cyclic-gmp and dihydroethidium

Previous studies indicated that estrogen could improve endothelial function. However, whether estrogen protects vascular complications of diabetes has yet to be clarified. The study was designed to investigate the action of 17ß-estradiol on vascular endothelium in streptozotocin (STZ)-induced diabetic rats. Ovariectomized female Sprague-Dawley rats were administered with streptozotocin to produce an ovariectomized-diabetic (OVS) model which manifested as dysfunction of aortic dilation and contraction ability. Meanwhile, OVS animals with 17ß-estradiol supplementation significantly improved aortic function. Accordingly, nitric oxide synthase-3 (NOS-3), Akt, PI3K and estrogen receptor α (ERα) protein expression in aorta declined in the OVS group. Such effects were partially restored by estrogen replacement. The presence of 17ß-estradiol similarly counteracted the reduction of cyclic guanosine monophosphate (cGMP), the enhanced expression of inducible NOS (NOS-2) and NO metabolites (nitrite and nitrate), as well as the increase of matrix metalloproteinase-9/tissue inhibitor of metalloproteinase-1 (MMP-9/TIMP-1), which is an index of arterial compliance. 17ß-estradiol could also decrease ROS production in vascular endothelium. In EA hy 926 cells we found that ER antagonist, wortmannin and Akt inhibitor could block improvement effects of 17ß-estradiol. These results strongly suggest that functional impairment of the ERα/NOS-3 signaling network in OVS animals was partially restored by 17ß-estradiol administration, which provides experimental support for estrogen recruitment to improve vascular outcomes in female diabetes after endogenous hormone depletion.

Topics: Animals; Aorta; Cell Line; Cyclic GMP; Diabetes Mellitus, Experimental; Down-Regulation; Endothelium, Vascular; Estradiol; Estrogen Receptor alpha; Estrogens; Ethidium; Extracellular Matrix; Female; Matrix Metalloproteinase 9; Nitric Oxide; Nitric Oxide Synthase Type III; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Signal Transduction; Tissue Inhibitor of Metalloproteinase-1; Vascular Endothelial Growth Factor A

T2238C ANP (atrial natriuretic peptide) gene variant has been associated with increased cardiovascular risk in humans and with a significant pharmacogenomic effect on cardiovascular disease outcome in hypertensive patients. We investigated the impact of T2238C ANP gene variant on oxidative stress production, cell proliferation and migration, angiogenesis and vascular remodeling in human umbilical vein endothelial cells in vitro.. Differentially expressed genes in human umbilical vein endothelial cells exposed to either wild-type (TT2238) or mutant (CC2238) alpha-ANP were characterized by real time-PCR-macroarray analysis using human oxidative stress, angiogenesis and matrix arrays. Reactive oxygen species (ROS) production was determined by dihydroethidium and by evaluation of dichlorofluorescein content. NADPH oxidase gp91phox subunit was investigated by western blotting. Endothelial cell proliferation, migration and tube formation were characterized both in the presence and in the absence of NADPH oxidase inhibition.. Compared with TT2238, CC2238 alpha-ANP altered the redox state balance of the cells in a more significant manner, favoring ROS production and reducing antioxidative stress response. Gene expression of molecules involved in atherogenesis and vascular remodeling was enhanced. In contrast to TT2238 peptide, CC2238 was unable to stimulate cell proliferation and it markedly inhibited endothelial cell tube formation. NADPH oxidase inhibition restored the cell proliferative properties under CC2238 peptide exposure.. CC2238 alpha-ANP led to ROS accumulation and increased expression of genes related to atherosclerosis and vascular remodeling in human umbilical vein endothelial cells. As a consequence of NADPH-derived ROS, blunted endothelial cell proliferation and impaired endothelial cell tube formation were observed. These in-vitro effects may link the T2238C alpha-ANP variant to enhanced susceptibility to vascular damage in vivo.

Topics: Atrial Natriuretic Factor; Blotting, Western; Cell Movement; Cell Proliferation; Cells, Cultured; Cyclic GMP; Endothelial Cells; Ethidium; Fluoresceins; Gene Expression Profiling; Humans; Membrane Glycoproteins; NADPH Oxidase 2; NADPH Oxidases; Neovascularization, Pathologic; Oxidative Stress; Reactive Oxygen Species

Oxygen radicals play an important role in signal transduction and have been shown to influence epithelial sodium channel (ENaC) activity. We show that aldosterone, the principal hormone regulating renal ENaC activity, increases superoxide (O2*) production in A6 distal nephron cells. Aldosterone (50 nM to 1.5 microM) induced increases in dihydroethidium fluorescence in a dose-dependent manner in confluent A6 epithelial cells. Using single-channel measurements, we showed that sequestering endogenous O2* (with the O2* scavenger 2,2,6,6-tetramethylpiperidine 1-oxyl) significantly decreased ENaC open probability from 0.10 +/- 0.03 to 0.03 +/- 0.01. We also found that increasing endogenous O2* in A6 cells, by applying a superoxide dismutase inhibitor, prevented nitric oxide (NO) inhibition of ENaC activity. ENaC open probability values did not significantly change from control values (0.23 +/- 0.05) after superoxide dismutase and 1.5 microM NO coincubation (0.21 +/- 0.04). We report that xanthine oxidase and hypoxanthine compounds increase local concentrations of O2* by approximately 30%; with this mix, an increase in ENaC number of channels times the open probability (from 0.1 to 0.3) can be achieved in a cell-attached patch. Our data also suggest that O2* alters NO activity in a cGMP-independent mechanism, since pretreating A6 cells with ODQ compound (a selective inhibitor of NO-sensitive guanylyl cyclase) failed to block 2,2,6,6-tetramethylpiperidine 1-oxyl inhibition of ENaC activity.

Topics: Aldosterone; Animals; Antioxidants; Cell Line; Cyclic GMP; Cyclic N-Oxides; Dose-Response Relationship, Drug; Drug Combinations; Enzyme Inhibitors; Epithelial Sodium Channel Blockers; Epithelial Sodium Channels; Ethidium; Fluorescent Dyes; Hypoxanthine; Nephrons; Nitric Oxide; Reactive Nitrogen Species; Superoxide Dismutase; Superoxides; Xanthine Oxidase; Xenopus laevis