Effects of pinacidil on guinea-pig airway smooth muscle contracted by asthma mediators

European Journal of Pharmacology, 157 (1988) 221-226

221

Elsevier EJP 50542

Effects of pinacidil on guinea-pig airway smooth muscle contracted by asthma mediators J e n s E r i k N i e l s e n - K u d s k *, S o r e n Mellemkj~er, C h a r l o t t e S i g g a a r d a n d C l a u s B r o c k n e r N i e l s e n Institute of Pharmacology, The Bartholin Building. University of Aarhus, DK-8000 Aarhus C, Denmark Received 19 May 1988, revised MS received 23 August 1988, accepted 6 September 1988

Pinacidil is a new antihypertensive, direct vasodilator drug which has been classified as a K + channel opener. The present study demonstrated a concentration-dependent relaxant activity of pinacidil in guinea-pig tracheal preparations. The potency and efficacy of pinacidil depended on the agent used to induce tracheal tone. Tracheal preparations with spontaneous tone or precontracted by different asthma mediators were completely relaxed by pinacidil. A high potency was found in spontaneously contracted preparations (ECs0 = 7.8 x 10-7 M). The ECs0 values ranged from 2.3 tO 5.4 X 10 -6 M in histamine-, PGFE,~- or LTC4-contracted preparations. When tone was induced by carbachol, the ECs0 was 2.1 x 10 -5 M. In contrast, pinacidil produced incomplete relaxation and had a low potency in preparations contracted by 30 or 124 mM K + Krebs solutions. This effect profile differed from that seen with f/E-receptor agonists, xanthines and Ca 2+ antagonists in guinea-pig trachealis and seems compatible with K + channel opening as a primary mode of relaxation for pinacidil in airway smooth muscle. Pinacidil; K + channel opening; Smooth muscle (airway); Trachea (guinea-pig)

1. Introduction Pinacidil, a pyridylcyanoguanidine derivative, has recently been marketed for clinical use as an antihypertensive drug. Its haemodynamic profile is characteristic of a direct vasodilator with preferential action on precapillary resistance vessels (Carlsen et al., 1981). Fluid retention and symptoms due to reflex tachycardia are c o m m o n sideeffects, as seen with other vasodilators. The drug may be especially suited for combination therapy with thiazide diuretics and, if necessary, fl-adrenoceptor blockers for control of side-effects and optimal blood pressure regulation (Carlsen et al., 1983; Nicholls et al., 1986). Pinacidil is structurally different from other antihypertensive agents.

* To whom all correspondence should be addressed.

Its primary mechanism of action is opening of potassium channels in vascular smooth muscle cell membranes. This leads to hyperpolarisation (towards the equilibrium potential for potassium) and smooth muscle relaxation by inhibition of depolarisation-induced contractile activity (Southerton et al., 1987; Weston, 1988; Hermsmeyer, 1988). fl-Adrenoceptor blockers are widely used in the treatment of hypertension. However, there is a risk of precipitation of bronchoconstriction and aggravated lung function in patients with coexisting bronchial asthma or other obstructive lung diseases (McNeill, 1964; Shinner et al., 1976). In contrast, calcium-entry blockers may have beneficial effects on lung function in these patients (Russi and Ahmed, 1984). The action of new antihypertensive drugs on airway smooth muscle contractility is of clinical interest.

0014-2999/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)

222 Cohen and Colbert (1986) have reported that pinacidil acted as a relaxant in isolated guinea-pig tracheas contracted by histamine or carbamylcholine. Potassium channel opening represents a new pharmacological principle for smooth muscle relaxation. It could possibly be of therapeutic interest in relation to bronchial asthma. We have now examined the effects of pinacidil on guinea-pig tracheal preparations contracted by different asthma mediators. The effects on spontaneously or potassium-contracted preparations were also studied.

