Divergent modulation of Rho-kinase and Ca2+ influx pathways by Src family kinases and focal adhesion kinase in airway smooth muscle

Yasin Shaifta, Nneka Irechukwu, Jesus Prieto-Lloret, Charles Mackay, Keisha A. Marchon, Jeremy P.T. Ward, Greg A. Knock

Research output: Contribution to journalArticle

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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 permeabilized 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 depolarization-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 languageEnglish (US)
Pages (from-to)5265-5280
Number of pages16
JournalBritish Journal of Pharmacology
Volume172
Issue number22
DOIs
StatePublished - Nov 1 2015

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Focal Adhesion Protein-Tyrosine Kinases
rho-Associated Kinases
src-Family Kinases
Smooth Muscle
Myosin-Light-Chain Phosphatase
Phosphorylation
Muscle Contraction
Bronchioles
Carbachol
Bradykinin
Myosin Light Chains
Fura-2
Protein-Tyrosine Kinases
Smooth Muscle Myocytes
Tyrosine

All Science Journal Classification (ASJC) codes

  • Pharmacology

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Divergent modulation of Rho-kinase and Ca2+ influx pathways by Src family kinases and focal adhesion kinase in airway smooth muscle. / Shaifta, Yasin; Irechukwu, Nneka; Prieto-Lloret, Jesus; Mackay, Charles; Marchon, Keisha A.; Ward, Jeremy P.T.; Knock, Greg A.

In: British Journal of Pharmacology, Vol. 172, No. 22, 01.11.2015, p. 5265-5280.

Research output: Contribution to journalArticle

Shaifta, Yasin ; Irechukwu, Nneka ; Prieto-Lloret, Jesus ; Mackay, Charles ; Marchon, Keisha A. ; Ward, Jeremy P.T. ; Knock, Greg A. / Divergent modulation of Rho-kinase and Ca2+ influx pathways by Src family kinases and focal adhesion kinase in airway smooth muscle. In: British Journal of Pharmacology. 2015 ; Vol. 172, No. 22. pp. 5265-5280.
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T1 - Divergent modulation of Rho-kinase and Ca2+ influx pathways by Src family kinases and focal adhesion kinase in airway smooth muscle

AU - Shaifta, Yasin

AU - Irechukwu, Nneka

AU - Prieto-Lloret, Jesus

AU - Mackay, Charles

AU - Marchon, Keisha A.

AU - Ward, Jeremy P.T.

AU - Knock, Greg A.

PY - 2015/11/1

Y1 - 2015/11/1

N2 - 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 permeabilized 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 depolarization-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.

AB - 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 permeabilized 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 depolarization-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.

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