Ablation of endothelial Pfkfb3 protects mice from acute lung injury in LPS-induced endotoxemia.

Acute lung injury (ALI) is one of the leading causes of death in sepsis. Endothelial inflammation and dysfunction play a prominent role in development of ALI. Glycolysis is the predominant bioenergetic pathway for endothelial cells (ECs). However, the role of EC glycolysis in ALI of sepsis remains unclear. Here we show that both the expression and activity of PFKFB3, a key glycolytic activator, were markedly increased in lipopolysaccharide (LPS)-treated human pulmonary arterial ECs (HPAECs) in vitro and in lung ECs of mice challenged with LPS in vivo. PFKFB3 knockdown significantly reduced LPS-enhanced glycolysis in HPAECs. Compared with LPS-challenged wild-type mice, endothelial-specific Pfkfb3 knockout (Pfkfb3ΔVEC) mice exhibited reduced endothelium permeability, lower pulmonary edema, and higher survival rate. This was accompanied by decreased expression of intracellular adhesion molecule-1 (Icam-1) and vascular cell adhesion molecule 1 (Vcam-1), as well as decreased neutrophil and macrophage infiltration to the lung. Consistently, PFKFB3 silencing or PFKFB3 inhibition in HPAECs and human pulmonary microvascular ECs (HPMVECs) significantly downregulated LPS-induced expression of ICAM-1 and VCAM-1, and monocyte adhesion to human pulmonary ECs. In contrast, adenovirus-mediated PFKFB3 overexpression upregulated ICAM-1 and VCAM-1 expression in HPAECs. Mechanistically, PFKFB3 silencing suppressed LPS-induced nuclear translocation of nuclear factor κB (NF-κB)-p65, and NF-κB inhibitors abrogated PFKFB3-induced expression of ICAM-1 and VCAM-1. Finally, administration of the PFKFB3 inhibitor 3PO also reduced the inflammatory response of vascular endothelium and protected mice from LPS-induced ALI. Overall, these findings suggest that targeting PFKFB3-mediated EC glycolysis is an efficient therapeutic strategy for ALI in sepsis.

Compounds of IL-6 Receptor Complex during Acute Lung Injury.

Soluble receptor of IL-6 (sIL-6R) and antagonist of the receptor complex, soluble glycoprotein 130 (sgp130) mediate opposite effects during inflammation. We measured the levels of these cytokines and their ratio in rat blood on the model of acute lung injury. The injury was modeled by the intratracheal administration of LPS. The levels of sgp130 and sIL-6R increased during the inflammatory process in the injured lungs. The sgp130/sIL-6R ratio increased or decreased depending on the intensity of the inflammatory process. sgp130/sIL-6R ratio might reflect the intensity of inflammation during lung injury.

[Endothelial cell cytoskeleton reorganization during functional monolayer formation in vitro].

The endothelium lining the inner surface of blood vessels regulates vascular permeability, permitting the exchange between the blood circulating in vessels and tissue fluid, and performs thereby the barrier function. Endothelial cells cultured in vitro retain the ability to perform a barrier function that is inherent in vascular endothelial cells in vivo. Endothelial monolayer in vitro is a unique model system that allows studying the interaction of cytoskeletal and adhesive structures of endothelial cells from the earliest stages of its formation. In this paper we describe and characterize quantitatively the changes in the cytoskeleton of endothelial cells from the time of endothelial cells spreading on the glass and formation of the first contacts between neighboring cells un- til the formation of a functional confluent monolayer. The main type of intermediate filaments of endothelial cells were vimentin filaments. The location of vimentin filaments and their number did not change at different stages of the endothelial monolayer formation, they occupied more than 80% of the cells. The system of actin filaments in endothelial cells was represented by the cortical actin at the cell periphery and bundles of actin stress fibers arranged in parallel. Upon the formation of contacts with neighboring cells the number and thickness of actin filaments increased. In addition, the formation of the endothelial monolayer led to changes in microtubule network, which was evident from the increase in the number of microtubules at the cell edge. Further, at all stages of assembling the endothelial monolayer, the number of microtubules formed at the cell margin in the area of cell-cell contacts exceeded the number of microtubules in the area of the free lamellae.

