The nitric oxide donor RuBPY does not induce in vitro cross-tolerance with acetylcholine

Purpose: We have demonstrated that RuBPY induces hypotensive effect in hypertensive rats, promotes vasodilation at low concentrations, and presents low cytotoxicity. This study aimed to verify whether the NO donor RuBPY synthesized in our laboratory induces in vitro tolerance and cross-tolerance to acetylcholine (ACh) and sodium nitroprusside (SNP) in rat cava vein. Methods: We compared the maximum relaxing effect (ME) and potency (pD(2)) of RuBPY and nitroglycerin (GTN) in cava vein rings. Exposure to RuBPY or GTN induced in vitro tolerance. Western Blotting helped to evaluate phosphorylation of endothelial nitric oxide synthase (NOS3/eNOS) at the Ser(1177) activation site and at the Thr(495) inactivation site and to determine the ratio between active eNOS dimers and inactive eNOS monomers. The NO and ROS ratio was assessed by flow citometry. Results: RuBPY did not induce cross-tolerance with ACh, and this NO donor took longer to induce tolerance than GTN. Only GTN elicited phosphorylation of eNOS at Ser(1177) and Thr(495). In contrast to results obtained with pre-exposure to GTN, pre-exposure to RuBPY did not reduce the formation of NO. The O-2(-) ratio increased in cells incubated with GTN. Conclusions: A major contribution of this work has been to evaluate the phenomenon of tolerance induced by GTN and by the new ruthenium complex RuBPY in a venous bed. RuBPY is more advantageous than GTN: RuBPY takes longer to induce tolerance, does not induce endothelial dysfunction or cross-tolerance to ACh, and generates lower amount of ROS. (C) 2017 Elsevier Inc. All rights reserved.