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Functional evidence for a glibenclamide-sensitive K+ channel in rat ileal smooth muscle
ejp ELSEVIER
European Journal of Pharmacology 271 (1994) 379-386
Functional evidence for a glibenclamide-sensitive K ÷ channel in rat ileal smooth muscle Hartmut Franck, Andreas Puschmann, Volker Schusdziarra, Hans-Dieter Allescher
*
Department of Internal Medicine II, Technical University of Munich, Ismaningerstr. 22, 81675 Munich, Germany Received 1 September 1994; revised MS received 22 September 1994; accepted 23 September 1994
Abstract The motor activity of gastrointestinal smooth muscle is closely related to the membrane potential. Controlling the membrane potential via modulation of K + channels is essential for the action of neurotransmitters on smooth muscle. In the present study the effect of the K + channel activator, lemakalim, on longitudinal smooth muscle of the rat ileum was investigated. Segments of rat ileum were stimulated by the muscarinic receptor agonist, carbachol (10 - 6 M). Lemakalim (10-10 to 3 x 10 -5 M) induced a dose-dependent inhibition of the carbachol-induced contraction. This inhibitory effect of lemakalim was not modified by neural blockade with tetrodotoxin (10 - 6 M, n = 9). Glibenclamide (10 - 7 to 10 -5 M), a specific blocker of ATP-dependent K + channels antagonized dose dependently the relaxant effect of lemakalim (IC50:3.4 x 10 - 6 M, n = 11, P < 0,001). In contrast, apamin (10 -7 M, n = 9, n.s.) and charybdotoxin (10 -7 M, n = 9, n.s.), specific blockers of Ca2+-dependent K ÷ channels and the non-specific K + channel blocker, tetraethylammonium (10 - 4 to 10-1 M), had no influence on the inhibitory effect of lemakalim. Contractions induced by the Ca 2÷ channel activator, Bay-K-8644, were completely inhibited by lemakalim (10 -5 M, n = 12). This inhibitory effect was also selectively antagonized by glibenclamide (10 -5 M). Potential non-adrenergic non-cholinergic (NANC) inhibitory mediators like ATP, nitric oxide (NO) or neurotensin showed no sensitivity to glibenclamide. These functional data indicate that the relaxant effect of lemakalim is due to a specific activation of glibenclamide-sensitive K ÷ channels, which in turn can modulate the activity of dihydropyridine-sensitive (voltage-dependent) Ca2÷ channels. A physiological or pathophysiological role of the glibenclamide-sensitive K + channels in intestinal smooth muscle is discussed; however, they seem not to be involved in the effect of the NANC inhibitory mediators tested.
Keywords: Lemakalim; Glibenclamide; ATP-sensitive Ca 2+ channel; Apamin; Charybdotoxin; Nitric oxide (NO)
1. Introduction Over the last few years several compounds have been identified which increase the permeability of the smooth muscle cell membrane to K ÷, thereby hyperpolarizing and relaxing the tissue (Cook and Hof, 1988; Cook et al., 1990). These K ÷ channel openers constitute a chemically diverse group of compounds including nicorandil, cromakalim (and its stereoisomer, lemakalim), pinacidil, diazoxide, minoxidil sulphate (the active metabolite of minoxidil) and RP-49356 (Cook et al., 1990; Cook and Hof, 1988; Fan et al., 1992). Poten-
* Corresponding author. II. Medizinische Klinik und Poliklinik der TU Miinchen, Ismaningerstr. 22, 81675 Miinchen, Germany. Tel. 089-4140-2481, fax 089-4180-5128. 0014-2999/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved
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tial clinical applications of the K ÷ channel openers include hypertension (Escande et al., 1989; Ogawa et al., 1992), asthma, irritable bladder syndrome. In addition K ÷ channel openers have been shown to dilate vessels preferentially in ischemic tissue (Samaha et al., 1992; Benndorf et al., 1992; Gwilt et al., 1992). Endogenous activation of K + channels seems to be one of the mechanisms, but not the principal ones underlying hypoxic vasodilation in myocardial muscle (Von-Beckerath et al., 1991; Liu and Downey, 1992; Grover et al., 1993; Thornton et al., 1993). Taking into account the immense potential of K ÷ channel openers in all different types of smooth muscle tissue, there may be potential additional clinical applications a n d / o r possible side-effects of K + channel openers in gastrointestinal smooth muscle. However, little information is available on the effect of K ÷ channel activators in intestinal
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smooth muscle (Post et al., 1991; D u e t al., 1994). Our preliminary work demonstrated an inhibitory response of the K + channel activator, lemakalim, on smooth muscle of rat ileum (Allescher et al., 1991). The aim of the present study was to determine the effect of the K ÷ channel activator, lemakalim, on intestinal smooth muscle of the rat ileum and to investigate the mechanism of action of lemakalim on intestinal smooth muscle. The results of the present study have been presented in part in preliminary form (Fick et al., 1993).
2. Material and methods
The experimental model and protocol for investigation of inhibitory responses was used as previously described (Allescher et al., 1992). In brief, male Wistar rats (400-500 g) were killed by injecting sodium pentobarbital intraperitoneally (100 mg/kg). The terminal ileum was immediately removed and kept in oxygenated Krebs-Ringer bicarbonate solution. Six segments of full thickness strips were prepared from the terminal ileum (length 1.5-2 cm), and one end was fixed to a hook on a holder. Holders with tissue were placed in a jacketed organ bath containing 3 ml KrebsRinger bicarbonate solution (KRS) (NaCI 115.5 mM, MgSO 4 1.16 mM, NaH2PO 4 1.16 mM, glucose 11.1 mM, NaHCO 3 21.9 mM, CaC12 2.5 mM, KCI 4.16 mM) gassed with 95% 02, 5% CO 2 and maintained at 37°C by circulating water through the jackets. The free end of the segment was connected with a thread to an isometric force transducer (Swegma force displacement transducer SG 4-500, Swegrna Sweden); 1 g of tension was applied to the muscle, and the preparation was allowed to equilibrate for at least 30 min. Changes in the tension were amplified by Hellige couplers and recorded on a Rikadenki chart recorder.
lemakalim concentration on carbachol- or KCl-induced contraction was tested for stability by repeating this protocol several times. The inhibitory effect of lemakalim remained stable for more than 3 h with this protocol. Each segment of rat ileum was used only for a single concentration-response curve. 2.3. Effect o f K + channel blockers on the lemakalim-induced inhibition
Following the initial stimulation with carbachol, lemakalim was added as described above, the inhibitory response serving as a control. After the respective washes, the blocker was added to the bath in microliter volumes, 2 min before stimulation with carbachol. The effect on the lemakalim response was tested in presence of the blocker. Again, after repeated washes, the next concentration of the blocker was added. Each segment was used only for a single concentration-response curve of a single blocker. 2.4. Data analysis and statistics
For data analysis the contraction level induced by the stimulus prior to the addition of lemakalim was determined. The inhibitory response to lemakalim, which reached a minimum usually within 20 s after application, was defined as the contraction remaining after application of lemakalim. The data are given as absolute values and the inhibition is expressed as percent of the agonist-induced contraction. The data are given as means + standard deviation (mean + S.D.). n indicates the number of independent observations in different strips. Each protocol was repeated with ileal segments of at least two different animal preparations. Analysis of variance was used to compare the mean values, and values of P < 0.05 or less were considered significant.
