A comparison of the relaxant effects of pinacidil in guinea-pig trachea, aorta and pulmonary artery

European Journal of Pharmacology. 167 (1989) 275-280

275

Elsevier EJP 50918

A comparison of the relaxant effects of pinacidil in guinea-pig trachea, aorta and pulmonary artery S o r e n Mellemkja~r, J e n s E r i k N i e l s e n - K u d s k *, C l a u s B r o c k n e r N i e l s e n a n d C h a r l o t t e S i g g a a r d Institute of Pharmacology, The Bartholin Building. University of Aarhus. DK-8000 Aarhus C, Denmark

Received 26 January 1989, revised MS received 8 May 1989, accepted 30 May 1989

The relaxant activity of pinacidil, a proposed K + channel opener, was compared in isolated guinea-pig trachea, aorta and pulmonary artery. In preparations precontracted by histamine or PGF2,, pinacidil produced complete tracheal relaxation but only partial relaxation of vascular tissues. The order of responsiveness was: pulmonary artery > trachea > aorta. The slope of the pinacidil concentration-effect (C/E) curve was much steeper in the tracheal than in the vascular preparations. The pinacidil C / E curves for relaxation were similar when the three types of preparations were precontracted by 124 mM K +. Pretreatment with pinacidil caused a parallel shift of the tracheal histamine C / E curve to the right, whereas the maximal response to histamine was markedly depressed in the pulmonary artery. Pinacidil; K + channels; Trachea; Smooth muscle (vascular)

1. Introduction Pinacidil represents a new class of smooth muscle relaxants that seem to act by increasing potassium conductance, leading to cell membrane hyperpolarisation. Evidence for this potassium channel opening activity was obtained recently with isolated vascular smooth muscle by using electrophysiological and ion flux techniques (Hermsmeyer, 1988; Southerton et al., 1988). Pinacidil is a potent direct vasodilator and has been introduced as an antihypertensive agent. Potassium channel opening properties might also be of interest outside the cardiovascular system (Cook, 1988). In a recent study pinacidil was shown to produce effective relaxation of guinea-pig airway smooth muscle contracted by different asthma mediators (Nielsen-Kudsk et al., 1988).

* To whom all correspondence should be addressed.

The aim of the present study was to simultaneously compare and quantitate the smooth muscle relaxant action profiles of pinacidil in tracheal, aortic and pulmonary artery preparations precontracted with histamine, PGF2~ or 124 m M K ÷Krebs solution. The effects of pinacidil pretreatment on the histamine-contracted isolated guineapig airway and vascular preparations were also studied.

2. Materials a n d m e t h o d s 2.1. Isolated tracheal and vascular preparations

Albino guinea-pigs of either sex (body weight 250-345 g) were killed b y a blow to the neck. The following ring preparations were obtained from each animal: two tracheal preparations, each comprising two adjoining cartilage tings; two tubular segments of the descending aorta (length 3 mm) and two segments of the proximal part of the left

0014-2999/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)

276 pulmonary artery (length 2 mm). The preparations were transferred to six temperature-regulated (37 o C) 5 ml organ baths containing Krebs solution (composition in mM: NaC1 118.0, KC1 4.6, CaC12 2.5, MgSO4 1.15, N a H C O 3 24.9, K H 2 P O 4 1.15, glucose 5.5, pH 7.4) aerated with a mixture of 95% 02 and 5% CO 2. Each preparation was mounted between two fine stainless steel pins in a precision myograph (Nielsen-Kudsk et al., 1986b) and isometric tension changes were recorded with a six-channel amplifier and recorded (Watanabe Linearcorder model WR 3101). All six preparations were run in parallel on each experimental day. The preparations were suspended under passive tensions of 0.6, 3.0 and 1.0 g (trachea; aorta; pulmonary artery). These values were determined by separate length-tension measurementsas being optimal for the contractile responses to 124 m M K+-Krebs. The preparations were allowed to equilibrate for 1 h, with frequent changing of the bathing solution, before the experiments.

