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A2741 - Protein Kinase C, L-Type Voltage-Gated Calcium Channels and Non-Muscle Myosin II Are Involved in ‘Force Adaptation’
Author Block: M. Gazzola1, A. Lee-Gosselin2, L. Auger2, K. Lortie2, Y. Bossé3; 1Pneumology, CRIUCPQ, Quebec, QC, Canada, 2Pneumology, CRIUCPQ, Québec, QC, Canada, 3Medicine, Université Laval, Québec, QC, Canada.
Rationale: We have previously demonstrated that tone (i.e., a sustained contraction) increases airway responsiveness in mice and humans by increasing the contractile capacity of airway smooth muscle (ASM). Since tone is elevated in asthma, we surmise that this phenomenon, dubbed 'force adaptation', contributes to airway hyperresponsiveness. Understanding the molecular basis underlying ‘force adaptation’ is thus paramount. We hypothesize that several intracellular pathways are involved in ‘force adaptation’, including the activation of protein kinase C (PKC), the handing of extracellular Ca2+, the activation of the non-muscle myosin II, the phosphorylation of the myosin light chain (MLC) and the polymerization of actin. Methods: A set of experiments is conducted with excised tracheal segments derived from C57BL/6 mice. As previously reported, the effect of tone on the contractile capacity of ASM is measured by simulating the tracheal segments with 10-4 M of methacholine (MCh) following a period of 30 minutes with or without tone elicited by the EC30 of MCh. These experiments are also conducted in the presence of BIM-X, an inhibitor of the classical PKC isoforms, Gö 6976, an inhibitor of the PKC isoforms α and β, nifedipine, an inhibitor of the L-type voltage-gated Ca2+ channels (LVGC), and blebbistatin, an inhibitor of the non-muscle myosin II. Other sets of experiments are conducted with isolated human bronchial smooth muscle cells. The cells are either left unstimulated or exposed to tone elicited by 10-7 M of MCh for 30 minutes and then stimulated or not with 10-4 M of MCh for 5 minutes prior to homogenization. The level of MLC phosphorylation is then measured by western blots. The level of actin polymerization is tested by measuring the magnitude by which the activity of the DNAse I is inhibited by the homogenates, which is a proxy to quantify the concentration of monomeric G-actin in the samples. Results: The inhibition of the conventional PKC isoforms, LVGC and the non-muscle myosin II seem to completely block ‘force adaptation’. Although the phosphorylation of MLC does not seem to be involved in ‘force adaptation’, preliminary results indicate that actin polymerization is required. Further experiments are needed to confirm these findings. Conclusion: Our preliminary results suggest that the activation of PKC, the presence of extracellular Ca2+, the activation of non-muscle myosin II and the polymerization of actin are required for ‘force adaptation’.