ojp European Journal of Pharmacology271 (1994) 453-459
K ÷ channel openers relax longitudinal muscle of guinea pig ileum Ya-Ding Sun, Christina G. Benishin
Department of Physiology, University of Alberta Faculty of Medicine, Edmonton, Alberta, Canada T6G 21-17
Received 21 September 1994; accepted 27 September 1994
The plexus-free longitudinal muscle was used to investigate the muscle-relaxing effects of the known K + channel openers, cromakalim, pinacidil and nicorandil, and compared with other known muscle relaxants, calcitonin gene-related peptide (CGRP) and isoprenaline. The three K + channel openers all decreased basal tension and inhibited the tonic tension evoked by 30 mM KCI, 0.5/~M histamine or 0.1 tzM oxotremorine in a dose-dependent manner. The order of potency is cromakalim > pinacidil > nicorandil in KCI or oxotremorine-precontracted muscle strip and nicorandil > cromakalim > pinacidil in histamine-precontracted muscle strip. Inhibition by cromakalim was completely reversed by glibenclamide, a blocker of ATP-sensitive K + channels, while inhibition by nicorandil or pinacidil was only partially antagonized. The tonic tension evoked by KCI, histamine or oxotremorine was relaxed by CGRP or isoprenaline. Inhibition by neither of these compounds was relieved by glibenclamide. These results suggest that while ATP-sensitive K ÷ channels may be present in the longitudinal muscle cells, they may not be involved in the actions of CGRP or isoprenaline on the longitudinal muscle. Keywords: K ÷ channel opener; Longitudinal muscle; CGRP (calcitonin gene-related peptide); K + channel, ATP-sensitive
A new class of synthetic compounds, K + channel openers, has been reported to relax different types of smooth muscle (Quast and Cook, 1989). The ATP-sensitive K ÷ channel (KATP channel) is the primary type of K ÷ channel on which K ÷ channel openers act. These channels are reported to exist in the cells of skeletal, cardiac and vascular smooth muscle (Quast and Cook, 1989). Thus far, there is no electrophysiological evidence for the presence of KAT P channels in the cells of the longitudinal muscle of the guinea pig ileum. The effects of these K + channel openers on the mechanical properties of intestinal muscle are still not fully understood. McPherson and Angus (1990) reported that cromakalim relaxed the KCl-induced contraction of the guinea pig ileum. Zini and co-workers (1991) found that cromakalim and other K ÷ channel openers inhibited the electrically induced contractions of guinea pig ileal muscle through a neuronal mechanism. Longitudinal muscle with attached myenteric
plexus or the whole segment preparation of guinea pig ileum were used in those studies. In this study, we examine the effect of three known K + channel openers to determine whether longitudinal smooth muscle cells have characteristic K ÷ channel opener receptors which may be associated with KATP channels. Nelson and co-workers (1990) have suggested that calcitonin gene-related peptide ( C G R P ) may induce relaxation of vascular smooth muscle via activation of KATP channels. Our previous studies have shown that C G R P can relax guinea pig longitudinal muscle. The muscle-relaxing effects of C G R P as well as another muscle relaxant, isoprenaline, were therefore studied for comparison with these K ÷ channel openers in the longitudinal muscle, to determine whether these compounds share a common mechanism of smooth muscle relaxation, via activation of KATP.
