Effect of cAMP/PKA signaling mechanism on barrier function of cultured endothelial cells : role of myosin light chain phosphatase
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Endothelial barrier dysfunction is often the underlying cause of capillary leakage during pathophysiological conditions like inflammation and ischemia reperfusion. Maneuvers increasing intracellular levels of cAMP protect against imminent failure of endothelial barrier function induced by inflammatory mediators or ischemia-reperfusion. This protective effect is mainly due to an inactivation of the contractile machinery, a primary determinant of endothelial barrier function. The activation of the contractile machinery is regulated by the phosphorylation state of the regulatory myosin light chains (MLC), which is controlled by balanced but antagonistic activities of myosin light chain phosphatase (MLCP) and myosin light chain kinase (MLCK). Here the molecular mechanisms by which cAMP/PKA induced the activation of the MLCP leading to inactivation of the contractile machinery were analyzed. In cultured human umbilical vein endothelial cells activation of adenylyl cyclase by forskolin (FSK) reduced basal macromolecule permeability and caused dephosphorylation of MLC. It also antagonized thrombin-induced increase of both parameters. FSK stimulated the formation of MLCP holoenzyme complex, i.e. it induced the recruitment of the protein phosphatase 1 (PP1) catalytic subunit and myosin phosphatase targeting subunit (MYPT1) to myosin, and activation of the PP1 catalytic subunit. FSK inhibited the RhoA/Rock pathway leading to dephosphorylation of MYPT1. It caused dephosphorylation of the PP1 inhibitory protein CPI-17 at threonine 38 and its detachment from the PP1 catalytic subunit. FSK also blunted the thrombin-induced effect on MYPT1 and CPI-17 phosphorylation. Down regulation of CPI-17 by siRNA attenuated the thrombin effect on endothelial permeability by 35 %. The data of the present study show that stimulation of adenylyl cyclase causes assembly and activation of the MLCP holoenzyme complex and thereby stabilizes endothelial barrier function.Verknüpfung zu Publikationen oder weiteren Datensätzen
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Giessen : VVB Laufersweiler 2007