[The effect of Rho-kinase inhibition depends on the nature of factors that modify endothelial permeability].

Endothelium lining the inner surface of all vessels plays barrier role and regulates permeability of vascular walls controling the exchange between circulating blood and tissue fluids. Disturbance of normal functions (endothelial dysfunction) can be caused by both internal, and external factors. Endothelial dysfunction is characterized by increased vascular wall permeability observed in many human diseases. Dysfunction is also a drug side effect of oncological diseases treatment by mitosis-blocking medications. Depolymerization of microtubules is the first step in the cascade of reactions leading to endothelial barrier dysfunction, and this stage is universal, it does not depend upon the nature of a factor provoking dysfunction. To develop the strategy of barrier dysfunction prevention, we are supposed here to find out to what stage the endothelial cell cytoskeleton reaction during the development of barrier dysfunction is universal. It has been found that the cascade stages, which follow the microtubule depolymerization and are connected with Rho-Rho-kinases activity, have the features depending on the factor provoking barrier dysfunction. Under suppression of Rho-kinase activity, the reaction of actin filaments does not depend on what substance caused dysfunction. But the microtubule system responds to the treatment varies depending on the dysfunction-provoking factor. Unlike thrombin, under the conditions of Rho-kinase activity suppression, nocodazole renders more strong effect, as much as possible destroying both dynamic, and stable microtubules. Thus, regardless of the dysfunction provoking factor, the initial stages of dysfunction connected with the depolymerization of microtubules appear to be unalterable. Consequently, endothelial cell defence strategy should be based on cytoplasmatic microtubules protectors application instead of employment of the factors involved in the cascade at later stages as we assumed earlier.

[The microtubule system in endothelial barrier dysfunction: disassembly of peripheral microtubules and microtubules reorganization in internal cytoplasm].

Endothelial cell barrier dysfunction is often associated with dramatic cytoskeletal reorganization, activation of actomyosin contraction and finally gap formation. At present time the role of microtubules in endothelial cell barrier regulation is not fully understood, however a number of observations allow to assume that microtubules reaction is the extremely important part in development of endothelial dysfunction. These observations have been forced us to examine the role of microtubule system reorganization in endothelial cell barrier regulation. In quiescent endothelial cells microtubule density is the highest in the centrosome region and insignificant near the cell margin. The analysis of microtubules distribution after specific antibodies staining using the method of measurement of their fluorescence intensity has shown that in control endothelial cells the reduction of fluorescence intensity from the cell center to its periphery is described by the equation of an exponential regression. The hormone agent, thrombin (25 nM), causes rapid increase of endothelial cell barrier permeability accompanied by fast decrease in quantity of peripheral microtubules and reorganization of microtubule system in internal cytoplasm of endothelial cells (the decrease of fluorescence intensity is described by the equation of linear regress already through 10 min after the beginning of the treatment). Both effects are reversible -- through 60 min after the beginning of the treatment the microtubule network does not differ from normal one, so the microtubule system is capable to adapt for influence of a natural regulator thrombin. The microtubules reaction develops more quickly, than reorganization of the actin filaments system, which responsible for the subsequent changes in the cell shape during barrier dysfunction. Apparently, the microtubules are the first part in a circuit of the reactions leading to the pulmonary endothelial cell barrier compromise.

Sphingosine 1-phosphate promotes endothelial cell barrier integrity by Edg-dependent cytoskeletal rearrangement.

