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Sarpogrelate protects against high glucose-induced endothelial dysfunction and oxidative stress
International Journal of Cardiology 147 (2011) 383–387
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International Journal of Cardiology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / i j c a r d
Sarpogrelate protects against high glucose-induced endothelial dysfunction and oxidative stress Yan-Ming Sun a,1, Ying Su b,1, Hong-Bo Jin c,⁎, Jia Li a, Sheng Bi d a
Department of Cardiac Care Unit, the First Affiliated Hospital of Harbin Medical University, Harbin, 150001, China Department of Endocrinology, the First Affiliated Hospital of Harbin Medical University, Harbin, 150001, China Department of Physiology, Harbin Medical University, Harbin, 150081, China d Central Laboratory, the First Affiliated Hospital of Harbin Medical University, Harbin, 150001, China b c
a r t i c l e
i n f o
Article history: Received 26 March 2009 Received in revised form 1 August 2009 Accepted 1 September 2009 Available online 2 November 2009 Keywords: Sarpogrelate hydrochloride Endothelium-dependent relaxation High glucose Superoxide dismutase L-arginine Nitric oxide
a b s t r a c t This study was designed to investigate the effect of sarpogrelate hydrochloride on impaired endotheliumdependent relaxation (EDR) induced by high glucose in isolated rat aorta. Both acetylcholine-induced EDR and sodium nitroprusside-induced endothelium-independent relaxation (EIR) were measured after the rings were exposed to high glucose in the absence and presence of sarpogrelate hydrochloride. Co-incubation of aortic rings with high glucose for 24 h resulted in a significant inhibition of EDR, but had no effects on EIR. After incubation of the rings in the co-presence of sarpogrelate hydrochloride with high glucose for 24 h, sarpogrelate hydrochloride significantly attenuated impaired EDR. This protective effect of sarpogrelate hydrochloride was abolished by NG-nitro-L-arginine methyl ester. Sarpogrelate hydrochloride significantly decreased superoxide anion (O− 2 ) production and increased superoxide dismutase (SOD) activity and the nitric oxide (NO) release. These results suggest that sarpogrelate hydrochloride can restore impaired EDR induced by high glucose in isolated rat aorta, which may be related to scavenging oxygen free radicals and enhancing NO production. © 2009 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Endothelial barrier dysfunction plays a pivotal role in the pathogenesis of diabetic vascular complications [1]. Serotonin (5-hydroxytryptamine, 5-HT), a major non-peptidergic substance released from activated platelets, mediates vasoconstriction and induces the activation of other platelets, which may promote coronary events by adhering to unstable atherosclerotic plaque and initiating thrombotic complications [2,3]. Serum 5-HT concentrations are elevated in diabetes [4,5], suggesting that it may be involved in the development of diabetic complications, although there is no clear clinical evidence for its involvement in the pathogenesis of diabetic complications. Recent studies have revealed that 5-HT has produced both contraction and relaxation of the vascular smooth muscles [6–9]. Multiple 5-HT receptors are involved in mediating these effects. 5-HT-induced vasoconstrictions of arteries are mainly mediated by 5-HT2A receptor subtype [10]. In porcine coronary and pulmonary artery, 5-HT causes endothelium-dependent relaxation (EDR) responses [11–14]. A selective 5-HT2A receptor antagonist (sarpogrelate) was introduced as a therapeutic agent for the treatment of ischemic diseases associated with ⁎ Corresponding author. Harbin Medical University, Department of Physiology, Harbin Medical University, 194 Xuefu Rd. Nangang District, Harbin, P.R., 150081, China. Tel.: +86 451 86641102. E-mail address: [email protected] (H.-B. Jin). 1 These authors contributed equally to this study. 0167-5273/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.ijcard.2009.09.539
thrombosis [15]. Sarpogrelate hydrochloride inhibits thrombus formation [16,17], suppresses platelet aggregation [18,19], inhibits 5-HT induced coronary artery spasm [20] and contraction of coronary artery in the porcine model mediated by 5-HT and α-methylserotonin [21] and also inhibits vascular smooth muscle cell proliferation [22]. High glucose in vitro or in vivo has been reported to inhibit acetylcholine (ACh)-mediated endothelium-dependent relaxation responses [23], to impair the biological synthesis pathway of nitric oxide (NO) [24], and to generate reactive oxygen species [25]. However, it is unclear whether sarpogrelate hydrochloride can improve impaired EDR evoked by high glucose. In the present study, we sought to examine whether sarpogrelate hydrochloride exerts beneficial effect on high glucose-induced endothelial dysfunction in isolated rat aorta. 2. Materials and methods 2.1. Drugs and chemicals Sarpogrelate hydrochloride was obtained from Mitsubishi Chemical Corporation (Tokyo, Japan). ACh, sodium nitroprusside (SNP), NG-nitro-L-arginine methyl ester (LNAME), phenylephrine, L-arginine, and superoxide dismutase (SOD) were purchased from Sigma Chemical Co (Saint Louis, Mo, USA). The rest of drugs were prepared in bidistilled water and diluted with Krebs buffer immediately before the experiment. 2.2. Organ chamber experiment Adult Sprague-Dawley rats of both genders (body weight: 180–200 g), which were supplied by the Medical Experimental Animal Center of Harbin Medical University,
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China, were used in accordance with the Guidelines on the Care and Use of Laboratory Animals issued by the Chinese Council on Animal Research and the Guidelines of the Animal Care. The study was approved by the ethical committee of Harbin Medical University. The rats were killed by exsanguination under anesthesia with pentobarbital sodium (30 mg/kg). The thoracic aorta was removed and placed immediately in Krebs bicarbonate buffer of the following composition (in mM): NaCl 118.3; KCl 4.7; CaCl2 2.5; MgSO4 1.2; KH2PO4 1.2; NaHCO3 25.0; glucose 5.5. The aortic segment was cut into 3–4 mm rings. The adhering perivascular tissue was carefully removed. The rings were suspended horizontally between two stainless-steel stirrups in organ chambers filled with 5 mL Krebs' solution at 37 °C, aerated continuously with a mixture of 95% O2 and 5% CO2. One stirrup was connected to an anchor and the other to a force transducer for recording of isometric tension. Rings were equilibrated for 90 min under 2 g resting tension. Then the rings were contracted with a submaximal concentration of phenylephrine (1 µM). After reaching a stable contraction plateau (approximately 15 min), rings were relaxed with cumulative concentrations of ACh (0.03–3 µM). The endothelium was considered to be intact when ACh caused at least a 75% relaxation of rings. 