title: Myosin phosphatase and cofilin mediate cAMP/cAMP-dependent protein kinase-induced decline in endothelial cell isometric tension and myosin II regulatory light chain phosphorylation.
abstract: This study determined the effects of increased intracellular cAMP and cAMP-dependent protein kinase activation on endothelial cell basal and thrombin-induced isometric tension development. Elevation of cAMP and maximal cAMP-dependent protein kinase activation induced by 10 microm forskolin, 40 microm 3-isobutyl-1-methylxanthine caused a 50% reduction in myosin II regulatory light chain (RLC) phosphorylation and a 35% drop in isometric tension, but it did not inhibit thrombin-stimulated increases in RLC phosphorylation and isometric tension. Elevation of cAMP did not alter myosin light chain kinase catalytic activity. However, direct inhibition of myosin light chain kinase with KT5926 resulted in a 90% decrease in RLC phosphorylation and only a minimal decrease in isometric tension, but it prevented thrombin-induced increases in RLC phosphorylation and isometric tension development. We showed that elevated cAMP increases phosphorylation of RhoA 10-fold, and this is accompanied by a 60% decrease in RhoA activity and a 78% increase in RLC phosphatase activity. Evidence is presented that it is this inactivation of RhoA that regulates the decrease in isometric tension through a pathway involving cofilin. Activated cofilin correlates with increased F-actin severing activity in cell extracts from monolayers treated with forskolin/3-isobutyl-1-methylxanthine. Pretreatment of cultures with tautomycin, a protein phosphatase type 1 inhibitor, blocked the effect of cAMP on 1) the dephosphorylation of cofilin, 2) the decrease in RLC phosphorylation, and 3) the decrease in isometric tension. Together, these data provide in vivo evidence that elevated intracellular cAMP regulates endothelial cell isometric tension and RLC phosphorylation through inhibition of RhoA signaling and its downstream pathways that regulate myosin II activity and actin reorganization.
authors.affiliation: Department of Pathology, St. Louis University School of Medicine, St. Louis, Missouri 63104, USA.
authors.forename: Zoe M
authors.initials: ZM
authors.lastname: Goeckeler
authors.forename: Robert B
authors.initials: RB
authors.lastname: Wysolmerski
mesh_terms: D000199:Actins / Q000737:chemistry / Q000378:metabolism; D000818:Animals; D002227:Carbazoles / Q000494:pharmacology; D002417:Cattle; D002478:Cells, Cultured; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism; D054746:Cyclic AMP-Dependent Protein Kinase Type II; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D004730:Endothelium, Vascular / Q000378:metabolism*; D047468:Immunoprecipitation; D007211:Indoles / Q000494:pharmacology; D008954:Models, Biological; D018994:Myosin Light Chains / Q000737:chemistry*; D024462:Myosin Type II / Q000378:metabolism*; D043263:Myosin-Light-Chain Phosphatase / Q000378:metabolism*; D010766:Phosphorylation; D011651:Pulmonary Artery / Q000378:metabolism; D011714:Pyrans / Q000494:pharmacology; D015398:Signal Transduction; D013141:Spiro Compounds / Q000494:pharmacology; D013997:Time Factors; D020742:rhoA GTP-Binding Protein / Q000378:metabolism
chemical_list: D000199:Actins; D002227:Carbazoles; D007211:Indoles; D018994:Myosin Light Chains; D011714:Pyrans; D013141:Spiro Compounds; C053079:tautomycin; C063769:KT 5926; D005576:Colforsin; D000242:Cyclic AMP; D054746:Cyclic AMP-Dependent Protein Kinase Type II; D017868:Cyclic AMP-Dependent Protein Kinases; D043263:Myosin-Light-Chain Phosphatase; D024462:Myosin Type II; D020742:rhoA GTP-Binding Protein