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Endothelium-dependent relaxation by cilostazol, a phosphodiesteras III inhibitor, on rat thoracic aorta
Life Sciences 69 (2001) 1709–1715
Endothelium-dependent relaxation by cilostazol, a phosphodiesteras III inhibitor, on rat thoracic aorta Toshimi Nakamuraa, Hitoshi Houchia, Asako Minamib, Sadaichi Sakamotob, Koichiro Tsuchiyac, Yasuharu Niwab, Kazuo Minakuchia, Yutaka Nakayab,* a
Department of Pharmacy, Tokushima University Hospital, 2-50-1 Kuramoto, Tokushima, Japan 770-8503 b Departments of Nutrition, The University of Tokushima, School of Medicine, 3-18-15 Kuramoto, Tokushima, Japan 770-8503 c Departments of Pharmacology, The University of Tokushima, School of Medicine, 3-18-15 Kuramoto, Tokushima, Japan 770-8503 Received 5 July 2000; accepted 12 March 2001
Abstract The relaxation effect of cilostazol, a phosphodiesterase III inhibitor, on the thoracic aorta was investigated. Cilostazol induced the relaxation of the thoracic aorta precontracted by phenylephrine in a concentration-dependent manner. The concentration-dependent relaxation was shifted to the right in the endothelium denuded aorta compared with that of intact endothelium, suggesting that this relaxation was partly dependent on endothelium. Cilostazol-induced relaxation of thoracic aorta tone was reversed by treatment with NG-nitro L-arginine (L-NNA), a competitive inhibitor of nitric oxide (NO) synthase. Cilostazol also significantly increased the NO level in the porcine thoracic aorta. In rats treated with cilostazol, the urinary excretion of nitrites, a stable metabolite of NO, and basal production of NO of the aortic ring were significantly greater than in those without treatment. These findings indicate that cilostazol-induced vasodilation of the rat thoracic aorta was dependent on the endothelium, which released NO from aortic endothelial cells. © 2001 Elsevier Science Inc. All rights reserved. Keywords: Cilostazol; Phosphodiesterase III inhibitor; Nitric oxide; Vasodilation
Introduction Cilostazol is a phosphodiesterase III inhibitor and is used in diabetic patients as an antiplatelet and antithrombotic drug [1–3]. It has been found that phosphodiesterase III inhibitors including cilostazol cause vasodilation leading to a concomitant reduction in arterial pressure [4–6]. Cyclic nucleotide phosphodiesterases catalyze the degradation of cAMP and cGMP * Corresponding author. Tel.: (181)-88-633-7090; fax: (181)-88-633-7113. E-mail address: [email protected] (Y. Nakaya) 0024-3205/01/$ – see front matter © 2001 Elsevier Science Inc. All rights reserved. PII: S 0 0 2 4 - 3 2 0 5 ( 0 1 )0 1 2 5 8 -9
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[3,7]. An increase in levels of cAMP or cGMP in smooth muscle cells was regulated by activation of adenylate cyclase or guanylate cyclase and inhibition of phosphodiesterases [3,7,8]. The endothelium plays an important role in regulating vascular tone, through the release of a variety of vasoactive substances. Endothelium-derived relaxing factor, identified as nitric oxide (NO) or as a nitric oxide compound, is one of the most important [9]. Cilostazol is suggested to relax smooth muscle cells by elevating cAMP. However, little is known about the role of cilostazol in relaxation of vascular tone, specially endothelium-dependent vascular relaxation. In the present study, we investigated a novel effect of cilostazol, namely, the endothelialdependent relaxation of the rat thoracic aorta. The present findings suggested that cilostazol relaxes aorta both endothelium -dependently and -independently. Methods Vascular relaxation by cilostazol in vitro; Twenty-five-week-old male Sprague-Dawley rats were anaesthetized with sodium pentobarbital (50 mg/kg) and were killed by exsanguination. The thoracic aorta was rapidly removed, freed of adhering fat and connective tissue under a binocular dissecting microscope, and cut with parallel razors into ring segments approximately 3 mm wide. The segments were soaked vertically in 3 ml of Krebs bicarbonate solution of the following composition (mM): NaCl 115.3, KCl 4.9, CaCl2 1.4, MgSO4 1.2, NaHCO3 25.0, glucose 11.1 and ascorbate. The