Gen. Pharmac. Vol. 23, No. 3, pp. 347-353, 1992 Printed in Great Britain. All rights reserved
0306-3623/92 $5.00 + 0.00 Copyright © 1992 Pergamon Press Ltd
EFFECTS OF LP-805, A NOVEL VASORELAXANT AGENT, A POTASSIUM CHANNEL OPENER, ON RAT THORACIC AORTA KEN-IcHI KISHll,t, TOSHIHIRO MORIMOTO,I NORIKO NAKAJIMA, ~KYOKO YAMAZAKi, l M1CHIHIKO TSUJITANI I a n d ISSEI TAKAYANAGI2 tPOLA Pharmaceutical R&D Laboratory, Totsuka-ku, Yokohama 244 and 2Department of Chemical Pharmacology, Toho University School of Pharmaceutical Sciences, Funabashi, Chiba 274, Japan (Received 3 October 1991) A ~ t r a c t - - l . In rat thoracic aorta, LP-805 (0.1-10#M) caused the marked reduction of NE-induced maximum response and relaxed the low K ÷ ( < 35.9 mM)-induced contraction, in a concentration-dependent manner, but failed to relax the high K ÷ (65.9 mM)-induced contraction. 2. Glibenclamide (0.3-1 #M) caused a parallel shift of concentration-response curve produced by LP-805 for 25.9 mM K+-induced contraction and prevented the LP-805-induced reduction in maximum response evoked by NE in a concentration-dependent manner. 3. Glibenclamide (10/~M) prevented the LP-805 (10/tM)-induced decrease in cytosolic Ca 2+ levels which was increased by 1 #M NE or 25.9 mM K ÷. 4. LP-805 (10 # M) increased basal 86Rb etttux, which was completely inhibited by 10/1 M glibenclamide. 5. These results suggest that LP-805 causes a vasorelaxation as a consequence of the decrease in cytosolic Ca 2+ levels due to the increase in K ÷ efflux via opening ATP-dependent K ÷ channels.
INTRODUCTION LP-805, 8-tert-butyl-6,7-dihydropyrolo[3,2-e]methylpyrazol[l,5-a]pyrimidine-3-carbonitril, is a novel vasodilating agent with b o t h antihypertensive and antianginal actions (Kishii et al., 1991; Y a n a k a et al., 1991). A vasodilating action o f LP-805 m a y be due to inhibiting the mobilization of external Ca 2+ and the Ca 2+ release from store sites (Kishii et al., 1991). However, the inhibitory action of LP-805 o n Ca 2+ influx was n o t clarified. It is well k n o w n that Ca 2+ entry blockers block voltage-dependent Ca 2+ influx a n d p o t e n t vasodilating properties ( G o d f r a i n d et al., 1986). It seemed t h a t LP-805 possessed a quite different vasorelaxant profile from Ca 2+ channel blockers (Kishii et aL, 1991). O u r preliminary observations suggest t h a t LP-805 decreases action potential duration ( A P D ) of canine ventricle, in a c o n c e n t r a t i o n d e p e n d e n t manner. The decrease in cardiac A P D is consistent with a n increase in potassium permeability (Findlay et al., 1989) or a decrease in calcium current ( M c D o n a l d , 1982). Therefore, it is expected that LP-805 would have an action of potassium channel opener. Recently, the pharmacological actions of p o t a s s i u m channel openers such as cromakalim, pinacidil a n d nicorandil have been studied, whose m e c h a n i s m of action is t h o u g h t to involve hyperpolarizing the resting m e m b r a n e as well as the memb r a n e depolarized by agonists or K + ( < 20 m M ) a n d this p r o d u c e d vasodilation (Palmer et al., 1988). Glibenclamide, a potent blocker of A T P - d e p e n d e n t K ÷ channels in pancreatic cells ( S c h m i d - A n t o m a r c h i *To whom all correspondence should be addressed.
et al., 1987), has been s h o w n to antagonize the vascular s m o o t h muscle relaxation caused by K + channel opener ( M a s u z a w a et al., 1990a,b; Q u a s t a n d Cook, 1988). The present experiments were intended to clarify the vasodilating action of LP-805 by studying changes in mechanical properties a n d simultaneously m o n i t o r i n g the muscle tension a n d cytosolic Ca z+ level in isolated rat thoracic aorta. Further, the effects of LP-805 on 86Rb efflux from rat a o r t a were also investigated.
MATERIALS AND METHODS
Measurement o f mechanical response Thoracic aorta was isolated from male Wistar rats (200-250 g), then cut into ring segments (4 mm length) and the endothelium was removed by gently rubbing with a stainless wire. The ring segments were suspended in organ baths filled with a normal physiological salt solution (PSS) which contained ll8.0mM NaC1, 4.TmM KCI, 2.5mM CaCl2, 1.2mM MgSO4, 1.2mM KH2PO4, 25.0mM NaHCO 3 and I 1.0 mM glucose. The solution was saturated with 95% O z and 5% CO z mixture at 37°C and pH7.4. After equilibration in normal PSS for 60 min, the response to agonist was recorded isometrically under an initial tension of 2.0 g. The complete removal of endothelial cells was confirmed by application of 10ktM acetylcholine (ACh), since ACh dose not relax the aortic preparations after removal of endothelial cells. The ring preparations were challenged with each agonist three times, and tension by the last challenge was used as a reference value to estimate the response magnitudes of the test compound. During K+-in duced contractions, guanethidine (3 #M) and tetrodotoxin (0.1/aM) were added into the bath solution to prevent the release of neurotransmitters. 347
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KEN-IcFII KISHII et al.
Measurement o f cytosolic Ca e+ and tension in fura-2-loaded preparation The thoracic aorta was cut into 2 x 10 mm helical strips and the endothelium was removed by gently rubbing with a small swab. The aortic strips were equilibrated for 60-90 min in a normal PSS containing 0.01 mM ethylendiamine tetraacetic acid (EDTA), the response to an agonist was recorded isometrically under an initial tension of 0.5 g. High K ÷ solution was made by substituting NaCI with equimolar KC1. These solutions were saturated with 95% O 2 and 5% CO 2 mixture at 37°C and pH 7.4. The strips were incubated with 5/~ M fura 2/AM in normal PSS containing 0.01 mM EDTA for 3 hr at room temp in the presence of 0•2% Cremophor EL, then rinsed with the same solution for 15min. Thereafter, experiments were performed with a double wavelength excitation fluorimeter (CAF-100, Japan spectroscopic). The mechanical activity was monitored isometrically, and simultaneously the ratio of 500 nm fluoresence emitted by 340 nm excitation (V~40) to that by 380 nm (F380) excitation was calculated automatically from successive illumination periods (48 Hz) and referred to as R3+0/380. In the muscle strips that were successfully loaded with fura-2, the increase in cytosolic Ca 2+ level resulted in an •increase in F340, a decrease in F3s 0 and an increase, in R34o/38 . . 2+ o The R340/380 w a s used to monitor the relative cytosohc Ca level (Sato et al., 1988; Karaki et al., 1988). 86Rb efflux In this experiment, 86Rb was used as a marker for K ÷ (Bolton and Clapp, 1984; Imaizumi and Watanabe, 1981; Weir and Weston, 1986). Rat aorta was cut into rings (10 mm length) and each ring was opened into a flat sheet by cutting it longitudinally. Endothelium-free segments were A 100
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Fig. 2. Dose--response curves for the relaxant responses to LP-805 on various concentrations of K+-induced contractions in rat thoracic aorta without intact endothelium. ( 0 ) 65.9mM K ÷, (O) 35.9mM K ÷, (A) 30.9mM K ÷, (A) 25•9 mM K ÷. Values are means ___SE for 4 preparations.