2. Materials and methods

2.1. Isometric tension measurements in guinea-pig isolated tracheal preparations Albino guinea-pigs of either sex (body weight 320-700 g) were used. They were killed by a blow to the neck and the trachea was rapidly excised and immersed in cold Krebs solution. The trachea was cleaned from adhering fat and connective tissue with the help of a microscope and was cut into tubular segments comprising two adjoining cartilage rings. Six preparations from each animal were transferred to temperature-regulated (37 o C) 5-ml organ baths containing Krebs solution (composition in mM: NaC1 118.0, KC1 4.6, CaC12 2.5, MgSO 4 1.15, N a H C O 3 24.9, K H 2 P O 4 1.15, glucose 5.5, p H = 7.4) and aerated with a mixture of 95% 02 and 5% CO 2. Each intact tubular tracheal preparation was mounted between two fine stainless steel pins in a newly constructed precision myograph (Nielsen-Kudsk et al., 1986a). One of the pins was connected to a highly isometric etched foil strain-gauge transducer, which like the other pin, was mounted on small adjustable precision rolling tables. The transducer signal was amplified and displayed on a Watanabe linearcorder model W R 3101. The tracheal preparations were initially suspended under a passive tension of 0.6 g. The bathing solution was exchanged frequently during equilibration (ca. 1 h). The preparations reached a mean spontaneous tone of 1.92 g + 0.13 S.E. (n = 14) during the equilibration period.

2.2. Experiments The effects of pinacidil were assessed from relaxant concentration-effect curves. The tracheal preparations were precontracted with isotonic high-K + Krebs solutions (30 m M K ÷ or 124 m M K ÷) or with different asthma mediators. Histamine 10 -6 M, LTC 4 10 -8 M, PGF2~ 10 -5 M or carbachol 2 × 10 -7 M (the latter as muscarinic receptor agonist) were used. The isotonic K + Krebs solutions were similar to the Krebs solution except that an equimolar amount of NaC1 was replaced by KCI to 30 or 124 m M K ÷. All of these experiments were carried out in the presence of indomethacin (10 -6 M). Indomethacin was added to the baths about 30 min before the addition of the contractile agent and resulted in about 80% inhibition of the spontaneous tone. A recent study has shown that the presence of indomethacin is essential for obtaining reproducible and consistent potassium-induced contractions in the guinea-pig trachealis (Nielsen-Kudsk et al., 1986b). When stable tonic contractions had developed in response to the agents used, pinacidil was added cumulatively by injection to the organ baths. The tension was allowed to stabilize between increases in the concentration of pinacicil. Relaxant responses are expressed as % decrease in the tension produced by the contractile agents. A control contraction, with only vehicle added, was run in parallel. The concentrations of mediators used were chosen to correspond to the submaximally effective concentrations (see Karlsson, 1984 for concentration-effect curves of asthma mediators in guinea-pig trachealis) and in order to reach about the same contraction level with the various contractile agents. Relaxant concentration-effect curves for pinacidil were also obtained in preparations with spontaneous tone.

2.3. Data analysis Relaxant mean values ___S.E. were calculated for each concentration of pinacidil. Continuous sigmoid log C / E curves and the corresponding ECs0 , Emax and Hill exponent (S) parameters were obtained from the mean data by non-linear iterative regression analysis by means of the Hill func-

223

tion E = Ema x C S / ( E C ~ 0 + C s) as pharmacodynamic model (cf. Holford and Sheiner, 1981). S expresses the slope of the C / E curve. S.E.s related to the parameters were determined simultaneously, as described by Waud (1975). Analysis of the data, calculation of the parameters, curve-fitting and plotting of the C / E curves were performed on an HP-85 personal computer using the computer programs in BASIC described by Nielsen-Kudsk (1983). Differences between the parameters calculated were evaluated statistically by means of Student's t-test for unpaired comparisons. A significance level of 5% was used.