2.1. Experimental protocol
2.L D ~ Each preparation was allowed to equilibrate for 30 min. At the beginning and the end of each experiment the response to the muscarinic receptor agonist, carbachol, was tested as a control. Carbachol was washed out after 5 min by three consecutive washes at 5-min intervals. The tissue was then incubated for 5 min in buffer prior to the application of the next stimulus. 2.2. Lemakalim-induced inhibition After washout and incubation a contractile response was induced by carbachol or the Ca z+ channel activator, Bay-K-8644. Lemakalim was added when the carbachol- or the Bay-K-8644-induced response had reached a plateau, 30 s after addition of the stimulus. In separate experiments the inhibitory effect of a given
The drugs used were: carbachol, tetrodotoxin, glibenclamide (Boehringer Mannheim, Germany), lemakalim (Beecham, UK), apamin (Sigma, Munich, Germany), charybdotoxin (Sigma, Munich, Germany), tetraethylammonium (Aldrich, Milwaukee, USA), BayK-8644 (Bayer, Heidelberg, Germany), ot,/3-methyleneATP (Sigma, Munich, Germany), neurotensin (Sigma, Munich, Germany), N-morpholino-N-nitroso-aminoacetonitril (SIN-l) (Cassella-Riedel, Frankfurt, Germany). The drugs were freshly dissolved in saline and further diluted with Krebs-Ringer buffer. Glibenclamide was dissolved in N-methyl-formamide. The drugs were added to the bath in microliter volumes and experiments were checked for the effects of the drug solvents.
H. Franck et al. / European Journal of Pharmacology 271 (1994) 379-386 120
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Fig. 1. (A) Representative tracing showing the response to lemakalim of longitudinal smooth muscle precontracted with carbachol (CCH) 10 -6 M. Repeated application gave an identical response. (B) Plot showing the dose-dependent inhibitory effect of lemakalim on longitudinal smooth muscle precontracted with carbachol (CCH) 10 -6 M.
application of lemakalim (10 -1° to 3 × 10 -5 M) caused an instantaneous and dose-dependent relaxation. The inhibitory effect of lemakalim started at a threshold concentration of 10 -8 M and increased dose dependently. The maximal inhibitory effect was obtained at 10 -5 M, amounting to 49.8 + 14.1% (n = 12) of the contraction induced by carbachol (10 -6 M). The halfmaximal inhibition of the carbachol-induced contraction was observed at 1.3 × 10 -6 M lemakalim. Repeated application of lemakalim (10 -5 M) on a single preparation revealed no change in relaxation on wash-
3. Results
3.1. Effect of lemakalim on carbachol-induced contraction The muscarinic receptor agonist, carbachol (10 -6 M), caused a tonic contractile response of the longitudinal smooth muscle. This effect was used as in previous studies (Allescher et al., 1991, 1992) in order to investigate actions of inhibitory mediators. As soon as the contractile response of carbachol reached a plateau,
Table 1 Effect of lemakalim 10 -5 M on carbachol- or Bay-K-8644-induced contraction % Inhibition of contraction
Contraction (mN)
CCH stimulation + T T X 10 -6 M Bay-K-8644 stimulation +TTX10-6M
Before lemakalim
After lemakalim
25.0 + 11.2 21.1 + 6.1 5.5 + 1.2 5 . 9 + 2.4
13.0 + 7.6 13.2 + 5.1 0.0 + 2.0 0.0+1.4
49.8 + 38.9 + 100.2 + 100.5+
14.1 16.3 3.1 3.6
Values are expressed as means + S.D. CCH = carbachol; T I ' X = tetrodotoxin.
Table 2 Effect of various K + channel blockers on the lemakalim-induced inhibiton Contraction (mN)
Control Apamin 10 -5 M Charybdotoxin 10 -7 M T E A 10 -2 M
% Inhibition of contraction
Before lemakalim
After lemakalim
25.0 21.7 25.9 6.9
13.0 11.1 16.6 0.0
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+ + + +
7.6 4.5 6.0 2.0
Values are expressed as means _ S.D. CCH = carbachol; TTX = tetrodotoxin.
49.8 48.7 36.5 100.5
+ + + +
14.1 14.7 7.7 3.6
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ing with Krebs-Ringer solution in between (1st application: 52.8%, 2nd: 49.9%, 3rd: 50.2% of the carbacholinduced response, n = 12, n.s.) (Fig. 1A and B). The lemakalim-induced relaxation showed a small decrease after neural blockade with tetrodotoxin, which was not significant (before tetrodotoxin 10 -6 M: 49.8 + 14.1%, after: 38.9 + 16.3%, n = 9, P > 0.05) (Table 1).
3.2. Effect of K ÷ channel blockers on the lemakalim-induced inhibition Several K ÷ channel blockers were tested against lemakalim to gain insight into the possible channel type functional during muscle relaxation (Table 2). Apamin (10 -7 M, n = 9, n.s.) and charybdotoxin (10 -7 M, n = 9, n.s.), specific blockers of Ca2÷-dependent K ÷ channels had no influence on the effect of lemakalim (Fig. 2A and B). Further the non-specific K ÷ channel blocker, tetraethylammonium (TEA) (10 -4 to 10 -1 M), did not modify the inhibitory action of lemakalim (Fig. 3C). In contrast, glibenclamide (10 -7 to 10 -5 M), a specific blocker of A T P - d e p e n d e n t K + channels antagonized dose dependently the relaxant effect of lemakalim (IC50:9.1 × 10 -8 M, n = 11) with complete blockade of the inhibitory effect at a concentration of 10 -5 M (n = 11, P < 0.001) (Fig. 3A and B). Glibenclamide (10 -5 M), which added after lemakalim (10 -5 M) had produced inhibition of the initial carbachol-induced contraction, was able to restore the carbachol response to a major extent, i.e. up to 90% of the pretreatment response.
