title: Regulation of endothelial cell gap formation and barrier function by myosin-associated phosphatase activities.
abstract: Thrombin-induced cultured bovine endothelial cell (EC) gap formation and albumin permeability is initiated by contraction, which is dependent upon myosin light chain kinase-mediated myosin light chain (MLC) phosphorylation. MLC are then rapidly dephosphorylated (J. G. N. Garcia, H. W. Davis, and C. E. Patterson, J. Cell. Physiol. 163: 510-522, 1995), suggesting a role for MLC dephosphorylation in regulation of EC barrier function. Therefore, we studied the effect of semiselective protein phosphatase (PPase) inhibitors, calyculin A and okadaic acid, on MLC phosphorylation status, myosin-associated PPase activity, and EC monolayer permeability. Calyculin A (0.1-10 nM), but not okadaic acid (1-100 nM) produced significant dose-dependent enhancement of both MLC phosphorylation (three- to four-fold) and EC permeability (eightfold). EC homogenates were utilized to assess Ser/Thr PPase activities using either [32P]phosphorylase A or 32P-labeled skeletal MLC as substrates. Calyculin A at 5 nM (sufficient to inhibit type 1 and type 2A PPase) produced approximately 95% inhibition of all EC PPase activity against both substrates, whereas 2 nM okadaic acid (selective for PPase 2A) only partially inhibited EC PPase activity (40-60%). Fractionation of EC homogenates produced a supernatant fraction containing < 10% of total myosin and a pellet fraction with > 90% of total myosin. PPase activity in the myosin-enriched pellet was insensitive to 2 nM okadaic acid (0% inhibition) but sensitive to 5 nM calyculin (> 95% inhibition). Immunoreactive PPase 1 was present in both fractions, whereas PPase 2A was present only in the myosin-depleted fraction. We conclude that a type 1 myosin-associated PPase is involved in regulation of EC contractility and barrier function.
authors.affiliation: Department of Medicine, Indiana University School of Medicine, Indianapolis, USA.
authors.forename: A D
authors.initials: AD
authors.lastname: Verin
authors.forename: C E
authors.initials: CE
authors.lastname: Patterson
authors.forename: M A
authors.initials: MA
authors.lastname: Day
authors.forename: J G
authors.initials: JG
authors.lastname: Garcia
mesh_terms: D000818:Animals; D015153:Blotting, Western; D002199:Capillary Permeability; D002417:Cattle; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000166:cytology / Q000502:physiology*; D004988:Ethers, Cyclic / Q000494:pharmacology; D017629:Gap Junctions / Q000502:physiology*; D008387:Marine Toxins; D009218:Myosins / Q000378:metabolism / Q000502:physiology*; D019319:Okadaic Acid; D010080:Oxazoles / Q000494:pharmacology; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D011817:Rabbits; D014018:Tissue Distribution
chemical_list: D004988:Ethers, Cyclic; D008387:Marine Toxins; D010080:Oxazoles; D019319:Okadaic Acid; C059041:calyculin A; D010749:Phosphoprotein Phosphatases; D010744:Phosphoric Monoester Hydrolases; D009218:Myosins