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Glibenclamide is a competitive antagonist of cromakalim, pinacidil and RP 49356 in guinea-pig pulmonary artery
European Journal of Pharmacology, 165 (1989) 231-239
231
Elsevier EJP 50824
Glibenclamide is a competitive antagonist of cromakalim, pinacidil and RIP 49356 in guinea-pig pulmonary artery M a n f r i d Eltze Department of Pharmacology, Byk Gulden Pharmaceuticals, D-7750 Konstanz, F.R.G.
Received 29 December 1988, revised MS received 23 February 1989, accepted 21 March 1989
The relaxant effect of cromakalim (BRL 34915), pinacidil and RP 49356 (N-methyl-2-(3-pyridyl)-tetrahydrothiopyran-2-carbothioamide-l-oxide) on the sustained contractions induced by 20 mM KC1 were compared with the effects of nicorandil. The preparation used was vascular smooth muscle of phenoxybenzamine-treated pulmonary artery tings from reserpinized guinea-pigs. Cromakalim, pinacidil, RP 49356 and nicorandil relaxed the tissues with - l o g ECs0 values of 6.78, 6.12, 6.02 and 5.46, respectively. The inhibitory effect of cromakalim, pinacidil and RP 49356, but not of nicorandil, was competitively antagonized by glibenclamide (10-7-3 × 10 -6 M), yielding uniform pA 2 values of 7.17-7.22 against all three relaxant drugs. The order of potency of other K ÷ channel blocking agents for the inhibition of vasorelaxation by cromakalim, pinacidil and RP 49356 was procaine > 4-aminopyridine > tetraethylammonium. The mainly competitive type of inhibition of the RP 49356-induced response was more comparable to that with pinacidil than with cromakalim. The relaxation caused by nicorandil was only attenuated by a high concentration of 4-aminopyridine or tetraethylammonium but was markedly antagonized by methylene blue (3 × 10-6-10 -5 M) and potentiated by M&B 22948 (3 × 10-6-10 -5 M). These results suggest that the vascular relaxation caused in guinea-pig pulmonary artery by cromakalim, pinacidil and RP 49356 is mediated through the same glibenclamide-sensitive K + channel whereas the major mechanism for the effect of nicorandil seems to involve stimulation of guanylate cyclase. K ÷ channel activators; Glibenclamide; Pulmonary artery; (Guinea-pig)
1. Introduction Cromakalim (BRL 34915) belongs to a novel class of antihypertensive agents which appear to produce vasorelaxation by activating an outward K + current (Allen et al., 1986; Hamilton et al., 1986; Cook, 1988). Studies on the mechanism of action of pinacidil (Arrigoni-Martelli et al., 1980; Southerton and Weston, 1987) and RP 49356 (Nmethyl-2-(3-pyridyl)-tetrahydrothiopyran-2-carbothioamide-l-oxide) (Cavero et al., 1988a; Escande et al., 1988; Mondot et al., 1988) have shown that these vasorelaxants share this membrane hyperpolarizing property. Although the antianginal agent, nicorandil, has been reported to open
potassium channels in vascular smooth muscle (Inoue et al., 1983; Kajiwara et al., 1984; Weston, 1987; Lefer and Lefer, 1988) it has been suggested that, due to the presence of a nitro group on the molecule, some of the smooth muscle relaxing effects of this agent are mediated in a manner characteristic of nitro vasodilators (Inoue et al., 1983; Inoue et al., 1984; Murakami et al., 1987). The insulin-releasing agent and potent inhibitor of ATP-sensitive K + channels in pancreatic fl-ceUs, glibenclamide (Schmid-Antomarchi et al., 1987; Ztinkler et al., 1988), has recently been shown to antagonize the vascular smooth muscle relaxation caused by cromakalim (Quast and Cook, 1988; Wilson et al., 1988a) and RP 49356 (Mondot et
0014-2999/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)
232
al., 1988) in vitro and to inhibit their hypotensive effect in vivo (Cavero et al., 1988b; Mondot et al., 1988). Thus, glibenclamide may also deactivate vascular K + channels that resemble pancreatic fl-cell or cardiac ATP-sensitive K + channels (Schrnid-Antomarchi et al., 1987; Escande et al., 1988). More recently, high concentrations of pinacidil have been shown to increase 86Rb+ outflow and to inhibit insulin release from rat pancreatic islets, suggesting that the ATP-sensitive K + channel in this tissue can be opened by a K + channel activator (Lebrun et al., 1988). The aim of the present study was therefore to investigate whether the inhibition, via K + channel blockade, by glibenclamide of the vasorelaxant response to cromakalim and RP 49356 would also be demonstrable for pinacidil and nicomndil, or whether these compounds act through a different mechanism. In order to explore these possibilities, the effects of three other agents with K + channel blocking activity, procaine (Jacobs and Keatinge, 1974), 4-aminopyridine (Glover, 1982) and tetraethylammonium (Stanfield, 1983) were tested for inhibition of vasorelaxation in the isolated guineapig pulmonary artery precontracted with low concentrations of KC1.
2. Materials and methods
2.1. Organ bath-suspended guinea-pig pulmonary artery Guinea-pigs (male, 350-450 g) were pretreated with reserpine (1 m g / k g i.p., 24 h before the experiment) to minimize possible interference by the release of endogenous catecholamines. Isolated ring segments of main pulmonary artery were set up as described by O'Donnell and Wanstall (1985) under a 1-g resting tension then were pre-exposed to phenoxybenzamine (10 -5 M for 30 min followed by washout) in order to exclude possible interactions with a-adrenoceptors. The bathing solution in the 10 ml organ baths consisted of (mM): NaC1 114, KC1 4.7, CaC12 2.5, K H 2 P O 4 1.2, MgSO4 1.2, NaHCO3 25.0 and glucose 11.1, maintained at 3 7 ° C and continuously bubbled with 95% Oz-5% CO 2. The contractions of four
tissues from two animals tested in parallel were measured isometrically (K-30, Hugo Sachs Elektronik) and recorded on multichannel recorders (Kipp and Zonen, BD 9).
2.2. Response curves to relaxant drugs After 20 min equihbration, the tissues were contracted by addition of 20 m M KC1 and the responses were allowed to reach a plateau (20-30 min). The relaxant drugs were then added to the preparations in half log unit increments until peak relaxation was achieved. The effects of the vasorelaxant drugs were expressed as % decrease in the tension produced by 20 mM KC1. The apparent potency of the drugs was expressed as their - l o g ECs0 value (median with 95% confidence limits), i.e. the - l o g molar concentration that had an effect equal to 50% of the individual maximal effect. In a second approach, the ECs0 values for the drugs were determined as the concentration producing a true 50% decrease in the contractile effect elicited by 20 m M KC1.
2.3. Effect of antagonists In each experiment, reproducible concentration-response curves for the relaxant drugs were obtained at 60-80 min intervals, the antagonists or enzyme inhibitors being equilibrated for either 20 min (glibenclamide, procaine, 4-aminopyridine, tetraethylammonium), 30 min ( M & B 22948) or 45 min (methylene blue) with the tissues before rechallenge with the same vasorelaxant drug. Schild plots for competitive antagonism were made from the dose ratios (x) of the relaxant drug for four different antagonist concentrations [B] so as to estimate the pA 2 value with 95% confidence limits and the slope of the regression line for each organ segment (Arunlakshana and Schild, 1959). The regression line for all organs was fitted by a conventional least squares method (Waud and Parker, 1971). The point estimator (mean) and its 95% confidence limits were calculated on a logtransformed scale. Statistical significance was assumed for the differences between the pA 2 values calculated or deviation of the slopes from 1.00 when P ~< 0.05.
