P283	Mechanism of myosin light chain phosphatase activation by extracellular ATP signaling.
1F.Härtel, 1Chr.Rodewald, 1M.Aslam, 1D.Gündüz, 2L.Hafer, 3J.Neumann, 1H-M.Piper, 1T.Noll
1Physiologisches Institut, Justus-Liebig-Universität, Gießen, DE; 2Institut für Pharmakologie und Toxikologie, Westfälische Wilhelms-Universität, Münster, DE; 3Institut für Pharmakologie und Toxikologie, Martin-Luther-Universität Halle-Wittenberg, Halle/Saale, DE.

Failure of endothelial barrier function can occur when endothelial cells are exposed to inflammatory mediators, e.g. generated during sepsis, or conditions of metabolic disturbance, e.g. in ischemia-reperfusion. Loss of barrier function results from the opening of gaps between adjacent cells as a consequence of both, loss of cell adhesion and activation of the endothelial contractile machinery. We recently showed that extracellular ATP stabilizes endothelial barrier and inactivates the contractile machinery of endothelial cells. Here we addressed the question, whether inactivation of the contractile machinery is due to an activation of the myosin light chain phosphatase holoenzyme (MLCP). The MLCP consists of a catalytic protein phosphatase 1 subunit (PP1) and a regulatory myosin targeting subunit (MYPT1), guiding PP1 to myosin. The mechanism of extracellular ATP-mediated activation of MLCP was analyzed in human umbilical vein endothelial cells transfected with adenovirus encoding the PP1-specific inhibitor-2 (Inh2). Overexpression of Inh2 leads to an inhibition of PP1 activity and abrogation of the ATP-induced dephosphorylation of MLC. This indicates that the PP1 catalytic subunit is the principal phosphatase catalyzing MLC dephosphorylation induced by extracellular ATP. As demonstrated by immunoprecipitation analysis, extracellular ATP causes the recruitment of PP1 catalytic subunit α and δ, and MYPT1 to myosin (1.7 ± 0.3, 1.5 ± 0.2, and 3.5 ± 0.5-fold increase of the components in the complex, respectively; n=3, P<0.05). Complex formation was accompanied by a 1.6-fold increase of PP1 activity. In addition, ATP caused dephosphorylation of the regulatory phosphorylation sites of MYPT1 at Thr850 and Thr696, as well as PP1 at Ser320. Extracellular ATP failed to dephosphorylate MYPT1 in Inh2-overexpressing cells, indicating that PP1 is not only involved in dephosphorylation of MLC but also of MYPT1. Conclusion: Signaling of extracellular ATP stimulates MLCP activation through assembly of the MLCP holoenzyme and reduction of inhibitory phosphorylation of the regulatory MYPT1 and catalytic PP1 subunit. These data may explain the mechanism by which extracellular ATP inactivates the contractile machinery and stabilizes endothelial barrier function.

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