Abstract
The biologically active gas, nitric oxide (NOÏ) is well recognized as an important modulator of vascular homeostasis with vasorelaxant, anti-aggregatory, and anti-proliferative properties. Interestingly, nitroxil (HNO), the one electron reduced and protonated form of NOÏ, is rapidly emerging as a new redox sibling of NOÏ, with distinct pharmacological actions and therapeutic advantages over NOÏ. Like NOÏ, HNO can be produced endogenously in the vessel and has similar vasoprotective properties, such as the ability to induce relaxation of blood vessels, inhibiting platelet aggregation and to inhibit the proliferation of vascular smooth muscle cells. In contrast to NOÏ, HNO targets distinct signaling pathways in the vessels that include direct activation of voltage-sensitive K+ channels (Kv) and the release of calcitonin-gene related peptide (CGRP). In addition, HNO is resistant to scavenging by superoxide anion (O2-) and is not susceptible to tolerance development (loss of hemodynamic properties and pharmacological effects of organic nitrates after continuous use). As such, the vascular actions of HNO may be preserved under disease conditions, whereas those of NO are compromised (i.e., during oxidative stress). Thus, HNO donors may offer an alternative to traditional nitrovasodilators for the treatment of cardiovascular diseases. In this context, the present study aims to study the relaxing effect of nitroxil released from Angeli's salt and to investigate the cellular mechanisms involved in this effect in rat cava vein. Cummulative concentration-effect relaxation curves will be constructed for the HNO donor, Angeli's salt, in preparations of rat cava vein with intact endothelium, contracted with phenylephrine. The scavenging species of NO, hydroxocobalamin and L-cysteine will be used to study the involvement in the relaxation of these species of nitric oxide, radical nitric oxide and nitroxil, respectively. Ion channel blockers TEA and nifedipine will be used to evaluate the involvement of ion channels for K+ and Ca2+. The activity of the enzymes NO-synthase, soluble guanylyl cyclase (sGC), protein kinase G, superoxide dismutase, phosphodiesterase 5, sarcoplasmic reticulum Ca2+-ATPase (SERCA), will be evaluated by their respective inhibitors L-NAME, ODQ, Rp-8-br-PET-cGMP (8-Br-PET), DDC, sildenafil and thapsigargin. By confocal microscopy, it will be evaluated the release of HNO/NO, the antioxidant activity of HNO and the cytosolic concentration of calcium in endothelial cells derived from human umbilical vein. We will also assess the activity of sGC by measuring its product, cGMP, using Elisa Immuno-Assay kit.
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