Mechanisms of ionomycin-induced endothelial cell barrier dysfunction.
Myosin light chain (MLC) phosphorylation catalyzed by the Ca(2+)- calmodulin-dependent MLC kinase (MLCK) is critical to thrombin-mediated endothelial cell gap formation and barrier dysfunction. We have tested the hypothesis that the Ca2+ ionophore ionomycin stimulates MLCK-dependent endothelial cell contraction and permeability. Ionomycin significantly increased albumin clearance and decreased electrical resistance across confluent bovine pulmonary microvascular and macrovascular endothelial cell monolayers in a concentration-dependent manner that was temporally similar to that produced by thrombin. In contrast, however, ionomycin produced a significant Ca(2+)-dependent reduction in the levels of phosphorylated MLC with evidence of serine/threonine phosphatase activation. Potential MLCK-independent mechanisms of endothelial cell permeability were examined with little evidence to support a role for stimulated nitric oxide synthase or phospholipase A2 activities. Importantly, ionomycin produced 1) reductions in the activities of the barrier protective adenylate cyclase and the adenosine 3',5'-cyclic monophosphate-dependent protein kinase A, 2) dramatic dose- and time-dependent inhibition of endothelial cell tyrosine kinase activities, and 3) marked decreases in the phosphotyrosine content of the p125 focal adhesion kinase. These data indicate that ionomycin produces endothelial cell barrier dysfunction by mechanisms that are independent of MLCK activation and may involve reductions in endothelial cell tethering forces via inhibition of protein kinase A and tyrosine kinase activities, especially the p125 focal adhesion kinase.
Garcia
J G
JG
Department of Medicine, Physiology, and Biophysics, Indiana University School of Medicine, Indianapolis, USA.
Schaphorst
K L
KL
Shi
S
S
Verin
A D
AD
Hart
C M
CM
Callahan
K S
KS
Patterson
C E
CE
D000262:Adenylyl Cyclases / Q000378:metabolism; D000818:Animals; D002417:Cattle; D015815:Cell Adhesion Molecules / Q000378:metabolism; D002462:Cell Membrane / Q000502:physiology; D002463:Cell Membrane Permeability / Q000187:drug effects / Q000502:physiology*; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D004553:Electric Conductivity; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; D006801:Humans; D015759:Ionomycin / Q000494:pharmacology*; D007700:Kinetics; D007770:L-Lactate Dehydrogenase; D008564:Membrane Potentials / Q000187:drug effects; D018994:Myosin Light Chains / Q000378:metabolism; D009219:Myosin-Light-Chain Kinase / Q000378:metabolism*; D009569:Nitric Oxide / Q000096:biosynthesis; D019001:Nitric Oxide Synthase / Q000378:metabolism; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D011651:Pulmonary Artery; D012710:Serum Albumin, Bovine / Q000493:pharmacokinetics; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013917:Thrombin / Q000494:pharmacology; D014471:Umbilical Veins
D015815:Cell Adhesion Molecules; D018994:Myosin Light Chains; D012710:Serum Albumin, Bovine; D009569:Nitric Oxide; D015759:Ionomycin; D007770:L-Lactate Dehydrogenase; D019001:Nitric Oxide Synthase; D011505:Protein-Tyrosine Kinases; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; C496050:PTK2 protein, human; D009219:Myosin-Light-Chain Kinase; D013917:Thrombin; D000262:Adenylyl Cyclases; D013755:Tetradecanoylphorbol Acetate