Substances released by platelets during blood clotting are essential participants in events that link hemostasis and angiogenesis and ensure adequate wound healing and tissue injury repair. We assessed the participation of sphingosine 1-phosphate (Sph-1-P), a biologically active phosphorylated lipid growth factor released from activated platelets, in the regulation of endothelial monolayer barrier integrity, which is key to both angiogenesis and vascular homeostasis. Sph-1-P produced rapid, sustained, and dose-dependent increases in transmonolayer electrical resistance (TER) across both human and bovine pulmonary artery and lung microvascular endothelial cells. This substance also reversed barrier dysfunction elicited by the edemagenic agent thrombin. Sph-1-P-mediated barrier enhancement was dependent upon G(ialpha)-receptor coupling to specific members of the endothelial differentiation gene (Edg) family of receptors (Edg-1 and Edg-3), Rho kinase and tyrosine kinase-dependent activation, and actin filament rearrangement. Sph-1-P-enhanced TER occurred in conjunction with Rac GTPase- and p21-associated kinase-dependent endothelial cortical actin assembly with recruitment of the actin filament regulatory protein, cofilin. Platelet-released Sph-1-P, linked to Rac- and Rho-dependent cytoskeletal rearrangement, may act late in angiogenesis to stabilize newly formed vessels, which often display abnormally increased vascular permeability.

RhoA S-nitrosylation as a regulatory mechanism influencing endothelial barrier function in response to G+-bacterial toxins.

Disruption of the endothelial barrier in response to Gram positive (G+) bacterial toxins is a major complication of acute lung injury (ALI) and can be further aggravated by antibiotics which stimulate toxin release. The integrity of the pulmonary endothelial barrier is mediated by the balance of disruptive forces such as the small GTPase RhoA, and protective forces including endothelium-derived nitric oxide (NO). How NO protects against the barrier dysfunction is incompletely understood and our goal was to determine whether NO and S-nitrosylation can modulate RhoA activity and whether this mechanism is important for G+ toxin-induced microvascular permeability. We found that the G+ toxin listeriolysin-O (LLO) increased RhoA activity and that NO and S-NO donors inhibit RhoA activity. RhoA was robustly S-nitrosylated as determined by biotin-switch and mercury column analysis. MS revealed that three primary cysteine residues are S-nitrosylated including cys16, cys20 and cys159. Mutation of these residues to serine diminished S-nitrosylation to endogenous NO and mutant RhoA was less sensitive to inhibition by S-NO. G+-toxins stimulated the denitrosylation of RhoA which was not mediated by S-nitrosoglutathione reductase (GSNOR), thioredoxin (TRX) or thiol-dependent enzyme activity but was instead stimulated directly by elevated calcium levels. Calcium-promoted the direct denitrosylation of WT but not mutant RhoA and mutant RhoA adenovirus was more effective than WT in disrupting the barrier integrity of human lung microvascular endothelial cells. In conclusion, we reveal a novel mechanism by which NO and S-nitrosylation reduces RhoA activity which may be of significance in the management of pulmonary endothelial permeability induced by G+-toxins.

Ca2+-induced conformational changes in cardiac troponin C as measured by N-(1-pyrene)maleimide fluorescence.

Bovine cardiac troponin C was modified by N-(1-pyrene)maleimide at Cys-35 and Cys-84; the Ca2+-induced conformational changes were followed by measuring pyrene fluorescence. In isolated troponin C, the saturation of Ca2+, Mg2+-sites leads to a simultaneous increase in the pyrene monomer as well as to a decrease in the pyrene excimer fluorescence, whereas the saturation of Ca2+-specific sites results in a slight decrease in the fluorescence of pyrene monomer. Troponin T does not influence the dependence of pyrene-troponin C fluorescence on Ca2+ concentration. Within the equimolar complex of troponin C and troponin I, the saturation of Ca2+, Mg2+-sites has no effect on pyrene fluorescence, whereas the saturation of Ca2+-specific sites leads to a simultaneous decrease of both pyrene monomer and pyrene excimer fluorescence. It is supposed that troponin I diminishes the conformational changes in troponin C that are induced by the saturation of Ca2+, Mg2+-sites and enhances the conformational changes induced by the saturation of Ca2+-specific sites of troponin C.

The Acute Respiratory Distress Syndrome: Mechanisms and Perspective Therapeutic Approaches.