233 --Guinea-pig
2.4. Drugs
pulmonary artery
_oC 100
(+)-Cromakalim (BRL 34915), (+)-pinacidil monohydrate, RP 49356 (N-methyl-2-(3-pyridyl)tetrahydrothiopyran-2-carbothioamide-l-oxide) and nicorandil were generous gifts from Beecham, Leo, Rh6ne-Poulenc and Merck, respectively. Glibenclamide and M & B 22948 (2-o-propoxyphenyl-8-azapurin-6-one) were kindly donated by Hoechst and May & Baker, respectively. All other drugs were purchased from Sigma (Munich).
3. Results
3.1. Vasorelaxant effects of cromakafim, pinacidil, RP 49356 and nicorandil on contractions induced by 20 mM KCl As shown in fig. 1, the cumulative application of cromakalim (3 × 10-8-3 x 10 -6 M), pinacidil (10-7-3 × 10 -5 M), RP 49356 (10-7-3 X 10 -5 M) and nicorandil (10-7-10 -4 M) inhibited the sustained contractions induced by 20 mM KC1 in a concentration-dependent manner. The rank order of relaxant potency calculated from the ECs0 values from the concentration-response curves related to their individual maximal effects (table 1) was cromakalim > pinacidil --- RP 49356 > nicorandil. However, the maximal response to cromakalim (64 + 7%) was lower than that to pinacidil, RP 49356 and nicorandil, ranging from 80 to 93% (table 1). The same order of potency was obtained
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Fig. 1. Cumulative concentration-response curves for the relaxant effect of cromakalim (CR), pinacidil (PI), RP 49356 (RP) and nicorandil (NI) in guinea-pig pulmonary artery precontracted with 20 m M KCI. The force developed in response to 20 mM KC1 was 0.79+0.17 g (mean+S.D., n = 3 2 ) . All responses are expressed as percent of the amplitude of the contraction due to KC1. The results are means-{-S.D, for 14-16 ring segments prepared from 7-8 animals for each drug.
when the ECs0 values were calculated on the basis of the true 50% decrease in contractile force elicited by 20 mM KC1. The - l o g ECs0 values (median, 95% confidence limits, n = 14-16) were: cromakal i m = 6 . 2 6 (5.43; 7.09), pinacidil=5.88 (5.21; 6.54), RP 49356 = 5.86 (5.19; 6.54) and nicorandil = 5.35 (4.90; 5.81).
3.2. Effect of glibenclarnide Pretreatment of the tissues for 20 min with glibenclamide (10-7-3 x 10 -6 M) produced parallel shifts to the right of the relaxant responses to cromakalim, pinacidil and RP 49356 (fig. 2) whereas the concentration-response curve to nicorandil was not affected. Thus, glibenclamide behaved as a competitive antagonist of the vaso-
TABLE 1 Activation and blockade of K ÷ channels in guinea-pig pulmonary artery: apparent potency of K ÷ channel activators to inhibit sustained contractions elicited by 20 mM KC1. ECs0 values (median with 95% confidence limits) were calculated from single concentration-response curves relative to individual maximal effects (mean+S.D.; left). Affinity for glibenclamide related to inhibition of drug-induced relaxation, pA 2 values (mean with 95% confidence limits) and regression slopes (mean_+S.D.) were calculated from Schild plots (right). Drug
Cromakalim Pinacidil RP 49356 Nicorandil
Activators: relaxation
Antagonist: glibenclamide
- log ECso (M)
Relative maximal effect (%)
n
pA 2
Slope
n
6.78 6.12 6.02 5.46
64+ 80+ 89 + 93 +
16 14 14 16
7.17 (6.67; 7.67) 7.22 (6.65; 7.79) 7.22 (6.91; 7.53) N o antagonism
1.01 +0.06 0.90+0.12 0.95 +0.06
28 32 38 8
(6.55; (5.56; (5.65; (5.11;
7.01) 6.67) 6.38) 5.82)
7 4 10 6
234
IO0.--Guineo-ptg pulmonary artery
3.3. Effect of other K + channel blockers
Cromakalim
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The inhibition of the vasorelaxant effect of cromakalim by procaine (3 × 10-4-3 X 10 -3 M) was characterized by a parallel shift and concurrent depression of the maximum effect of the concentration-response curve, whereas procaine produced an apparently competitive antagonism of the vasorelaxation induced by pinacidil and RP
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Fig. 2. Cumulative concentration-response curves for the relaxant effect of cromakalim, pinacidil, RP 49356 and nicorandil in guinea-pig pulmonary artery in the presence of glibenclamide (rn, 10 -7 M; zx, 3 x 1 0 -7 M; ~ , 10 -6 M; v, 3 x 10 -6 M) added 20 min before the drugs (O, control). Each point represents the mean 4-S.D. from typical experiments with four organ segments from two animals. Ordinate: % of maximal drug control response.
relaxant action of cromakalim, pinacidil and RP 49356. The pA z values (7.17-7.22) were not significantly different from each other (P > 0.05). Also, the slopes of the regression lines were not significantly different from unity (P > 0.05). The data obtained from the Schild plots are summarized in table 1. Glibenclamide did not increase the vascular tone of the tissues at concentrations effective for antagonizing the response to the dilator drugs (not shown).
IO0
7
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0i p agonist Fig. 3. Cumulative concentration-response curves for the relaxant effect of cromakalim, pinacidil, RP 49356 and nicorandil on guinea-pig pulmonary artery in the presence of procaine (12, 3 x 1 0 -4 M; t,, 10 -3 M; O, 3 × 1 0 - 3 M) added 20 min before the drugs (o, control). Each point represents the mean +S.D. from typical experiments with four organ segments from two animals. Ordinate: % of maximal control response to the drug.
235
49356. Procaine was almost ineffective against nicorandil-induced vasorelaxation (fig. 3). Similar effects were observed using 4-aminopyridine (10-3-10 -2 M) as antagonist of the vasorelaxation due to the various drugs. Again, the concentration-response curves for the relaxant effect of cromakalim were shifted to the right and depressed by 4-aminopyridine. In the case of pinacidil and RP 49356, concentrations of 10-3-3 × 10 -3 M produced horizontal displacement
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Fig. 4. Cumulative concentration-response curves for the relaxant effect of cromakalim, pinacidil, RP 49356 and nicorandil in guinea-pig pulmonary artery in the presence of 4aminopyridine (El, 10 - 3 M; zx, 3 × 1 0 - 3 M; ~ , 10 - 2 M) added 20 min before the drugs (O, control). Each point represents the m e a n + S . D , from typical experiments with four organ segments from two animals. Ordinate: % of maximal drug control response to the drug.