lumen of the artery was gently rubbed with cotton thread to remove the endothelium. Two stainless steel wires were inserted into the lumen of the aortic ring. One wire was connected to a transducer and the other anchored to a plastic holder. The segment was then placed in a 5 ml organ bath at 348C containing oxygenated (95% O2 and 5% CO2) Krebs bicarbonate solution. The preparations were equilibrated for 120 min prior to the start of the experiments and the resting tension was maintained at 1.0 g. To measure relaxation, the segment was contracted initially with phenylephrine at a concentration designed to induce 80% of maximal contraction (EC80: 0.3 mM for arteries with endothelium and 0.1 mM for those without endothelium). Responses were recorded isometrically with a force displacement transducer (SB 1TH, Nihon Kohden, Tokyo, Japan). A 7 – 20 ml volume of cilostazol was added to the 5 ml organ bath, and relaxations were measured as percentages of the contractions induced by the EC80 concentration of phenylephrine. Relaxations of aortic rings, with or without endothelium, were compared following cumulative application of cilostazol. Male diabetic (Otsuka Long-Evans Tokushima Fatty; OLETF) rats were fed standard rat chow (Oriental Yeast, Tokyo, Japan) and tap water ad libitum until the age of 16 weeks, when they were randomly assigned to two groups of 10 rats each: those with normal diet (OLETF-Normal group) and those with a cilostazol (40 mg/kg/day) supplemented diet (OLETF-Cilostazol group). The serum concentration of cilostazol in rats was about 1.5 mg/ml, which is equivalent to those of patients treated with cilostazol. Body weight was not significantly different among groups at the beginning of the experiment. Non-diabetic (Long-Evans Tokushima Otsuka; LETO) rat strain was used as the age-matched control (LETO-Normal and LETOCilostazol groups, respectively). NO measurement was by the electron paramagnetic resonance spin trapping method. Porcine thoracic aorta in rings were used in this study. Porcine serum albumin (33 mg/ml, final
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concentration), FeSO4 (3.3 mM), sodium N,N-diethyldithiocarbamate trihydrate (3.3 mM), the sample, and excess Na2S2O4 (2 M) were introduced into a 4 mm diameter electron paramagnetic resonance (EPR) tube (LST-5HS, Labotec Co.), and after 5 min, the mixture was frozen at liquid nitrogen temperature (77 K). EPR spectra were measured in a JEOL FE1X spectrometer under the following conditions: microwave frequency, 9.15 GHz, microwave power, 20 mW, modulation frequency, 100 kHz, modulation amplitude, 0.63 mT, magnetic field, 320 6 25 mT, response time, 0.1 s, sweep time 50 mT/4 min, temperature 77 K. The concentration of NO trapped was determined by comparing the intensities of the first lowfield derivative EPR signal heights and their amplitudes [10]. NO22 measurement was by the modified Griess method. To determine the urinary NO22 nitrite excretion rate, 24 h urine samples from 25-week-old rats were collected in bottles containing an antibiotic solution (1 mg/ml of penicillin G, 1 mg/ml of streptomycin and 0.25 mg/ ml of amphotericin B). NO32 in urine was converted into NO22 with nitrate reductase and measured with the Griess reagent (Cyman Chemical, Ann Arbor, MI). Data in figures are expressed as the mean 6 S.E.. Data were analysed by post hoc test (Scheffe’s test and Bonferroni’s test) followed by the one way analysis of variance (ANOVA) and/or Student’s t-test. A value at ,0.05 was accepted statistically significant. Results Figure 1 shows the relaxant effects of cilostazol in vitro on rat thoracic aorta with or without endothelium. Vasodilative effects of cilostazol were dose-dependent at concentrations of 0.3 mM to 100 mM in the rings with or without endothelium, suggesting that cilostazol-
Fig. 1. Effects of endothelium on cilostazol-induced relaxation on ring segments of the rat thoracic aorta. The preparations were contracted with phenylephrine at concentrations corresponding to the EC80. Vasodilative responses are expressed as percentages of the phenylephrine-induced contractions. Values are means 6 S.E. for 4 separate preparations.