Acute Respiratory Distress Syndrome (ARDS) is a severe lung inflammatory disorder with a 30-50% mortality. Sepsis and pneumonia are the leading causes of ARDS. On the cellular level there is pulmonary capillary endothelial cell permeability and fluid leakage into the pulmonary parenchyma, followed by neutrophils, cytokines and an acute inflammatory response. When fluid increases in the interstitium then the outward movement continues and protein rich fluid floods the alveolar spaces through the tight junctions of the epithelial cells. Neutrophils play an important role in the development of pulmonary edema associated with acute lung injury or ARDS. Animal studies have shown that endothelial injury appears within minutes to hours after Acute Lung Injury (ALI) initiation with resulting intercellular gaps of the endothelial cells. The Endothelial Cell (EC) gaps allow for permeability of fluid, neutrophils and cytokines into the pulmonary parenchymal space. The neutrophils that infiltrate the lungs and migrate into the airways express pro-inflammatory cytokines such as tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1ß), and contribute to both the endothelial and epithelial integrity disruption of the barriers. Pharmacological treatments have been ineffective. The ARDS Network trial identified low tidal volume mechanical ventilation, positive end expiratory pressure and fluid management guidelines that have improved outcomes for patients with ARDS. Extracorporeal membrane oxygenation is used in specialized centers for severe cases. Prone positioning has recently proven to have significantly decreased ventilator days and days in the intensive care unit. Current investigation includes administration of mesenchymal stem cell therapy, partial fluid ventilation, TIP peptide nebulized administration and the continued examination of pharmacologic drugs.

Regulation of endothelial barrier function by the cAMP-dependent protein kinase.

Elevation of cAMP promotes the endothelial cell (EC) barrier and protects the lung from edema development. Thus, we tested the hypothesis that both increases and decreases in PKA modulate EC function and coordinate distribution of regulatory, adherence, and cytoskeletal proteins. Inhibition of PKA activity by RpcAMPS and activation by cholera toxin was verified by assay of kemptide phosphorylation in digitonin permeabilized EC. Inhibition of PKA by RpcAMPS or overexpression of the endogenous inhibitor, PKI, decreased monolayer electrical impedance and exacerbated the decreases produced by agonists (thrombin and PMA). RpcAMPS directly increased F-actin content and organization into stress fibers, increased co-staining of actin with both phosphatase 2B and myosin light chain kinase (MLCK), caused reorganization of focal adhesions, and decreased catenin at cell borders. These findings are similar to those evoked by thrombin. In contrast, cholera toxin prevented the agonist-induced resistance decrease and protein redistribution. Although PKA activation attenuated thrombin-induced myosin light chain (MLC) phosphorylation, PKA inhibition per se did not cause MLC phosphorylation or affect [Ca2+]i. These studies indicate that a decrease in PKA activity alone can produce disruption of barrier function via mechanisms not involving MLCK and support a central role for cAMP/PKA in regulation of cytoskeletal and adhesive protein function in EC which correlates with altered barrier function.

Regulation of interleukin-1-stimulated GMCSF mRNA levels in human endothelium.

The regulation of interleukin-1 (IL-1)-mediated increases in GMCSF mRNA levels in human endothelium was examined and determined to occur in a time- and protein kinase C (PKC)-dependent manner. IL-1beta induced the early activation and translocation of PKC isotypes alpha and beta2 to the nucleus and PKC inhibition attenuated the IL-1-mediated increase in GMCSF mRNA levels. PKC activation by PMA alone, in the absence of IL-1beta activation, however, was insufficient to allow GMCSF mRNA detection. Increasing cyclic adenosine nucleotide (cAMP) levels suppressed IL-1beta-induced increases in GMCSF mRNA levels. In contrast, botulinum toxin C, which mediates the ADP ribosylation of a 21 kD ras-related G protein, augmented IL-1beta-induced GMCSF mRNA expression. Inhibition of protein synthesis (with cycloheximide) raised basal GMCSF mRNA transcripts to detectable levels, augmented IL-1-induced increases in GMCSF mRNA levels, and exhibited negative regulation by cAMP. Finally, disruption of either microtubules (with colchicine) or microfilaments (with cytochalasin B) resulted in reduced GMCSF mRNA expression in response to IL-1beta. These results are compatible with a model wherein IL-1-mediated increases in human endothelial cell GMCSF mRNA may be linked to both nuclear protein kinase C activation and activation of a low molecular weight G-protein, although neither activity alone is sufficient to increase the levels of GMCSF mRNA.