Fig. 5. Cumulative concentration-response curves for the relaxant effect of cromakalim, pinacidil, R P 49356 and nicorandil in guinea-pig pulmonary artery in the presence of tetraethy l a m m o n i u m 03, 3 × 1 0 -3 M; zx, 10 - 2 M; ~ , 3 × 1 0 - 2 M) added 20 min before the drugs (e, control). Each point represents the mean + S.D. from typical experiments with four organ segments from two animals. Ordinate: % of maximal control response to the drug.
without depressing the maximal effect of either drug. Nicorandil-induced vasorelaxation was only affected by 4-aminopyridine concentrations of 10 -2 M (fig. 4). Tetraethylammonium (3 × 10-3-3 x 10 -2 M) was the least potent K + channel blocker as regards inhibition of the vasorelaxation produced by the relaxant drugs: concentrations of 10 -2 M or higher were necessary to produce depression of the maximal response to cromakalim whereas there
236 Guinea-pig pulmonary artery
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100 M*8 2 2 9 50-
~__
j
~
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Methylene blue
p Nicorandit
Fig. 6. Cumulative concentration-response curves for the relaxant effect of nicorandil (O, control) in guinea-pig pulmonary artery in the presence of M&B 22948 or methylene blue added 30 or 45 min before nicorandil, respectively. Figures refer to - l o g molar concentrations. Each point represents the mean + S.D. from typical experiments with four organ segments from two animals. Ordinate: % of maximal control response to nicorandil.
was a near parallel shift to the right of both pinacidil and RP 49356 concentration-response curves. The relaxation due to nicorandil was concentration-dependently antagonized by 3 × 10 -310 -2 M tetraethylammonium and higher concentrations (3 × 10 -2 M) failed to further increase the inhibition (fig. 5). 3.4. Effect of methylene blue and M & B 22948 on nicorandil-induced relaxation As shown in fig. 6, the vasorelaxant effect of nicorandil on the 20 mM KCl-induced sustained contraction was reduced by a 45-min exposure to 3 x 10-6-10 -5 M methylene blue but was significantly potentiated by a 30-rain preincubation of the tissues with 3 × 10-6-10 -5 M M & B 22948.
4. Discussion
The study showed, that the K + channel openers, cromakalim (Allen et al., 1986; Hamilton et al., 1986; Hollingsworth et al., 1987; Cook, 1988), pinacidil (Bray et al., 1987; Cook et al., 1988) and RP 49356 (Cavero et al., 1988a; Escande et al., 1988; Mondot et al., 1988) are effective concentration-dependent inhibitors of the contractions induced in the guinea-pig isolated pulmonary artery by low KC1 concentrations. Inhibition of the KCl-induced vascular contractions by cromakalim
has been shown to depend on the KC1 concentration used; the higher the KC1 concentration, the less the inhibition (Weir and Weston, 1986a; Cook et al., 1988). The difference between membrane potential and K ÷ equilibrium potential, especially at a low KC1 concentration, allows the K + efflux inducible by a putative K ÷ channel opener to occur, resulting in membrane hyperpolarization, reduced probability of Ca 2÷ channel opening and vascular relaxation. The pulmonary artery of the guinea-pig is known for its high K ÷ flux as are the ear artery, aorta and pulmonary artery of the rabbit (Bolton and Clapp, 1984; Den Hertog et al., 1984). Raising the external potassium ([K÷]o) in rabbit pulmonary artery is accompanied by a large increase in 86Rb+ efflux which serves as a marker for potassium permeability. In the present experiments, 20 mM KC1 was the concentration selected, since this concentration elicits sustained and reproducible contractions of guinea-pig pulmonary artery which are consistently relaxed by cromakalim, pinacidil, RP 49356 and nicorandil. Cromakalim proved to be 3-5 times more potent in terms of ability to relax KCl-induced contractions, than the nearly equieffective pinacidil and RP 49356, which were 3-fold more potent than nicorandil. These results are in good agreement with those of similar experiments on aorta and portal vein preparations from rats (Weir and Weston, 1986b; Bray et al., 1987) and rabbits (Hamilton et al., 1987). Of the drugs investigated, nicorandil produced near maximal relaxation (93%) of the precontracted vessel whereas cromakalim elicited a maximal relaxation of only approximately 64%. RP 49356 and pinacidil exhibited an activity intermediate between that of the former two drugs. When the tissues were in contact with glibenclamide, competitive antagonism was observed against the relaxant responses to cromakalim, pinacidil and RP 49356 whereas the vasorelaxant effect of nicorandil was resistant. One of the most reliable techniques used to differentiate between receptors and receptor subtypes is comparison of the dissociation constants of antagonists (Furchgott, 1972). Extending this approach to putative K + channel binding sites on smooth muscle of
237
guinea-pig pulmonary artery, it seems clear that the action of glibenclamide can be explained simply by blockade of one K ÷ channel with a uniform affinity constant, irrespective of the K ÷ channel activators used for its assessment. These findings suggest convincingly that the vasorelaxant effects of cromakalim, pinacidil and RP 49356 are medicated by the same type of K + channel in this particular preparation. The pA 2 values obtained for glibenclamide from shifts of the dose-response curves of cromakalim, pinacidil and RP 49356 (7.17-7.22) were in very good agreement with the pA 2 obtained for cromakalim and glibenclamide on rat portal vein (pA 2 = 7.20; Quast, 1988). This suggests a common site of competition between activators and glibenclamide on a similar K ÷ channel in both guinea-pig pulmonary artery and rat portal vein. The ability of relatively high glibenclamide concentrations (10 -7 M) to inhibit competitively the opening of K + channel induced by cromakalim or other K + channel activators in both vascular tissues contrasts with the high potency of glibenclamide (in the nanomolar range) to inhibit ATP-dependent K + currents in rat insulinoma cells (Schmid-Antomarchi et al., 1987) or mouse pancreatic fl-cells (Ziinkler et al., 1988). Nevertheless, it can be concluded that glibenclamide is a potent inhibitor of the vasorelaxant effects of cromakalim (Quast and Cook, 1988; Wilson et al., 1988a), RP 49356 (Mondot et al., 1988) and pinacidil (this study) and thus may represent a valuable pharmacological tool for investigating the mechanism of K + channel opening. The rank order of potency of other K + channel blockers to antagonize the relaxant responses to cromakalim, pinacidil and RP 49356 was procaine (threshold concentration 3 × 10 -4 M) > 4-aminopyridine (threshold concentration 10 -3 M ) > tetraethylammonium (threshold concentration 3 × 10 -3 M). Competitive antagonism was generally observed with pinacidil and RP 49356 whereas inhibition of cromakalim-induced vasodilatation was non-competitive. Recent pharmacological studies on rabbit mesenteric artery preparations gave the same rank order of antagonist potency and different types of inhibition of these K ÷ channel antagonists against cromakalim and pinacidil (Wilson et al., 1988b).
