Agonist-induced Ca 2 +-sensitization in smooth muscle : redundancy of RhoGEFs and response kinetics ; a caged compound study

Ca/calmodulin-dependent myosin light chain kinase (MLCK) phosphorylates myosin regulatory light chain (RLC) to initiate smooth muscle contraction while myosin light chain phosphatase (MLCP) containing regulatory (MYPT1) and catalytic (PP1cδ) subunits dephosphorylates RLC for relaxation. Activation of a small fraction of MLCK by limiting amounts of free Ca/calmodulin combined with MLCP inhibition is sufficient for robust RLC phosphorylation and contractile responses in bladder smooth muscle. As predicted, haploinsufficiency of MLCK had no effect on RLC phosphorylation and contraction. However, MLCK activity is limiting in aorta and Ca sensitization responses are modest. Haploinsufficiency of MLCK in aorta attenuates RLC phosphorylation and contractile responses which may contribute to dissections of human thoracic aorta. In bladder smooth muscle neurostimulation leads to purinergic-mediated Ca signals for the initial Ca/calmodulin activation of MLCK with muscarinic receptors supporting sustained RLC phosphorylation and contractile responses. Neurostimulation also leads to phosphorylation of CPI-17, but not MYPT1, to enhance Ca sensitivity while also initiating phosphorylation-dependent Ca desensitization of MLCK. The interplay between Ca sensitization and desensitization mechanisms fine tunes the contractile signaling module for force development. Smooth muscle-specific knockout of MYPT1 in mice shows it is not essential for smooth muscle function. Alterations in contractile and RLC phosphorylation responses in isolated bladder tissues were modest. Quantitative measurements of MYPT1 phosphorylation showed significant constitutive phosphorylation which predicts significant inhibition of MLCP activity. Thus, the knockout of MYPT1 resulting in a 70% reduction of PP1cδ expression appears to result in similar phosphatase activity compared to wildtype tissues containing constitutively phosphorylated MYPT1. Discovery of HS38, a Novel Potent and Selective inhibitor of ZIPK and the DAPKs.