Role of tyrosine phosphorylation in thrombin-induced endothelial cell contraction and barrier function.

Thrombin-induced endothelial cell (EC) barrier dysfunction is highly dependent upon phosphorylation of serine and threonine residues present on myosin light chains (MLC) catalyzed by a novel EC myosin light chain kinase (MLCK) isoform. In this study, we examined the participation of tyrosine protein phosphorylation in EC contraction, gap formation and barrier dysfunction. We first determined that thrombin significantly increases protein tyrosine kinase activity and protein tyrosine phosphorylation in bovine pulmonary artery EC. Tyrosine kinase inhibitors, genistein and 2,5 DHC, reduced EC tyrosine kinase activities, however, only genistein significantly attenuated thrombin-mediated increases in albumin clearance and reductions in transendothelial electrical resistance. Similarly, genistein but not 2,5 DHC, decreased basal and thrombin-induced Ca2+ increases and MLC phosphorylation in the absence of alterations in Type 1 or 2A serine/threonine phosphatase activities. Immunoprecipitation of the EC MLCK isoform revealed a 214 kD immunoreactive phosphotyrosine protein and genistein pretreatment significantly reduced MLCK activity in MLCK immunoprecipitates. Although thrombin induced the translocation of p60src from the cytosol to the EC cytoskeleton, a detectable increase in the level of MLCK tyrosine phosphorylation was not noted after thrombin challenge. Taken together, our data suggest that genistein-sensitive tyrosine kinase activities are involved in thrombin-mediated EC MLCK activation, MLC phosphorylation, and barrier dysfunction.

Adherent neutrophils activate endothelial myosin light chain kinase: role in transendothelial migration.

Increased vascular endothelial cell (EC) permeability and neutrophilic leukocyte (PMN) diapedesis through paracellular gaps are cardinal features of acute inflammation. Activation of the EC contractile apparatus is necessary and sufficient to increase vascular permeability in specific models of EC barrier dysfunction. However, it is unknown whether EC contraction with subsequent paracellular gap formation is required for PMN transendothelial migration in response to chemotactic factors. To test this possibility, we assessed migration of human PMNs across confluent bovine pulmonary arterial EC monolayers. Transendothelial PMN migration in the absence of a chemotactic gradient was minimal, whereas abluminal addition of leukotriene B4 (LTB4; 5 microM) resulted in significantly increased PMN migration. Reductions in EC myosin light chain kinase (MLCK) activity by EC monolayer pretreatment with specific MLCK inhibitors (KT-5926 or ML-7) or by increases in cAMP-protein kinase A activity (cholera toxin) significantly reduced PMN transmigration (30-70% inhibition). In contrast, pretreatment with the myosin-associated phosphatase inhibitor calyculin resulted in the accumulation of phosphorylated myosin light chains, EC contraction, and significantly enhanced PMN migration. Finally, the interaction of PMNs with 32P-labeled EC monolayers was shown to directly increase EC myosin phosphorylation in a time-dependent fashion. Taken together, these results are consistent with the hypothesis that the phosphorylation status of EC myosin regulates PMN migration and further indicate that EC MLCK is activated by chemoattractant-stimulated PMNs. Neutrophil-dependent activation of the EC contractile apparatus with subsequent paracellular gap formation may be a key determinant of transendothelial PMN migration responses to chemotactic agents.

Diperoxovanadate alters endothelial cell focal contacts and barrier function: role of tyrosine phosphorylation.

