SrcFK is a key mediator of oxidant signalling pathways in Pulmonary Vascular Smooth Muscle

Abstract

Oxidative stress is associated with a number of cardiovascular diseases such as atherosclerosis and pulmonary hypertension. Previously, our group has shown that prostaglandin–F2α and hypoxia enhance total tyrosine phosphorylation, activate Src and cause SrcFK-dependent constriction. Combining these observations with the ROS-sensitivity of Src, lead me to investigate whether interaction between SrcFK and ROS makes a central contribution to vascular smooth muscle function.
Intra-pulmonary Arteries (IPA) were dissected from rat lungs, protein was extracted and phosphorylation of known targets of Rho-kinase (MYPT-1) and MLCK (MLC20) as well as Src auto-phosphorylation (Tyr416) was measured. Protein-protein interactions were examined by immune-precipitation/co-immuno-precipitation. Contraction was measured in IPA mounted on a wire myograph. Primary smooth muscle cell lines, generated from IPAs were used to measure ROS production using the luminescent dye L-012 or were transfected with GFP-tagged RhoA or ARHGEF1 to visualise protein translocation in live cells. In this study, I demonstrate that U46619 and LY83583 induce ROS production in PASMCs. I also demonstrate that U46619 and LY83583 contractile responses in IPA are sensitive to both antioxidants and SrcFK inhibition. U46619, hypoxia and LY83583 also enhance SrcFK autophosphorylation, ROCK activity and MLC20 phosphorylation which are blocked by SrcFK inhibition, antioxidants (U46619 and LY83583) and mitochondrial inhibitors (hypoxia). Finally, U46619 and LY83583 promote reversible translocation of eGFP-RhoA or eGFPARHGEF1 in a SrcFK dependent and ROS dependent manner. U46619 also enhanced coimmunoprecipitation of ARHGEF1 with SrcFK. Taken together these results suggest SrcFK’s play a key role as ROS sensitive intermediates which enhance contractile responses via interactions with ARHGEF1 and enhanced ROCK activity.

Date of Award	2016
Original language	English
Awarding Institution	King's College London
Supervisor	Gregory Knock (Supervisor) & Jeremy Ward (Supervisor)