title: Rho and rho kinase modulation of barrier properties: cultured endothelial cells and intact microvessels of rats and mice.
abstract: Previous experiments using cultured endothelial monolayers indicate that Rho-family small GTPases are involved in modulation of endothelial monolayer permeability by regulating assembly of the cellular actin filament scaffold, activity of myosin-based contractility and junctional distribution of the Ca2+-dependent endothelial cell adhesion molecule, VE-cadherin. We investigated these mechanisms using both cultured endothelial cells (from porcine pulmonary artery and mouse heart) and vascular endothelium in situ (mouse aorta, and individually perfused venular microvessels of mouse and rat mesentery). Exposure to Clostridium difficile toxin B (100 ng x ml(-1)) inactivated 50-90% of all endothelial Rho proteins within 60-90 min. This was accompanied by considerable reduction of actin filament stress fibres and junctional F-actin in cultured endothelial monolayers and in mouse aortic endothelium in situ. Also, VE-cadherin became discontinuous along endothelial junctions. Inhibition of Rho kinase with Y-27632 (30 microM) for 90-120 min induced F-actin reduction both in vitro and in situ but did not cause redistribution or reduction of VE-cadherin staining. Perfusion of microvessels with toxin B increased basal hydraulic permeability (L(p)) but did not attenuate the transient increase in L(p) of microvessels exposed to bradykinin. Perfusion of microvessels with Y-27632 (30 microM) for up to 100 min reduced basal L(p) but did not attenuate the permeability increase induced by platelet activating factor (PAF) or bradykinin. These results show that toxin B-mediated reduction of endothelial barrier properties is due to inactivation of small GTPases other than RhoA. Rho proteins as well as RhoA-mediated contractile mechanisms are not involved in bradykinin- or PAF-induced hyperpermeability of intact microvessels.
authors.affiliation: Department of Human Physiology, University of California, Davis, CA 95616, USA.
authors.forename: R H
authors.initials: RH
authors.lastname: Adamson
authors.forename: F E
authors.initials: FE
authors.lastname: Curry
authors.forename: G
authors.initials: G
authors.lastname: Adamson
authors.forename: B
authors.initials: B
authors.lastname: Liu
authors.forename: Y
authors.initials: Y
authors.lastname: Jiang
authors.forename: K
authors.initials: K
authors.lastname: Aktories
authors.forename: H
authors.initials: H
authors.lastname: Barth
authors.forename: A
authors.initials: A
authors.lastname: Daigeler
authors.forename: N
authors.initials: N
authors.lastname: Golenhofen
authors.forename: W
authors.initials: W
authors.lastname: Ness
authors.forename: D
authors.initials: D
authors.lastname: Drenckhahn
mesh_terms: D000209:Acute-Phase Proteins / Q000502:physiology*; D000577:Amides / Q000494:pharmacology; D000818:Animals; D001011:Aorta / Q000187:drug effects; D001426:Bacterial Proteins; D001427:Bacterial Toxins / Q000494:pharmacology; D001920:Bradykinin / Q000494:pharmacology; D002199:Capillary Permeability / Q000502:physiology*; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006031:Glycosylation; D018836:Inflammation Mediators / Q000494:pharmacology; D047908:Intracellular Signaling Peptides and Proteins; D051379:Mice; D008833:Microcirculation; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000502:physiology*; D011725:Pyridines / Q000494:pharmacology; D051381:Rats; D013552:Swine; D014699:Venules / Q000187:drug effects; D054460:rho-Associated Kinases
chemical_list: D000209:Acute-Phase Proteins; D000577:Amides; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D004791:Enzyme Inhibitors; D018836:Inflammation Mediators; D047908:Intracellular Signaling Peptides and Proteins; D011725:Pyridines; C010781:acute-phase protein rho; C031815:toxB protein, Clostridium difficile; C108830:Y 27632; D017346:Protein Serine-Threonine Kinases; D054460:rho-Associated Kinases; D001920:Bradykinin