2.2. Experiments The relaxant activity of pinacidil was assessed from concentration-effect ( C / E ) curves obtained with preparations precontracted by the agonists (histamine, PGF2,, 124 mM K+-Krebs) or from determination of the histamine C / E curves before and after pinacidil pretreatment for 20 min. Histamine and PGF2~ are asthma mediators and were used because they produce effective and stable contractions of both airway and vascular smooth muscle. The concentrations of these agonists were chosen to reach the same level of contraction as that produced by 124 mM K ÷Krebs solution. Pinacidil was added cumulatively to the organ baths when stable contractions to the agonists had developed. The tension was allowed to stabilize before the concentration of pinacidil was increased. Indomethacin (10 - 6 M) was present in the organ baths containing the tracheal preparations. The presence of indomethacin is essential to obtain reproducible and consistent potassiuminduced contractions of the guinea-pig trachealis (Nielsen-Kudsk et al., 1986a). It was added 30 min before the contractile agonist and resulted in 78 +

2% S.E. (n = 16) inhibition of the spontaneous tone of the trachea. Indomethacin was without effect on the pinacidil-induced vasorelaxation.

2.3. Data analysis The mean values + S.E. of the relaxant activity were calculated for each concentration of pinacidil. Continuous sigmoid log C / E curves and the corresponding ECs0, Ema x and Hill exponent (S) values were obtained from the mean data by nonlinear iterative regression analysis with the Hill function E = EmaxfS/(ECs0S + C s) as the pharmacodynamic model (cf. Holford and Sheiner, 1981). The C / E curves for histamine before and after pinacidil treatment were made in a similar way. S is the slope of the C / E curve. The S.E.s of the values were determined simultaneously, as described by Waud (1975). Analysis of the data, calculation of the values, curve-fitting and plotting of the C / E curves were done on a HP-85 personal computer using the computer programs in BASIC described by Nielsen-Kudsk (1983). Differences between calculated parameters 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, Copenhagen, Denmark), histamine and indomethacin (Sigma Chemical Co., St. Louis, MO, USA), PGF2a (Amoglandin, KabiVitrum AB, Stockholm, Sweden). Pinacidil (21.1 mg) was dissolved either in a mixture of 96% ethanol, propylene glycol and water (2 + 2 + 4 ml) to a stock solution of 10 -2 M or in 0.1 M HC1 to make a stock solution of 10 - 1 M which was used in most experiments. Neither vehicle had any effect. Indomethacin was dissolved in 5% N a H C O 3 and diluted with 0.9% NaC1. Other drugs were dissolved directly in 0.9% NaC1. The isotonic K+-Krebs solution was similar to the normal Krebs solution except that an equimolar amount of NaC1 was replaced with KC1 (124 mM K+).

277

3. Results

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3.1. The effects of contractile agents

IZZ

The tracheal and vascular preparations were precontracted by histamine, PGF2~ or isotonic 124 m M K+-Krebs solution. The mean tensions in g _+ S.E. (n = 8) before the addition of pinacidil were: (1) trachea: histamine (10 -6 M) 2.19 + 0.19; PGF2~ (10 -6 M) 2.79 + 0.19; K + (124 mM) 2.42 + 0.19; (2) aorta: histamine (10 -5 M) 2.34 + 0.16; PGF2~ (10 -5 M) 2.83 + 0.19; K + (124 mM) 2.50 ___0.12; and (3) pulmonary artery: histamine (10 -5 M) 0.71 ___0.06; PGF2~ (3 × 10 -5 M) 0.61 ___0.06; K ÷ (124 m M ) 1 . 0 8 _ 0.08. The control contractions, where vehicle was added instead of pinacidil, were stable during the experiments except for the K÷-contracted aorta. A gradual loss of tone (8% per h) was seen in this preparation (corrections were made when calculating the relaxant effect of pinacidil). The tracheal response to high K + was biphasic, as described previously (NielsenKudsk et al., 1986a). Small phasic oscillations (amplitude 0.14 g; frequency 5.7 min -1) were superimposed on the contraction induced by PGF2~ in the pulmonary artery.

