The Src-family of non-receptor tyrosine kinases (SrcFK) are important regulators of cell migration and proliferation, but also contribute to smooth muscle contractility. In asthma, airway hyper-responsiveness has been associated with the actions of inflammatory mediators, such as transforming growth-factor-β (TGF-β), on smooth muscle function. We hypothesised that SrcFK may contribute to airway smooth muscle responses to acute bronchoconstrictor stimuli, and to the longer term enhancement of these responses by TGF-β.Isometric tension was measured in isolated bronchioles of male Wistar rats. The component of contraction not dependent on changes in intracellular Ca2+ concentration ([Ca2+]i), was measured in α-toxin permeabilised bronchioles. Measurement of [Ca2+]i (Fluo-4 kit, Molecular Probes), protein phosphorylation and protein expression, were all performed in primary cultures of human airway smooth muscle cells (ASM), obtained from healthy volunteers by deep endobronchial biopsy. Statistical comparisons were by one-way ANOVA unless otherwise stated.The bronchoconstrictors carbachol (Cch) and bradykinin (BK) both induced concentration-dependent (0.01-100µM) contractile responses in isolated rat bronchioles. Maximum contraction (Bmax) and LogM[EC50] (PD2) values were obtained using Hill curve for CCh data and 2-site saturation curve for BK data. These responses were variably sensitive to inhibition by antagonists of Rho-kinase (Y27632, 10µM) and SrcFK (PP2, 30µM), with Cch-induced responses being partially but significantly sensitive to Y27632 (Bmax: control= 210 ±26% vs. Y27632= 160 ± 26%, p<0.01; PD2: control= -5.82 ± 0.1 vs. Y27632= -5.4 ± 0.1, p<0.01; n=6, paired t-tests) and PP2 (Bmax: control= 178 ± 7.6% vs. PP2= 141 ±9.8%, p<0.01; PD2: control= -5.8 ± 0.1 vs. PP2= -5.55 ± 0.1, p<0.05; n=8, paired t-tests). BK-induced contractions were abolished by Y27632 (n=4) while PP2 significantly reduced amplitude of both high and low affinity components (Bmax1: control= 12 ± 4% vs. PP2= 1.7 ± 0.5%, p<0.05; Bmax2: control= 24 ± 6% vs. PP2= 6.8 ± 2%, p<0.05; n=11, paired t-test).SrcFK auto-phosphorylation at tyr416 was enhanced in human ASM by both 1µM BK (323 ± 91% of control, n=7, p<0.05 vs. control) and 100µM CCh (170 ± 30% of control, n=8, p<0.05 vs. control). This phosphorylation was nearly abolished by PP2 in both cases (n=6-8, p<0.01 vs. BK or Cch alone). BK also enhanced phosphorylation of myosin phosphatase targeting subunit-1 (MYPT-1) at thr696 (206 ± 33% of control, n=20, p<0.01 vs. control) and myosin light-chain-20 (MLC20) at ser-19 (172 ± 31% of control, n=16, p<0.01 vs. control). Both phosphorylation responses were significantly inhibited by Y27632 (MYPT-1: n=13, p<0.01 vs. BK alone; MLC20: n=9, p<0.05 vs. BK alone) and PP2 (MYPT-1: n=15, p<0.05 vs. BK alone; MLC20: n=15, p<0.05 vs. BK alone). Cch did not alter phosphorylation of either protein.Cch also induced concentration-dependent contractile responses in α-toxin permeabilised rat bronchioles and these were abolished by Y27632 (n=6) and inhibited by PP2 (Bmax: control= 61 ± 8% vs. PP2= 38 ± 6%, p<0.05; PD2: control= -5.1 ± 0.1 vs. PP2= -4.6 ± 0.1, p<0.05; n=9, unpaired t-test). Both BK (n=12) and Cch (n=12) induced increases in [Ca2+]i in human ASM. However, neither response was significantly affected by SrcFK inhibition with PP2 (paired t-test). Prior Incubation of bronchioles with TGF-β (30ng/ml, 18hr) in serum-free media specifically enhanced the low affinity component of the BK-induced contraction when compared to incubation in media alone (Bmax2: media= 21 ± 5%, n=17 vs. TGF-β= 49 ± 12%, n=15, p<0.05, unpaired t-test). Cch-induced contraction was not significantly altered by TGF-β incubation (TGF-β n=8 vs. media only n=13). SrcFK inhibition did not prevent the potentiating effect of TGF-β on BK contraction, in fact, contractile responses to both BK and Cch were generally less sensitive to PP2 after 18hr incubation (with or without TGF-β). In human ASM, incubation with TGF-β (10ng/ml, 24hr) enhanced the protein expression of MLC20 (253 ± 29% of control, n=6, P<0.05 vs. control), MYPT-1 (199 ± 25% of control, n=6, p<0.05 vs. control) and the RhoA guanine nucleotide exchange factor ARHGEF-1 (201 ± 35% of control, n=6, p<0.05 vs. control). It also significantly reduced the protein expression of Src (83 ± 12% of control, n=16, p<0.05 vs. control), but had no effect on RhoA (n=6) or Rho-kinase (n=6) expression.Our results suggest that Src contributes to the bronchoconstrictor effects of BK and Cch, acting primarily via Rho-kinase dependent Ca2+-sensitization, involving enhanced MYPT-1 and MLC20 phosphorylation. Prior treatment with TGF-β enhances BK-induced constriction and the expression of several contractile proteins, but this is not associated with an enhanced dependence of contractile responses on SrcFK.