2.3. Experimental protocol Rings were incubated with normal (5.5 mM) and high concentrations (11 mM or 25 mM) of glucose for 24 h. In the sarpogrelate hydrochloride groups, rings were incubated with various concentrations of sarpogrelate hydrochloride (0.1–10 µM) plus high glucose (25 mM) for 24 h, respectively. To investigate whether increased production of superoxide anion and decreased NO synthesis exert detrimental effects and whether oxygen free radicals and enhanced NO production contribute to the protective effect of sarpogrelate hydrochloride, some rings were co-incubated in high glucose in the presence of SOD (200 U/mL), L-arginine (3 mM) or D-arginine (3 mM) for 24 h, the others were incubated with L-NAME (10 µM) for 24 h. We also compared the effects of SOD, L-arginine, or D-arginine alone or in a combination of SOD and L-arginine on EDR of aortic rings with those of sarpogrelate. After the above incubations periods, all rings were washed repeatedly and then recontracted with phenylephrine (1 µM) and relaxation responses to ACh (0.03–3 µM) were repeated. Before finishing the experiment, relaxation to SNP at the plateau phase of the phenylephrine contraction was also tested. 2.4. Determination of aortic superoxide dismutase Aortic superoxide production was determined as described previously [26]. Briefly, equilibrated segments of thoracic aorta were incubated at 37 °C in albuminbuffer ( pH7.4 ) of the following composition (in mM): Na+ 144.93, K+ 7.23, Cl− 2− 138.77, H2PO− 4 4.55, HPO4 8.03, glucose 5.5 and bovine serum albumine (0.1%, weight volume− 1). This buffer was enriched with lucigenin (0.5 mM) and superoxide production was calculated from chemiluminescence measurements. 2.5. Measurement of NO production Quantitative determination of NO production was carried out as described by Zhu et al [27]. Aortic segments were loaded with the indicator by incubating them for 30 min at 37 °C in HEPES-buffered PSS (pH 7.4) containing 5 µM 4,5-diaminofluorescein. Once loading was finished, the vessels were rinsed three times and placed in a chamber containing HEPES-buffered PSS maintained at 37 °C with a water bath. Larginine (100 µM) was added to the chamber during measurements to ensure adequate substrate availability for NO synthase.
3. Results 3.1. Effect of high glucose on relaxation of aortic rings There were no significant differences in relaxation responses to ACh in rat aortic rings between any of the pretreated groups (data not shown). After incubation with rings for 24 h, ACh evoked a significant concentration-dependent relaxation in control group (Fig. 1 and Table 1). As shown in Fig. 1 and Table 1, the maximal relaxation (Emax) value was decreased (p < 0.01), and the EC50 values were increased (p < 0.01) significantly in the high glucose group (25 mM) compared with the control group. Exposure of aortic rings to high glucose for 24 h significantly attenuated relaxation responses to ACh in a dosedependent manner compared with the control group (p < 0.05) (Fig. 1). However, there were no significant differences between mannitol (25 mM) group and the control group in Emax and EC50 (p > 0.05) (Fig. 1 and Table 1). The SNP-induced EIR was not different between the high glucose group and the control group (p > 0.05) (Fig. 2). 3.2. Effects of sarpogrelate hydrochloride on the inhibition of EDR induced by high glucose As shown in Fig. 3 and Table 1, treatment of aortic rings with sarpogrelate hydrochloride (0.1–10 µM) significantly ameliorated the impaired EDR elicited by high glucose in a dose-dependent manner. However, treatment of the rings with L-arginine (3 mM) partially prevented the high glucose-induced impairment of EDR. A similar effect was observed after treatment of aortic rings with SOD (200 U/ mL, Fig. 4 and Table 1). However, in the combined presence of SOD and L-arginine, the high glucose-induced inhibition of EDR was almost completely abolished, which was very similar to the effect of sarpogrelate hydrochloride (10 µM). In addition, incubation of aortic rings in the presence of both L-NAME (10 µM) and sarpogrelate hydrochloride (10 µM) significantly abolished the protective effect of sarpogrelate hydrochloride on the impairment of EDR induced by high glucose (Fig. 5 and Table 1). The relaxation responses to ACh of aortic rings were not significantly different among the three groups (the control group, sarpogrelate group, and SOD plus L-arginine group) (Fig. 6 and Table 1). All these results suggested that both the deleterious effects of high glucose and the protective effects of sarpogrelate hydrochloride was correlated with oxidative stress and NO production/release in endothelial cells.
2.6. Detection of superoxide anion Superoxide anion (O− 2 ) production by endothelium functional segments was measured as lucigenin-derived chemiluminescence in the presence of 5 μM lucigenin after stimulation with 100 μM nicotinamine adenine dinucleotide phosphate reduced form (NADPH) [28]. Each tissue sample was placed into 2 mL modified Krebs–Ringer solution, pH 7.40, and prewarmed to 37 °C for 1 h under a supply of carbogen. Immediately before measurement, rings were transferred into scintillation tubes filled with 500 µL Krebs–Hepes solution, pH 7.40, at 37 °C. Coelenterazine was added to give a final concentration of 5 µmol/L. L-NAME (1 mM) was used to inhibit the reaction of O− 2 with NO. Other NOS inhibitors interfere with NADPH-dependent reduction [28]. To − estimate the true O2 production, the values with SOD were subtracted from those obtained in its absence. 2.7. Statistical analysis All data were expressed as mean ± SEM. Relaxation was expressed as percentage of the active tension generated by phenylephrine. The half maximum effective concentration (EC50) response to ACh was estimated by linear regression from log concentration-effect curves. Two-group mean comparisons were made using twotailed Student's t test. Comparisons between every two groups were performed using one-way ANOVA. Differences were considered statistically significant at p < 0.05.
Fig. 1. Effects of high glucose on endothelium-dependent relaxation in response to acetylcholine. Values are mean±SEM. n=8. ⁎p<0.05, ⁎⁎p<0.01 versus the control group.