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Fig. 2. Effects of cilostazol, nitroprusside and L-NNA on ring segments of rat thoracic aorta. Cilostazol was supplemented for nine-weeks at 40 mg/kg/day on rats. The ring segments were preincubated for 30 min presence or absence of 1024 M sodium nitroprusside(SNP) or 5 3 1024 M NG-nitro L-arginine (L-NNA). The preparations were contracted with phenylephrine at concentrations corresponding to the EC80. Vasodilative responses are expressed as percentages of phenylephrine-induced contraction. Values are means 6S.E. for 7–10 separate preparations. * P,0.05, ** P,0.01, Significantly less than the control value. *** P,0.01, Significantly greater than the control value. (ANOVA, Scheffe’s test)
induced relaxation was, in part, dependent on endothelium. The EC50 value was 5 6 0.3 mM in preparations with endothelium, and 10 6 0.7 mM in those without (n 5 8). To determine whether cilostazol activates NO/cGMP pathway, we examined the effects of cilostazol on guanylate cyclase activator nitroprusside-induced relaxation of thoracic aorta tone. As shown in Figure 2, cilostazol failed to potentiate the relaxation of thoracic aorta tone induced by 1024 M sodium nitroprusside(SNP). Moreover, phenylephrine-induced contraction of the thoracic aorta was potentiated by 5 3 1024 M NG-nitro L-arginine (L-NNA), a competitive inhibitor of NO synthase. Phenylephrine-induced contraction was increased in the presence of L-NNA, which blocks the basal secretion of NO. Thus, the difference between contraction in the presence and the absence of L-NNA can be assumed to be due to basal NO secretion. Basal NO secretion tended to be greater in the rings from rats treated with cilostazol compared with those without treatment. To determine whether cilostazol can produce NO in thoracic aorta we measured NO in porcine thoracic aortic ring segments, in vitro, using electron paramagnetic resonance (EPR) spectroscopy. In the preliminary experiments, we tried the production of NO in the thoracic aortic rings of rats using EPR spectroscopy. However, it was failed to detect the NO levels in these ring segments because of too small ring segments from rats to detect by our EPR spectroscopy method. As shown in Figure 3, cilostazol at concentration of 1024 M increased NO production from the thoracic aorta. However, it’s production was not significantly stimulated
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Fig. 3. Effects of cilostazol on nitric oxide production in porcine thoracic aortic ring segments. The porcine thoracic aortic ring segments were incubated for 30 min at 348C with or without 102621024M cilostazol. NO levels were determined as described in Materials and Methods. Values are mean 6 S.E. for 6 separate experiments. * P,0.01, Significantly greater than the control value. (ANOVA, Bonferroni’s test)
by up to 1025 M cilostazol. Cilostazol induced NO production from the thoracic aortic ring segments was inhibited by L-NNA (data not shown). These findings indicated that the production of NO induced by cilostazol may partially regulate the vasodilation of rat thoracic aorta. We also measured the urinary secretion of NO and its metabolites by the Griess method. The NO22 secretion was not significantly greater in those treated with cilostazol than in those without treatment in non-diabetic rats. We also treated diabetic rats, because this drug is used in patients with diabetes and decreased endothelial function. The basal production of NO was significantly greater in non-diabetic (LETO) rats than in the diabetic (OLETF) rats. The urinary secretion of NO22 was significantly decreased in diabetic rats compared with the nondiabetic rats. Cilostazol treatment significantly increased the urinary secretion of NO in diabetic rats, although it was still smaller than that in non-diabetic rats (Fig. 4). Discussion The findings of present study indicated that cilostazol induced endothelium-dependent relaxation of the thoracic aorta, in addition to its direct relaxant effect on smooth muscle. The contribution of the endothelium was more evident at lower concentrations, which corresponds to clinical doses. In diabetic rats, in which the endothelial function was partly disturbed, cilostazol also increased NO production, suggesting the usefulness of this drug in diabetic patients. Cilostazol is an inhibitor of phosphodiesterase III and therefore prevents the breakdown of cAMP. The mechanisms of vasodilation by increased cAMP in vascular smooth muscle cells are thought to be the following: (1) stimulation of cAMP-dependent protein kinase, which activates a sarcolemmal calcium pump, (2) stimulation of Na1/K1-ATPase, which results in hyperpolarization and removal of intracellular sodium and calcium, (3) augmantation of dephosphorylation of the myosin light chain [11]. In the current findings (Fig. 1), cilostazol produced relaxation of