2.4. Drugs The drugs used were: pinacidil (Leo Pharmaceutical Products, C o p e n h a g e n , D e n m a r k ) , histamine, carbamylcholine chloride (carbachol), indomethacin (Sigma chemical Co., St. Louis, MO, USA), PGF2~ (Amoglandin, KabiVitrum AB, S t o c k h o l m , Sweden), L T C 4 ( M e r c k - F r o s s t , Canada). Pinacidil (21.1 mg) was dissolved in a mixture of 96% ethanol, propylene glycol and water (2 + 2 + 4 ml) to a stock solution of 10 -2 M and was diluted with 0.9% NaC1. LTC 4 was dissolved in 96% ethanol and was diluted with 0.9% NaC1. Other drugs were dissolved directly in 0.9% NaC1.

scribed (Nielsen-Kudsk et al., 1986b). An initial phasic contraction was followed by a larger and sustained contraction with a mean tension of 2.64 + 0.39 g (n = 8). Addition of the isotonic 30 m M K ÷ Krebs solution resulted in a monophasic tonic contraction with the same tension level (2.60 + 0.20 g, n = 11). Control contractions, where vehicle was added instead of pinacidil, showed that all contractile agents produced highly stable contractions during the time of experimentation. There was no effect of vehicle up to concentrations corresponding to 10 - 4 M pinacidil, which was the highest concentration used. The mean tension before the addition of pinacidil to preparations with spontaneous tone was 1.59 + 0.16 S.E. (n = 12).

3.2. Effects of pinacidil Pinacidil produced concentration-dependent relaxation of all types of contracted tracheal preparations studied. The relaxant concentration-effect curves obtained are shown in fig. la,b and the derived ECs0, Ema x and Hill exponent values in table 1. All the contractions induced by asthma mediators were completely relaxed by pinacidil. There TABLE 1

3. Results

3.1. Effects of mediators and isotonic high-K + Krebs solutions The tubular tracheal preparations were treated with indomethacin (10 -6 M) and precontracted by asthma mediators or high-K + solutions before the addition of pinacidil. All asthma mediators produced tonic contractions with the following mean tracheal tensions in g + S.E.: histamine (10 - 6 M) 2.30 + 0.26 (n = 14), PGF2~ (10 -5 M) 2.98 + 0.30 (n = 14), L T C 4 (10 -8 M) 2.18 + 0.21 (n = 6) and carbachol (2 × 10 - 7 M ) 3.06 + 0.23 (n = 11). When the bathing solution was changed to the isotonic 124 m M K ÷ Krebs solution a biphasic contractile response developed as previously de-

ECs0 (mol/1), Ema~ (% tracheal relaxation of tension produced by contractile agents) and Hill exponent (S) values_+S.E, derived by non-linear iterative regression analysis from experimental concentration-effect data for the relaxation of guineapig tracheal preparations produced by pinacidil. Except for experiments in preparations with spontaneous tone, the tracheal preparations were treated with indomethacin (10 -6 M) and precontracted by isotonic high-K + Krebs solutions (30 or 124 mM K +) or by different asthma mediators: histamine 10 6 M, PGF2,,10 -5 M, LTC 410 8 Morcarbacho12×10 7 M . n equals number of animals used. Pinacidil

ECs0

Spontaneous Histamine PGF2, LTC4 Carbachol K +(30mM) K+(124mM)

7.84_+0.27×10 2.34_+0.16x10 3.66_+0.20x10 5.43_+0.58×10 2.09_+0.30×10 2.15_+0.17×10 3.00_+0.70×10

Emax 7 -6 -6 -6 -5 -5 5

S

99.0_+0.9 1.51_+0.07 100.0_+1.9 1.24_+0.09 94.7_+1.8 2.08_+0.22 104.7_+3.6 1.33_+0.14 101.3_+5.9 1.16_+0.11 74.9+2.4 1.09-+0.05 74.2_+7.2 1.07_+0.13

n 12 14 14 6 14 11 9

224 125-

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b.

100

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o--c

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0 7-

0

~ z5

75-

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50

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25-

~ 50-

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0 .