The vasorelaxation elicited by nicorandil was resistant to glibenclamide blockade, was hardly affected by procaine but was affected to a small extent by 4-aminopyridine and tetraethylammonium, indicating a predominant involvement of mechanisms other than K + channel activation for vasorelaxation. The responses to nicorandil were potentiated by M & B 22948, a selective inhibitor of cGMP-phosphodiesterase (Bowman and Drummond, 1984; Kukovetz et al., 1981), and inhibited by methylene blue, an inhibitor of guanylate cyclase (Gruetter et al., 1981). Thus, the results of the present study support the view that nicorandilinduced vasodilatation in guinea-pig pulmonary artery involves mainly the generation of cGMP, an effect which has been shown previously in rabbit aorta (Murakami et al., 1987); other mechanisms of action, e.g. inhibition of Ca 2+ mobilization in the cell (Itoh et al., 1981; Kreye et al., 1975; Satake and Shibata, 1987) may also be involved. Recent pharmacological studies showed that the tetrahydrothiopyrane derivative, RP 49356, behaved like cromakalim in inhibiting spontaneous contractile activity and increasing the effiux of 86Rb+ in rat portal vein (Cavero et al., 1988a). RP 49356 inhibited the contractions evoked in rat isolated aortic rings by 4-5 mM KCI but those evoked by 50 mM KC1 were not modified. Additionally, the relaxation of rat aortic rings contracted with 20 mM KC1 by RP 49356 or cromakalim was blocked by glibenclamide (Mondot et al., 1988). This suggests that RP 49356 might act in a manner similar to cromakalim in activating K + channels in vascular smooth muscle (Cavero et al., 1988a). Confirmation of this general assumption was now obtained from the finding that glibenclamide antagonizes the vasorelaxation caused by both drugs. Comparing the inhibition by other K + channel blockers, e.g. procaine, 4-aminopyridine and tetraethylammonium, of the vasorelaxation due to RP 49356 and of the vasorelaxation caused by pinacidil, evidences a mainly competitive antagonism. However the inhibition of cromakalim responses by these blockers is non-competitive at higher concentrations. Whether this effect, in addition to the predominant activation of glibenclamide-sensitive K ÷
238 c h a n n e l s , p o i n t s to a n o t h e r m e c h a n i s m for t h e production of vascular relaxation by cromakalim, p o s s i b l y n o t s h a r e d w i t h p i n a c i d i l a n d R P 49356, c a n n o t yet b e a n s w e r e d . I n c o n c l u s i o n , t h e p r e s e n t studies w i t h g u i n e a pig isolated pulmonary artery demonstrate that g l i b e n c l a m i d e is a n e q u a l l y p o t e n t c o m p e t i t i v e a n t a g o n i s t o f t h e v a s c u l a r r e l a x a n t effects o f c r o m a k a l i m , p i n a c i d i l a n d R P 49356 b u t n o t o f t h e effects o f n i c o r a n d i l , s u g g e s t i n g t h a t t h e v a s o r e l a x a t i o n c a u s e d b y the t h r e e d r u g s m a y p r e d o m i n a n t l y i n v o l v e o p e n i n g of t h e s a m e K ÷ channel. This property could make glibenclamide a useful d r u g f o r t h e r e l i a b l e d e t e c t i o n o f h e t e r o g e n o u s K ÷ c h a n n e l s m e d i a t i n g a p a r t i c u l a r response. T h e v a s o d i l a t a t i o n c a u s e d b y t h e s e d r u g s w a s c o m p a r a t i v e l y less a f f e c t e d b y o t h e r K ÷ c h a n nel b l o c k e r s , e.g. p r o c a i n e , 4 - a m i n o p y r i d i n e a n d t e t r a e t h y l a m m o n i u m . N e v e r t h e l e s s , t h e l a t t e r exhibit an inhibitory mechanism against pinacidil a n d R P 49356, w h i c h is m a i n l y c o m p e t i t i v e a n d different from that against cromakalim. Whether this p h e n o m e n o n c a n b e f u r t h e r u s e d t o d i f f e r e n t i ate b e t w e e n p o s s i b l e K ÷ c h a n n e l h e t e r o g e n e i t y in v a s c u l a r s m o o t h m u s c l e r e m a i n s to b e e l u c i d a t e d .
Acknowledgement The author gratefully acknowledges the skillful technical assistance of Miss Caroline Biirkle.
References Alien, S.L., J.P. Boyle, J. Cortijo, R.W. Foster, G.P. Morgan and R.C. Small, 1986, Electrical and mechanical effects of BRL 34915 in guinea-pig isolated trachealis, Br. J. Pharmacol. 89, 395. Arunlakshana, O. and H.O. Schild, 1959, Some quantitative uses of drug antagonists, Br. J. Pharmacol. 14, 48. Arrigoni-Martelli, E., C.K. Nielsen, U.B. Olsen and H.J. Petersen, 1980, Na-cyano-N-4-pyridyl-N'-l,2,3-trimethylpropylguanidine monohydrate (P 1134): A new, potent vasodilator, Experientia 36, 445. Bolton, T.B. and L.H. Clapp, 1984, The diverse effects of noradrenaline and other stimulants on S6Rb and 42K efflux in rabbit and guinea-pig arterial muscle, J. Physiol. (London) 355, 43. Bowman, A. and A.H. Drummond, 1984, Cyclic GMP mediates neurogenic relaxations in the bovine retractor penis muscle, Br. J. Pharmacol. 81, 665.
Bray, K.M., D.T. Newgreen and A.H. Weston, 1987, Some effects of the enantiomers of the potassium channel openers, BRL 34915 and pinacidil, on rat blood vessels, Br. J. Pharmacol. 91 (Suppl.), 357P. Cavero, I., S. Mondot, K. Lawson, M. Mestre, D. Escande and C.G. Calllard, 1988a, 49356 RP: A potent antihypertensive agent with potassium channel activating properties, 12th Scientific Meeting of the International Society of Hypertension, Kyoto, Abstract 1192. Cavero, I., S. Mondot, M. Mestre and D. Escande, 1988b, Haemodynamic and pharmacological mechanisms of the hypotensive effects of cromakalim in rats: Blockade by glibenclamide, Br. J. Pharmacol. 95 (Suppl.), 643P. Cook, N.S., 1988, The pharmacology of potassium channels and their therapeutic potential, Trends Pharmacol. Sci. 9, 21. Cook, N.S., U. Quast, R.P. Hof, Y. Baumlin and C. Pally, 1988, Similarities in the mechanism of action of two new vasodilator drugs: pinacidil and BRL 34915, J. Cardiovasc. Pharmacol. 11, 90. Den Hertog, A., J. Pielkenrood, R. Ras and J. Van den Akker, 1984, The contribution of calcium and potassium to the a-action of adrenahne on smooth muscle cells of the portal vein, pulmonary artery, and taenia caeci of the guinea-pig, European J. Pharmacol. 98, 223. Escande, D., D. Thuringer, M. Laville, J. Courteix and I. Cavero, 1988, RP 49356 is a potent opener of ATP-modulated potassium channels in cardiac myocytes, Br. J. Pharmacol. 95 (Suppl.), 814P. Furchgott, R.F., 1972, The classification of adrenoceptors (adrenergic receptors). An evaluation from the standpoint of receptor theory, in: Catecholamines, eds. H. Blaschko and E. Muscholl (Springer-Verlag, Berlin) p. 283. Glover, W.E., 1982, The aminopyridines, Gen. Pharmacol. 13, 259. Gruetter, C.A., P.J. Kadowitz and L.J. Ignarro, 1981, Methylene blue inhibits coronary arterial relaxation and guanylate cyclase activity by nitroglycerin, sodium nitrate, and amyl nitrate, Can. J. Physiol. Pharmacol. 59, 150. Hamilton, T.C., S.W. Weir and A.H. Weston, 1986, Comparison of the effects of BRL 34915 and verapamil on electrical and mechanical activity in rat portal vein, Br. J. Pharmacol. 88, 103. Hamilton, T.C., R.E. Buckingham, J.C. Clapham, M.C. Coldwell, D.R. Howlett and C. Wilson, 1987, BRL 34915, a novel anti-hypertensive agent with potassium channel activating properties, Cardiovasc. Drugs. Ther. 1, 244. Hollingsworth, M., T. Amrdre, D. Edwards, J. Mironneau, J.P. Savineau, R.C. Small and A.H. Weston, 1987, The releases action of BRL 34915 in rat uterus, Br. J. Pharmacol. 91, 803. Inoue, T., Y. Ito and K. Takeda, 1983, The effects of 2-nicotinamidoethyl nitrate on smooth muscle cells of the dog mesenteric artery and trachea, Br. J. Pharmacol. 80, 459. Inoue, T., Y. Kaumura, T. Fujisawa, T. Itoh and H. Kuriyama, 1984, Effects of 2-nicotinamidoethyl nitrate (nicorandil; SG-75) and its derivatives on smooth muscle cells of the canine mesenteric artery, J. Pharmacol. Exp. Ther. 229, 793.
