Effects of different antioxidant agents on 6-nitrodopamine production/release by human umbilical artery in vitro

Abstract

Introduction: It is well established that endothelial cells modulate vascular reactivity through the release of mediators such as prostacyclin (Moncada et al., 1976), nitric oxide (Furchgott and Zawadzki, 1980) and endothelin (Yanagisawa et al., 1988). Catecholamines modulate vascular tone through actions on ±- and ²-adrenergics (Ahlquist, 1948); meanwhile, the production and release of catecholamines are associated with the existence of nerve endings in our vessels (Kadowitz et al., 1976; Matsuyama et al., 1985).The vasodilator activity of 6-nitrodopamine (6-ND) in human umbilical cord vessels is mediated by selective dopamine D2 receptor antagonism (Britto-Júnior J, 2021). Endothelial 6-ND production/release is dependent on NO production. Here we intend to characterize the participation of reactive species derived from oxygen and nitrogen (ROS and RNS, respectively) in the production/release of 6-ND by the human umbilical artery (HUA) in vitro.Methods: The umbilical cords of 35 normotensive volunteers, aged 18 to 40 years, without regular medication and without pre-eclampsia, pre-gestational or gestational diabetes mellitus, natural childbirth or cesarean section will be collected by obstetrician. Only cords originating from pregnant women who consent in writing to collaborate with the studio will be included in the study. Immediately the tissue will be placed in Krebs-Henselei solution and will be kept under conduction until transport to the study laboratory. In it, it will be manually dissected from the human umbilical artery, which will be kept in the organ vat with constant heating and oxygenation, and an initial tension of 10mN will be applied. After stabilization, the paired rings of the same HUA will be incubated for 30 min in an adjuvant or in the presence of: superoxide dismutase (SOD; 250U/ml), catalase (1000U/ml), resveratrol (100M), uric acid ( 1mM), ebselen (100M), GSK2795039 (1M) or GKT137831 (1M). At the end of the incubation period, the supernatant will be separated and stored at -20C until analyzed by HPLC-MS/MS.