Biotransformation of RuBPY

Abstract

For many years the nitrite was considered an inert metabolite of nitric oxide (NO) pathway. However, it is present in abundance in the blood and tissues and may be the largest intravascular and tissue source of NO storage. NO is one of the smallest and simplest molecules with characteristics that confer high diffusion capacity through the cells. It is classified as a messenger that does not depend on specific transporters. Endogenous production is important to the vascular tone modulation, blood pressure control, inhibition of platelet aggregation and immune responses. The ruthenium nitrocomplexes have been extensively studied as NO donors. They are attractive as potential therapeutic agents because they have low cytotoxicity, which may be due to the similarity between ruthenium and iron. Although several compounds of ruthenium have been synthesized in our laboratory, the complex - [Ru(bpy)2(py)NO2](PF6), (RuBPY), seems to be promising as a nitrite donor that is intracellularly converted. The main clinical limitation of NO donors is the development of tolerance that is characterized by the loss of its vasodilator and hemodynamic effects. The mechanisms that lead to tolerance are still poorly understood, but it is believed that it is a multifactorial process that involves increased production of reactive oxygen species (ROS) due to endothelial NO-synthase (NOS3) uncoupling, reduction of the enzyme soluble guanylyl cyclase (sGC) activity and increased expression and activity of phosphodiesterases. The process of tolerance has been widely associated with organic nitrates, however the occurrence of this phenomenon with nitrite donors remain understudied. In the present work we hypothesized that RuBPY and nitrite (NaNO2) do not induce tolerance because these compounds release nitrite instead of NO, and it is released in the vascular smooth muscle cells and not in the endothelial cells. Thus, these compounds would not induce NOS3 uncoupling and ROS production. Therefore, this study aims to evaluate if the compound RuBPY induces tolerance in intact or denuded rat aorta. Nitrite (NaNO2) will be used for comparison. In order to study the cellular mechanisms involved on the tolerance development after the continuous exposure to nitrites, we will use functional vascular studies, microscopy, western blot and other molecular and cellular techniques. Therefore, the objective of developing the proposed split PhD project is to evaluate whether the compound RuBPY releases nitrite and the cellular mechanism by which it occurs. In the laboratory of Dr. A. Ahluwalia, we will be able to perform experiments to evaluate the formation of NO and nitrite, the activity of enzymes that reduce nitrite and the formation of NO from nitrite. In addition, we will also evaluate whether RuBPY inhibits platelet aggregation. (AU)