3.3. Effect of lemakalim on contraction induced by the Ca 2 + channel activator Bay-K-8644 The Ca 2÷ channel activator, Bay-K-8644 (10 -7 M), caused a tonic contraction of the longitudinal smooth muscle segments, which reached 2 0 - 2 5 % of the maxim u m carbachol-induced response. This contractile response was completely inhibited by lemakalim (10 -5 M) (100.2 + 3.1% of the Bay-K-8644 response, n = 9). Application of tetrodotoxin led to no modification of the lemakalim-induced response in these experiments (before tetrodotoxin 10 -6 M: 100.2-t-3.1%, after: 100.5 _ 3.6%, n = 9, P > 0.05) (Table 1). Analogous to the studies using carbachol as stimulus, glibenclamide (10 -5 M) selectively antagonized the lemakalim-induced inhibitory response (n = 6, P < 0.001).
3.4. Effect of glibenclamide on the inhibitory effect of putative non-adrenergic non-cholinergic mediators (ATP, nitric oxide, neurotensin) a/3-Methylene-ATP (10 -4 M) and the N O donor, N-morpholino-N-nitroso-amino-acetonitril (SIN-l) (10 -3 M), caused an instantaneous potent inhibition of
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akalim
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nium (TEA) 10-4 to 10-2 M, on the lemakalim-induced inhibition. The tissue was precontracted with carbachol (10 -6 M).
the carbachol-induced contraction. The inhibitory effects of both inhibitory agents were unaffected by glibenclamide, whereas a-methylene-ATP was blocked by tetraethylammonium (10 -1 M) and the Ca 2÷activated K ÷ channel blocker, apamin (10 -7 M). Neurotensin showed an instantaneous dose-dependent inhibition of the carbachol-induced contraction (IC50:2.8 × 10-10 M ) w i t h a maximum inhibitory effect of 52.4 ___7.9% (n = 12) of the carbachol-induced con-
H. Franck et aL / European Journal of Pharmacology 271 (1994) 379-386
383
hibitory responses of the guinea pig gall-bladder were blocked by glibenclamide and that glibenclamide-sensitive responses might be involved in responses evoked from capsaicin-sensitive afferent nerves. Therefore the effect of glibenclamide on capsaicin- and CGRP-induced effects was tested. Application of capsaicin (10 - 6 M) caused only a small, short-lasting relaxation of the carbachol-induced contraction, which reached 7.7 + 9.3% of the carbachol-induced contraction. This small inhibitory effect of capsaicin was not affected by preincubation with glibenclamide (10 -5 M). Similarly C G R P (3 x 10 -8 to 10 -7 M) caused a small inhibition (13.4 + 5.5%) of the carbachol-induced contraction (n = 8). Glibenclamide (10 -5 M) had no effect on the CGRPinduced inhibition (12.1 + 3.7%, n = 8, P > 0.05).
traction, which was in a range similar to that of the maximum lemakalim-induced inhibition. Glibenclamide (n = 9) did not affect this neurotensin-induced response. As shown previously the neurotensin response was blocked by the Ca2+-activated K ÷ channel blocker, apamin, whereas other K + channel blockers (tetraethylammonium, 4-aminopyridine, 9-aminoacridine and diazoxide) had no influence (Allescher et al., 1992).
3.5. Effect of glibenclamide on capsaicin- and CGRP-induced responses It has been suggested in a preliminary report that calcitonin gene-related peptide (CGRP)-induced in-
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Fig. 3. (A) Representative tracing showing the effect of the K ÷ channel blocker glibenclamide 10 -5 M, on the lemakalim-induced inhibition. (B) Plot showing the effect of the K + channel blocker, glibenclamide 10 -8 to 10 -5 M, on the lemakalim-induced inhibition. The contraction after application of lemakalim with preincubation with glibenclamide is shown.
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4. Discussion
ATP-sensitive K ÷ (KAxe) channels are present in pancreatic beta cells where they play an important role in insulin secretion (Ashcroft et al., 1984; Cook and Hales, 1984; Dunne and Petersen, 1986, 1991; SchmidAntomarchi et al., 1987). Closure of KATP channels following glucose superfusion (and subsequent variation of the A T P / A D P ratio) leads via membrane depolarization to Ca 2+ entry and insulin release (Ashcroft et al., 1984; Cook and Hales, 1984; Dunne and Petersen, 1986, 1991). Well known antidiabetic sulfonylureas such as glibenclamide release insulin by blockade of KATP channels (Schmid-Antomarchi et al., 1987). ATP-sensitive K ÷ channels have been identified in cardiac muscle (Noma, 1983), skeletal muscle (Spruce et al., 1985), neurons (Ashford et al., 1988), and recently in vascular smooth muscle (Standen et al., 1989; Kajioka et al., 1991; Kovacs and Nelsen, 1991; Silberberg and Van Breemen, 1992). The KAXP channels in all these tissues are blocked by sulfonylureas like glibenclamide. The chemically diverse group of K ÷ channel openers including nicorandil, cromakalim (and its stereoisomer, lemakalim), pinacidil, diazoxide, minoxidil sulphate (the active metabolite of minoxidil) and RP-49356 (Cook et al., 1990; Cook and Hof, 1988; Fan et al., 1992) are potent vasodilatators and relaxant agents in smooth muscle. Recently the K ÷ openers, cromakalim (and its more active isomer, lemakalim), diazoxide and RP 49356, have been shown to open KATP channels in vascular smooth muscle, cardiac and skeletal muscle (Quast and Cook, 1989). There are only few functional data on the effect of K ÷ channel openers in intestinal smooth muscle, especially the small intestine. Den Hertog et al. (1989) showed that, in guinea-pig taenia caeci, cromakalim induced a dose-dependent hyperpolarization which was inhibited by glibenclamide. Sanders and coworkers demonstrated in colonic smooth muscle a cromakalimand lemakalim-induced K ÷ outward current (Post et al., 1991; Carl et al., 1992), responsible for the noticed hyperpolarization. Sanders and coworkers provided evidence that this effect could be due to activation of a glibenclamide-sensitive KATP channel (Du et al., 1994). In the present study the effect of the K ÷ channel activator, lemakalim, on longitudinal smooth muscle of rat ileum was investigated. This system has been utilized previously in our laboratory to characterize the cellular mechanism of action of neurotensin by our laboratory (Allescher et al., 1992). Application of lemakalim on precontracted longitudinal smooth muscle of rat ileum caused a marked dose-dependent relaxation, which showed no decline with repeated application after wash-out. Neural blockade by tetrodotoxin (10 -6 M) did not significantly