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Fig. 4. Effects of cilostazol on nitrites secretion in diabetic and non-diabetic rat urine. Cilostazol was supplemented for nine-weeks at 40 mg/kg/day on the diabetic (OLETF) and non-diabetic (LETO) rats. Nitrites levels were determined as described in Materials and Methods. Values are means 6 S.E. from 7 separate experiments, expressed as mmol/10hr/100g body weight. Urinary excretion of nitrites was decreased in OLETF rats (* P,0.01). Cilostazol supplementation improved urinary excretion of nitrites in OLETF rats (P,0.05), but not in LETO (Student’s t-test). * P,0.01 OLETF vs. LETO. (ANOVA, Bonferroni’s test)
the thoracic aorta without endothelium. Therefore, it is possible that vasodilation of the thoracic aorta may be induced by an increase in cAMP levels in vascular smooth muscle cells. It has not previously been established whether the endothelium contributes to the effect of cilostazol. The findings of the present study indicated that in the presence of endothelium relaxation occurs at lower concentrations, and its effect could be suppressed by the addition of NO synthase inhibitor, L-NNA. Thus, the involvement of the NO pathway in the vasodilative effect of cilostazol is suggested. In addition, in rats chronically treated with cilostazol, basal NO production as well as urinary secretion of NO22 was greater than those without treatment, suggesting that cilostazol increased NO secretion from endothelium. This effect was also observed in diabetic rats although improvement was not complete. Previous studies have reported that isoprenaline acts through a beta-adrenoceptor on the endothelium to raise cAMP in rat thoracic aorta, and that this may, directly or indirectly, release NO to evoke vascular relaxation [12]. Uncontrast, cilostazol increases the cellular concentration of cAMP, but not that of cGMP, in rabbit arterial tissues [1, 5]. Furthermore cilostazol has a beneficial effect on diabetic neuropathy by improving Na1/K1-ATPase activity via directly increasing cAMP and NO production in human neuroblastoma cells [13]. The findings of the present study showed that cilostazol induced NO production and that their vasodilative effect in the presence of endothelium was reversed by L-NNA (Figs. 3 and 4). The changes in urinary excretion of NO22 1 NO32 as a measurement of changes in NO production has been guestiond in a previous study [14]. However, our previous report have suggested that an in-
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crease in urinary excretion of NO22 is dependent on the elevation of NO production in diabetic rats [15]. Moreover, cilostazol enhanced the urinary secretion of NO22 1 NO32 in diabetic rats (Fig. 4). An increase in the NO level in the endothelium induces production of cGMP [16]. In the present study, we did not measure cGMP production in the rat thoracic aorta. Therefore, the role of cGMP in cilostazol-induced relaxation of the rat thoracic aorta remains to be clarified. NO has a variety of functions, including as a vasorelaxant, an anticoagulant and also antiatherogenic actions. Increased production of NO by cilostazol is expected to enhance these functions and inhibit smooth muscle proliferation and development of atherosclerosis. References 1.
2.
3. 4.
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16.
Umekawa H, Tanaka T, Kimura Y, Hidaka H. Purification of cyclic adenosine monophosphate phosphodiesterase from human platelets using new-inhibitor Sepharose chromatography. Biochemical Pharmacology 1984; 33(21): 3339–44. Caldicott LD, Hawley K, Heppell R, Woodmansey PA, Channer KS. Intravenous enoximone or dobutamine for severe heart failure after acute myocardial infarction: a randomized double-blind trial. European Heart Journal 1993; 14(5): 696–700. Polson JB, Strada SJ. Cyclic nucleotide phosphodiesterases and vascular smooth muscle. Annual Review Pharmacology and Toxicology 1996; 36: 403–27. Shintani S, Watanabe K, Kawamura K, Mori T, Tani T, Toba Y, Sasabe H, Nakagiri N, Hongoh O, Fujita S, Aogi T, Kido Y, Umezato M, Ishikawa M, Hiyama T. General pharmacological properties of cilostazol, a new antithrombotic drug. Part II:Effect on the peripheral organs. Arzneimittelforschung 1985; 35(II)7A: 1163–72. Tanaka T, Ishikawa T, Hagiwara M, Onoda K, Itoh H, Hidaka H. Effects of cilostazol, a selective cAMP phosphodiesterase inhibitor on the contraction of vascular smooth muscle. Pharmacology 