOVA) followed by Duncan's (Duncan 1955) or by Wilcoxon rank test, as appropriate. Differences were considered significantwhen P < 0.05.
Results Effects o f cromakalim on spontaneous and stimulated tone C r o m a k a l i m ( 0 . 1 - 3 0 gmol/l), i s o p r e n a l i n e (I n m o l / 1 1 ~tmol/1) a n d t h e o p h y l l i n e (10 n m o l / 1 - 1 mmol/1) each caused c o n c e n t r a t i o n - d e p e n d e n t i n h i b i t i o n o f s p o n t a n e o u s tone o f h u m a n b r o n c h i (Fig. 1 a). I s o p r e n a l i n e a n d t h e o p h y l l i n e each p r o d u c e d full relaxation, whereas r e l a x a t i o n to c r o m a k a l i m (30 gmol/1) was 78 _+4% (1136 +_ 167 mg, n = 7): o f t h a t p r o d u c e d b y t h e o p h y l l i n e (10 mmol/l). R e l a x a t i o n s evoked b y c r o m a k a l i m reached
464 120 mmol/1 (1058 +_ 193 mg, n = 6), cromakalim (0.1 to 30 gmol/1) lacked any relaxant effect.
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Fig. 1. Relaxant effect of cromakalim (BRL 34915) (ll), isoprenaline (0) and theophylline (O) in human bronchi with (a) spontaneous tone or (b) tone raised by addition of histamine (0.1 mmol/ 1). The spontaneous tone (baseline) was 1892 _+27 mg (n = 25) and histamine-induced tone above baseline was 735 _+66 mg (n = 23). The ordinates represent relaxation expressed as a percentage of the maximal response to theophylline (10 mmol/1). The abscissae show the negative loglo of the molar concentration of relaxant drugs. Points are mean values of 7 to 10 experiments with SEM shown by vertical bars
equilibration within 8 - 1 0 min whilst those produced by isoprenaline and theophylline required 4 and 6 rain respectively to equilibrate. Values for - l o g ICso (M) were: 5.89 _+ 0.12 (n = 7) for cromakalim, 7.29 +_ 0.27 (n = 8) for isoprenaline, and 4.07 _+ 0.13 (n = 10) for theophylline. The efficacy and potency of cromakalim was substantially reduced in tissues where tone had been raised by submaximal concentrations of spasmogens. The contractions above baseline evoked by KC1 (25mmol/1), acetylcholine (0.1 mmol/1), histamine (0.1 retool/l) and leukotriene D 4 (0.1 pmol/1) were 890 _+ 152 mg (n = 6), 658 -t- 8 9 m g (n = 7), 729_+175mg (n = 7), and 714 _+ 198 mg (n = 6), respectively. The contractile responses of the different spasmogens tested were not significantly different. Cromakalim (1 ~tmol/1) did not inhibit tone raised by these spasmogens except for a minor inhibition (2.3 + 1.7%) of leukotriene D4-induced tone. Cromakalim (30 p.mol/1) produced small reductions in the tone raised by KC1 (31.2 + 16.4%), acetylcholine (18.8-t_ 4.5%), histamine (36.3-t- 4.1%, as shown in Fig. lb) and leukotriene D4 (17.2 + 4.3%). There was no significant correlation between the amount of tone (mg) induced by a certain spasmogen in a tissue and the subsequent relaxation to cromakalim (30 pmol/1) (data not shown). In contrast, isoprenaline and theophylline were equally effective and potent as relaxants of histamine (0.1 mmol/1)-induced tone ( - l o g ICso values were 7.37 + 0.14, n = 9, and 4.01 + 0.19, n = 7, respectively) compared with their actions against spontaneous tone (Fig. l b). When tone had been raised with KC1
As shown above, cromakalim (0.1 - 10 gmol/1) inhibited spontaneous tone of human bronchi. In time matched control tissues the concentration-effect curves for cromakalim moved slightly to the left after tissue incubation in Krebs solution (Fig. 2a). In the first curve cromakalim (10gmol/1) produced a relaxation of 1093 _4- 188 mg (n = 7), and of 1142 _-t-237 mg (n = 7) in the second curve. TEA (8 retool/l) per se caused no change in bronchial tone except in 1 out of 7 bronchi where a minor contraction was produced. TEA (8 mmol/1) did not modify the relaxant action of cromakalim (Fig. 2b). Procaine (0.25 to 5 retool/l) per se had no effect on bronchial tone but produced a concentration-related depression of the concentration-effect curves of cromakalim (Fig. 2c). Glibenclamide (0.3 pmol/1) caused no change in tone but antagonised cromakalim shifting the concentration-effect curve to the right as shown in Fig. 2d. This antagonism was specific since glibenclamide (0.3 pmol/1) did not alter the concentration-effect curve to isoprenaline (data not shown).