attached to the micro wire clamps, and after a 30 min equilibration period in PSS at 37°C bubbled with 95% 02 and 5% CO2, they were loaded with 86RbC1 5 #Ci/ml for 90 min. 86Rb was then allowed to efflux from the tissues into 2 ml aliquots of PSS using 2 min collection periods. After 7 such periods, preparations were exposed to PSS alone (control) or to PSS containing LP-805 for the next 5 collection periods. Glibenclamide was applied after 5 collection periods. For the remaining periods, the tubes contained PSS alone. At the end of the efflux period, the radioactivity in the tubes and that remaining in tissues were measured• The effiux data were expressed in terms of the rate coefficient (fractional loss of 86Rb from the tissue standardized for I min period) expressed as a percentage. Drugs and chemicals LP-805 was synthesized in POLA Pharmaceutical R&D Laboratory. The following drugs were used: /-norepinephrine bitartrate (Wako Pure Chemicals, Tokyo, Japan); diltiazem hydrochloride, glibenclamide, guanethidine (Sigma Chemicals, St Louis, Mo.); fura 2/AM (Dojindo Laboratories, Kumamoto, Japan); Cremophor EL (Nakalai Tesque, Kyoto, Japan); tetrodotoxin (Seikagaku Kogyo, Tokyo, Japan), 86RbCI (NEN Research Products). Other chemicals used were analytical grade•
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Statistics Numerical results are presented as the mean + SE with the number of the observations in parentheses. Values were considered to be significantly different when P < 0•05 by Duncan's multiple range test and Student's t-test•
RESULTS
Effects o f L P - 8 0 5 on mechanical response
0 ¢ +Sin's" N E I -IogM )
Fig. 1. Effects of various concentrations of LP-805 and diltiazem on (A) 65•9 mM K + and (B) NE-induced contractions in rat thoracic aorta without intact endothelium. (A) O, LP-805; @, diltiazem. (B) ©, control; A, 1/~M diltiazem; &, 10#M diltiazem; I-1, 1/~M LP-805; I , 10/tM LP-805. Values are means + SE for 5qi preparations.
The c o n c e n t r a t i o n - r e s p o n s e curve for the vasorelaxant effects o f LP-805 and diltiazem on isolated rat thoracic aorta are shown in Fig. 1. The rings were precontracted with high K ÷ (65.9 m M ) and relaxed in a c o n c e n t r a t i o n ~ l e p e n d e n t m a n n e r when diltiazem (0.01-1 # M ) was applied, but LP-805 (0.1-10 /~M) failed to relax those. Whereas, LP-805 ( I - 1 0 / ~ M ) caused a marked reduction in NE-induced m a x i m u m response in a c o n c e n t r a t i o n - d e p e n d e n t manner, but diltiazem (1-10/~M) slightly reduced that. The vasodilating action o f LP-805 was different from that o f Ca 2+ entry blocker•
LP-805, a potassium channel opener Figure 2 shows the effects of LP-805 on various concentrations of K+-induced tonic contractions. LP-805 relaxed the contraction induced by < 35.9 mM K ÷, in a concentration-dependent manner, but not the contraction evoked by higher concentration of K ÷. Glibenclamide (0.3-1#M) caused a parallel shift of the concentration-response curve produced by LP-805 for 25.9 mM K+-induced tonic contraction, in a concentration-dependent manner [Fig. 3(A)]. A schild plot of these results gave a straight line with a slope of unity, suggesting a competitive antagonism. The pA: value was 7.10 + 0.05 (n = 6). LP-805 (10 #M) caused a marked reduction in NE-induced maximum response. This LP-805-induced reduction was prevented by pretreatment of glibenclamide (0.3-3 pM), in a concentration-dependent manner [Fig. 3(B)],
Effects o f LP-805 and glibenclarnide on changes in cytosolic Ca 2+ level and contractions by NE and high K + Figure 4(A) shows the effect of LP-805 on NE (1 pM)-induced contraction and cytosolic Ca 2÷ level in normal PSS. LP-805 (10 pM) markedly inhibited the sustained increase in cytosolic Ca 2+ level, and
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Fig. 4. Effects of LP-805 and glibenclamide on changes in cytosolic Ca2+ levels and contractions induced by NE. (A) Effect of LP-805 (10 #M) on 1 itM NE-induced contraction and increase in cytosolic Ca2+ level. (B) Effect of glibenclamide (10/IM) on the inhibition produced by LP805 (10 pM) for 1/aM NE-induced contraction and increase in cytosolic Ca2+ level.
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NE( -IogMI Fig. 3. (A) Effects of glibenclamide on concentration-response curves produced by LP-805 for 25.9 mM K ÷-induced contractions in rat thoracic aorta. (C)) Without glibenclamide, (A) 0,3pM glibenclamide, (A) l llM glibenclamide. Values are means + SE for 10-11 preparations. (B) Effects of glibenclamideon LP-805 (I0 # M)-induced reductions in maximum responses evoked by NE in rat thoracic aorta. (O) Control, (©) I0 pM LP-805, (A) 0.3pM glibenclamide plus 10#M LP-805, (111) l pM glibenclamideplus 10 pM LP-805, (E]) 3 pM glibenclamide plus 10#M LP-805. Glibenclamide applied 5min before application of LP-805. Values are means _ SE for 6 preparations.
both the phasic and sustained contraction evoked by NE, but not those induced by high K + (65.9 mM). LP-805-induced these inhibitions were prevented by pretreatment with 10/~M glibenclamide [Fig. 4(B)]. Figure 5 shows the effect of LP-805 and glibenclamide on K+-induced contraction and cytosolic Ca 2÷ level in normal PSS. LP-805 (10 pM) had no effect on 65.9 mM K+-induced sustained increase in cytosolic Ca 2+ level and contraction [Fig. 5(A)]. While LP-805 (10 pM) caused the marked reduction in 25.9mM K+-induced maximum response, but slightly inhibited the sustained increase in cytosolic Ca 2÷ level [Fig. 5(B)]. LP-805-induced these changes also prevented by pretreatment with 10pM glibenclamide [Fig. 5(C)].
