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

Dmitry Kondrikov, Hye Rim Han, Edward R. Block, Yunchao Su

Research output: Contribution to journalArticle

26 Citations (Scopus)

Abstract

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 β-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 β-actin by silencing β-actin expression using β-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 β-actin. These results indicate increase in eNOS association with actin is responsible for greater eNOS activity in confluent PAEC.

Original languageEnglish (US)
Pages (from-to)L41-L50
JournalAmerican Journal of Physiology - Lung Cellular and Molecular Physiology
Volume290
Issue number1
DOIs
StatePublished - Jan 1 2006

Fingerprint

Actin Cytoskeleton
Nitric Oxide Synthase
Pulmonary Artery
Actins
Endothelial Cells
Growth
jasplakinolide
Cytochalasin D
Oxygenases
Proteins
Octoxynol
Fluorescence Microscopy
Confocal Microscopy
Small Interfering RNA
Cell Count
Yeasts
Cell Membrane
Wounds and Injuries

Keywords

  • Angiogenesis
  • Endothelium
  • Nitric oxide
  • Proliferation
  • Regeneration

ASJC Scopus subject areas

  • Physiology
  • Pulmonary and Respiratory Medicine
  • Physiology (medical)
  • Cell Biology

Cite this

Growth and density-dependent regulation of NO synthase by the actin cytoskeleton in pulmonary artery endothelial cells. / Kondrikov, Dmitry; Han, Hye Rim; Block, Edward R.; Su, Yunchao.

In: American Journal of Physiology - Lung Cellular and Molecular Physiology, Vol. 290, No. 1, 01.01.2006, p. L41-L50.

Research output: Contribution to journalArticle

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