1988; 36(5): 313–20. Mori K, Takeuchi S, Moritoki H, Tsuchiya K, Nakaya Y, Matsuoka S, Kuroda Y. Endothelium-dependent relaxation of rat thoracic aorta by amrinone-induced nitric oxide release. European Heart Journal 1996; 17(2): 308–16. Beavo JA. Cyclic nucleotide phosphodiesterases: functional implications of multiple isoforms. Physiological Reviews 1995; 75(4): 725–48. Weishaar RE, Burrows SD, Kobylarz DC, Quade MM, Evans DB. Multiple molecular forms of cyclic nucleotide phosphodiesterase in cardiac and smooth muscle and in platelets. Isolation, characterization, and effects of various reference phosphodiesterase inhibitors and cardiotonic agents. Biochemical Pharmacology 1986; 35(5): 787–800. Palmer RM, Ferrige AG, Moncada S. Nitric oxide release accounts for the biological activity of endotheliumderived relaxing factor. Nature 1987; 327(6122): 524–6. Tsuchiya K, Takasugi M, Minakuchi K, Fukuzawa K. Sensitive quantitation of nitric oxide by EPR spectroscopy. Free Radical Biology and Medicine 1996; 21(5): 733–7. Honerjager P. Pharmacology of positive inotropic phosphodiesterase III inhibitors. European Heart Journal 1989; 10(Suppl C): 25–31. Gray DW, Marshall I. Novel signal transduction pathway mediating endothelium-dependent beta-adrenoceptor vasorelaxation in rat thoracic aorta. British Journal of Pharmacology 1992; 107(3): 684–90. Inada H, Shindo H, Tawata M, Onaya T. Cilostazol, a cyclic AMP phosphodiesterase inhibitor, stimulates nitric oxide production and sodium potassium adenosine triphosphatase activity in SH-SY5Y human neuroblastoma cells. Life Sciences 1999; 65(13): 1413–22. Suto T, Losonczy G, Qiu C, Hill C, Samsell L, Ruby J, Charon N, Venuto R, Baylis C. Acute changes in urinary excretion of nitrite 1 nitrate do not necessarily predict renal vascular NO production. Kidney International 1995; 48(4): 1272–7. Sakamoto S, Minami K, Niwa Y, Ohnaka M, Nakaya Y, Mizuno A, Kuwajima M, Shima K. Effect of exercise training and food restriction on endothelium-dependent relaxation in the Otsuka Long-Evans Tokushima Fatty rat, a model of spontaneous NIDDM. Diabetes 1998; 47(1): 82–6. Walter U, Waldmann R, Nieberding M. Intracellular mechanism of action of vasodilators. European Heart Journal 1988; 9(Suppl H): 1–6.
Endothelium-dependent relaxation by cilostazol, a phosphodiesteras III inhibitor, on rat thoracic aorta Toshimi Nakamuraa, Hitoshi Houchia, Asako Minamib, Sadaichi Sakamotob, Koichiro Tsuchiyac, Yasuharu Niwab, Kazuo Minakuchia, Yutaka Nakayab,* a
Department of Pharmacy, Tokushima University Hospital, 2-50-1 Kuramoto, Tokushima, Japan 770-8503 b Departments of Nutrition, The University of Tokushima, School of Medicine, 3-18-15 Kuramoto, Tokushima, Japan 770-8503 c Departments of Pharmacology, The University of Tokushima, School of Medicine, 3-18-15 Kuramoto, Tokushima, Japan 770-8503 Received 5 July 2000; accepted 12 March 2001
Abstract The relaxation effect of cilostazol, a phosphodiesterase III inhibitor, on the thoracic aorta was investigated. Cilostazol induced the relaxation of the thoracic aorta precontracted by phenylephrine in a concentration-dependent manner. The concentration-dependent relaxation was shifted to the right in the endothelium denuded aorta compared with that of intact endothelium, suggesting that this relaxation was partly dependent on endothelium. Cilostazol-induced relaxation of thoracic aorta tone was reversed by treatment with NG-nitro L-arginine (L-NNA), a competitive inhibitor of nitric oxide (NO) synthase. Cilostazol also significantly increased the NO level in the porcine thoracic aorta. In rats treated with cilostazol, the urinary excretion of nitrites, a stable metabolite of NO, and basal production of NO of the aortic ring were significantly greater than in those without treatment. These findings indicate that cilostazol-induced vasodilation of the rat thoracic aorta was dependent on the endothelium, which released NO from aortic endothelial cells. © 2001 Elsevier Science Inc. All rights reserved. Keywords: Cilostazol; Phosphodiesterase III inhibitor; Nitric oxide; Vasodilation
Introduction Cilostazol is a phosphodiesterase III inhibitor and is used in diabetic patients as an antiplatelet and antithrombotic drug [1–3]. It has been found that phosphodiesterase III inhibitors including cilostazol cause vasodilation leading to a concomitant reduction in arterial pressure [4–6]. Cyclic nucleotide phosphodiesterases catalyze the degradation of cAMP and cGMP * Corresponding author. Tel.: (181)-88-633-7090; fax: (181)-88-633-7113. E-mail address: [email protected] (Y. Nakaya) 0024-3205/01/$ – see front matter © 2001 Elsevier Science Inc. All rights reserved. PII: S 0 0 2 4 - 3 2 0 5 ( 0 1 )0 1 2 5 8 -9