239 Itoh, T., K. Furukawa, M. Kajiwara, K. Kitamura, H. Suzuki, Y. Ito and H. Kuriyama, 1981, Effects of 2-nicotinamidoethyl nitrate on smooth muscle cells and on adrenergic transmission on guinea-pig and porcine mesenteric arteries, J. Pharmacol. Exp. Ther. 218, 260. Jacobs, A. and W.R. Keatinge, 1974, Effects of procaine and lignocaine on electrical and mechanical activity of smooth muscle of sheep carotid arteries, Br. J. Pharmacol. 51, 405. Kajiwara, M., G. Droogmans and R. Casteels, 1984, Effects of 2-nicotinamidoethyl nitrate (nicorandil) on excitation contraction coupling in the smooth muscle cells of the rabbit ear artery, J. Pharmacol. Exp. Ther. 230, 462. Kreye, V.A.M., G.D. Baron, J.B. Luth and H. Schmidt-Gayk, 1975, Mode of action of sodium nitroprusside on vascular smooth muscle, Naunyn-Schrniedeb. Arch. Pharmacol. 288, 381. Kukovetz, W.R., G. PSch and S. Holzmann, 1981, Cyclic nucleotides and relaxation of vascular smooth muscle, in: Vasodilatation, eds. P.M. Vanhoutte and I. Leusen (Raven Press, New York) p. 339. Lebrun, P., V. Devreux, M. Hermann and A. Herchuelz, 1988, Pinacidil inhibits insulin release by increasing K ÷ outflow from pancreatic B-cells, European J. Pharmacol. 156, 283. Lefer, D.J. and A.M. Lefer, 1988, Studies on the mechanism of the vasodilator action of nicorandil, Life Sci. 42, 1907. Mondot, S., M. Mestre, C.G. Caillard and I. Cavero, 1988, RP 49356: A vasorelaxant agent with potassium channel activating properties, Br. J. Pharmacol. 95 (Suppl.), 813P. Murakami, K., H. Karaki and N. Urakawa, 1987, Comparison of the inhibitory effects of nicorandil, nitroglycerin and isosorbide dinitrate on vascular smooth muscle of rabbit aorta, European J. Pharmacol. 141,195. O'Donnell, S.R. and J.C. Wanstall, 1985, Responses to the fl2-selective agonist procaterol of vascular and atrial preparations with different functional fl-adrenoceptor populations, Br. J. Pharmacol. 84, 227. Quast, U., 1988, Inhibition of the effects of the K + channel stimulator cromakalim (BRL 34915) in vascular smooth muscle by glibenclamide and forskolin, Naunyn-Schmiedeb. Arch. Pharmacol. 337 (Suppl.), R72. Quast, U. and N.S. Cook, 1988, Potent inhibitors of the effect of the K + channel opener BRL 34915 in vascular smooth muscle, Br. J. Pharmacol. 93 (Suppl.), 204P.
Satake, N. and S. Shibata, 1987, The mechanism of inhibitory effects of nicorandil, a new antianginal agent, on contractile responses to a 1- and a2-adrenoceptor agonists in isolated vascular smooth muscles, J. Cardiovasc. Pharmacol. 10 (Suppl. 8), $38. Schmid-Antomarchi, H., J. De Weille, M. Fosset and M. Lazdunski, 1987, The receptor for antidiabetic sulfonylureas controls the activity of the ATP-modulated K + channel in insulin-secreting cells, J. Biol. Chem. 262, 15840. Southerton, J.S. and A.H. Weston, 1987, Some effects of Ca 2+and K+-channel blocking agents on responses to BRL 34915 and pinacidil in the isolated rat portal vein, J. Physiol: 391, 77P. Stanfield, P.R., 1983, Tetraethylammonium ions and the potassium permeability of excitable cells, Rev. Physiol. Biochem. Pharmacol. 97, 1. Waud, D.R. and R.B. Parker, 1971, Pharmacological estimation of drug-receptor dissociation constants. Statistical evaluation. II. Competitive antagonists, J. Pharmacol. Exp. Ther. 177, 13. Weir, S.W. and A.H. Weston, 1986a, Effect of apamin on responses to BRL 34915, nicorandil and other relaxants in the guinea-pig taenia caeci, Br. J. Pharmacol. 88, 113. Weir, S.W. and A.H. Weston, 1986b, The effects of BRL 34915 and nicorandil on electrical and mechanical activity and on S6Rb efflux in rat blood vessels, Br. J. Pharmacol. 88, 121. Weston, A.H., 1987, Some effects of nicorandil on the smooth muscle of the rat and guinea pig, J. Cardiovasc. Pharmacol. 10 (Suppl. 8), $56. Wilson, C., R.E. Buckingham, S. Mootoo, L.S. Parrott, T.C. Hamilton, S.C. Pratt and M.A. Cawthorne, 1988a, In vivo and in vitro studies of cromakalim (BRL 34915) and glibenclamide in the rat, Br. J. Pharmacol. 93 (Suppl.), 126P. Wilson, C., M.C. Coldwell, D.R. Howlett, S.M. Cooper and T.C. Hamilton, 1988b, Comparative effects of K + channel blockade on the vasorelaxant activity of cromakalim, pinacidil and nicorandil, European J. Pharmacol. 152, 331. Ziinkler, B.J., S. Lenzen, K. M~_ner, U. Panten and G. Trube, 1988, Concentration-dependent effects of tolbutamide, meglitinide, glipizide, glibenclamide and diazoxide on ATP-regulated K + currents in pancreatic B-cells, NaunynSchmiedeb. Arch. Pharmacol. 337, 225.
231
Elsevier EJP 50824
Glibenclamide is a competitive antagonist of cromakalim, pinacidil and RIP 49356 in guinea-pig pulmonary artery M a n f r i d Eltze Department of Pharmacology, Byk Gulden Pharmaceuticals, D-7750 Konstanz, F.R.G.