2. Materials and methods 2.1. Materials
* Corresponding author. Tel. 403-492-5284, fax 403-492-8915. 0014-2999/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0014-2999(94)00592-3
Drugs used were: histamine and oxotremorine (Sigma Chemical Co., St. Louis, MO, USA), rat
Y.-D. Sun, C.G. Benishin/ European Journal of Pharmacology 271 (1994) 453-459
a C G R P (Bachem, Essex, UK), glibenclamide (Research Biochemicals Int., Natick, MA, USA). Cromakalim, pinacidil a nd nicorandil were generously donated by Beecham Pharmaceuticals (Sussex, UK), Zenyaku Kogyo Co. (Tokyo, Japan) and Leo Pharmaceutical Products (Copenhagen, Denmark), respectively. Glibenclamide was dissolved in dimethyl sulfoxide (DMSO). Cromakalim, pinacidil and nicorandil were first dissolved in 100% ethanol at a concentration of 100 mM or 200 mM and then diluted to different concentrations with water. 2.2. Tissue preparation The plexus-free longitudinal muscle of the guinea pig ileum was prepared as described previously (Paton and Aboo Zar, 1968; Sun and Benishin, 1991). In brief, to isolate the plexus-free longitudinal muscle, a gentle incision was made along the length of the isolated ileum, and the longitudinal muscle was dissected free. The free end was held and a long piece of strip (20-30 cm) was detached by pulling. Our previous study has shown that the distal portion of the strip contained no functional plexus since the strip did not respond to electrical field stimulation or to the nicotinic agonist, dimethylphenylpiperazinium (Sun and Benishin, 1991). Furthermore, visual examination of the preparation under the dissecting microscope verified that the plexus was absent in this preparation (Sun, 1993). This preparation was used in the experiments. 2.3. Tension measurement The tissue strip was fixed between a force displacement transducer (Grass FT.03, Quincy, USA) and the bottom of the incubation chamber and equilibrated for 1 h (initial tension 0.7 g) in 10 ml of oxygenated Krebs solution having the following composition in mM: NaC1, 117; KC1, 4.7; NaH2PO 4, 1.2; CaC12, 2.5; MgSO 4, 1.3; NaHCO 3, 25 and glucose, 11, bubbled with 95% 02-5% CO 2. Tension was increased by addition of KCI (30 mM), oxotremorine (0.1 /~M) or histamine (0.5 ~M). The possible effects of KCI on the tension due to the increased osmolarity of the bathing fluid were examined. NaC1 or sucrose at the same osmolar concentrations as the added KCI did not affect the tension of the muscle strip (data not shown). K + channel openers were added cumulatively to non-pre-contracted or pre-contracted muscle to achieve a muscle relaxing effect. After the muscle had been maximally relaxed by K + channel openers, glibenclamide was added cumulatively to reverse the relaxation. At a concentration of 10 /xM, glibenclamide itself induced a transient contraction of 0.1 g which lasted 3 min, and had no effect on the precontracted muscle strip. The effects of solvents used to dissolve K + channel
openers (ethanol) and glibenclamide (DMSO) on both the basal and the stimulated tension were examined and the final solvent concentration in the chamber was controlled so that the solvents themselves had no effect on either the basal or the stimulated tension. Ethanol decreased basal tension at concentrations higher than 0.2%, and relaxed the tonic tension induced by KC1 (30 mM), histamine (0.5 /~M) and oxotremorine (0.1 ~M) at a concentration of 0.5% and greater. Therefore the final concentration of ethanol in the bath was kept below 0.2%. DMSO did not contract the muscle at concentrations less than 0.3%. This concentration also did not reverse the K ÷ channel opener-induced relaxation of precontracted muscle. DMSO at concentrations up to 0.5% did not depress the tension evoked by the stimulants. Therefore the final concentration of DMSO in the bath was kept below 0.3%. 2.4. Calculations and statistics Each K ÷ channel opener was added 3 min after the addition of KCI, oxotremorine or histamine. The values of tension 3 min after the addition of the stimulant when the K ÷ channel opener was added were calculated as 100% and the other values were calculated as percentages accordingly in both K ÷ channel openertreated or non-treated tissues. The percentages obtained in K + channel opener-treated tissues were then calculated again as the percentage of that in K ÷ channel opener-non-treated tissue strip at the same time point. IC50 values were calculated using the P C S / PHARM computer program.