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Table 1 Effects of drugs on the Emax and EC50 values for acetylcholine-induced relaxation in isolated aortic rings. Groups
n
ACh Emax (%)
ACh EC50 (nM)
Control Mannitol (25 mM) HG (11 mM) HG (25 mM) Sarpogrelate (10 μM) HG (25 mM) + Sarpogrelate (0.1 μM) HG (25 mM) + Sarpogrelate (1.0 μM) HG (25 mM) + Sarpogrelate (10 μM) HG (25 mM) + SOD (200 U/mL) HG (25 mM) + L-arginine (3 mM) HG (25 mM) + D-arginine (3 mM) HG (25 mM) + L-NAME (10 μM) + Sarpogrelate (10 μM) HG (25 mM) + SOD (200 U/mL) + L-arginine (3 mM)
8 8 8 8 8 8 8 8 8 8 8 8 8
90.87 ± 1.97 87.64 ± 1.86 73.18 ± 1.51† 49.21 ± 1.65‡ 80.23 ± 1.49 50.07 ± 1.87 60.32 ± 1.51⁎ 88.23 ± 2.05⁎⁎ 78.61 ± 1.96⁎⁎§ 74.12 ± 1.85⁎⁎§ 52.67 ± 1.59 52.21 ± 2.08‡ 86.29 ± 2.31⁎⁎
101.43 ± 1.12 108.16 ± 1.23 156.71 ± 1.66† 169.29 ± 1.36‡ 107.37 ± 1.36 142.64 ± 1.38 128.61 ± 1.73⁎ 109.25 ± 1.46⁎⁎ 124.33 ± 1.62⁎⁎§ 127.54 ± 1.71⁎⁎§ 167.49 ± 1.53 169.82 ± 1.49‡ 112.56 ± 1.69⁎⁎
The Emax to acetylcholine (3 mM) was expressed as percentage of phenylephrine-induced contraction. EC50 reaction to acetylcholine was calculated by linear regression from log concentration-effect curves of acetylcholine. Data are expressed as means± SEM. n = 8. †p < 0.05, ‡p < 0.01 versus the control group; *p < 0.05, **p < 0.01 versus the high glucose group (25 mM); §p < 0.05 versus SOD plus L-arginine group. HG, high glucose; SOD, superoxide dismutase; L-NAME, NG-nitro-L-arginine methyl ester; ACh, acetylcholine.
3.3. Effects of sarpogrelate hydrochloride on biochemical index High glucose decreased SOD activity and the NO release, and increased O2− production in aortic tissue (p< 0.05). Treatment with sarpogrelate hydrochloride significantly prevented the increase in O2− production, and increased SOD activity and the NO release (p< 0.05), however, mannitol had no effects on SOD, NO and O2− production (p> 0.05) (Table 2).
subjects and its improvement seems to predict treatment-induced risk reduction [31]. However, oxidative stress that occurs in diabetes may result from a variety of other abnormalities that can emerge with
4. Discussion The aim of this study was to determine whether sarpogrelate hydrochloride exerts beneficial effect on high glucose-induced endothelial dysfunction in isolated rat aorta. The results from this study confirmed that sarpogrelate was able to protect vascular endothelium from dysfunction induced by high glucose. The results of this study showed the following a new finding: sarpogrelate hydrochloride attenuated the impairment of EDR elicited by high glucose partially through scavenging oxygen free radicals. Diabetes mellitus is associated with endothelial dysfunction and oxidative stress [29]. Endothelial dysfunction represents a very early step in the development of atherosclerosis [30]. The reduced NO mediated, endothelium-dependent vasodilatation occurring in endothelial dysfunction is a predictor of cardiovascular risk in high risk
Fig. 2. Effects of high glucose on endothelium-independent relaxation in response to sodium nitroprusside. Values are mean ± SEM. n = 8.
Fig. 3. Effects of sarpogrelate on the impaired endothelium-dependent relaxation in isolated aortic rings. Values are mean ± SEM. n = 8. #p < 0.05, ##p < 0.01 versus the control group; ⁎p < 0.05, ⁎⁎p < 0.01 versus the high glucose group.
Fig. 4. Effects of SOD and L-arginine on the impaired endothelium-dependent relaxation in isolated aortic rings. Values are mean ± SEM. n = 8. #p < 0.05, ##p < 0.01 versus the control group; ⁎p < 0.05, ⁎⁎p < 0.01 versus the high glucose group.
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Fig. 5. Effects of L-NAME on the protection of sarpogrelate against impairment of endothelium-dependent relaxation induced by high glucose. Values are mean ± SEM. n = 8. ⁎p < 0.05, ⁎⁎p < 0.01 versus the high glucose group; #p < 0.05, ##p < 0.01 versus the control group.
Fig. 6. Effects of L-arginine and SOD on the impaired endothelium-dependent relaxation in isolated aortic rings. Data are the mean ± SEM. n = 8. #p < 0.05, ##p < 0.01 versus the control group; ⁎p < 0.05, ⁎⁎p < 0.01 versus the high glucose group.
a more rapid time course, including decreased activities of antioxidant enzymes and increased superoxide production [32]. The vascular endothelium releases various vasodilators, such as NO, prostacyclin and endothelium-derived hyperpolarizing factor, in addition to a variety of vasoconstrictors [33]. NO plays an important role in the regulation of vascular tone, inhibition of platelet aggregation, and suppression of smooth muscle cell proliferation [34].
Table 2 Biochemical measurements of isolated aortic segments in the different groups. Groups
SOD activity (kNU·g− 1 protein)
NO (mM·g− 1 protein)
O− 2 (cm2 min− 1)
Control (5.5 mmol/L) Mannitol (25 mmol/L) Sarpogrelate (10 μmol/L) High Glucose (25 mmol/L) HG + Sarpogrelate (10 μmol/L)
171.29 ± 1.53 167.32 ± 1.71 173.08 ± 1.79
0.88 ± 0.011 0.85 ± 0.009 0.84 ± 0.008
5.09 ± 0.041 5.31 ± 0.062 5.23 ± 0.059
0.41 ± 0.006⁎
9.27 ± 0.053⁎⁎
0.91 ± 0.009§
6.03 ± 0.072§
91.84 ± 1.45 ⁎⁎ 163.15 ± 1.58§
Values are expressed as mean ± SEM. *p < 0.05, **p < 0.01 versus the control group; §p < 0.05 versus the high glucose group. HG, high glucose; SOD, superoxide dismutase; NO, nitric oxide; O− 2 , superoxide anion.
