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O36 Endothelium-dependent contractions depending on biased sGC activation: The Janus face of thymoquinone
Abstracts / Biochemical Pharmacology 139 (2017) 105–141
O35 An anthocyanin-rich blackcurrant extract induced NO-mediated relaxation in coronary artery rings and eNOS phosphorylation in cultured endothelial cells: Role of sodium-glucose cotransporters 1 and 2 Hyunho Lee a, Sonia Khemais-Benkhiat a, Philippe Chabert a, Cyril Auger a, Sin-Hee Park a, Claire Kevers b, Joel Pincemail c, Min-Ho Oak a,d, Valérie B. Schini-Kerth a a UMR CNRS 7213, Faculty of Pharmacy, Strasbourg University, Strasbourg, France, b Plant Molecular Biology and Biotechnology, University of Liège, Liège, Belgium, c CHU of Liège, Department of Cardiovascular Surgery/CREDEC, Liège, Belgium, d College of Pharmacy, Mokpo National University, Mokpo, Republic of Korea Introduction: The beneficial cardiovascular effect of polyphenolrich food and beverages has been attributed, at least in part, to an improved vascular function through an increased endothelial formation of nitric oxide (NO) and endothelium-dependent hyperpolarization (EDH), two major vasoprotective mechanisms. Black currant is a rich source of anthocyanins, and, in particular, of glucoside- and rutinoside-conjugated delphinidin and cyanidin. This study evaluated the role of sodium-glucose cotransporters (SGLT) 1 and 2 in the blackcurrant extract (BCE)-induced NO-mediated endothelium-dependent relaxation and activation of endothelial NO synthase (eNOS). Methods: The reactivity of porcine coronary artery rings was assessed in organ chambers, and the expression and phosphorylation level of proteins in cultured porcine coronary artery endothelial cells by Western blot analysis. Results: BCE (310.0 ± 0.1 mg gallic acid equivalent/g) was prepared from a blackcurrant juice containing 2.7 g gallic acid equivalent/L of polyphenols using a Sephadex LH-20 column. BCE caused potent concentration-dependent relaxations of coronary artery rings with endothelium, which were significantly reduced by the dual SGLT1 and SGLT2 inhibitor LX4211, and also to some extent by the SGLT2 inhibitor canagliflozin but not dapagliflozin. In contrast, LX4211 did not affect relaxations to bradykinin, sodium nitroprusside and epigallocatechin gallate. BCE induced the phosphorylation of eNOS at the activator site Ser1177 in cultured endothelial cells, which was significantly prevented by LX4211, dapagliflozin and canagliflozin. Conclusion: These observations indicate that BCE is a potent activator of eNOS and inducer of NO-mediated vasorelaxation possibly subsequent to the entry of conjugated anthocyanins via SGLT1/2 into endothelial cells. doi:10.1016/j.bcp.2017.06.100
O36 Endothelium-dependent contractions depending on biased sGC activation: The Janus face of thymoquinone Susan W.S. Leung, Charlotte M.S. Detremmerie, Paul M. Vanhoutte University of Hong Kong, Pokfulam, Hong Kong, Hong Kong Objective: Thymoquinone, a major component of black seed oil, has beneficial (vasodilator, antioxidative and anti-inflammatory) effects in the cardiovascular system. Previous experiments in isolated arteries indicate that thymoquinone causes augmentations of contraction. The present study aims to identify the mechanisms underlying the counterintuitive augmentations caused by thymoquinone. Methods: Isometric tension was measured in rings of porcine coronary arteries, and rat aortae and mesenteric arteries. Precontracted preparations were exposed to increasing concentrations of thymoquinone, in the absence/presence of endothelium and of pharmacological inhibitors/activators. Cyclic nucleotide levels in rings were
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measured with ultra-high performance liquid chromatography coupled with mass spectrometry (UPLC–MS/MS). Results: Thymoquinone-induced augmentations in the three arteries were not observed in rings without endothelium, and were abolished by inhibitors of nitric oxide (NO) synthase and soluble guanylyl cyclase (sGC), L-NAME and ODQ respectively. Rho-kinase inhibitor and L-type voltage-dependent calcium channel (VDCC) blockers inhibited the augmentations in porcine, but not in rat arteries, while T-type VDCC blockers did so only in the latter. In L-NAMEtreated rings, the NO donor detaNONOate and sGC activator YC-1 restored the augmentations; by contrast, in ODQ-treated rings, cell-permeable cyclic GMP did not. UPLC–MS/MS measurements showed that thymoquinone increased intracellular levels of cyclic IMP. Conclusion: Thymoquinone causes endothelium-dependent augmentations that depend on a biased sGC activation by NO to produce cyclic IMP instead of cyclic GMP. Cyclic IMP causes contraction of the vascular smooth muscle through alteration of calcium handling. Hence, thymoquinone is the first pharmacological agent reported to cause endothelium-dependent contractions with similar characteristics as hypoxic vasoconstrictions. doi:10.1016/j.bcp.2017.06.101