3. Results A preliminary study showed that all three K + channel openers produced a maximal decrease in the basal tension of about 0.2 g. The decrease in tension was observed at concentrations from 0.1 to 1 /~M for cromakalim, or 1 ~M for pinacidil and 1 to 10 /~M for nicorandil. Glibenclamide at concentrations up to 20 /xM totally reversed the inhibition induced by pinacidil and nicorandil, but only partially reversed the inhibition induced by cromakalim. At a concentration of 10 /zM, glibenclamide itself induced a transient contraction of 0.1 g which lasted 3 min. The K ÷ channel openers were found to totally relax the tonic tension induced by KC1 at the concentration for maximal contraction (30 mM) while they could not inhibit the maximal tension induced by oxotremorine or histamine (5/~M, data not shown). In the following experiments, the longitudinal muscle was precontracted by KCI at a concentration of 30 mM, or oxotremorine or histamine at concentrations of approximately the EDs0.
Y.-D. Sun, C.G. Benishin/ European Journal of Pharmacology 271 (1994) 453-459 125
tu Ia tu .J <
< :E nO Z
Fig. 1. K + channel openers inhibit KCl-induced tonic contraction of the longitudinal muscle. Cromakalim (e), pinacidil (11) and nicorandil ( • ) relaxed the longitudinal muscle pre-contracted by 30 mM KCI in a dose-dependent manner. Data are mean + S.E. n = 4. Cromakalim, pinacidil and nicorandil relaxed the longitudinal muscle precontracted by KCI (30 mM), oxotremorine (0.1 /zM) or histamine (0.5 /zM) in a concentration-dependent m a n n e r (Fig. 1, 2 and 3). The IC50 values are 1.8 + 0.3 /xM for cromakalim, 3 + 1 /zM for pinacidil and 28 + 1 /xM for nicorandil in the KCl-precontracted longitudinal muscle (Fig. 1), and 1.3 + 0 . 4 / x M for cromakalim, 4 + 1 /zM for pinacidil, and 11 + 2 / z M for nicorandil, in oxotremorine-precontracted muscle (Fig. 2). However, the rank order of potency of these K ÷ channel openers is different in histamine-stimulated tissue: 4 + 1 /zM for nicorandil, 7 + 4 / x M for cromakalim and 20 + 3 IzM for pinacidil (Fig. 3). Glibenclamide at concentrations from 0.1 to 1 0 / z M was found to reverse the inhibitory effects of K + 125 Z
u) z ,,, Ia "' _N
100 75 5O
Fig. 3. K + channel openers inhibit histamine-induced tonic contraction of the longitudinal muscle. Cromakalim (e), pinacidil ( • ) and nicorandil ( • ) relaxed the longitudinal muscle pre-contracted by 0.5 /zM histamine in a dose-dependent manner. Data represent mean+ S.E. n=5.
channel openers (Fig. 4 and Table 1) in a dose-dependent manner. The cromakalim-induced relaxation was totally reversed by glibenclamide at a concentration less than 10 /zM. In contrast, the nicorandil-induced relaxation was only partially reversed by glibenclamide. The pinacidil-induced relaxation of oxotremorine precontracted muscle was completely antagonized while the induced relaxation of KCI- or histamine-stimulated muscle was only partially reversed by glibenclamide. C G R P at a concentration of 0.26 /xM maximally relaxed the tension induced by KC1 or histamine by 40% or 100%, respectively. Both Ba 2+ and glibenclamide were reported to reverse the relaxation induced by C G R P in vascular smooth muscle (Nelson et al., 1990). Ba 2+, at a concentration of 100/zM reversed the C G R P - i n d u c e d inhibition by 86%. However, Ba 2+ itself was found in the present study to increase the basal tension and enhance the contractile effect of KC[ on the longitudinal muscle by 80%. Glibenclamide at concentrations up to 10 /xM had little effect on the CGRP-induced inhibition of tension induced by KC1 or histamine (Fig. 5). Inhibition of the tension by isoprenaline (10 /xM) on oxotremorine- or KCl-stimulated tissue was also not reversed by glibenclamide (Fig. 5).