Influence of cromakalim on concentration-effect curves of KCl, acetylcholine, histamine and leukotriene D 4 KC1 (1 - 1 0 0 mmol/l), acetylcholine (1 nmol/1-1 retool/l), histamine (1 nmol/1-1 mmol/1) and leukotriene D4 (0.1 nmol/1-0.1 p~mol/1) each caused concentration-dependent contractions of human bronchi. The use of time matched control tissues showed that the concentrationeffect curves of the spasmogens moved slightly to the left (KC1, acetylcholine) or to the right (histamine, leukotriene D4) when reconstructed following further tissue incubation in Krebs solution. Treatment of test tissues with cromakalim (10 ~tmol/1) decreased basal tone by 1137 -t- 79 mg (n = 28) and caused upward shifts in the log concentration-effect curves of KC1, acetylcholine and histamine whilst the concentration-effect curve of leukotriene D4 remained unchanged (Fig. 3). The sensitivity of the preparation to the spasmogens in the presence of cromakalim (10 p.mol/1) was not significantly altered. Thus, potency ( - l o g ECso) of KC1 was 1.88-t-0.07 (n = 6) in the absence vs. 2.01 _ 0.06 (n = 6) in the presence of cromakalim (10 ~tmol/1). The corresponding values for acetylcholine and histamine were 5.57 +_ 0.59 (n = 6) vs. 5.13_+0.29 (n = 8) and 4.36 +_0.23 (n = 7) vs. 4.50 + 0.10 (n = 8), respectively. The concentration-effect curves for spasmogens constructed in the presence of cromakalim (10 gmol/1) started from a lower tone than those generated in the absence of this drug. To ensure that changes in responsiveness and sensitivity were not due to the decrease in basal tone, concentration-effect curves to the same spasmogens were
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Fig. 2 a - d. The effect of three K + channel inhibitors on the relaxant action of cromakalim against spontaneous tone of human bronchi. An initial concentration-effect curve for cromakalim was constructed. After washing, baseline tone recovered and tissues were allocated to test or time matched control groups. Panel a shows initial log concentration-effect curve of cromakalim in control tissues ( 0 ) (n = 7) and subsequent log concentration-effect curve constructed in control tissues after further incubation with Krebs solution (O) (n = 7). Panels 5, c and d show pooled initial log concentration-effect curves of cromakalim constructed in the ab-
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sence of inhibitors (O), and the subsequent curves constructed in test tissues after exposure to (b) TEA (8 mmol/1) (O) (n = 7) or (e) procaine (0.25 mmol/1, %, n = 6; 0.5 mmol/1, • , n = 6; 1 mmol/1, [3, n = 6; or 5mmol/1, A, n = 4) or (d) glibenclamide (0.3 ~Lmol/1, O, n = 5). The ordinate represents relaxation expressed as a percentage of the maximal response to theophylline (10 mmol/1). The abscissae show the negative logto of molar concentration of cromakalim. Points are mean values with SEM shown by vertical bars
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Fig. 3. The effects of cromakalim and papaverine on the log concentration-effect curves of (a) KC1, (b) acetyleholine (ACh), (c) histamine (HA) and (d) leukotriene D4 (LTD,~) in human bronchi. The ordinate scale represents contraction (rag) and the abscissa scales represent concentrations of the spasmogens as - l o g l o tool/1. ( 0 ) Pooled initial log concentration-effect curves for the test and control tissues; (O) subsequent time matched control log concentration-
effect curves constructed in control tissues after further incubation in Krebs solution; (A) log concentration-effect curves constructed in test tissues treated with cromakalim (10 gmol/1); ( • ) log concentration-effect curves constructed in test tissues treated with papaverine (I0 grnol/1). Points are mean values of at least 6 experiments with SEM shown by vertical bars. * indicates a significant difference (P < 0.05) from the corresponding point for control tissues
r e p r o d u c e d i n the p r e s e n c e o f p a p a v e r i n e (10 gmol/1), w h i c h d i m i n i s h e s the b a s a l t o n e b y 1 3 4 4 + 1 0 8 m g , (n = 26; P > 0.05 vs. c r o m a k a l i m at the s a m e c o n c e n t r a t i o n ) . P a p a v e r i n e d e p r e s s e d o r d i d n o t m o d i f y the c o n c e n t r a t i o n - e f f e c t c u r v e s to the s p a s m o g e n s tested as s h o w n i n Fig. 3.
Influence o f cromakalim on responses to spasmogens in calcium-free medium or in the presence o f verapamil C r o m a k a l i m (10 gmol/1), C a 2 + - f r e e s o l u t i o n , a n d v e r a p a m i l (10 gmol/1) e a c h c a u s e d i n h i b i t i o n o f s p o n t a n e o u s tone. T h e r e l a x a t i o n a m o u n t e d to 1037 + 8 9 r a g for
466 with acetylcholine or histamine in either a Ca 2 +-free medium or in the presence of verapamil.
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Fig. 4. The effect of cromakalim on the responses to a maximal concentration of KC1 (upper panel), acetylcholine (middle panel) and histamine (lower panel) in human bronchi. Two consecutive challenges to KC1 (100 mmol/1),acetylcholine(1 mmol/1)and histamine (1 mmol/1)were generated. The ordinates indicate the contractile responses to the second challenge as per cent of the response to the first challenge with the spasmogen. Second challenges in time matched control tissues were generated in normal Krebs (a), Ca2+free (plus EGTA 0.1 mmol/1) Krebs (e) or Krebs containing verapamil (I0 gmol/1) (e). Second challenges in test tissues were generated as mentioned for control tissues but in the presence of cromakalim (10 Ixmol/1)(b, d and f, respectively). Column heights indicate the mean values of at least 6 experiments and vertical lines indicate the SEM. * indicates a significant difference (P < 0.05) compared with its corresponding control tissues. -k indicates a significant difference (P < 0.05) compared with a
cromakalim (n = 21), 547 4-78 mg for Ca2+-free solution (n = 18; P < 0.05 vs. cromakalim) and 492 _+ 77 mg for verapamil (n = 18; P < 0.05 vs. cromakalim). The relaxations observed for Ca2+-free solution and verapamil in the presence of cromakalim were 1150 -t- 85 mg (n = 18) and 1127 + 71 (n = 18), respectively (P < 0.05 vs. their corresponding values in the absence of cromakalim). Cromakalim augmented contractions produced by challenges with KC1 (100 mmol/1), acetylcholine (1 mmol/1) or histamine (1 mmol/1) in normal Krebs (Fig. 4). The contractile responses to KC1, acetylcholine or histamine were unaltered by 20 min prior incubation in a Ca 2 +-free medium. Verapamil depressed responses to KC1 and histamine without affecting those to acetylcholine. Cromakalim augmented responses to KC1 in a Ca2+-free medium or in the presence of verapamil but differences only reached statistical significance in verapamil-treated tissues. This enhancement by cromakalim was absent when tissues were challenged