modify the lemakalim-induced relaxation, indicating that this effect is probably caused by a direct action on the smooth muscle. The specific ATP-sensitive K ÷ channel blocker, glibenclamide (Schmid-Antomarchi et al., 1987; Liu and Downey, 1992; Grover et al., 1993; Thornton et al., 1993), antagonized dose dependently the relaxant effect of lemakalim whereas other K ÷ channel blockers such as apamin (10 -7 M) or charybdotoxin (10 -7 M), specific blockers of Ca2÷-dependent K ÷ channels, or the non-specific K ÷ channel blocker, tetraethylammonium (TEA) (10 -4 to 10 -1 M), had no influence on the lemakalim-induced relaxation. The results indicate that lemakalim activates a K + channel which is blocked by glibenclamide, a specific KATP channel blocker. This feature is characteristic of KATP channels in other tissues. Therefore the data provide functional evidence for the presence of KATP channels in the smooth muscle of rat ileum, which modulate the contractile activity. The concentration of glibenclamide which reduced the inhibitory effect of lemakalim by 50% in the present study was 3.4 x 10 -6 M. Even though a comparison has to be made with great care this concentration is in a range similar to that of the IC50 values reported in electrophysiological studies on vascular smooth muscle and the heart (0.11.5 x 10 -6 M) (Escande, 1989) but is slightly higher than the IC50 reported for KATP channels expressed in Xenopus oocytes (IC50:2.9 × 10 -7 M) (Sakuta et al., 1992; Sakuta and Okamoto, 1993) and about 100-fold higher than reported for pancreatic beta cells (Dunne and Petersen, 1991). The physiological role of these KATP channels in intestinal smooth muscle is unclear. The functional and pharmacological data presented in this study together with the electrophysiological data reported from colonic smooth muscle (Carl et al., 1992) provide strong evidence for the existence of these channels. From the present experiments there is no evidence that these channels are involved in the inhibitory effect of non-adrenergic non-cholinergic (NANC) neurotransmitters such as the NO donor, SIN-l, or the ATP analogue, aft-methylene ATP. In a recently presented preliminary electrophysiological study on guinea pig gallbladder smooth muscle cells it was speculated that KATP channels might be involved in the inhibitory action of calcitonin gene-related peptide (CGRP) and subsequently in the action of neurotransmitters released from afferent nerves (Zhang et al., 1993). In the present study capsaicin and CGRP elicited only a small inhibitory effect, which was not influenced by glibenclamide. Hence there is no functional evidence for a physiological role of KATP channels in capsaicin or CGRP-induced responses in the small intestine. ATP-sensitive K+-channels have been shown to be involved in ischemia responses in cardiac tissue (Benndorf et al., 1992; Escande, 1989) and to maintain
H. Franck et al. / European Journal of Pharmacology 271 (1994) 379-386
vascular tone in coronary vessels under certain conditions (Samaha et al., 1992). These channels have also been associated with vascular function in the intestine (Silberberg and Van Breemen, 1992) and haemodynamic changes and a reduction of portal pressure have been reported for an experimental model of portal hypertension in the rat (Moreau et al., 1992). Whether KATP channels in intestinal smooth muscle could also be modulated under ischemic conditions cannot be answered from the present study; however, it would be in line with changes observed in other organ systems. Additionally these channels could offer another pharmacological approach for direct spasmolytic agents in the gastrointestinal tract.
Acknowledgements The authors acknowledge the courtesy of Prof. Dr. BliJmel and his collaborators, Dept. Exp. Surgery, Technical University of Munich. The study was supported by Deutsche Forschungsgemeinschaft A1 245/2-2 and DFG A1-245/8-1.
References Allescher, H.D., H. Fick, V. Schusdziarra and M. Classen, 1991, Inhibitory action of neurotensin in rat ileum is due to modulation of L-type Ca z÷ channels via apamin-sensitive K ÷ channels, Gastroenterology 100, 155A. Allescher, H.D., H. Fick, V. Schusdziarra and M. Classen, 1992, Mechanisms of neurotensin-induced inhibition in rat ileal smooth muscle, Am. J. Physiol. 263, G767. Ashcroft, F.M., D.E. Harrison and S.J.H. Ashcroft, 1984, Glucose induces closure of single potassium channels in isolated rat pancreatic fl cells, Nature 312, 446. Ashford, M.L.J., N.C. Sturgess, N.J. Trout, N.J. Gardner and C.N. Hales, 1988, Adenosine-5'-triphosphate-sensitive ion channels in neonatal rat cultured central neurones, Pfliig. Arch. 412, 297. Benndorf, K., G. Bollmann, M. Friedrich and H. Hirche, 1992, Anoxia induces time-independent K + current through KATP channels in isolated heart cells of the guinea-pig, J. Physiol. (London) 454, 339. Carl, A., S. Bowen, C.H. Gelband, K.M. Sanders and J.R. Hume, 1992, Cromakalim and lemakalim activate Ca 2+ dependent K + channels in canine colon, Pfliig. Arch. 421, 67. Cook, D.L. and C.N. Hales, 1984, Intracellular ATP directly blocks K + channels in pancreatic/3-cells, Nature 311, 271. Cook, N.S., D. Bertrand, D.H. Jenkinson, M.L.J. Ashford, R.E. Buckingham, A.H. Weston, J. Longmore, U. Quast, L.D. Partridge, S.A. Siegelbaum, D.G. Haylett, D.J. Adams and P.N. Strong, 1990, Potassium Channels (Ellis Horwood, New York, Chichester, Brisbane, Toronto) p. 1. Cook, N.S. and R.P. Hof, 1988, Cardiovascular effects of apamin and BRL 34915 in rats and rabbits, Br. J. Pharmacol. 93, 121. Den Hertog, A., J. Van den Akker and A. Nelemans, 1989, Effect of cromakalim on smooth muscle cells of guinea-pig taenia caeci, Eur. J. Pharmacol. 174, 287.