Received 29 December 1988, revised MS received 23 February 1989, accepted 21 March 1989
The relaxant effect of cromakalim (BRL 34915), pinacidil and RP 49356 (N-methyl-2-(3-pyridyl)-tetrahydrothiopyran-2-carbothioamide-l-oxide) on the sustained contractions induced by 20 mM KC1 were compared with the effects of nicorandil. The preparation used was vascular smooth muscle of phenoxybenzamine-treated pulmonary artery tings from reserpinized guinea-pigs. Cromakalim, pinacidil, RP 49356 and nicorandil relaxed the tissues with - l o g ECs0 values of 6.78, 6.12, 6.02 and 5.46, respectively. The inhibitory effect of cromakalim, pinacidil and RP 49356, but not of nicorandil, was competitively antagonized by glibenclamide (10-7-3 × 10 -6 M), yielding uniform pA 2 values of 7.17-7.22 against all three relaxant drugs. The order of potency of other K ÷ channel blocking agents for the inhibition of vasorelaxation by cromakalim, pinacidil and RP 49356 was procaine > 4-aminopyridine > tetraethylammonium. The mainly competitive type of inhibition of the RP 49356-induced response was more comparable to that with pinacidil than with cromakalim. The relaxation caused by nicorandil was only attenuated by a high concentration of 4-aminopyridine or tetraethylammonium but was markedly antagonized by methylene blue (3 × 10-6-10 -5 M) and potentiated by M&B 22948 (3 × 10-6-10 -5 M). These results suggest that the vascular relaxation caused in guinea-pig pulmonary artery by cromakalim, pinacidil and RP 49356 is mediated through the same glibenclamide-sensitive K + channel whereas the major mechanism for the effect of nicorandil seems to involve stimulation of guanylate cyclase. K ÷ channel activators; Glibenclamide; Pulmonary artery; (Guinea-pig)
1. Introduction Cromakalim (BRL 34915) belongs to a novel class of antihypertensive agents which appear to produce vasorelaxation by activating an outward K + current (Allen et al., 1986; Hamilton et al., 1986; Cook, 1988). Studies on the mechanism of action of pinacidil (Arrigoni-Martelli et al., 1980; Southerton and Weston, 1987) and RP 49356 (Nmethyl-2-(3-pyridyl)-tetrahydrothiopyran-2-carbothioamide-l-oxide) (Cavero et al., 1988a; Escande et al., 1988; Mondot et al., 1988) have shown that these vasorelaxants share this membrane hyperpolarizing property. Although the antianginal agent, nicorandil, has been reported to open
potassium channels in vascular smooth muscle (Inoue et al., 1983; Kajiwara et al., 1984; Weston, 1987; Lefer and Lefer, 1988) it has been suggested that, due to the presence of a nitro group on the molecule, some of the smooth muscle relaxing effects of this agent are mediated in a manner characteristic of nitro vasodilators (Inoue et al., 1983; Inoue et al., 1984; Murakami et al., 1987). The insulin-releasing agent and potent inhibitor of ATP-sensitive K + channels in pancreatic fl-ceUs, glibenclamide (Schmid-Antomarchi et al., 1987; Ztinkler et al., 1988), has recently been shown to antagonize the vascular smooth muscle relaxation caused by cromakalim (Quast and Cook, 1988; Wilson et al., 1988a) and RP 49356 (Mondot et
0014-2999/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)
232
al., 1988) in vitro and to inhibit their hypotensive effect in vivo (Cavero et al., 1988b; Mondot et al., 1988). Thus, glibenclamide may also deactivate vascular K + channels that resemble pancreatic fl-cell or cardiac ATP-sensitive K + channels (Schrnid-Antomarchi et al., 1987; Escande et al., 1988). More recently, high concentrations of pinacidil have been shown to increase 86Rb+ outflow and to inhibit insulin release from rat pancreatic islets, suggesting that the ATP-sensitive K + channel in this tissue can be opened by a K + channel activator (Lebrun et al., 1988). The aim of the present study was therefore to investigate whether the inhibition, via K + channel blockade, by glibenclamide of the vasorelaxant response to cromakalim and RP 49356 would also be demonstrable for pinacidil and nicomndil, or whether these compounds act through a different mechanism. In order to explore these possibilities, the effects of three other agents with K + channel blocking activity, procaine (Jacobs and Keatinge, 1974), 4-aminopyridine (Glover, 1982) and tetraethylammonium (Stanfield, 1983) were tested for inhibition of vasorelaxation in the isolated guineapig pulmonary artery precontracted with low concentrations of KC1.
2. Materials and methods
2.1. Organ bath-suspended guinea-pig pulmonary artery Guinea-pigs (male, 350-450 g) were pretreated with reserpine (1 m g / k g i.p., 24 h before the experiment) to minimize possible interference by the release of endogenous catecholamines. Isolated ring segments of main pulmonary artery were set up as described by O'Donnell and Wanstall (1985) under a 1-g resting tension then were pre-exposed to phenoxybenzamine (10 -5 M for 30 min followed by washout) in order to exclude possible interactions with a-adrenoceptors. The bathing solution in the 10 ml organ baths consisted of (mM): NaC1 114, KC1 4.7, CaC12 2.5, K H 2 P O 4 1.2, MgSO4 1.2, NaHCO3 25.0 and glucose 11.1, maintained at 3 7 ° C and continuously bubbled with 95% Oz-5% CO 2. The contractions of four
tissues from two animals tested in parallel were measured isometrically (K-30, Hugo Sachs Elektronik) and recorded on multichannel recorders (Kipp and Zonen, BD 9).
2.2. Response curves to relaxant drugs After 20 min equihbration, the tissues were contracted by addition of 20 m M KC1 and the responses were allowed to reach a plateau (20-30 min). The relaxant drugs were then added to the preparations in half log unit increments until peak relaxation was achieved. The effects of the vasorelaxant drugs were expressed as % decrease in the tension produced by 20 mM KC1. The apparent potency of the drugs was expressed as their - l o g ECs0 value (median with 95% confidence limits), i.e. the - l o g molar concentration that had an effect equal to 50% of the individual maximal effect. In a second approach, the ECs0 values for the drugs were determined as the concentration producing a true 50% decrease in the contractile effect elicited by 20 m M KC1.
2.3. Effect of antagonists In each experiment, reproducible concentration-response curves for the relaxant drugs were obtained at 60-80 min intervals, the antagonists or enzyme inhibitors being equilibrated for either 20 min (glibenclamide, procaine, 4-aminopyridine, tetraethylammonium), 30 min ( M & B 22948) or 45 min (methylene blue) with the tissues before rechallenge with the same vasorelaxant drug. Schild plots for competitive antagonism were made from the dose ratios (x) of the relaxant drug for four different antagonist concentrations [B] so as to estimate the pA 2 value with 95% confidence limits and the slope of the regression line for each organ segment (Arunlakshana and Schild, 1959). The regression line for all organs was fitted by a conventional least squares method (Waud and Parker, 1971). The point estimator (mean) and its 95% confidence limits were calculated on a logtransformed scale. Statistical significance was assumed for the differences between the pA 2 values calculated or deviation of the slopes from 1.00 when P ~< 0.05.
233 --Guinea-pig
2.4. Drugs
pulmonary artery
_oC 100
(+)-Cromakalim (BRL 34915), (+)-pinacidil monohydrate, RP 49356 (N-methyl-2-(3-pyridyl)tetrahydrothiopyran-2-carbothioamide-l-oxide) and nicorandil were generous gifts from Beecham, Leo, Rh6ne-Poulenc and Merck, respectively. Glibenclamide and M & B 22948 (2-o-propoxyphenyl-8-azapurin-6-one) were kindly donated by Hoechst and May & Baker, respectively. All other drugs were purchased from Sigma (Munich).
3. Results
3.1. Vasorelaxant effects of cromakafim, pinacidil, RP 49356 and nicorandil on contractions induced by 20 mM KCl As shown in fig. 1, the cumulative application of cromakalim (3 × 10-8-3 x 10 -6 M), pinacidil (10-7-3 × 10 -5 M), RP 49356 (10-7-3 X 10 -5 M) and nicorandil (10-7-10 -4 M) inhibited the sustained contractions induced by 20 mM KC1 in a concentration-dependent manner. The rank order of relaxant potency calculated from the ECs0 values from the concentration-response curves related to their individual maximal effects (table 1) was cromakalim > pinacidil --- RP 49356 > nicorandil. However, the maximal response to cromakalim (64 + 7%) was lower than that to pinacidil, RP 49356 and nicorandil, ranging from 80 to 93% (table 1). The same order of potency was obtained
o× o
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6
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Fig. 1. Cumulative concentration-response curves for the relaxant effect of cromakalim (CR), pinacidil (PI), RP 49356 (RP) and nicorandil (NI) in guinea-pig pulmonary artery precontracted with 20 m M KCI. The force developed in response to 20 mM KC1 was 0.79+0.17 g (mean+S.D., n = 3 2 ) . All responses are expressed as percent of the amplitude of the contraction due to KC1. The results are means-{-S.D, for 14-16 ring segments prepared from 7-8 animals for each drug.
when the ECs0 values were calculated on the basis of the true 50% decrease in contractile force elicited by 20 mM KC1. The - l o g ECs0 values (median, 95% confidence limits, n = 14-16) were: cromakal i m = 6 . 2 6 (5.43; 7.09), pinacidil=5.88 (5.21; 6.54), RP 49356 = 5.86 (5.19; 6.54) and nicorandil = 5.35 (4.90; 5.81).
