Abstract. Cytoplasmic dynein is a multisubunit, microtubule-dependent mechanochemical enzyme that has been proposed to function in a variety of intracellular movements, including minus-end-directed transport of organelles. Dynein-mediated vesicle transport is stimulated in vitro by addition of the Glued/ dynactin complex raising the possibility that these two complexes interact in vivo. We report here that a class of phenotypically identical mutants of the filamentous fungus Neurospora crassa are defective in genes encoding subunits of either cytoplasmic dynein or the Glued/dynactin complex. These mutants, defined as ropy, have cured hyphae with abnormal nuclear distribution, ro4 encodes the heavy chain of cytoplasmic dynein, while ro-4 encodes an actin-related protein that is a probable homologue of the actin-related protein Arpl (formerly referred to as actin-RPV or centractin), the major component of the glued/dynactin complex. The phenotypes of ro-1 and ro-4 mutants suggest that cytoplasmic dynein, as well as the Glued/dynactin complex, are required to maintain uniform nuclear distribution in fungal hyphae. We propose that cytoplasmic dynein maintains nuclear distribution through sliding of antiparallel microtubules emanating from neighboring spindle pole bodies.
We previously demonstrated that vascular endothelial growth factor (VEGF)-elicited increase in the permeability of coronary venules was blocked by the nitric oxide (NO) synthase inhibitor N G-monomethyl-l-arginine (l-NMMA). The aim of this study was to delineate in more detail the signaling pathways upstream from NO production in VEGF-induced venular hyperpermeability. The apparent permeability coefficient of albumin ( P a) and endothelial cytosolic Ca2+concentration ([Ca2+]i) were measured in intact perfused porcine coronary venules using fluorescence microscopy. VEGF (10−10 M) induced a two- to threefold increase in P a, which was blocked by a monoclonal antibody directed against the VEGF receptor Flk-1/KDR, the phospholipase C (PLC) antagonist U-73122, or the protein kinase C (PKC) antagonist bisindolylmaleimide (BIM). In 12 venules that displayed the [Ca2+]iresponse to bradykinin (10−6M) and ionomycin (10−6 M), only 4 vessels responded to VEGF with a transient increase in [Ca2+]i. Furthermore, Western blot analysis of cultured human umbilical vein endothelial cells showed that VEGF increased tyrosine phosphorylation of PLC-γ and serine phosphorylation of endothelial constitutive NO synthase (ecNOS). The hyperphosphorylation of PLC-γ was greatly attenuated by the KDR receptor antibody and U-73122, but not by BIM orl-NMMA. In contrast, U-73122 and BIM were able to inhibit VEGF-elicited serine phosphorylation of ecNOS. The results suggest that VEGF induces venular hyperpermeability through a KDR receptor-mediated activation of PLC. In turn, ecNOS is activated by PLC-mediated PKC and/or cytosolic Ca2+ elevation stimulation.
The endothelial adherens junction is formed by complexes of transmembrane adhesive proteins, of which -catenin is known to connect the junctional protein vascular endothelial (VE)-cadherin to the cytoskeleton and to play a signaling role in the regulation of junctioncytoskeleton interaction. In this study, we investigated the effect of neutrophil activation on endothelial monolayer integrity and on -catenin and VE-cadherin modification. Treatment of cultured bovine coronary endothelial monolayers with C5a-activated neutrophils resulted in an increase in permeability as measured by albumin clearance across the monolayer. Furthermore, large scale intercellular gap formation was observed in coincidence with the hyperpermeability response. Immunofluorescence analysis showed that -catenin and VE-cadherin staining changed from a uniform distribution along the membrane of control cells to a diffuse pattern for both proteins and finger-like projections for -catenin in neutrophil-exposed monolayers. Correlatively, there was an increase in actin stress fiber formation in treated cells. Finally, -catenin and VE-cadherin from neutrophil-treated endothelial cells showed a significant increase in tyrosine phosphorylation. Our results are the first to link neutrophil-mediated changes in adherens junctions with intercellular gap formation and hyperpermeability in microvascular endothelial cells. These data suggest that neutrophils may regulate endothelial barrier function through a process conferring conformational changes to -catenin and VE-cadherin.The wall of exchange vessels consists of a layer of endothelial cells that connect to each other with closely opposed intercellular junctions. A major function of the junctional connection is to maintain the semi-permeable property of the endothelial barrier and to control the transvascular passage of solutes, fluid, and blood cells. Four types of junctions associated with endothelial cells have been identified: adherens junctions (AJ), 1 tight junctions, gap junctions, and complexus adherentes (1, 2). AJ, formed by transmembrane adhesive proteins called cadherins, appear to be the main complex regulating macromolecular permeability in microvascular endothelium. Cadherins, specifically vascular endothelial (VE)-cadherin, are associated with the actin cytoskeleton through a family of proteins called catenins, including ␣-catenin, -catenin, and plakoglobin (3, 4). The endothelial permeability is affected by many agonists including ␣-thrombin, histamine, and phorbol esters (5-9) as well as by a group of inflammatory cells, namely polymorphonuclear leukocytes (PMNs) (10 -14). At the site of injury or inflammation, circulating PMNs often adhere to and subsequently migrate through the endothelium and enter surrounding tissues (15). It has long been documented that the process of PMN adherence and migration is associated with an increase in endothelial permeability (10, 11). Although much work has been dedicated to identify PMN-derived hyperpermeability factors (11-14), little is k...