The vascular endothelium releases various vasodilators, such as NO, prostacyclin and endothelium-derived hyperpolarizing factor, in addition to a variety of vasoconstrictors [33]. Previous studies have demonstrated that 5-HT is sequestered and released by endothelial cells, acts as an endothelial cell mitogen, promotes the release of NO and increases eNOS mRNA, which may mean the increase of NO and the 2+ scavenge of O− and caveolin-1 in 2 [35]. eNOS is controlled by Ca caveolae. eNOS is located within the plasma membrane microdomain caveolae, where it is complexed with the coat protein for this organelle, termed CAV-1. CAV-1 is a 22-kDa protein that decorates the cytoplasmic surface of caveolae and serves as the primary structural component of caveolae (50 to 100 nm invaginations of the plasma membrane), where it acts as a physiological inhibitor of eNOS [36]. Thus, the reduction of NO release in diabetes can be ascribed to a reduction in eNOS activity through an enhanced expression of caveolin-1 [37]. Sarpogrelate hydrochloride is a selective 5-HT2A antagonist and is clinically used for the treatment of cutaneous ulcers and ischemic changes associated with atherosclerosis [38,39]. It strongly inhibits the effects of serotonin such as platelet aggregation, vasoconstriction and vascular smooth muscle proliferation [39]. Sarpogrelate hydrochloride inhibits human platelet aggregation induced by collagen and secondary aggregation by adenosine diphosphate or adrenaline by 50% (IC50) at a concentration of 1 μM, and this effect was potentiated for aggregation induced by collagen plus serotonin, with an IC50 of 0.1 mM [40]. Furthermore, a major metabolite of sarpogrelate, M-1, was approximately 10 times more potent than the unaltered substance [40]. Sarpogrelate hydrochloride may act to increase NO production and hence improve blood flow via NO dependant vasodilatation [41]. The previous studies have shown that the relaxations induced by 5-HT in the arteries including porcine coronary arteries were mediated by activation of 5-HT receptors localized on the endothelial cells [11,12]. In addition, the recent studies indicated that sarpogrelate improved endothelial function in balloon-injured porcine coronary artery [42] and aorta in cholesterolfed rabbits [43] and enhanced ACh-induced, endothelium-dependent NO-mediated relaxation in the vein grafts [44]. The present study demonstrated that high glucose significantly inhibited EDR, and this inhibition of EDR was completely abolished by treating the aortic rings with sarpogrelate (10 µM). Furthermore, our data showed that the inhibition of EDR caused by high glucose was partially improved by treatment of aortic rings with L-arginine or SOD alone, and D-arginine had no effect on the impaired EDR. But treating of the aortic rings with SOD and L-arginine together, the inhibition of EDR was also completely prevented, which was very similar to the effect of sarpogrelate hydrochloride. In addition, incubation of the rings with high glucose had no effect on the SNP-induced EIR. These results indicated that increasing production of oxygen free radicals and reducing NO may contribute to the deleterious effects of high glucose on vascular endothelial function and sarpogrelate hydrochloride was an effective pharmacological approach to prevent the endothelial dysfunction induced by high glucose, and may exert its beneficial effect by both scavenging oxygen free radicals and increasing NO production in endothelial cells. In the present work, we showed the evidence that sarpogrelate hydrochloride can restore impaired EDR induced by high glucose in isolated rat aorta. We postulated that this beneficial effect may be related to scavenging oxygen free radicals and enhancing NO production. It was found that sarpogrelate enhanced eNOS expression and increased NO production in aorta of hypercholesterolemic rabbits [45], while it was found that sarpogrelate reduced superoxide production in neutrophils in vitro [46]. In the present experiments, we found that superoxide production was much smaller in the sarpogrelate-treated group than in the high glucose group. Superoxide is thought to generate peroxynitrite by reaction with NO, which oxidizes tetrahydrobiopterin, leading to“eNOS uncoupling”. Once “eNOS uncoupling” has been developed, eNOS becomes dysfunctional and produces superoxide rather than NO
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[47,48]. We found that sarpogrelate may inhibit the development of “eNOS uncoupling”, thus leading to an increase in the endotheliumderived NO function. However, further studies are required to definitively determine whether sarpogrelate works though an NOS dependent or independent mechanism. In conclusion, the results of the study showed for the first time that sarpogrelate hydrochloride attenuated the impairment of EDR elicited by high glucose partially through scavenging oxygen free radicals. These results provided a possible new and intriguing target for the prevention and treatment of the vascular complications of diabetes. Acknowledgement The author of this manuscript has certified that he complies with the Principles of Ethical Publishing in the International Journal of Cardiology [49]. This work was supported by grants from the Foundation of the First Affiliated of Harbin Medical University (Q08-002). References [1] Liang KW, Lee WJ, Lee WL, Chen YT, Ting CT, Sheu WH. Diabetes exacerbates angiographic coronary lesion progression in subjects with metabolic syndrome independent of CRP levels. Clin Chim Acta 2008;388:41–5. [2] Suguro T, Watanabe T, Kanome T, et al. Serotonin acts as an up-regulator of acylcoenzyme A:cholesterol acyltransferase-1 in human monocyte-macrophages. Atherosclerosis 2006;186:275–81. [3] Hayashi T, Sumi D, Matsui-Hirai H, et al. Sarpogrelate HCl, a selective 5-HT2A antagonist, retards the progression of atherosclerosis through a novel mechanism. Atherosclerosis 2003;168:23–31. [4] Jagroop IA, Mikhailidis DP. Doxazosin, an alpha1-adrenoceptor antagonist, inhibits serotonin-induced shape change in human platelets. J Hum Hypertens 2001;15:203–7. [5] Hasegawa Y, Suehiro A, Higasa S, Namba M, Kakishita E. Enhancing effect of advanced glycation end products on serotonin-induced platelet aggregation in patients with diabetes mellitus. Thromb Res 2002;107:319–23. [6] Saxena PR, Villalon CM. Cardiovascular effects of serotonin agonists and antagonists. J Cardiovasc Pharmacol 1990;15:S17–34. [7] Andersson KE. Neurophysiology/pharmacology of erection. Int J Impot Res 2001;13:S8–S17. [8] Furukawa K, Chairungsrilerd N, Ohta T, Nozoe S, Ohizumi Y. Novel types of receptor antagonists from the medicinal plant Garcinia mangostana. Nippon Yakurigaku Zasshi 1997;110:153–8. [9] Komalavilas P, Mehta S, Wingard CJ, et al. PI3-kinase/Akt modulates vascular smooth