3.2. Effect of glibenclarnide Pretreatment of the tissues for 20 min with glibenclamide (10-7-3 x 10 -6 M) produced parallel shifts to the right of the relaxant responses to cromakalim, pinacidil and RP 49356 (fig. 2) whereas the concentration-response curve to nicorandil was not affected. Thus, glibenclamide behaved as a competitive antagonist of the vaso-
TABLE 1 Activation and blockade of K ÷ channels in guinea-pig pulmonary artery: apparent potency of K ÷ channel activators to inhibit sustained contractions elicited by 20 mM KC1. ECs0 values (median with 95% confidence limits) were calculated from single concentration-response curves relative to individual maximal effects (mean+S.D.; left). Affinity for glibenclamide related to inhibition of drug-induced relaxation, pA 2 values (mean with 95% confidence limits) and regression slopes (mean_+S.D.) were calculated from Schild plots (right). Drug
Cromakalim Pinacidil RP 49356 Nicorandil
Activators: relaxation
Antagonist: glibenclamide
- log ECso (M)
Relative maximal effect (%)
n
pA 2
Slope
n
6.78 6.12 6.02 5.46
64+ 80+ 89 + 93 +
16 14 14 16
7.17 (6.67; 7.67) 7.22 (6.65; 7.79) 7.22 (6.91; 7.53) N o antagonism
1.01 +0.06 0.90+0.12 0.95 +0.06
28 32 38 8
(6.55; (5.56; (5.65; (5.11;
7.01) 6.67) 6.38) 5.82)
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234
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3.3. Effect of other K + channel blockers
Cromakalim
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The inhibition of the vasorelaxant effect of cromakalim by procaine (3 × 10-4-3 X 10 -3 M) was characterized by a parallel shift and concurrent depression of the maximum effect of the concentration-response curve, whereas procaine produced an apparently competitive antagonism of the vasorelaxation induced by pinacidil and RP
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Fig. 2. Cumulative concentration-response curves for the relaxant effect of cromakalim, pinacidil, RP 49356 and nicorandil in guinea-pig pulmonary artery in the presence of glibenclamide (rn, 10 -7 M; zx, 3 x 1 0 -7 M; ~ , 10 -6 M; v, 3 x 10 -6 M) added 20 min before the drugs (O, control). Each point represents the mean 4-S.D. from typical experiments with four organ segments from two animals. Ordinate: % of maximal drug control response.
relaxant action of cromakalim, pinacidil and RP 49356. The pA z values (7.17-7.22) were not significantly different from each other (P > 0.05). Also, the slopes of the regression lines were not significantly different from unity (P > 0.05). The data obtained from the Schild plots are summarized in table 1. Glibenclamide did not increase the vascular tone of the tissues at concentrations effective for antagonizing the response to the dilator drugs (not shown).
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6
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235
49356. Procaine was almost ineffective against nicorandil-induced vasorelaxation (fig. 3). Similar effects were observed using 4-aminopyridine (10-3-10 -2 M) as antagonist of the vasorelaxation due to the various drugs. Again, the concentration-response curves for the relaxant effect of cromakalim were shifted to the right and depressed by 4-aminopyridine. In the case of pinacidil and RP 49356, concentrations of 10-3-3 × 10 -3 M produced horizontal displacement
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Fig. 4. Cumulative concentration-response curves for the relaxant effect of cromakalim, pinacidil, RP 49356 and nicorandil in guinea-pig pulmonary artery in the presence of 4aminopyridine (El, 10 - 3 M; zx, 3 × 1 0 - 3 M; ~ , 10 - 2 M) added 20 min before the drugs (O, control). Each point represents the m e a n + S . D , from typical experiments with four organ segments from two animals. Ordinate: % of maximal drug control response to the drug.
Fig. 5. Cumulative concentration-response curves for the relaxant effect of cromakalim, pinacidil, R P 49356 and nicorandil in guinea-pig pulmonary artery in the presence of tetraethy l a m m o n i u m 03, 3 × 1 0 -3 M; zx, 10 - 2 M; ~ , 3 × 1 0 - 2 M) added 20 min before the drugs (e, control). Each point represents the mean + S.D. from typical experiments with four organ segments from two animals. Ordinate: % of maximal control response to the drug.
without depressing the maximal effect of either drug. Nicorandil-induced vasorelaxation was only affected by 4-aminopyridine concentrations of 10 -2 M (fig. 4). Tetraethylammonium (3 × 10-3-3 x 10 -2 M) was the least potent K + channel blocker as regards inhibition of the vasorelaxation produced by the relaxant drugs: concentrations of 10 -2 M or higher were necessary to produce depression of the maximal response to cromakalim whereas there
236 Guinea-pig pulmonary artery
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Fig. 6. Cumulative concentration-response curves for the relaxant effect of nicorandil (O, control) in guinea-pig pulmonary artery in the presence of M&B 22948 or methylene blue added 30 or 45 min before nicorandil, respectively. Figures refer to - l o g molar concentrations. Each point represents the mean + S.D. from typical experiments with four organ segments from two animals. Ordinate: % of maximal control response to nicorandil.
was a near parallel shift to the right of both pinacidil and RP 49356 concentration-response curves. The relaxation due to nicorandil was concentration-dependently antagonized by 3 × 10 -310 -2 M tetraethylammonium and higher concentrations (3 × 10 -2 M) failed to further increase the inhibition (fig. 5). 3.4. Effect of methylene blue and M & B 22948 on nicorandil-induced relaxation As shown in fig. 6, the vasorelaxant effect of nicorandil on the 20 mM KCl-induced sustained contraction was reduced by a 45-min exposure to 3 x 10-6-10 -5 M methylene blue but was significantly potentiated by a 30-rain preincubation of the tissues with 3 × 10-6-10 -5 M M & B 22948.