-8

-7

~6

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3

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PINACIDIL LOG CONC., ( m o l / l )

-7

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PINACIDIL LOG CONC., ( m o l / l )

Fig. 1. Cumulative concentration-effect curves for the relaxation of guinea-pig tracheal preparations produced by pinacidil. The curves were computer-fitted by non-linear regression analysis. (a) Curves obtained in preparations with spontaneous tone (©, n = 12) or precontracted by isotonic high-K + K_rebs solutions: 30 mM K + (zx, n =11) or 124 mM K + (A, n = 8). (b) Curves obtained in preparations precontracted by various asthma mediators: histamine 10 -6 M (O, n = 14), PGF2,<10 -5 M (O, n = 14), LTC4 10 -8 M (D, n = 6) or carbachol 2 x 10-7 M (11,n = 11). Preparations contracted by high-K + solutions or asthma mediators were treated with indomethacin (10 -6 M). Ordinate shows the % relaxation of tension produced by contractile agents. Vertical bars show S.E. values, n equals number of animals used.

were significant differences b e t w e e n the r e l a x a n t ECs0 values for pinacidil, d e p e n d i n g on which m e d i a t o r was used. T h e o r d e r of p o t e n c y was: h i s t a m i n e > PGF2~ > L T C 4 > c a r b a c h o l . Pinacidil was 9 times m o r e p o t e n t to relax h i s t a m i n e - i n d u c e d c o n t r a c t i o n s (ECs0 = 2.34 × 10 - 6 M ) t h a n c a r b a c h o l - i n d u c e d c o n t r a c t i o n s (ECs0 = 2.09 × 1 0 - 5 M) whereas the differences in p o t e n c y for the effect on histamine-, PGF2~- a n d L T C 4 - i n d u c e d c o n t r a c t i o n s were small. T h e highest p o t e n c y of p i n a c i d i l was f o u n d for the r e l a x a t i o n of tracheal p r e p a r a t i o n s with s p o n t a n e o u s tone, which were c o m p l e t e l y relaxed. T h e ECs0 value was 7.84 × 10 - 7 M. In contrast, p i n a c i d i l h a d a very low p o t e n c y against c o n t r a c t i o n s i n d u c e d b y isotonic h i g h - K + K r e b s solutions a n d caused i n c o m p l e t e relaxation. T h e K + - c o n t r a c t e d p r e p a r a t i o n s were r e l a x e d to a b o u t 60% at the highest c o n c e n t r a t i o n of p i n a c i dil ( 1 0 _4 M ) . T h e c a l c u l a t e d Ema x values were a b o u t 75% relaxation. Pinacidil was 40 times m o r e p o t e n t to relax s p o n t a n e o u s l y c o n t r a c t e d t h a n 124 m M K + - c o n t r a c t e d p r e p a r a t i o n s . N o significant difference was f o u n d b e t w e e n ECs0 a n d Ema x values for p i n a c i d i l in m a x i m a l l y . K + - d e p o l a r i z e d p r e p a r a t i o n s (124 m M K +) c o m p a r e d to 30 m M K +-contracted p r e p a r a t i o n s .

T h e time n e e d e d to p r o d u c e d stable r e l a x a t i o n at each i n c r e a s e in the b a t h c o n c e n t r a t i o n of p i n a c i d i l was a b o u t 5 m i n in s p o n t a n e o u s l y a n d m e d i a t o r - c o n t r a c t e d p r e p a r a t i o n s a n d twice this ( a b o u t 10 min) in K + - d e p o l a r i z e d p r e p a r a t i o n s . T h e lowest Hill e x p o n e n t values (which express the slopes of the C / E curves) were f o u n d in K + - c o n t r a c t e d p r e p a r a t i o n s (cf. table 1).

4. Discussion The present study demonstrated a concentrat i o n - d e p e n d e n t r e l a x a n t activity of pinacidil in g u i n e a - p i g a i r w a y s m o o t h muscle. I n d o m e t h a c i n , in a c o n c e n t r a t i o n that inhibits c y c l o o x y g e n a s e (Flower, 1974), was p r e s e n t in the organ b a t h s w h e n p i n a c i d i l was a d d e d to tracheal p r e p a r a t i o n s contracted by asthma mediators or high-K + Krebs solutions (cf. M e t h o d s ) . Previous studies have shown that i n d o m e t h a c i n does n o t influence the s m o o t h m u s c l e r e l a x a t i o n or v a s o d i l a t o r effect p r o d u c e d b y p i n a c i d i l (Olsen a n d A r r i g o n i Martelli, 1983; K a w a s h i m a a n d Liang, 1985). T h e p o t e n c y a n d efficacy of p i n a c i d i l d e p e n d e d on which agent was used to i n d u c e tracheal tone. Pinacidil r e l a x e d s p o n t a n e o u s l y a n d m e d i a t o r -