muscle tone via cAMP signaling pathways. J Appl Physiol 2001;91:1819–27. [10] Tsurumaki T, Nagai S, Bo X, Toyosato A, Higuchi H. Potentiation by neuropeptide Y of 5HT2A receptor-mediated contraction in porcine coronary artery. Eur J Pharmacol 2006;544:111–7. [11] Yang Q, Scalbert E, Delagrange P, Vanhoutte PM, O'Rourke ST. Melatonin potentiates contractile responses to serotonin in isolated porcine coronary arteries. Am J Physiol Heart Circ Physiol 2001;280:H76–82. [12] Lv PP, Fan Y, Chen WL, et al. COX-2 inhibitor nimesulide protects rat heart against oxidative stress by improving endothelial function and enhancing NO production. Sheng Li Xue Bao 2007;59:674–80. [13] Glusa E, Pertz HH. Further evidence that 5-HT-induced relaxation of pig pulmonary artery is mediated by endothelial 5-HT(2B) receptors. Br J Pharmacol 2000;130:692–8. [14] Li HF, Zhang P, Tian ZF, et al. Differential mechanisms involved in effects of genistein and 17-beta-estradiol on porcine coronary arteries. Pharmazie 2006;61:461–5. [15] Nagatomo T, Rashid M, Abul Muntasir H, Komiyama T. Functions of 5-HT2A receptor and its antagonists in the cardiovascular system. Pharmacol Ther 2004;104:59–81. [16] Ozawa H, Abiko Y, Akimoto T. A 50-year history of new drugs in Japan-the development and trends of hemostatics and antithrombotic drugs. Yakushigaku Zasshi 2003;38:93–105. [17] Yamashita T, Kitamori K, Hashimoto M, Watanabe S, Giddings JC, Yamamoto J. Conjunctive effects of the 5ht2 receptor antagonist, sarpogrelate, on thrombolysis with modified tissue plasminogen activator in different laser-induced thrombosis models. Haemostasis 2000;30:321–32. [18] Nishihira K, Yamashita A, Tanaka N, et al. Inhibition of 5-hydroxytryptamine receptor prevents occlusive thrombus formation on neointima of the rabbit femoral artery. J Thromb Haemost 2006;4:247–55. [19] Uchiyama S, Ozaki Y, Satoh K, Kondo K, Nishimaru K. Effect of sarpogrelate, a 5-HT (2A) antagonist, on platelet aggregation in patients with ischemic stroke: clinicalpharmacological dose-response study. Cerebrovasc Dis 2007;24:264–70. [20] Doggrell SA. Sarpogrelate: cardiovascular and renal clinical potential. Expert Opin Investig Drugs 2004;13:865–74.
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Contents lists available at ScienceDirect
International Journal of Cardiology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / i j c a r d
Sarpogrelate protects against high glucose-induced endothelial dysfunction and oxidative stress Yan-Ming Sun a,1, Ying Su b,1, Hong-Bo Jin c,⁎, Jia Li a, Sheng Bi d a
Department of Cardiac Care Unit, the First Affiliated Hospital of Harbin Medical University, Harbin, 150001, China Department of Endocrinology, the First Affiliated Hospital of Harbin Medical University, Harbin, 150001, China Department of Physiology, Harbin Medical University, Harbin, 150081, China d Central Laboratory, the First Affiliated Hospital of Harbin Medical University, Harbin, 150001, China b c
a r t i c l e
i n f o
Article history: Received 26 March 2009 Received in revised form 1 August 2009 Accepted 1 September 2009 Available online 2 November 2009 Keywords: Sarpogrelate hydrochloride Endothelium-dependent relaxation High glucose Superoxide dismutase L-arginine Nitric oxide
a b s t r a c t This study was designed to investigate the effect of sarpogrelate hydrochloride on impaired endotheliumdependent relaxation (EDR) induced by high glucose in isolated rat aorta. Both acetylcholine-induced EDR and sodium nitroprusside-induced endothelium-independent relaxation (EIR) were measured after the rings were exposed to high glucose in the absence and presence of sarpogrelate hydrochloride. Co-incubation of aortic rings with high glucose for 24 h resulted in a significant inhibition of EDR, but had no effects on EIR. After incubation of the rings in the co-presence of sarpogrelate hydrochloride with high glucose for 24 h, sarpogrelate hydrochloride significantly attenuated impaired EDR. This protective effect of sarpogrelate hydrochloride was abolished by NG-nitro-L-arginine methyl ester. Sarpogrelate hydrochloride significantly decreased superoxide anion (O− 2 ) production and increased superoxide dismutase (SOD) activity and the nitric oxide (NO) release. These results suggest that sarpogrelate hydrochloride can restore impaired EDR induced by high glucose in isolated rat aorta, which may be related to scavenging oxygen free radicals and enhancing NO production. © 2009 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Endothelial barrier dysfunction plays a pivotal role in the pathogenesis of diabetic vascular complications [1]. Serotonin (5-hydroxytryptamine, 5-HT), a major non-peptidergic substance released from activated platelets, mediates vasoconstriction and induces the activation of other platelets, which may promote coronary events by adhering to unstable atherosclerotic plaque and initiating thrombotic complications [2,3]. Serum 5-HT concentrations are elevated in diabetes [4,5], suggesting that it may be involved in the development of diabetic complications, although there is no clear clinical evidence for its involvement in the pathogenesis of diabetic complications. Recent studies have revealed that 5-HT has produced both contraction and relaxation of the vascular smooth muscles [6–9]. Multiple 5-HT receptors are involved in mediating these effects. 5-HT-induced vasoconstrictions of arteries are mainly mediated by 5-HT2A receptor subtype [10]. In porcine coronary and pulmonary artery, 5-HT causes endothelium-dependent relaxation (EDR) responses [11–14]. A selective 5-HT2A receptor antagonist (sarpogrelate) was introduced as a therapeutic agent for the treatment of ischemic diseases associated with ⁎ Corresponding author. Harbin Medical University, Department of Physiology, Harbin Medical University, 194 Xuefu Rd. Nangang District, Harbin, P.R., 150081, China. Tel.: +86 451 86641102. E-mail address: [email protected] (H.-B. Jin). 1 These authors contributed equally to this study. 0167-5273/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.ijcard.2009.09.539
