Abstract 659: NO Enhances Endothelial Barrier Function by Inactivating the Contractile Machinery and Stabilizing Cell-Adhesions
Failure of endothelial barrier function induced by inflammatory mediators causes severe edema that impedes functional recovery of the organ. NO by activating cGMP/PKG pathway protects against imminent barrier failure.
Here the hypothesis was tested whether NO protects endothelial barrier by inhibiting the contractile machinery and stabilizing cell-cell adhesions, two major determinants of endothelial barrier function.
Methods and Results: Exposure of cultured monolayers of HUVEC to Spermine-NONOate [NO] (10μM, 20 minutes) reduced permeability (P, albumin flux) by 35±7%, isometric tension (IT, cells cultured on collagen gels) by 80±9%, contractile activation, (myosin light chain [MLC] phosphorylation, Western blot) by 31±6% (P<0.05, n=5 for all following parameters). During the same time period NO caused a 2-fold increase in phosphorylation of vasodilator-stimulated phosphoprotein (VASP). All these NO effects could be blunted by KT5823 (1μM), a specific PKG inhibitor. Thrombin (0.2 U/ml) increased permeability by 210±12%, IT (130±8%), MLC~P (90±8%). These effects were attenuated significantly when cells were preincubated with NO. NO induced assembly and activation of the myosin light chain phosphatase (MLCP) holoenzyme complex (3-fold increase in translocation of PP1 catalytic subunit to myosin phosphatase targeting subunit [MYPT1], immunoprecipitation, IP), and 1.5-fold increase in PP1 activity. It also induced a 3-fold increase in translocation of PKG to MYPT1 (IP). NO induced translocation of VE-cadherin, β-catenin, and β-catenin to cell-cell junctions. Thrombin induced loss of VE-cadherin, γ-catenin, and γ-catenin from cell-cell junctions. These thrombin effects were attenuated significantly by preincubation with NO. Moreover, NO induced phosphorylation of the focal adhesion kinase (FAK) and increased formation of focal adhesions (cell fractionation, confocal microscopy).
Conclusion: These data show that NO stabilizes endothelial barrier by activation of MLCP, leading to an inactivation of contractile machinery, and enhancement of cell-cell and cell-matrix adhesions. Co-immunoprecipitation of PKG and MYPT1 indicate that PKG may mediate its effect on MLCP by a direct interaction with MYPT1.