3.2. The effects of pinacidil on precontracted preparations

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All contracted preparations were relaxed by pinacidil in a concentration-dependent way. The C / E curves are shown in fig. la-c and the derived ECs0, Emax and Hill exponent values in table 1. Characteristic differences in the response to pinacidil between the trachea, a o r t a and pulmonary artery were seen when these preparations were contracted by histamine or PGF2~ (fig. la-b) The trachea was the only preparation that was relaxed completely by pinacidil. The aorta and pulmonary artery were only partly relaxed, to about 65-70%. Further addition of papaverin (10 -4 M) produced 100% relaxation of these vascular preparations. The slopes of the pinacidil C / E curves obtained with tracheal preparations were much steeper (Hill-coefficients about 2) than the slopes of the vascular preparations (Hill-coefficients about 0.8). Pinacidil was 2-4 times more

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Fig. 1. Relaxant concentration-effect curves for pinacidil obtained with isolated' guinea-pig trachea (o), aorta ( O ) and pulmonary artery (I). The preparations were precontracted by histamine (a), PGF2,, (b) or 124 mM K+-Krebs solution (c). The derived ECso , E ~ and Hill exponent values are given in table 1. The pinacidil effect is expressed as % relaxation of the initial precontraction tension level. Vertical bars show S.E. values (n = 8).

potent in the trachea than in the aorta. The pulmonary artery was the most sensitive preparation (ECs0 values of 0.5 and 0.9/xM), pinacidil was 20 times more potent in this tissue than in the

278 TABLE 1 Relaxant ECs0 (/~mol/1), Emax (% relaxation of tension produced by contractile agents) and Hill exponent (S) values for pinacidil obtained with isolated guinea-pig trachea, aorta and pulmonary artery (PA). The preparations were precontracted by histamine, PGF2~ or isotonic 124 mM K+-Krebs solution. The parameters were derived from the corresponding concentration-effect curves (Hill functions) determined by computer-fitting of the mean data plotted in fig. 1. S.E.s (n = 8) are given in parentheses. Histamine

PGF2~

124 mM K +

Trachea

ECso 3.29 (0.21) 4.83 (0.18) 45.0 (1.35) Ema~ 95.7 (2.11) 99.4 (1.29) 101.3 (0.95) S 1.97 (0.24) 2.05 (0.12) 1.08 (0.03)

Aorta

ECs0 12.3 (4.32) 10.5 (5.31) E max 64.4 (6.16) 70.7 (9.94) S 0.70 (0.08) 0.74 (0.14)

PA

p i n a c i d i l was m o r e p o t e n t on P G F 2 ~ - c o n t r a c t e d p r e p a r a t i o n s t h a n o n h i s t a m i n e - c o n t r a c t e d preparations.

3.3. The effects of pinacidil pretreatment T h e C / E curves for h i s t a m i n e o b t a i n e d with tracheal a n d p u l m o n a r y a r t e r y p r e p a r a t i o n s b e f o r e (control) a n d after 20 rain p r e t r e a t m e n t with p i n a c i d i l (10 -5 M ) are s h o w n in fig. 2. T h e derived ECs0, Ema x a n d Hill e x p o n e n t values are given in t a b l e 2. T h e m e a n c o n t r o l m a x i m a l tension i n d u c e d b y h i s t a m i n e was 2.75 g + 0.25 S.E.