Effects o f LP-805 on 86Rb efflux Effects of LP-805 on the 86Rb effiux in rat aorta were determined. In resting strips, LP-805 (10pM) increased the 86Rb efflux rate coefficient from the basal value of 2.34 +_ 0.36% per min (n = 5, measured between the 10th and 14th min of the efflux period) to the peakvalue of 4.14 +_0.36% per min (n = 5) [Fig. 6(B)]. While, in the absence of LP-805 [control, Fig. 6(A)], there was no significant increase in the s6Rb efflux rate coefficient between the 10th and 32nd min of the efflux period [Fig. 6(A)]. Glibenclamide (10#M) completely inhibited the LP-805-induced increase in the S6Rb effiux [Fig. 6(C)].
350
KEN-Icm K1smI et al.
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Fig. 5. Effects of LP-805 and glibenclamide on changes in cytosolic Ca2÷ levels and contractions induced by high K +. Effect of (A) LP-805 (10#M) on 65.9mM K ÷ and (B) 25.9 mM KS-induced contraction and increase in cytosolic Ca2÷ level. (C) Effect of glibenclamide (10/zM) on the inhibition produced by LP-805 (10 pM) for 25.9 mM KS-induced contraction and increase in cytosolic Ca2+ level. DISCUSSION
The results of these experiments provided a vasorelaxant profile as a K ÷ channel opener with regard to the effects of LP-805 on 86Rb efflux and contractile responses in vascular smooth muscle. LP-805 produces the arterial relaxation via the increase in the K + efflux, possibly by opening ATP-dependent K + channels. It has been reported that the vascular relaxant effects of K ÷ channel opener such as pinacidil are highly correlated with both reductions in cardiac APD and contractility (Steinberg et al., 1988). Further, a similar correlation between APD reduction and antihypertensive activity was reported in spontaneously hypertensive rats (Smallwood and Steinberg, 1988). The results taken together strongly suggest that the vascular relaxant and cardiac electrophysiological effects of this group of compounds are mediated by a common mechanism. LP-805 (1-10pM) markedly decreased APD in ventricular muscle without much effect on other measured parameters. The maximum decrease in APD10, APD50 and APD90 were 55.2_ 12.1, 75.4_+ 7.7 and 85.1 _+ 5.4%, respectively (data not shown). The decrease of cardiac APD is consistent with an increase
in potassium permeability in cardiac tissue (6). APD is also maintained at least partly by influx of calcium ions in ventricular muscle (7). Indeed, calcium agonist substances have been shown to cause large increase in APD of ventricular muscle (Thomas et al., 1985). LP-805 had no effect on voltage-dependent 45Ca2+ influx by 65.9 mM K ÷ in rat thoracic aorta (unpublished data). Therefore, the basis for the vascular relaxant effects of LP-805 may involve an increase in membrane permeability to potassium ions resulting in hyperpolarization. In rat thoracic aorta, LP-805 caused the marked reduction of NE-induced maximum response in a concentration-dependent manner, and diltiazem slightly reduced that. On the other hand, diltiazem completely relaxed the aortic rings precontracted with high K ÷ (65.9 mM) in a concentration-dependent manner, but LP-805 slightly relaxed that with its high concentration. Therefore, it seemed that LP-805 possessed a quite different vasorelaxant profile from Ca 2÷ entry blockers (Fig. 1). The aortic rings were precontracted with low K ÷ ( < 35.9 mM) and relaxed in a concentration-dependent manner when LP-805 was applied (Fig. 2). In vascular smooth muscle cells the resting membrane potential is less negative than the K ÷ equilibrium potential (Furukawa et al., 1981). In the present study, when arterial rings were contracted with low concentration of K + ( < 35.9 mM), the equilibrium potential for K s would be equivalent to or more negative than the normal resting membrane potential. Under such conditions, it is unlikely that extracellular Ca 2÷ can enter through voltage-dependent Ca 2÷ channels, Whereas, LP-805 failed to relax the high K ÷ (65.9 mM)-induced contraction, presumably because the membrane potential remains at a level of depolarization which required to open voltage-dependent Ca 2+ channels. Under these conditions, the LP-805-induced increase in the K ÷ efflux would not hyperpolarize arterial smooth muscle sufficiently to inhibit transmembrane Ca :+ influx. When the membrane is hyperpolarized towards the K ÷ equilibrium potential by K + channel openers, Ca 2+ influx via voltage-dependent Ca 2÷ channels is thought to decrease (Cook, 1988; Weston, 1989). Therefore, LP-805 cannot block directly the Ca 2+ entry through voltage-dependent Ca 2÷ channels. These experiments allow Ca 2+ entry blocker to be distinguished from vasodilators with K s channel opening activity as already demonstrated in other investigations (Bray et al., 1987; Hamilton et al., 1986; Hamilton and Weston, 1989; Weston et al., 1988). Glibenclamide is a potent blocker of ATP-dependent K + channels. These channels are activated when intracellular ATP levels are low. A number of pharmacological studies have demonstrated a competitive interaction of glibenclamide with the response to K + channel openers in vascular tissues (Buckingham et al., 1989; Cavero et al., 1989; Winquist et al., 1989). These channels have also been identified in cardiac cells (Noma, 1983). In the present study, glibenclamide was also found to antagonize the relaxant responses to LP-805. As shown in Fig. 3, glibenclamide prevented the LP-805-produced reduction in NE-induced maximum response. Moreover, glibenclamide caused a parallel shift of the
LP-805, a potassium channel opener
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Fig. 6. Effects of LP-805 and glibenclamide on the loss of s6Rb from rat thoracic aorta. 86Rb efflux rate coefficient expressed as a % per min. (A) Control (physiological salt solution alone). (B) Tissues were exposed to LP-805 (10/tM) between the 14th and 24th min of the effiux period. (C) Tissues were exposed to LP-805 (I0 #M) between the 14th and 24th min of the efflux period, and glibenclamide (10 #M) was applied between 10th min and 28th min of the efllux period. Values are means + SE for 4-7 experiments. *P < 0.05, **P < 0.01 vs basal value.
