Effect Of M3 MAchR Dysfunction On VE-cadherin Mediated Adherens Junctions And Related Signaling Pathway

PartⅠConstitutive M3 mAChR Stimulation is a Determinant of Endothelial Barrier FunctionObjective:To investigate whether dysfunction of type 3 muscarinic acetylcholine receptor (M3 mAChR) will induce vascular hyperpermeability.Methods and Results:Immunofluorescence staining analysis shows that confluent VE-cadherin andβ-catenin were located at cell borders. EA.hy926 cells exhibit an increase in Ca2+ fluorescence intensity in response to acetylcholine chloride (1×10-6 M); this response is inhibited by 4-DAMP (1×10-6M) pretreatment under laser confocal microscopy. These results suggest that EA.hy926 endothelial cells express VE-cadherin,β-catenin, and functional M3 mAChR and are satisfactory for our next experiments. Transwell system analysis shows that the M3 mAChR selective antagonist 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP) and M3 mAChR siRNA increase endothelial permeability. Because cellular injury also induces endothelial permeability, we measured cell viability by the MTT assay. Endothelial cell (EC) monolayers were exposed for 10 h to 4-DAMP (1×10-4 M), M3 mAChR siRNA, control siRNA or media alone. Significant differences in cell viability were not observed in any of these samples.Conclusions:The basal physiological stimulation of M3 mAChR preserves the endothelial barrier function.PartⅡM3 mAChR dysfunction disrupt VE-cadherin mediated adherens junctionsObjective:To investigate the impact of dysfunction of type 3 muscarinic acetylcholine receptor (M3 mAChR) on vascular endothelial (VE)-cadherin mediated adherens junctions in atherosclerosis.Methods and Results:Using coimmunoprecipitation and a western blotting assay, it was determined that M3 mAChR inhibition increases VE-cadherin andβ-catenin tyrosine phosphorylation but does not affect their expression. Confocal microscopy analysis shows that 4-DAMP reduces the staining of VE-cadherin andβ-catenin at cell borders. Western blotting revealed that a selective M3 mAChR antagonist,4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP), and M3 mAChR siRNA decrease VE-cadherin andβ-catenin in Triton X-100-insoluble fractions, indicating that M3 mAChR inhibition weakens the linkage between the VE-cadherin/β-catenin complex and the actin cytoskeleton.Conclusions:The basal physiological stimulation of M3 mAChR preserves the endothelial barrier function and the integrity of the adherens junctions. PartⅢRole of PTP1B in M3 mAChR inhibition-related tyrosine phosphorylation of adherens junction proteinsObjective:To determine the role of protein-tyrosine phosphatase 1B (PTP1B) in M3 mAChR inhibition-related tyrosine phosphorylation of adherens junction proteins.Methods and Results:Using PTP1B siRNA, we find that PTP1B is required to maintain VE-cadherin andβ-catenin proteins in the dephosphorylated state in unstimulated cells and that PTP1B siRNA augments M3 mAChR inhibition, which is related to tyrosine phosphorylation. In addition,4-DAMP suppresses PTP1B activity by reducing cyclic adenosine monophosphate (cAMP) but not protein kinase Cα(PKCα).Conclusions:The basal physiological stimulation of M3 mAChR preserves the endothelial barrier function and the integrity of the adherens junctions through a mechanism, which potentially maintains PTP1B activity and retains the adherens junction proteins in the dephosphorylated state.PartⅣM3 mAChR Dysfunction Inhibits Rac1 Activity and Disrupts VE-cadherin/β-catenin-Actin Cytoskeleton Interaction.Objective:To determine the role of Ras-related C3 botulinum toxin substrate 1 (Rac1) in M3 mAChR inhibition-induced breakdown of the adherens junction proteins and the actin cytoskeleton connection.Methods and Results:Western blotting revealed that a selective M3 mAChR antagonist, 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP), and M3 mAChR siRNA decrease VE-cadherin andβ-catenin in Triton X-100-insoluble fractions, indicating that M3 mAChR inhibition weakens the linkage between the VE-cadherin/β-catenin complex and the actin cytoskeleton. Co-immunoprecipitation assays showed that M3 mAChR inhibition reduces Rac1 activity and the association of IQ motif-containing GTPase-activating protein 1 (IQGAP1) with Ras-related C3 botulinum toxin substrate 1 (Rac1), while increasing the interaction between IQGAP1 and p-catenin. Using IQGAP1 siRNA, we found that IQGAP1 is required for stable interaction between VE-cadherin/β-catenin and the actin cytoskeleton in quiescent endothelial cells; IQGAP1 siRNA augments the M3 mAChR inhibition-induced dissociation between them. Moreover, S-nitroso-N-acetylpenicillamine (SNAP), a nitric oxide (NO) donor, attenuates this disassociation and Racl inhibition.Conclusions:By maintaining Racl activity, thereby regulating the interaction between IQGAP1/Rac1 and IQGAP1/β-catenin, M3 mAChR facilitates interaction of the VE-cadherin-based adherens junctional complex and the actin-based cytoskeleton, which may contribute endothelial barrier function under physiological conditions.