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Du, C., A. Carl, T.K. Smith, K.M. Sanders and K.D. Keef, 1994, Mechanism of cyclic AMP-induced hyperpolarization in canine colon, J. Pharmacol. Exp. Ther. 268, 208. Dunne, M.J. and O.H. Petersen, 1986, Intracellular ADP activates K ÷ channels that are inhibited by ATP in an insulin-secreting cell line, FEBS Lett. 208, 59. Dunne, M.J. and O.H. Petersen, 1991, Potassium selective ion channels in insulin-secreting cells: physiology, pharmacology and their role in stimulus-secretion coupling, Biochim. Biophys. Acta 1071, 67. Escande, D., 1989, The pharmacology of ATP-sensitive K ÷ channels in the heart, Pfli~g. Arch. 414 (Suppl.), $93. Escande, D., D. Thuringer, S. Le-Guern, J. Courteix, M. Laville and I. Cavero, 1989, Potassium channel openers act through an activation of ATP-sensitive K + channels in guinea-pig cardiac myocytes, Pfliig. Arch. 414, 669. Fan, Z., K. Nakayama, T. Sawanobori and M. Hiraoka, 1992, Aromatic aldehydes and aromatic ketones open ATP-sensitive K + channels in guinea-pig ventricular myocytes, Pfliig. Arch. 421, 409. Fick, H., A. Puschmann, H.D. AUescher, V. Schusdziarra and M. Classen, 1993, Functional evidence for a specific glibenclamide sensitive K + channel in rat ileum smooth muscle. J. Gastrointest. Motil. 5, 191. Grover, G.J., S. Dzwonczyk, P.G. Sleph and C.A. Sargent, 1993, The ATP-sensitive potassium channel blocker glibenclamide (glyburide) does not abolish preconditioning in isolated ischemic rat hearts, J. Pharmacol. Exp. Ther. 265, 559. Gwilt, M., C.G. Henderson, J. Orme and J.D. Rourke, 1992, Effects of drugs on ventricular fibrillation and ischemic K + loss in a model of ischaemia in perfused guinea-pig hearts in vitro, Eur. J. Pharmacol. 220, 231. Kajioka, S., K. Kitamura and H. Kuriyama, 1991, Guanosine diphosphate activates an adenosine 5'-triphosphate-sensitive K + channel in the rabbit portal vein, J. Physiol. (London) 444, 397. Kovacs, R.J. and M.T. Nelson, 1991, ATP-sensitive K + channels from aortic smooth muscle incorporated into planar lipid bilayers, Am. J. Physiol. 261, H604. Liu, Y. and J.M. Downey, 1992, Ischemic preconditioning protects against infarction in rat heart, Am. J. Physiol. 263, Hl107. Moreau, R., H. Komeichi, S. Cailmail and D. Lebrec, 1992, Blockade of ATP-sensitive K ÷ channels by glibenclamide reduces portal pressure and hyperkinetic circulation in portal hypertensive rats, J. Hepatol. 16, 215. Noma, A., 1983, ATP-regulated K + channels in cardiac muscle, Nature 305, 147. Ogawa, N., Y. Fukata, S. Kaneta, Y. Jinno, H. Fukushima and K. Nishikori, 1992, Comparison of KRN2391 with nicorandil and nifedipine on canine coronary blood flow: antagonism by glibenclamide, J. Cardiovasc. Pharmacol. 20, 11. Post, J.M., R.J. Stevens, K.M. Sanders and J.R. Hume, 1991, Effect of cromakalim and lemakalim on slow waves and membrane currents in colonic smooth muscle, Am. J. Physiol. 260, C375. Quast, U. and N.S. Cook, 1989, Moving together, K ÷ channel openers and ATP-sensitive K + channels, Trends Pharmacol. Sci. 10, 431. Sakuta, H. and K. Okamoto, 1993, Activation by KRN2391 and nicorandil of glibenclamide-sensitive K + channels in Xenopus oocytes, Eur. J. Pharmacol. 244, 277. Sakuta, H., K. Okamoto and Y. Watanabe, 1992, Blockade by antiarrhythmic drugs of glibenclamide-sensitive K ÷ channels in Xenopus oocytes, Br. J. Pharmacol. 107, 1061. Samaha, F.F., F.W. Heineman, C. Ince, J. Fleming and R.S. Balaban, 1992, ATP-sensitive potassium channel is essential to maintain basal coronary vascular tone in vivo, Am. J. Physiol. 262, C1220.
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Schmid-Antomarchi, H., J.R. 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. Silberberg, S.D. and C. Van-Breemen, 1992, A potassium current activated by lemakalim and metabolic inhibition in rabbit mesenteric artery, Pfliig. Arch. 420, 118. Spruce, A.E., N.B. Standen and P.R. Stanfield, 1985, Voltage-dependent, ATP-sensitive potassium channels of skeletal muscle membrane, Nature 316, 736. Standen, N.B., J.M. Quayle, N.W. Davies, J.E. Brayden, Y. Huang and M.T. Nelson, 1989, Hyperpolarizing vasodilators activate ATP-sensitive K ÷ channels in arterial smooth muscle, Science 245, 177.
Thornton, J.D., C.S. Thornton, D.L. Sterling and J.M. Downey, 1993, Blockade of ATP-sensitive potassium channels increases infarct size but does not prevent preconditioning in rabbit hearts, Circ. Res. 72, 44. Von-Beckerath, N., S. Cyrys, A. Dischner and J. Daut, 1991, Hypoxic vasodilatation in isolated, perfused guinea-pig heart: an analysis of the underlying mechanisms, J. Physiol. (London) 442, 297. Zhang, L., A. Bonev, M.T. Nelson and G.M Mawe, 1993, Calcitonin gene-related peptide activates an ATP-sensitive potassium current in guinea pig gallbladder smooth muscle cells, Am. J. Physiol. 104, A1069.
European Journal of Pharmacology 271 (1994) 379-386
Functional evidence for a glibenclamide-sensitive K ÷ channel in rat ileal smooth muscle Hartmut Franck, Andreas Puschmann, Volker Schusdziarra, Hans-Dieter Allescher
*
Department of Internal Medicine II, Technical University of Munich, Ismaningerstr. 22, 81675 Munich, Germany Received 1 September 1994; revised MS received 22 September 1994; accepted 23 September 1994
Abstract The motor activity of gastrointestinal smooth muscle is closely related to the membrane potential. Controlling the membrane potential via modulation of K + channels is essential for the action of neurotransmitters on smooth muscle. In the present study the effect of the K + channel activator, lemakalim, on longitudinal smooth muscle of the rat ileum was investigated. Segments of rat ileum were stimulated by the muscarinic receptor agonist, carbachol (10 - 6 M). Lemakalim (10-10 to 3 x 10 -5 M) induced a dose-dependent inhibition of the carbachol-induced contraction. This inhibitory effect of lemakalim was not modified by neural blockade with tetrodotoxin (10 - 6 M, n = 9). Glibenclamide (10 - 7 to 10 -5 M), a specific blocker of ATP-dependent K + channels antagonized dose dependently the relaxant effect of lemakalim (IC50:3.4 x 10 - 6 M, n = 11, P < 0,001). In contrast, apamin (10 -7 M, n = 9, n.s.) and charybdotoxin (10 -7 M, n = 9, n.s.), specific blockers of Ca2+-dependent K ÷ channels and the non-specific K + channel blocker, tetraethylammonium (10 - 4 to 10-1 M), had no influence on the inhibitory effect of lemakalim. Contractions induced by the Ca 2÷ channel activator, Bay-K-8644, were completely inhibited by lemakalim (10 -5 M, n = 12). This inhibitory effect was also selectively antagonized by glibenclamide (10 -5 M). Potential non-adrenergic non-cholinergic (NANC) inhibitory mediators like ATP, nitric oxide (NO) or neurotensin showed no sensitivity to glibenclamide. These functional data indicate that the relaxant effect of lemakalim is due to a specific activation of glibenclamide-sensitive K ÷ channels, which in turn can modulate the activity of dihydropyridine-sensitive (voltage-dependent) Ca2÷ channels. A physiological or pathophysiological role of the glibenclamide-sensitive K + channels in intestinal smooth muscle is discussed; however, they seem not to be involved in the effect of the NANC inhibitory mediators tested.