Cromakalim inhibits spontaneous tone of human airway smooth muscle (Taylor et al. 1988; Black et al. 1990; this study). In this respect, cromakalim (30 ~tmol/1) was almost as efficacious as isoprenaline and theophylline. The order of potencies was isoprenaline > cromakalim > theophylline. When tested against tone raised by the addition of a variety of spasmogens, the ability of cromakalim to relax human airway muscle was substantially diminished. In contrast, isoprenaline and theophylline were fully competent in relaxing preparations precontracted with histamine (0.1 mmol/1). Concentrations of the spasmogens used in this study represent near-maximally effective concentrations. Taylor et al. (1988) and Black et al. (1990) have reported that when smaller concentrations (below ECso) of the spasmogens were used, cromakalim had an efficacy as a relaxant similar to that exerted against spontaneous tone, although potency tended to diminish mainly against cholinomimetic drugs. Relaxation by cromakalim is attributed to its activity at potassium channels (Hamilton et al. 1986). Cromakalim (30 gmol/1) relaxes KC1 (25 mmol/1)-induced tone but failed to relax KC1 (120 mmol/1)-induced spasm. In the present study we have tested TEA, procaine and glibenclamide as K+-channel inhibitors. TEA (8 mmol/ 1) did not alter cromakalim-induced inhibition of spontaneous tone. Conversely, procaine (0.25 to 5 mmol/1) produced a concentration-related suppression of relaxation by cromakalim. However, procaine is a non-selective inhibitor of smooth muscle K +-channels (Yamanaka et al. 1985). These results are similar to those reported by Allen et al. (1986) in the guinea-pig trachea. Black et al. (1990) found that glibenclamide, a purported blocker of ATP-sensitive K+-channels (Schmid-Antomarchi et al. 1987), inhibited relaxations of human bronchi elicited by lemakalim (BRL 38227), the L-enantiomer of cromakalim. In the present study, glibenclamide (0.3 gmol/1) antagonised responses to cromakalim but not those to isoprenaline. These findings suggest that cromakalim is in fact acting as a K+-channel opener, producing hyperpolarisation (not yet demonstrated in human tissues) and suppression of spontaneous tone of human airway muscle. This supports data obtained in studies of guinea-pig and bovine trachealis (Allen et al. 1986; Longmore et al. 1991). Other actions (inhibition of Ca 2 + uptake, stimulation of extrusion of Ca 2 + and/or effects on intracellular Ca 2+ release) not well characterised (Bray et al. 1991) could also contribute to the relaxant effects of cromakalim in human airways. Another aspect to explore in the action of cromakalim is the possibility of an antispasmogenic effect. Incubation with cromakalim did not depress the concentration-effect curves of KC1, acetylcholine or histamine in guinea-pig isolated trachealis bathed in Krebs solution containing indomethacin (Allen et al. 1986). Cromakalim produced an upward displacement of log concentration-effect
467 curves to KC1, acetylcholine and histamine in h u m a n bronchi. The concentration-effect curve o f leukotriene D4 was not affected by c r o m a k a l i m but concentrations of leukotriene D4 greater than 0.1 pmol/1 which would be necessary to produce maximal effects (Buckner et al. 1990) were not tested. Although papaverine (10 ~tmol/1) reduces basal tension to the same extent as cromakalim (10 I.tmol/1) and did not augment the responses to the spasmogens, the possibility that the enhancing effect of cromakalim m a y be due to the previous reduction of resting tone cannot be completely ruled out. This enhancement by cromakalim was absent when preparations were challenged with acetylcholine or histamine in either a Ca 2 +-free medium or in the presence of verapamil. Large conductance Ca 2 +-activated K + channels are present in airway smooth muscle cells but their precise role is u n k n o w n (Green et al. 1991). Charybdotoxin, a specific inhibitor of Ca2+-activated K + channels, did not affect cromakalim-induced relaxation of guinea-pig trachea (Jones et al. 1990; M u r r a y et al. 1991). This indicates that Ca2+-activated K + channels do not contribute to the inhibitory effect of cromakalim (Berry et al. 1991). In fact, it is possible that cromakalim m a y have opposing effects on ATP-dependent K + channels (opening) and on CaE+-activated K + channels (closing), as reported in vascular smooth muscle (Masuzawa et al. 1991). The closing of Ca2+-activated K + channels by cromakalim when they are being activated by increased cytosolic free Ca 2+ concentration due to cellular excitation by spasmogens m a y lead to enhancement of the contractile responses of these spasmogens. The previous inactivation of Ca 2 +-activated K + channels by CaZ+-free medium or verapamil (Masuzawa et al. 1991) m a y interfere with the action o f cromakalim on the same channels thus inhibiting the cromakalim-induced enhancement of contractile responses to acetylcholine and histamine. Nevertheless, the enhancement by cromakalim (10 ~tmol/1) of responses to KC1, acetylcholine and histamine in h u m a n airway muscle is probably irrelevant from the clinical point of view since 10 ~tmol/1 of cromakalim is a higher concentration than the therapeutic plasma levels (Davies et al. 1988). In contrast with these in vitro findings, cromakalim protected conscious guinea-pigs f r o m asphyxic collapse in response to histamine aerosol (Arch et al. 1988), and clinical studies have shown that cromakalim inhibited histamine-induced bronchoconstriction in normal volunteers (Baird et al. 1988) and attenuates early morning bronchoconstriction in asthmatics (Williams et al. 1990). The reason for differences between results in vivo and in vitro is not apparent. Besides direct inhibitory effects on smooth muscle contraction, c r o m a k a l i m modulates cholinergic neuroeffector transmission in the guinea-pig trachea, chiefly by a prejunctional mechanism (McCaig and De Jonckheere 1989) and also caused inhibitory effects on the release of other neurotransmitters (Ichinose and Barnes 1990; Burka et al. 1991) or mediators f r o m the wide array of nerve endings and cell types existing at the airways wall. M o r e basic and clinical research is needed to establish the place of this new approach to the treatment of asthma.
Acknowledgements. The present work was supported in part by a grant from the C.I.C.Y.T. (FAR-90-0680) of Ministerio de Industria y Energia (Spain). Cromakalim (BRL 34915) was kindly provided by Dr. T. C. Hamilton of Beechams Research Laboratories (England). The collaboration of Mrs. P. Bordes, C. Nieves, V. Salanova and all the surgical group in obtaining human lung samples is gratefully acknowledged.
References
