Oxidative burst and NO generation as initial response to ischemia in flow-adapted endothelial cells.
Shear stress modulates endothelial physiology, yet the effect(s) of flow cessation is poorly understood. The initial metabolic responses of flow-adapted bovine pulmonary artery endothelial cells to the abrupt cessation of flow (simulated ischemia) was evaluated using a perfusion chamber designed for continuous spectroscopy. Plasma membrane potential, production of reactive O2 species (ROS), and intracellular Ca(2+) and nitric oxide (NO) levels were measured with fluorescent probes. Within 15 s after flow cessation, flow-adapted cells, but not cells cultured under static conditions, showed plasma membrane depolarization and an oxidative burst with generation of ROS that was inhibited by diphenyleneiodonium. EGTA-inhibitable elevation of intracellular Ca(2+) and NO were observed at approximately 30 and 60 s after flow cessation, respectively. NO generation was decreased in the presence of inhibitors of NO synthase and calmodulin. Thus flow-adapted endothelial cells sense the altered hemodynamics associated with flow cessation and respond by plasma membrane depolarization, activation of NADPH oxidase, Ca(2+) influx, and activation of Ca(2+)/calmodulin-dependent NO synthase. This signaling response is unrelated to cellular anoxia.
Manevich
Y
Y
Institute for Environmental Medicine, University of Pennsylvania Medical Center, Philadelphia, Pennsylvania 19104-6068, USA.
Al-Mehdi
A
A
Muzykantov
V
V
Fisher
A B
AB
D000222:Adaptation, Physiological / Q000502:physiology; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002147:Calmodulin / Q000378:metabolism; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000166:cytology / Q000201:enzymology*; D007511:Ischemia / Q000378:metabolism*; D008564:Membrane Potentials / Q000502:physiology; D008856:Microscopy, Fluorescence; D019255:NADPH Oxidases / Q000378:metabolism; D009569:Nitric Oxide / Q000096:biosynthesis*; D019001:Nitric Oxide Synthase / Q000378:metabolism; D052250:Nitric Oxide Synthase Type III; D010101:Oxygen Consumption / Q000502:physiology; D011651:Pulmonary Artery / Q000166:cytology; D011673:Pulsatile Flow / Q000502:physiology; D016897:Respiratory Burst / Q000502:physiology*; D013314:Stress, Mechanical; D013481:Superoxides / Q000378:metabolism
D002147:Calmodulin; D013481:Superoxides; D009569:Nitric Oxide; D019001:Nitric Oxide Synthase; D052250:Nitric Oxide Synthase Type III; D019255:NADPH Oxidases; D002118:Calcium