title: Uric acid enhances PKC-dependent eNOS phosphorylation and mediates cellular ER stress: A mechanism for uric acid-induced endothelial dysfunction.
abstract: The mechanism by which hyperuricemia induced-endothelial dysfunction contributes to cardiovascular diseases (CVDs) is not yet fully understood. In the present study, we used uric acid (UA) to trigger endothelial dysfunction in cultured endothelial cells, and investigated the effects of induced reactive oxygen species (ROS) generation, endoplasmic reticulum (ER) stress induction, and the protein kinase C (PKC)-dependent endothelial nitric oxide synthase (eNOS) signaling pathway. Human umbilical vein endothelial cells (HUVECs) were incubated with 6, 9 or 12 mg/dl UA, ROS scavenger polyethylene glycol-superoxide dismutase (PEG‑SOD), ER stress inhibitor 4-phenylbutyric acid (4-PBA), and PKC inhibitor polymyxin B for 6-48 h. Nitric oxide (NO) production, eNOS activity, intracellular ROS, ER stress levels, and the interaction between eNOS and calmodulin (CaM) and cytosolic calcium levels were assessed using fluorescence microscopy and western blot analysis. Apoptosis was assessed by annexin V staining. UA increased HUVEC apoptosis and reduced eNOS activity and NO production in a dose- and time-dependent manner. Intracellular ROS was elevated after 3 h, while ER stress level increased after 6 h. UA did not alter intracellular Ca2+, CaM, or eNOS concentration, or eNOS Ser1177 phosphorylation. However, PKC-dependent eNOS phosphorylation at Thr495 was greatly enhanced, and consequently interaction between eNOS and CaM was reduced. Cellular ROS depletion, ER stress inhibition and PKC activity reduction inhibited the effect of UA on eNOS activity, NO release and apoptosis in HUVECs. Thus, we concluded that UA induced HUVEC apoptosis and endothelial dysfunction by triggering oxidative and ER stress through PKC/eNOS-mediated eNOS activity and NO production.
authors.affiliation: The Key Laboratory of Cardiovascular Remodeling and Function Research, Chinese Ministy of Education and Chinese Ministy of Public Health, Qilu Hospital of Shandong University, Jinan, Shandong, P.R. China.
authors.forename: Peng
authors.initials: P
authors.lastname: Li
authors.affiliation: Department of Ophthalmology, The Affiliated Hospital of Qingdao University, Qingdao, Shandong, P.R. China.
authors.forename: Lina
authors.initials: L
authors.lastname: Zhang
authors.affiliation: The Key Laboratory of Cardiovascular Remodeling and Function Research, Chinese Ministy of Education and Chinese Ministy of Public Health, Qilu Hospital of Shandong University, Jinan, Shandong, P.R. China.
authors.forename: Mei
authors.initials: M
authors.lastname: Zhang
authors.affiliation: Department of Emergency Room, The Affiliated Hospital of Qingdao University, Qingdao, Shandong, P.R. China.
authors.forename: Changyong
authors.initials: C
authors.lastname: Zhou
authors.affiliation: Department of Emergency Room, The Affiliated Hospital of Qingdao University, Qingdao, Shandong, P.R. China.
authors.forename: Nan
authors.initials: N
authors.lastname: Lin
mesh_terms: D017209:Apoptosis; D059865:Endoplasmic Reticulum Stress; D042783:Endothelial Cells / Q000378:metabolism* / Q000473:pathology; D061307:Human Umbilical Vein Endothelial Cells; D006801:Humans; D009569:Nitric Oxide / Q000378:metabolism; D052250:Nitric Oxide Synthase Type III / Q000378:metabolism*; D018384:Oxidative Stress; D010766:Phosphorylation; D011092:Polyethylene Glycols / Q000378:metabolism; D011493:Protein Kinase C / Q000378:metabolism*; D017382:Reactive Oxygen Species / Q000378:metabolism; D013482:Superoxide Dismutase / Q000378:metabolism; D014527:Uric Acid / Q000378:metabolism*
chemical_list: D017382:Reactive Oxygen Species; D014527:Uric Acid; D009569:Nitric Oxide; D011092:Polyethylene Glycols; D052250:Nitric Oxide Synthase Type III; D013482:Superoxide Dismutase; C063471:polyethylene glycol-superoxide dismutase; D011493:Protein Kinase C