Contribution of extra-endothelial eNOS in regulation of blood pressure

Drugs and devices / Endothelial function and vascular remodelling the other hand, medetomidine activates cardiac vagal nerve without gastric vagal activation. Selective vagal activation of medetomidine may be beneficial for vagal activation therapy in the HF patients. P576 | BEDSIDE Dopamine withdrawal causes chemoreflex-related increase in minute ventilation S. Tubek, P. Niewinski, W. Banasiak, P. Ponikowski. Heart disease Center - 4th Military Clinical Hospital, Wroclaw, Poland ENDOTHELIAL FUNCTION AND VASCULAR REMODELLING P578 | BENCH The EPA:DHA 6:1-evoked endothelium-dependent NO-mediated relaxation in the coronary artery involves a copper-dependent pro-oxidant response triggering the PI3-kinase/Akt-mediated activation of eNOS F. Zgheel, M. Alhosin, S. Rashid, C. Auger, V.B. Schini-Kerth. University of Strasbourg, Faculty of Pharmacy, UMR CNRS 7213, Illkirch, France Purpose: Omega-3 fatty acid products containing Eicosapentaenoic Acid (EPA) and Docosahexaenoic Acid (DHA) have been shown to reduce the risk of cardiovascular disease, in part, by stimulating the endothelial formation of Nitric Oxide (NO), a potent vasoprotective factor. This study determined the mechanism leading to Endothelial NO Synthase (eNOS) activation in response to the highly active EPA:DHA 6:1 product. Methods: Vascular reactivity was assessed using porcine coronary artery rings suspended in organ chambers, the level of oxidative stress in coronary artery sections using the redox-sensitive probe, dihydroethidine, and the phosphorylation level of target proteins in cultured coronary artery endothelial cells by Western blot analysis. Results: EPA:DHA 6:1 caused pronounced relaxations in porcine coronary artery rings with endothelium but only minor effects in those without endothelium. Relaxations to EPA:DHA 6:1 were slightly but significantly reduced by an eNOS inhibitor, not affected by inhibition of endothelium-dependent hyperpolarization and abolished by both treatments. Relaxations to EPA:DHA 6:1 were reduced by inhibitors of oxidative stress (MnTMPyP, PEG-catalase), an inhibitor of either Src kinase (PP2) or PI3-kinase (wortmannin), and intracellular copper chelating agents (neocuproine, tetrathiomolybdate) and were insensitive to cyclooxygenase inhibition (indomethacin), an iron chelating agent (desferroxamine), a zinc chelating agent (histidine), and an extracellular copper chelating agent (bathocuproine). EPA:DHA 6:1 induced phosphorylation of Src, Akt and eNOS at Ser 1177; these effects were inhibited by MnTMPyP and PEG-catalase. EPA:DHA 6:1 induced the endothelial formation of ROS in coronary artery sections, this effect was inhibited by MnTMPyP, PEG-catalase, and intracellular copper chelating agents. Conclusion: EPA:DHA 6:1 causes endothelium-dependent NO-mediated relaxations in coronary artery rings, and this effect involves an intracellular copperdependent event triggering the redox-sensitive PI3-kinase/Akt pathway to activate eNOS by phosphorylation at Ser 1177. P579 | BENCH A role of eNOS dimer stability in essential hypertension? S. Pick 1 , T. Suvorava 1 , M. Oppermann 1 , M. Weber 2 , G. Kojda 1 . 1 Institute of Pharmacology and Clinical Pharmacology, University Hospital HHU Duesseldorf, Duesseldorf, Germany; 2 Emory University School of Medicine, Division of Cardiology, Atlanta, United States of America Purpose: Essential hypertension is associated with endothelial dysfunction. However, if endothelial dysfunction contributes to the pathophysiology of essential hypertension remains unclear. Methods: Wildtype eNOS (eNOS-tg) or a destabilized eNOS (C101A-eNOS-tg) were introduced into the endothelium of C57BL/6 mice using the endothelial specific tie-2 promoter. Destabilization of eNOS was induced via replacement of cysteine by alanine. Transfection in HEK cells demonstrated decreased dimer stability and NO formation and increased superoxide (O2-) generation. Results: Western blot analysis proved the vascular specific overexpression of eNOS in aortic tissue of C101A-eNOS-tg (150±8.4%, n=8, p<0.05) and eNOS-tg (229±15.5%, n=6, p<0.01; both vs. C57BL/6). Destabilization in C101A-eNOS-tg resulted in elevated phosphorylation of aortic eNOS at Ser1176/9 (153±13.8%, n=6, p<0.05, vs. C57BL/6). In keeping with destabilized eNOS being a source of ROS, O2- levels were increased in C101A-eNOS-tg (223±30.4%, n=10, p<0.01) but not in eNOS-tg (109±6.3%, n=3; myocardial tissue, both vs. C57BL/6). Incubation with the NOS-inhibitor L-NA of aortic, myocardial and skeletal muscle tissue completely inhibited the increase of O2-. S-glutathionylation of aortic eNOS was increased only in C101A-eNOS-tg (178±23.1%, n=6, p<0.05, vs. C57BL/6) indicating augmented oxidative and nitrosative stress. Peroxynitrite levels were increased in myocardial tissue of C101A-eNOS-tg (153±8.1%, n=7, p<0.01) but unchanged in eNOS-tg (103±11.9%, n=5; both vs. C57BL/6). PKG activity was evaluated by VASP phosphorylation at Ser239 showing no difference in both strains vs. C57BL/6 suggesting unchanged NO-cGMP pathway activation. Organ bath experiments revealed normal aortic reactivity to acetylcholine, phenylephrine and the NO-donor SNAP in both strains vs. C57BL/6 (n=5-7). Systolic blood pressure (sBP) was significantly decreased in eNOS-tg (109±1.9mmHg, n=4, p<0.05). In striking contrast and although dimer destabilization did reduce but not impair NO formation, sBP in C101A-eNOS-tg (118±2.8mmHg, n=8) was identical to C57BL/6 (118±3.1mmHg, n=6). Plotting aortic eNOS expression against sBP using 3 different transgenic strains revealed a highly significant approximation by one phase exponential decay equation (R2 =0.9992). According to the expression level in C101A-eNOS-tg a sBP reduction of at least 4mmHg would have been expected. Conclusion: These data demonstrate that a decrease of eNOS dimer stability attenuates the effect of endothelial eNOS on sBP. This finding might contribute to explain the pathophysiology of essential hypertension. P580 | BENCH Contribution of extra-endothelial eNOS in regulation of blood pressure T. Suvorava 1 , J. Stegbauer 2 , S. Friedrich 2 , C. Rump 2 , T. Hohlfeld 1 , G. Kojda 1 . 1 Institute of Pharmacology and Clinical Pharmacology, University Hospital, Duesseldorf, Germany; 2 Departhment of Nephrology, University Hospital, Düsseldorf, Germany Purpose: We sought to investigate whether endothelial-specific targeting of endothelial NO-syntase (eNOS) in eNOS-deficient mice (eNOS-KO) normalizes vascular reactivity and blood pressure (BP). Methods: By crossing mice with endothelial specific overexpression of eNOS on C57BL/6 background (eNOS-Tg) with eNOS-KO, a double transgenic strain expressing eNOS exclusively in the vascular endothelium has been generated (eNOS-Tg/KO). Results: Western blot analysis confirmed eNOS expression in aorta, myocardium, kidney, brain stem and skeletal muscle of eNOS-Tg/KO. Organ bath studies revealed a complete normalization of aortic reactivity to acetylcholine (...truncated)