concentration-response curve produced by LP-805 for 25.9mM K+-induced contraction in a concentration-dependent manner (Fig. 2). The Schild plot for glibenclamide antagonism of the response to LP-805 showed a typical competitive antagonism with the slope of unity and the pA 2 value of 7.10 _ 0.05 (n = 6). These results suggest that LP-805 produces the arterial relaxation through the increase in K ÷ permeability via the opening of ATP-dependent K ÷ channels. Smooth muscle contraction is initiated by the increase in cytosolic free Ca 2÷ levels through stimulation of Ca z÷ release from intracellular stores as well as by promoting Ca 2+ influx through Ca 2+ channels (Rasmussen and Barrett, 1984; Somlyo and Somlyo, 1986). LP-805 inhibits Ca 2+ influx which is mediated by a receptor stimulated pathway, and moreover Ca 2÷ release from store sites, resulting the decrease in cytosolic Ca 2+ levels (Kishii et al., 1991). Then we studied the effects of glibenclamide on LP-805-induced reduction in cytosolic Ca 2+ levels and muscle tension. As shown in Figs 4(A) and 5(A), LP-805 (10/~M) markedly inhibited the sustained increase in cytosolic Ca 2÷ level, and both the phasic and sustained contraction evoked by NE, but not those induced by 65.9mM K +. While LP-805 (10#M) caused the reduction in 25.9 mM K+-induced maximum response, but slightly inhibited the increase in cytosolic Ca 2÷ level [Fig. 5(B)]. LP-805-induced changes were prevented by pretreatment with 10 #M
glibenclamide [Figs 4(B), 5(C)]. Similar results have been reported with cromakalim (Yanagisawa et al., 1990). From these results, the inhibitory action of LP-805 on NE- or 25.9 mM K÷-induced contraction may be accounted for a reduction of cytosolic Ca 2÷ level due to the closure of voltage-dependent Ca 2÷ channels by hyperpolarization through the opening of ATP-dependent K ÷ channels. S6Rb was used as a marker for K ÷ permeability in smooth muscle (Bolton and Clapp, 1984; Hamilton et al., 1986; Imaizumi and Watanabe, 1981). In resting strips, LP-805 (10 #M) increased the basal 86Rb efflux. The increase in 86Rb efflux induced by LP-805 was completely inhibited by 10pM glibenclamide (Fig. 6). Therefore, LP-805 possesses the characteristics of ATP-dependent K ÷ channel opener resulting the increase in K ÷ permeability. In conclusion, present study shows that LP-805 possesses a mechanism of action as a K ÷ channel opener, and that vasorelaxation produced by LP-805, in part, is due to the decrease in cytosolic Ca 2÷ levels via the closure of voltage dependent Ca 2÷ channels by hyperpolarization through the increase in K ÷ permeability, possibly by opening ATP-dependent K ÷ channels. A vasodilating action of LP-805 is due to inhibiting the mobilization of external Ca 2+ and the Ca 2÷ release from store sites (Kishii et al., 1991). Therefore, inhibitory action of LP-805 on Ca 2+ influx accounts for the increase in K ÷ permeability through ATP-dependent K ÷ channels.
352
KEN-ICHI KISHn et al. SUMMARY
The characteristics o f LP-805, a novel vasodilating agent, were investigated in isolated rat thoracic aorta. LP-805 (0. I - 1 0 # M) caused the m a r k e d reduction of N E - i n d u c e d m a x i m u m response a n d relaxed the low K + ( < 35.9 m M ) - i n d u c e d contraction, in a concent r a t i o n - d e p e n d e n t manner, but failed to relax the high K + (65.9 m M ) - i n d u c e d contraction. Therefore, it is t h o u g h t t h a t LP-805 possesses a quite different vasorelaxant profile from Ca 2+ entry blockers, such as diltiazem. Glibenclamide (0.3-1/~M) caused a parallel shift of the c o n c e n t r a t i o n - r e s p o n s e curve produced by LP-805 for 25.9 m M K + - i n d u c e d contraction a n d prevented the LP-805-induced reduction in m a x i m u m response evoked by NE, in a concent r a t i o n - d e p e n d e n t m a n n e r . Further, glibenclamide (10/~M) prevented the LP-805 (10/~M)-induced decrease in cytosolic Ca 2+ levels which was increased by 1 / I M N E or 2 5 . 9 m M K +. LP-805 (10/~M) also increased basal 86Rb efflux, which was completely inhibited by 10/~M glibenclamide. These results suggest that the vasorelaxant action induced by LP805 involves in opening A T P - d e p e n d e n t K ÷ channels, which is due to the reduction in cytosolic Ca 2+ levels via the closure of voltage-dependent Ca 2÷ channels by hyperpolarization t h r o u g h K ÷ permeability.
REFERENCES Bolton T. B. and Clapp L. H. (1984) The diverse effects of norepinephrine and other stimulants on 86Rb and 42K efftux in rabbit and guinea-pig arterial muscle. J. Physiol. 355, 43~53. Bray K. M., Newgreen D. T., Small R. C., Southerton J. S., Taylor S. G., Weir S. W. and Weston A. H. (1987) Evidence that the mechanism of the inhibitory action of pinacidil in rat and guinea pig smooth muscle differs from that of glyceryltrinitrate. Br. J. Pharmac. 91, 421-429. Buckingham R. E., Hamilton T. C., Howlett D. R., Mootoo S. and Wilson C. (1989) Inhibition by glibenclamide of the vasorelaxant action of cromakalim in the rat. Br. J. Pharmac. 97, 57~4. Cavero I., Mondot S. and Mestre M. (1989) Vasorelaxant effects of cromakalim in rats are mediated by glibenclamide-sensitive potassium channels. J. Pharmac. exp. Ther. 248, 1261-1268. Cook N. S. (1988) The pharmacology of potassium channels and their therapeutic potential. Trends Pharmac. Sci. 9, 21-28. Findlay I., Deroubaix E., Guiraudou P. and Coraboeuf E. (1989) Effects of activation of ATP-sensitive K channels in mammalian ventricular myocytes. Am. J. Physiol. 26, HI551-H1559. Frukawa K., Itoh T., Kajiwara M., Kitamura K., Suzuki H., Ito Y. and Kuriyama H. (1981) Vasodilating action of 2-nicotinamidoethyl nitrate on porcine and guinea-pig coronary arteries. J. Pharmac. exp. Ther. 218, 248-259. Godfraind T., Miller R. and Wibo M. (1986) Calcium antagonism and calcium entry blockade. Pharmac. Rev. 38, 324-416. Hamilton T. C. and Weston A. H. (1989) Cromakalim, nicorandil and pinacidil: novel drugs which open potassium channels in smooth muscle. Gen. Pharmac. 20, 1-9. Hamilton T. C., Weir S. W. and Weston T. H. (1986) Comparison of the effects of BRL34915 and verapamil on
electrical and mechanical activity in rat portal vein. Br. J. Pharmac. 88, 103-111.
