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Am J Physiol Heart Circ Physiol 280: H2126-H2135, 2001;
0363-6135/01 $5.00
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Vol. 280, Issue 5, H2126-H2135, May 2001

Oxidative burst and NO generation as initial response to ischemia in flow-adapted endothelial cells

Yefim Manevich, Abu Al-Mehdi, Vladimir Muzykantov, and Aron B. Fisher

Institute for Environmental Medicine, University of Pennsylvania Medical Center, Philadelphia, Pennsylvania 19104-6068

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 Ca2+ 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 Ca2+ and NO were observed at ~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, Ca2+ influx, and activation of Ca2+/calmodulin-dependent NO synthase. This signaling response is unrelated to cellular anoxia.

reduced nicotinamide adenine dinucleotide phosphate oxidase; plasma membrane potential; shear stress; intracellular calcium; O2 consumption


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