Allen SL, Boyle JP, Cortijo J, Forster RW, Morgan GP, Small RC (1986) Electrical and mechanical effects of BRL34915 in guineapig isolated trachealis. Br J Pharmacol 89:395-405 Arch JRS, Buckle DR, Bumstead J, Clarke GD, Taylor JF, Taylor SG (1988) Evaluation of the potassium channel activator cromakalim (BRL34915) as a bronchodilatator in the guineapig: comparison with nifedipine. Br J Pharmacol 95:763- 770 Baird A, Hamilton TC, Richards DH, Tasker T, Williams AJ (1988) Inhibition of histamine induced bronchoconstriction in normal healthy volunteers by potassium channel activator, cromakalim BRL34915. Br J Clin Pharmacol 25:114P Berry JL, Elliott KRF, Foster RW, Green KA, Murray MA, Small RC (1991) Mechanical, biochemical and eleetrophysiological studies of RP 49356 and cromakalim in guinea-pig and bovine trachealis muscle. Pulmonary Pharmacol 4:91 - 9 8 Black JL, Armour CL, Johnson PRA, Alouan LA, Barnes PJ (1990) The action of a potassium channel activator, BRL 38227 (Lemakalim), on human airway smooth muscle. Am Rev Respir Dis 142:1384-1389 Bray KM, Weston AH, Duty S, Newgreen DT, Longmore J, Edwards G, Brown TJ (1991) Differences between the effects of cromakalim and nifedipine on agonist-induced responses in rabbit aorta. Br J Pharmacol 102: 337- 344 Buckner CK, Fedyna JS, Robertson JL, Will JA, England DM, Krell RD, Saban R (1990) An examination of the influence of the epithelium on contractile responses to peptidoleukotrienes and blockade by ICI 204,219 in isolated guinea-pig trachea and human intralobar airways. J Pharmacol Exp Ther 252:76-85 Burka JF, Berry JL, Foster RW, Small RC, Watt AJ (1991) Effects of cromakalim on neurally-mediated responses of guinea-pig tracheal smooth muscle. Br J Pharmacol 104: 263 - 269 Davies BE, Dierdorf D, Eckl KM, Mellows G, Thomsen T (1988) The pharmacokinetics ofcromakalim BRL 34915, a new antihypertensive agent, in healthy male subjects. Br J Clin Pharmacol 25:136P - 137P Duncan DB (1955) Multiple range and multiple-F tests. Biometrics 11:1-42 Green KA, Foster RW, Small RC (1991) A patch-clamp study of K+-channel activity in bovine isolated tracheal smooth muscle cells. Br J Pharmacol 102: 871 - 878 Hamilton TC, Weston AH (1989) Cromakalim, nicorandil and pinacidil: novel drugs which open potassium channels in smooth muscle. Gen Pharmacol 20:1 - 9 Hamilton TC, Weir SW, Weston 'AH (1986) Comparison of the effects of BRL 34915 and verapamil on electrical and mechanical activity in rat portal vein. Br J Pharmacol 88 : 103 - 111 Ichinose M, Barnes PJ (1990) A potassium channel activator modulates both excitatory noncholinergic and cholinergic neurotransmission in guinea-pig airways. J Pharmacol Exp Ther 252:1207-1212 Jones TR, Charette L, Garcia ML, Kaczorowski GJ (1990) Selective inhibition of relaxation of guinea-pig trachea by charybdotoxin, a potent CaZ+-activated K + channel inhibitor. J Pharmacol Exp Ther 255:697-706 Longmore J, Bray KM, Weston AH (1991) The contribution of Rbpermeable potassium channels to the relaxant and membrane hyperpolarizing actions of cromakalim, RP 49356 and diazoxide in bovine tracheal smooth muscle. Br J Pharmacol 102:979985
468 Masuzawa K, Matsuda T, Asano M (1991) The diverse effects of cromakalim on tension and 86Rb efflux in canine arterial smooth muscle. Br J Pharmacol 103 : 1033 - 1040 McCaig DJ, De Jonckheere B (1989) Effects of cromakalim on bronchoconstriction evoked by cholinergic nerve stimulation in guinea-pig isolated trachea. Br J Pharmacol 98 : 662- 668 Murray MA, Boyle JP, Small RC (1989) Cromakalim-inducedrelaxation of guinea-pig isolated trachealis: antagonism by glibenclamide and phentolamine. Br J Pharmacol 98: 865 - 874 Murray MA, Berry JL, Cook NS, Foster RW, Green KA, Small RC (1991) Guinea-pig isolated trachealis: the effects of charybdotoxin on mechanical activity, membrane potential changes and the activity of plasmalemmal K+-channels. Br J Pharmacol 103:1814-1818 Schmid-Antomarchi H, Dewille J, Fosset M, Lazdunski M (1987) The receptor for antidiabetic sulphonylureas controls the ac-
tivity of the ATP-modulated K+-channel in insulin-secreting cells. J Biol Chem 262:15 840-15 844 Taylor SG, Bumstead J, Morris JEJ, Shaw DJ, Taylor JF (1988) Cromakalim inhibits cholinergic-mediated responses in human isolated bronchioles but not in guinea-pig airways. Br J Pharmacol 95 : 795P Williams AJ, Lee TH, Cochrane GM, Hopkirk A, Vyse T, Chiew F, Lavender E, Richards DH, Owen S, Stone P, Church S, Woodcock A (1990) Attenuation of nocturnal asthma by cromakalim. Lancet II: 334- 336 Yamanaka K, Furukawa K, Kitamura K (1985) The different mechanisms of action of nicorandil and adenosine triphosphate on potassium channels of circular smooth muscle of the guineapig small intestine. Naunyn-Schmiedeberg's Arch Pharmacol 331:96-103
Naunyn-Schmiedeberg's
Archivesof Pharmacology © Springer-Verlag 1992
The relaxant effects of cromakalim (BRL 34915) on human isolated airway smooth muscle J. Cortijo 1, B. Sarrifi 1, C. Pedr6s 1, M. Perpififi 1, F. Paris 2 and E. Moreillo t 1 Department of Pharmacology, Faculty of Medicine, University of Valencia, Av. Blasco Ibafiez 15, E-46010 Valencia, Spain 2 Department of ThoraCic Surgery, C. S. "La Fe", Valencia, Spain Received February 26, 1992/Accepted June 16, 1992
Summary. Cromakalim (BRL 34915) is a potassium channel opener with therapeutic potential as a bronchodilator in asthma. Cromakalim (0.1-30~tmol/1) inhibited the spontaneous tone of human isolated bronchi in a concentration-related manner being nearly as effective as isoprenaline or theophylline. The order of relaxant potencies (expressed as -logxo IC5o mol/1; mean + SEM) was isoprenaline (7.29+0.27; n = 8) > cromakalim (5.89+0.12; n = 7) > theophylline (4.07_+ 0.13; n = 10). In human bronchi where tone had been raised by addition of histamine (0.1 mmol/l), acetylcholine (0.1 mmol/1) or leukotriene D4 (LTD4, 0.1 gmol/1), the relaxant effect of cromakalim was substantially reduced. Cromakalim suppressed the contraction produced by KC1 (25 mmol/1) but not that produced by KC1 (120 mmol/1). Tetraethylammonium (8 mmol/1) was without effect against the relaxant action of cromakalim but procaine ( 0 . 5 - 5 mmol/1) and glibenclamide (0.3 gmol/1) antagonised it. Cromakalim (10 gmol/1) produced an upward displacement of concentration-effect curves for KC1 (1 - 100 mmol/1), acetylcholine (1 nmol/l-1 mmol/1) and histamine (1 nmol/1-1 mmol/l) but it did not alter the concentration-effect curve for LTD4 (0.1 nmol/1-0.1 lamol/1). When tissues were challenged in the presence of cromakalim (10 lamol/1) with KC1 (100 mmol/1), acetylcholine (1 mmol/1) or histamine (1 mmol/1), an enhanced contraction was observed compared to control tissues. This enhancement by cromakalim was absent when tissues were challenged with acetylcholine or histamine in either a Ca2+-free medium (plus EGTA 0.1 mmol/1) or in the presence of verapamil (10 gmol/1). It is concluded that cromakalim is an effective relaxant of human airway smooth muscle in vitro and this activity may depend on the opening of K + channels in the plasma membrane of smooth muscle cells but other actions cannot be ruled out.