48.1 (2.88) 99.3 (2.27) 1.64 (0.12)

125

ECso 0.88 (0.11) 0.51 (0.05) 48.5 (2.27) Emax 65.7 (1.83) 72.1 (1.39) 100.2 (1.61) S 0.86 (0.08) 0.84 (0.06) 1.31 (0.06)

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a o r t a (cf. table 1). T h e tension oscillations t h a t were s u p e r i m p o s e d o n the c o n t r a c t i o n s i n d u c e d b y PGF2~ in the p u l m o n a r y artery were a b o l i s h e d at a m e a n p i n a c i d i l c o n c e n t r a t i o n of 2.4 /~M. This i n h i b i t i o n was p r e c e d e d b y a g r a d u a l r e d u c t i o n in the frequency w i t h o u t there b e i n g changes in the a m p l i t u d e , starting at a 10-fold lower p i n a c i d i l concentration. I n sharp contrast, the p i n a c i d i l C / E curves were a l m o s t identical when the three different s m o o t h muscle p r e p a r a t i o n s were c o n t r a c t e d b y isotonic 124 m M K + - K r e b s s o l u t i o n (fig. lc). T h e r e l a x a n t p o t e n c y of p i n a c i d i l was m u c h lower in these p r e p a r a t i o n s (ECs0 values a b o u t 50 /xM). T h e Hill coefficients were a b o u t 1.1-1.6, a n d differ f r o m those o b t a i n e d with PGF2~- or h i s t a m i n e c o n t r a c t e d p r e p a r a t i o n s . T h e t i m e n e e d e d to p r o duce stable r e l a x a t i o n at bach increase 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 m u c h longer for the K + - d e p o l a r i z e d p r e p a r a t i o n s ( a b o u t 20 rain) t h a n for the p r e p a r a t i o n s c o n t r a c t e d b y h i s t a m i n e or PGF2~ (5 rain). A s r e p o r t e d p r e v i o u s l y ( N i e l s e n - K u d s k et al., 1988), p i n a c i d i l was f o u n d to b e m o r e p o t e n t in relaxing histamine-contracted tracheas than PGF2~-contracted tracheas. T h e o p p o s i t e was seen in b o t h the a o r t a a n d p u l m o n a r y artery, where

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Fig. 2. Contractile concentration-effect curves for histamine obtained with isolated guinea-pig trachea (a) and pulmonary artery (b) before (open symboJs; control curves) and after 20 rain pretreatment with pinacidi110 -3 M (dosed symbols; % of control). The derived ECs0, Em~ and Hill exponent values are given in table 2. The contractile effects of histamine are expressed as a % of its own maximum in the control experiments. Vertical bars show S.E. values (n = 5).

279 TABLE 2 Contractile ECs0 (~mol/1), Emax (% contraction) and Hill exponent (S) values for histamine obtained with isolated guinea-pig trachea and pulmonary artery (PA) before (control values) and after 20 rain pretreatment with pinacidil 10-5 M (Ema x expressed as % of control). The parameters were derived from the corresponding concentration-effectcurves (Hill functions), which were computer-fitted to the mean data plotted in fig. 2. S.E.s (n = 5) are given in parentheses. Histamine (control)

Histamine (after pinacidil)

Trachea

EC50 Emax S

0.53 (0.03) 99.2 (1.86) 1.32 (0.08)

2.19 (0.05) 86.6 (0.80) 2.49 (0.11)

PA

ECs0 Emax S

1.33 (0.08) 103.2 (2.84) 1.57 (0.12)

2.11 (0.10) 51.7 (0.87) 1.51 (0.09)

(n = 5) in the trachea an d 0.74 g _+ 0.04 S.E. (n = 5) in the pulmonary artery. The C / E curves for histamine were not reproducible in the aorta and prevented the study of pinacidil pretreatment in this particular preparation. In the trachealis muscle, pinacidil shifted the histamine C / E curve to the right, with only a minor depression (12.7%) of the maximal response to histamine. However, in the pulmonary artery, pinacidil depressed the maximal histamine response by 50% whereas the potency of histamine was little affected.