Abstract-Neutrophil-induced coronary microvascular leakage represents an important pathophysiological consequence of ischemic and inflammatory heart diseases. The precise mechanism by which neutrophils regulate endothelial barrier function remains to be established. The aim of this study was to examine the microvascular endothelial response to neutrophil activation with a focus on myosin light chain kinase (MLCK)-mediated myosin light chain (MLC) phosphorylation, a regulatory process that controls cell contraction. The apparent permeability coefficient of albumin (Pa) was measured in intact isolated porcine coronary venules. Incubation of the vessels with C5a-activated neutrophils induced a time-and concentration-dependent increase in Pa. The hyperpermeability response was significantly attenuated during inhibition of endothelial MLC phosphorylation with the selective MLCK inhibitor ML-7 and transfection of a specific MLCK-inhibiting peptide. In contrast, transfection of constitutively active MLCK elevated Pa, which was abolished by ML-7. In addition to the vessel study, albumin transendothelial flux was measured in cultured bovine coronary venular endothelial monolayers, which displayed a hyperpermeability response to neutrophils and MLCK in a pattern similar to that in venules. Importantly, neutrophil stimulation caused MLC phosphorylation in endothelial cells in a time course closely correlated with that of the hyperpermeability response. Consistently, the MLCK inhibitors abolished neutrophil-induced MLC phosphorylation. Furthermore, immunohistochemical observation of neutrophil-stimulated endothelial cells revealed an increased staining for phosphorylated MLC in association with contractile stress fiber formation and intercellular gap development. Taken together, the results suggest that endothelial MLCK activation and MLC phosphorylation play an important role in mediating endothelial barrier dysfunction during neutrophil activation.
Abstract-The functional disturbance of microvasculature is recognized as an initiating mechanism that underlies the development of various diabetic complications. Although a causal relationship between microvascular leakage and tissue damage has been well documented in diabetic kidneys and eyes, there is a lack of information regarding the barrier function of coronary exchange vessels in the disease state. The aim of the present study was to evaluate the permeability property of coronary microvessels during the early development of experimental diabetes with a focus on the protein kinase C (PKC)-dependent signaling mechanism. The apparent permeability coefficient of albumin (Pa) was measured in isolated and perfused porcine coronary venules. The administration of high concentrations of D-glucose induced a dose-dependent increase in the Pa value, which was prevented by blockage of PKC with its selective inhibitors bisindolylmaleimide and Goe 6976. More importantly, an elevated basal permeability to albumin was observed in coronary venules at the early onset of streptozotocin-induced diabetes. The hyperpermeability was corrected with bisindolylmaleimide and the selective PKC inhibitor hispidin. Concomitantly, protein kinase assay showed a high PKC activity in isolated diabetic venules. Immunoblot analysis of the diabetic heart revealed a significant subcellular translocation of PKCII and PKC⑀ from the cytosol to the membrane, indicating that the specific activity of these isoforms was preferentially elevated. The results suggest that endothelial barrier dysfunction attributed to the activation of PKC occurs at the coronary exchange vessels in early diabetes. (Circ Res. 2000;87:412-417.)Key Words: diabetes Ⅲ microcirculation Ⅲ permeability Ⅲ protein kinases C ardiovascular complications represent the major cause of morbidity and mortality in patients with diabetes mellitus. A functional disturbance in the microvasculature independent of large-vessel atherosclerosis has been implicated in the development of end-organ damage and clinical abnormalities manifested as nephropathy, retinopathy, neuropathy, and skin ulceration. 1 Characteristic changes in the microcirculation during the early stages of diabetes include autoregulatory dysfunction and increased endothelial permeability. 1,2 Capillary leakage has been documented in various tissues of experimentally diabetic animals, including the retina, 3 aorta, 4 skin, intestine, kidney, 5 and heart. 6 Endothelial barrier dysfunction, occurring before microvascular sclerosis and other structural alterations, is considered to be one of the initiating mechanisms that underlies the pathogenesis of microangiopathic complications.The precise cause of vascular hyperpermeability in diabetes has not been established. Recent evidence suggests that hyperglycemia-induced de novo synthesis of diacylglycerol and the subsequent activation of protein kinase C (PKC) constitute an important signaling pathway that leads to endothelial dysfunction. 7,8 The PKC activity has been found to be...
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