4. D i s c u s s i o n 0 -7
-6 Log [OPENER]
Fig. 2. K + channel openers inhibit oxotremorine-induced tonic contraction of the longitudinal muscle. Cromakalim (e), pinacidil (11) and nicorandil ( • ) relaxed the longitudinal muscle pre-contracted by 0.1 p.M oxotremorine in a dose-dependent manner. Each value is mean + S.E. n = 5.
One of the common mechanisms of action of many synthetic smooth muscle relaxants as well as some endogenous compounds is their ability to hyperpolarize smooth muscle (Cook, 1988; Brayden, 1990; Brayden et al., 1991). M e m b r a n e hyperpolarization can, in turn, reduce influx of calcium via the deactivation of voltage-gated calcium channel activity and lead to va-
Y.-D. Sun, C.G. Benishin/ European Journal of Pharmacology 271 (1994) 453-459
KCI 30 mM
Pin 0.3 1
KCI 30 mM
ford et al., 1988), and vascular smooth muscle cells (Standen et al., 1989). A primary characteristic of this ion channel is its inhibition by intracellular ATP, as well as by sulfonylurea compounds such as glibenclamide, tolbutamide, and by low concentrations of Ba 2+ (Standen et al., 1989). Recent evidence shows that K + channel openers can activate KATv channels on different types of tissues (Quast and Cook, 1989). Electrophysiological studies have provided the evidence for existence of KAT P channels in vascular smooth muscle cells and K + channel openers have been shown to open these channels (Inoue et al., 1989, 1990; Kovacs and Nelson, 1991; Kajioka et al., 1991). To date there has been no electrophysiological evidence for the presence of KAT P channels in cells of guinea pig ileal longitudinal smooth muscle. Both high and low affinity binding sites for [3H]glibenclamide in the guinea pig longitudinal muscle-myenteric plexus preparation have been recently reported (Gopalakrishnan et al., 1991; Zini et al., 1991) although the
Nic ._= ¢Q
KCI 30 mM
1 Glib rCGRP
Fig. 4. Glibenclamide reversed the K + channel opener-induced relaxation of KCl-precontracted longitudinal muscle. Cromakalim (crom, upper panel), pinacidil (pin, middle panel) and nicorandil (nic, lower panel) induced inhibitions were reversed by glibenclamide.
0.31 3 Glib
sodilation. Opening of K + channels or increasing the membrane permeability to K + is one mechanism underlying hyperpolarization. The KAT P channel is one type of K + channel present in some smooth muscle cells, as well as in the cells of cardiac muscle (Noma, 1983), skeletal muscle (Spruce et al., 1985), pancreatic beta cells (Cook and Hales, 1984), neuronal cells (Ash-
KCI 30 mM
Table 1 Reversal effect of glibenclamide on the relaxation induced by potassium channel openers in longitudinal muscle pre-contracted by KCI (30 mM), oxotremorine (0.1 /zM) or histamine (0.5/zM) Cromakalim
KCI Oxotremorine Histamine
96 5:18 (0.3) 101 _+10 (3) 146 _ 17 (5)
80 5 : 7 (10) 110 + 13 (10) 48 + 14 (3)
67 5:6 (1) 33 + 2 (1) 59_ 7 (5)
Oxo 0.1 pM
Values presented are percentage of the tension in the control tissue strip. Numbers in parentheses are the lowest concentration of glibenclamide (in /zM) for the maximal reversal of the inhibition by K + channel openers, n = 3-5 for each combination.
0.3 3 Glib
0.3 3 10/~M KCI 3 0 mM
Fig. 5. CGRP and isoprenaline (iso) inhibit the tonic tension evoked by spasmogens and the effect of glibenclamide on the inhibition.
