P284	Stimulation of adenylyl cyclase enhances endothelial barrier property by activation of the myosin light chain phosphatase and Inhibition of CPI-17.
M.Aslam, F.Haertel, D.Gündüz, T.Noll
Institute of Physiology, Justus Liebig University, Giessen, Germany, Gießen, DE.

Failure of endothelial barrier function during inflammation or ischemia-reperfusion causes severe edema that impedes functional recovery of the organ. Maneuvers increasing intracellular levels of cAMP protect against imminent failure of endothelial barrier function by inactivating contractratile machinery, a primary determinant of endothelial barrier. The activation state of the contractile apparatus is controlled by phosphorylation of the myosin light chains (MLC), which are regulated by a balance between the activities of myosin light chain kinase and phosphatase (MLCP). Here we analyzed whether the cAMP-induced stabilization of endothelial barrier function is due to an activation of MLCP. In cultured human umbilical vein endothelial cells, the influence of an adenylyl cyclase activator forskolin (FSK) was analyzed on (i) assembly of the MLCP complex (recruitment of PP1 catalytic subunit and myosin phosphatase targeting subunit [MYPT] to myosin; immunoprecipitation) and activity of the MLCP holoenzyme, (ii) dephosphorylation of MYPT at Thr 850, and (iii) dephosphorylation of the PP1 inhibitory protein CPI-17 at Thr38 and its detachement from PP1. FSK (10µM for 10 min) reduced macromolecule permeability (albumin passage) by 70 ± 9 % (n = 5; P < 0.05, for all further parameters), dephosphorylated MLC by 79 ± 15 % (Western blot) and reduced isometric force in endothelial monolayers by 80 ± 9 % (endothelial cells cultured on collagen lattices). Under the same conditons, FSK dephosphorylated CPI-17 by 45 ± 10 % compared to control and induced detachment of CPI-17 from PP1. FSK also led to dephosphorylation of MYPT by 69 ± 10 % and enhanced translocation of PP1 catalytic subunit and MYPT to myosin by 2.5 ± 0.5, and 4.5 ± 0.5 fold, respectively. These effects of FSK were accompanied by a 2-fold increase in activity of the MLCP holoenzyme. Conclusion: Stimulation of adenylyl cyclase causes assembly and activation of the MLCP holoenzyme complex through dephosphorylation of the myosin targeting subunit and inactivation of the PP1 inhibitor CPI-17. These data demonstrate the mechanism by which cAMP signaling inactivates the contractile machinery and thus stabilizes endothelial barrier function.

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