Diperoxovanadate (DPV), a potent tyrosine kinase activator and protein tyrosine phosphatase inhibitor, was utilized to explore bovine pulmonary artery endothelial cell barrier regulation. DPV produced dose-dependent decreases in transendothelial electrical resistance (TER) and increases in permeability to albumin, which were preceded by brief increases in TER (peak TER effect at 10-15 min). The significant and sustained DPV-mediated TER reductions were primarily the result of decreased intercellular resistance, rather than decreased resistance between the cell and the extracellular matrix, and were reduced by pretreatment with the tyrosine kinase inhibitor genistein but not by inhibition of p42/p44 mitogen-activating protein kinases. Immunofluorescent analysis after DPV challenge revealed dramatic F-actin polymerization and stress-fiber assembly and increased colocalization of tyrosine phosphoproteins with F-actin in a circumferential pattern at the cell periphery, changes that were abolished by genistein. The phosphorylation of focal adhesion and adherens junction proteins on tyrosine residues was confirmed in immunoprecipitates of focal adhesion kinase and cadherin-associated proteins in which dramatic dose-dependent tyrosine phosphorylation was observed after DPV stimulation. We speculate that DPV enhances endothelial cell monolayer integrity via focal adhesion plaque phosphorylation and produces subsequent monolayer destabilization of adherens junctions initiated by adherens junction protein tyrosine phosphorylation catalyzed by p60(src) or Src-related tyrosine kinases.

[Role of phosphorylation of myosin and actin-binding proteins in endothelial permeability induced by thrombin]. / Rol' fosforilirovania miozina na aktinsviazyvaiushchikh belkov v indutsirovannoi trombinom pronitsaemosti éndoteliia.

The thrombin-induced dysfunction of the barrier function of the blood vessel endothelium, which manifests itself in increased permeability, is largely mediated via the initiation of specific receptors that trigger multiple signaling cascades, including the activation of some protein kinases and the phosphorylation of their cytoskeletal targets. The role of the phosphorylation of myosin and actin-binding proteins in the thrombin-induced permeability of the endothelium and possible mechanisms of the regulation of the endothelium barrier function are discussed.

[Reorganization of microtubule system in pulmonary endothelial cells in response to thrombin treatment]. / Reorganizatsiia sistemy mikrotrubochek v kletkakh legochnogo éndoteliia v otvet na vozdeistvie trombina.

Thrombin induces rapid and reversible increase of endothelial (EC) barrier permeability associated with actin cytoskeleton remodeling and contraction. The role of microtubules (Mts) in EC barrier regulation compared with actin systems is poorly understood. In this work we studied pathways of Mt and actin regulation in response to thrombin treatment in cultured EC, and the involvement of trimeric G-proteins and in this process. Cells were treated with thrombin, and further analysed using immunofluorescent staining of actin and Mts, digital microscopy and morphometric analysis. In normal cells actin network consists of thin bundles basically located in the cell periphery, Mt density decreases from the cell center to the cell edge. Thrombin (25 nM) induced endothelial dysfunction associated with a rapid (within 5 min) decrease of peripheral Mt network and a slower actin stress fiber formation in the cytoplasm. Pretreatment with Pertussis toxin, which is Gi protein inhibitor, attenuated thrombin-induced stress fiber formation and Mt disassembly. Overexpression of activated G12, G13, Gi and Gq proteins, which are involved in thrombin receptor-mediated signaling, resulted in increasing stress fibers thickness and density and complete Mt disassembly. From the results obtained we suggest that thrombin regulates actin cytoskeleton of EC using local Mt depolymerization at the cell edge.

Suppression of endotoxin-induced inflammation by taxol.

The pathogenesis of acute lung injury includes transendothelial diapedesis of leukocytes into lung tissues and disruption of endothelial/epithelial barriers leading to protein-rich oedema. In vitro studies show that the microtubule network plays a role in the regulation of endothelial permeability as well as in neutrophil locomotion. It was hypothesised that the microtubule-stabilising agent, taxol, might attenuate inflammation and vascular leak associated with acute lung injury in vivo. The effect of intravenously delivered taxol was assessed using a model of murine lung injury induced by intratracheal lipopolysaccharide (LPS) administration. Parameters of lung injury and inflammation were assessed 18 h after treatment. Intravenously delivered taxol significantly reduced inflammatory histological changes in lung parenchyma and parameters of LPS-induced inflammation: infiltration of proteins and inflammatory cells into bronchoalveolar lavage fluid, lung myeloperoxidase activity, and extravasation of Evans blue-labelled albumin into lung tissue. Taxol alone (in the absence of LPS) had no appreciable effect on these parameters. In addition to lung proteins, intravenous taxol reduced accumulation of leukocytes in ascitic fluid in a model of LPS-induced peritonitis. Taken together, the present data demonstrate that microtubule stabilisation with taxol systemically attenuates lipopolysaccharide-induced inflammation and vascular leak.