Imaizumi Y. and Watanabe M. (1981) The effect of tetraethylammonium chloride on potassium permeability in the smooth muscle cell membrane of canine trachea. J. Physiol. 316, 33-46. Karaki H., Sato K., Ozaki H. and Murakami K. (1988) Effects of sodium nitroprusside on cytosolic calcium level in vascular smooth muscle. Eur. J. Pharmac. 156, 259-266. Kishii K., Inazu M., Morimoto T., Tsujitani M. and Takayanagi I. (1991) Mode of actions of LP-805, a new vasodilating agent, on rat thoracic aorta. Jap. J. Pharmac. Suppl. 55, 261. Masuzawa K., Matsuda T. and Asano M. (1990a) Evidence that pinacidil may promote the opening of ATP-sensitive K ÷ channels yet inhibit the opening of Ca2+-activated K ÷ channels in K+-contracted canine mesenteric artery. Br. J. Pharmac. 100, 143-149. Masuzawa K., Asano M., Matsuda T., Imaizumi Y. and Watanabe M. (1990b) Comparison of the effects of cromakalim and pinacidil on mechanical activity and on 86Rb efflux in dog coronary arteries. J. Pharmac. exp. Ther. 253, 586-593. McDonald T. F. (1982) The slow inward calcium current in the heart. A. Rev. Physiol. 44, 425-434. Noma A. (1983) ATP-regulated K ÷ channels in cardiac muscle. Nature 305, 147-148. Palmer P. M., Ashton D. S. and Moncada S. (1988) Vascular endothelial cells synthesize nitric oxide from L-arginine. Nature 333, 664q566. Quast U. and Cook N. S. (1988) Potent inhibitors of the effect of the K ÷ channel opener BRL 34915 in vascular smooth muscle. Br. J. Pharmac. Suppl. 93, 204. Rasmussen H. and Barrett P. Q. (1984) Calcium messenger system: an integrated view. Physiol. Rev. 64, 938-984. Sato K., Ozaki H. and Karaki H. (1988) Changes in cytosolic calcium level in vascular smooth muscle strip measured simultaneously with contraction using fluorescent calcium indicator fura 2. d. Pharmac. exp. Ther. 246, 294-300. Schmid-Antomarchi H., De Weille J., Fosset M. and Lazdunski M. (1987) The receptor for antidiabetic sulfonylureas controls the activity of the ATP-modulated K + channel in insulin-secreting cells. J. biol. Chem. 262, 15,840015,844. Smallwood J. K. and Steinberg M. (1988) Cardiac electrophysiological effects of pinacidil and related pyridylcyanoguanides: relationship to antihypertensive activity. J. cardiovasc. Pharmac. 12, 102-109. Somlyo A. P. and Somlyo A. V. (1986) In The Heart and Cardiovascular System (Edited by Fozzard H. A.), pp. 845-864. Raven Press, New York. Steinberg M. I., Ertel P., Smallwood J. K., Wyss V. and Zimmerman K. (1988) The relation between vascular relaxant and cardiac electrophysiological effects of pinacidil. J. cardiovasc. Pharmac. Suppl. 12, $30-$40. Thomas G., Chung M. and Cohen C. J. (1985) A dihydropyridine (Bay K8644) that enhances calcium currents in guinea pig calf myocaldial cells. Circulation Res. 56, 87-96. Weir S. W. and Weston A. H. (1986) The effects of BRL 34915 and pinacidil on electrical and mechanical activity and on 86Rb efflux in rat blood vessels. Br. J. Pharmac. 88, 121 128. Weston A. H. (1989) Smooth muscle K ÷ channel openers; their pharmacology and clinical potential. Pflfigers Arch. ges. Physiol. Suppl. 44, $99-S105. Weston A. H., Southerton J. S., Bray K. M., Newgreen D. T. and Taylor S. G. (1988) The mode of action of pinacidil and its analogs PI060 and P1368: results of studies in rat brood vessels. J. cardiovasc. Pharmac. Suppl. 12, S10-S16.
LP-805, a potassium channel opener Winquist R. J., Heaney L. A., Wallace A. A., Baskin E. P., Stein R. B., Garcia M. L. and Kaczorowski G. J. (1989) Glybride blocks and relaxation response to BRL34915 (cromakalim), minoxidil sulfate and diazoxide in vascular smooth muscle. J. Pharmac. exp. Ther. 248, 149-156. Yanagisawa T., Teshigawara T. and Taira N. (1990) Cytoplasmic calcium and the relaxation of canine coronary
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arterial smooth muscle produced by cromakalim, pinacidil and nicorandil. Br. J. Pharmac. 101, 157-165. Yanaka M., Shikata Y., Sugiyama Y., Shiba T., Nishiki K., Morimoto T., Momota Y., Tsujitani M. and Furukawa Y. (1991) Antihypertensive and antianginal actions of LP-805, a new vasodilating agent. Jpn. J. Pharmac. Suppl. $5, 330.
0306-3623/92 $5.00 + 0.00 Copyright © 1992 Pergamon Press Ltd
EFFECTS OF LP-805, A NOVEL VASORELAXANT AGENT, A POTASSIUM CHANNEL OPENER, ON RAT THORACIC AORTA KEN-IcHI KISHll,t, TOSHIHIRO MORIMOTO,I NORIKO NAKAJIMA, ~KYOKO YAMAZAKi, l M1CHIHIKO TSUJITANI I a n d ISSEI TAKAYANAGI2 tPOLA Pharmaceutical R&D Laboratory, Totsuka-ku, Yokohama 244 and 2Department of Chemical Pharmacology, Toho University School of Pharmaceutical Sciences, Funabashi, Chiba 274, Japan (Received 3 October 1991) A ~ t r a c t - - l . In rat thoracic aorta, LP-805 (0.1-10#M) caused the marked reduction of NE-induced maximum response and relaxed the low K ÷ ( < 35.9 mM)-induced contraction, in a concentration-dependent manner, but failed to relax the high K ÷ (65.9 mM)-induced contraction. 2. Glibenclamide (0.3-1 #M) caused a parallel shift of concentration-response curve produced by LP-805 for 25.9 mM K+-induced contraction and prevented the LP-805-induced reduction in maximum response evoked by NE in a concentration-dependent manner. 3. Glibenclamide (10/~M) prevented the LP-805 (10/tM)-induced decrease in cytosolic Ca 2+ levels which was increased by 1 #M NE or 25.9 mM K ÷. 4. LP-805 (10 # M) increased basal 86Rb etttux, which was completely inhibited by 10/1 M glibenclamide. 5. These results suggest that LP-805 causes a vasorelaxation as a consequence of the decrease in cytosolic Ca 2+ levels due to the increase in K ÷ efflux via opening ATP-dependent K ÷ channels.
INTRODUCTION LP-805, 8-tert-butyl-6,7-dihydropyrolo[3,2-e]methylpyrazol[l,5-a]pyrimidine-3-carbonitril, is a novel vasodilating agent with b o t h antihypertensive and antianginal actions (Kishii et al., 1991; Y a n a k a et al., 1991). A vasodilating action o f LP-805 m a y be due to inhibiting the mobilization of external Ca 2+ and the Ca 2+ release from store sites (Kishii et al., 1991). However, the inhibitory action of LP-805 o n Ca 2+ influx was n o t clarified. It is well k n o w n that Ca 2+ entry blockers block voltage-dependent Ca 2+ influx a n d p o t e n t vasodilating properties ( G o d f r a i n d et al., 1986). It seemed t h a t LP-805 possessed a quite different vasorelaxant profile from Ca 2+ channel blockers (Kishii et aL, 1991). O u r preliminary observations suggest t h a t LP-805 decreases action potential duration ( A P D ) of canine ventricle, in a c o n c e n t r a t i o n d e p e n d e n t manner. The decrease in cardiac A P D is consistent with a n increase in potassium permeability (Findlay et al., 1989) or a decrease in calcium current ( M c D o n a l d , 1982). Therefore, it is expected that LP-805 would have an action of potassium channel opener. Recently, the pharmacological actions of p o t a s s i u m channel openers such as cromakalim, pinacidil a n d nicorandil have been studied, whose m e c h a n i s m of action is t h o u g h t to involve hyperpolarizing the resting m e m b r a n e as well as the memb r a n e depolarized by agonists or K + ( < 20 m M ) a n d this p r o d u c e d vasodilation (Palmer et al., 1988). Glibenclamide, a potent blocker of A T P - d e p e n d e n t K ÷ channels in pancreatic cells ( S c h m i d - A n t o m a r c h i *To whom all correspondence should be addressed.