Correspondence to." J. Cortijo at the above address
Key words: Cromakalim muscle
Human airway smooth
Introduction Potassium channel activators represent a new class of smooth muscle relaxant agents (Hamilton and Weston 1989). One of these substances, cromakalim (BRL 34915; Hamilton et al. 1986) is currently being evaluated for the treatment of nocturnal asthma (Williams et al. 1990). Cromakalim relaxes guinea-pig (Allen et al. 1986) and human (Taylor et al. 1988; Black et al. 1990) isolated airways and prevents bronchospasm in vivo both in the guinea-pig (Arch et al. 1988) and in man (Baird et al. 1988). Pharmacomechanical studies with selective antagonists (Murray et al. 1989, 1991; Jones et al. 1990) and results from electrophysiological (Green et al. 1991) and radioisotopic (Longmore et al. 1991) experiments have confirmed the presence of K + channels in the plasmalemma of airway smooth muscle cells although their characterisation has not been completed. The effects of cromakalim on airway smooth muscle appear to be mainly the consequence, as in vascular smooth muscle, of the opening of a single or different populations of K + channels (Longmore et al. 1991). The subsequent membrane hyperpolarization is considered crucial for relaxation (Allen et al. 1986; Jones et al. 1990). However, the precise links between this action and Ca 2+ movements in the airway smooth muscle cells is mostly unknown and other direct (Bray et al. 1991) and indirect (McCaig and De Jonckheere 1989) mechanisms may contribute to the final relaxation. Cromakalim has been extensively studied in the guinea-pig trachea (Allen et al. 1986; Arch et al. 1988; McCaig and De Jonckheere 1989; Murray et al. 1989; Jones et al. 1990) but there are fewer studies in human isolated airways (Taylor et al. 1988; Black et al. 1990).
463 T h e a i m o f the p r e s e n t s t u d y was to investigate the effects o f c r o m a k a l i m o n s p o n t a n e o u s t o n e a n d a g a i n s t v a r i o u s s p a s m o g e n s i n h u m a n isolated airway s m o o t h muscle, c o m p a r i n g its activity with t h a t o f other r e l a x a n t agents.
Methods Tissue preparation. Human lung tissue was obtained from patients who, in the majority of cases, were undergoing surgery for bronchial carcinoma. Within a few minutes after lung resection, part of the bronchus was dissected free from macroscopically abnormal lung tissue and placed in Krebs solution (composition, mmol/l: NaC1 118.4, KC1 4.7, CaCI22.5, MgSO4 1.2, KHzPO4 1.2, NaHCO3 25.0, glucose 11.1). After rapid transport to the laboratory, bronchi of 2 to 3 mm internal diameter were dissected from the specimen. The tissues were either immediately set up (fresh tissue) or were first stored in Krebs solution, equilibrated with 5% COz in Oz, at 4° C for 1 2 - 2 4 h (stored tissue). Previous experiments have shown that tissue reactivity is not altered by storage for this period of time. Several bronchial rings, 3 to 4 mm in length, were prepared and suspended on tissue hooks in 10 ml double-jacketed organ baths containing Krebs solution, gassed with 5% CO/: 95% 02, at 37°C (pH = 7.35). Each ring was connected to a force-displacement transducer (Grass FT03) and isometric tension changes recorded on a Grass polygraph (model 7P). The preparations were equilibrated for 1 - 2 h and the medium changed every 20 min. The extensive washing of the tissue will remove anaesthetics. An optimal load of 2 g was maintained throughout the equilibration period. A steady resting level of tone (between 1.8 and 2.0 g) was present at the end of the equilibrationperiod and before any drug addition to the bath. Effects of drugs on spontaneous and induced bronchial tone. The relaxant effects of cromakalim (0.1 to 30 txmol/l), theophylline (10 nmol/1 to 1 mmol/l) and isoprenaline (1 nmol/1 to 1 I.tmol/1)on the spontaneous tone were studied by constructing cumulative concentration-effect curves for each relaxant agent. When testing the effects of K+-channel inhibitors an initial concentration-effect curve for cromakalim (0.1-10 ~tmol/1) was constructed. After washing, baseline tone was re-established and tissues were allocated randomly to test or time matched control groups. Test tissues were exposed to Krebs solution containing tetraethylammonium (TEA, 8 mmol/1), procaine (0.25, 0.5, 1 or 5 retool/l) or glibenclamide (0.3 gmol/1) for 20 min before and during the generation of a second concentration-effect curve to cromakalim. Control tissues were treated similarly but not exposed to the inhibitors. Concentration-effect curves for isoprenaline (1 nmol/1-1 gmol/l) were also generated in the absence or presence of glibenclamide (0.3 gmol/1). In other experiments increased tone was induced by addition of a spasmogen (KC1 25 mmol/1, acetylcholine 0.1 mmol/1, histamine 0.1 retool/1 or leukotriene D4 0.1 gmol/1) and then cromakalim was cumulatively added (0.1 to 30 gmol/1). Cromakalim was also tested against KCI (120mmol/1)-induced spasm. Cumulative concentration-effect curves for theophylline (10 nmol/1 to 1 mmol/1) and isoprenaline (1 nmol/1 to 1 gmol/1) were also generated in preparations contracted with histamine (0.1 mmol/1). Only one concentration-effect curve was constructed in each ring. Experiments were terminated by the addition of theophylline (10 retool/l), the effect of which was taken to represent the maximum relaxation. The molar concentration required to produce 50% of maximum relaxation (ICso) was calculated graphically from plots of individual log concentration-effect curves and expressed as lOglo ICso. Cumulative concentration-effect curves were constructed for KC1 (i -- i00 mmol/1), acetylcholine (1 nmol/1-- 1 mmol/1), histamine (1 nmol/1-1 mmol/1) or leukotriene D4 (0.1 nmol/1-0.1 ~tmol/1). Following the construction of an initial concentration-
effect curve for one of these spasmogens, tissues were allocated randomly to test or time matched control groups. In test tissues, concentration-effect curves for spasmogens were reconstructed following 20 min incubation with and in the presence of cromakalim (10 gmol/1). Time matched control tissues were treated similarly but were not exposed to cromakalim. In other experiments the same protocol was followed except that tissues were treated with papaverine (10 gmol/1) instead of cromakalim (10 gmol/l). Efficacy (estimated as Emax) and potency (assessed as -loglo molar ECso) of the spasmogens was calculated from individualconcentration-effect curves. In an additional set of experiments tissues were challenged two times with acetylcholine (1 mmol/1), histamine (1 mmol/1) or KC1 (100 mmol/1) with a 30 rain interval between challenges. In test tissues, cromakalim (10 gmol/l) was present for 20 min before and throughout the second challenge with the spasmogens. Time matched control tissues were not exposed to cromakalim but were otherwise treated identically. In other experiments the same protocol was followed except that after the first challenge with a spasmogen, the test tissues were exposed to a Ca 2+-free Krebs solution containing EGTA (0.I mmol/1); cromakalim (10 gmol/1) was present for 20 min before and throughout the second challenge. Time matched control tissues were treated similarly but were not exposed to cromakalim. The same protocol was followed in other experiments but after the first challenge with a spasmogen, the test tissues were incubated with Krebs solution containig verapamil (10 ~mol/1) and cromakalim (10 ~tmol/1) for 20 min before and throughout the second challenge whereas time matched control tissues were treated similarly but were not exposed to cromakalim. In all experiments the response to the second challenge was expressed as a percentage of that to the first challenge.
