Endothelial nitric oxide synthase (eNOS) is tightly regulated by a variety of transcriptional, posttranscriptional, and posttranslational mechanisms. We have found that eNOS is associated with the actin protein and that this association increases eNOS activity. We propose a novel hypothesis that eNOS-actin association regulates electron transfer of eNOS resulting in increased catalytic activity and that alterations in eNOS-actin interaction mediate the regulation of eNOS in lung endothelium exposed to different oxygen tensions.
Aim #1 : Determine whether actin interacts with the putative actin binding sequences (ABSs) in the eNOS protein resulting in increased electron transfer and catalytic activity of eNOS. We will use a yeast two-hybrid system and the peptides with sequences of the three putative ABSs of eNOS to pinpoint the actin-binding site in the eNOS protein. We will evaluate the effects of ABS peptides of eNOS on eNOS activity, eNOS-actin interaction, the enzyme kinetics, and on cytochrome c and ferricyanide reductase activities in the mixture of purified eNOS and actin to clarify the mechanism for increased eNOS activity by actin association.
Aim #2 : Determine whether alterations of eNOS activity in lung endothelium exposed to different oxygen tensions are due to changes in eNOS-actin association. We will manipulate the availability of actin in endothelium using siRNA and adenovirus gene transfer technology and introduce the ABS peptides of eNOS into endothelial cells to block eNOS-actin interaction using microinjection technique. Then we will observe hypoxia/hyperoxia-induced alterations in eNOS activity, the actin cytoskeleton, eNOS-actin association, NO release, and endothelium-dependent vasorelaxation. The successful completion of these aims will advance our understanding of the biology of NO, and in turn, have major implications for pulmonary diseases characterized by impaired eNOS activity and vascular NO production, such as primary and secondary pulmonary hypertension, COPD, and cor pulmonale. Determination of the effect of short-term hyperoxia will help to advance our understanding of the mechanism of oxygen therapy and will lead to better management strategy for patients receiving supplemental oxygen therapy.

Public Health Relevance

This proposal is to study a novel hypothesis that eNOS-actin association regulates electron transfer of eNOS resulting in increased catalytic activity and that alterations in eNOS-actin interaction mediate the regulation of eNOS in lung endothelium exposed to different oxygen tensions. Proof of the role of eNOS-actin interaction in hypoxia/hyperoxia-induced alteration of eNOS function would advance our understanding of the biology of NO, and in turn, have major implications for pulmonary diseases characterized by impaired eNOS activity and vascular NO production, such as primary and secondary pulmonary hypertension, COPD, and cor pulmonale. Determination of the effect of short-term hyperoxia will help to advance our understanding of the mechanism of oxygen therapy and will lead to better management strategy for patients receiving supplemental oxygen therapy.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL088261-04
Application #
8097443
Study Section
Respiratory Integrative Biology and Translational Research Study Section (RIBT)
Program Officer
Moore, Timothy M
Project Start
2008-07-01
Project End
2013-06-30
Budget Start
2011-07-01
Budget End
2012-06-30
Support Year
4
Fiscal Year
2011
Total Cost
$367,500
Indirect Cost
Name
Georgia Regents University
Department
Pharmacology
Type
Schools of Medicine
DUNS #
966668691
City
Augusta
State
GA
Country
United States
Zip Code
30912
Kovacs, Laszlo; Han, Weihong; Rafikov, Ruslan et al. (2016) Activation of Calpain-2 by Mediators in Pulmonary Vascular Remodeling of Pulmonary Arterial Hypertension. Am J Respir Cell Mol Biol 54:384-93
Abeyrathna, Prasanna; Su, Yunchao (2015) The critical role of Akt in cardiovascular function. Vascul Pharmacol 74:38-48
Guo, Lu; Tian, Shuang; Chen, Yuguo et al. (2015) CAT-1 as a novel CAM stabilizes endothelial integrity and mediates the protective actions of L-Arg via a NO-independent mechanism. J Mol Cell Cardiol 87:180-91
Kondrikov, Dmitry; Fulton, David; Dong, Zheng et al. (2015) Heat Shock Protein 70 Prevents Hyperoxia-Induced Disruption of Lung Endothelial Barrier via Caspase-Dependent and AIF-Dependent Pathways. PLoS One 10:e0129343
Kondrikov, Dmitry; Gross, Christine; Black, Stephen M et al. (2014) Novel peptide for attenuation of hyperoxia-induced disruption of lung endothelial barrier and pulmonary edema via modulating peroxynitrite formation. J Biol Chem 289:33355-63
Chen, Feng; Barman, Scott; Yu, Yanfang et al. (2014) Caveolin-1 is a negative regulator of NADPH oxidase-derived reactive oxygen species. Free Radic Biol Med 73:201-13
Du, Ying; Zhao, Jianhong; Li, Xi et al. (2014) Dissociation of FK506-binding protein 12.6 kD from ryanodine receptor in bronchial smooth muscle cells in airway hyperresponsiveness in asthma. Am J Respir Cell Mol Biol 50:398-408
Su, Yunchao (2014) Regulation of endothelial nitric oxide synthase activity by protein-protein interaction. Curr Pharm Des 20:3514-20
Sun, Xutong; Kumar, Sanjiv; Sharma, Shruti et al. (2014) Endothelin-1 induces a glycolytic switch in pulmonary arterial endothelial cells via the mitochondrial translocation of endothelial nitric oxide synthase. Am J Respir Cell Mol Biol 50:1084-95
Yang, Ping; Zhang, Yushan; Xu, Junfa et al. (2013) SUMO1 regulates endothelial function by modulating the overall signals in favor of angiogenesis and homeostatic responses. Am J Transl Res 5:427-40

Showing the most recent 10 out of 20 publications