Keywords: Lemakalim; Glibenclamide; ATP-sensitive Ca 2+ channel; Apamin; Charybdotoxin; Nitric oxide (NO)
1. Introduction Over the last few years several compounds have been identified which increase the permeability of the smooth muscle cell membrane to K ÷, thereby hyperpolarizing and relaxing the tissue (Cook and Hof, 1988; Cook et al., 1990). These K ÷ channel openers constitute a chemically diverse group of compounds including nicorandil, cromakalim (and its stereoisomer, lemakalim), pinacidil, diazoxide, minoxidil sulphate (the active metabolite of minoxidil) and RP-49356 (Cook et al., 1990; Cook and Hof, 1988; Fan et al., 1992). Poten-
* Corresponding author. II. Medizinische Klinik und Poliklinik der TU Miinchen, Ismaningerstr. 22, 81675 Miinchen, Germany. Tel. 089-4140-2481, fax 089-4180-5128. 0014-2999/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved
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tial clinical applications of the K ÷ channel openers include hypertension (Escande et al., 1989; Ogawa et al., 1992), asthma, irritable bladder syndrome. In addition K ÷ channel openers have been shown to dilate vessels preferentially in ischemic tissue (Samaha et al., 1992; Benndorf et al., 1992; Gwilt et al., 1992). Endogenous activation of K + channels seems to be one of the mechanisms, but not the principal ones underlying hypoxic vasodilation in myocardial muscle (Von-Beckerath et al., 1991; Liu and Downey, 1992; Grover et al., 1993; Thornton et al., 1993). Taking into account the immense potential of K ÷ channel openers in all different types of smooth muscle tissue, there may be potential additional clinical applications a n d / o r possible side-effects of K + channel openers in gastrointestinal smooth muscle. However, little information is available on the effect of K ÷ channel activators in intestinal
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smooth muscle (Post et al., 1991; D u e t al., 1994). Our preliminary work demonstrated an inhibitory response of the K + channel activator, lemakalim, on smooth muscle of rat ileum (Allescher et al., 1991). The aim of the present study was to determine the effect of the K ÷ channel activator, lemakalim, on intestinal smooth muscle of the rat ileum and to investigate the mechanism of action of lemakalim on intestinal smooth muscle. The results of the present study have been presented in part in preliminary form (Fick et al., 1993).
2. Material and methods
The experimental model and protocol for investigation of inhibitory responses was used as previously described (Allescher et al., 1992). In brief, male Wistar rats (400-500 g) were killed by injecting sodium pentobarbital intraperitoneally (100 mg/kg). The terminal ileum was immediately removed and kept in oxygenated Krebs-Ringer bicarbonate solution. Six segments of full thickness strips were prepared from the terminal ileum (length 1.5-2 cm), and one end was fixed to a hook on a holder. Holders with tissue were placed in a jacketed organ bath containing 3 ml KrebsRinger bicarbonate solution (KRS) (NaCI 115.5 mM, MgSO 4 1.16 mM, NaH2PO 4 1.16 mM, glucose 11.1 mM, NaHCO 3 21.9 mM, CaC12 2.5 mM, KCI 4.16 mM) gassed with 95% 02, 5% CO 2 and maintained at 37°C by circulating water through the jackets. The free end of the segment was connected with a thread to an isometric force transducer (Swegma force displacement transducer SG 4-500, Swegrna Sweden); 1 g of tension was applied to the muscle, and the preparation was allowed to equilibrate for at least 30 min. Changes in the tension were amplified by Hellige couplers and recorded on a Rikadenki chart recorder.
lemakalim concentration on carbachol- or KCl-induced contraction was tested for stability by repeating this protocol several times. The inhibitory effect of lemakalim remained stable for more than 3 h with this protocol. Each segment of rat ileum was used only for a single concentration-response curve. 2.3. Effect o f K + channel blockers on the lemakalim-induced inhibition
Following the initial stimulation with carbachol, lemakalim was added as described above, the inhibitory response serving as a control. After the respective washes, the blocker was added to the bath in microliter volumes, 2 min before stimulation with carbachol. The effect on the lemakalim response was tested in presence of the blocker. Again, after repeated washes, the next concentration of the blocker was added. Each segment was used only for a single concentration-response curve of a single blocker. 2.4. Data analysis and statistics
For data analysis the contraction level induced by the stimulus prior to the addition of lemakalim was determined. The inhibitory response to lemakalim, which reached a minimum usually within 20 s after application, was defined as the contraction remaining after application of lemakalim. The data are given as absolute values and the inhibition is expressed as percent of the agonist-induced contraction. The data are given as means + standard deviation (mean + S.D.). n indicates the number of independent observations in different strips. Each protocol was repeated with ileal segments of at least two different animal preparations. Analysis of variance was used to compare the mean values, and values of P < 0.05 or less were considered significant.
2.1. Experimental protocol
2.L D ~ Each preparation was allowed to equilibrate for 30 min. At the beginning and the end of each experiment the response to the muscarinic receptor agonist, carbachol, was tested as a control. Carbachol was washed out after 5 min by three consecutive washes at 5-min intervals. The tissue was then incubated for 5 min in buffer prior to the application of the next stimulus. 2.2. Lemakalim-induced inhibition After washout and incubation a contractile response was induced by carbachol or the Ca z+ channel activator, Bay-K-8644. Lemakalim was added when the carbachol- or the Bay-K-8644-induced response had reached a plateau, 30 s after addition of the stimulus. In separate experiments the inhibitory effect of a given
The drugs used were: carbachol, tetrodotoxin, glibenclamide (Boehringer Mannheim, Germany), lemakalim (Beecham, UK), apamin (Sigma, Munich, Germany), charybdotoxin (Sigma, Munich, Germany), tetraethylammonium (Aldrich, Milwaukee, USA), BayK-8644 (Bayer, Heidelberg, Germany), ot,/3-methyleneATP (Sigma, Munich, Germany), neurotensin (Sigma, Munich, Germany), N-morpholino-N-nitroso-aminoacetonitril (SIN-l) (Cassella-Riedel, Frankfurt, Germany). The drugs were freshly dissolved in saline and further diluted with Krebs-Ringer buffer. Glibenclamide was dissolved in N-methyl-formamide. The drugs were added to the bath in microliter volumes and experiments were checked for the effects of the drug solvents.