et al., 1987), has been s h o w n to antagonize the vascular s m o o t h muscle relaxation caused by K + channel opener ( M a s u z a w a et al., 1990a,b; Q u a s t a n d Cook, 1988). The present experiments were intended to clarify the vasodilating action of LP-805 by studying changes in mechanical properties a n d simultaneously m o n i t o r i n g the muscle tension a n d cytosolic Ca z+ level in isolated rat thoracic aorta. Further, the effects of LP-805 on 86Rb efflux from rat a o r t a were also investigated.
MATERIALS AND METHODS
Measurement o f mechanical response Thoracic aorta was isolated from male Wistar rats (200-250 g), then cut into ring segments (4 mm length) and the endothelium was removed by gently rubbing with a stainless wire. The ring segments were suspended in organ baths filled with a normal physiological salt solution (PSS) which contained ll8.0mM NaC1, 4.TmM KCI, 2.5mM CaCl2, 1.2mM MgSO4, 1.2mM KH2PO4, 25.0mM NaHCO 3 and I 1.0 mM glucose. The solution was saturated with 95% O z and 5% CO z mixture at 37°C and pH7.4. After equilibration in normal PSS for 60 min, the response to agonist was recorded isometrically under an initial tension of 2.0 g. The complete removal of endothelial cells was confirmed by application of 10ktM acetylcholine (ACh), since ACh dose not relax the aortic preparations after removal of endothelial cells. The ring preparations were challenged with each agonist three times, and tension by the last challenge was used as a reference value to estimate the response magnitudes of the test compound. During K+-in duced contractions, guanethidine (3 #M) and tetrodotoxin (0.1/aM) were added into the bath solution to prevent the release of neurotransmitters. 347
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KEN-IcFII KISHII et al.
Measurement o f cytosolic Ca e+ and tension in fura-2-loaded preparation The thoracic aorta was cut into 2 x 10 mm helical strips and the endothelium was removed by gently rubbing with a small swab. The aortic strips were equilibrated for 60-90 min in a normal PSS containing 0.01 mM ethylendiamine tetraacetic acid (EDTA), the response to an agonist was recorded isometrically under an initial tension of 0.5 g. High K ÷ solution was made by substituting NaCI with equimolar KC1. These solutions were saturated with 95% O 2 and 5% CO 2 mixture at 37°C and pH 7.4. The strips were incubated with 5/~ M fura 2/AM in normal PSS containing 0.01 mM EDTA for 3 hr at room temp in the presence of 0•2% Cremophor EL, then rinsed with the same solution for 15min. Thereafter, experiments were performed with a double wavelength excitation fluorimeter (CAF-100, Japan spectroscopic). The mechanical activity was monitored isometrically, and simultaneously the ratio of 500 nm fluoresence emitted by 340 nm excitation (V~40) to that by 380 nm (F380) excitation was calculated automatically from successive illumination periods (48 Hz) and referred to as R3+0/380. In the muscle strips that were successfully loaded with fura-2, the increase in cytosolic Ca 2+ level resulted in an •increase in F340, a decrease in F3s 0 and an increase, in R34o/38 . . 2+ o The R340/380 w a s used to monitor the relative cytosohc Ca level (Sato et al., 1988; Karaki et al., 1988). 86Rb efflux In this experiment, 86Rb was used as a marker for K ÷ (Bolton and Clapp, 1984; Imaizumi and Watanabe, 1981; Weir and Weston, 1986). Rat aorta was cut into rings (10 mm length) and each ring was opened into a flat sheet by cutting it longitudinally. Endothelium-free segments were A 100
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Fig. 2. Dose--response curves for the relaxant responses to LP-805 on various concentrations of K+-induced contractions in rat thoracic aorta without intact endothelium. ( 0 ) 65.9mM K ÷, (O) 35.9mM K ÷, (A) 30.9mM K ÷, (A) 25•9 mM K ÷. Values are means ___SE for 4 preparations.
attached to the micro wire clamps, and after a 30 min equilibration period in PSS at 37°C bubbled with 95% 02 and 5% CO2, they were loaded with 86RbC1 5 #Ci/ml for 90 min. 86Rb was then allowed to efflux from the tissues into 2 ml aliquots of PSS using 2 min collection periods. After 7 such periods, preparations were exposed to PSS alone (control) or to PSS containing LP-805 for the next 5 collection periods. Glibenclamide was applied after 5 collection periods. For the remaining periods, the tubes contained PSS alone. At the end of the efflux period, the radioactivity in the tubes and that remaining in tissues were measured• The effiux data were expressed in terms of the rate coefficient (fractional loss of 86Rb from the tissue standardized for I min period) expressed as a percentage. Drugs and chemicals LP-805 was synthesized in POLA Pharmaceutical R&D Laboratory. The following drugs were used: /-norepinephrine bitartrate (Wako Pure Chemicals, Tokyo, Japan); diltiazem hydrochloride, glibenclamide, guanethidine (Sigma Chemicals, St Louis, Mo.); fura 2/AM (Dojindo Laboratories, Kumamoto, Japan); Cremophor EL (Nakalai Tesque, Kyoto, Japan); tetrodotoxin (Seikagaku Kogyo, Tokyo, Japan), 86RbCI (NEN Research Products). Other chemicals used were analytical grade•
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Statistics Numerical results are presented as the mean + SE with the number of the observations in parentheses. Values were considered to be significantly different when P < 0•05 by Duncan's multiple range test and Student's t-test•
RESULTS
Effects o f L P - 8 0 5 on mechanical response
0 ¢ +Sin's" N E I -IogM )
Fig. 1. Effects of various concentrations of LP-805 and diltiazem on (A) 65•9 mM K + and (B) NE-induced contractions in rat thoracic aorta without intact endothelium. (A) O, LP-805; @, diltiazem. (B) ©, control; A, 1/~M diltiazem; &, 10#M diltiazem; I-1, 1/~M LP-805; I , 10/tM LP-805. Values are means + SE for 5qi preparations.