thrombosis [15]. Sarpogrelate hydrochloride inhibits thrombus formation [16,17], suppresses platelet aggregation [18,19], inhibits 5-HT induced coronary artery spasm [20] and contraction of coronary artery in the porcine model mediated by 5-HT and α-methylserotonin [21] and also inhibits vascular smooth muscle cell proliferation [22]. High glucose in vitro or in vivo has been reported to inhibit acetylcholine (ACh)-mediated endothelium-dependent relaxation responses [23], to impair the biological synthesis pathway of nitric oxide (NO) [24], and to generate reactive oxygen species [25]. However, it is unclear whether sarpogrelate hydrochloride can improve impaired EDR evoked by high glucose. In the present study, we sought to examine whether sarpogrelate hydrochloride exerts beneficial effect on high glucose-induced endothelial dysfunction in isolated rat aorta. 2. Materials and methods 2.1. Drugs and chemicals Sarpogrelate hydrochloride was obtained from Mitsubishi Chemical Corporation (Tokyo, Japan). ACh, sodium nitroprusside (SNP), NG-nitro-L-arginine methyl ester (LNAME), phenylephrine, L-arginine, and superoxide dismutase (SOD) were purchased from Sigma Chemical Co (Saint Louis, Mo, USA). The rest of drugs were prepared in bidistilled water and diluted with Krebs buffer immediately before the experiment. 2.2. Organ chamber experiment Adult Sprague-Dawley rats of both genders (body weight: 180–200 g), which were supplied by the Medical Experimental Animal Center of Harbin Medical University,
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China, were used in accordance with the Guidelines on the Care and Use of Laboratory Animals issued by the Chinese Council on Animal Research and the Guidelines of the Animal Care. The study was approved by the ethical committee of Harbin Medical University. The rats were killed by exsanguination under anesthesia with pentobarbital sodium (30 mg/kg). The thoracic aorta was removed and placed immediately in Krebs bicarbonate buffer of the following composition (in mM): NaCl 118.3; KCl 4.7; CaCl2 2.5; MgSO4 1.2; KH2PO4 1.2; NaHCO3 25.0; glucose 5.5. The aortic segment was cut into 3–4 mm rings. The adhering perivascular tissue was carefully removed. The rings were suspended horizontally between two stainless-steel stirrups in organ chambers filled with 5 mL Krebs' solution at 37 °C, aerated continuously with a mixture of 95% O2 and 5% CO2. One stirrup was connected to an anchor and the other to a force transducer for recording of isometric tension. Rings were equilibrated for 90 min under 2 g resting tension. Then the rings were contracted with a submaximal concentration of phenylephrine (1 µM). After reaching a stable contraction plateau (approximately 15 min), rings were relaxed with cumulative concentrations of ACh (0.03–3 µM). The endothelium was considered to be intact when ACh caused at least a 75% relaxation of rings. 2.3. Experimental protocol Rings were incubated with normal (5.5 mM) and high concentrations (11 mM or 25 mM) of glucose for 24 h. In the sarpogrelate hydrochloride groups, rings were incubated with various concentrations of sarpogrelate hydrochloride (0.1–10 µM) plus high glucose (25 mM) for 24 h, respectively. To investigate whether increased production of superoxide anion and decreased NO synthesis exert detrimental effects and whether oxygen free radicals and enhanced NO production contribute to the protective effect of sarpogrelate hydrochloride, some rings were co-incubated in high glucose in the presence of SOD (200 U/mL), L-arginine (3 mM) or D-arginine (3 mM) for 24 h, the others were incubated with L-NAME (10 µM) for 24 h. We also compared the effects of SOD, L-arginine, or D-arginine alone or in a combination of SOD and L-arginine on EDR of aortic rings with those of sarpogrelate. After the above incubations periods, all rings were washed repeatedly and then recontracted with phenylephrine (1 µM) and relaxation responses to ACh (0.03–3 µM) were repeated. Before finishing the experiment, relaxation to SNP at the plateau phase of the phenylephrine contraction was also tested. 2.4. Determination of aortic superoxide dismutase Aortic superoxide production was determined as described previously [26]. Briefly, equilibrated segments of thoracic aorta were incubated at 37 °C in albuminbuffer ( pH7.4 ) of the following composition (in mM): Na+ 144.93, K+ 7.23, Cl− 2− 138.77, H2PO− 4 4.55, HPO4 8.03, glucose 5.5 and bovine serum albumine (0.1%, weight volume− 1). This buffer was enriched with lucigenin (0.5 mM) and superoxide production was calculated from chemiluminescence measurements. 2.5. Measurement of NO production Quantitative determination of NO production was carried out as described by Zhu et al [27]. Aortic segments were loaded with the indicator by incubating them for 30 min at 37 °C in HEPES-buffered PSS (pH 7.4) containing 5 µM 4,5-diaminofluorescein. Once loading was finished, the vessels were rinsed three times and placed in a chamber containing HEPES-buffered PSS maintained at 37 °C with a water bath. Larginine (100 µM) was added to the chamber during measurements to ensure adequate substrate availability for NO synthase.
3. Results 3.1. Effect of high glucose on relaxation of aortic rings There were no significant differences in relaxation responses to ACh in rat aortic rings between any of the pretreated groups (data not shown). After incubation with rings for 24 h, ACh evoked a significant concentration-dependent relaxation in control group (Fig. 1 and Table 1). As shown in Fig. 1 and Table 1, the maximal relaxation (Emax) value was decreased (p < 0.01), and the EC50 values were increased (p < 0.01) significantly in the high glucose group (25 mM) compared with the control group. Exposure of aortic rings to high glucose for 24 h significantly attenuated relaxation responses to ACh in a dosedependent manner compared with the control group (p < 0.05) (Fig. 1). However, there were no significant differences between mannitol (25 mM) group and the control group in Emax and EC50 (p > 0.05) (Fig. 1 and Table 1). The SNP-induced EIR was not different between the high glucose group and the control group (p > 0.05) (Fig. 2). 3.2. Effects of sarpogrelate hydrochloride on the inhibition of EDR induced by high glucose As shown in Fig. 3 and Table 1, treatment of aortic rings with sarpogrelate hydrochloride (0.1–10 µM) significantly ameliorated the impaired EDR elicited by high glucose in a dose-dependent manner. However, treatment of the rings with L-arginine (3 mM) partially prevented the high glucose-induced impairment of EDR. A similar effect was observed after treatment of aortic rings with SOD (200 U/ mL, Fig. 4 and Table 1). However, in the combined presence of SOD and L-arginine, the high glucose-induced inhibition of EDR was almost completely abolished, which was very similar to the effect of sarpogrelate hydrochloride (10 µM). In addition, incubation of aortic rings in the presence of both L-NAME (10 µM) and sarpogrelate hydrochloride (10 µM) significantly abolished the protective effect of sarpogrelate hydrochloride on the impairment of EDR induced by high glucose (Fig. 5 and Table 1). The relaxation responses to ACh of aortic rings were not significantly different among the three groups (the control group, sarpogrelate group, and SOD plus L-arginine group) (Fig. 6 and Table 1). All these results suggested that both the deleterious effects of high glucose and the protective effects of sarpogrelate hydrochloride was correlated with oxidative stress and NO production/release in endothelial cells.