H. Franck et al. / European Journal of Pharmacology 271 (1994) 379-386 120
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Fig. 1. (A) Representative tracing showing the response to lemakalim of longitudinal smooth muscle precontracted with carbachol (CCH) 10 -6 M. Repeated application gave an identical response. (B) Plot showing the dose-dependent inhibitory effect of lemakalim on longitudinal smooth muscle precontracted with carbachol (CCH) 10 -6 M.
application of lemakalim (10 -1° to 3 × 10 -5 M) caused an instantaneous and dose-dependent relaxation. The inhibitory effect of lemakalim started at a threshold concentration of 10 -8 M and increased dose dependently. The maximal inhibitory effect was obtained at 10 -5 M, amounting to 49.8 + 14.1% (n = 12) of the contraction induced by carbachol (10 -6 M). The halfmaximal inhibition of the carbachol-induced contraction was observed at 1.3 × 10 -6 M lemakalim. Repeated application of lemakalim (10 -5 M) on a single preparation revealed no change in relaxation on wash-
3. Results
3.1. Effect of lemakalim on carbachol-induced contraction The muscarinic receptor agonist, carbachol (10 -6 M), caused a tonic contractile response of the longitudinal smooth muscle. This effect was used as in previous studies (Allescher et al., 1991, 1992) in order to investigate actions of inhibitory mediators. As soon as the contractile response of carbachol reached a plateau,
Table 1 Effect of lemakalim 10 -5 M on carbachol- or Bay-K-8644-induced contraction % Inhibition of contraction
Contraction (mN)
CCH stimulation + T T X 10 -6 M Bay-K-8644 stimulation +TTX10-6M
Before lemakalim
After lemakalim
25.0 + 11.2 21.1 + 6.1 5.5 + 1.2 5 . 9 + 2.4
13.0 + 7.6 13.2 + 5.1 0.0 + 2.0 0.0+1.4
49.8 + 38.9 + 100.2 + 100.5+
14.1 16.3 3.1 3.6
Values are expressed as means + S.D. CCH = carbachol; T I ' X = tetrodotoxin.
Table 2 Effect of various K + channel blockers on the lemakalim-induced inhibiton Contraction (mN)
Control Apamin 10 -5 M Charybdotoxin 10 -7 M T E A 10 -2 M
% Inhibition of contraction
Before lemakalim
After lemakalim
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13.0 11.1 16.6 0.0
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7.6 4.5 6.0 2.0
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49.8 48.7 36.5 100.5
+ + + +
14.1 14.7 7.7 3.6
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ing with Krebs-Ringer solution in between (1st application: 52.8%, 2nd: 49.9%, 3rd: 50.2% of the carbacholinduced response, n = 12, n.s.) (Fig. 1A and B). The lemakalim-induced relaxation showed a small decrease after neural blockade with tetrodotoxin, which was not significant (before tetrodotoxin 10 -6 M: 49.8 + 14.1%, after: 38.9 + 16.3%, n = 9, P > 0.05) (Table 1).
3.2. Effect of K ÷ channel blockers on the lemakalim-induced inhibition Several K ÷ channel blockers were tested against lemakalim to gain insight into the possible channel type functional during muscle relaxation (Table 2). Apamin (10 -7 M, n = 9, n.s.) and charybdotoxin (10 -7 M, n = 9, n.s.), specific blockers of Ca2÷-dependent K ÷ channels had no influence on the effect of lemakalim (Fig. 2A and B). Further the non-specific K ÷ channel blocker, tetraethylammonium (TEA) (10 -4 to 10 -1 M), did not modify the inhibitory action of lemakalim (Fig. 3C). In contrast, glibenclamide (10 -7 to 10 -5 M), a specific blocker of A T P - d e p e n d e n t K + channels antagonized dose dependently the relaxant effect of lemakalim (IC50:9.1 × 10 -8 M, n = 11) with complete blockade of the inhibitory effect at a concentration of 10 -5 M (n = 11, P < 0.001) (Fig. 3A and B). Glibenclamide (10 -5 M), which added after lemakalim (10 -5 M) had produced inhibition of the initial carbachol-induced contraction, was able to restore the carbachol response to a major extent, i.e. up to 90% of the pretreatment response.
3.3. Effect of lemakalim on contraction induced by the Ca 2 + channel activator Bay-K-8644 The Ca 2÷ channel activator, Bay-K-8644 (10 -7 M), caused a tonic contraction of the longitudinal smooth muscle segments, which reached 2 0 - 2 5 % of the maxim u m carbachol-induced response. This contractile response was completely inhibited by lemakalim (10 -5 M) (100.2 + 3.1% of the Bay-K-8644 response, n = 9). Application of tetrodotoxin led to no modification of the lemakalim-induced response in these experiments (before tetrodotoxin 10 -6 M: 100.2-t-3.1%, after: 100.5 _ 3.6%, n = 9, P > 0.05) (Table 1). Analogous to the studies using carbachol as stimulus, glibenclamide (10 -5 M) selectively antagonized the lemakalim-induced inhibitory response (n = 6, P < 0.001).
3.4. Effect of glibenclamide on the inhibitory effect of putative non-adrenergic non-cholinergic mediators (ATP, nitric oxide, neurotensin) a/3-Methylene-ATP (10 -4 M) and the N O donor, N-morpholino-N-nitroso-amino-acetonitril (SIN-l) (10 -3 M), caused an instantaneous potent inhibition of
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akalim
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nium (TEA) 10-4 to 10-2 M, on the lemakalim-induced inhibition. The tissue was precontracted with carbachol (10 -6 M).
the carbachol-induced contraction. The inhibitory effects of both inhibitory agents were unaffected by glibenclamide, whereas a-methylene-ATP was blocked by tetraethylammonium (10 -1 M) and the Ca 2÷activated K ÷ channel blocker, apamin (10 -7 M). Neurotensin showed an instantaneous dose-dependent inhibition of the carbachol-induced contraction (IC50:2.8 × 10-10 M ) w i t h a maximum inhibitory effect of 52.4 ___7.9% (n = 12) of the carbachol-induced con-
H. Franck et aL / European Journal of Pharmacology 271 (1994) 379-386
383
hibitory responses of the guinea pig gall-bladder were blocked by glibenclamide and that glibenclamide-sensitive responses might be involved in responses evoked from capsaicin-sensitive afferent nerves. Therefore the effect of glibenclamide on capsaicin- and CGRP-induced effects was tested. Application of capsaicin (10 - 6 M) caused only a small, short-lasting relaxation of the carbachol-induced contraction, which reached 7.7 + 9.3% of the carbachol-induced contraction. This small inhibitory effect of capsaicin was not affected by preincubation with glibenclamide (10 -5 M). Similarly C G R P (3 x 10 -8 to 10 -7 M) caused a small inhibition (13.4 + 5.5%) of the carbachol-induced contraction (n = 8). Glibenclamide (10 -5 M) had no effect on the CGRPinduced inhibition (12.1 + 3.7%, n = 8, P > 0.05).
traction, which was in a range similar to that of the maximum lemakalim-induced inhibition. Glibenclamide (n = 9) did not affect this neurotensin-induced response. As shown previously the neurotensin response was blocked by the Ca2+-activated K ÷ channel blocker, apamin, whereas other K + channel blockers (tetraethylammonium, 4-aminopyridine, 9-aminoacridine and diazoxide) had no influence (Allescher et al., 1992).
