Role of src-family kinases in hypoxic vasoconstriction of rat pulmonary artery.
OBJECTIVE
We investigated the role of src-family kinases (srcFKs) in hypoxic pulmonary vasoconstriction (HPV) and how this relates to Rho-kinase-mediated Ca(2+) sensitization and changes in intracellular Ca(2+) concentration ([Ca(2+)](i)).


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 (MLC(20)) 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). [Ca(2+)](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 MLC(20) 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 [Ca(2+)](i) that accompany HPV were also inhibited by PP2.


CONCLUSIONS
Hypoxia activates srcFKs and triggers protein tyrosine phosphorylation in IPA. Hypoxia-mediated Rho-kinase activation, Ca(2+) sensitization, and [Ca(2+)](i) responses are depressed by srcFK inhibitors and/or siRNA knockdown, suggesting a central role of srcFKs in HPV.
Knock
Greg A
GA
Department of Asthma, Allergy and Respiratory Science, School of Medicine, King's College London, Room 3.20, Franklin Wilkins Building, Stamford Street, London SE1 9NH, UK. greg.knock@kcl.ac.uk
Snetkov
Vladimir A
VA
Shaifta
Yasin
Y
Drndarski
Svetlana
S
Ward
Jeremy P T
JP
Aaronson
Philip I
PI
D000577:Amides / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D000860:Hypoxia / Q000378:metabolism*; D007211:Indoles / Q000494:pharmacology; D008297:Male; D023421:Models, Animal; D018994:Myosin Light Chains / Q000378:metabolism; D043263:Myosin-Light-Chain Phosphatase / Q000378:metabolism; D010766:Phosphorylation; D011651:Pulmonary Artery / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D011725:Pyridines / Q000494:pharmacology; D051381:Rats; D017208:Rats, Wistar; D013449:Sulfonamides / Q000494:pharmacology; D014661:Vasoconstriction / Q000502:physiology*; D054460:rho-Associated Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D019061:src-Family Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*
D000577:Amides; D004791:Enzyme Inhibitors; D007211:Indoles; D018994:Myosin Light Chains; D011725:Pyridines; C416927:SU 6656; D013449:Sulfonamides; C108830:Y 27632; D019061:src-Family Kinases; D054460:rho-Associated Kinases; D043263:Myosin-Light-Chain Phosphatase; D002118:Calcium