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

Abstract

AIMS:

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)).

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 (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.

CONCLUSION:

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.

• Authors: Greg A Knock, Vladimir A Snetkov, Yasin Shaifta, Svetlana Drndarski, Jeremy P T Ward, Philip I Aaronson
• Journal: Cardiovascular research (Cardiovasc Res) Vol. 80 Issue 3 Pg. 453-62 (Dec 01 2008) ISSN: 1755-3245 [Electronic] England
• PMID: 18682436 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• Amides
• Enzyme Inhibitors
• Indoles
• Myosin Light Chains
• Pyridines
• SU 6656
• Sulfonamides
• Y 27632
• src-Family Kinases
• rho-Associated Kinases
• Myosin-Light-Chain Phosphatase
• Calcium

Topics

• Amides (pharmacology)
• Animals
• Calcium (metabolism)
• Enzyme Inhibitors (pharmacology)
• Hypoxia (metabolism)
• Indoles (pharmacology)
• Male
• Models, Animal
• Myosin Light Chains (metabolism)
• Myosin-Light-Chain Phosphatase (metabolism)
• Phosphorylation
• Pulmonary Artery (cytology, drug effects, metabolism)
• Pyridines (pharmacology)
• Rats
• Rats, Wistar
• Sulfonamides (pharmacology)
• Vasoconstriction (physiology)
• rho-Associated Kinases (antagonists & inhibitors, metabolism)
• src-Family Kinases (antagonists & inhibitors, metabolism)