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This Article Full Text Full Text (PDF) All Versions of this Article: 290/1/L41    most recent 00444.2004v2 00444.2004v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via ISI Web of Science (2) Citing Articles via Google Scholar Google Scholar Articles by Kondrikov, D. Articles by Su, Y. Search for Related Content PubMed PubMed Citation Articles by Kondrikov, D. Articles by Su, Y.

Growth and density-dependent regulation of NO synthase by the actin cytoskeleton in pulmonary artery endothelial cells

¹Department of Medicine, University of Florida College of Medicine, Gainesville; and ²Malcom Randall Veterans Affairs Medical Center, Gainesville, Florida

Submitted 29 November 2004 ; accepted in final form 12 August 2005

We previously reported association of eNOS with actin increases eNOS activity. In the present study, regulation of activity of eNOS by actin cytoskeleton during endothelial growth was studied. We found eNOS activity in PAEC increased when cells grew from preconfluence to confluence. eNOS activity was much greater in PAEC in higher density than those in lower density, suggesting increase in eNOS activity during cell growth is caused by increase in cell density. Although eNOS protein contents were also increased when endothelial cells grew from preconfluence to confluence, magnitude of increase in eNOS activity was much higher than increase in eNOS protein content, suggesting posttranslational mechanisms played an important role in regulation of eNOS activity during endothelial growth. Confocal fluorescence microscopy revealed eNOS was colocalized with G-actin in preconfluent cells in perinuclear region, with both G-actin in perinuclear area and cortical F-actin in plasma membrane in confluent cells. There was more -actin coimmunoprecipitated with eNOS in Triton X-100-soluble fraction in confluent cells in later growth phase and in high density. Decrease in eNOS association with -actin by silencing -actin expression using -actin siRNA causes inhibition of eNOS activity, NO production, and endothelial monolayer wound repair in PAEC. Moreover, PAEC incubation with cytochalasin D and jasplakinolide resulted in increases in eNOS/actin association and in eNOS activity without changes in eNOS protein content. Yeast two-hybrid experiments suggested strong association between eNOS oxygenase domain and -actin. These results indicate increase in eNOS association with actin is responsible for greater eNOS activity in confluent PAEC.

angiogenesis; regeneration; proliferation; endothelium; nitric oxide

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