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[3,7]. An increase in levels of cAMP or cGMP in smooth muscle cells was regulated by activation of adenylate cyclase or guanylate cyclase and inhibition of phosphodiesterases [3,7,8]. The endothelium plays an important role in regulating vascular tone, through the release of a variety of vasoactive substances. Endothelium-derived relaxing factor, identified as nitric oxide (NO) or as a nitric oxide compound, is one of the most important [9]. Cilostazol is suggested to relax smooth muscle cells by elevating cAMP. However, little is known about the role of cilostazol in relaxation of vascular tone, specially endothelium-dependent vascular relaxation. In the present study, we investigated a novel effect of cilostazol, namely, the endothelialdependent relaxation of the rat thoracic aorta. The present findings suggested that cilostazol relaxes aorta both endothelium -dependently and -independently. Methods Vascular relaxation by cilostazol in vitro; Twenty-five-week-old male Sprague-Dawley rats were anaesthetized with sodium pentobarbital (50 mg/kg) and were killed by exsanguination. The thoracic aorta was rapidly removed, freed of adhering fat and connective tissue under a binocular dissecting microscope, and cut with parallel razors into ring segments approximately 3 mm wide. The segments were soaked vertically in 3 ml of Krebs bicarbonate solution of the following composition (mM): NaCl 115.3, KCl 4.9, CaCl2 1.4, MgSO4 1.2, NaHCO3 25.0, glucose 11.1 and ascorbate. The lumen of the artery was gently rubbed with cotton thread to remove the endothelium. Two stainless steel wires were inserted into the lumen of the aortic ring. One wire was connected to a transducer and the other anchored to a plastic holder. The segment was then placed in a 5 ml organ bath at 348C containing oxygenated (95% O2 and 5% CO2) Krebs bicarbonate solution. The preparations were equilibrated for 120 min prior to the start of the experiments and the resting tension was maintained at 1.0 g. To measure relaxation, the segment was contracted initially with phenylephrine at a concentration designed to induce 80% of maximal contraction (EC80: 0.3 mM for arteries with endothelium and 0.1 mM for those without endothelium). Responses were recorded isometrically with a force displacement transducer (SB 1TH, Nihon Kohden, Tokyo, Japan). A 7 – 20 ml volume of cilostazol was added to the 5 ml organ bath, and relaxations were measured as percentages of the contractions induced by the EC80 concentration of phenylephrine. Relaxations of aortic rings, with or without endothelium, were compared following cumulative application of cilostazol. Male diabetic (Otsuka Long-Evans Tokushima Fatty; OLETF) rats were fed standard rat chow (Oriental Yeast, Tokyo, Japan) and tap water ad libitum until the age of 16 weeks, when they were randomly assigned to two groups of 10 rats each: those with normal diet (OLETF-Normal group) and those with a cilostazol (40 mg/kg/day) supplemented diet (OLETF-Cilostazol group). The serum concentration of cilostazol in rats was about 1.5 mg/ml, which is equivalent to those of patients treated with cilostazol. Body weight was not significantly different among groups at the beginning of the experiment. Non-diabetic (Long-Evans Tokushima Otsuka; LETO) rat strain was used as the age-matched control (LETO-Normal and LETOCilostazol groups, respectively). NO measurement was by the electron paramagnetic resonance spin trapping method. Porcine thoracic aorta in rings were used in this study. Porcine serum albumin (33 mg/ml, final
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concentration), FeSO4 (3.3 mM), sodium N,N-diethyldithiocarbamate trihydrate (3.3 mM), the sample, and excess Na2S2O4 (2 M) were introduced into a 4 mm diameter electron paramagnetic resonance (EPR) tube (LST-5HS, Labotec Co.), and after 5 min, the mixture was frozen at liquid nitrogen temperature (77 K). EPR spectra were measured in a JEOL FE1X spectrometer under the following conditions: microwave frequency, 9.15 GHz, microwave power, 20 mW, modulation frequency, 100 kHz, modulation amplitude, 0.63 mT, magnetic field, 320 6 25 mT, response time, 0.1 s, sweep time 50 mT/4 min, temperature 77 K. The concentration of NO trapped was determined by comparing the intensities of the first lowfield derivative EPR signal heights and their amplitudes [10]. NO22 measurement was by the modified Griess method. To determine the urinary NO22 nitrite excretion rate, 24 h urine samples from 25-week-old rats were collected in bottles containing an antibiotic solution (1 mg/ml of penicillin G, 1 mg/ml