Y.-D. Sun, C.G. Benishin / European Journal of Pharmacology 271 (1994) 453-459
relationship between the sulfonylurea receptors and KATP channels still needs to be clarified. K ÷ channel openers have been reported to influence the mechanical properties of guinea pig ileum. In the longitudinal muscle-myenteric plexus preparation, Zini and coworkers (1991) found that cromakalim and other K ÷ channel openers had no effect on the dose-response curve of carbachol but depressed the electrically evoked contraction. This latter action was antagonized by glibenclamide. Electrical stimulation primarily releases acetylcholine from myenteric neurons (Paton and Aboo Zar, 1968). Both acetylcholine and carbachol activate cholinergic muscarinic receptors on longitudinal smooth muscle ceils. The different effects of K + channel openers on these contractions led the authors to conclude that these compounds regulate the muscle activity indirectly through presynaptic mechanisms. On the other hand, McPherson and Angus (1990) reported that in experiments using the whole segment of guinea pig ileum, cromakalim inhibited the KCl-induced contraction which is reversed by glibenclamide. KC1 could act on both the muscle and myenteric neurons to affect the mechanical activity of the muscle. To eliminate the neuronal and other extrinsic factors and study the direct effect of K + channel openers on the smooth muscle, we used the plexus-free longitudinal muscle of guinea pig ileum in the present study. This preparation has been shown to contain no detectable amount of acetylcholine, and to be unresponsive to electrical field stimulation and dimethylphenylpiperazinium which release neurotransmitters from nerve endings (Paton and Zar, 1968; Sun and Benishin, 1991). We found that all three K + channel openers tested in this study decreased basal tension of the longitudinal muscle. Furthermore all three K ÷ channel openers totally relax the contraction induced by KCI at the concentration for maximal contraction. Because the tonic contraction induced by KCI at a concentration close to its EDs0 was not sustained, a higher concentration of KC1 was used in the experiment. One of the results of the activation of either histamine n I or muscarinic receptors is depolarization of the cell membrane of guinea pig longitudinal muscle (Bolton and Clark, 1981a,b). It is, therefore, reasonable to conclude that K ÷ channel openers could inhibit the contractions which are induced by activation of these two types of receptors and that the inhibition induced by K ÷ channel openers could be antagonized by glibenclamide if KAa-P is present on the longitudinal muscle. All three K + channel openers tested inhibited the tension developed by KC1, histamine and oxotremorine, providing evidence for the direct effect of K + channel openers on smooth muscle cells. The rank order of potency of these K ÷ channel openers for inhibiting the longitudinal muscle precontracted by KCI or oxotremorine is consistent with that for relaxation of vascular smooth muscle
(Newgreen et al., 1990; Ksoll et al., 1991; Kamijo et al., 1992; Zhang et al., 1992) and different from that for inhibition of insulin release as reported by other investigators (Garrino et al., 1989). KCI and contractants other than oxotremorine and histamine were used by those investigators to contract the vascular smooth muscle. It is interesting to note that in the present study elevated KCI did not significantly alter the potencies of cromakalim and pinacidil (KCI vs. oxotremorine induced contractions) as noted by others (e.g. Weir and Weston, 1986), which may suggest that the compounds may act by mechanisms other than opening K ÷ channels. However, nicorandil was less potent in the presence of elevated KCI. The rank order of potency for these K ÷ channel openers apparently changed when histamine was used as a contractant in the longitudinal muscle. The mechanisms are not clear. The discrepancy between the rank order of potency based on the average of individual calculated ECs0 values and the rank order of potency displayed in Fig. 3 may be due to the relatively large standard errors of the nicorandil data points. The reason for the large standard errors is not clear. Glibenclamide totally or partially reversed the inhibition of the tension by all three K + channel openers, suggesting that