4. Discussion

It has been demonstrated previously that the relaxant activity of pinacidil depends on the type of agonist used to induce contraction. This is seen both in guinea-pig trachealis (Nielsen-Kudsk et al., 1988) and vascular smooth muscle (Nielsen and Arrigoni-Martelli, 1981). The potency and intrinsic activity of bronchodilators in guinea-pig trachealis are dependent on the actual level of contraction (Karlsson, 1981). The different types of preparations used in this study (trachea, aorta, pulmonary artery) were taken from the same animal in each experiment and were studied in parallel. They were precontracted by the same agents (histamine, PGF2~ or 124 m M K+-Krebs)

in concentrations producing equal levels of tension within each type of preparation. This experimental approach was aimed at creating favourable conditions to allow comparison of the relaxant activity of pinacidil in the different types of smooth muscle. The results presented demonstrate that the action profile of pinacidil in guinea-pig trachealis differed both qualitatively and quantitatively from that seen in the aorta and pulmonary artery. Several observations in vascular smooth muscle (cf. Southerton et al., 1988) support the view that the opening of potassium channels and hyperpolarisation of the cell membrane may be the cellular mechanism of pinacidil-induced vasorelaxation. It is not know at present if this mechanism of action is relevant for the airway smooth muscle r e l a x a t i o n p r o d u c e d b y pinacidil. Cromakalim (BRL 34915) is another antihypertensive agent that has been classified as a K + channel opener (Cook, 1988). The in vitro pharmacology of this drug is similar to that of pinacidil (Cook et al., 1988). Allen et al. (1986) demonstrated that chromakalim caused relaxation, membrane hyperpolarisation and stimulated 86Rb+ efflux in guinea-pig trachealis, indications of K + channel opening activity. Our findings that pinacidil had little relaxant activity and that its C / E curves were nearly identical in the K+-depolarized tracheal and vascular preparations may also point to a role of K + channel opening in the airway smooth muscle relaxation induced by pinacidil. Increased K + conductance has also been suggested to be relevant for the inhibitory action of pinacidil on isolated human bladder preparations (Andersson et al., 1988). It is thus less likely, although theoretically possible, that the differences between the effect of pinacidil in airway compared to vascular tissues observed in the present study could reflect different cellular mechanisms of action. Several different types of K +selective channels have been characterized (Latorre and Miller, 1983). Pinacidil may act preferentially on C a / +-dependent K + channels (Hermsmeyer, 1988). Differences in the number, types, distribution or function of K + channels between the tissues could provide an explanation for the dissimilarities observed.

280

Pinacidil had a low potency (ECs0 values about 50/~M) but clearly relaxed both airway and vascular tissues contracted by 124 mM K ÷. The three C / E curves were nearly identical (cf. fig. lc). This action of pinacidil cannot be explained by K ÷ channel opening. It is unlikely that pinacidil could cause hyperpolarisation in 124 mM K+-stimulated preparations, since the electrochemical gradient for K ÷ is practically eliminated. Furthermore, the different Hill coefficients and equilibration times for pinacidil in these preparations indicate a different mechanism of action. Although the pinacidil ECs0 values were higher in the K+-contracted than in the PGF2~- and histamine-contracted preparations, the maximal relaxations were greater in the aortas and pulmonary arteries contracted with excess K ÷. This cannot be explained by K ÷ channel opening or by non-specific inhibition of smooth muscle contractility. It has been suggeste d that pinacidil could also act by causing a redistribution of intracellular Ca 2+ (Erne and Hermsmeyer, 1987). Pinacidil was very potent (ECs0 values about 0.5 ~tM) in relaxing the pulmonary artery: it was about 15-20 times more potent than in the aorta. In isolated dog arteries, Toda et al. (1985) reported differences between the relaxant potency of pinacidil in coronary, mesenteric and cerebral arteries. The relaxant action of pinacidil thus seems to depend on the particular vascular region studied, and could reflect tissue differences in the population of K ÷ channelg. The finding that pinacidil had a greater efficacy in airway compared to vascular preparations and a greater relaxant potency in trachealis than in the aorta was striking. This, together with the previous observation of the relaxant action of pinacidil in trachealis contracted by several different asthma mediators (Nielsen-Kudsk et al., 1988), may focus interest on K ÷ channel opening drugs as possible bronchodilators.