of streptomycin and 0.25 mg/ ml of amphotericin B). NO32 in urine was converted into NO22 with nitrate reductase and measured with the Griess reagent (Cyman Chemical, Ann Arbor, MI). Data in figures are expressed as the mean 6 S.E.. Data were analysed by post hoc test (Scheffe’s test and Bonferroni’s test) followed by the one way analysis of variance (ANOVA) and/or Student’s t-test. A value at ,0.05 was accepted statistically significant. Results Figure 1 shows the relaxant effects of cilostazol in vitro on rat thoracic aorta with or without endothelium. Vasodilative effects of cilostazol were dose-dependent at concentrations of 0.3 mM to 100 mM in the rings with or without endothelium, suggesting that cilostazol-
Fig. 1. Effects of endothelium on cilostazol-induced relaxation on ring segments of the rat thoracic aorta. The preparations were contracted with phenylephrine at concentrations corresponding to the EC80. Vasodilative responses are expressed as percentages of the phenylephrine-induced contractions. Values are means 6 S.E. for 4 separate preparations.
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Fig. 2. Effects of cilostazol, nitroprusside and L-NNA on ring segments of rat thoracic aorta. Cilostazol was supplemented for nine-weeks at 40 mg/kg/day on rats. The ring segments were preincubated for 30 min presence or absence of 1024 M sodium nitroprusside(SNP) or 5 3 1024 M NG-nitro L-arginine (L-NNA). The preparations were contracted with phenylephrine at concentrations corresponding to the EC80. Vasodilative responses are expressed as percentages of phenylephrine-induced contraction. Values are means 6S.E. for 7–10 separate preparations. * P,0.05, ** P,0.01, Significantly less than the control value. *** P,0.01, Significantly greater than the control value. (ANOVA, Scheffe’s test)
induced relaxation was, in part, dependent on endothelium. The EC50 value was 5 6 0.3 mM in preparations with endothelium, and 10 6 0.7 mM in those without (n 5 8). To determine whether cilostazol activates NO/cGMP pathway, we examined the effects of cilostazol on guanylate cyclase activator nitroprusside-induced relaxation of thoracic aorta tone. As shown in Figure 2, cilostazol failed to potentiate the relaxation of thoracic aorta tone induced by 1024 M sodium nitroprusside(SNP). Moreover, phenylephrine-induced contraction of the thoracic aorta was potentiated by 5 3 1024 M NG-nitro L-arginine (L-NNA), a competitive inhibitor of NO synthase. Phenylephrine-induced contraction was increased in the presence of L-NNA, which blocks the basal secretion of NO. Thus, the difference between contraction in the presence and the absence of L-NNA can be assumed to be due to basal NO secretion. Basal NO secretion tended to be greater in the rings from rats treated with cilostazol compared with those without treatment. To determine whether cilostazol can produce NO in thoracic aorta we measured NO in porcine thoracic aortic ring segments, in vitro, using electron paramagnetic resonance (EPR) spectroscopy. In the preliminary experiments, we tried the production of NO in the thoracic aortic rings of rats using EPR spectroscopy. However, it was failed to detect the NO levels in these ring segments because of too small ring segments from rats to detect by our EPR spectroscopy method. As shown in Figure 3, cilostazol at concentration of 1024 M increased NO production from the thoracic aorta. However, it’s production was not significantly stimulated
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Fig. 3. Effects of cilostazol on nitric oxide production in porcine thoracic aortic ring segments. The porcine thoracic aortic ring segments were incubated for 30 min at 348C with or without 102621024M cilostazol. NO levels were determined as described in Materials and Methods. Values are mean 6 S.E. for 6 separate experiments. * P,0.01, Significantly greater than the control value. (ANOVA, Bonferroni’s test)
by up to 1025 M cilostazol. Cilostazol induced NO production from the thoracic aortic ring segments was inhibited by L-NNA (data not shown). These findings indicated that the production of NO induced by cilostazol may partially regulate the vasodilation of rat thoracic aorta. We also measured the urinary secretion of NO and its metabolites by the Griess method. The NO22 secretion was not significantly greater in those treated with cilostazol than in those without treatment in non-diabetic rats. We also treated diabetic rats, because this drug is used in patients with diabetes and decreased endothelial function. The basal production of NO was significantly greater in non-diabetic (LETO) rats than in the diabetic (OLETF) rats. The urinary