Session 13: Translating novel pathways – Cardiovascular disease II O37 Salidroside prevents toxicity induced by delayed treatment of tPA in a rat model of focal cerebral ischemia involving PKA-and PI3Kdependent Akt activation Wei Zuo a,b, Dan Mei a, Naihong Chen b a Peking Union Medical College Hospital, Beijing, China, b Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China Ischemic stroke is currently treated with thrombolytic therapy with the drawback that it induces hemorrhagic transformation (HT) if applied beyond its relatively narrow treatment time window. The present study was designed to examine the role of Salidroside, one of the major phenolic glycosides in Rhodiola, in recombinant tissue plasminogen activator (tPA)-induced HT. Rats subjected to 6 h of thromboembolic occlusion or middle cerebral artery occlusion received tPA with or without Salidroside. Delayed tPA intervention drastically increased the risk of HT and exaggerated ischemic injury. To assess the effect of Salidroside on delayed treatment of tPAinduced toxicity after ischemia and reperfusion, various approaches were used, including behaviour tests, TTC-staining, determination of HT, laser speckle imaging, Western blot, gelatin zymogram, immunohistochemistry and immunofluorescence staining. Experiments were also conducted in vitro in human brain microvascular endothelial cells (HBMECs) and PC12 cells to explore the mechanisms involved in the effects of glycosides. Combination therapy of tPA and Salidroside prevented the development of HT, and reduced the mortality rate, infarct volume and brain edema. Salidroside also exerted protective roles by decreasing matrix metalloproteinases, the co-localization of matrix metalloproteinase-2 with astrocytes and increasing occludin. Experiments in HBMECs and PC12 cells revealed an elevation in ATP level and a protein kinase A- and PI3K-dependent activation of Akt by glycosides after tPA administration. These results suggest Salidroside as a promising adjuvant to alleviate the detrimental side effects of tPA in the clinical therapy of ischemic stroke, and contribute to a better understanding
O35 An anthocyanin-rich blackcurrant extract induced NO-mediated relaxation in coronary artery rings and eNOS phosphorylation in cultured endothelial cells: Role of sodium-glucose cotransporters 1 and 2 Hyunho Lee a, Sonia Khemais-Benkhiat a, Philippe Chabert a, Cyril Auger a, Sin-Hee Park a, Claire Kevers b, Joel Pincemail c, Min-Ho Oak a,d, Valérie B. Schini-Kerth a a UMR CNRS 7213, Faculty of Pharmacy, Strasbourg University, Strasbourg, France, b Plant Molecular Biology and Biotechnology, University of Liège, Liège, Belgium, c CHU of Liège, Department of Cardiovascular Surgery/CREDEC, Liège, Belgium, d College of Pharmacy, Mokpo National University, Mokpo, Republic of Korea Introduction: The beneficial cardiovascular effect of polyphenolrich food and beverages has been attributed, at least in part, to an improved vascular function through an increased endothelial formation of nitric oxide (NO) and endothelium-dependent hyperpolarization (EDH), two major vasoprotective mechanisms. Black currant is a rich source of anthocyanins, and, in particular, of glucoside- and rutinoside-conjugated delphinidin and cyanidin. This study evaluated the role of sodium-glucose cotransporters (SGLT) 1 and 2 in the blackcurrant extract (BCE)-induced NO-mediated endothelium-dependent relaxation and activation of endothelial NO synthase (eNOS). Methods: The reactivity of porcine coronary artery rings was assessed in organ chambers, and the expression and phosphorylation level of proteins in cultured porcine coronary artery endothelial cells by Western blot analysis. Results: BCE (310.0 ± 0.1 mg gallic acid equivalent/g) was prepared from a blackcurrant juice containing 2.7 g gallic acid equivalent/L of polyphenols using a Sephadex LH-20 column. BCE caused potent concentration-dependent relaxations of coronary artery rings with endothelium, which were significantly reduced by the dual SGLT1 and SGLT2 inhibitor LX4211, and also to some extent by the SGLT2 inhibitor canagliflozin but not dapagliflozin. In contrast, LX4211 did not affect relaxations to bradykinin, sodium nitroprusside and epigallocatechin gallate. BCE induced the phosphorylation of eNOS at the activator site Ser1177 in cultured endothelial cells, which was significantly prevented by LX4211, dapagliflozin and canagliflozin. Conclusion: These observations indicate that BCE is a potent activator of eNOS and inducer of NO-mediated vasorelaxation possibly subsequent to the entry of conjugated anthocyanins via SGLT1/2 into endothelial cells. doi:10.1016/j.bcp.2017.06.100