The c o n c e n t r a t i o n - r e s p o n s e curve for the vasorelaxant effects o f LP-805 and diltiazem on isolated rat thoracic aorta are shown in Fig. 1. The rings were precontracted with high K ÷ (65.9 m M ) and relaxed in a c o n c e n t r a t i o n ~ l e p e n d e n t m a n n e r when diltiazem (0.01-1 # M ) was applied, but LP-805 (0.1-10 /~M) failed to relax those. Whereas, LP-805 ( I - 1 0 / ~ M ) caused a marked reduction in NE-induced m a x i m u m response in a c o n c e n t r a t i o n - d e p e n d e n t manner, but diltiazem (1-10/~M) slightly reduced that. The vasodilating action o f LP-805 was different from that o f Ca 2+ entry blocker•
LP-805, a potassium channel opener Figure 2 shows the effects of LP-805 on various concentrations of K+-induced tonic contractions. LP-805 relaxed the contraction induced by < 35.9 mM K ÷, in a concentration-dependent manner, but not the contraction evoked by higher concentration of K ÷. Glibenclamide (0.3-1#M) caused a parallel shift of the concentration-response curve produced by LP-805 for 25.9 mM K+-induced tonic contraction, in a concentration-dependent manner [Fig. 3(A)]. A schild plot of these results gave a straight line with a slope of unity, suggesting a competitive antagonism. The pA: value was 7.10 + 0.05 (n = 6). LP-805 (10 #M) caused a marked reduction in NE-induced maximum response. This LP-805-induced reduction was prevented by pretreatment of glibenclamide (0.3-3 pM), in a concentration-dependent manner [Fig. 3(B)],
Effects o f LP-805 and glibenclarnide on changes in cytosolic Ca 2+ level and contractions by NE and high K + Figure 4(A) shows the effect of LP-805 on NE (1 pM)-induced contraction and cytosolic Ca 2÷ level in normal PSS. LP-805 (10 pM) markedly inhibited the sustained increase in cytosolic Ca 2+ level, and
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NE( -IogMI Fig. 3. (A) Effects of glibenclamide on concentration-response curves produced by LP-805 for 25.9 mM K ÷-induced contractions in rat thoracic aorta. (C)) Without glibenclamide, (A) 0,3pM glibenclamide, (A) l llM glibenclamide. Values are means + SE for 10-11 preparations. (B) Effects of glibenclamideon LP-805 (I0 # M)-induced reductions in maximum responses evoked by NE in rat thoracic aorta. (O) Control, (©) I0 pM LP-805, (A) 0.3pM glibenclamide plus 10#M LP-805, (111) l pM glibenclamideplus 10 pM LP-805, (E]) 3 pM glibenclamide plus 10#M LP-805. Glibenclamide applied 5min before application of LP-805. Values are means _ SE for 6 preparations.
both the phasic and sustained contraction evoked by NE, but not those induced by high K + (65.9 mM). LP-805-induced these inhibitions were prevented by pretreatment with 10/~M glibenclamide [Fig. 4(B)]. Figure 5 shows the effect of LP-805 and glibenclamide on K+-induced contraction and cytosolic Ca 2÷ level in normal PSS. LP-805 (10 pM) had no effect on 65.9 mM K+-induced sustained increase in cytosolic Ca 2+ level and contraction [Fig. 5(A)]. While LP-805 (10 pM) caused the marked reduction in 25.9mM K+-induced maximum response, but slightly inhibited the sustained increase in cytosolic Ca 2÷ level [Fig. 5(B)]. LP-805-induced these changes also prevented by pretreatment with 10pM glibenclamide [Fig. 5(C)].
Effects o f LP-805 on 86Rb efflux Effects of LP-805 on the 86Rb effiux in rat aorta were determined. In resting strips, LP-805 (10pM) increased the 86Rb efflux rate coefficient from the basal value of 2.34 +_ 0.36% per min (n = 5, measured between the 10th and 14th min of the efflux period) to the peakvalue of 4.14 +_0.36% per min (n = 5) [Fig. 6(B)]. While, in the absence of LP-805 [control, Fig. 6(A)], there was no significant increase in the s6Rb efflux rate coefficient between the 10th and 32nd min of the efflux period [Fig. 6(A)]. Glibenclamide (10#M) completely inhibited the LP-805-induced increase in the S6Rb effiux [Fig. 6(C)].
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Fig. 5. Effects of LP-805 and glibenclamide on changes in cytosolic Ca2÷ levels and contractions induced by high K +. Effect of (A) LP-805 (10#M) on 65.9mM K ÷ and (B) 25.9 mM KS-induced contraction and increase in cytosolic Ca2÷ level. (C) Effect of glibenclamide (10/zM) on the inhibition produced by LP-805 (10 pM) for 25.9 mM KS-induced contraction and increase in cytosolic Ca2+ level. DISCUSSION
The results of these experiments provided a vasorelaxant profile as a K ÷ channel opener with regard to the effects of LP-805 on 86Rb efflux and contractile responses in vascular smooth muscle. LP-805 produces the arterial relaxation via the increase in the K + efflux, possibly by opening ATP-dependent K + channels. It has been reported that the vascular relaxant effects of K ÷ channel opener such as pinacidil are highly correlated with both reductions in cardiac APD and contractility (Steinberg et al., 1988). Further, a similar correlation between APD reduction and antihypertensive activity was reported in spontaneously hypertensive rats (Smallwood and Steinberg, 1988). The results taken together strongly suggest that the vascular relaxant and cardiac electrophysiological effects of this group of compounds are mediated by a common mechanism. LP-805 (1-10pM) markedly decreased APD in ventricular muscle without much effect on other measured parameters. The maximum decrease in APD10, APD50 and APD90 were 55.2_ 12.1, 75.4_+ 7.7 and 85.1 _+ 5.4%, respectively (data not shown). The decrease of cardiac APD is consistent with an increase
in potassium permeability in cardiac tissue (6). APD is also maintained at least partly by influx of calcium ions in ventricular muscle (7). Indeed, calcium agonist substances have been shown to cause large increase in APD of ventricular muscle (Thomas et al., 1985). LP-805 had no effect on voltage-dependent 45Ca2+ influx by 65.9 mM K ÷ in rat thoracic aorta (unpublished data). Therefore, the basis for the vascular relaxant effects of LP-805 may involve an increase in membrane permeability to potassium ions resulting in hyperpolarization. In rat thoracic aorta, LP-805 caused the marked reduction of NE-induced maximum response in a concentration-dependent manner, and diltiazem slightly reduced that. On the other hand, diltiazem completely relaxed the aortic rings precontracted with high K ÷ (65.9 mM) in a concentration-dependent manner, but LP-805 slightly relaxed that with its high concentration. Therefore, it seemed that LP-805 possessed a quite different vasorelaxant profile from Ca 2÷ entry blockers (Fig. 1). The aortic rings were precontracted with low K ÷ ( < 35.9 mM) and relaxed in a concentration-dependent manner when LP-805 was applied (Fig. 2). In vascular smooth muscle cells the resting membrane potential is less negative than the K ÷ equilibrium potential (Furukawa et al., 1981). In the present study, when arterial rings were contracted with low concentration of K + ( < 35.9 mM), the equilibrium potential for K s would be equivalent