2.6. Detection of superoxide anion Superoxide anion (O− 2 ) production by endothelium functional segments was measured as lucigenin-derived chemiluminescence in the presence of 5 μM lucigenin after stimulation with 100 μM nicotinamine adenine dinucleotide phosphate reduced form (NADPH) [28]. Each tissue sample was placed into 2 mL modified Krebs–Ringer solution, pH 7.40, and prewarmed to 37 °C for 1 h under a supply of carbogen. Immediately before measurement, rings were transferred into scintillation tubes filled with 500 µL Krebs–Hepes solution, pH 7.40, at 37 °C. Coelenterazine was added to give a final concentration of 5 µmol/L. L-NAME (1 mM) was used to inhibit the reaction of O− 2 with NO. Other NOS inhibitors interfere with NADPH-dependent reduction [28]. To − estimate the true O2 production, the values with SOD were subtracted from those obtained in its absence. 2.7. Statistical analysis All data were expressed as mean ± SEM. Relaxation was expressed as percentage of the active tension generated by phenylephrine. The half maximum effective concentration (EC50) response to ACh was estimated by linear regression from log concentration-effect curves. Two-group mean comparisons were made using twotailed Student's t test. Comparisons between every two groups were performed using one-way ANOVA. Differences were considered statistically significant at p < 0.05.
Fig. 1. Effects of high glucose on endothelium-dependent relaxation in response to acetylcholine. Values are mean±SEM. n=8. ⁎p<0.05, ⁎⁎p<0.01 versus the control group.
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Table 1 Effects of drugs on the Emax and EC50 values for acetylcholine-induced relaxation in isolated aortic rings. Groups
n
ACh Emax (%)
ACh EC50 (nM)
Control Mannitol (25 mM) HG (11 mM) HG (25 mM) Sarpogrelate (10 μM) HG (25 mM) + Sarpogrelate (0.1 μM) HG (25 mM) + Sarpogrelate (1.0 μM) HG (25 mM) + Sarpogrelate (10 μM) HG (25 mM) + SOD (200 U/mL) HG (25 mM) + L-arginine (3 mM) HG (25 mM) + D-arginine (3 mM) HG (25 mM) + L-NAME (10 μM) + Sarpogrelate (10 μM) HG (25 mM) + SOD (200 U/mL) + L-arginine (3 mM)
8 8 8 8 8 8 8 8 8 8 8 8 8
90.87 ± 1.97 87.64 ± 1.86 73.18 ± 1.51† 49.21 ± 1.65‡ 80.23 ± 1.49 50.07 ± 1.87 60.32 ± 1.51⁎ 88.23 ± 2.05⁎⁎ 78.61 ± 1.96⁎⁎§ 74.12 ± 1.85⁎⁎§ 52.67 ± 1.59 52.21 ± 2.08‡ 86.29 ± 2.31⁎⁎
101.43 ± 1.12 108.16 ± 1.23 156.71 ± 1.66† 169.29 ± 1.36‡ 107.37 ± 1.36 142.64 ± 1.38 128.61 ± 1.73⁎ 109.25 ± 1.46⁎⁎ 124.33 ± 1.62⁎⁎§ 127.54 ± 1.71⁎⁎§ 167.49 ± 1.53 169.82 ± 1.49‡ 112.56 ± 1.69⁎⁎
The Emax to acetylcholine (3 mM) was expressed as percentage of phenylephrine-induced contraction. EC50 reaction to acetylcholine was calculated by linear regression from log concentration-effect curves of acetylcholine. Data are expressed as means± SEM. n = 8. †p < 0.05, ‡p < 0.01 versus the control group; *p < 0.05, **p < 0.01 versus the high glucose group (25 mM); §p < 0.05 versus SOD plus L-arginine group. HG, high glucose; SOD, superoxide dismutase; L-NAME, NG-nitro-L-arginine methyl ester; ACh, acetylcholine.
3.3. Effects of sarpogrelate hydrochloride on biochemical index High glucose decreased SOD activity and the NO release, and increased O2− production in aortic tissue (p< 0.05). Treatment with sarpogrelate hydrochloride significantly prevented the increase in O2− production, and increased SOD activity and the NO release (p< 0.05), however, mannitol had no effects on SOD, NO and O2− production (p> 0.05) (Table 2).
subjects and its improvement seems to predict treatment-induced risk reduction [31]. However, oxidative stress that occurs in diabetes may result from a variety of other abnormalities that can emerge with
4. Discussion The aim of this study was to determine whether sarpogrelate hydrochloride exerts beneficial effect on high glucose-induced endothelial dysfunction in isolated rat aorta. The results from this study confirmed that sarpogrelate was able to protect vascular endothelium from dysfunction induced by high glucose. The results of this study showed the following a new finding: sarpogrelate hydrochloride attenuated the impairment of EDR elicited by high glucose partially through scavenging oxygen free radicals. Diabetes mellitus is associated with endothelial dysfunction and oxidative stress [29]. Endothelial dysfunction represents a very early step in the development of atherosclerosis [30]. The reduced NO mediated, endothelium-dependent vasodilatation occurring in endothelial dysfunction is a predictor of cardiovascular risk in high risk
Fig. 2. Effects of high glucose on endothelium-independent relaxation in response to sodium nitroprusside. Values are mean ± SEM. n = 8.
Fig. 3. Effects of sarpogrelate on the impaired endothelium-dependent relaxation in isolated aortic rings. Values are mean ± SEM. n = 8. #p < 0.05, ##p < 0.01 versus the control group; ⁎p < 0.05, ⁎⁎p < 0.01 versus the high glucose group.