References Allen, S.L., J.P. Boyle, J. Cortijo, R.W. Foster, G.P. Morgan and R.C. Small, 1986, Electrical and mechanical effects of BRL34915 in guinea-pig isolated trachealis, Br. J. Pharmacol. 89, 395. Andersson, K.-E., P.-O. Andersson, M. Fovaeus, H. Hedlund, A. Malmgren and C. Sj~Sgren, 1988, Effects of pinacidil on bladder muscle, Drugs 36 (Suppl. 7), 41.

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 t w o n e w vasodilator drugs: Pinacidil and BRL 34915, J. Cardiovasc. Pharmacol. 11, 90. Erne, P. and R.K. Hermsmeyer, 1987, Actions of pinacidil on calcium release in single vascular muscle cells of rat veins, Physiologist 30, 129. Hermsmeyer, R.K., 1988, Pinacidil actions on ion channels in vascular muscle, J. Cardiovasc. Pharmacol. 12 (Suppl. 2), S17. Holford, N.H.G. and L.B. Sheiner, 1981, Understanding the dose-effect relationship: Clinical application of pharmacokinetic-pharmacodynamic models, Clin. Pharmacokin. 6, 429. Karlsson, J.-A., 1981, Influence of tracheal contraction on relaxant effects in vitro of theophylline and isoprenaline, Br. J. Pharmacol. 74, 73. Latorre R. and C. Miller, 1983, Conduction and selectivity in potassium channels, J. Membr. Biol. 71, 11. Nielsen, C.K. and E. Arrigoni-Martelli, 1981, Effect of a n e w vasodilator, pinacidil (Pl134), on potassium, noradrenaline and serotonin induced contractions in rabbit vascular tissues, Acta Pharmacol. Toxicol. 49, 427. Nielsen-Kudsk, F., 1983, Application of microcomputers in analysis and simulation of drug kinetics. A program package for use with the personal microcomputer HP-85, in: Proc. of the 1983 Summer Computer Simulation Conference (Vancouver, Canada) p. 646. Nielsen-Kudsk, J.E., J.-A. Karlsson and C.G.A. Persson, 1986a, Relaxant effects of xanthines, a fl2-receptor agonist and Ca2+-antagonists in guinea-pig tracheal preparations contracted by potassium or carbachol, European J. Pharmacol. 128, 33. Nielsen-Kudsk, J.E., S. Mellemkjaer, C. Siggaard and C.B. Nielsen, 1988, Effects of pinacidil in guinea-pig airway smooth muscle contracted by asthma mediators, European J. Pharmacol. 157, 221. Nielsen-Kudsk, F., B. Poulsen, C. Ryom and J.E. NielsenKudsk, 1986b, A strain-gauge myograph for isometric measurements of tension in isolated small blood vessels and o t h e r muscle preparations, J. Pharmacol. Meth. 16, 215. Southerton, J.S., A.H. Weston, K.M. Bray, D.T. Newgreen and S.G. Tayler, 1988, The potassium channel opening action of pinacidil; studies using biochemical, ion flux and microelectrode techniques, Naunyn-Schmiedeb. Arch. Pharmacol. 338, 310. Toda, N., S. Nakajima, M. Miyazaki and M. Ueda, 1985, Vasodilation induced by pinacidil in dogs. Comparison with hydralazin and nifedipine, J. Cardiovasc. Pharmacol. 7, 1118. Wand, D.R., 1975, Analysis of dose-response curves, in: M e t h ods in Pharmacology, Vol. 3, Smooth Muscle, eds. E.E. Daniel and D.M. Paton (Plenum Press, New York and London) p. 471.