to or more negative than the normal resting membrane potential. Under such conditions, it is unlikely that extracellular Ca 2÷ can enter through voltage-dependent Ca 2÷ channels, Whereas, LP-805 failed to relax the high K ÷ (65.9 mM)-induced contraction, presumably because the membrane potential remains at a level of depolarization which required to open voltage-dependent Ca 2+ channels. Under these conditions, the LP-805-induced increase in the K ÷ efflux would not hyperpolarize arterial smooth muscle sufficiently to inhibit transmembrane Ca :+ influx. When the membrane is hyperpolarized towards the K ÷ equilibrium potential by K + channel openers, Ca 2+ influx via voltage-dependent Ca 2÷ channels is thought to decrease (Cook, 1988; Weston, 1989). Therefore, LP-805 cannot block directly the Ca 2+ entry through voltage-dependent Ca 2÷ channels. These experiments allow Ca 2+ entry blocker to be distinguished from vasodilators with K s channel opening activity as already demonstrated in other investigations (Bray et al., 1987; Hamilton et al., 1986; Hamilton and Weston, 1989; Weston et al., 1988). Glibenclamide is a potent blocker of ATP-dependent K + channels. These channels are activated when intracellular ATP levels are low. A number of pharmacological studies have demonstrated a competitive interaction of glibenclamide with the response to K + channel openers in vascular tissues (Buckingham et al., 1989; Cavero et al., 1989; Winquist et al., 1989). These channels have also been identified in cardiac cells (Noma, 1983). In the present study, glibenclamide was also found to antagonize the relaxant responses to LP-805. As shown in Fig. 3, glibenclamide prevented the LP-805-produced reduction in NE-induced maximum response. Moreover, glibenclamide caused a parallel shift of the
LP-805, a potassium channel opener
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Fig. 6. Effects of LP-805 and glibenclamide on the loss of s6Rb from rat thoracic aorta. 86Rb efflux rate coefficient expressed as a % per min. (A) Control (physiological salt solution alone). (B) Tissues were exposed to LP-805 (10/tM) between the 14th and 24th min of the effiux period. (C) Tissues were exposed to LP-805 (I0 #M) between the 14th and 24th min of the efflux period, and glibenclamide (10 #M) was applied between 10th min and 28th min of the efllux period. Values are means + SE for 4-7 experiments. *P < 0.05, **P < 0.01 vs basal value.
concentration-response curve produced by LP-805 for 25.9mM K+-induced contraction in a concentration-dependent manner (Fig. 2). The Schild plot for glibenclamide antagonism of the response to LP-805 showed a typical competitive antagonism with the slope of unity and the pA 2 value of 7.10 _ 0.05 (n = 6). These results suggest that LP-805 produces the arterial relaxation through the increase in K ÷ permeability via the opening of ATP-dependent K ÷ channels. Smooth muscle contraction is initiated by the increase in cytosolic free Ca 2÷ levels through stimulation of Ca z÷ release from intracellular stores as well as by promoting Ca 2+ influx through Ca 2+ channels (Rasmussen and Barrett, 1984; Somlyo and Somlyo, 1986). LP-805 inhibits Ca 2+ influx which is mediated by a receptor stimulated pathway, and moreover Ca 2÷ release from store sites, resulting the decrease in cytosolic Ca 2+ levels (Kishii et al., 1991). Then we studied the effects of glibenclamide on LP-805-induced reduction in cytosolic Ca 2+ levels and muscle tension. As shown in Figs 4(A) and 5(A), LP-805 (10/~M) markedly inhibited the sustained increase in cytosolic Ca 2÷ level, and both the phasic and sustained contraction evoked by NE, but not those induced by 65.9mM K +. While LP-805 (10#M) caused the reduction in 25.9 mM K+-induced maximum response, but slightly inhibited the increase in cytosolic Ca 2÷ level [Fig. 5(B)]. LP-805-induced changes were prevented by pretreatment with 10 #M
glibenclamide [Figs 4(B), 5(C)]. Similar results have been reported with cromakalim (Yanagisawa et al., 1990). From these results, the inhibitory action of LP-805 on NE- or 25.9 mM K÷-induced contraction may be accounted for a reduction of cytosolic Ca 2÷ level due to the closure of voltage-dependent Ca 2÷ channels by hyperpolarization through the opening of ATP-dependent K ÷ channels. S6Rb was used as a marker for K ÷ permeability in smooth muscle (Bolton and Clapp, 1984; Hamilton et al., 1986; Imaizumi and Watanabe, 1981). In resting strips, LP-805 (10 #M) increased the basal 86Rb efflux. The increase in 86Rb efflux induced by LP-805 was completely inhibited by 10pM glibenclamide (Fig. 6). Therefore, LP-805 possesses the characteristics of ATP-dependent K ÷ channel opener resulting the increase in K ÷ permeability. In conclusion, present study shows that LP-805 possesses a mechanism of action as a K ÷ channel opener, and that vasorelaxation produced by LP-805, in part, is due to the decrease in cytosolic Ca 2÷ levels via the closure of voltage dependent Ca 2÷ channels by hyperpolarization through the increase in K ÷ permeability, possibly by opening ATP-dependent K ÷ channels. A vasodilating action of LP-805 is due to inhibiting the mobilization of external Ca 2+ and the Ca 2÷ release from store sites (Kishii et al., 1991). Therefore, inhibitory action of LP-805 on Ca 2+ influx accounts for the increase in K ÷ permeability through ATP-dependent K ÷ channels.
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KEN-ICHI KISHn et al. SUMMARY
The characteristics o f LP-805, a novel vasodilating agent, were investigated in isolated rat thoracic aorta. LP-805 (0. I - 1 0 # M) caused the m a r k e d reduction of N E - i n d u c e d m a x i m u m response a n d relaxed the low K + ( < 35.9 m M ) - i n d u c e d contraction, in a concent r a t i o n - d e p e n d e n t manner, but failed to relax the high K + (65.9 m M ) - i n d u c e d contraction. Therefore, it is t h o u g h t t h a t LP-805 possesses a quite different vasorelaxant profile from Ca 2+ entry blockers, such as diltiazem. Glibenclamide (0.3-1/~M) caused a parallel shift of the c o n c e n t r a t i o n - r e s p o n s e curve produced by LP-805 for 25.9 m M K + - i n d u c e d contraction a n d prevented the LP-805-induced reduction in m a x i m u m response evoked by NE, in a concent r a t i o n - d e p e n d e n t m a n n e r . Further, glibenclamide (10/~M) prevented the LP-805 (10/~M)-induced decrease in cytosolic Ca 2+ levels which was increased by 1 / I M N E or 2 5 . 9 m M K +. LP-805 (10/~M) also increased basal 86Rb efflux, which was completely inhibited by 10/~M glibenclamide. These results suggest that the vasorelaxant action induced by LP805 involves in opening A T P - d e p e n d e n t K ÷ channels, which is due to the reduction in cytosolic Ca 2+ levels via the closure of voltage-dependent Ca 2÷ channels by hyperpolarization t h r o u g h K ÷ permeability.
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