opening of the KAa-P channel could be one of the mechanisms of action of these K ÷ channel openers. The inhibition induced by cromakalim was abolished by glibenclamide while that induced by pinacidil and nicorandil was only partially reversed. This phenomenon was also observed in vascular smooth muscle by other investigators (Yanagisawa et al., 1990; Kamijo et al., 1992). It has been reported that in addition to the K ÷ channel opening effect, nicorandil increases intracellular levels of guanosine 3',5'-cyclic monophosphate by activating soluble guanylate cyclase in various vascular smooth muscle tissues (Endoh and Taira, 1983; Holzman, 1983), and in cardiac muscle (Yanagisawa et al., 1988). The latter mechanism also contributes to the relaxation of smooth muscle by nicorandil and is not antagonized by glibenclamide. This also might be the case in the longitudinal muscle. Yanagisawa et al. (1990) found cromakalim, nicorandil or pinacidil all inhibited the tension and [Ca2+]i; only the inhibition by cromakalim could be completely reversed by glibenclamide, suggesting that pinacidil, like nicorandil, might have mechanisms other than the KAXP channel-opening effect underlying its action. The effects of the K + channel openers on the mechanical properties of the longitudinal muscle seem very similar to those on vascular smooth muscle where the existence of KATP channels has been confirmed by electrophysiological studies. This tension study suggests the possibility of the existence of KA-rt; channels in longitudinal smooth muscle cells of the guinea pig ileum. However, the final recognition of the presence of KATP channels will rely on evidence from electro-
Y.-D. Sun, C G. Benishin/ European Journal of Pharmacology 271 (1994) 453-459
physiological studies. In addition the ability of glibenclamide to increase basal tone in the longitudinal muscle may suggest that under resting conditions there is a small proportion of KATP channels which are activated. In rabbit mesenteric arterial muscle, Nelson et al. (1990) found that (a) the relaxing effect of CGRP on norepinephrine pre-contracted strips is partially reversed, and (b) CGRP-produced hyperpolarization of the arterial muscle cell membrane is totally prevented by glibenclamide. Finally, this peptide opened single K ÷ channels in isolated muscle cells. In the longitudinal muscle, we found that glibenclamide could not reverse the CGRP-induced inhibition or the isoprenaline-induced inhibition of the tension evoked by either KCI or histamine. These results differ from the effect of CGRP on rabbit mesenteric arterial muscle (Nelson et al., 1990) but are consistent with the effect of CGRP on rat coronary arterial muscle (Prieto et al., 1991), human mammary arterial muscle and saphenous vein (Boyle and Brown, 1991), and porcine coronary arterial muscle (Kageyama et al., 1993). Ba 2÷ has been reported to be a blocker of KAT P channels in skeletal muscle (Quayle et al., 1988). In vascular smooth muscle, Ba 2+ was found to have no effect on norepinephrine-precontracted muscle and to reverse the inhibition by CGRP (Nelson et ak, 1990). In the longitudinal muscle, Ba 2+ also slightly reversed the inhibition by CGRP. However, it is difficult to conclude that KAT P might be involved in the actions of CGRP and Ba 2+ considering the fact that Ba 2+ itself affected the histamine-precontracted longitudinal muscle. Both isoprenaline and CGRP seem to inhibit smooth muscle through mechanisms other than the opening of KAT P channels in the cells of the longitudinal muscle. Isoprenaline increases intracellular cyclic AMP levels in the muscle and relaxes the muscle (Seamon and Daly, 1981). In a previous study, we demonstrated that CGRP also increases the cyclic AMP content in the longitudinal muscle and this correlates with the muscle relaxing effect (Sun and Benishin, unpublished data). CGRP did not influence the [Ca2+] i elevated by KCI in the longitudinal muscle (Sun and Benishin, unpublished data) and in porcine coronary arterial smooth muscle (Kageyama et al., 1993). This evidence suggests that the KAT P channel opening effect may not be involved in the mechanism of action of CGRP on the longitudinal muscle. In addition, the mechanisms of the muscle-relaxing effects of CGRP appear to be tissuedependent.
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