Diverse effects of vascular endothelial growth factor on human pulmonary endothelial barrier and migration.

Increased endothelial permeability is involved in the pathogenesis of many cardiovascular and pulmonary diseases. Vascular endothelial growth factor (VEGF) is a permeability-increasing cytokine. At the same time, VEGF is known to have a beneficial effect on endothelial cells (EC), increasing their survival. Pulmonary endothelium, particularly, may be exposed to higher VEGF concentrations, since the VEGF level is the higher in the lungs than in any other organ. The purpose of this work was to evaluate the effects of VEGF on barrier function and motility of cultured human pulmonary EC. Using transendothelial resistance measurements as an indicator of permeability, we found that 10 ng/ml VEGF significantly improved barrier properties of cultured human pulmonary artery EC (118.6+/-0.6% compared with 100% control, P<0.001). In contrast, challenge with 100 ng/ml VEGF decreased endothelial barrier (71.6+/-1.0% compared with 100% control, P<0.001) and caused disruption of adherens junctions. VEGF at both concentrations increased cellular migration; however, 10 ng/ml VEGF had a significantly stronger effect. VEGF caused a dose-dependent increase in intracellular Ca2+ concentration; however, phosphorylation of myosin light chain was detectably elevated only after treatment with 100 ng/ml. In contrast, 10 ng/ml but not 100 ng/ml VEGF caused a significant increase in intracellular cAMP (known barrier-protective stimulus) compared with nonstimulated cells (1,096+/-157 and 610+/-86 fmol/mg, respectively; P<0.024). Y576-specific phosphorylation of focal adhesion kinase was also stimulated by 10 ng/ml VEGF. Our data suggest that, depending on its concentration, VEGF may cause diverse effects on pulmonary endothelial permeability via different signaling pathways.

[The role of the N-terminal fragment of troponin T in the interaction with troponin-tropomyosin complex components of the heart]. / Izuchenie roli N-kontsevogo fragmenta troponina T vo vzaimodeistvii s komponentami troponintropomiozinovogo kompleksa serdtsa.

Bovine heart troponin T was hydrolyzed at the single cysteine residue. This procedure resulted in two peptides--a short N-terminal peptide (40-50 amino acid residues) and a long C-terminal peptide (240 amino acid residues). The C-terminal peptide was purified to homogeneity by ion-exchange chromatography; its properties were compared to those of intact troponin T. Data from circular dichroism spectroscopy suggest that the short N-terminal peptide cleavage was unaccompanied by any conspicuous changes in the secondary structure of the large C-terminal peptide of troponin T. Unlike intact troponin T, its C-terminal peptide can interact with troponin C in the presence of Ca2+. Data from affinity chromatography demonstrated that troponin I and tropomyosin more strongly interacted with native troponin T than with its C-terminal peptide. It is concluded that the short N-terminal peptide (40-50 residues) plays an essential role in cardiac troponin T interaction with troponin and tropomyosin components.

[Various properties of cardiac troponin C tryptic peptides]. / Izuchenie nekotorykh svoistv tripticheskikh peptidov troponina C serdtsa.

Using chromatography and preparative polyacrylamide gel electrophoresis, tryptic peptides TP 1 (residues 47-83), TP 2 (residues 84-118) and TP 3 (residues 119-161) were isolated in a highly homogeneous state from cardiac troponin C. Peptides TP 1, TP 2 and TP 3 were found to contain isolated cation-binding sites II, III and IV of cardiac troponin C. The interaction of these peptides with troponins I and T was studied. It was found that only peptide TP 2 could interact with troponin I. Neither of the peptides isolated interacted with troponin T. The cation-binding properties and structural peculiarities of peptide TP 1 were investigated. It was shown that despite its small size (37 amino acid residues), peptide TP 1 retained its ability to bind Ca2+ which caused conformational changes in the peptide structure. This was accompanied by changes in the electrophoretic mobility and absorption of TP 1 on phenyl-Sepharose.