3.5. Effect of glibenclamide on capsaicin- and CGRP-induced responses It has been suggested in a preliminary report that calcitonin gene-related peptide (CGRP)-induced in-
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4. Discussion
ATP-sensitive K ÷ (KAxe) channels are present in pancreatic beta cells where they play an important role in insulin secretion (Ashcroft et al., 1984; Cook and Hales, 1984; Dunne and Petersen, 1986, 1991; SchmidAntomarchi et al., 1987). Closure of KATP channels following glucose superfusion (and subsequent variation of the A T P / A D P ratio) leads via membrane depolarization to Ca 2+ entry and insulin release (Ashcroft et al., 1984; Cook and Hales, 1984; Dunne and Petersen, 1986, 1991). Well known antidiabetic sulfonylureas such as glibenclamide release insulin by blockade of KATP channels (Schmid-Antomarchi et al., 1987). ATP-sensitive K ÷ channels have been identified in cardiac muscle (Noma, 1983), skeletal muscle (Spruce et al., 1985), neurons (Ashford et al., 1988), and recently in vascular smooth muscle (Standen et al., 1989; Kajioka et al., 1991; Kovacs and Nelsen, 1991; Silberberg and Van Breemen, 1992). The KAXP channels in all these tissues are blocked by sulfonylureas like glibenclamide. The chemically diverse group of K ÷ channel openers including nicorandil, cromakalim (and its stereoisomer, lemakalim), pinacidil, diazoxide, minoxidil sulphate (the active metabolite of minoxidil) and RP-49356 (Cook et al., 1990; Cook and Hof, 1988; Fan et al., 1992) are potent vasodilatators and relaxant agents in smooth muscle. Recently the K ÷ openers, cromakalim (and its more active isomer, lemakalim), diazoxide and RP 49356, have been shown to open KATP channels in vascular smooth muscle, cardiac and skeletal muscle (Quast and Cook, 1989). There are only few functional data on the effect of K ÷ channel openers in intestinal smooth muscle, especially the small intestine. Den Hertog et al. (1989) showed that, in guinea-pig taenia caeci, cromakalim induced a dose-dependent hyperpolarization which was inhibited by glibenclamide. Sanders and coworkers demonstrated in colonic smooth muscle a cromakalimand lemakalim-induced K ÷ outward current (Post et al., 1991; Carl et al., 1992), responsible for the noticed hyperpolarization. Sanders and coworkers provided evidence that this effect could be due to activation of a glibenclamide-sensitive KATP channel (Du et al., 1994). In the present study the effect of the K ÷ channel activator, lemakalim, on longitudinal smooth muscle of rat ileum was investigated. This system has been utilized previously in our laboratory to characterize the cellular mechanism of action of neurotensin by our laboratory (Allescher et al., 1992). Application of lemakalim on precontracted longitudinal smooth muscle of rat ileum caused a marked dose-dependent relaxation, which showed no decline with repeated application after wash-out. Neural blockade by tetrodotoxin (10 -6 M) did not significantly
modify the lemakalim-induced relaxation, indicating that this effect is probably caused by a direct action on the smooth muscle. The specific ATP-sensitive K ÷ channel blocker, glibenclamide (Schmid-Antomarchi et al., 1987; Liu and Downey, 1992; Grover et al., 1993; Thornton et al., 1993), antagonized dose dependently the relaxant effect of lemakalim whereas other K ÷ channel blockers such as apamin (10 -7 M) or charybdotoxin (10 -7 M), specific blockers of Ca2÷-dependent K ÷ channels, or the non-specific K ÷ channel blocker, tetraethylammonium (TEA) (10 -4 to 10 -1 M), had no influence on the lemakalim-induced relaxation. The results indicate that lemakalim activates a K + channel which is blocked by glibenclamide, a specific KATP channel blocker. This feature is characteristic of KATP channels in other tissues. Therefore the data provide functional evidence for the presence of KATP channels in the smooth muscle of rat ileum, which modulate the contractile activity. The concentration of glibenclamide which reduced the inhibitory effect of lemakalim by 50% in the present study was 3.4 x 10 -6 M. Even though a comparison has to be made with great care this concentration is in a range similar to that of the IC50 values reported in electrophysiological studies on vascular smooth muscle and the heart (0.11.5 x 10 -6 M) (Escande, 1989) but is slightly higher than the IC50 reported for KATP channels expressed in Xenopus oocytes (IC50:2.9 × 10 -7 M) (Sakuta et al., 1992; Sakuta and Okamoto, 1993) and about 100-fold higher than reported for pancreatic beta cells (Dunne and Petersen, 1991). The physiological role of these KATP channels in intestinal smooth muscle is unclear. The functional and pharmacological data presented in this study together with the electrophysiological data reported from colonic smooth muscle (Carl et al., 1992) provide strong evidence for the existence of these channels. From the present experiments there is no evidence that these channels are involved in the inhibitory effect of non-adrenergic non-cholinergic (NANC) neurotransmitters such as the NO donor, SIN-l, or the ATP analogue, aft-methylene ATP. In a recently presented preliminary electrophysiological study on guinea pig gallbladder smooth muscle cells it was speculated that KATP channels might be involved in the inhibitory action of calcitonin gene-related peptide (CGRP) and subsequently in the action of neurotransmitters released from afferent nerves (Zhang et al., 1993). In the present study capsaicin and CGRP elicited only a small inhibitory effect, which was not influenced by glibenclamide. Hence there is no functional evidence for a physiological role of KATP channels in capsaicin or CGRP-induced responses in the small intestine. ATP-sensitive K+-channels have been shown to be involved in ischemia responses in cardiac tissue (Benndorf et al., 1992; Escande, 1989) and to maintain
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vascular tone in coronary vessels under certain conditions (Samaha et al., 1992). These channels have also been associated with vascular function in the intestine (Silberberg and Van Breemen, 1992) and haemodynamic changes and a reduction of portal pressure have been reported for an experimental model of portal hypertension in the rat (Moreau et al., 1992). Whether KATP channels in intestinal smooth muscle could also be modulated under ischemic conditions cannot be answered from the present study; however, it would be in line with changes observed in other organ systems. Additionally these channels could offer another pharmacological approach for direct spasmolytic agents in the gastrointestinal tract.
Acknowledgements The authors acknowledge the courtesy of Prof. Dr. BliJmel and his collaborators, Dept. Exp. Surgery, Technical University of Munich. The study was supported by Deutsche Forschungsgemeinschaft A1 245/2-2 and DFG A1-245/8-1.
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