Fig. 4. Effects of SOD and L-arginine on the impaired endothelium-dependent relaxation in isolated aortic rings. Values are mean ± SEM. n = 8. #p < 0.05, ##p < 0.01 versus the control group; ⁎p < 0.05, ⁎⁎p < 0.01 versus the high glucose group.
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Fig. 5. Effects of L-NAME on the protection of sarpogrelate against impairment of endothelium-dependent relaxation induced by high glucose. Values are mean ± SEM. n = 8. ⁎p < 0.05, ⁎⁎p < 0.01 versus the high glucose group; #p < 0.05, ##p < 0.01 versus the control group.
Fig. 6. Effects of L-arginine and SOD on the impaired endothelium-dependent relaxation in isolated aortic rings. Data are the mean ± SEM. n = 8. #p < 0.05, ##p < 0.01 versus the control group; ⁎p < 0.05, ⁎⁎p < 0.01 versus the high glucose group.
a more rapid time course, including decreased activities of antioxidant enzymes and increased superoxide production [32]. The vascular endothelium releases various vasodilators, such as NO, prostacyclin and endothelium-derived hyperpolarizing factor, in addition to a variety of vasoconstrictors [33]. NO plays an important role in the regulation of vascular tone, inhibition of platelet aggregation, and suppression of smooth muscle cell proliferation [34].
Table 2 Biochemical measurements of isolated aortic segments in the different groups. Groups
SOD activity (kNU·g− 1 protein)
NO (mM·g− 1 protein)
O− 2 (cm2 min− 1)
Control (5.5 mmol/L) Mannitol (25 mmol/L) Sarpogrelate (10 μmol/L) High Glucose (25 mmol/L) HG + Sarpogrelate (10 μmol/L)
171.29 ± 1.53 167.32 ± 1.71 173.08 ± 1.79
0.88 ± 0.011 0.85 ± 0.009 0.84 ± 0.008
5.09 ± 0.041 5.31 ± 0.062 5.23 ± 0.059
0.41 ± 0.006⁎
9.27 ± 0.053⁎⁎
0.91 ± 0.009§
6.03 ± 0.072§
91.84 ± 1.45 ⁎⁎ 163.15 ± 1.58§
Values are expressed as mean ± SEM. *p < 0.05, **p < 0.01 versus the control group; §p < 0.05 versus the high glucose group. HG, high glucose; SOD, superoxide dismutase; NO, nitric oxide; O− 2 , superoxide anion.
The vascular endothelium releases various vasodilators, such as NO, prostacyclin and endothelium-derived hyperpolarizing factor, in addition to a variety of vasoconstrictors [33]. Previous studies have demonstrated that 5-HT is sequestered and released by endothelial cells, acts as an endothelial cell mitogen, promotes the release of NO and increases eNOS mRNA, which may mean the increase of NO and the 2+ scavenge of O− and caveolin-1 in 2 [35]. eNOS is controlled by Ca caveolae. eNOS is located within the plasma membrane microdomain caveolae, where it is complexed with the coat protein for this organelle, termed CAV-1. CAV-1 is a 22-kDa protein that decorates the cytoplasmic surface of caveolae and serves as the primary structural component of caveolae (50 to 100 nm invaginations of the plasma membrane), where it acts as a physiological inhibitor of eNOS [36]. Thus, the reduction of NO release in diabetes can be ascribed to a reduction in eNOS activity through an enhanced expression of caveolin-1 [37]. Sarpogrelate hydrochloride is a selective 5-HT2A antagonist and is clinically used for the treatment of cutaneous ulcers and ischemic changes associated with atherosclerosis [38,39]. It strongly inhibits the effects of serotonin such as platelet aggregation, vasoconstriction and vascular smooth muscle proliferation [39]. Sarpogrelate hydrochloride inhibits human platelet aggregation induced by collagen and secondary aggregation by adenosine diphosphate or adrenaline by 50% (IC50) at a concentration of 1 μM, and this effect was potentiated for aggregation induced by collagen plus serotonin, with an IC50 of 0.1 mM [40]. Furthermore, a major metabolite of sarpogrelate, M-1, was approximately 10 times more potent than the unaltered substance [40]. Sarpogrelate hydrochloride may act to increase NO production and hence improve blood flow via NO dependant vasodilatation [41]. The previous studies have shown that the relaxations induced by 5-HT in the arteries including porcine coronary arteries were mediated by activation of 5-HT receptors localized on the endothelial cells [11,12]. In addition, the recent studies indicated that sarpogrelate improved endothelial function in balloon-injured porcine coronary artery [42] and aorta in cholesterolfed rabbits [43] and enhanced ACh-induced, endothelium-dependent NO-mediated relaxation in the vein grafts [44]. The present study demonstrated that high glucose significantly inhibited EDR, and this inhibition of EDR was completely abolished by treating the aortic rings with sarpogrelate (10 µM). Furthermore, our data showed that the inhibition of EDR caused by high glucose was partially improved by treatment of aortic rings with L-arginine or SOD alone, and D-arginine had no effect on the impaired EDR. But treating of the aortic rings with SOD and L-arginine together, the inhibition of EDR was also completely prevented, which was very similar to the effect of sarpogrelate hydrochloride. In addition, incubation of the rings with high glucose had no effect on the SNP-induced EIR. These results indicated that increasing production of oxygen free radicals and reducing NO may contribute to the deleterious effects of high glucose on vascular endothelial function and sarpogrelate hydrochloride was an effective pharmacological approach to prevent the endothelial dysfunction induced by high glucose, and may exert its beneficial effect by both scavenging oxygen free radicals and increasing NO production in endothelial cells. In the present work, we showed the evidence that sarpogrelate hydrochloride can restore impaired EDR induced by high glucose in isolated rat aorta. We postulated that this beneficial effect may be related to scavenging oxygen free radicals and enhancing NO production. It was found that sarpogrelate enhanced eNOS expression and increased NO production in aorta of hypercholesterolemic rabbits [45], while it was found that sarpogrelate reduced superoxide production in neutrophils in vitro [46]. In the present experiments, we found that superoxide production was much smaller in the sarpogrelate-treated group than in the high glucose group. Superoxide is thought to generate peroxynitrite by reaction with NO, which oxidizes tetrahydrobiopterin, leading to“eNOS uncoupling”. Once “eNOS uncoupling” has been developed, eNOS becomes dysfunctional and produces superoxide rather than NO
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