Hyper-activation of pp60Src limits nitric oxide signaling by increasing asymmetric dimethylarginine levels during acute lung injury

Sanjiv Kumar, Xutong Sun, Satish Kumar Noonepalle, Qing Lu, Evgeny Zemskov, Ting Wang, Saurabh Aggarwal, Christine Gross, Shruti Sharma, Ankit Desai, Yali Hou, Sridevi Dasarathy, Ning Qu, Vijay Reddy, Sung Gon Lee, Mary Cherian-Shaw, Jason Yuan, John D. Catravas, Ruslan Rafikov, Joe GN Garcia & 1 others Stephen M. Black

Research output: Contribution to journalArticle

2 Citations (Scopus)

Abstract

The molecular mechanisms by which the endothelial barrier becomes compromised during lipopolysaccharide (LPS) mediated acute lung injury (ALI) are still unresolved. We have previously reported that the disruption of the endothelial barrier is due, at least in part, to the uncoupling of endothelial nitric oxide synthase (eNOS) and increased peroxynitrite-mediated nitration of RhoA. The purpose of this study was to elucidate the molecular mechanisms by which LPS induces eNOS uncoupling during ALI. Exposure of pulmonary endothelial cells (PAEC) to LPS increased pp60Src activity and this correlated with an increase in nitric oxide (NO) production, but also an increase in NOS derived superoxide, peroxynitrite formation and 3-nitrotyrosine (3-NT) levels. These effects could be simulated by the over-expression of a constitutively active pp60Src (Y527FSrc) mutant and attenuated by over-expression of dominant negative pp60Src mutant or reducing pp60Src expression. LPS induces both RhoA nitration and endothelial barrier disruption and these events were attenuated when pp60Src expression was reduced. Endothelial NOS uncoupling correlated with an increase in the levels of asymmetric dimethylarginine (ADMA) in both LPS exposed and Y527FSrc over-expressing PAEC. The effects in PAEC were also recapitulated when we transiently over-expressed Y527FSrc in the mouse lung. Finally, we found that the pp60-Src-mediated decrease in DDAH activity was mediated by the phosphorylation of DDAH II at Y207 and that a Y207F mutant DDAH II was resistant to pp60Src-mediated inhibition. We conclude that pp60Src can directly inhibit DDAH II and this is involved in the increased ADMA levels that enhance eNOS uncoupling during the development of ALI.

Original languageEnglish (US)
Pages (from-to)217-228
Number of pages12
JournalFree Radical Biology and Medicine
Volume102
DOIs
StatePublished - Jan 1 2017

Fingerprint

Acute Lung Injury
Lipopolysaccharides
Nitric Oxide
Chemical activation
Nitric Oxide Synthase Type III
Endothelial cells
Nitration
Lung
Peroxynitrous Acid
Endothelial Cells
Phosphorylation
Superoxides
N,N-dimethylarginine

Keywords

  • Acute lung injury
  • eNOS uncoupling
  • Signal transduction

ASJC Scopus subject areas

  • Biochemistry
  • Physiology (medical)

Cite this

Hyper-activation of pp60Src limits nitric oxide signaling by increasing asymmetric dimethylarginine levels during acute lung injury. / Kumar, Sanjiv; Sun, Xutong; Noonepalle, Satish Kumar; Lu, Qing; Zemskov, Evgeny; Wang, Ting; Aggarwal, Saurabh; Gross, Christine; Sharma, Shruti; Desai, Ankit; Hou, Yali; Dasarathy, Sridevi; Qu, Ning; Reddy, Vijay; Lee, Sung Gon; Cherian-Shaw, Mary; Yuan, Jason; Catravas, John D.; Rafikov, Ruslan; Garcia, Joe GN; Black, Stephen M.

In: Free Radical Biology and Medicine, Vol. 102, 01.01.2017, p. 217-228.