Drugs and solutions. Drugs concentrations are expressed in terms of the molar concentration of the active substances. The following drugs were used: acetylcholine chloride, glibenclamide, histamine phosphate, isoprenaline hydrochloride, papaverine hydrochloride, procaine hydrochloride, theophylline, and tetraethylammonium bromide; all were purchased from Sigma (St. Louis, Mo., USA). Cromakalim (BRL 34915; (+) 6-cyano-3,4-dihydro-2,2-dimethyltrans-4-(2-oxo-l-pyrrolidyl)-2H-benzo[b]pyran-3-ol)was obtained from Smith Kline Beecham Pharmaceuticals (Surrey, UK). KC1 was of analytical grade. Stock solutions of cromakalim and isoprenaline were prepared in 70% v/v ethanol and 0.1 M HC1 respectively, those of glibenclamide in absolute ethanol, and those of other drugs in twice-distilled water. Dilutions of isoprenaline were prepared in distilled water containing 0.57 mmol/1 ascorbic acid as an antioxidant. The final concentrations of vehicles in the bath did not alter either baseline tension or drug-induced responses.
Statistical analysis of results. Results are given as means _ SEM of observations in tissues fron different patients. Occasionally two observations were derived from the same patient. Statistical analysis of the results was performed by analysis of variance (ANOVA) followed by Duncan's (Duncan 1955) or by Wilcoxon rank test, as appropriate. Differences were considered significantwhen P < 0.05.
Results Effects o f cromakalim on spontaneous and stimulated tone C r o m a k a l i m ( 0 . 1 - 3 0 gmol/l), i s o p r e n a l i n e (I n m o l / 1 1 ~tmol/1) a n d t h e o p h y l l i n e (10 n m o l / 1 - 1 mmol/1) each caused c o n c e n t r a t i o n - d e p e n d e n t i n h i b i t i o n o f s p o n t a n e o u s tone o f h u m a n b r o n c h i (Fig. 1 a). I s o p r e n a l i n e a n d t h e o p h y l l i n e each p r o d u c e d full relaxation, whereas r e l a x a t i o n to c r o m a k a l i m (30 gmol/1) was 78 _+4% (1136 +_ 167 mg, n = 7): o f t h a t p r o d u c e d b y t h e o p h y l l i n e (10 mmol/l). R e l a x a t i o n s evoked b y c r o m a k a l i m reached
464 120 mmol/1 (1058 +_ 193 mg, n = 6), cromakalim (0.1 to 30 gmol/1) lacked any relaxant effect.
a
O- ~ _
Effect of K+-channel inhibitors on the relaxant action of cromakalim ~J
=c=
50.
100i
l
l
l
i
i i
i
9 8 7 6 5 4 3 Cromakalim
~
l
9
8
l
l
7
l
6
l
5
l
4
3
( -logmol/I)
Fig. 1. Relaxant effect of cromakalim (BRL 34915) (ll), isoprenaline (0) and theophylline (O) in human bronchi with (a) spontaneous tone or (b) tone raised by addition of histamine (0.1 mmol/ 1). The spontaneous tone (baseline) was 1892 _+27 mg (n = 25) and histamine-induced tone above baseline was 735 _+66 mg (n = 23). The ordinates represent relaxation expressed as a percentage of the maximal response to theophylline (10 mmol/1). The abscissae show the negative loglo of the molar concentration of relaxant drugs. Points are mean values of 7 to 10 experiments with SEM shown by vertical bars
equilibration within 8 - 1 0 min whilst those produced by isoprenaline and theophylline required 4 and 6 rain respectively to equilibrate. Values for - l o g ICso (M) were: 5.89 _+ 0.12 (n = 7) for cromakalim, 7.29 +_ 0.27 (n = 8) for isoprenaline, and 4.07 _+ 0.13 (n = 10) for theophylline. The efficacy and potency of cromakalim was substantially reduced in tissues where tone had been raised by submaximal concentrations of spasmogens. The contractions above baseline evoked by KC1 (25mmol/1), acetylcholine (0.1 mmol/1), histamine (0.1 retool/l) and leukotriene D 4 (0.1 pmol/1) were 890 _+ 152 mg (n = 6), 658 -t- 8 9 m g (n = 7), 729_+175mg (n = 7), and 714 _+ 198 mg (n = 6), respectively. The contractile responses of the different spasmogens tested were not significantly different. Cromakalim (1 ~tmol/1) did not inhibit tone raised by these spasmogens except for a minor inhibition (2.3 + 1.7%) of leukotriene D4-induced tone. Cromakalim (30 p.mol/1) produced small reductions in the tone raised by KC1 (31.2 + 16.4%), acetylcholine (18.8-t_ 4.5%), histamine (36.3-t- 4.1%, as shown in Fig. lb) and leukotriene D4 (17.2 + 4.3%). There was no significant correlation between the amount of tone (mg) induced by a certain spasmogen in a tissue and the subsequent relaxation to cromakalim (30 pmol/1) (data not shown). In contrast, isoprenaline and theophylline were equally effective and potent as relaxants of histamine (0.1 mmol/1)-induced tone ( - l o g ICso values were 7.37 + 0.14, n = 9, and 4.01 + 0.19, n = 7, respectively) compared with their actions against spontaneous tone (Fig. l b). When tone had been raised with KC1
As shown above, cromakalim (0.1 - 10 gmol/1) inhibited spontaneous tone of human bronchi. In time matched control tissues the concentration-effect curves for cromakalim moved slightly to the left after tissue incubation in Krebs solution (Fig. 2a). In the first curve cromakalim (10gmol/1) produced a relaxation of 1093 _4- 188 mg (n = 7), and of 1142 _-t-237 mg (n = 7) in the second curve. TEA (8 retool/l) per se caused no change in bronchial tone except in 1 out of 7 bronchi where a minor contraction was produced. TEA (8 mmol/1) did not modify the relaxant action of cromakalim (Fig. 2b). Procaine (0.25 to 5 retool/l) per se had no effect on bronchial tone but produced a concentration-related depression of the concentration-effect curves of cromakalim (Fig. 2c). Glibenclamide (0.3 pmol/1) caused no change in tone but antagonised cromakalim shifting the concentration-effect curve to the right as shown in Fig. 2d. This antagonism was specific since glibenclamide (0.3 pmol/1) did not alter the concentration-effect curve to isoprenaline (data not shown).
