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Gainor JP Morton CA Roberts JT Vincent PA Minnear FL 《American journal of physiology. Heart and circulatory physiology》2001,281(5):H1992-H2001
Platelets release a soluble factor into blood and conditioned medium (PCM) that decreases vascular endothelial permeability. The objective of this study was to determine the signal-transduction pathway that elicits this decrease in permeability. Permeability-decreasing activity of PCM was assessed by the real-time measurement of electrical resistance across cell monolayers derived from bovine pulmonary arteries and microvessels. Using a desensitization protocol with cAMP/protein kinase A (PKA)-enhancing agents and pharmacological inhibitors, we determined that the activity of PCM is independent of PKA and PKG. Genistein, an inhibitor of tyrosine kinases, prevented the increase in endothelial electrical resistance. Because lysophosphatidic acid (LPA) has been proposed to be responsible for this activity of PCM and is known to activate the G(i) protein, inhibitors of the G protein pertussis toxin and of the associated phosphatidylinositol 3-kinase (PI3K) wortmannin were used. Pertussis toxin and wortmannin caused a 10- to 15-min delay in the characteristic rise in electrical resistance induced by PCM. Inhibition of phosphorylation of extracellular signal-regulated kinase with the mitogen-activated kinase kinase inhibitors PD-98059 and U-0126 did not prevent the activity of PCM. Similar findings with regard to the cAMP protocols and inhibition of G(i) and PI3K were obtained for 1-oleoyl-LPA. These results demonstrate that PCM increases endothelial electrical resistance in vitro via a novel, signal transduction pathway independent of cAMP/PKA and cGMP/PKG. Furthermore, PCM rapidly activates a signaling pathway involving tyrosine phosphorylation, the G(i) protein, and PI3K. 相似文献
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Xu M Waters CL Hu C Wysolmerski RB Vincent PA Minnear FL 《American journal of physiology. Cell physiology》2007,293(4):C1309-C1318
Sphingosine 1-phosphate (S1P) rapidly increases endothelial barrier function and induces the assembly of the adherens junction proteins vascular endothelial (VE)-cadherin and catenins. Since VE-cadherin contributes to the stabilization of the endothelial barrier, we determined whether the rapid, barrier-enhancing activity of S1P requires VE-cadherin. Ca(2+)-dependent, homophilic VE-cadherin binding of endothelial cells, derived from human umbilical veins and grown as monolayers, was disrupted with EGTA, an antibody to the extracellular domain of VE-cadherin, or gene silencing of VE-cadherin with small interfering RNA. All three protocols caused a reduction in the immunofluorescent localization of VE-cadherin at intercellular junctions, the separation of adjacent cells, and a decrease in basal endothelial electrical resistance. In all three conditions, S1P rapidly increased endothelial electrical resistance. These findings demonstrate that S1P enhances the endothelial barrier independently of homophilic VE-cadherin binding. Junctional localization of VE-cadherin, however, was associated with the sustained activity of S1P. Imaging with phase-contrast and differential interference contrast optics revealed that S1P induced cell spreading and closure of intercellular gaps. Pretreatment with latrunculin B, an inhibitor of actin polymerization, or Y-27632, a Rho kinase inhibitor, attenuated cell spreading and the rapid increase in electrical resistance induced by S1P. We conclude that S1P rapidly closes intercellular gaps, resulting in an increased electrical resistance across endothelial cell monolayers, via cell spreading and Rho kinase and independently of VE-cadherin. 相似文献
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Minnear FL Zhu L He P 《American journal of physiology. Heart and circulatory physiology》2005,289(2):H840-H844
