Proceedings of The Physiological Society
University of Oxford (2011) Proc Physiol Soc 23, C58
Role of Rho-kinase and Src-Family Kinases in carbachol-induced contraction of isolated rat bronchioles.
N. Irechukwu1, G. A. Knock1, J. P. Ward1
1. King's College London, London, United Kingdom.
Asthma is associated with remodelling of airway smooth muscle and hyper-responsiveness to constrictor stimuli. Potential therapeutic targets for the treatment of asthma are Rho-kinase and its principle activator RhoA. They have been implicated in both remodelling and constrictor hyper-responsiveness due to their involvement in smooth muscle migration, proliferation and constriction, the latter primarily through inhibition of myosin phosphatase (Ca2+-sensitization). The signalling pathways that control activity of RhoA/Rho-kinase however, such as the Src-family of tyrosine kinases (SrcFK), are largely unexplored. We studied carbachol-induced contraction in isolated wire myograph-mounted rat bronchioles (1260± 68µm), bathed in bicarbonate-buffered physiological salt solution, pH 7.4 at 37°C, and investigated the effects of inhibitors of Rho-Kinase (Y27632, 10µM) and SrcFK (PP2, 30µM) on these contractile responses. Carbachol (0.001-100µM) caused a concentration-dependent constriction (EC50 -5.84 ± 0.06 Log[M], n=18]), which was reproducible upon repetition (both EC50 and max unchanged, P>0.05, n=6). Y27632 significantly increased the EC50 (from -5.81 ± 0.09 to -5.32 ± 0.03, P<0.001, paired t-test, n=6) and decreased the maximal contractile response (by 17.3 ± 6.5%, p<0.05, paired t-test, n=6) (fig a). PP2 also significantly increased the EC50 (from -5.91 ± 0.10 to -5.64 ± 0.10, P<0.01, paired t-test, n=6) and decreased the maximal contractile response (by 10.1 ± 2.4%, p<0.01 paired t-test, n=6) (fig b). Carbachol also caused constriction in α-toxin permeabilised rat bronchioles with [Ca2+]i clamped at pCa 6.4 and in the presence of 10µM cyclopiazonic acid and 1µM GTP. This constriction was also inhibited by Y27632 (n =3). These results suggest the involvement of both Rho-kinase and SrcFK in contraction of rat bronchioles. We aim to further characterise these responses and determine whether SrcFK are acting upstream of Rho-kinase, as shown previously in pulmonary artery (1).
Where applicable, experiments conform with Society ethical requirements