Role of src-family kinases in hypoxic vasoconstriction of rat pulmonary artery

• Published in: Cardiovascular research Vol. 80; no. 3; pp. 453 - 462
• Main Authors: Knock, Greg A., Snetkov, Vladimir A., Shaifta, Yasin, Drndarski, Svetlana, Ward, Jeremy P.T., Aaronson, Philip I.
• Format: Journal Article
• Language: English
• Published: Oxford Oxford University Press 01-12-2008
• Subjects: Amides - pharmacology; Animals; Biological and medical sciences; Calcium - metabolism; Cardiology. Vascular system; Enzyme Inhibitors - pharmacology; Hypoxia; Hypoxia - metabolism; Indoles - pharmacology; Male; Medical sciences; Models, Animal; Myosin Light Chains - metabolism; Myosin-Light-Chain Phosphatase - metabolism; Original; Phosphorylation; Pulmonary; Pulmonary Artery - cytology; Pulmonary Artery - drug effects; Pulmonary Artery - metabolism; Pyridines - pharmacology; Rats; Rats, Wistar; rho-Associated Kinases - antagonists & inhibitors; rho-Associated Kinases - metabolism; Rho-kinase; src-family kinase; src-Family Kinases - antagonists & inhibitors; src-Family Kinases - metabolism; Sulfonamides - pharmacology; Tyrosine kinase; Vasoconstriction; Vasoconstriction - physiology; Rho-kinase; Tyrosine kinase; Hypoxia; family kinase; Pulmonary; Vasoconstriction; Oxygen; Rat; Enzyme; Family study; Transferases; Lung; Rodentia; Phlebology; Pulmonary artery; Vertebrata; Mammalia; Kinase; Animal; Family environment; src-famity kinase; Circulatory system; Cardiology; Protein-tyrosine kinase
• ISSN: 0008-6363; 1755-3245
• DOI: 10.1093/cvr/cvn209

Aims We investigated the role of src-family kinases (srcFKs) in hypoxic pulmonary vasoconstriction (HPV) and how this relates to Rho-kinase-mediated Ca2+ sensitization and changes in intracellular Ca2+ concentration ([Ca2+]i). Methods and results Intra-pulmonary arteries (IPAs) were obtained from male Wistar rats. HPV was induced in myograph-mounted IPAs. Auto-phosphorylation of srcFKs and phosphorylation of the regulatory subunit of myosin phosphatase (MYPT-1) and myosin light-chain (MLC20) in response to hypoxia were determined by western blotting. Translocation of Rho-kinase and effects of siRNA knockdown of src and fyn were examined in cultured pulmonary artery smooth muscle cells (PASMCs). [Ca2+]i was estimated in Fura-PE3-loaded IPA. HPV was inhibited by two blockers of srcFKs, SU6656 and PP2. Hypoxia enhanced phosphorylation of three srcFK proteins at Tyr-416 (60, 59, and 54 kDa, corresponding to src, fyn, and yes, respectively) and enhanced srcFK-dependent tyrosine phosphorylation of multiple target proteins. Hypoxia caused a complex, time-dependent enhancement of MYPT-1 and MLC20 phosphorylation, both in the absence and presence of pre-constriction. The sustained component of this enhancement was blocked by SU6656 and the Rho-kinase inhibitor Y27632. In PASMCs, hypoxia caused translocation of Rho-kinase from the nucleus to the cytoplasm, and this was prevented by anti-src siRNA and to a lesser extent by anti-fyn siRNA. The biphasic increases in [Ca2+]i that accompany HPV were also inhibited by PP2. Conclusion Hypoxia activates srcFKs and triggers protein tyrosine phosphorylation in IPA. Hypoxia-mediated Rho-kinase activation, Ca2+ sensitization, and [Ca2+]i responses are depressed by srcFK inhibitors and/or siRNA knockdown, suggesting a central role of srcFKs in HPV.