ROS-dependent activation of RhoA/Rho-kinase in pulmonary artery

Role of Src-family kinases and ARHGEF1

Charles Mackay, Yasin Shaifta, Vladimir V. Snetkov, Asvi A. Francois, Jeremy P.T. Ward, Greg A. Knock

Research output: Contribution to journalArticle

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Abstract

The role of reactive oxygen species (ROS) in smooth muscle contraction is poorly understood. We hypothesised that G-protein coupled receptor (GPCR) activation and hypoxia induce Rho-kinase activity and contraction in rat intra-pulmonary artery (IPA) via stimulation of ROS production and subsequent Src-family kinase (SrcFK) activation. The T-type prostanoid receptor agonist U46619 induced ROS production in pulmonary artery smooth muscle cells (PASMC). U46619 also induced c-Src cysteine oxidation, SrcFK auto-phosphorylation, MYPT-1 and MLC20 phosphorylation and contraction in IPA, and all these responses were inhibited by antioxidants (ebselen, Tempol). Contraction and SrcFK/MYPT-1/MLC20 phosphorylations were also inhibited by combined superoxide dismutase and catalase, or by the SrcFK antagonist PP2, while contraction and MYPT-1/MLC20 phosphorylations were inhibited by the Rho guanine nucleotide exchange factor (RhoGEF) inhibitor Y16. H2O2 and the superoxide-generating quinoledione LY83583 both induced c-Src oxidation, SrcFK auto-phosphorylation and contraction in IPA. LY83583 and H2O2-induced contractions were inhibited by PP2, while LY83583-induced contraction was also inhibited by antioxidants and Y16. SrcFK auto-phosphorylation and MYPT-1/MLC20 phosphorylation was also induced by hypoxia in IPA and this was blocked by mitochondrial inhibitors rotenone and myxothiazol. In live PASMC, sub-cellular translocation of RhoA and the RhoGEF ARHGEF1 was triggered by both U46619 and LY83583 and this translocation was blocked by antioxidants and PP2. RhoA translocation was also inhibited by an ARHGEF1 siRNA. U46619 enhanced ROS-dependent co-immunoprecipitation of ARHGEF1 with c-Src. Our results demonstrate a link between GPCR-induced cytosolic ROS or hypoxia-induced mitochondrial ROS and SrcFK activity, Rho-kinase activity and contraction. ROS and SrcFK activate RhoA via ARHGEF1.

Original languageEnglish (US)
Pages (from-to)316-331
Number of pages16
JournalFree Radical Biology and Medicine
Volume110
DOIs
StatePublished - Sep 1 2017

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Rho Guanine Nucleotide Exchange Factors
rho-Associated Kinases
src-Family Kinases
Phosphorylation
Pulmonary Artery
Reactive Oxygen Species
6-anilino-5,8-quinolinedione
Chemical activation
15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid
Muscle
Antioxidants
G-Protein-Coupled Receptors
Smooth Muscle Myocytes
Rotenone
Oxidation
Muscle Contraction
Immunoprecipitation
Superoxides
Catalase
Small Interfering RNA

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Physiology (medical)

Cite this

ROS-dependent activation of RhoA/Rho-kinase in pulmonary artery : Role of Src-family kinases and ARHGEF1. / Mackay, Charles; Shaifta, Yasin; Snetkov, Vladimir V.; Francois, Asvi A.; Ward, Jeremy P.T.; Knock, Greg A.

In: Free Radical Biology and Medicine, Vol. 110, 01.09.2017, p. 316-331.

Research output: Contribution to journalArticle

Mackay, Charles ; Shaifta, Yasin ; Snetkov, Vladimir V. ; Francois, Asvi A. ; Ward, Jeremy P.T. ; Knock, Greg A. / ROS-dependent activation of RhoA/Rho-kinase in pulmonary artery : Role of Src-family kinases and ARHGEF1. In: Free Radical Biology and Medicine. 2017 ; Vol. 110. pp. 316-331.
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