cAMP/PKA antagonizes thrombin-induced inactivation of endothelial myosin light chain phosphatase: role of CPI-17.
OBJECTIVE
Activation of cAMP signalling abrogates thrombin-induced hyperpermeability. One of the mechanisms underlying this protective effect is the inactivation of endothelial contractile machinery, one of the major determinants of endothelial barrier function, mainly via the activation of myosin light chain phosphatase (MLCP). To date, the mechanisms of cAMP-mediated MLCP activation are only partially understood. Here the contribution of two cAMP effectors, PKA and Epac, in the regulation of endothelial contractile machinery and barrier function was studied.


RESULTS
Endothelial contractile machinery and barrier function were analysed in cultured human umbilical vein endothelial cells (HUVEC). The cAMP analogues 8-CPT-cAMP and 6-Bnz-cAMP were used to activate Epac and PKA, respectively, and forskolin (FSK) was used to activate adenylyl cyclase. The cells were challenged by thrombin to inhibit MLCP via the RhoA/Rock pathway. Activation of either PKA or Epac partially blocked thrombin-induced hyperpermeability. Simultaneous activation of PKA and Epac had additive effects that were comparable to that of FSK. Activation of PKA but not Epac inhibited thrombin-induced phosphorylation of MLC and the MLCP regulatory subunit MYPT1, partly via inhibition of the RhoA/Rock pathway. FSK activated the MLCP catalytic subunit PP1 via dephosphorylation and dissociation of the PP1 inhibitory protein CPI-17. FSK blunted thrombin-induced CPI-17 phosphorylation, CPI-17/PP1 complex formation, and PP1 inactivation. Down-regulation of CPI-17 attenuated thrombin-induced hyperpermeability and abolished the antagonistic effect of the PKA activator, whereas the Epac activator retained its antagonistic effect.


CONCLUSIONS
cAMP/PKA regulates the endothelial barrier via inhibition of the contractile machinery, mainly by the activation of MLCP via inhibition of CPI-17 and RhoA/Rock. The permeability-lowering effect of the cAMP/Epac pathway is independent of CPI-17.
Aslam
Muhammad
M
Physiologisches Institut, Justus-Liebig-Universität, Aulweg 129, Giessen, Germany.
Härtel
Frauke V
FV
Arshad
Muhammad
M
Gündüz
Dursun
D
Abdallah
Yaser
Y
Sauer
Heinrich
H
Piper
Hans Michael
HM
Noll
Thomas
T
D000262:Adenylyl Cyclases / Q000378:metabolism; D002199:Capillary Permeability / Q000187:drug effects; D002478:Cells, Cultured; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000031:analogs & derivatives / Q000378:metabolism* / Q000494:pharmacology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D042783:Endothelial Cells / Q000201:enzymology*; D004789:Enzyme Activation; D020536:Enzyme Activators / Q000494:pharmacology; D020662:Guanine Nucleotide Exchange Factors / Q000378:metabolism; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D009124:Muscle Proteins; D018994:Myosin Light Chains / Q000378:metabolism; D043263:Myosin-Light-Chain Phosphatase / Q000378:metabolism*; D010749:Phosphoprotein Phosphatases / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D054645:Protein Phosphatase 1 / Q000378:metabolism; D034622:RNA Interference; D015398:Signal Transduction; D013873:Thionucleotides / Q000494:pharmacology; D013917:Thrombin / Q000378:metabolism*; D013997:Time Factors; D054460:rho-Associated Kinases / Q000378:metabolism; D020742:rhoA GTP-Binding Protein / Q000378:metabolism
D020536:Enzyme Activators; D020662:Guanine Nucleotide Exchange Factors; D047908:Intracellular Signaling Peptides and Proteins; D009124:Muscle Proteins; D018994:Myosin Light Chains; C107507:PPP1R14A protein, human; C116122:RAPGEF3 protein, human; D013873:Thionucleotides; C066737:RHOA protein, human; D005576:Colforsin; C050086:N(6)-benzoyl-cyclic AMP; C015929:8-((4-chlorophenyl)thio)cyclic-3',5'-AMP; D000242:Cyclic AMP; D054460:rho-Associated Kinases; D017868:Cyclic AMP-Dependent Protein Kinases; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1; D043263:Myosin-Light-Chain Phosphatase; C490372:PPP1R12A protein, human; D013917:Thrombin; D020742:rhoA GTP-Binding Protein; D000262:Adenylyl Cyclases