4. Discussion
The study showed, that the K + channel openers, cromakalim (Allen et al., 1986; Hamilton et al., 1986; Hollingsworth et al., 1987; Cook, 1988), pinacidil (Bray et al., 1987; Cook et al., 1988) and RP 49356 (Cavero et al., 1988a; Escande et al., 1988; Mondot et al., 1988) are effective concentration-dependent inhibitors of the contractions induced in the guinea-pig isolated pulmonary artery by low KC1 concentrations. Inhibition of the KCl-induced vascular contractions by cromakalim
has been shown to depend on the KC1 concentration used; the higher the KC1 concentration, the less the inhibition (Weir and Weston, 1986a; Cook et al., 1988). The difference between membrane potential and K ÷ equilibrium potential, especially at a low KC1 concentration, allows the K + efflux inducible by a putative K ÷ channel opener to occur, resulting in membrane hyperpolarization, reduced probability of Ca 2÷ channel opening and vascular relaxation. The pulmonary artery of the guinea-pig is known for its high K ÷ flux as are the ear artery, aorta and pulmonary artery of the rabbit (Bolton and Clapp, 1984; Den Hertog et al., 1984). Raising the external potassium ([K÷]o) in rabbit pulmonary artery is accompanied by a large increase in 86Rb+ efflux which serves as a marker for potassium permeability. In the present experiments, 20 mM KC1 was the concentration selected, since this concentration elicits sustained and reproducible contractions of guinea-pig pulmonary artery which are consistently relaxed by cromakalim, pinacidil, RP 49356 and nicorandil. Cromakalim proved to be 3-5 times more potent in terms of ability to relax KCl-induced contractions, than the nearly equieffective pinacidil and RP 49356, which were 3-fold more potent than nicorandil. These results are in good agreement with those of similar experiments on aorta and portal vein preparations from rats (Weir and Weston, 1986b; Bray et al., 1987) and rabbits (Hamilton et al., 1987). Of the drugs investigated, nicorandil produced near maximal relaxation (93%) of the precontracted vessel whereas cromakalim elicited a maximal relaxation of only approximately 64%. RP 49356 and pinacidil exhibited an activity intermediate between that of the former two drugs. When the tissues were in contact with glibenclamide, competitive antagonism was observed against the relaxant responses to cromakalim, pinacidil and RP 49356 whereas the vasorelaxant effect of nicorandil was resistant. One of the most reliable techniques used to differentiate between receptors and receptor subtypes is comparison of the dissociation constants of antagonists (Furchgott, 1972). Extending this approach to putative K + channel binding sites on smooth muscle of
237
guinea-pig pulmonary artery, it seems clear that the action of glibenclamide can be explained simply by blockade of one K ÷ channel with a uniform affinity constant, irrespective of the K ÷ channel activators used for its assessment. These findings suggest convincingly that the vasorelaxant effects of cromakalim, pinacidil and RP 49356 are medicated by the same type of K + channel in this particular preparation. The pA 2 values obtained for glibenclamide from shifts of the dose-response curves of cromakalim, pinacidil and RP 49356 (7.17-7.22) were in very good agreement with the pA 2 obtained for cromakalim and glibenclamide on rat portal vein (pA 2 = 7.20; Quast, 1988). This suggests a common site of competition between activators and glibenclamide on a similar K ÷ channel in both guinea-pig pulmonary artery and rat portal vein. The ability of relatively high glibenclamide concentrations (10 -7 M) to inhibit competitively the opening of K + channel induced by cromakalim or other K + channel activators in both vascular tissues contrasts with the high potency of glibenclamide (in the nanomolar range) to inhibit ATP-dependent K + currents in rat insulinoma cells (Schmid-Antomarchi et al., 1987) or mouse pancreatic fl-cells (Ziinkler et al., 1988). Nevertheless, it can be concluded that glibenclamide is a potent inhibitor of the vasorelaxant effects of cromakalim (Quast and Cook, 1988; Wilson et al., 1988a), RP 49356 (Mondot et al., 1988) and pinacidil (this study) and thus may represent a valuable pharmacological tool for investigating the mechanism of K + channel opening. The rank order of potency of other K + channel blockers to antagonize the relaxant responses to cromakalim, pinacidil and RP 49356 was procaine (threshold concentration 3 × 10 -4 M) > 4-aminopyridine (threshold concentration 10 -3 M ) > tetraethylammonium (threshold concentration 3 × 10 -3 M). Competitive antagonism was generally observed with pinacidil and RP 49356 whereas inhibition of cromakalim-induced vasodilatation was non-competitive. Recent pharmacological studies on rabbit mesenteric artery preparations gave the same rank order of antagonist potency and different types of inhibition of these K ÷ channel antagonists against cromakalim and pinacidil (Wilson et al., 1988b).
The vasorelaxation elicited by nicorandil was resistant to glibenclamide blockade, was hardly affected by procaine but was affected to a small extent by 4-aminopyridine and tetraethylammonium, indicating a predominant involvement of mechanisms other than K + channel activation for vasorelaxation. The responses to nicorandil were potentiated by M & B 22948, a selective inhibitor of cGMP-phosphodiesterase (Bowman and Drummond, 1984; Kukovetz et al., 1981), and inhibited by methylene blue, an inhibitor of guanylate cyclase (Gruetter et al., 1981). Thus, the results of the present study support the view that nicorandilinduced vasodilatation in guinea-pig pulmonary artery involves mainly the generation of cGMP, an effect which has been shown previously in rabbit aorta (Murakami et al., 1987); other mechanisms of action, e.g. inhibition of Ca 2+ mobilization in the cell (Itoh et al., 1981; Kreye et al., 1975; Satake and Shibata, 1987) may also be involved. Recent pharmacological studies showed that the tetrahydrothiopyrane derivative, RP 49356, behaved like cromakalim in inhibiting spontaneous contractile activity and increasing the effiux of 86Rb+ in rat portal vein (Cavero et al., 1988a). RP 49356 inhibited the contractions evoked in rat isolated aortic rings by 4-5 mM KCI but those evoked by 50 mM KC1 were not modified. Additionally, the relaxation of rat aortic rings contracted with 20 mM KC1 by RP 49356 or cromakalim was blocked by glibenclamide (Mondot et al., 1988). This suggests that RP 49356 might act in a manner similar to cromakalim in activating K + channels in vascular smooth muscle (Cavero et al., 1988a). Confirmation of this general assumption was now obtained from the finding that glibenclamide antagonizes the vasorelaxation caused by both drugs. Comparing the inhibition by other K + channel blockers, e.g. procaine, 4-aminopyridine and tetraethylammonium, of the vasorelaxation due to RP 49356 and of the vasorelaxation caused by pinacidil, evidences a mainly competitive antagonism. However the inhibition of cromakalim responses by these blockers is non-competitive at higher concentrations. Whether this effect, in addition to the predominant activation of glibenclamide-sensitive K ÷
238 c h a n n e l s , p o i n t s to a n o t h e r m e c h a n i s m for t h e production of vascular relaxation by cromakalim, p o s s i b l y n o t s h a r e d w i t h p i n a c i d i l a n d R P 49356, c a n n o t yet b e a n s w e r e d . I n c o n c l u s i o n , t h e p r e s e n t studies w i t h g u i n e a pig isolated pulmonary artery demonstrate that g l i b e n c l a m i d e is a n e q u a l l y p o t e n t c o m p e t i t i v e a n t a g o n i s t o f t h e v a s c u l a r r e l a x a n t effects o f c r o m a k a l i m , p i n a c i d i l a n d R P 49356 b u t n o t o f t h e effects o f n i c o r a n d i l , s u g g e s t i n g t h a t t h e v a s o r e l a x a t i o n c a u s e d b y the t h r e e d r u g s m a y p r e d o m i n a n t l y i n v o l v e o p e n i n g of t h e s a m e K ÷ channel. This property could make glibenclamide a useful d r u g f o r t h e r e l i a b l e d e t e c t i o n o f h e t e r o g e n o u s K ÷ c h a n n e l s m e d i a t i n g a p a r t i c u l a r response. T h e v a s o d i l a t a t i o n c a u s e d b y t h e s e d r u g s w a s c o m p a r a t i v e l y less a f f e c t e d b y o t h e r K ÷ c h a n nel b l o c k e r s , e.g. p r o c a i n e , 4 - a m i n o p y r i d i n e a n d t e t r a e t h y l a m m o n i u m . N e v e r t h e l e s s , t h e l a t t e r exhibit an inhibitory mechanism against pinacidil a n d R P 49356, w h i c h is m a i n l y c o m p e t i t i v e a n d different from that against cromakalim. Whether this p h e n o m e n o n c a n b e f u r t h e r u s e d t o d i f f e r e n t i ate b e t w e e n p o s s i b l e K ÷ c h a n n e l h e t e r o g e n e i t y in v a s c u l a r s m o o t h m u s c l e r e m a i n s to b e e l u c i d a t e d .
Acknowledgement The author gratefully acknowledges the skillful technical assistance of Miss Caroline Biirkle.
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