Abstract
Background and PurposeThe 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 ApproachContraction 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 ResultsContractile 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 ImplicationsSrcFK 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.
Highlights
Airway smooth muscle (ASM) tone is subject to regulation by cholinergic, catecholamine and NANC neurotransmitters as well as local inflammatory mediators
We found that Src-family kinases (SrcFK), most likely c-Src itself, modulate Rho-kinase dependent Ca2+-sensitization, but focal adhesion kinase (FAK) does not, and that the two tyrosine kinases differentially regulate store-operated Ca2+ entry (SOCE)/receptor-operated Ca2+ entry (ROCE) and voltage-operated Ca2+ entry (VOCE)
The maximum response to CCh was significantly reduced by PP2 (P < 0.01, paired t-test, n = 8), Y27632 (P < 0.01, paired t-test, n = 6) and PF-573228 (P < 0.05, paired t-test, n = 8), and the PD2 was significantly increased by PP2 (À5.55 ± 0.09 vs. control À5.8 ± 0.14, P < 0.05, paired t-test, n = 8), Y27632 (À5.4 ± 0.07 vs. control À5.82 ± 0.07, P < 0.01, paired t-test, n = 6) and PF-573228 (À5.21 ± 0.08 vs. control À5.69 ± 0.07, P < 0.001, paired t-test, n = 8) (Figure 1A–D)
Summary
Airway smooth muscle (ASM) tone is subject to regulation by cholinergic, catecholamine and NANC neurotransmitters as well as local inflammatory mediators. Smooth muscle contractile force depends on the degree of myosin light-chain-20 (MLC20) phosphorylation, which is in turn determined by the balance between Ca2+-dependent activation of myosin light-chain kinase (MLCK) and Ca2+-independent inhibition of myosin light-chain phosphatase (MLCP), as well as the formation and recruitment of myofilaments (Gunst et al, 2003; Somlyo and Somlyo, 2003). Inhibition of MLCP occurs via phosphorylation of myosin phosphatase targeting subunit-1 (MYPT-1), primarily by Rho-kinase (Feng et al, 1999), resulting in a further increase in MLC20 phosphorylation and contraction without the need for a further increase in [Ca2+]i (Somlyo and Somlyo, 2003). The importance of tyrosine kinases in airway smooth muscle (ASM) contraction is not fully understood. Phosphorylation of SrcFK, FAK, myosin light-chain (MLC20) and myosin phosphatase targeting subunit-1 (MYPT-1) was evaluated in cultured human ASM cells (hASMC). Responses to carbachol (CCh) and bradykinin (BK) and the contribution of SrcFK and FAK to these responses were determined
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