225 contracted preparations completely but produced incomplete relaxation when the contractions were induced by high-K + (calculated Ema x values were about 75% relaxation). We found a high potency of pinacidil in preparations with intrinsic tone. The ECs0 value was 7.84 × 10-7 M, the same level or even lower than ECs0 values reported for pinacidil in isolated vascular smooth muscle preparations contracted by various agonists (Toda et al., 1985; Cohen and Colbert, 1986). In contrast, pinacidil was poorly effective on both 30 and 124 mM K+-depolarized preparations (about 40 times less potent). The time needed to produce stable relaxation was twice as long and the C / E curves were less steep than for spontaneously and mediator-contracted preparations relaxed by pinacidil. The ECs0 values obtained in preparations contracted by asthma mediators (histamine, PGF2~, LTC4, carbachol) ranged from 2.3 to 20.9 x 10 -6 M with pinacidil being most potent against histamine-induced contractions and least potent against carbachol-induced contractions. This effect profile for pinacidil in the isolated guinea-pig trachealis differs from that seen with flz-receptor agonists, xanthines and Ca 2+ antagonists, indicating that the mechanism for tracheal relaxation by pinacidil is different. Both flzagonists and xanthines show the same potency irrespective of whether tracheal tone are spontaneous or induced by several different asthma mediators (Karlsson, 1984). The drugs are only slightly less potent against high-K+-contracted preparations whereas Ca 2+ antagonists preferentially relax K+-induced compared to mediatorinduced contractions (Nielsen-Kudsk et al., 1986b). Pinacidil has been classified as a K + channel opener based on reports that the drug produces concentration-dependent hyperpolarisation (close to the K + equilibrium potential) and stimulates 86Rb (a K + marker) efflux in vascular smooth muscle (Weston, 1988). The present finding of poor efficiency for pinacidil in K+-contracted tracheal preparations seems consistent with, but not necessarily decisive evidence for, opening of K + channels as a primary mode of action of the drug in airway smooth muscle also. However, pinacidil produced clear-cut concentration-depen-

dent relaxation even in maximally K+-depolarized tracheal preparations, as also reported for several vascular preparations (Nielsen and ArrigoniMartelli, 1981; Mikkelsen and Pedersen, 1982; Matsui et al., 1986). Opening of K + channels in such preparations cannot evoke cellular hyperpolarization because the K + equilibrium potential approaches the membrane potential. The relaxant activity of pinacidil on high-K + (124 mM)-induced contractions could thus be attributed to a mechanism different from K + channel opening. It also cannot be excluded that the effect of pinacidil on mediator-contracted preparations may involve mechanisms other than K + channel opening. The results presented make it possible to assume that treatment with pinacidil of patients with coexisting hypertension and bronchial asthma will not aggravate lung function, which is a possible risk in antihypertensive therapy with B-adrenoceptor blockers. K + channel opening, which is a new pharmacological principle for smooth muscle relaxation, may possibly prove to be of therapeutic interest in relation to bronchial asthma. Action potential discharges are not normally seen in airway smooth muscle exposed to depolarizing stimuli, due to strong rectifying properties of the cell membrane (Kirkpatrick, 1981). Opening of potential-dependent K + channels seems to be the basis for these rectifying properties (Small and Foster, 1986). It has recently been theorized by Allen et al. (1986) that loss of rectifying properties through inhibition of K + channel opening might account for the hyperreactivity seen in asthma. Drugs like pinacidil would thus theoretically tend to restore rectification and counteract airway smooth muscle hyperreactivity. Further studies with K + channel openers in airway smooth muscle including future studies in patients with bronchial asthma seem justified.

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