O36 Endothelium-dependent contractions depending on biased sGC activation: The Janus face of thymoquinone Susan W.S. Leung, Charlotte M.S. Detremmerie, Paul M. Vanhoutte University of Hong Kong, Pokfulam, Hong Kong, Hong Kong Objective: Thymoquinone, a major component of black seed oil, has beneficial (vasodilator, antioxidative and anti-inflammatory) effects in the cardiovascular system. Previous experiments in isolated arteries indicate that thymoquinone causes augmentations of contraction. The present study aims to identify the mechanisms underlying the counterintuitive augmentations caused by thymoquinone. Methods: Isometric tension was measured in rings of porcine coronary arteries, and rat aortae and mesenteric arteries. Precontracted preparations were exposed to increasing concentrations of thymoquinone, in the absence/presence of endothelium and of pharmacological inhibitors/activators. Cyclic nucleotide levels in rings were
121
measured with ultra-high performance liquid chromatography coupled with mass spectrometry (UPLC–MS/MS). Results: Thymoquinone-induced augmentations in the three arteries were not observed in rings without endothelium, and were abolished by inhibitors of nitric oxide (NO) synthase and soluble guanylyl cyclase (sGC), L-NAME and ODQ respectively. Rho-kinase inhibitor and L-type voltage-dependent calcium channel (VDCC) blockers inhibited the augmentations in porcine, but not in rat arteries, while T-type VDCC blockers did so only in the latter. In L-NAMEtreated rings, the NO donor detaNONOate and sGC activator YC-1 restored the augmentations; by contrast, in ODQ-treated rings, cell-permeable cyclic GMP did not. UPLC–MS/MS measurements showed that thymoquinone increased intracellular levels of cyclic IMP. Conclusion: Thymoquinone causes endothelium-dependent augmentations that depend on a biased sGC activation by NO to produce cyclic IMP instead of cyclic GMP. Cyclic IMP causes contraction of the vascular smooth muscle through alteration of calcium handling. Hence, thymoquinone is the first pharmacological agent reported to cause endothelium-dependent contractions with similar characteristics as hypoxic vasoconstrictions. doi:10.1016/j.bcp.2017.06.101
Session 13: Translating novel pathways – Cardiovascular disease II O37 Salidroside prevents toxicity induced by delayed treatment of tPA in a rat model of focal cerebral ischemia involving PKA-and PI3Kdependent Akt activation Wei Zuo a,b, Dan Mei a, Naihong Chen b a Peking Union Medical College Hospital, Beijing, China, b Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China Ischemic stroke is currently treated with thrombolytic therapy with the drawback that it induces hemorrhagic transformation (HT) if applied beyond its relatively narrow treatment time window. The present study was designed to examine the role of Salidroside, one of the major phenolic glycosides in Rhodiola, in recombinant tissue plasminogen activator (tPA)-induced HT. Rats subjected to 6 h of thromboembolic occlusion or middle cerebral artery occlusion received tPA with or without Salidroside. Delayed tPA intervention drastically increased the risk of HT and exaggerated ischemic injury. To assess the effect of Salidroside on delayed treatment of tPAinduced toxicity after ischemia and reperfusion, various approaches were used, including behaviour tests, TTC-staining, determination of HT, laser speckle imaging, Western blot, gelatin zymogram, immunohistochemistry and immunofluorescence staining. Experiments were also conducted in vitro in human brain microvascular endothelial cells (HBMECs) and PC12 cells to explore the mechanisms involved in the effects of glycosides. Combination therapy of tPA and Salidroside prevented the development of HT, and reduced the mortality rate, infarct volume and brain edema. Salidroside also exerted protective roles by decreasing matrix metalloproteinases, the co-localization of matrix metalloproteinase-2 with astrocytes and increasing occludin. Experiments in HBMECs and PC12 cells revealed an elevation in ATP level and a protein kinase A- and PI3K-dependent activation of Akt by glycosides after tPA administration. These results suggest Salidroside as a promising adjuvant to alleviate the detrimental side effects of tPA in the clinical therapy of ischemic stroke, and contribute to a better understanding