secretion of NO22 was significantly decreased in diabetic rats compared with the nondiabetic rats. Cilostazol treatment significantly increased the urinary secretion of NO in diabetic rats, although it was still smaller than that in non-diabetic rats (Fig. 4). Discussion The findings of present study indicated that cilostazol induced endothelium-dependent relaxation of the thoracic aorta, in addition to its direct relaxant effect on smooth muscle. The contribution of the endothelium was more evident at lower concentrations, which corresponds to clinical doses. In diabetic rats, in which the endothelial function was partly disturbed, cilostazol also increased NO production, suggesting the usefulness of this drug in diabetic patients. Cilostazol is an inhibitor of phosphodiesterase III and therefore prevents the breakdown of cAMP. The mechanisms of vasodilation by increased cAMP in vascular smooth muscle cells are thought to be the following: (1) stimulation of cAMP-dependent protein kinase, which activates a sarcolemmal calcium pump, (2) stimulation of Na1/K1-ATPase, which results in hyperpolarization and removal of intracellular sodium and calcium, (3) augmantation of dephosphorylation of the myosin light chain [11]. In the current findings (Fig. 1), cilostazol produced relaxation of
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Fig. 4. Effects of cilostazol on nitrites secretion in diabetic and non-diabetic rat urine. Cilostazol was supplemented for nine-weeks at 40 mg/kg/day on the diabetic (OLETF) and non-diabetic (LETO) rats. Nitrites levels were determined as described in Materials and Methods. Values are means 6 S.E. from 7 separate experiments, expressed as mmol/10hr/100g body weight. Urinary excretion of nitrites was decreased in OLETF rats (* P,0.01). Cilostazol supplementation improved urinary excretion of nitrites in OLETF rats (P,0.05), but not in LETO (Student’s t-test). * P,0.01 OLETF vs. LETO. (ANOVA, Bonferroni’s test)
the thoracic aorta without endothelium. Therefore, it is possible that vasodilation of the thoracic aorta may be induced by an increase in cAMP levels in vascular smooth muscle cells. It has not previously been established whether the endothelium contributes to the effect of cilostazol. The findings of the present study indicated that in the presence of endothelium relaxation occurs at lower concentrations, and its effect could be suppressed by the addition of NO synthase inhibitor, L-NNA. Thus, the involvement of the NO pathway in the vasodilative effect of cilostazol is suggested. In addition, in rats chronically treated with cilostazol, basal NO production as well as urinary secretion of NO22 was greater than those without treatment, suggesting that cilostazol increased NO secretion from endothelium. This effect was also observed in diabetic rats although improvement was not complete. Previous studies have reported that isoprenaline acts through a beta-adrenoceptor on the endothelium to raise cAMP in rat thoracic aorta, and that this may, directly or indirectly, release NO to evoke vascular relaxation [12]. Uncontrast, cilostazol increases the cellular concentration of cAMP, but not that of cGMP, in rabbit arterial tissues [1, 5]. Furthermore cilostazol has a beneficial effect on diabetic neuropathy by improving Na1/K1-ATPase activity via directly increasing cAMP and NO production in human neuroblastoma cells [13]. The findings of the present study showed that cilostazol induced NO production and that their vasodilative effect in the presence of endothelium was reversed by L-NNA (Figs. 3 and 4). The changes in urinary excretion of NO22 1 NO32 as a measurement of changes in NO production has been guestiond in a previous study [14]. However, our previous report have suggested that an in-
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crease in urinary excretion of NO22 is dependent on the elevation of NO production in diabetic rats [15]. Moreover, cilostazol enhanced the urinary secretion of NO22 1 NO32 in diabetic rats (Fig. 4). An increase in the NO level in the endothelium induces production of cGMP [16]. In the present study, we did not measure cGMP production in the rat thoracic aorta. Therefore, the role of cGMP in cilostazol-induced relaxation of the rat thoracic aorta remains to be clarified. NO has a variety of functions, including as a vasorelaxant, an anticoagulant and also antiatherogenic actions. Increased production of NO by cilostazol is expected to enhance these functions and inhibit smooth muscle proliferation and development of atherosclerosis. References 1.
2.
3. 4.
5. 6. 7. 8.
9. 10. 11. 12. 13.
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