Research output: Contribution to journalArticle

Kumar, S, Sun, X, Noonepalle, SK, Lu, Q, Zemskov, E, Wang, T, Aggarwal, S, Gross, C, Sharma, S, Desai, A, Hou, Y, Dasarathy, S, Qu, N, Reddy, V, Lee, SG, Cherian-Shaw, M, Yuan, J, Catravas, JD, Rafikov, R, Garcia, JGN & Black, SM 2017, 'Hyper-activation of pp60Src limits nitric oxide signaling by increasing asymmetric dimethylarginine levels during acute lung injury', Free Radical Biology and Medicine, vol. 102, pp. 217-228. https://doi.org/10.1016/j.freeradbiomed.2016.11.008
Kumar, Sanjiv ; Sun, Xutong ; Noonepalle, Satish Kumar ; Lu, Qing ; Zemskov, Evgeny ; Wang, Ting ; Aggarwal, Saurabh ; Gross, Christine ; Sharma, Shruti ; Desai, Ankit ; Hou, Yali ; Dasarathy, Sridevi ; Qu, Ning ; Reddy, Vijay ; Lee, Sung Gon ; Cherian-Shaw, Mary ; Yuan, Jason ; Catravas, John D. ; Rafikov, Ruslan ; Garcia, Joe GN ; Black, Stephen M. / Hyper-activation of pp60Src limits nitric oxide signaling by increasing asymmetric dimethylarginine levels during acute lung injury. In: Free Radical Biology and Medicine. 2017 ; Vol. 102. pp. 217-228.
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abstract = "The molecular mechanisms by which the endothelial barrier becomes compromised during lipopolysaccharide (LPS) mediated acute lung injury (ALI) are still unresolved. We have previously reported that the disruption of the endothelial barrier is due, at least in part, to the uncoupling of endothelial nitric oxide synthase (eNOS) and increased peroxynitrite-mediated nitration of RhoA. The purpose of this study was to elucidate the molecular mechanisms by which LPS induces eNOS uncoupling during ALI. Exposure of pulmonary endothelial cells (PAEC) to LPS increased pp60Src activity and this correlated with an increase in nitric oxide (NO) production, but also an increase in NOS derived superoxide, peroxynitrite formation and 3-nitrotyrosine (3-NT) levels. These effects could be simulated by the over-expression of a constitutively active pp60Src (Y527FSrc) mutant and attenuated by over-expression of dominant negative pp60Src mutant or reducing pp60Src expression. LPS induces both RhoA nitration and endothelial barrier disruption and these events were attenuated when pp60Src expression was reduced. Endothelial NOS uncoupling correlated with an increase in the levels of asymmetric dimethylarginine (ADMA) in both LPS exposed and Y527FSrc over-expressing PAEC. The effects in PAEC were also recapitulated when we transiently over-expressed Y527FSrc in the mouse lung. Finally, we found that the pp60-Src-mediated decrease in DDAH activity was mediated by the phosphorylation of DDAH II at Y207 and that a Y207F mutant DDAH II was resistant to pp60Src-mediated inhibition. We conclude that pp60Src can directly inhibit DDAH II and this is involved in the increased ADMA levels that enhance eNOS uncoupling during the development of ALI.",
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AU - Kumar, Sanjiv

AU - Sun, Xutong

AU - Noonepalle, Satish Kumar

AU - Lu, Qing

AU - Zemskov, Evgeny

AU - Wang, Ting

AU - Aggarwal, Saurabh

AU - Gross, Christine

AU - Sharma, Shruti

AU - Desai, Ankit

AU - Hou, Yali

AU - Dasarathy, Sridevi

AU - Qu, Ning

AU - Reddy, Vijay

AU - Lee, Sung Gon

AU - Cherian-Shaw, Mary

AU - Yuan, Jason

AU - Catravas, John D.

AU - Rafikov, Ruslan

AU - Garcia, Joe GN

AU - Black, Stephen M.

PY - 2017/1/1

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N2 - The molecular mechanisms by which the endothelial barrier becomes compromised during lipopolysaccharide (LPS) mediated acute lung injury (ALI) are still unresolved. We have previously reported that the disruption of the endothelial barrier is due, at least in part, to the uncoupling of endothelial nitric oxide synthase (eNOS) and increased peroxynitrite-mediated nitration of RhoA. The purpose of this study was to elucidate the molecular mechanisms by which LPS induces eNOS uncoupling during ALI. Exposure of pulmonary endothelial cells (PAEC) to LPS increased pp60Src activity and this correlated with an increase in nitric oxide (NO) production, but also an increase in NOS derived superoxide, peroxynitrite formation and 3-nitrotyrosine (3-NT) levels. These effects could be simulated by the over-expression of a constitutively active pp60Src (Y527FSrc) mutant and attenuated by over-expression of dominant negative pp60Src mutant or reducing pp60Src expression. LPS induces both RhoA nitration and endothelial barrier disruption and these events were attenuated when pp60Src expression was reduced. Endothelial NOS uncoupling correlated with an increase in the levels of asymmetric dimethylarginine (ADMA) in both LPS exposed and Y527FSrc over-expressing PAEC. The effects in PAEC were also recapitulated when we transiently over-expressed Y527FSrc in the mouse lung. Finally, we found that the pp60-Src-mediated decrease in DDAH activity was mediated by the phosphorylation of DDAH II at Y207 and that a Y207F mutant DDAH II was resistant to pp60Src-mediated inhibition. We conclude that pp60Src can directly inhibit DDAH II and this is involved in the increased ADMA levels that enhance eNOS uncoupling during the development of ALI.

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