Sphingosine 1-phosphate (S1P) is a biologically active lipid. In vitro, S1P tightens the endothelial barrier, as assessed by a rapid increase in electrical resistance and a decrease in solute permeability. We hypothesized that this activity of S1P would also occur in vivo. Hydraulic conductivity (Lp), an assessment of endothelial barrier function, was measured in individually perfused venules in rat mesenteries. S1P (1 microM) decreased basal Lp by 63% when basal Lp was between 3.6 and 4.1 x 10(-7) cm x s(-1) x cmH2O(-1) but showed no effect when basal Lp was below 2 x 10(-7) cm x s(-1) x cmH2O(-1). Under either condition, S1P blocked the sixfold increase in Lp induced by platelet-activating factor (PAF, 10 nM). Perfusion of venules with pertussis toxin (0.1 microg/ml), a specific inhibitor of the inhibitory G protein, Gi, for 3 h did not affect basal Lp or the increased Lp induced by PAF. Pertussis toxin, however, significantly attenuated the inhibitory action of S1P on the PAF-induced increase in Lp, indicating the involvement of the Gi protein. Measurement of endothelial cytoplasmic Ca2+ concentration ([Ca2+]i) in venules loaded with fura-2 AM showed that S1P alone transiently increased basal endothelial [Ca2+]i (from 89 nM to 193 nM) but had no effect on the magnitude and time course of the PAF-induced increase in endothelial [Ca2+]i. These results indicate that S1P functions in vivo to prevent the PAF-induced increase in microvessel permeability. The inhibitory action of S1P involves the pertussis toxin-sensitive Gi protein and is not mediated by prevention of the PAF-induced increase in endothelial [Ca2+]i. 相似文献
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Ferreri DM Minnear FL Yin T Kowalczyk AP Vincent PA 《Cell communication & adhesion》2008,15(4):333-349
Endothelial cells (ECs) express VE-cadherin and N-cadherin, and recent data suggest that VE-cadherin levels are dependent on N-cadherin expression. While investigating changes in N-cadherin levels during endothelial monolayer maturation, the authors found that VE-cadherin levels are maintained in ECs despite a decrease in N-cadherin, suggesting that VE-cadherin levels may not depend on N-cadherin. Knockdown of N-cadherin did not affect VE-cadherin levels in ECs with low endogenous N-cadherin expression. Surprisingly, however, knockdown of N-cadherin in ECs with high endogenous N-cadherin expression increased VE-cadherin levels, suggesting an inverse relationship between the two. This was further supported by a decrease in VE-cadherin following overexpression of N-cadherin. Experiments in which p120, a catenin that binds N- and VE-cadherin, was knocked down or overexpressed indicate that these two cadherins compete for p120. These data demonstrate that VE-cadherin levels are not directly related to N-cadherin levels but may be inversely related due to competition for p120. 相似文献
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Rodrigues PC Aguiar O Serpieri F Lima AP Uetanebaro M Recco-Pimentel SM 《Journal of genetics》2011,90(1):31-37
Dendrobatid frogs have undergone an extensive systematic reorganization based on recent molecular findings. The present work
describes karyotypes of the Brazilian species Adelphobates castaneoticus, A. quinquevittatus, Ameerega picta, A. galactonotus and Dendrobates tinctorius which were compared to each other and with previously described related species. All karyotypes consisted of 2n = 18 chromosomes, except for A. picta which had 2n = 24. The karyotypes of the Adelphobates and D. tinctorius species were highly similar to each other and to the other 2n = 18 previously studied species, revealing conserved karyotypic characteristics in both genera. In recent phylogenetic studies,
all Adelphobates species were grouped in a clade separated from the Dendrobates species. Thus, we hypothesized that their common karyotypic traits may have a distinct origin by chromosome rearrangements
and mutations. In A. picta, with 2n = 24, chromosome features of pairs from 1 to 8 are shared with other previously karyotyped species within this genus. Hence,
the A. picta data reinforced that the C-banding pattern and the NOR location are species-specific traits in the genus Ameerega. Moreover, the Ameerega monophyletism proposed by previous phylogenetic studies indicates that the karyotypic differences among species in this genus
result from a long divergence time. 相似文献