Influence of cromakalim on concentration-effect curves of KCl, acetylcholine, histamine and leukotriene D 4 KC1 (1 - 1 0 0 mmol/l), acetylcholine (1 nmol/1-1 retool/l), histamine (1 nmol/1-1 mmol/1) and leukotriene D4 (0.1 nmol/1-0.1 p~mol/1) each caused concentration-dependent contractions of human bronchi. The use of time matched control tissues showed that the concentrationeffect curves of the spasmogens moved slightly to the left (KC1, acetylcholine) or to the right (histamine, leukotriene D4) when reconstructed following further tissue incubation in Krebs solution. Treatment of test tissues with cromakalim (10 ~tmol/1) decreased basal tone by 1137 -t- 79 mg (n = 28) and caused upward shifts in the log concentration-effect curves of KC1, acetylcholine and histamine whilst the concentration-effect curve of leukotriene D4 remained unchanged (Fig. 3). The sensitivity of the preparation to the spasmogens in the presence of cromakalim (10 p.mol/1) was not significantly altered. Thus, potency ( - l o g ECso) of KC1 was 1.88-t-0.07 (n = 6) in the absence vs. 2.01 _ 0.06 (n = 6) in the presence of cromakalim (10 ~tmol/1). The corresponding values for acetylcholine and histamine were 5.57 +_ 0.59 (n = 6) vs. 5.13_+0.29 (n = 8) and 4.36 +_0.23 (n = 7) vs. 4.50 + 0.10 (n = 8), respectively. The concentration-effect curves for spasmogens constructed in the presence of cromakalim (10 gmol/1) started from a lower tone than those generated in the absence of this drug. To ensure that changes in responsiveness and sensitivity were not due to the decrease in basal tone, concentration-effect curves to the same spasmogens were
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Fig. 2 a - d. The effect of three K + channel inhibitors on the relaxant action of cromakalim against spontaneous tone of human bronchi. An initial concentration-effect curve for cromakalim was constructed. After washing, baseline tone recovered and tissues were allocated to test or time matched control groups. Panel a shows initial log concentration-effect curve of cromakalim in control tissues ( 0 ) (n = 7) and subsequent log concentration-effect curve constructed in control tissues after further incubation with Krebs solution (O) (n = 7). Panels 5, c and d show pooled initial log concentration-effect curves of cromakalim constructed in the ab-
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sence of inhibitors (O), and the subsequent curves constructed in test tissues after exposure to (b) TEA (8 mmol/1) (O) (n = 7) or (e) procaine (0.25 mmol/1, %, n = 6; 0.5 mmol/1, • , n = 6; 1 mmol/1, [3, n = 6; or 5mmol/1, A, n = 4) or (d) glibenclamide (0.3 ~Lmol/1, O, n = 5). The ordinate represents relaxation expressed as a percentage of the maximal response to theophylline (10 mmol/1). The abscissae show the negative logto of molar concentration of cromakalim. Points are mean values with SEM shown by vertical bars
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Fig. 3. The effects of cromakalim and papaverine on the log concentration-effect curves of (a) KC1, (b) acetyleholine (ACh), (c) histamine (HA) and (d) leukotriene D4 (LTD,~) in human bronchi. The ordinate scale represents contraction (rag) and the abscissa scales represent concentrations of the spasmogens as - l o g l o tool/1. ( 0 ) Pooled initial log concentration-effect curves for the test and control tissues; (O) subsequent time matched control log concentration-
effect curves constructed in control tissues after further incubation in Krebs solution; (A) log concentration-effect curves constructed in test tissues treated with cromakalim (10 gmol/1); ( • ) log concentration-effect curves constructed in test tissues treated with papaverine (I0 grnol/1). Points are mean values of at least 6 experiments with SEM shown by vertical bars. * indicates a significant difference (P < 0.05) from the corresponding point for control tissues
r e p r o d u c e d i n the p r e s e n c e o f p a p a v e r i n e (10 gmol/1), w h i c h d i m i n i s h e s the b a s a l t o n e b y 1 3 4 4 + 1 0 8 m g , (n = 26; P > 0.05 vs. c r o m a k a l i m at the s a m e c o n c e n t r a t i o n ) . P a p a v e r i n e d e p r e s s e d o r d i d n o t m o d i f y the c o n c e n t r a t i o n - e f f e c t c u r v e s to the s p a s m o g e n s tested as s h o w n i n Fig. 3.
Influence o f cromakalim on responses to spasmogens in calcium-free medium or in the presence o f verapamil C r o m a k a l i m (10 gmol/1), C a 2 + - f r e e s o l u t i o n , a n d v e r a p a m i l (10 gmol/1) e a c h c a u s e d i n h i b i t i o n o f s p o n t a n e o u s tone. T h e r e l a x a t i o n a m o u n t e d to 1037 + 8 9 r a g for
466 with acetylcholine or histamine in either a Ca 2 +-free medium or in the presence of verapamil.
