Annual Scientific Meeting, Edmonton, September 27-30, 2007

S. Martin, S. Basu, D. Schaafsma, A.J. Halayko, Department of Physiology, Faculty of Medicine, University of Manitoba and Manitoba Institute of Child Health, Winnipeg, MB Caveolin-1 (Cav-1) can modulate intracellular signaling pathways in airway smooth muscle (ASM) that may mediate inflammation, contraction, and/or proliferation. Previously, we observed enhanced methacholine (MCh)induced ASM contraction ex vivo and enhanced airway resistance in vivo in mice lacking Cav-1. In the present study, we investigated the possible role of Rho kinase, protein kinase C (PKC), and p42/p44 mitogen-activated protein kinase (MAPK) in the enhanced MCh-induced ASM contraction and airway resistance in Cav-1 knockout mice (Cav-1 KO). Tracheal rings from naive, 8-week-old, female Cav-1 KO and genetically matched B6129SF2/J mice were isolated and mounted on a wire myograph. Isometric contraction in response to MCh was measured in the presence or absence of selective inhibitors of Rho kinase (Y-27632, 1 mM and 10 mM), PKC (bisindolylmaleimide, 3 mM), and p42/p44 MAPK (U0126, 3 mM). The role of Rho kinase in MCh-induced airway resistance (Raw) was also investigated in vivo using a Scireq ventilator. Thirty minutes prior to measuring respiratory mechanics, Cav-1 KO and B6129SF2/J mice were exposed to aerosolized saline or Rho kinase inhibitor (5 mM Y27632, 4 minutes). Using excised tracheal rings, maximum MCh-induced contraction was increased significantly in preparations from Cav-1 KO (8.93 6 0.77 mN) compared to B6129SF2/J mice (6.45 6 0.64 mN). Pretreatment with 10 mM Y-27632 reduced sensitivity and maximum response to MCh in both tissues and normalized the difference in contractile responses between mouse strains. Notably, whereas tracheas from Cav-1 KO exhibited concentration-dependent responses to Y-27632, maximum suppression was achieved with 1 mM of inhibitor in B6129SF2/J mice. Though we did observe a modest effect in suppressing MCh-induced contractile force with bisindolylmaleimide and U0126 treatment, the effect was of equal magnitude on Cav-1 KO and B6129SF2/J mice, suggesting that the contribution of PKC and p42/p44 MAPK to contractile responses is not changed in Cav-1 KO mice. As we previously observed in vivo, Cav-1 KO mice exhibited a significant increase in Raw and tissue resistance (G). However, consistent with our ex vivo experiments, inhaled Y-27632 both decreased Raw and G and normalized these parameters between mouse strains. Collectively, the data suggest that Cav-1 modulates the contribution of Rho kinase in MCh-mediated ASM contraction, which is a principal determinant of Raw.