Ca2+- and Phosphatidylinositol 3-Kinase-dependent Nitric Oxide Generation in Lung Endothelial Cells in Situ with Ischemia

Abstract

Endothelial cells generate nitric oxide (NO) in response to agonist stimulation or increased shear stress. In this study, we evaluated the effects of abrupt cessation of shear stress on pulmonary endothelial NO generation and its relationship to changes in intracellular Ca 2؉ . In situ endothelial generation of NO and changes in intracellular Ca 2؉ in isolated, intact rat lungs were evaluated using fluorescence microscopy with diaminofluorescein diacetate, an NO probe, and Fluo-3, a Ca 2؉ probe. The onset of increased NO generation in endothelial cells of subpleural microvessels in situ occurred between 30 and 90 s after onset of ischemia and was preceded by an increase in intracellular Ca 2؉ due to both influx of extracellular Ca 2؉ and release from intracellular stores. Flow cessation-induced NO generation in endothelial cells in situ was Ca 2؉ -, calmodulin-, and PI3-kinase-dependent. The similarity of endothelial cell response (increased NO generation) to either increased flow or cessation of flow suggests that cells respond to an imposed alteration from a state of adaptation. This response to flow cessation may constitute a compensatory vasodilatatory mechanism and may play a role in signaling for cell proliferation and vascular remodeling.Nitric oxide (NO) 1 is a potent regulator of vascular tone in systemic and pulmonary vessels and plays an important role in cellular signaling and respiration (1-4). This mediator is generated by endothelial cells in response to agonist stimulation and also as a response to increased shear stress (5). Although the effect of increase in flow on endothelial cell NO generation has been characterized, the effect of abrupt cessation of shear stress (i.e. acute ischemia as in pulmonary embolism or donor lung isolation for transplantation) on pulmonary endothelial NO generation in situ is unknown.Endothelial cells in situ are constantly exposed to shear stress associated with blood flow and thus become flowadapted. These cells respond with an increase in cytosolic Ca 2ϩ and generation of reactive oxygen species when flow is stopped but oxygenation is maintained (6, 7). A similar response with reactive oxygen species generation subsequent to the abrupt cessation of flow has been demonstrated with endothelial cells adapted to flow in vitro (8). We hypothesized that the basis for this early response is mechanotransduction related to removal of endothelial cell shear stress (9). Increased intracellular Ca 2ϩ is known to activate endothelial nitric-oxide synthase (eNOS) resulting in increased NO generation and has been demonstrated in cells exposed to increased shear stress (10,11). In these nonadapted endothelial cells, shear-stress-induced NOS activation was biphasic, with an initial Ca 2ϩ -dependent phase and a second, sustained phase that was Ca 2ϩ -independent and phosphorylation-dependent (10 -12). In this study, we evaluated whether the increased intracellular Ca 2ϩ associated with the early response to flow cessation leads to increased NO generation. This paradigm constitu...

Greater Effectiveness of ε-Viniferin in Red Wine Than Its Monomer Resveratrol for Inhibiting Vascular Smooth Muscle Cell Proliferation and Migration

“…The DAF-2DA fluorescence reagent was used to measure NO production. 30) Briefly, VSMCs (3 Â 10 3 cells/well) plated on to a 96-well plate were loaded with 10 mM DAF-2DA for 30 min and then incubated for 5 h with 100 mM L-arginine in the presence of different concentrations of resveratrol or "-viniferin. The production of NO was evaluated by recording the fluorescence at 485 nm as excitation and 530 nm as emission with a luminescence spectrophotometer (PerkinElmer, Waltham, MA, USA).…”

Arrest of B16 Melanoma Cells in the Mouse Pulmonary Microcirculation Induces Endothelial Nitric Oxide Synthase-Dependent Nitric Oxide Release that Is Cytotoxic to the Tumor Cells

“…NO and its higher oxides, such as NO x or nitrous anhydride (N 2 O 3 ), provide the third nitrogen to form a triazo ring from the two amino groups of the nonfluorescent DAF-2 and convert it to diaminotriazolofluorescein (DAF-2T) that is fluorescent and can be monitored at 490 nm excitation and 530 nm emission. 13 We used an established intact organ microscopy technique to image microvascular endothelial cells in situ in isolated, ventilated, blood-free mouse lungs in real time using an epifluorescence microscope. 14 Briefly, female WT mice, weighing 20 to 22 g were anesthetized with intraperitoneal sodium pentobarbital (50 mg/kg).…”

Acute Lung Injury and Pulmonary Vascular Permeability: Use of Transgenic Models

“…Depolarization was followed by translocation of NADPH oxidase components to the cell membrane and production of ROS (9), and superoxide production could be blocked by knockout of gp91phox, the flavoprotein component of NADPH oxidase in endothelial cells. Depolarization appears to induce calcium entry through the T-type voltage-gated calcium channels that subsequently activates eNOS and NO production (8,537). These initial responses resulted in downstream activation of MAPK and NF-κB even in oxygenated pulmonary endothelial cells when flow was suddenly stopped.…”

“…K f values were calculated using W/ t at one of the following time points: 1 ( W/ t) t = 0 zero time extrapolation using slope data from 3 to 10 min; 2 ( W/ t) t = 0 zero time extrapolation using data from 6 to 15 min; 3 ( W/ t) t = 3 min ; 4 ( W/ t) t = 10 min ; 5 ( W/ t) t = 15 min ; 6 ( W/ t) t = 20 min ; 7 ( W/ t) t = 0 exponential curve fit extrapolated using 3 to 4 min; 8 Changes in ( W/ t) fitted to step changes in P c . Lung injury was induced by NEM, N-ethylmaleimide; ischemia-reperfusion; VILI, ventilator-induced lung injury; PAR-1, protease-activated receptor-1 (thrombin); PAO, phenylarsine oxide.…”