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Fig. 4. The effect of cromakalim on the responses to a maximal concentration of KC1 (upper panel), acetylcholine (middle panel) and histamine (lower panel) in human bronchi. Two consecutive challenges to KC1 (100 mmol/1),acetylcholine(1 mmol/1)and histamine (1 mmol/1)were generated. The ordinates indicate the contractile responses to the second challenge as per cent of the response to the first challenge with the spasmogen. Second challenges in time matched control tissues were generated in normal Krebs (a), Ca2+free (plus EGTA 0.1 mmol/1) Krebs (e) or Krebs containing verapamil (I0 gmol/1) (e). Second challenges in test tissues were generated as mentioned for control tissues but in the presence of cromakalim (10 Ixmol/1)(b, d and f, respectively). Column heights indicate the mean values of at least 6 experiments and vertical lines indicate the SEM. * indicates a significant difference (P < 0.05) compared with its corresponding control tissues. -k indicates a significant difference (P < 0.05) compared with a
cromakalim (n = 21), 547 4-78 mg for Ca2+-free solution (n = 18; P < 0.05 vs. cromakalim) and 492 _+ 77 mg for verapamil (n = 18; P < 0.05 vs. cromakalim). The relaxations observed for Ca2+-free solution and verapamil in the presence of cromakalim were 1150 -t- 85 mg (n = 18) and 1127 + 71 (n = 18), respectively (P < 0.05 vs. their corresponding values in the absence of cromakalim). Cromakalim augmented contractions produced by challenges with KC1 (100 mmol/1), acetylcholine (1 mmol/1) or histamine (1 mmol/1) in normal Krebs (Fig. 4). The contractile responses to KC1, acetylcholine or histamine were unaltered by 20 min prior incubation in a Ca 2 +-free medium. Verapamil depressed responses to KC1 and histamine without affecting those to acetylcholine. Cromakalim augmented responses to KC1 in a Ca2+-free medium or in the presence of verapamil but differences only reached statistical significance in verapamil-treated tissues. This enhancement by cromakalim was absent when tissues were challenged
Cromakalim inhibits spontaneous tone of human airway smooth muscle (Taylor et al. 1988; Black et al. 1990; this study). In this respect, cromakalim (30 ~tmol/1) was almost as efficacious as isoprenaline and theophylline. The order of potencies was isoprenaline > cromakalim > theophylline. When tested against tone raised by the addition of a variety of spasmogens, the ability of cromakalim to relax human airway muscle was substantially diminished. In contrast, isoprenaline and theophylline were fully competent in relaxing preparations precontracted with histamine (0.1 mmol/1). Concentrations of the spasmogens used in this study represent near-maximally effective concentrations. Taylor et al. (1988) and Black et al. (1990) have reported that when smaller concentrations (below ECso) of the spasmogens were used, cromakalim had an efficacy as a relaxant similar to that exerted against spontaneous tone, although potency tended to diminish mainly against cholinomimetic drugs. Relaxation by cromakalim is attributed to its activity at potassium channels (Hamilton et al. 1986). Cromakalim (30 gmol/1) relaxes KC1 (25 mmol/1)-induced tone but failed to relax KC1 (120 mmol/1)-induced spasm. In the present study we have tested TEA, procaine and glibenclamide as K+-channel inhibitors. TEA (8 mmol/ 1) did not alter cromakalim-induced inhibition of spontaneous tone. Conversely, procaine (0.25 to 5 mmol/1) produced a concentration-related suppression of relaxation by cromakalim. However, procaine is a non-selective inhibitor of smooth muscle K +-channels (Yamanaka et al. 1985). These results are similar to those reported by Allen et al. (1986) in the guinea-pig trachea. Black et al. (1990) found that glibenclamide, a purported blocker of ATP-sensitive K+-channels (Schmid-Antomarchi et al. 1987), inhibited relaxations of human bronchi elicited by lemakalim (BRL 38227), the L-enantiomer of cromakalim. In the present study, glibenclamide (0.3 gmol/1) antagonised responses to cromakalim but not those to isoprenaline. These findings suggest that cromakalim is in fact acting as a K+-channel opener, producing hyperpolarisation (not yet demonstrated in human tissues) and suppression of spontaneous tone of human airway muscle. This supports data obtained in studies of guinea-pig and bovine trachealis (Allen et al. 1986; Longmore et al. 1991). Other actions (inhibition of Ca 2 + uptake, stimulation of extrusion of Ca 2 + and/or effects on intracellular Ca 2+ release) not well characterised (Bray et al. 1991) could also contribute to the relaxant effects of cromakalim in human airways. Another aspect to explore in the action of cromakalim is the possibility of an antispasmogenic effect. Incubation with cromakalim did not depress the concentration-effect curves of KC1, acetylcholine or histamine in guinea-pig isolated trachealis bathed in Krebs solution containing indomethacin (Allen et al. 1986). Cromakalim produced an upward displacement of log concentration-effect
467 curves to KC1, acetylcholine and histamine in h u m a n bronchi. The concentration-effect curve o f leukotriene D4 was not affected by c r o m a k a l i m but concentrations of leukotriene D4 greater than 0.1 pmol/1 which would be necessary to produce maximal effects (Buckner et al. 1990) were not tested. Although papaverine (10 ~tmol/1) reduces basal tension to the same extent as cromakalim (10 I.tmol/1) and did not augment the responses to the spasmogens, the possibility that the enhancing effect of cromakalim m a y be due to the previous reduction of resting tone cannot be completely ruled out. This enhancement by cromakalim was absent when preparations were challenged with acetylcholine or histamine in either a Ca 2 +-free medium or in the presence of verapamil. Large conductance Ca 2 +-activated K + channels are present in airway smooth muscle cells but their precise role is u n k n o w n (Green et al. 1991). Charybdotoxin, a specific inhibitor of Ca2+-activated K + channels, did not affect cromakalim-induced relaxation of guinea-pig trachea (Jones et al. 1990; M u r r a y et al. 1991). This indicates that Ca2+-activated K + channels do not contribute to the inhibitory effect of cromakalim (Berry et al. 1991). In fact, it is possible that cromakalim m a y have opposing effects on ATP-dependent K + channels (opening) and on CaE+-activated K + channels (closing), as reported in vascular smooth muscle (Masuzawa et al. 1991). The closing of Ca2+-activated K + channels by cromakalim when they are being activated by increased cytosolic free Ca 2+ concentration due to cellular excitation by spasmogens m a y lead to enhancement of the contractile responses of these spasmogens. The previous inactivation of Ca 2 +-activated K + channels by CaZ+-free medium or verapamil (Masuzawa et al. 1991) m a y interfere with the action o f cromakalim on the same channels thus inhibiting the cromakalim-induced enhancement of contractile responses to acetylcholine and histamine. Nevertheless, the enhancement by cromakalim (10 ~tmol/1) of responses to KC1, acetylcholine and histamine in h u m a n airway muscle is probably irrelevant from the clinical point of view since 10 ~tmol/1 of cromakalim is a higher concentration than the therapeutic plasma levels (Davies et al. 1988). In contrast with these in vitro findings, cromakalim protected conscious guinea-pigs f r o m asphyxic collapse in response to histamine aerosol (Arch et al. 1988), and clinical studies have shown that cromakalim inhibited histamine-induced bronchoconstriction in normal volunteers (Baird et al. 1988) and attenuates early morning bronchoconstriction in asthmatics (Williams et al. 1990). The reason for differences between results in vivo and in vitro is not apparent. Besides direct inhibitory effects on smooth muscle contraction, c r o m a k a l i m modulates cholinergic neuroeffector transmission in the guinea-pig trachea, chiefly by a prejunctional mechanism (McCaig and De Jonckheere 1989) and also caused inhibitory effects on the release of other neurotransmitters (Ichinose and Barnes 1990; Burka et al. 1991) or mediators f r o m the wide array of nerve endings and cell types existing at the airways wall. M o r e basic and clinical research is needed to establish the place of this new approach to the treatment of asthma.
Acknowledgements. The present work was supported in part by a grant from the C.I.C.Y.T. (FAR-90-0680) of Ministerio de Industria y Energia (Spain). Cromakalim (BRL 34915) was kindly provided by Dr. T. C. Hamilton of Beechams Research Laboratories (England). The collaboration of Mrs. P. Bordes, C. Nieves, V. Salanova and all the surgical group in obtaining human lung samples is gratefully acknowledged.
References
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