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Abstract
BACKGROUND AND PURPOSE
The importance of tyrosine kinases in airway smooth muscle (ASM) contraction is not fully understood. The aim of this study was to investigate the role of Src-family kinases (SrcFK) and focal adhesion kinase (FAK) in GPCR-mediated ASM contraction and associated signalling events.
EXPERIMENTAL APPROACH
Contraction was recorded in intact or α-toxin permeabilised rat bronchioles. Phosphorylation of SrcFK, FAK, myosin light-chain-20 (MLC20) and myosin phosphatase targeting subunit-1 (MYPT-1) was evaluated in cultured human ASM cells (hASMC). [Ca2+]i was evaluated in Fura-2 loaded hASMC. Responses to carbachol (CCh) and bradykinin (BK) and the contribution of SrcFK and FAK to these responses were determined.
KEY RESULTS
Contractile responses in intact bronchioles were inhibited by antagonists of SrcFK, FAK and Rho-kinase, while after α-toxin permeabilization, they were sensitive to inhibition of SrcFK and Rho-kinase, but not FAK. CCh and BK increased phosphorylation of MYPT-1 and MLC20 and auto-phosphorylation of SrcFK and FAK. MYPT-1 phosphorylation was sensitive to inhibition of Rho-kinase and SrcFK, but not FAK. Contraction induced by SR Ca2+ depletion, and equivalent [Ca2+]i responses in hASMC, were sensitive to inhibition of both SrcFK and FAK, while depolarisation-induced contraction was sensitive to FAK inhibition only. SrcFK auto-phosphorylation was partially FAK-dependent while FAK auto-phosphorylation was SrcFK independent.
CONCLUSIONS AND IMPLICATIONS
SrcFK mediates Ca2+-sensitization in ASM, while SrcFK and FAK together and individually influence multiple Ca2+ influx pathways. Tyrosine phosphorylation is therefore a key upstream signalling event in ASM contraction, and may be a viable target for modulating ASM tone in respiratory disease.
The importance of tyrosine kinases in airway smooth muscle (ASM) contraction is not fully understood. The aim of this study was to investigate the role of Src-family kinases (SrcFK) and focal adhesion kinase (FAK) in GPCR-mediated ASM contraction and associated signalling events.
EXPERIMENTAL APPROACH
Contraction was recorded in intact or α-toxin permeabilised rat bronchioles. Phosphorylation of SrcFK, FAK, myosin light-chain-20 (MLC20) and myosin phosphatase targeting subunit-1 (MYPT-1) was evaluated in cultured human ASM cells (hASMC). [Ca2+]i was evaluated in Fura-2 loaded hASMC. Responses to carbachol (CCh) and bradykinin (BK) and the contribution of SrcFK and FAK to these responses were determined.
KEY RESULTS
Contractile responses in intact bronchioles were inhibited by antagonists of SrcFK, FAK and Rho-kinase, while after α-toxin permeabilization, they were sensitive to inhibition of SrcFK and Rho-kinase, but not FAK. CCh and BK increased phosphorylation of MYPT-1 and MLC20 and auto-phosphorylation of SrcFK and FAK. MYPT-1 phosphorylation was sensitive to inhibition of Rho-kinase and SrcFK, but not FAK. Contraction induced by SR Ca2+ depletion, and equivalent [Ca2+]i responses in hASMC, were sensitive to inhibition of both SrcFK and FAK, while depolarisation-induced contraction was sensitive to FAK inhibition only. SrcFK auto-phosphorylation was partially FAK-dependent while FAK auto-phosphorylation was SrcFK independent.
CONCLUSIONS AND IMPLICATIONS
SrcFK mediates Ca2+-sensitization in ASM, while SrcFK and FAK together and individually influence multiple Ca2+ influx pathways. Tyrosine phosphorylation is therefore a key upstream signalling event in ASM contraction, and may be a viable target for modulating ASM tone in respiratory disease.
| Original language | English |
|---|---|
| Pages (from-to) | 5265-5280 |
| Journal | Br J Pharmacol |
| Volume | 172 |
| Issue number | 22 |
| Early online date | 23 Aug 2015 |
| DOIs | |
| Publication status | Published - 1 Nov 2015 |
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Src as a key mediator of multiple oxidant signalling pathways in vascular smooth muscle
Knock, G. (Primary Investigator) & Ward, J. (Co-Investigator)
1/01/2013 → 31/12/2015
Project: Research